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Being a supersaturated solution of sugar, honey pulls the water out of cells it comes in contact with via osmosis - killing the cells. It also contains the inactive enzyme glucose oxidase, which when diluted with water activates and then converts glucose into gluconic acid and hydrogen peroxide - both of which are bad news for cells.
Honey also likely contains an antibiotic protein Bee Defensin-1, which is also bad news for bacteria. I didn't yet look into how it works so I don't know if it affects our epithelial cells.
Then some honey types (manuka) contain Methylglyoxal. Damage by methylglyoxal to low-density lipoprotein through glycation causes a fourfold increase of atherogenesis in diabetics.  Methylglyoxal binds directly to the nerve endings and by that increases the chronic extremity soreness in diabetic neuropathy.
How is it safe to eat?
I'll change my initial comment into a real answer.
The human digestive tract has evolved to allow us to be broadly omnivorous, safely consuming a wide range of vegetable, animal, and mineral-based foods. While honey can be damaging to exposed cells, the epithelial cells lining the digestive tract are not exposed like the endothelial cells that line the circulatory system. Eating honey is not the same as injecting it into your veins.
A layer of mucous covers the cells of the digestive tract from mouth to colon and provides both lubrication as well as protection to the cells underneath it. It is protective to varying degrees against such challenges as acid, osmotic stress, mechanical damage, and foreign microorganisms. Additionally, the cells themselves are layered and are continually being sloughed off and regenerated, so even if the mucosal lining isn't 100% effective, damaged cells will be replaced fairly quickly.
Food quickly travels from the mouth through the esophagus to the stomach, not allowing enough time for osmotic stress or enzymes to damage anything to any significant degree. (Obviously, if you swallow something extremely harsh like bleach, lye, or concentrated acid, that's a different story.)
Once in the stomach, a food like honey is diluted by the digestive juices and any other food or drink already present -- it's not like the stomach is totally empty and globs of honey can just sit in direct contact with cells for an extended period of time, allowing for osmotic or enzymatic damage. Digestive enzymes and the acidic environment quickly attack proteins and other substrates, so even if the glucose oxidase can become activated, it will be cleaved by proteases into inactive fragments long before damaging levels of H2O2 can build up.
Low-density lipoprotein (LDL) is found in the bloodstream, not in the digestive tract, so any methylglyoxal present in food would need to be actively transported into the blood to do any damage. The work in the paper you cited was done completely in vitro, with no evidence that MG-mediated glycation as a result of eating honey actually occurs in people.
Many foods we commonly eat would appear to be dangerous to our bodies in one way or another, yet we have evolved strong mechanisms to minimize harm. However, it's pretty clear that some foods are damaging in various ways over time, so we still need to moderate what we eat. I don't know if honey (or certain kinds of it) is one of those foods, so more research is needed.
Defective epithelial barriers linked to two billion chronic diseases
Epithelial cells form the covering of most internal and external surfaces of the human body. This protective layer acts as a defense against invaders -- including bacteria, viruses, environmental toxins, pollutants and allergens. If the skin and mucosal barriers are damaged or leaky, foreign agents such as bacteria can enter into the tissue and cause local, often chronic inflammation. This has both direct and indirect consequences.
Chronic diseases due to defective epithelial barriers
Cezmi Akdis, Director of the Swiss Institute of Allergy and Asthma Research (SIAF), which is associated with the University of Zurich (UZH), has now published a comprehensive summary of the research on epithelial barrier damage in Nature Reviews Immunology. "The epithelial barrier hypothesis proposes that damages to the epithelial barrier are responsible for up to two billion chronic, non-infectious diseases," Professor Akdis says. In the past 20 years, researchers at the SIAF alone published more than 60 articles on how various substances damage the epithelial cells of a number of organs.
Rise in allergic and autoimmune conditions
The epithelial barrier hypothesis provides an explanation as to why allergies and autoimmune diseases have been increasing for decades -- they are linked to industrialization, urbanization and westernized lifestyle. Today many people are exposed to a wide range of toxins, such as ozone, nanoparticles, microplastics, household cleaning agents, pesticides, enzymes, emulsifiers, fine dust, exhaust fumes, cigarette smoke and countless chemicals in the air, food and water. "Next to global warming and viral pandemics such as COVID-19, these harmful substances represent one of the greatest threats to humankind," emphasizes Akdis.
Asthma, Alzheimer's and more
Local epithelial damage to the skin and mucosal barriers lead to allergic conditions, inflammatory bowel disorders and celiac disease. But disruptions to the epithelial barrier can also be linked to many other diseases that are characterized by changes in the microbiome. Either the immune system erroneously attacks "good" bacteria in healthy bodies or it targets pathogenic -- i.e. "bad" -- invaders. In the gut, leaky epithelial barriers and microbial imbalance contribute to the onset or development of chronic autoimmune and metabolic diseases such as diabetes, obesity, rheumatoid arthritis, multiple sclerosis or ankylosing spondylitis. Moreover, defective epithelial barriers have also been linked to neurodegenerative and psychiatric diseases such as Parkinson's disease, Alzheimer's disease, autism spectrum disorders and chronic depression, which may be triggered or aggravated by distant inflammatory responses and changes in the gut's microbiome.
Prevention, intervention -- and more research
"There is a great need to continue research into the epithelial barrier to advance our understanding of molecular mechanisms and develop new approaches for prevention, early intervention and therapy," says Akdis. Novel therapeutic approaches could focus on strengthening tissue-specific barriers, blocking bacteria or avoiding colonization by pathogens. Other strategies to reduce diseases may involve the microbiome, for example through targeted dietary measures. Last but not least, the focus must also be on avoiding and reducing exposure to harmful substances and developing fewer toxic products.
How is honey not toxic to our epithelial cells? - Biology
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Honey and Cancer: Sustainable Inverse Relationship Particularly for Developing Nations—A Review
Honey and cancer has a sustainable inverse relationship. Carcinogenesis is a multistep process and has multifactorial causes. Among these are low immune status, chronic infection, chronic inflammation, chronic non healing ulcers, obesity, and so forth. There is now a sizeable evidence that honey is a natural immune booster, natural anti-inflammatory agent, natural antimicrobial agent, natural cancer “vaccine,” and natural promoter for healing chronic ulcers and wounds. Though honey has substances of which the most predominant is a mixture of sugars, which itself is thought to be carcinogenic, it is understandable that its beneficial effect as anticancer agent raises skeptics. The positive scientific evidence for anticancer properties of honey is growing. The mechanism on how honey has anticancer effect is an area of great interest. Among the mechanisms suggested are inhibition of cell proliferation, induction of apoptosis, and cell-cycle arrest. Honey and cancer has sustainable inverse relationship in the setting of developing nations where resources for cancer prevention and treatment are limited.
1. Cancer: The Global Epidemic
Cancer is a global epidemic. In 2008, it was estimated there were 12,332,300 cancer cases of which 5.4 million were in developed countries and 6.7 million were in developing countries  (Figure 1). Over half of the incident cases occurred in residents of four WHO regions. The world population increased from 6.1 billion in 2000 to 6.7 billion in 2008 . The increase in populations was much more in developing countries than in developed countries. Even if the age-specific rates of cancer remain constant, developing countries would have a higher cancer burden than developed countries.
Cancer trends are showing upward trends in many developing countries [3–5] and a mixed pattern in developed countries [6–8]. By 2050, the cancer burdencould reach 24 million cases per year worldwide, with 17 million cases occurring in developing countries . Cancers which are associated with diet and life style are seen more in developed countries while cancers which are due to infections are more in developing countries. According to the World Health Organization (WHO), death from cancer is expected to increase to 104% worldwide by 2020.
While the number of total cancer is increasing, the trend of certain cancers is changing in developed and developing countries. In developed countries, the trend is declining  since infections by microorganisms are declining and screening facilities are available. In Singapore, there was an average annual increase of 3.6% for breast cancers in women in the 1988–1992 period . In Qatar, there was a 57.1% rise of cancers 1991–2006 , and in Netherlands, there was an increase between 1.9% (females) and 3.4% (males) per year for oesophageal cancer 1989–2003 .
In order to understand the usefulness of honey in cancer, we need to understand the various factors which could cause cancer. Carcinogenesis is a multi-step process and has multi-factorial causes. Development of cancers takes place long after initiation, promotion, and progression steps (Figure 2) have taken place. The cellular damage could be by one factor or multiplicity of these factors. The latter is more frequent. Cancer development could occur 10–15 years after exposure to the risk factors.
1.1. Life-Style Habits/Diseases as Risks to Cancer Development
Cancer is caused by genetic damage in the genome of cells. This damage is either inherited or acquired throughout life. The acquired genetic damage is often “self-inflicted” through unhealthy lifestyles. Essentially one-third of cancer is due to tobacco use, one-third due to dietary and lifestyle factors, and one-fifth due to infections. Other factors include chemical carcinogens, environmental pollutants, and alcohol (Figure 3). In the developing countries, cancers caused by infections by microorganisms such as cervical (by human papilloma virus) , liver (by hepatitis viruses) , nasopharynx (by Epstein-Barr virus) , and stomach (by Helicobacter pylori)  are more common than those in developed countries . While cancers of the prostate, breasts, and colorectal are clearly more prevalent in developed than developing countries, the distinction is not very apparent as that for cancer of the lung which is as prevalent as that in more or less developed nations. Except for breast cancers, the top 5 cancers in males and females of developing nations are due to life-styles or infections .
1.1.1. Smoking and Tobacco Use
Association of cancer to cigarette smoking is beyond doubt. The prevalence of smoking is higher in developing than that in developed countries . Smoking is associated with a number of cancers such as larynx, bladder, breasts, oesophagus, and cervix. While in developed countries the prevalence of smoking is decreasing , the scenario is the reverse in developing countries. The initiation and the influence to start smoking are similar to those in developed countries . Smoking increases the risk of colorectal carcinomas by 43% . Ever-smokers were associated with an 8.8-fold increased risk of colorectal cancers (95% confidence interval, 1.7–44.9) when fed on well-done red meat diet if they have NAT2 and CYP1A2 rapid phenotypes . No similar association was found in never-smokers .
1.1.2. Obesity and Physical Inactivity
Obese subjects have an approximately 1.5–3.5-fold increased risk of developing cancers compared with normal-weight subjects . Obesity is associated in a number of cancers [25, 26] particularly endometrium [27, 28], breasts [29, 30], and colorectal cancers . Adipocytes have the ability to enhance the proliferation of colon cancer cells in vitro . The trend of prevalence of overweight/obesity is rising in many developed and developing countries . In a study conducted in 2005  in the Kota Bharu district in the state of Kelantan Malaysia, the overall prevalence of overweight/obesity was 49.1% , much higher than the figure reported earlier in 1996 . In this community, the rise of cancer is exponential in the period from year 2002 to 2007 (143.6% increment) compared to the previous 5-year period of 1996–2001 .
Obesity is not a social problem but a disease. The greatest risk is for obese persons who are also diabetic, particular those whose body mass index is above 35 kg/m 2 . The increase in risk is by 93-fold in women and by 42-fold in men .
1.1.3. Diabetes Particularly Type 2 as Risk for Cancer Development
Obesity is closely related with diabetes . A community that has high prevalence of obesity also has high prevalence of diabetes . In Kelantan, Malaysia, the prevalence of diabetes in 1999 was 10.5%, and impaired glucose tolerance was 16.5% . Kelantan is ranked highest in prevalence of diabetes in Malaysia in which the overall national prevalence is 8.3% , thus it was not a surprise to see a rapid rise of cancer prevalence in the state . According to a review on diabetes, the WHO has estimated that, by 2030, there would be 2.48 million diabetics in Malaysia, a jump of 164% from 0.94 million in 2002 . One of the most common cancers noted in community that has high diabetics and obesity is colorectal cancer [42–45].
In a study of 138 colorectal cancers (CRC) seen in Hospital Universiti Sains Malaysia, 47.8% had metabolic diseases, of which 13.8% were diabetes type 2 . Those diabetics with CRC often have distal cancers .
1.2. Chronic Infections as Risk for Cancer Development
There are a number of microorganisms which could cause cancer. Common viruses causing cancers  are Epstein-Barr virus (EBV)  (nasopharyngeal carcinomas), human papilloma virus (cervical cancers and other squamous cancers) and Hepatitis B viruses (liver cancers). Viruses are oncogenic after long period of latency .
Bacteria which has been studied to have associations with cancer are Helicobacter pylori infections (stomach cancer) , Ureaplasma urealyticum (prostate cancer) , and chronic typhoid carrier (gall bladder cancer) . Chronic fungi infections have also been studied to be associated with cancer . Parasites such as Schistosoma haematobium are associated with carcinoma of the urinary bladder liver flukes Opisthorchis viverrini and Clonorchis sinensis associated with cholangiocarcinoma and hepatocellular carcinoma. There are three main mechanisms by which infections can cause cancer. They appear to involve initiation as well as promotion of carcinogenesis . Persistent infection within host induces chronic inflammation accompanied by formation of reactive oxygen and nitrogen species (ROS and RNOS) . ROS and RNOS have the potential to damage DNA, proteins, and cell membranes. Chronic inflammation often results in repeated cycles of cell damage leading to abnormal cell proliferation . DNA damage promotes the growth of malignant cells. Secondly, infectious agents may directly transform cells, by inserting active oncogenes into the host genome, inhibiting tumour suppressors or stimulating mitosis . Thirdly, infectious agents, such as human immunodeficiency virus (HIV), may induce immunosuppression .
2. Low Immune Status as Risk of Cancer Development
2.1. Cancer and Aging
The most important change that would occur in the world population in the next 50 years is the change in the proportion of elderly people (more than 65 years): 7% in 2000 to 16% in 2050 . Many cancers are associated with aging. Although age per se is not an important determinant of cancer risk, it implies prolonged exposure to carcinogen . By the year 2050, 27 million people are projected to have cancer. More than half of the estimated number will be residents of developing countries . Aging is also associated with reduced immune system.
2.2. Low Immune Status due to Chronic Diseases
Patients who have low immune system are at risk for cancer development. This explains why diabetics are more at risk than non-diabetics to get epithelial cancers. HIV patients are at risk to develop epithelial and nonepithelial cancers. These persons are also at risk to develop multiple chronic infections implying the multiplicity in cancer genesis. Patients with autoimmune diseases are also at risk to develop cancers such as colorectal carcinomas in ulcerative colitis and Crohn’s disease and thyroid cancer in autoimmune thyroiditis.
2.3. Chronic Ulcers and Wounds
Chronic ulcers have risk to develop cancer. The most common is Marjolin’s ulcer , and they are common in developing nations especially in rural areas with poor living conditions . This risk factor is related to chronic infections as most if not all chronic ulcers are not healing because of persistent infections.
3. What Is Honey and Why Is It Useful against Cancer? (See Figure 4)
Honey is known for centuries for its medicinal and health-promoting properties. It contains various kinds of phytochemicals with high phenolic and flavonoid content which contribute to its high antioxidant activity [58–60]. Agent that has strong antioxidant property may have the potential to prevent the development of cancer as free radicals and oxidative stress play a significant role in inducing the formation of cancers . Phytochemicals available in honey could be narrowed down into phenolic acids and polyphenols. Variants of polyphenols in honey were reported to have antiproliferative property against several types of cancer .
4. Honey As a Natural Immune Booster
Honey stimulates inflammatory cytokine production from monocytes . Manuka, pasture, and jelly bush honey were found to significantly increase TNF-α, IL-1β, and IL-6 release from MM6 cells (and human monocytes) when compared with untreated and artificial honey-treated cells (
) . A 5.8 kDa component of manuka honey was found to stimulate cytokine production from immune cells via TLR4 . Honey stimulates antibody production during primary and secondary immune responses against thymus-dependent and thymus-independent antigens in mice injected with sheep red blood cells and E. coli antigen . Consumption of 80 g daily of natural honey for 21 days showed that prostaglandin levels compared with normal subjects were elevated in patient with AIDS . Natural honey has been shown to decrease prostaglandin level, elevated NO production in patients with a long history of AIDS . It was reported that oral intake of honey augments antibody productions in primary and secondary immune responses against thymus-dependent and thymus-independent antigens .
These studies suggest that daily consumption of honey improves one’s immune system.
5. Honey As Natural Anti-Inflammatory Agent
In routine everyday life, our cells may be injured by irritants from outside or within our bodies (by microbes or nonmicrobes). Cellular/molecular injuries result in inflammatory response, the body defense mechanisms in trying to rid of the irritants. In general inflammatory responses are beneficial and protective to us, but at times, inflammatory responses are detrimental to health. Honey is a potent anti-inflammatory agent. Infants suffering from diaper dermatitis improved significantly after topical application of a mixture containing honey, olive oil, and beeswax after 7 days . Honey provides significant symptom relief of cough in children with an upper respiratory tract infection (URTI) . It has been shown to be effective in management of dermatitis and Psoriasis vulgaris . Eight out of 10 patients with dermatitis and five of eight patients with psoriasis showed significant improvement after 2 weeks on honey-based ointment . Honey at dilutions of up to 1 : 8 reduced bacterial adherence from 25.6 ± 6.5 (control) to 6.7 ± 3.3 bacteria per epithelial cell ( ) in vitro . Volunteers who chewed “honey leather” showed that there were statistically highly significant reductions in mean plaque scores (0.99 reduced to 0.65
) in the manuka honey group compared to the control group suggesting a potential therapeutic role for honey for gingivitis, periodontal disease , mouth ulcers, and other problems of oral health .
A case report of a patient who had chronic dystrophic epidermolysis bullosa (EB) for 20 years healed with honey impregnated dressing in 15 weeks  after conventional dressings and creams failed. This illustrates the usefulness of honey as an anti-inflammatory agent. Chronic inflammatory process has risk of cancer development.
6. Honey As Natural Antimicrobials
Everyday we are exposed to all kinds of microbial insults from bacteria, viruses, parasites, and fungi. Honey is a potent natural antimicrobial. The most common infections humans get are from staphylococcal infection. Antibacterial effect of honey is extensively studied. The bactericidal mechanism is through disturbance in cell division machinery . The minimum inhibitory concentration (MIC) for Staphylococcus aureus by A. mellifera honey ranged from 126.23 to 185.70 mgml −1 . Honey is also effective against coagulase-negative staphylococci . Local application of raw honey on infected wounds reduced signs of acute inflammation , thus alleviating symptoms. Antimicrobial activity of honey is stronger in acidic media than in neutral or alkaline media . The potency of honey is comparable to some local antibiotics. Honey application into infective conjunctivitis reduced redness, swelling, pus discharge, and time for eradication of bacterial infections . When honey is used together with antibiotics, gentamycin, it enhances anti-Staphylococcus aureus activity, by 22% . When honey is added to bacterial culture medium, the appearance of microbial growth on the culture plates is delayed . Mycobacteria did not grow in culture media containing 10% and 20% honey while it grew in culture media containing 5%, 2.5%, and 1% honey, suggesting that honey could be an ideal antimycobacterial agent  at certain concentrations.
Honey is also effective in killing hardy bacteria such as Pseudomonas aeruginosa (PA) and could lead to a new approach in treating refractory chronic rhinosinusitis . Daily consumption of honey reduces risk of chronic infections by microorganisms. Chronic infections have risk for cancer development.
There are three main mechanisms by which infections can cause cancer. They appear to involve initiation as well as promotion of carcinogenesis . Persistent infection within host induces chronic inflammation accompanied by formation of reactive oxygen and nitrogen species (ROS and RNOS) . ROS and RNOS have the potential to damage DNA, proteins, and cell membranes. Chronic inflammation often results in repeated cycles of cell damage leading to abnormal cell proliferation . DNA damage promotes the growth of malignant cells. Secondly, infectious agents may directly transform cells, by inserting active oncogenes into the host genome, inhibiting tumour suppressors . Thirdly, infectious agents, such as human immunodeficiency virus (HIV), may induce immunosuppression .
The effectiveness of honey is best when used at room temperature. Heating honey to 80 degrees for 1 hour decreased antimicrobial activity of both new and stored honey. Storage of honey for 5 years decreased its antimicrobial activity, while ultraviolet light exposure increased its activity against some of microorganisms .
Honey also has been shown to have antiviral properties. In a comparative study topical application of honey was found to be better than acyclovir treatment on patients with recurrent herpetic lesions . Two cases of labial herpes and one case of genital herpes remitted completely with the use of honey while none with acyclovir treatment .
7. Honey As Possible Agent for Controlling Obesity
Obese individuals are at risk to develop cancer. There is a close link among obesity, a state of chronic low-level inflammation, and oxidative stress . Obese subjects have an approximately 1.5–3.5-fold increased risk of developing cancers compared with normal-weight subjects [24–26] particularly endometrium [27, 28], breasts [29, 30], and colorectal cancers . Adipocytes have the ability to enhance the proliferation of colon cancer cells in vitro . The greatest risk is for obese persons who are also diabetic, particularly those whose body mass index is above 35 kg/m 2 . The increase in risk is by 93-fold in women and by 42-fold in men . One of the most common cancers noted in community that has high diabetics and obesity is colorectal cancer [42–45].
In a clinical study on 55 overweight or obese patients, the control group (17 subjects) received 70 g of sucrose daily for a maximum of 30 days and patients in the experimental group (38 subjects) received 70 g of natural honey for the same period. Results showed that honey caused a mild reduction in body weight (1.3%) and body fat (1.1%) . Beneficial effect of honey on obesity is not well established thus far.
8. Honey as “Fixer” for Chronic Ulcers and Wounds
Increasing numbers of antibiotic-resistant bacteria has made simple wounds become chronic and non-healing and as such honey provides alternative treatment options . Honey absorbs exudates released in wounds and devitalized tissue . Honey is effective in recalcitrant surgical wounds . It increases the rate of healing by stimulation of angiogenesis, granulation, and epithelialization, making skin grafting unnecessary and giving excellent cosmetic results . In a randomized control trial, Manuka honey improved wound healing in patients with sloughy venous leg ulcers . Honey was shown to eradicate MRSA (Methylene resistant Staphylococcus aureus) infection in 70% of chronic venous ulcers . Honey is acidic and chronic non healing wounds have an elevated alkaline environment. Manuka honey dressings is associated with a statistically significant decrease in wound pH . Available evidence in meta-analysis studies indicates markedly greater efficacy of honey compared with alternative dressings for superficial or partial thickness burns . Honey is an inexpensive moist dressing with antibacterial and tissue-healing properties suitable for diabetic foot . The average cost of treatment per patient using honey dressing is much cheaper with conventional dressing .
9. Honey As Natural Cancer “Vaccine”
Synthetic vaccines like BCG or polio vaccine work by preventing vaccinated subjects from contracting tuberculosis and poliomyelitis. Honey has the element of a “natural cancer vaccine” as it can reduce chronic inflammatory processes, improve immune status, reduce infections by hardy organisms and so forth. Some simple and polyphenols found in honey, namely, caffeic acid (CA), caffeic acid phenyl esters (CAPE), chrysin (CR), galangin (GA), quercetin (QU), kaempferol (KP), acacetin (AC), pinocembrin (PC), pinobanksin (PB), and apigenin (AP), have evolved as promising pharmacological agents in prevention and treatment of cancer . The antioxidant activity of Trigona carbonaria honey from Australia is high at 233.96±50.95 microM Trolox equivalents . The antioxidant activity of four honey samples from different floral sources showed high antioxidant properties tested by different essay methods . Dark honey had higher phenolic compounds and antioxidant activity than clear honey . The amino acid composition of honey is an indicator of the toxic radical scavenging capacity .
10. Honey as Potential Use in “Cancer Therapy”
Honey may provide the basis for the development of novel therapeutics for patients with cancer and cancer-related tumors. Jungle honey fragments were shown to have chemotactic induction for neutrophils and reactive oxygen species (ROS), proving its antitumor activity . Recent studies on human breast , cervical , oral , and osteosarcoma  cancer cell lines using Malaysian jungle honey showed significant anticancer activity. Honey has been shown to have antineoplastic activity in an experimental bladder model in vivo and in vitro .
Honey is rich in flavonoids [62, 103]. Flavanoids have created a lot of interests among researchers because of its anticancer properties. The mechanisms suggested are rather diverse such as various signaling pathways , including stimulation of TNF-alpha (tumor necrosis factor-alpha) release , inhibition of cell proliferation, induction of apoptosis , and cell cycle arrest  as well as inhibition of lipoprotein oxidation . Honey is thought to mediate these beneficial effects due to its major components such as chrysin  and other flavonoids . These differences are explainable as honeys are of various floral sources, and each floral source may exhibit different active compounds. Though honey has other substances of which the most predominant are a mixture of sugars (fructose, glucose, maltose, and sucrose)  which itself is carcinogenic , it is understandable that its beneficial effect on cancer raises skeptics. The mechanism on how honey has anti-cancer effect is an area of great interest recently. The effects of honeys on hormone-dependent cancers such as breast, endometrial, and prostate cancer and tumors remain largely unknown. There is a lot we can learn from nature . For example, phytochemicals, such as genistein, lycopene, curcumin, epigallocatechin-gallate, and resveratrol have been studied to be used for treatment of prostate cancer . Phytoestrogens constitute a group of plant-derived isoflavones and flavonoids, and honey belongs to plant phytoestrogen [112, 114].
There is now a sizeable evidence that honey is a natural immune booster, natural anti-inflammatory agent, natural antimicrobial agent, natural cancer “vaccine,” and natural promoter for healing chronic ulcers and wounds some of the risk factors for cancer development. Bee farming is a lucrative business. Honey and cancer have sustainable inverse relationship in the setting of developing nations where resources for cancer prevention and treatment are limited.
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Copyright © 2012 Nor Hayati Othman. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Human Cells Recognize Conserved Features of Pathogens
Microorganisms do occasionally breach the epithelial barricades. It is then up to the innate and adaptive immune systems to recognize and destroy them, without harming the host. Consequently, the immune systems must be able to distinguish self from nonself. We discuss in Chapter 24 how the adaptive immune system does this. The innate immune system relies on the recognition of particular types of molecules that are common to many pathogens but are absent in the host. These pathogen-associated molecules (called pathogen-associated immunostimulants) stimulate two types of innate immune responses—inflammatory responses (discussed below) and phagocytosis by cells such as neutrophils and macrophages. Both of these responses can occur quickly, even if the host has never been previously exposed to a particular pathogen.
The pathogen-associated immunostimulants are of various types. Procaryotic translation initiation differs from eucaryotic translation initiation in that formylated methionine, rather than regular methionine, is generally used as the first amino acid. Therefore, any peptide containing formylmethionine at the N-terminus must be of bacterial origin. Formylmethionine-containing peptides act as very potent chemoattractants for neutrophils, which migrate quickly to the source of such peptides and engulf the bacteria that are producing them (seeFigure 16-96).
In addition, the outer surface of many microorganisms is composed of molecules that do not occur in their multicellular hosts, and these molecules also act as immunostimulants. They include the peptidoglycan cell wall and flagella of bacteria, as well as lipopolysaccharide (LPS) on Gram-negative bacteria (Figure 25-40) and teichoic acids on Gram-positive bacteria (see Figure 25-4D). They also include molecules in the cell walls of fungi such as zymosan, glucan, and chitin. Many parasites also contain unique membrane components that act as immunostimulants, including glycosylphosphatidylinositol in Plasmodium.
Structure of lipopolysaccharide (LPS). On the left is the 3-dimensional structure of a molecule of LPS with the fatty acids shown in yellow and the sugars in blue. The molecular structure of the base of LPS is shown on the right. The hydrophobic membrane (more. )
Short sequences in bacterial DNA can also act as immunostimulants. The culprit is a 𠇌pG motif”, which consists of the unmethylated dinucleotide CpG flanked by two 5′ purine residues and two 3′ pyrimidines. This short sequence is at least twenty times less common in vertebrate DNA than in bacterial DNA, and it can activate macrophages, stimulate an inflammatory response, and increase antibody production by B cells.
The various classes of pathogen-associated immunostimulants often occur on the pathogen surface in repeating patterns. They are recognized by several types of dedicated receptors in the host, that are collectively called pattern recognition receptors. These receptors include soluble receptors in the blood (components of the complement system) and membrane-bound receptors on the surface of host cells (members of the Toll-like receptor family). The cell-surface receptors have two functions: they initiate the phagocytosis of the pathogen, and they stimulate a program of gene expression in the host cell for stimulating innate immune responses. The soluble receptors also aid in the phagocytosis and, in some cases, the direct killing of the pathogen.
Boletus edulis biologically active biopolymers induce cell cycle arrest in human colon adenocarcinoma cells
The use of biologically active compounds isolated from edible mushrooms against cancer raises global interest. Anticancer properties are mainly attributed to biopolymers including mainly polysaccharides, polysaccharopeptides, polysaccharide proteins, glycoproteins and proteins. In spite of the fact that Boletus edulis is one of the widely occurring and most consumed edible mushrooms, antitumor biopolymers isolated from it have not been exactly defined and studied so far. The present study is an attempt to extend this knowledge on molecular mechanisms of their anticancer action. The mushroom biopolymers (polysaccharides and glycoproteins) were extracted with hot water and purified by anion-exchange chromatography. The antiproliferative activity in human colon adenocarcinoma cells (LS180) was screened by means of MTT and BrdU assays. At the same time fractions' cytotoxicity was examined on the human colon epithelial cells (CCD 841 CoTr) by means of the LDH assay. Flow cytometry and Western blotting were applied to cell cycle analysis and protein expression involved in anticancer activity of the selected biopolymer fraction. In vitro studies have shown that fractions isolated from Boletus edulis were not toxic against normal colon epithelial cells and in the same concentration range elicited a very prominent antiproliferative effect in colon cancer cells. The best results were obtained in the case of the fraction designated as BE3. The tested compound inhibited cancer cell proliferation which was accompanied by cell cycle arrest in the G0/G1-phase. Growth inhibition was associated with modulation of the p16/cyclin D1/CDK4-6/pRb pathway, an aberration of which is a critical step in the development of many human cancers including colon cancer. Our results indicate that a biopolymer BE3 from Boletus edulis possesses anticancer potential and may provide a new therapeutic/preventive option in colon cancer chemoprevention.
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Biology Finally Explains Why Honey Badger Don’t Care
It’s official: Honey badger don’t care. This “crazy nasty-ass” critter—the subject of a National Geographic documentary transformed into a viral meme through satirical overdubbing—“really don’t give a shit.” Not about snarky documentaries, not about stinging bees, and especially not about venomous snakes.
Venomous snakes kill up to 94,000 people every year, on top of the millions of other animals that make up their diet. And death by venomous snakebite isn’t pretty: The toxins in venom can paralyze muscles, break down tissue, and even make victims bleed uncontrollably.
So why don’t honey badgers care about venoms that can kill almost any other animal?
Danielle Drabeck, a University of Minnesota grad student, wanted to study this question on a molecular level, but she ran into a problem: Honey badgers aren’t found in Minnesota or even the Western Hemisphere, but only in Africa, the Middle East, and India.
“The hardest part, honest to God, was finding honey badger tissue” to study, says Drabeck—which likely explains why no other biologists ever investigated how honey badgers resist cobra venom. Working with biologist Sharon Jansa and biochemist Antony Dean, Drabeck obtained some precious honey badger blood from the zoos of San Diego and Fort Wayne, Indiana.
With this blood, the scientists figured out, for the first time, how the honey badger defends itself on the molecular level against its venomous prey. The blood also revealed clues of an evolutionary arms race. And it might help us design better antivenoms for humans bitten by venomous snakes.
But why would a honey badger need venom resistance in the first place? Why doesn’t it avoid venomous snakes, like more sensible mammals?
“Snakes,” says Drabeck, “are an excellent source of meat.” Up to 25 percent of the honey badger’s omnivorous diet consists of venomous snakes. But the honey badger doesn’t eat snakes out of desperation. Evolving to withstand snake venom is like being the only person at a party who can eat the extra-hot salsa: You get it all to yourself. Plus, Drabeck says, this means the honey badger gets to hunt fairly slow-moving prey with only one pointy end, rather than fast prey with one pointy end plus four sets of claws.
But it’s one hell of a pointy end. Venom has more than 100 proteins and other molecules that could potentially poison a snake’s victim—meaning that honey badgers need multiple defenses. To narrow the field, Drabeck guessed that the honey badger had probably evolved a defense similar to that used by other venom-resistant critters like mongooses. She focused on a defense against a nasty class of molecules in cobra venom called alpha-neurotoxins that paralyze the muscles used for breathing. These neurotoxins essentially park in a muscle cell’s nicotinic acetylcholine receptor, preventing the cell from receiving the nervous system’s signals to keep working.
Drabeck figured that the receptor targeted by cobra neurotoxin had probably changed to prevent the neurotoxin from parking there. Once she had the blood from the zoos’ honey badgers, Drabeck extracted DNA and sequenced part of the gene that contains the blueprint for making the receptor. Drabeck discovered several mutations in that gene that tweak the receptor. Cobra neurotoxin fits as well in the tweaked receptor as an SUV in a compact’s parking spot—and therefore it can’t paralyze the honey badger’s breathing.
Drabeck wasn’t surprised by these mutations, but she was surprised when she compared the honey badger’s tweaks to those found in other mammals. These tweaks had evolved independently in at least four species: honey badgers, mongooses, hedgehogs, and pigs. The hedgehog—which loves to eat venomous snakes—wasn’t a surprise. But the pig? “We were pretty excited by that,” says Drabeck. She hadn’t expected pigs to have molecular defenses against venom biologists knew wild pigs could survive snakebites but assumed that was because their thick skin and fat acts like armor against fangs. But wild pigs, like honey badgers, have long shared the same parts of the world as venomous snakes—giving them an incentive to evolve venom resistance. And that in turn has given the snakes an incentive to evolve more toxic venom.
Venomous snakes and resistant honey badgers, it turns out, are locked in what Jansa describes as a “tit-for-tat arms race.” This co-evolution is an unending cycle of one-upmanship between predators and prey. When venomous snakes are attacked by venom-resistant honey badgers, the snakes need to evolve more toxic venom to protect themselves.
But what does this research mean for the 1.8 million unfortunate people bitten by venomous snakes every year? Drabeck suggests that figuring out these molecular tweaks in the honey badger’s resistant receptor could suggest new ways to create better antivenoms. “That’s one of the important questions” about this research into honey badgers, says biologist James Biardi, an expert on venom resistance at Fairfield University in Connecticut. “What does this mean for people?”
Right now, many antivenom infusions are made of antibodies—molecules produced by the immune systems of horses and sheep exposed to venom, which can neutralize the venom in bitten people. But whenever someone gets treated with these antivenoms, they run the risk of having an allergic reaction as dangerous as the venom itself. By understanding more about the targets of venom—targets like the honey badger’s neurotoxin receptor—scientists can hopefully design safer treatments. Because unlike the mongoose, hedgehog, pig, and honey badger, we humans with our puny neurotoxin receptors do care—especially about venomous snakes.
Honey Bee Viruses, the Deadly Varroa Mite Associates
If your bees have Varroa, your bees have viruses.
Authors: Philip A. Moore, Michael E. Wilson, and John A. Skinner
Department of Entomology and Plant Pathology, the University of Tennessee, Knoxville TN
Originally Published: August 21, 2014
Varroa mites (Varroa spp.) are a ubiquitous parasite of honey bee (Apis spp.) colonies. They are common nearly everywhere honey bees are found, and every beekeeper should assume they have a Varroa infestation, if they are in a geographic area that has Varroa (Varroa mites are not established in Australia as of spring 2014). Varroa mites were first introduced to the western honey bee (Apis mellifera) about 70 years ago after bringing A. mellifera to the native range of the eastern honey bee (Apis cerana). Varroa mites (Varroa jacobsoni) in eastern honey bee colonies cause little damage. But after switching hosts and being dispersed across the world through natural and commercial transportation of honey bee colonies, Varroa has became a major western honey bee pest since the 1980’s. Varroa mites (Varroa destructor) are now the most serious pest of western honey bee colonies and one of the primary causes of honey bee decline (Dietemann et al. 2012). A western honey bee colony with Varroa, that is not treated to kill the pest, will likely die within one to three years (Korpela et al. 1993 Fries et al. 2006).
Varroa Life History
Varroa mites attack honey bee colonies as an external parasite of adult and developing bees, by feeding on hemolymph (fluid of the circulatory system similar to blood), spreading disease, and reducing their lifespan. Evidence suggests that Varroa and their vectored viruses affect the immune response of honey bees, making them more susceptible to disease agents (Yang and Cox-Foster 2005). For more information on this topic see here. Mature female Varroa mites survive on immature and adult honey bees (worker, drone, and rarely queen), are reddish brown, and about the size of a pin head. Male mites are a smaller size and tan color, do not feed on bees, and are only found inside brood cells (Rosenkranz et al. 2010).
Varroa have two life stages, phoretic and reproductive. The phoretic stage is when a mature Varroa mite is attached to an adult bee and survives on the bee’s hemolymph. During this stage the mite may change hosts often transmitting viruses by picking up the virus on one individual and injecting it to another during feeding. Phoretic mites may fall off the host, sometimes being bitten when bees groom each other, or it may die of old age. Mites found on the bottom board of the hive or that fall though a screen bottom board are called the “natural mite drop”. But these mites that fall off of bees represent a small portion of the total mite population because the reproductive mites are hidden under cell cappings.
Image 1: Reproductive Varroa mite on a developing pupa (reddish oval) and two immature Varroa (opaque ovals). Credit: Abdullah Ibrahim (arrows added for emphasis)
The reproductive life stage of Varroa begins when an adult female mite is ready to lay eggs and moves from an adult bee into the cell of a developing larval bee. After the brood cell is capped and the larva begins pupating, the mite begins to feed. After about three days from capping, the mite lays its eggs, one unfertilized egg (male) and four to six fertilized (female) eggs (Rosenkranz et al. 2010). After the eggs hatch, the female mites feed on the pupa, mate with the male mite and the surviving sexually mature female mites stay attached to the host bee when it emerges as an adult. It takes six to seven days for a female mite to mature from egg to adult and it can live two to three months in the summer and five to eight months in the fall. Only mature female mites can survive outside of a brood cell (the phoretic stage), and on average a mite will produce 1.2 viable mature female offspring per worker cell invaded (Schulz 1984 Fuchs & Langenbach 1989). However, since the development time is longer for drone brood, the average viable offspring for a mite in a drone cell increases to 2.2 per cell invaded (Schulz 1984 Fuchs & Langenbach 1989). For more on Varroa life history see here.
One of the serious problems caused by Varroa is the transmission of viruses to honey bees which cause deadly diseases. Viruses found in honey bees have been known to scientists for 50 years and were generally considered harmless until the 1980’s when Varroa became a widespread problem. Since then, nearly twenty honey bee viruses have been discovered and the majority of them have an association with Varroa mites, which act as a physical and or biological vector (Kevan et al. 2006). Therefore controlling Varroa populations in a hive will often control the associated viruses and finding symptoms of the viral diseases is indicative of a Varroa epidemic in the colony. Viruses are however, the least understood of honey bee diseases. Emerging information of honey bee viruses continue to alter our understanding of the role viruses play in honey bee colonies (Genersch and Aubert 2010).
Viral Life History
Viruses are microscopic organisms that consist of genetic material (RNA or DNA) contained in a protein coat. Viruses do not acquire their own nutrients or live independently, and can only multiply within living cells of a host. An individual virus unit is called a virus particle or virion and the abundance of these particles in a host is called the virus titer. A virus particle injects itself in to a host cell and uses the cells’ organelles to make copies of itself. This process will continue without obvious change to the cell, until the host cell becomes damaged or dies, releasing large amounts of infective virus particles. All forms of life are attacked by viruses and most are host specific.
Honey Bee Viruses
Viruses of the honey bee typically infect the larval or pupa stage, but the symptoms are often most obvious in adult bees. Many of these viruses are consumed in pollen or the jelly produced by nurse bees that are fed to developing bees. Many viruses are also transmitted by Varroa. Varroa, when feeding on the hemolymph transfer the viruses directly into the open circulatory system, which reaches every cell in the insect body.
Honey bee viruses are not limited to honey bees. Honey bee viruses have been found in other non-Apis bee species, other colony inhabitants like small hive beetle, and in pollen and nectar (Andersen 1991 Bailey and Gibbs 1964 Genersch et al. 2006 Singh et al 2010). For more on honey bee pathogens found in native bees see here. Transfer of honey bee viruses from infected colonies to non-infected individuals or colonies can occur during foraging on common flowers or through robbing of weak or collapsed colonies (Singh et al 2010).
Identification of a virus is difficult due to the small size of particles. Expensive and often uncommon laboratory equipment is required for accurate diagnosis. However, symptoms of some viral diseases are more visible, especially with overt infection. A lack of symptoms does not rule out the presence of a virus. Viruses can remain in a latent form within the host, acting as a reservoir of infection, complicating diagnosis and control, and only becoming an outbreak when conditions are right.
Viral Prevalence in the United States
The USDA-APHIS National Honey Bee Pests and Diseases Survey has taken samples from honey bee colonies in over 27 states since the year 2009. Data from these surveys and other data are complied into a database with the Bee Informed Partnership and used to determine baseline disease level, determine the absence of exotic honey bee pests that have not yet been found in America, and to gauge the overall health of U.S. honey bee colonies. Results of virus presence from the 2013 survey are below (Figure 1). Deformed wing virus (DWV) and Black queen cell virus (BQCV) were present in over 80% of sampled colonies. Other viruses were much less common, but still present in 10-20 percent of colonies sampled. Of the viruses tested for presence, only slow bee paralysis virus (SBPV) was not found in the U.S
Figure 1: 2013 USDA-APHIS National Honey Bee Pests and Diseases Survey, Virus Prevalence Results (Virus abbreviations: BQCV=Black queen cell virus DWV= Deformed wing virus LSV2= Lake Sinai virus 2 ABPV= Acute bee paralysis virus KBV= Kashmir bee virus IAPV= Israel acute paralysis virus CBPV= Chronic bee paralysis virus SBPV= Slow bee paralysis virus)
Sacbrood, a disease cause by a virus, was the first honey bee virus to be discovered in the early 20 th century and now has a recognized widespread distribution. It is perhaps the most common honey bee virus (Shen et al. 2005). This disease has been found in adult, queen, egg, and larval bees, in all forms of food, and in Varroa mites, suggesting a wide range of transmission routes. Although it is commonly found without serious outbreak, sacbrood is more likely to cause disease when the division of labor is less defined, in the early parts of the year before the nectar flow, or during prolong dearth (Bailey 1981). It often goes unnoticed since it usually infects only a small portion of brood, and adult bees will usually detect and remove infected larvae.
Image 2: Sacbrood infected pupa. Credit: Michael E. Wilson
The disease causes larvae to fail to shed their final skin prior to pupation, after the larva has spun its cocoon. Infected larvae remain on their back with their head towards the cell capping. Fluid accumulates in the body and the color will change from pearly white to pale yellow, with the head changing color first. Then, after the larva dies, it becomes dark brown with the head black (Image 2). Larvae that have ingested sufficient quantities of sacbrood in their food die after being sealed in their comb.
Sacbrood multiplies in several body tissues of young larvae but these larvae appear normal until cell capping. Each larva that dies from sacbrood contains enough virus particles to infect every larva in 1000 colonies (Bailey 1981). But in most instances, diseased larvae are quickly removed in the early stages of the disease by nurse bees. The cell cappings are first punctured to detect the disease, which a good sign of infection for the beekeeper look for (Image 6). Then, young worker bees remove the diseased larvae from the colony. Adult bees, although not susceptible to infection, become a harbor as the virus collects in the bee’s hypopharyngeal glands, which are used to produce larval jelly (Bailey 1981). These infected adult bees, however, cease to eat pollen and soon stop tending larvae. They will become foragers more quickly in life than usual and tend to collect nectar instead of pollen (Bailey 1981). Nectar that contains the virus becomes diluted in the colony when mixed with nectar from other foragers. Whereas pollen, is collected and compacted into the “pollen basket” and deposited intact into a cell. Dilute virus containing nectar is less likely to cause infection than when the virus is concentrated in a pollen pellet. Therefore use caution when transferring frames with pollen among colonies. Little is known of the other transmission routes: through Varroa mites, between workers, from bee feces or through transovarial transmission (from queen to egg). Sacbrood usually subsides in late spring when the honey flow begins, but if symptoms persist, requeening with hygienic stock is recommended (Frazier et al. 2011).
Deformed Wing Virus (DWV)
Deformed wing virus is common, widely distributed, and closely associated with Varroa mites. Both the virus titers and prevalence of the virus in colonies are directly linked to Varroa infestations (Bowens-Walker et al. 1999). In heavily Varroa infested colonies, nearly 100 percent of adult workers may be infected with DWV and have high virus titers even without showing symptoms (de Miranda et al. 2012). DWV is strongly associated with winter colony mortality (Highfield et al 2009 Genersch et al 2010). Control of DWV is usually achieved by treatment against Varroa, After treatment a gradual decrease in virus titers occurs as infected bees are replaced by healthy ones (Martin et al 2010). DWV can be found in all castes and life stages of honey bees and will persist in adults without obvious symptoms. DWV is also transmitted through food, feces, from queen to egg, and from drone to queen (de Miranda et al. 2012).
Image 3: Adult bees with deformed wings resulting from DWV. Credit: Katherine Aronstein
Acute infections of DWV are typically linked to high Varroa infestation levels (Martin et al 2010). Covert infections (a detectable level of virus without damaging symptoms) can occur through transovarial transmission (Chen et al. 2004), and through larval food (Chen et al. 2006). Symptoms noted in acute infections include early death of pupae, deformed wings, shortened abdomen, and cuticle discoloration in adult bees, which die within 3 days causing the colony to eventually collapse. Not all mite infested pupae develop these symptoms, but all adult honey bees with symptoms develop from parasitized pupae. Bees infected as adults can have high virus titers but do not develop symptoms. DWV may also affect aggression (Fujiyuki et al. 2004) and learning behaviors of adult bees (Iqbal and Muller 2007). DWV appears to replicate in Varroa, making it a biological as well as physical vector. Infection of pupae may be dependent on DWV replication in Varroa prior to transmission. Winter colony mortality is strongly associated with DWV presence, irrespective of Varroa infestation. This suggests that Varroa infection should be reduced in a colony far in advance of producing overwintering bees, to ensure reduction in DWV titers. DWV is closely related to Kakugo Virus and Varroa destructor Virus 1, which together form the Deformed Wing Virus Complex (de Miranda et al. 2012).
Black Queen Cell Virus (BQCV)
Black queen cell virus is a widespread and common virus that persists as asymptomatic infections of worker bees and brood. Although generally understood as being asymptomatic in adult bees, Shutler et al. (2014) found BQCV to be associated with the symptom K-wing, where the wing pair is disjointed and more perpendicular to one another. Queen pupae with symptoms display a pale yellow sac-like skin similar to sacbrood. The pupae rapidly darken after death and turn the wall of the queen cell dark brown to black. Symptomatic drone pupae have also been observed. Unlike other viruses that are associated with Varroa, BQCV is strongly associated with Nosema apis and little evidence supports its co-occurrence with Varroa, although, BQCV has been isolated from Varroa (Ribière et al. 2008). Nosema disease affects a bee’s mid gut, increasing susceptibility of the alimentary tract to infection by BQCV. BQCV can be orally transmitted to adults only when Nosema has co-infected (Ribière et al. 2008). It can also be transmitted by injection to pupae. BQCV has a seasonal relationship similar to Nosema, with a strong peak in spring. Because of the seasonal occurrence with Nosema, queen rearing operations who produce queens in the spring are susceptible to BQCV (Ribière et al. 2008).
Image 4: Dysentery on the front of a hive is a symptom but not indicative of Nosema disease. Credit: Michael E. Wilson
Chronic Bee Paralysis Virus (CBPV)
Chronic bee paralysis virus was one of the first honey bee viruses to be isolated. It is unique among honey bee viruses in that it has a distinct particle size and genome composition. It is also the only common honey bee virus to have both visual behavior and physiological modifications resulting from infection. Symptoms of the disease are observed in adult bees displaying one of two sets of symptoms called syndromes (Genersch & Aubert 2010). Type 1 symptoms include trembling motion of the wings and bodies of adult bees, who are unable to fly, and crawl along the ground or up plant stems, often clustering together. The bees may also have a bloated abdomen, causing dysentery and will die within a few days after displaying symptoms.
Type 2 symptoms are greasy, hairless, black adult bees that can fly, but within a few days, become flightless, trembling, and soon die (Image 5). Both of these syndromes can occur within the same colony. Severely affected colonies, often the strongest in an apiary (Ribiere at al. 2010), quickly lose adult workers, causing collapse and often leaving few adult bees with the queen on unattended comb (Bailey & Ball 1991). These symptoms, however, are similar and often confused with other honey bee maladies including Nosema apis, colony collapse disorder (CCD), tracheal mites, chemical toxicity, and other viruses.
Image 5: Bees with CBPV type 2 symptoms: greasy and hairless. Credit: The Food and Environment Research Agency (Fera), Crown Copyright
Transmission of the virus primarily occurs through direct body contact, although oral transmission also occurs but is much less virulent. Direct body transmission happens when bees are either crowded or confined within the hive for a long period of time (due to poor weather or during long-distance transportation) or when too many colonies are foraging within a limited area, such as a monoculture of sunflower with high honey bee colony density (Genersch & Aubert 2010). In both instances, small cuts from broken hairs on an adult bee’s cuticle and direct contact with infected adult bees spreads the virus through their exposed pores if this occurs rapidly and enough adult bees are infected, an outbreak with colony mortality will occur. Feces from infected bees within a colony can also spread the disease, and other transmission routes are still being investigated, including possible Varroa transmission. The virus is widespread and an outbreak can occur at any time of year. Spring and summer are the most common seasons for mortality from the virus, but it will persist in a colony year-round without displaying any overt symptoms (de Miranda et al. 2012).
Two new viruses related to CBPV with no yet described symptoms are Lake Sinai virus 1 (LSV1) and Lake Sinai virus 2 (LSV2) (Runckel et al. 2011). New molecular tools have allowed researchers to identify the presence of these and other new viruses and their seasonality in test colonies. Little else is know of the Lake Sinai viruses, including its pathogenic or epidemiological significance. Other described honey bee viruses that were discovered before the advent of molecular techniques have no genomic data to reference therefore newly discovered viruses may in fact be the already discovered viruses of the past such as Bee virus X and Y, Arkansas Bee Virus or Berkeley Bee Virus (Runckel et al. 2011).
Acute Bee Paralysis Virus Complex
Acute bee paralysis virus (ABPV), Kashmir bee virus (KBV), and Israel acute paralysis virus (IAPV) are a complex of associated viruses with similar transmission routes and affect similar life stages. These viruses are widespread at low titers and can quickly develop high titers due to extremely virulent pathology. Frequently associated with colony loss, this virus complex is especially deadly when colonies are heavily infested with Varroa mites. (Ball 1989 Genersch 2010, Genersh et al. 2010). These viruses have not been shown to cause symptoms in larval life stages, but show quick mortality in pupae and adult bees.
Acute Bee Paralysis Virus (ABPV)
Acute bee paralysis virus was accidentally discovered when CBPV was first isolated. ABPV displays similar symptoms as CBPV however the acute adjective describes a bees’ more rapid mortality compared to CBPV. Unlike CBPV, ABPV virulence is directly related to Varroa infestation. APBV is transmitted in larval jelly from asymptomatic infected adult bees to developing larva or when vectored by Varroa mites to larvae and pupae. ABPV is common and typically cause covert infections (no obvious symptoms) when transmitted orally from adult to developing bee. It takes about one billion viral particles to cause death via ingestion, but when vectored by Varroa and directly injected into the developing bee’s hemolymph, only 100 virus particles will cause death (Genersch & Aubert 2010). When the virus is picked up by Varroa, the transmission rate to pupae is between 50 and 90 percent. The longer the feeding period of Varroa, the greater the transmission rate will become. (Genersch & Aubert 2010). Pupae infected with ABPV die before emerging, making the appearance of paralysis symptoms less obvious. The decline in emerging bees causes a colony to dwindle towards collapse. A colony infected with an ABPV epidemic will die within one season (Sumpter and Martin 2004).
Kashmir Bee Virus (KBV)
Kashmir bee virus has widespread distribution and is considered the most virulent of honey bee viruses under laboratory condition (Allen and Ball 1996). When KBV is injected in to adult bee hemolymph, death occurs in just 3 days (de Miranda et al. 2012). KBV does not cause infection when fed to developing bees, but does persist in adult and developing bees without any obvious symptoms. When Varroa mites transmit the virus, it becomes deadly to all forms of the bee lifecycle but displays no clearly defined symptoms. Even with moderate levels of mite infestation, KBV, like ABPV, can kill colonies (Todd et al. 2007). Control of Varroa mites is necessary to prevent colony losses from KBV.
Israeli Acute Paralysis Virus (IAPV)
Symptoms of IAPV are similar to ABPV and CBPV including: shivering wings, darkened hairless abdomens and thoraxes, progressing into paralysis and death. IAPV is found in all life stages and castes of bees. IAPV and other viruses were found to be strongly associated with colony collapse disorder (CCD) in the United States, but no direct relationship between the viruses and CCD has yet been shown (Cox-Foster et al. 2007). IAPV is extremely virulent at high titers, as when vectored by Varroa and is covert at low titers.
Slow Bee Paralysis Virus
In contrast to ABPV, which produces symptoms in a few days after infection, SBPV induces paralysis after 12 days, and only on the two fore (anterior) legs. SBPV persists as a covert infection and is transmitted by Varroa to adults and pupae. The disease will kill adult bees and eventually the entire colony (de Miranda et al. 2012). Prevalence of the virus is limited. It has not been found in the U.S., but has been found in England, Switzerland, Fiji and Western Samoa and only in Britain has SBPV been associated with colony deaths (Carreck et al. 2010).
Most pathogens invade the digestive system through oral ingestion of inoculated food. These pathogens infect the mid gut epithelial cells, which are constantly being replaced and are protected by membranes and filters which confine the pathogen to gut tissues. Parasites that infect gut tissue like Nosema apis and Nosema cerana can create lesions in the epithelium that allow a virus like BQCV to pass into the hemolymph and infect other cells in the body. In contrast the external parasite Varroa destructor feeds directly on bee hemolymph providing an opening in the cuticle for viruses to enter. Most virus infections rarely cause infection when ingested orally, but only a few virus particles are necessary to cause infection when injected directly into the hemolymph. Many viruses can be directly transmitted by Varroa mites, such as: DWV, those in the acute bee paralysis virus complex, and slow bee paralysis virus. Other viruses, like sacbrood, have been detected in Varroa mites but Varroa has not been shown to directly transmit the virus. Some viruses, like DWV, have been shown to directly multiply in Varroa mites, however in most cases we don’t know the exact relationship of Varroa mites to viruses or enough about how transmission occurs from mites to bees. Knowledge about the presence, role, and transmitting routes of these viruses in native bees, and other potential non-Varroa transmission routes is also lacking in detail, complicating recommendations for control. Research does show viruses clearly affect honey bee health and warrant attention from the beekeeper and researcher alike.
Viruses persist in normal, healthy colonies, only to explode during times of stress. Many viruses are only damaging when in combination with another stressor like Varroa or Nosema. Active, integrated management of Varroa and other stressors is essential to minimizing virus titers. To learn more about reducing stressors with best management practices see here.
Routinely inspect your colonies for possible disease. Have a thorough knowledge of symptoms and identify when colonies are slow to build up or have sporadic brood patterns, indicating brood has been pulled out and removed (Image 6). If you suspect you have a disease, take a sample and send it to be identified. For more information on submitting a sample for diagnosis see here.
Image 6: Punctured cell cappings that indicate adult bees have detected a brood disease (note DWV infected adult bee). Credit: The Food and Environment Research Agency (Fera), Crown Copyright (Arrows added for emphasis)
Other future avenues of control include breeding hygienic bee strains that detect brood diseases and remove infected individuals from colonies or breeding of resistance to Varroa infestation. Specific resistance to viruses are not yet considered in most breeding programs. There is evidence of specific viral resistance in honey bees, and there has been at least some attempt to breed resistance to IAPV. For more on this topic see here.
Another promising research area for controlling honey bee viruses in the use of gene silencing called RNAi. The private bee research company Beeologics, as well as public and private university researchers are developing this method and a consumer product may be available in the near future as RNAi technology continues to become more efficient and inexpensive. For more on this topic see here.
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Regulated Necrosis in Pulmonary Disease. A Focus on Necroptosis and Ferroptosis
To date, increasing evidence suggests the possible involvement of various types of cell death in lung diseases. The recognized regulated cell death includes necrotic cell death that is immunogenic, releasing damage-associated molecular patterns and driving tissue inflammation. Necroptosis is a well-understood form of regulated necrosis that is executed by RIPK3 (receptor-interacting protein kinase 3) and the pseudokinase MLKL (mixed lineage kinase domain–like protein). Ferroptosis is a newly described caspase-independent form of regulated necrosis that is characterized by the increase of detrimental lipid reactive oxygen species produced via iron-dependent lipid peroxidation. The role of these two cell death pathways differs depending on the disease, cell type, and microenvironment. Moreover, some experimental cell death models have demonstrated shared ferroptotic and necroptotic cell death and the synergistic effect of simultaneous inhibition. This review examines the role of regulated necrotic cell death, particularly necroptosis and ferroptosis, in lung disease pathogenesis in the context of recent insights into the roles of the key effector molecules of these two cell death pathways.
Cell death is a crucial process for dealing with injury or stress caused by perturbations of the cellular microenvironment in multicellular organisms. Over the past decade, the mechanism known as “regulated cell death” (RCD) has been recognized as an integral part of tissue homeostasis, specifically in response to stress conditions (1, 2). Apoptosis, known as a prototypical form of RCD, has been well studied and is considered to eliminate unnecessary and harmful cells, including virus-infected cells, senescent cells, and cancer cells (3–5). However, the mechanism by which apoptosis, an immunologically silent form of cell death, affects disease pathogenesis, including inflammatory diseases, has not been well understood. Conversely, necrosis has long been considered a form of merely accidental cell death triggered by severe stimuli and is thus conceived of as an uncontrollable process. Over the past decade, a spectrum of investigations have disclosed regulated necrosis (RN) as a nonapoptotic form of RCD that relies on genetically encoded molecular machinery (6). Thus far, various forms of RN pathways have been established that emit abnormal signals such as damage-associated molecular patterns (DAMPs) to alert environmental cells of danger (7) (Table 1). Necrotic forms of RCD appear to be connected to each other via the release of DAMPs and form a necroinflammatory autoamplification loop, resulting in tissue damage and organ dysfunction (7). Among the several types of morphologically classified RN, such as necroptosis, parthanatos, mitochondrial permeability transition, and ferroptosis, the most comprehensively established subroutine of RN is necroptosis (8). Necroptosis was confirmed in 2005 as a possible nonapoptotic cell death pathway triggered by TNFR1 (TNF-α receptor 1) in the absence of caspase 8–dependent intracellular apoptotic signaling (9). Nec-1 (necrostatin 1) was discovered at the same time as a specific small-molecule inhibitor of necroptosis (9), followed by RIPK1 (receptor-interacting kinase 1) as the cellular target of Nec-1 (10, 11), leading to significant advancements in the understanding of the mechanism of necroptosis (Table 1). Subsequent research has revealed that the multiprotein complex termed the “necrosome”—the complex of RIPK1/RIPK3/MLKL (mixed lineage kinase domain–like protein)—can effectively trigger necroptosis in response to activation of the death receptors of TNFR1 (12) (Table 1). Necroptosis is observed in a variety of disease states, such as stroke (13), myocardial infarction (14), ischemia-reperfusion injury (IRI) (15), and other inflammatory diseases (16), via releasing DAMPs from dying cells.
Ferroptosis is a newly described form of caspase-independent RCD characterized by cellular accumulation of reactive oxygen species (ROS) driven through iron-dependent lipid peroxidation (17), which is induced by erastin and Ras synthetic lethality molecule 3 (RSL3). Because the cell membrane is damaged directly by lethal lipid peroxidation and subsequent lipid ROS production, ferroptosis shows a necrotic morphotype, and dying cells release DAMPs and immunogenic metabolites (7, 18), resulting in the inflammatory disease pathogenesis. Accumulating evidence has demonstrated the involvement of ferroptosis in experimental models of multiple human diseases, including acute renal injury (19), neurodegeneration (20–22), IRI (23, 24), and Parkinson’s disease (21).
In this review, we present increasing evidence supporting the involvement of necroptosis and ferroptosis in the pathogenic process of various lung diseases, and we recapitulate the molecular mechanism of necroptosis and ferroptosis in the lung. Understanding the cellular mechanism by which small molecules protect from RCD in the lung will help in the development of specific therapeutic strategies targeting different forms of cell death in lung diseases (Table 2).
Table 2. Role of Regulated Necrosis in Lung Disease Pathogenesis
Definition of abbreviations: 3T3 = 3-day transfer, inoculum 3 × 10 5 cells COPD = chronic obstructive pulmonary disease DAMP = damage-associated molecular pattern IPF = idiopathic pulmonary fibrosis MEFs = mouse embryonic fibroblasts N/A = not available RN = regulated necrosis Th2 = T-helper cell type 2.
Necroptosis is a form of RN that has gained special attention because its inhibition, including genetic modification of RIPK1, RIPK3, and MLKL or small-molecule inhibitor Nec-1, can regulate disease progression in several mouse models (13–16). Necroptosis is triggered by signaling from various death receptors, such as TNFR, Toll-like receptor–Toll-IL-1 receptor domain–containing adapter-inducing IFN-β (TRIF) pathway, TRAILR (TNF-related apoptosis-inducing ligand receptor), FAS (known as CD95 and TNFRSF6 [TNF receptor superfamily member 6]), IFN receptor–RIPK1 pathway, and DNA-dependent activator of IFN-regulatory factors (DAI) pathway (7, 25–28) (Table 1) ( Figure 1 ). Activation of RIPK is a crucial step in the induction of necroptosis signaling. RIPK1 and RIPK3 are serine/threonine kinases, and both have a receptor-interacting protein homotypic interaction motif (RHIM) in the C-terminal domain. RIPK1 combines with RIPK3 via the RHIM domain, forming an amyloid-like complex called the “necrosome” (29). The necrosome is now considered to contain four proteins: RIPK1, RIPK3, TRIF, and DAI (30). As a consequence of necrosome formation, phosphorylation of RIPK3 at Ser345 occurs, enabling the phosphorylation of MLKL (31). The phosphorylation of MLKL elicits an oligomer formation by binding to phosphatidylinositol lipids and cardiolipin, which allows MLKL to translocate from the cytosol to the plasma membranes and form a hole in the membrane. These properties disrupt the integrity of the plasma membrane, resulting in necrotic cell death (32) ( Figure 1 ). A novel and interesting mechanism to regulate necroptosis has emerged whereby the E3 ubiquitin ligase of Parkin, known as a mitophagy activator, regulates necroptosis and inflammation via inhibiting necrosome formation (33). Parkin activated by AMPK (AMP-activated protein kinase) prevents the formation of the RIPK1–RIPK3 complex by promoting polyubiquitination of RIPK3. Parkin-knockout mice show increased inflammation and spontaneous tumor formation via activation of RIPK3 phosphorylation and necroptosis (33) ( Figure 1 ). This AMPK–Parkin–RIPK3 pathway is a promising regulatory mechanism of necroptosis, and therefore further experiments based on lung inflammation models should be conducted in the near future.
Figure 1. The involvement of necroptosis and ferroptosis in lung diseases. In each cell death, the release of damage-associated molecular patterns (DAMPs) after plasma membrane rupture is involved in the pathogenesis of lung disease. Necroptosis is triggered by signaling from various death receptors (e.g., TNFR1, Toll-like receptors [TLRs], and INFR) and viruses. These signaling receptors include complex I (TRADD, RIPK1, cIAP, and TNF receptor–associated factors [TRAFs]), TRIF, and ZBP1/DNA-dependent activator of IFN-regulatory factors (DAI). Signaling via these death receptors causes formation of the complex IIc necrosome by phosphorylated (p)-RIPK1, p-RIPK3, and p-MLKL. p-MLKL then forms an oligomer and moves to the cell surface, causing plasma membrane disruption. Some necroptosis-related molecules, such as the RIPK1–RIPK3 complex, elicit inflammasome activation and IL-1β processing, leading to inflammatory lung diseases. Ferroptosis inducer erastin and Ras synthetic lethality molecule 3 (RSL3) downregulate the glutathione (GSH)–glutathione peroxidase 4 (GPx4) axis, leading to phospholipid peroxidation (PL-OOH). Labile iron accumulation induced by TFR, DMT1, and ferritinophagy evoke Fenton reactions, resulting in production of lipid reactive oxygen species (ROS) and plasma membrane rupture. Cigarette smoke reduces GPx4 activity and induces autophagic degradation of ferritin, which together contribute to labile iron accumulation and ferroptosis. cIAP = cellular inhibitor of apoptosis protein DMT1 = divalent metal transporter 1 INFR = interferon receptor MLKL = mixed lineage kinase domain–like protein NLRP3 = NLR family pyrin domain containing 3 PL-OH = nontoxic lipid alcohol RIPK = receptor-interacting protein kinase TNFR1 = TNF receptor 1 TRADD = TNFR1-associated death domain protein TRIF = TIR domain-containing adaptor induced IFN-β ZBP1 = Z-DNA-binding protein 1.
Ferroptosis, like necroptosis, is a form of necrotic cell death because it is potently regulated by lipid repair enzymes, including glutathione (GSH) and glutathione peroxidase 4 (GPx4) (Table 1). The term “ferroptosis” was coined in 2012 (17) to define a form of cell death elicited by the small molecule erastin in Ras-mutant cells, which suppresses the import of cystine, leading to GSH reduction and downregulation of the phospholipid peroxidase GPx4 (34). GPx4 efficiently shifts toxic phospholipid hydroperoxides to nontoxic lipid alcohols. Downregulation of GPx4 via inhibition of GSH with erastin, or directly with (1S,3R)-RSL3, leads to an increase in detrimental lipid peroxidation, resulting in induction of cell death. During ferroptosis, labile iron accumulation induced by the disrupted iron metabolism regulation system, such as transferrin receptor and divalent metal transporter 1, evokes the Fenton reaction, resulting in phospholipid peroxidation of plasma membranes caused by ROS. These two representative characteristics of ferroptosis—disrupted iron homeostasis and accelerated lipid peroxidation—are associated with different pathologies across many animal species during various stages of life ( Figure 1 ).
Emerging evidence suggests the involvement of both ferroptosis and necroptosis in diverse experimental models. An experimental neuronal death model after hemorrhagic stroke in vivo and in vitro shows shared ferroptotic and necroptotic cell death but not caspase-dependent apoptosis or autophagy (35). Mitochondrial complex I inhibition triggers mitophagy-dependent ROS production via depolarization of the mitochondrial membrane potential, leading to activation of combined necroptotic and ferroptotic cell death in melanoma cells (36). Treatment of 1-methyl-4-phenylpyridium on the human neuroblastoma cell line (a widely used model of Parkinson’s disease) reportedly elicits necrotic and nonapoptotic cell death, which is sensitive to both Nec-1 and ferrostatin 1 (Fer-1) (37). In addition, we have demonstrated that cigarette smoke extract–induced epithelial cell death is significantly inhibited by Fer-1, marginally inhibited by Nec-1 and pan-caspase inhibitor zVAD-FMK treatment, and significantly inhibited by Fer-1 (38). Several different cell death pathways or multiple cell death pathways may be detected on the basis of cell type, time point, and triggers of cell death. Collectively, these findings indicate a possible involvement of ferroptosis and necroptosis in shared disease pathogenesis, such as stroke, cancer, neurodegenerative disease, and chronic obstructive pulmonary disease (COPD).
The cross-talk between several cell death pathways, including ferroptosis and necroptosis, via the release of DAMPs has been proposed in regulation of inflammatory diseases (7). In addition, ferroptosis mediates synchronized cell death regulated by Nec-1 and compounds that inhibit mitochondrial permeability transition in an IRI and acute kidney injury model, triggering a toxic immune response (39). Ferroptosis and necroptosis act as alternative cell death pathways and notably show synergism in vivo in acute ischemic kidney injury (40). Further studies are needed to clarify whether dual targeting of these pathways by combination therapies is necessary for efficient clinical intervention.
In contrast to apoptosis, RN is more immunogenic because of plasma membrane rupture and release of DAMPs from dying cells. DAMPs are host-derived molecules, including ATP, HMGB1 (high mobility group box 1 protein), IL-33, and heat shock proteins, by which the immune response is triggered with binding to pattern recognition receptors, such as Toll-like receptors (41). Releasing DAMPs has been widely implicated in various lung inflammatory diseases, such as acute lung injury (42) and COPD (43–45) ( Figure 1 ). Accordingly, DAMPs could potentially become a common therapeutic target because they are downstream of the ferroptosis and necroptosis pathways in lung inflammation.
Autophagy is a lysosome-mediated catabolic pathway that maintains cellular viability and homeostasis by eliminating unnecessary and harmful components. During the process of autophagy, intracellular substrates such as proteins, molecules, lipids, and organelles are sequestered into double-membraned autophagosomes that are subsequently degraded after autophagosome–lysosome fusion. Selective autophagy that recycles specific components (e.g., mitochondria/mitophagy, ferritin/ferritinophagy, and lipids/lipophagy) has been employed in response to various cellular stressors. An increase of autophagosomes is often shown in dying cells, which is morphologically classified as type 2 programmed cell death and termed “autophagic cell death.” Cell death often occurs concomitantly with autophagy rather than being induced by autophagy (46). Hence, this term has been changed to “autophagy-dependent cell death” in the presence of experimental evidence of a mechanistic or functional link between RCD and the autophagy apparatus. Moreover, pharmacological or genetic manipulation of autophagy can modulate cell death (47). Autophagic inhibition promotes cell death in various pathological disorders, such as cancer, cardiovascular disease, and inflammatory disorders, indicating the cytoprotective ability of autophagy to maintain cell viability and homeostasis (48, 49). In contrast, selective autophagy has been shown to contribute to RCD in various models. Selective degradation of Fap-1 (Fas-associated phosphatase 1) by autophagy accelerates FAS apoptosis in a cell-type–specific manner (50). Cigarette smoke elicits mitophagy and autophagic degradation of mitochondria, contributing to necroptosis in COPD (51). Ferritinophagy, the autophagic degradation of ferritin to free iron mediated by specific adapter NCOA4 (nuclear receptor coactivator 4), contributes to ferroptosis development via lipid peroxidation (52). GPx4, a selenoprotein that inhibits ferroptosis by deoxidization of lipid ROS, is diminished by chaperone-mediated autophagic degradation, resulting in increased ferroptosis (53). Collectively, although autophagy plays a protective role against cell death by maintaining cellular metabolic homeostasis in the steady state, organelle- and protein-selective autophagy appear to induce RCD in various experimental models, including cigarette smoke exposure.
COPD is characterized by irreversible airway narrowing and distal airspace destruction as a result of prolonged smoke exposure it is refractory to currently available therapies and is now one of the leading causes of morbidity and mortality worldwide (54–56). Increasing studies suggest that necrotic forms of RCD are implicated in the pathogenesis of COPD. Airway epithelial cell necroptosis via mitophagy is implicated in COPD pathogenesis (51) (Table 2). In the same set of studies, the increase of RIPK3 expression is observed in the lungs of cigarette smoke–exposed wild-type mice. Another group has also revealed that cigarette smoke exposure induces necrotic cell death and DAMP release in the BEAS2B cell line. Furthermore, cigarette smoke exposure of wild-type mice increases neutrophils and DAMPs in BAL, which is attenuated by Nec-1 treatment (43). However, both of these studies seem to have potential limitations because they lack experiments with mice with knockout of RIPK3 or MLKL (major necroptosis regulators). Thus, further in vivo experiments are needed to evaluate the involvement of necroptosis in COPD pathogenesis.
Alternatively, we have previously demonstrated that lung epithelial cell ferroptosis induced via autophagic degradation of ferritin plays an important role in the generation of a COPD phenotype (39). Labile iron accumulation and augmented lipid peroxidation are shown with concomitant necrotic cell death during cigarette smoke exposure, which is enhanced by GPx4 downregulation in in vivo and in vitro experiments (39). Treatment with deferoxamine and Fer-1 and GPx4-knockdown experiments also support the role of ferroptosis in cigarette smoke–treated lung epithelial cells. Ferroptosis caused by labile iron accumulation in epithelial cells is initiated by autophagic ferritin degradation (ferritinophagy) via NCOA4 (nuclear receptor coactivator 4) in response to cigarette smoke treatment (39). Moreover, cigarette smoke–exposed GPx4 +/− mice show significantly higher degrees of lipid peroxidation, nonapoptotic cell death, DAMP release, and enhanced COPD phenotypes than wild-type mice, all of which were attenuated in GPx4-transgenic mice (39). These data indicate the important role of cigarette smoke in epithelial cell ferroptosis in COPD pathogenesis. Thus, suppression of lipid peroxidation by GPx4 and antioxidants such as Fer-1 and of iron chelation by deferoxamine is a possible target of an antiferroptotic treatment to prevent COPD progression.
To date, there are no successful clinical trials using antioxidant therapy for COPD. Unsaturated lipids lead to constant propagation of free radical autoxidation because of the weak carbon–hydrogen bond in the methylene group. Hence, the targeting of lipid peroxidation is a potential antioxidant therapy. This is supported by our data showing that both the presence of the GPx4 transgene and treatment with Fer-1 rescued COPD phenotype. Our data indicate that it is necessary to develop lipid-specific antioxidant drugs for treatment of COPD.
Idiopathic pulmonary fibrosis (IPF) is a refractory and inexorably progressive lung fibrotic disease pathologically characterized by lung epithelial injury and subsequent fibroblastic proliferation and deposition of extracellular matrix in lung parenchyma (57, 58). The lung epithelium acts as the first barrier to prevent access to inspired external stimuli. Alveolar epithelial cell (AEC) or airway epithelial cell destruction during lung injury leads to dysregulated wound healing, including proinflammatory stress response, thereby resulting in myofibroblast differentiation and aberrant collagen deposition in the lung interstitium (58, 59). Epithelial cell apoptosis that is considered a major form of RCD in IPF pathogenesis was first described in 1996 (60). There are significantly higher numbers of TUNEL-positive bronchiolar epithelial cells and AECs in the lungs of patients with IPF than in normal lungs and lungs of patients with COPD (60) Table 2. Furthermore, the Fas–Fas ligand pathway, a novel inducer of apoptosis, is upregulated in the BAL and frozen lung sections of patients with IPF compared with those of healthy control subjects, indicating the involvement of apoptosis in IPF pathogenesis (61). Lung epithelial cells have been considered a key player within the context of apoptosis in IPF pathogenesis (62). However, the mechanisms by which apoptotic epithelial cells contribute to lung fibrosis are still poorly understood. Nonapoptotic DNA fragmentation was detected by TUNEL assay, indicating that TUNEL-positive cells include not only apoptotic cells but also necrotic cells. Epithelial–mesenchymal interactions are believed to be important in IPF pathogenesis, as shown by AEC apoptosis/necrosis adjacent to underlying myofibroblasts in the lungs of human patients (63). In a previous study, IL-1β secretion from senescent bronchial epithelial cells was shown to induce myofibroblast differentiation in fibroblasts (58). DAMPs secreted by dying necrotic cells are involved in some lung diseases (64, 65), indicating the potential role of necrotic cell death in IPF. Earlier, we found that RIPK3-mediated necroptosis in AECs plays a role in IPF development via the release of DAMPs (66) (Table 2). Immunohistochemistry and Western blotting show that RIPK3 and p-MLKL expression levels are significantly higher in the lungs of patients with IPF than in lungs of healthy control subjects. Bleomycin (BLM)-treated AECs isolated from RIPK3-knockout mice show attenuation of cell death with decreased p-MLKL expression. RIPK3-knockout mice efficiently inhibit BLM-induced DAMP secretion, cell death, and lung fibrosis without decrease of cleaved caspase 3 expression level. According to the in vitro experimental results using zVAD-fmk and Nec-1, both apoptosis and necroptosis coexist in BLM-treated AECs. BLM injection into wild-type murine lung elicits equally both a cleaved caspase 3–positive cell death that is efficiently inhibited by zVAD-fmk and a cleaved caspase 3–negative cell death that is efficiently inhibited by Nec-1 in AECs, suggesting that both apoptosis and necroptosis are important steps during BLM-induced lung fibrosis. These two RCD pathways are believed to suppress each other, but necroptosis works as an alternative when caspase-dependent apoptosis is absent (67). However, given that the aforementioned in vitro experiment shows that zVAD-fmk and Nec-1 synergistically suppress cell death, we speculate that the simultaneous blockage of apoptosis and necroptosis may be a promising target for IPF treatment.
RIPK3-mediated necroptosis and subsequent activation of the NLRP3 (nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3) inflammasome are observed in neonatal mice with hypoxia-induced lung injury, which is attenuated by genetic deletion in RIPK3 (68). Similarly, contributions of RIPK3-mediated necroptosis and inflammasome pathways in the development of an LPS-induced acute lung injury mouse model have been reported. GSK872, a selective inhibitor of RIPK3, significantly reduces LPS-induced necroptosis and NLRP inflammasome activation, with concomitant attenuation of IL-1β and IL-18 production and inflammatory cell infiltration (69). RIPK3-deficient mice are protected against ventilator-induced lung injury in a fatty acid β-oxidation–dependent manner (70). Inhibition of RIPK1 by Nec-1 is shown to decrease systemic and lung inflammation and increase survival in neonatal mice with sepsis (71). Thus, these studies suggest that necroptosis and inflammasome activation contribute to various forms of acute lung injury.
Cell death pathways, including apoptosis and RN, have evolved as a first-line host defense system against virus-encoded cell death suppressors during viral infection (72). As a preliminary stage to RN, apoptosis plays a defensive role to eliminate virus-infected cells, subsequently providing the adaptation for some viruses to acquire virus-encoded cell death suppressors. RN is considered an adaptation against caspase 8–targeted apoptosis suppressors in a RIP3-dependent manner (72). Some viruses, including vaccinia, are susceptible to RIP3-dependent RN (73), whereas other viruses, including murine cytomegalovirus, are no longer susceptible because of cytomegalovirus-encoded RIP3 suppressors that limit the efficacy of cell death pathways (74). Mice deficient in RIPK3 activity are reportedly more susceptible to influenza A infection than their wild-type counterparts (75). Thus, the prevailing consensus is that necroptosis plays a protective role during viral pneumonia by eliminating infected cells, thus reducing viral replication (Table 2).
With respect to the association between RCD and bacterial infections, RIPK1, caspase 8, and RIPK3 contribute to Yersinia pestis infection–induced macrophage cell death, and caspase 8– and RIPK3-knockout mice are highly susceptible to Y. pestis infection (76). These knockout mouse models also reduce caspase 1–associated inflammasome activity, which is crucial to host responses against Y. pestis and other infections (76). These results suggest that necrosis plays a protective role in innate immunity during bacterial infections. Likewise, necroptosis promotes Staphylococcus aureus elimination by suppressing excessive inflammatory signaling (77). Although several groups have pointed out the detrimental role of necroptosis during bacterial infection (78), the vast majority of studies have indicated a protective role for necroptosis via inhibition of bacterial load and inflammation.
In stark contrast, some recent research studies have shed light on the adverse role of ferroptosis in bacterial infection and polymicrobial sepsis (79). Human epithelial cell ferroptosis is initiated by the prokaryotic bacterium Pseudomonas aeruginosa via oxidizing host membrane arachidonic acid phosphatidylethanolamines by lipoxygenase (pLoxA). Furthermore, P. aeruginosa–induced ferroptosis of host epithelial cells promotes biofilm formation, enhancing colonization by the pathogen (80). Similarly, Mycobacterium tuberculosis (Mtb) elicits macrophage necrosis associated with reduced concentrations of GPx4 concomitantly with increased free iron, mitochondrial superoxide, and lipid peroxidation, all of which are notable features of ferroptosis. Interestingly, Fer-1 treatment remarkably reduces lung pathology, including pulmonary necrosis and Mtb bacterial burden, in Mtb-infected mice (81). Moreover, ferroptosis occurs in T cells during lymphocyte-responsive infections such as lymphocytic choriomeningitis virus and Leishmania major parasite infection. GPx4, a scavenger of phospholipid hydroperoxide, prevents T-cell ferroptosis, which plays a crucial role in the immune response. T-cell–specific GPx4-deficient mouse experiments reveal that GPx4-deficient T cells rapidly accumulate lipid peroxides and die by ferroptosis (82). GPx4-deficient T cells fail to prevent viral and parasitic infection and are rescued by high-dose vitamin E exposure. This finding suggests the detrimental role of ferroptosis by weakening T-cell immunity and subsequently suggests a beneficial role for vitamin E and GPx4 inhibition of ferroptosis during viral and parasitic infection (82). Taken together, whether the role of RN in infectious disease is protective or injurious may depend on cell type and on the kinds of pathogens involved (Table 2).
In 2013, Saddoughi and colleagues were the first to report a role for necroptosis in lung tumor growth inhibition. In this study, targeting of oncoprotein I2PP2A/SET using the sphingosine analog drug FTY720 suppressed lung tumor growth via PP2A (protein phosphatase 2A) activation and necroptosis mediated by RIPK1 (83) (Table 2). Accumulated evidence suggests that anticancer drugs trigger necroptosis in lung cancer cells. Cisplatin and paclitaxel still play a central role in lung cancer chemotherapy. Paclitaxel induces necroptosis in lung adenocarcinoma, which is promoted by dasatinib, a c-Src inhibitor, via dephosphorylation of caspase 8 by c-Src (84). Long-term cisplatin treatment is known to enhance tumor resistance to cell death induction via diverse mechanisms. Overexpression of PDIA6 (protein disulfide isomerase, family A, member 6) is observed in cisplatin-resistant non–small cell lung cancer (NSCLC) cells and in the lungs of patients with adenocarcinoma. PDIA6-targeting siRNA reverses the cisplatin-resistant phenotype by restoring a noncanonical cell death pathway that overlaps with some necroptosis pathways (85).
Despite an increasing number of studies reporting the close relationship of ferroptosis with progression of various tumors, a paucity of material is available with respect to lung tumors. Alvarez and colleagues revealed the mechanism by which lung adenocarcinoma is protected from ferroptosis. The iron-sulfur cluster biosynthetic enzyme NFS1 that lies in a region of genomic amplification plays an important role in surviving the high-oxygen environment of early-stage lung tumors. Suppression of NFS1 cooperates with the inhibition of cysteine transport to trigger ferroptosis and aid in the gradual progression of lung adenocarcinoma (86) (Table 2). Therefore, lung adenocarcinoma highly expressed NFS1 to protect from ongoing ferroptosis in response to oxidative damage (81, 86). GPx4 is highly expressed in radioresistant A549 and H460 cells (NSCLC cell line). Erastin, a representative ferroptosis inducer, partially suppresses radioresistance of NSCLC cells by inducing GPx4-mediated ferroptosis (87). The network of long noncoding RNA and miRNA regulation of ferroptosis is reported to be an important mechanism in NSCLC tumorigenesis (88). Collectively, these observations indicate an inhibitory role for ferroptosis in the development and progression of lung cancer (Table 2). Despite the presence of in vitro reports suggesting the clinical importance of RN targeting therapy, the in vivo evidence is scarce. Therefore, further evidence including in vivo studies is needed to elucidate the role of RN in human lung cancer.
Recent studies indicate the involvement of RN in the pathogenesis of bronchial asthma. Virus-induced asthma exacerbation, mimicked by IFN-β–knockout mice treated with house dust mite, is associated with necroptosis in terms of increased necroptosis markers, pMLKL, and lactate dehydrogenase in BAL fluid (89). IL-33, a major proinflammatory cytokine in the type 2 immune response in inflammatory diseases, including asthma, is released in response to necroptosis, resulting in activation of basophils and eosinophils (90). Furthermore, the necroptosis inhibitor GW806742X abrogates necroptosis and IL-33 reaction in vitro and attenuates eosinophilia in a mouse model of Aspergillus fumigatus extract–induced asthma, which is potently dependent on IL-33 (90). TNF-α–induced necroptosis in human bronchial epithelial cells, which partially mimics severe asthma, is enhanced by MUC1 (mucin 1) knockdown, which is attenuated by Nec-1 (91). The same research group proposed that the resistance of glucocorticoids against asthma may depend on blocked glucocorticoid receptor-α nuclear translocation and p-p65 phosphorylation induced by MUC1 ablation in TNF-α–induced necroptosis in human bronchial epithelial cells (92). The phenotype of bronchial asthma is known to be heterogeneous with respect to T-helper cell type 2 (Th2) inflammation dominance. Necroptosis seems to be involved in multiple aspects of asthma, including Th2 inflammation via release of IL-33 from necrotic cells, as well as TNF-α–induced non-Th2 inflammation, often observed in steroid-refractory asthma.
With respect to ferroptosis in lung epithelial cells, little is known about its role in the pathogenesis of asthma. The PEBP1 (phosphatidylethanolamine-binding protein 1)–15-lipoxygenase (LO) complex, known to stimulate IL-13/IL-4–induced Th2 inflammation, is found to be a master regulator of ferroptosis as well as GPx4. Higher degrees of colocalization of PEBP1 with 15-LO are seen in human bronchial epithelial cells from patients with asthma than in those from healthy individuals. In addition, a strong correlation has been reported between this colocalization in asthma and the fractional exhaled nitric oxide in human bronchial epithelial cells, suggesting the importance of PEBP1/15-LO–driven ferroptosis in Th2 inflammation during asthma pathogenesis (93).
In summary, evidence from these research studies suggests the involvement of RN in allergic airway inflammation and asthma exacerbation. Therapeutic targeting of necroptotic and ferroptotic signaling may lead to future developments in asthma treatment.
With respect to necroptosis and ferroptosis, the involved signaling pathways and induction mechanisms of the identified RN have been well documented by virtue of steady progress in related research. However, the role of RN in diverse lung diseases remains incompletely understood in spite of mounting evidence revealing that RN is also implicated in several other organ diseases. In addition, compared with necroptosis, there is less evidence to support a clinical role for ferroptosis in lung disease pathogenesis, which can most likely be attributed to its recent discovery. Accordingly, accumulation of clinically relevant evidence for ferroptosis is expected in the future.
In this review, we have attempted to clarify that RN plays contradictory roles—disease protection or disease progression—depending on the type of cell and pathogenesis. In addition, some in vivo or in vitro experimental cell death models have demonstrated shared roles for ferroptotic and necroptotic cell death and the synergistic effects of Nec-1 and Fer-1. Therefore, it would be useful to further investigate the therapeutic potential of simultaneous inhibition of both cell death pathways in complex disease models. We believe that necroptosis and ferroptosis will become the new target for treating various lung diseases that currently have no cure.
The authors thank Stephanie Cambier for comments on the manuscript and thoughtful suggestions.