Vitamin D3 is so important in the treatment of COVID, that some hospitals in Europe made it part of a mandatory protocol for every COVID patient. But sadly, they aren't doing that in the US. Tony Fauci admitted he uses Vitamin D3 himself, because he knows how effective it is.... but did he bother to tell the rest of us?? NOPE! You can see him telling Jennifer Garner, in a video chat shown on THIS PAGE, that Vitamin D3 can be beneficial.... sure would have been nice if he could have told the REST of us!!!
Dr Michael Cohen, a General Practitioner, prescribed Vitamin D for al of his COVID patients, knowing how well it worked, to prevent COVID deaths. He has not had a single patient admitted to the hospital! This is a really compelling interview.
Vitamin D in Israel
CAN VITAMIN D PREVENT COVID DEATHS?
Vitamin D + Immunity: How it Helps Stamp Out Over-Active Immune Responses
At least HALF of the people in the US are Vitamin D3 deficient, but I guessed it was just because people don't go outside enough, or because they use sunscreen more often. I had no idea it could be related to your skin. If you watch the video, you will see why many health practitioners urge people to make sure they aren't D3 deficient, during this COVID crisis (also keep in mind, that what it says to take on the bottle is not necessarily what you, personally, should take).
You may want to get your blood levels checked. You can read more about that, HERE. Dr. Weil wrote an article about COVID-19 and D3 supplementation, HERE.
Vitamin D deficiency and co-morbidities in COVID-19 patients – A fatal relationship?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7276229/
1.2. Vitamin D and immune system
3. Conclusion
Vitamin D3 is GREATLY undervalued in our culture that is severely deficient. Did you know that 40,000 IU of vitamin D is just equivalent to 1 milligram? (You can see a video with Dr. Eric Berg, talking about this, at the bottom of this post).
This next video below (another by Tom Coffee) is not about Baking soda or Lime juice, but I thought it was worth sharing during this COVID crisis, because he gives some EYE OPENING facts about Vitamin D.
He learned that people with dark skin are less able to absorb Vitamin D3 from the sun, than people with fair skin. Wow. I had no idea, and I am guessing that most people don't know this either, so please share this information with others if you think it could help them. I thought I am a huge fan of Vitamin D3, and have told SO many people about it, but I had never heard that before.
I've been taking about 10,000 Vitamin D3 for seasonal affective disorder and back pain for over 10 years (this stuff is a lifesaver for me). I took it on a whim, thinking maybe it would help with Seasonal Affective Disorder, but couldn't believe how much it helped me with lower back pain. I can't tell you what to do, or how much you should take, but I can tell you that I, personally, don't feel a change in my back stiffness unless I take at least 5,000 or 10,000 IU (2,000 just doesn't cut it for me, personally). The reason I went up to 10,000 IU is because I wanted to take the maximum amount that could be considered safe, and Dr. Andrew Weil says this on his page about Vitamin D3:
"No adverse effects have been seen with supplemental vitamin D intakes up to 10,000 IU daily"
I know about HALF of the people in the US are Vitamin D3 deficient, but I guessed it was just because people don't go outside enough, or because they use sunscreen more often. I had no idea it could be related to your skin. If you watch the video, you will see why many health practitioners urge people to make sure they aren't D3 deficient, during this COVID crisis (also keep in mind, that what it says to take on the bottle is not necessarily what you, personally, should take).
You may want to get your blood levels checked. You can read more about that, HERE. Dr. Weil wrote an article about COVID-19 and D3 supplementation, HERE.
Vitamin D for Covid, What’s the Catch? - DarkHorse Podcast Gruff Davies and Linda Benskin
IAN CLARK DISCUSSES WHY YOU NEED 'K2D3' FOR YOUR IMMUNE SYSTEM WITH NICHOLAS VENIAMIN
THE TRUTH ABOUT CANCER PRESENTS: HEALTH NUGGETS - VITAMIN D - NATURE'S MEDICINE CHEST
If You Get COVID 19: Optimize Immune System (Vitamin D, Monoclonal Antibodies, NAC, Quercetin etc.)
STUDY SHOWS VITAMIN D3 SUPPLEMENTATION REDUCES COVID DEATHS BY 64%
Vitamin D is a steroid hormone that has a lot of different effects on the body. At 5-10,000 units a day it can switch aon and off
Vitamin D is found in virtuallly every cell.
Vitamin D deficiency and co-morbidities in COVID-19 patients – A fatal relationship?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7276229/
1.2. Vitamin D and immune system
3. Conclusion
This is a pretty good article from USA Today:
https://www.usatoday.com/in-depth/news/2020/06/09/vitamin-d-and-covid-19-could-low-vitamin-d-levels-affect-coronavirus/5274331002/
Can vitamin D help with symptoms of COVID-19? Possibly, it's key to helping your immune system function
Research shows vitamin D helps prevent acute respiratory distress syndrome (ARDS), a common killer in COVID-19 patients.
Vitamin D, Immune System & SARS-CoV-2 (COVID-19) | Mechanism of Vit D Immune Regulation & Overview
UK doctors review use of vitamin D's effects on coronavirus
Dosage For Vitamin D, K2, and Calcium
The Truth About VITAMIN D
Latest Research on Vitamin D | The Best Ways to Boost Vitamin D Level
| REVIEW ARTICLE | ||
|
Respiratory infections: Role of Vitamin D and surfactant proteins A and D
Rachel Poorna, Niranjan Biswal
Department of Pediatrics, JIPMER, Puducherry, India
| Date of Submission | 12-Sep-2018 |
| Date of Decision | 19-Apr-2019 |
| Date of Acceptance | 03-May-2019 |
| Date of Web Publication | 31-Aug-2020 |
Correspondence Address:
Dr. Niranjan Biswal
Department of Pediatrics, JIPMER, Puducherry - 605 006
India
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/lungindia.lungindia_369_18
 Abstract |
Respiratory tract infection is the common viral infection and the principal cause of death among children under 5 years of age. It damages lung epithelium and increases mucus production and inflammation, leading to dyspnea. The sunshine vitamin (Vitamin D) and surfactant protein (SP) A and D along with their usual function play an important role in host defense. This article reviews with immune role of Vitamin D and SP A and D which aids excessive cytokines production, boosts phagocytosis, hinders inflammatory activity, and thus acts as a first-line defense against lung pathogens.
Keywords: Innate immunity, respiratory tract infection, surfactant protein, Vitamin D
| How to cite this article: Poorna R, Biswal N. Respiratory infections: Role of Vitamin D and surfactant proteins A and D. Lung India 2020;37:421-4 |
| How to cite this URL: Poorna R, Biswal N. Respiratory infections: Role of Vitamin D and surfactant proteins A and D. Lung India [serial online] 2020 [cited 2020 Dec 6];37:421-4. Available from: https://www.lungindia.com/text.asp?2020/37/5/421/293975 |
| Introduction |  |
Respiratory tract infection (RTI) is the primary cause of death in children under 5 years of age. It is usually caused by various viruses; sometimes, <20% are caused by bacteria or both. Inhaled microbes damage the epithelial lining of the tract and increase the mucus production and inflammation, leading to breathing difficulty. It is the main reason for the financial burden on healthcare services and accounts for a huge proportion of daily consultations of physicians.[1]
Vitamin D and surfactant proteins (SPs) have recently been found to play an important role in preventing infections of the respiratory tract by increasing immunity. Therefore, it is necessary to enhance our current knowledge about the immunological functions of the Vitamin D and SP in the prevention of RTI. This review deals with the information on Vitamin D and SPs immune function.
| Vitamin D | |
Vitamin D is usually referred as “sunlight hormone” or “sunshine vitamin” as it is derived mainly from sunlight. It is a secosteroid hormone that is vital to homeostasis of calcium and a pluripotent hormone with vast immunological function. Vitamin D is the only micronutrient which is synthesized by the skin and utilized as a hormone. Skin exposed to ultraviolet-B spectrum coverts 7-dehydrocholesterol in subcutaneous fat to produce an inactive form of Vitamin D. This inactive form is hydroxylated in the liver to form 25(OH) D and converted into the active form 1,25 dihydroxyvitamin D in the kidney.[2],[3],[4]
Synthesis of Vitamin D is affected by latitude, lack of sun exposure, season, use of sunscreen, indoor lifestyle, clothing, air pollution, low intake of calcium, intestinal malabsorption, deficiency of maternal Vitamin D, and obesity (Vitamin D is hidden in adipose tissue).[3],[5],[6]
Recent studies show that Vitamin D deficiency and insufficiency have a potential role in developing respiratory infection, and ample level of Vitamin D in the body is linked with better lung function. The prevalence of respiratory infection during the winter season is probably due to decrease Vitamin D production resource from sun exposure. Infants with low Vitamin D level in cord blood and low Vitamin D content in breast milk are more prone to developing respiratory infection.[7]
Immune function of Vitamin D in lungs
The antiricketic Vitamin D alongside with its role in skeletal health is now known for strengthening the immunity by increasing the production of natural antibodies.[8] The innate immunity will take action quickly against the entered pathogen according to the level of immune cells and proteins in the body. Vitamin D builds up the innate immune system by increasing the amount of good immune protein.[9],[10] It also operates as immune system modulator, averts excessive production of inflammatory cytokines, and boosts the macrophages activity. Vitamin D that is mediated by Vitamin D receptor (VDR) has wide range of effects on different cells of immune system and expresses innate defense against viruses and bacteria.[6],[7] Vitamin D and its receptor manipulate the three foremost immunity troupes of the lungs (airway epithelium, alveolar macrophages, and dendritic cells) in identifying the pathogen and encountering them.[4]
As mentioned earlier, the Vitamin D is converted to active form by two hydroxylation steps. The airway epithelium expresses high levels of 1α-hydroxylase which converts inactive Vitamin D to active form. The active form of Vitamin D kindles cathelicidin secretion and other peptides in the epithelial cell that guard against bacterial and viral infections. Thus, locally generated Vitamin D promotes innate immunity and controls inflammation and tissue damage in the respiratory tract.[4],[11],[12] It also promotes the adaptive immune response by initiating macrophages and triggering the cells responsible for antigen recognition, the T- and B-lymphocytes.[13] Alveolar macrophages generate active Vitamin D which plays a significant intracrine role in macrophage response to infection. The enzyme 1α-hydroxylase expressed by stimulation of macrophages has favorable effects on host defense.[4],[14] Vitamin D has been found to have direct effect on T- and B-cells. Both T- and B-lymphocytes express VDR and 1α-hydroxylase. Vitamin D helps in producing cytokine interleukin (IL)-10 which plays a major role in anti-inflammation and immunosuppressant. Monocyte-derived dendritic cells boost up the function of 1α-hydroxylase and metabolize Vitamin D originator to active Vitamin D. Dendritic cells initiate and regulate the adaptive immunity against the inhaled microbes by maturation. This maturation is exemplified by controlling antigen uptake and activation of inhabitant T-cells. Vitamin D generated by dendritic cells hinders cell differentiation and maturation and increases IL-10 secretion. The inhibition dendritic cell maturation and T-cell hyperresponsiveness had immunosuppressive actions.[4] The enzyme matrix metalloproteinases (MMPs) in the lung is concerned with the inflammation and cell movement. Vitamin D is found to lessen the level of circulating MMPs and thus reduces the lung inflammation.[15] Thus, locally generated Vitamin D in theairways and lungs will minimize the tissue damage and inflammation by clearing the microbes.[4]
Vitamin D and respiratory viruses
Inhaled respiratory viruses unite first to the nonspecific receptors such as glycolipids or glycoproteins on the respiratory epithelium and endocytosis occur which assists the virus for subsequent reproduction, transcription, and translation of new viruses to infect new cells. The infected cells are predicted by intracellular intrinsic pathogen recognition receptors in the lung epithelium and commence a brisk immune response against viral invasion.[16] At the same time, viral infection amplifies the activation of Vitamin D in the airway and boosts cathelicidin production in the form of LL-37. LL-37 disrupts viral membrane through electrostatic interactions and blocks the viral entry. Vitamin D also slows down proinflammatory cytokine release by macrophages and upregulates the antimicrobial peptides to exhibit antiviral activity.[17]
| Vitamin D Supplementation | |
Adequate Vitamin D level lessens the risk of lower RTI.[18] Hence, it is necessary to modify the dietary habits (including oily fish, cod liver oil, organ meats, and egg yolks), exposure to sunlight, and Vitamin D supplementation for overcoming the deficiency. The recommended dose for Vitamin D supplementation is 400–1000 IU for children <1 year and 600–1000 IU for children >1–18 years of age. It is available in the form of drops for infants and chewable tablets for children and adolescence.[19],[20]
| Pulmonary Surfactant | |
The surface tension between two media is decreased by a thin film of amphiphilic molecules are called surfactant or surface active agent. The surfactant present in the lung is called pulmonary surfactant which facilitates gas diffusion by reducing the surface tension at gaseous-aqueous interphase, maintains alveolar size, lung compliance, lung tissue elasticity, keeps alveolar dry, and also plays a host defense role.[21]
In humans, the pulmonary surfactant is discriminated between 24 and 34 weeks of gestation. It is a fusion of 70%–80% of phospholipids (dipalmitoylphosphatidylcholine) and phosphatidylglycerol, 10% of proteins (SP A, SP B, SP C, and SP D), and 10% of neutral lipids (mainly cholesterol).[22] The different compounds of surfactant are synthesized by multivesicular bodies from endoplasmic reticulum and Golgi apparatus. The synthesized surfactant is transferred to prelamellar bodies and gathered to form lamellar bodies. The lamellar bodies endure exocytosis form alveolar type II cells to secrete the surfactant into extracellular matrix.[23]
Immune function of surfactant protein A and D in lungs
Among the four types SP A, B, C, and D, SP A and SP D are a huge glycosylated protein with hydrophilic property and they are the molecular factors of inherent host defense immune system.[21] These proteins contain collagen C-type lectin and belong to collectin family with carbohydrate-binding properties. They attach to the particular carbohydrates and lipid structure on the surface of pathogens (bacteria, virus, fungal, and protozoa) through calcium-dependent interaction and prevent them from penetrating the target cell. It also stimulates alveolar macrophages for opsonization of pathogens. These protein control inflammation and regulate the immune cell activity in lungs.[22] The SP A and SP D make the innate host defense by altering cytokine production, enhancing immune cell chemotaxis and function, and regulating cell proliferation and apoptosis.[24]
Surfactant protein A and D and respiratory viruses
SP A and SP D in the mucus layer and alveolar surface bind to glycoproteins such as G protein and F protein of respiratory viruses, assist in pathogen elimination by neutralization and clumping, and boost phagocytosis.[25] The G protein is accountable for viral attachment, and the F protein is responsible for infiltration of the virus into the host cells and spreading from cell to cell.[26] SP A binds with oligosaccharides through sialic acid deposits and C-type lectin of SP D binds with carbohydrate structure on the viruses, which inactivates and inhibits hemagglutination activity, thus preventing inflammation, and enhances viral clearance. The immunologic environment of both SP A and SP D aids in host defense and at the same time hinders the inflammatory activities that injury the lung and impair gas exchange.[27]
| Conclusion | |
Vitamin D and SP A and D other than their traditional functions also play a vital role in lung innate immunity. They act as a first-line defense against respiratory viruses, both individually and dependently. They optimize the lung function by improving viral clearance, inhibit inflammation, and boost phagocytosis along with the reverse defense mechanism in the respiratory tract [Figure 1].
 | Figure 1: Immune function of Vitamin D and surfactant protein A and D Click here to view |
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
| References | |
| 1. | Bhasin SM, Budden EH, Ketkar AR, Pawar AP. Current trends in the treatment of upper respiratory tract infections in neonates, infants and children: A survey. Indian J Pharmacol 2002;34:62-3.  [Full text] |
| 2. | Kochupillai N. The physiology of Vitamin D: Current concepts. Indian J Med Res 2008;127:256-62. [PUBMED] [Full text] |
| 3. | Finklea JD, Grossmann RE, Tangpricha V. Vitamin D and chronic lung disease: A review of molecular mechanisms and clinical studies. Adv Nutr 2011;2:244-53. |
| 4. | Hansdottir S, Monick MM. Vitamin D effects on lung immunity and respiratory diseases. Vitam Horm 2011;86:217-37. |
| 5. | Harinarayan CV, Joshi SR. Vitamin D status in India – Its implications and remedial measures. J Assoc Physicians India 2009;57:40-8. |
| 6. | Aranow C. Vitamin D and the immune system. J Investig Med 2011;59:881-6. |
| 7. | Leis KS, McNally JD, Montgomery MR, Sankaran K, Karunanayake C, Rosenberg AM. Vitamin D intake in young children with acute lower respiratory infection. Transl Pediatr 2012;1:6-14. |
| 8. | Charan J, Goyal JP, Saxena D, Yadav P. Vitamin D for prevention of respiratory tract infections: A systematic review and meta-analysis. J Pharmacol Pharmacother 2012;3:300-3. [PUBMED] [Full text] |
| 9. | Cannell JJ, Vieth R, Umhau JC, Holick MF, Grant WB, Madronich S, et al. Epidemic influenza and Vitamin D. Epidemiol Infect 2006;134:1129-40. |
| 10. | Mascitelli L, Grant WB, Goldstein MR. Obesity, influenza virus infection, and hypovitaminosis D. J Infect Dis 2012;206:1481-2. |
| 11. | Brockman-Schneider RA, Pickles RJ, Gern JE. Effects of Vitamin D on airway epithelial cell morphology and rhinovirus replication. PLoS One 2014;9:e86755. |
| 12. | Hansdottir S, Monick MM, Hinde SL, Lovan N, Look DC, Hunninghake GW. Respiratory epithelial cells convert inactive Vitamin D to its active form: Potential effects on host defense. J Immunol 2008;181:7090-9. |
| 13. | Bikle DD. Vitamin D metabolism, mechanism of action, and clinical applications. Chem Biol 2014;21:319-29. |
| 14. | Gerke AK, Pezzulo AA, Tang F, Cavanaugh JE, Bair TB, Phillips E, et al. Effects of Vitamin D supplementation on alveolar macrophage gene expression: Preliminary results of a randomized, controlled trial. Multidiscip Respir Med 2014;9:18. |
| 15. | Gilbert CR, Arum SM, Smith CM. Vitamin D deficiency and chronic lung disease. Can Respir J 2009;16:75-80. |
| 16. | Greiller CL, Martineau AR. Modulation of the immune response to respiratory viruses by vitamin D. Nutrients 2015;7:4240-70. |
| 17. | Hughes DA, Norton R. Vitamin D and respiratory health. Clin Exp Immunol 2009;158:20-5. |
| 18. | Martineau AR, Jolliffe DA, Hooper RL, Greenberg L, Aloia JF, Bergman P, et al. Vitamin D supplementation to prevent acute respiratory tract infections: Systematic review and meta-analysis of individual participant data. BMJ 2017;356:i6583. |
| 19. | Lee JY, So TY, Thackray J. A review on vitamin d deficiency treatment in pediatric patients. J Pediatr Pharmacol Ther 2013;18:277-91. |
| 20. | Casey CF, Slawson DC, Neal LR. VItamin D supplementation in infants, children, and adolescents. Am Fam Physician 2010;81:745-8. |
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| 27. | Pastva AM, Wright JR, Williams KL. Immunomodulatory roles of surfactant proteins A and D: Implications in lung disease. Proc Am Thorac Soc 2007;4:252-7. |
From the United States
I've been using it daily in the morning to give me the boost I need of said vitamin, since i'm a bit deficient. I feel healthier, much more alert, less tired and this vitamin gives me a great appetite as well. Once again, thanks!! I hope you guys keep making safe, all natural products.. It doesn't have to be the strongest, just consistently good and wholesome for all to consume and be healthier. amen
Switched from the vitamin D spray I used the past 4 years and the difference in how I feel now is remarkable! I think it is better absorbed by my body because of the coconut oil and just the overall quality of this product . Started my husband on it too due to the supporting research regarding Covid. Definitely buying again!
From the United States
From the United States
From the United States
From the United States
Vitamin D3 is important because COVID impact lung.
Research for lung from latest Regeneron Antibodies Phase 3 trials for COVID show the need for vitamin D3 in lung.
I got high blood pressure to 212/120 for day 5 to day 7 July 16 to July 18 because I sat in my car alone and virus in the air trapped in my car.
after one month using supplement Forskolin my high blood pressure normal now
so I need K2 vitamin and D3 to prevent lung infection
During COVID I have no other symptom besides lung high blood pressure
update June 2020
using it for a vitamin D supplement , as it has Vitamin K blended it improves absorption.the supplement keeps me energized all day and my immunity high against flu/covid etc
From the United States
From the United States
Get YOUR level checked.. mine was 17 ng/ml! Now it sits between 60 and 100 ng/ml which is OPTIMAL for good health. It has cured my Seasonal Depression. My Multiple Sclerosis is 100% under control with NO drugs. My PAIN is no longer crippling me.. consider taking a Magnesium supplement too!
From the United States
The Immunomodulatory Potential of tolDCs Loaded with Heat Shock Proteins
Introduction
Regulatory T cells (Tregs) downmodulate unwanted immune responses. The induction or expansion of Tregs with the use of antigen-loaded tolerogenic dendritic cells (tolDCs) is a novel and attractive therapeutic possibility. Tregs are predominantly immunosuppressive CD4+ T cells, selected in the thymus on the basis of relatively high-affinity interactions with self-antigens (1). These cells are called natural Tregs (nTregs). Alternative populations of Tregs may become induced by peripheral antigen presentation in tolerance promoting tissues leading to what is called peripheral or induced Tregs (pTreg). Comparisons between T cell receptor (TCR) repertoires of thymus-derived nTregs and gut-residing pTregs have not led to direct conclusions on the relative significance of either nTregs or pTregs. Some studies, such as those for the colon, have reported a relatively unique nature of TCRs present on gut Tregs (2), whereas others have emphasized the presence of shared TCRs between thymic and colon Tregs (3, 4). Nonetheless, in both situations, it is proposed that gut-residing Tregs expand after recognition of microbiota-associated microbial antigens. The cognate interactions with these foreign antigens would fit well with a unique nature of colon Treg TCRs, whereas a driving activity by self-cross-reactive microbial antigens would be more compatible with the shared TCR idea.
Microbial heat shock proteins (HSPs) are antigens with a well-established tolerance promoting capacity. Since the identification of mycobacterial HSP60 as the driving antigen for modulatory T cells in the adjuvant arthritis model ( 5), immunization with microbial HSPs, mainly HSP60 and HSP70, was shown to inhibit disease in various inflammatory models (6–8). Subsequent analysis of the specificities of the microbial antigen responding T cells led to the hypothesis that conservation of HSPs was critical in the tolerance promoting character of HSPs (9). T cells with specificity for conserved microbial sequences are cross-reactive with (mammalian) self-HSPs and therefore have potential to regulate by targeting a regulatory effect to upregulated self-HSP in inflamed tissues (6, 10). A possible sequence of mechanistic events (see Table Table1)1) would be that T cells, or Tregs in this case, are selected in the thymus on the basis of self-HSP recognition (Table (Table1).1). For example, HSP70 is abundantly expressed in normal thymic epithelial cells (11). In addition, HSP70 epitopes were found on thymic dendritic cells (DCs) which demonstrates that these epitopes are presented in the healthy human thymus (12). For these reasons, central tolerance may be assisted by HSP-driven thymic-positive selection. In the periphery, HSP recognizing T cells are maintained or expanded in the tolerizing gut environment through recognition of cross-reactive microbiota HSPs. Several of the following factors could add to the efficiency of this immune imprinting of HSP reactivity at the Treg level. When microbes are being taken up by macrophages or DCs lining the gut, phagocytosis and exposure of the ingested bacteria to the hostile intracellular environment of the phagocyte will lead to a microbial HSP upregulating stress response (13). In addition, the continuous contact with a variable set of microbiota-associated bacterial species may emphasize the driving nature of just the conserved and therefore repeatedly encountered bacterial sequences. Similarly, stress in host tissues as seen in inflammation will also lead to the enhanced expression of self-HSPs and thereby enhance attractiveness as a target for T cell regulation.
Table 1
Mechanistic sequence of events leading to anti-inflammatory activity of heat shock protein (HSP)-specific regulatory T cell (Tregs).
|
In the next sections, we will explain how the anti-inflammatory effects of HSPs can work in synergy with the cell therapy approach with tolDC.
Different Approaches to Induce tolDCs and Their Function in Experimental Models
Experimental disease models have been used to explore the abilities of tolDCs to induce Tregs and their possible therapeutic application in vivo (14–16). Among these studies, tolDCs were also tested in an arthritis model. In this study, tolDCs were generated in vitro from murine bone marrow with dexamethasone and 1α,25-dihydroxyvitamin D3 (the active form of vitamin D3). Subsequently, they were pulsed with collagen type II (17). These tolDCs, showing a semi-mature phenotype, were able to reduce T cell proliferation and diminish arthritis severity. Unpulsed tolDCs were not able to reduce arthritis. This suggests that antigen is needed to suppress disease. Choosing the right antigen is important since most autoimmune diseases are caused by a deviant lymphocytic response against a self-antigen. Whereas rheumatoid arthritis (RA) affects one body component (the joint), other autoimmune diseases such as systemic lupus erythematosus (SLE) affect multiple organs. Can tolDC therapy also be applied in these type of autoimmune diseases? And could we induce tolDCs in vivo if the autoantigen is unknown?
Systemic lupus erythematosus is a multisystem autoimmune disease in which autoantibodies play an important role. Another feature of SLE is dysregulation of DCs as these cells continuously display a mature phenotype with high expression of costimulatory molecules and chemokine receptors (18, 19). Because dysregulated DCs are involved in the pathogenesis of SLE, DC therapy could contribute to the welfare of SLE patients. Monocyte-derived DCs (moDCs) from SLE patients were isolated as a first step in investigating the possibility of tolDC therapy. The moDCs were stimulated with iC3b-opsonized apoptotic cells or the combination of dexamethasone and vitamin D3. In both cases, the DCs gained tolerogenic properties (20, 21). This indicates that SLE DCs can be modified. Up until now, no in vivo studies have been performed with in vitro generated tolDCs in SLE but blocking NF-κB activity in DC-induced tolerogenic characteristics. SLE mice treated with these NF-κB blockers showed a reduction in circulating autoantibodies (22). These results suggest that inducing tolDCs in vivo could be a solution in SLE. The same group showed that this method is also successful in experimental immune encephalomyelitis (EAE) (23). Antigen-specific effects of NF-κB activity-blocked DCs in EAE were studied with MOG as the autoantigen. In this model also bone marrow-derived tolDCs loaded with MOG peptide (40–55) were found to reduce disease by the induction of Treg (15).
Next to using NF-κB blockers in SLE and EAE, they have also been used to generate tolDCs in vitro with regard to RA. More specifically, addition of the NF-κB inhibitor Bay11-7082 to bone marrow- or peripheral blood-derived DCs caused a lower expression of costimulatory molecules and weak stimulation of T cells (24, 25). These DCs, when pulsed with antigen and injected into mice, attenuated inflammatory arthritis via the induction of Tregs. To test if this could also be achieved in vivo without modulating DCs in vitro, solely liposomes containing antigen and a NF-κB inhibitor were infused into arthritic mice. Arthritic symptoms were reduced only when the antigen was codelivered with the NF-κB inhibitor. Merely the delivery of antigen or NF-κB inhibitor did not reduce arthritis (26). This shows that liposomes carrying both antigen and NF-κB inhibitor can target DCs in vivo to induce antigen-specific tolerance.
Other drug delivery systems have also been used to influence the status of a DC in vivo. DC stimulation with intranasal antigen encapsulated by polylactic-co-glycolic acid (PLGA) nanoparticles resulted in increased antigen uptake of the DCs and induction of CD4+FoxP3+ cells. Furthermore, PLGA nanoparticle treatment was tested in a delayed type hypersensitivity (DTH) model and an arthritis model. In the DTH model, the mice were treated with PLGA nanoparticles or control and subsequently sensitized with ovalbumin (OVA) in combination with incomplete Freund’s adjuvant and after 24 h challenged with OVA. PLGA nanoparticle treatment resulted in a reduced sensitivity reaction, whereas the controls did not (27). Next to this, nasal application with PLGA nanoparticles encapsulating mB29a (a mammalian HSP70 peptide) reduced arthritis severity for 30 days after disease development, suggesting that chronic inflammatory responses can also be modulated by tolDC (27). All in all, substantial evidence has been collected in preclinical models for an effective tolerance promoting effect of tolDC in autoimmunity.
A Treg-Inducing HSP70 Peptide
Since the autoantigen in many autoimmune diseases is unknown, surrogate autoantigens could be used to restore tolerance. Among mammalian HSP, the HSP70 family of proteins contains some of the most stress-inducible HSPs, besides constitutive family members. In addition, some HSP70 family members are involved in chaperone-mediated autophagy (CMA). CMA contributes to maintenance of cellular homeostasis by facilitating recycling of degraded proteins and by eliminating abnormal or damaged proteins. HSP70, in combination with HSP90, is responsible for the targeting of proteins to the lysosome during CMA. MHC class II elution studies have shown that autophagy, as a consequence of cell stress, contributes to preferential loading of MHC II with HSP70 peptides (28). In the latter study, nutrient-deprived human HLA-DR4+ B cells were used for the analysis. Among the more abundant peptides present in the elution profile of the stressed B cells, there was a peptide that had been previously discovered by us as a dominant T cell epitope in Balb/c mice previously (29). This peptide was discovered, when immunizations with mycobacterial HSP70 were found to protect against disease in the proteoglycan-induced arthritis (PGIA) model. This particular peptide, called B29, triggered disease protective T cell responses and consisted of a highly conserved sequence (29). This mycobacterial HSP70-B29 peptide had mammalian homologs (counterparts), called mB29a, mB29b, and mB29c in both constitutive and stress-inducible HSP70 family members. Exactly the mB29b variant was present in the elution profile of these HLA-DR4+ B cells. Interestingly enough, the same mB29b variant was also reported to be present in the MHC-II clefts of human thymic antigen-presenting cells (12).
Our interest in the B29 peptides developed from the observations that nasal application of the peptide-suppressed PGIA in mice (29). Follow-up experiments made clear that B29 and its mammalian homologs were capable of inducing Tregs. Immunizations with B29 or an ovalbumin peptide (pOVA) as a control were performed, and CD25+ T cells were sorted by FACS from the responding CD4+ T cell population. Adoptive transfer of these CD25+ T cells led to reduction of PGIA in recipients, whereas CD25− T cells did not. Also CD25+ and CD25− populations obtained from pOVA immunized animals were not having any effect on arthritis. The cell numbers needed for reaching these effects were relatively low: 3 × 105 cells sufficed for prevention of disease by adoptive transfer prior to disease induction, whereas only 1 × 106 T cells were needed to suppress ongoing disease.
By the use of a congenic T cell marker (CD90.1), exclusively present on the transferred T cells, we were able to track the transferred T cells in vivo (13). Our transferred, disease suppressing, T cells were still found present 50 days after transfer in the spleen, draining lymph nodes, joints, bone marrow, and blood. In addition, they were found to have kept their Treg phenotype, as they expressed CD25, Foxp3, NRP-1 (neuropilin 1), and lymphocyte activation gene-3 (LAG-3). When we infused a depleting CD90.1 antibody during the phase of disease suppression, after transfer of the CD25+ T cells from B29 immunized donors, disease relapsed, reaching a severity identical with that in CD25− T cell-transferred animals. Altogether, these experiments showed the potential of conserved HSP70 peptides to induce a Treg response, which is long-lived and actively engaged in suppression of disease. When we sorted the LAG-3 positive CD4+ T cells from our CD25+ population and transferred these cells before induction of PGIA, we prevented induction of disease with the very small number of 4,000 CD4+ T cells (29). As far as we know, this has never been seen in mouse models before, and it may indicate the superiority of antigen-selected Tregs as compared to non-antigen-selected Tregs. Furthermore, it also shows the strong potential of targeting Tregs to HSPs since the pOVA-induced Tregs never suppressed disease. Herewith, the example of HSP70 peptide B29 shows the potential of HSP peptides to effectively modulate immunity by the induction of Tregs.
HSP-Mediated DC Modulation
Given the protective effects of mycobacterial HSP70 in arthritis models, the immune modulatory activities of this molecule were also studied in RA. Bonorino and her group have demonstrated that mycobacterial HSP70 is capable of inducing IL-10 production in cells obtained from the inflamed synovium of arthritis patients. In addition, this was found in peripheral blood mononuclear cells from RA patients and healthy controls. Besides this, TNF-α and INF-γ production in these cells decreased and IL-10 production was raised. Cell-separation studies showed that the cells that produced IL-10 were monocytes (30).
When mouse bone marrow-derived DC were exposed to mycobacterial HSP70, maturation markers MHC class II and CD86 remained suppressed, indicating a tolerogenic phenotype (31, 32). Also in the presence of lipopolysaccharide (LPS), HSP70 reduced the upregulation of these markers. As in human cells, mycobacterial HSP70 induced IL-10 and not TNF-α. Altogether, it was concluded that DC maturation was halted by mycobacterial HSP70. Furthermore, LPS-free mycobacterial HSP70 was seen to inhibit phytohemagglutinin-induced T cell proliferation and was not seen to induce CD86 expression on splenic DCs in vivo, whereas LPS did (31). Although various alternative receptors were claimed to act as cellular receptors for HSP70, the signaling leading to IL-10 production in these studies may have involved TLR2 triggering with MyD88 activation and ERK phosphorylation (33).
When HSP70-treated DCs were tested in the proteoglycan (PG)-induced arthritis model, suppression of disease induction was seen when the treated DCs were loaded in addition with PG (32). And interestingly, when OVA-specific (TCR transgenic) T cells were cotransferred together with OVA-pulsed HSP70-treated DCs, the OVA-specific T cells were producing increased levels of IL-10 when re-stimulated in vitro with OVA. This regulatory cytokine induction in DCs was seen for both mycobacterial and mammalian HSP70 (32).
Thus, it may be concluded that part of the tolerance-promoting effects of HSP is mediated through their capacity to induce a regulatory mode in DCs.
Coinduction of Endogenous HSPs
Cell stress, caused by environmental factors or endogenous factors such as accumulation of unfolded proteins in the cytosol, is the primary trigger for upregulation of endogenous HSPs in cells. Such upregulation of HSPs lead, among others as the result of stress induced autophagy as discussed earlier, to further routing of HSP peptides to the MHC class II-binding grooves for recognition by T cells.
One possibility for enhancing HSP expression during stress is through the application of the so-called HSP coinducers. These are compounds that help to enhance production of HSP during stress, but are not capable of initiating a stress response on their own. When heat shock factor has become activated, this transcription factor induces the HSP synthesis, and coinducers just boost the level of production, through as yet unknown mechanisms.
A first and well-studied HSP coinducer, which is rather selective for the coinduction of HSP70 and not the others, is geranylgeranylacetone (GGA), an acyclic polyisoprenoid. Originally, GGA was developed as an effective anti-gastric ulcer drug. It has been tested now in various inflammatory diseases, including experimental autoimmune uveoretinitis (34). In this model, oral GGA inhibited disease and local HSP70 mRNA expression in the eyes was transiently upregulated. The autoantigen-specific T cell proliferation was also suppressed in GGA-treated mice (34).
A more recent example of an effective HSP coinducer is carvacrol, an essential oil present in Oregano species. Carvacrol was known to have antibacterial activity but upon testing on mammalian cells, carvacrol was found to have strong stress protein coinducing capacity (35). Carvacrol promoted HSP70 expression in human cell lines and mouse spleen cells during stress in vitro (caused by a raised temperature or exposure to arsenite). Upon intragastric administration, it resulted in raised HSP70 gene expression in Peyer’s patches of mice in vivo. As a consequence, the same intragastric administration of carvacrol specifically promoted T cell recognition of endogenous HSP70, as demonstrated by amplified T cell responses to HSP70. It also systemically increased the number of CD4+CD25+FoxP3+ T cells in the spleen, and almost completely suppressed PG-induced experimental arthritis.
Very similar to what was seen for HSP-treated DCs loaded with PG in the PGIA model, as mentioned in the previous section, heat stress (42.5°C for 1 h) and carvacrol treated DCs loaded with PG also protected against PGIA. In addition, these DCs induced expansions of Foxp3+ cells in vivo (17).
Herewith, it can be concluded that apart from the loading of DCs with HSP peptides, it would be possible in theory to use upregulated cell-endogenous HSPs for getting HSP peptides presented in tolDCs.
The Attractive Possibility of HSP Peptide-Loaded tolDCs
Anti-inflammatory interventions using antigen-loaded tolDCs are already being developed for several chronic inflammatory diseases, such as diabetes type I and RA. First clinical trials indicated safety and have suggested clinical benefits (36, 37). In these cases, presumed relevant autoantigens were used to load the DCs.
In multiple sclerosis (MS), tolerogenic moDCs from relapsing-remitting (RR) MS patients, loaded with myelin peptides as specific antigen, were studied. The RR-MS tolDCs expressed a stable semi-mature phenotype and induced a stable antigen-specific hyporesponsiveness in myelin-reactive T cells from RR-MS patients in vitro (38).
A recent clinical trial in RA has attempted to induce tolerance for self-antigens present in the synovial fluids of inflamed joints (37). Nevertheless, the target group here was patients with active disease, which may have hindered the chances for real tolerance induction. In addition, not a well-defined antigen for immune monitoring was available in this study.
In the case of HSP-loaded tolDCs (see Figure Figure1),1), the use of a well-defined HSP antigen will help the exact monitoring of induced HSP-specific Tregs with defined specificity in clinical experiments. In addition, patients can be selected on the basis of their antigen-specific response profiles for inclusion in the trial. Given the fact that molecules such as HSP70 are upregulated in inflamed tissues, therapeutic tolerance can be achieved through the induction of HSP70-specific Tregs, which then via bystander suppression reduce inflammation irrespective of the disease or inciting autoantigens. Therefore, when shown to be effective, HSP peptide-loaded tolDCs could be of use for therapies directed toward inflammatory diseases in the broadest possible sense.
The basic principle that underpins the therapy is to educate tolerance-invoking regulatory T-cells. An orchestrated series of events needs to happen to achieve this and hence to allow it to be an effective approach to treatment of rheumatoid arthritis. (1) Tolerogenic heat shock protein (B29)-specific regulatory T cells (Tregs) are present in the patient, which can be verified with antigen-specific T cell assays. (2) The patient is treated with anti-TNF (or similar) to induce a state of disease remission. (3) Dendritic cells (DCs) are obtained from the patient by expanding peripheral blood obtained monocytes with GM-CSF/IL-4 as growth factors ①, (4) The DCs are ex vivo made into tolerogenic DCs with vitamin D3/dexamethasone and loaded with B29 ②, so that they can present this epitope to regulatory T cells. (5) The cells are re-introduced into the patient ③ (remission allows for better tolerance induction). (6) The epitope is presented to Tregs by the tolerogenic DCs ④, to activate the regulatory T cells. (7) The patient now has a Treg repertoire that naturally suppresses inflammation ⑤ (in the joint).
Author Contributions
WE wrote most of the paper. MJ wrote part of the paper. IL, CW, PL, and FB were essential for discussing the content of the paper.
Conflict of Interest Statement
WE and PL have shares in Trajectum Pharma. The co-authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Acknowledgments
We thank the Dutch Reumafonds for their support in the preclinical development of tolDCs loaded with HSP 70 peptides for the induction of tolerance.
References
The basic principle that underpins the therapy is to educate tolerance-invoking regulatory T-cells. An orchestrated series of events needs to happen to achieve this and hence to allow it to be an effective approach to treatment of rheumatoid arthritis. (1) Tolerogenic heat shock protein (B29)-specific regulatory T cells (Tregs) are present in the patient, which can be verified with antigen-specific T cell assays. (2) The patient is treated with anti-TNF (or similar) to induce a state of disease remission. (3) Dendritic cells (DCs) are obtained from the patient by expanding peripheral blood obtained monocytes with GM-CSF/IL-4 as growth factors ①, (4) The DCs are ex vivo made into tolerogenic DCs with vitamin D3/dexamethasone and loaded with B29 ②, so that they can present this epitope to regulatory T cells. (5) The cells are re-introduced into the patient ③ (remission allows for better tolerance induction). (6) The epitope is presented to Tregs by the tolerogenic DCs ④, to activate the regulatory T cells. (7) The patient now has a Treg repertoire that naturally suppresses inflammation ⑤ (in the joint).
Vitamin D and the Immune System
The immune system defends the body from foreign, invading organisms, promoting protective immunity while maintaining tolerance to self. The implications of vitamin D deficiency on the immune system have become clearer in recent years and in the context of vitamin D deficiency, there appears to be an increased susceptibility to infection and a diathesis, in a genetically susceptible host to autoimmunity.
The classical actions of vitamin D are to promote calcium homeostasis and to promote bone health. Vitamin D enhances absorption of calcium in the small intestine and stimulates osteoclast differentiation and calcium reabsorption of bone. Vitamin D additionally promotes mineralization of the collagen matrix in bone. In humans, vitamin D is obtained from the diet or it is synthesized it in the skin (reviewed in [1]). As vitamin D is cutaneously produced after exposure to UV B light, its synthesis is influenced by latitude, season, use of sunblock and skin pigmentation. Melanin absorbs UVB radiation inhibiting the synthesis of vitamin D from 7-dihydrocholesterol. This initial vitamin D compound is inactive and it is next hydroxylated in the liver to form 25 OH vitamin D3 (25 D). 25 D is also an inactive compound, but is the most reliable measurement of an individual’s vitamin D status. It is converted in the kidney to the active compound 1,25 dihydroxy vitamin D (1,25 D) or calcidiol by 1-α-hydroxylase (CYP27B1), an enzyme which is stimulated by PTH . 1,25 D may be further metabolized to the inactive 1,24,25 vitamin D by 24-hydroxylase (CYP24). 1,25 D levels are tightly regulated in a negative feedback loop. 1,25 D both inhibits renal 1-α-hydroxylase and stimulates the 24-hydroxylase enzymes, thus maintaining circulating levels within limited boundaries and preventing excessive vitamin D activity/signaling.
1,25 D acts on the intestine where it stimulates calcium reabsorption, and upon bone, where it promotes osteoblast differentiation and matrix calcification. The active hormone exerts its effects on these tissues by binding to the vitamin D receptor (VDR). This complex dimerizes with the retinoid X receptor (RXR) and the 1,25D-VDR-RXR heterodimer translocates to the nucleus where it binds vitamin D responsive elements (VDRE) in the promoter regions of vitamin D responsive genes and induces expression of these vitamin D responsive genes.
Many tissues other than the skeletal and intestine express the VDR including cells in the bone marrow, brain, colon, breast and malignant cells and immune cells suggesting that vitamin D may have functions other than calcium and bone homeostasis[2]. Additionally, tissues other than the kidney express 1-α-hydroxylase and are capable of converting 25 D to 1,25 D, in non-renal compartments[1, 3-4]. Therefore, in addition to its endocrine functions, vitamin D may act in a paracrine or autocrine manner. Some of the more recently recognized non-classical actions of vitamin D include effects upon cell proliferation and differentiation as well immunologic effects resulting in an ability to maintain tolerance and to promote protective immunity. As antigen presenting cells (macrophages and dendritic cells), T cells and B cells have the necessary machinery to synthesize and respond to 1,25 D, vitamin D may act in a paracrine or autocrine manner in an immune environment. Moreover, local levels of 1,25 D may differ from systemic, circulating levels as local regulation of the enzymes synthesizing and inactivating vitamin D are different from the controls originating in the kidney. The extrarenal 1-α-hydroxylase enzyme in macrophages differs from the renal hydroxylase as it is not regulated by PTH[5]. Instead, it is dependent upon circulating levels of 25 D or it may be induced by cytokines such as IFN-γ, IL-1 or TNF-α[6]. Furthermore, the macrophage 24 hydroxylase enzyme is a non-functional splice variant, so there is no negative feedback of local 1,25 D production by 1,25 D.
Vitamin D and Protective Immunity
Vitamin D has been used (unknowingly) to treat infections such as tuberculosis before the advent of effective antibiotics. Tuberculosis patients were sent to sanatoriums where treatment included exposure to sunlight which was thought to directly kill the tuberculosis. Cod liver oil, a rich source of vitamin D has also been employed as a treatment for tuberculosis as well as for general increased protection from infections[7].
There have been multiple cross-sectional studies associating lower levels of vitamin D with increased infection. One report studied almost 19,000 subjects between 1988 and 1994. Individuals with lower vitamin D levels (<30 ng/ml) were more likely to self-report a recent upper respiratory tract infection than those with sufficient levels, even after adjusting for variables including season, age, gender, body mass and race[8]. Vitamin D levels fluctuate over the year. Although rates of seasonal infections varied, and were lowest in the summer and highest in the winter, the association of lower serum vitamin D levels and infection held during each season. Another cross-sectional study of 800 military recruits in Finland stratified men by serum vitamin D levels[9]. Those recruits with lower vitamin D levels lost significantly more days from active duty secondary to upper respiratory infections than recruits with higher vitamin D levels (above 40nmol). There have been a number of other cross-sectional studies looking at vitamin D levels and rates of influenza [10] as well as other infections including bacterial vaginosis[11] and HIV[12-13]. All have reported an association of lower vitamin D levels and increased rates of infection.
Results of studies looking at potential benefits of administering vitamin D to decrease infection have not been consistent, most likely secondary to a number of methodologic concerns[14]. One recent well-designed prospective, double blind placebo study using an objective outcome, nasopharyngeal swab culture (and not self report), and a therapeutic dose of vitamin D showed that vitamin D administration resulted in a statistically significant (42%) decrease in the incidence of influenza infection[15].
The beneficial effects of vitamin D on protective immunity are due in part to its effects on the innate immune system. It is known that macrophages recognize lipopolysacharide LPS, a surrogate for bacterial infection, through toll like receptors (TLR). Engagement of TLRs leads to a cascade of events that produce peptides with potent bacterialcidal activity such as cathelocidin and beta defensin 4[16]. These peptides colocalize within phagosomes with injested bacteria where they disrupt bacterial cell membranes and have potent anti-microbacterial activity [17].
Vitamin D plays an important part in the innate antimicrobial response. TLR binding leads to increased expression of both the 1-α-hydroxylase and the VDR[17-18]. This results in binding of the 1,25 D-VDR-RXR heterodimer to the VDREs of the genes for cathelocidin and beta defensin 4 and subsequent transcription of these proteins. Transcription of cathelocidin is absolutely dependent on sufficient 25 D[17]. It is now clear that transcription of beta defensin 4 requires binding of NFkB to appropriate response elements on the beta defensin 4 RNA[19]. TLR 2-1 signaling facilitates IL-1 receptor engagement which results in translocation of NFkB to its binding site[19].
Vitamin D and Autoimmune Disease
There is increasing epidemiologic evidence linking vitamin D deficiency and autoimmune diseases including multiple sclerosis (MS), rheumatoid arthritis (RA), diabetes mellitus (DM), inflammatory bowel disease and systemic lupus erythematosus (SLE) (reviewed in reference[20]. Reports of low serum vitamin D predicting development of autoimmune disease in the future have been published for MS, autoimmune DM and RA[21-23]. There is also data linking decreased in utero exposure to vitamin D and islet cell autoimmunity[24]. Lower in utero exposure assessed by a lower maternal intake of vitamin D during pregnancy in women whose prospective child was at risk of developing autoimmune DM is associated with a statistically increased risk of the child developing pancreatic autoimmunity.
Vitamin D has also been shown to facilitate progression of existing autoimmune disease. In one study, 161 patients with an early undifferentiated connective tissue disease were followed for a mean of over 2 years[25]. Most patients did not progress and remained in an undifferentiated state. Thirty-five (21%) patients went on to develop a defined rheumatologic diagnosis including RA, SLE, Mixed Connective Tissue Disease, and Sjogren’s Disease while 126 did not progress. Baseline characteristics of the two groups were similar. Importantly, the mean vitamin D level was significantly lower in the group that progressed to a definitive disease.
There have been many studies of vitamin D status in lupus patients from across the globe (reviewed in [26]). Vitamin D levels are typically lower in patients than in disease or normal controls. Deficiency of vitamin D is extremely common, often with more than 50% of lupus patients with deficient levels and severe deficiency (vitamin D levels less than 10ng/ml) is not uncommon. Disease activity has been shown to correlate inversely with vitamin D in many but not all studies. Similar correlations between low levels of vitamin D and disease activity and severity have been observed in other autoimmune diseases such as MS and RA[27-30].
Vitamin D and Immunologic Function
Vitamin D has numerous effects on cells within the immune system. It inhibits B cell proliferation and blocks B cell differentiation and immunoglobulin secretion[31-32]. Vitamin D additionally suppresses T cell proliferation[33] and results in a shift from a Th1 to a Th2 phenotype[34-35]. Furthermore, it affects T cell maturation with a skewing away from the inflammatory Th17 phenotype[36-37] and facilitates the induction of T regulatory cells[38-41]. These effects result in decreased production of inflammatory cytokines (IL-17, IL-21) with increased production of anti-inflammatory cytokines such as IL-10 (Figure 1A). Vitamin D also has effects on monocytes and dendritic cells (DCs). It inhibits monocyte production of inflammatory cytokines such as IL-1, IL-6, IL-8, IL-12 and TNFα[42]. It additionally inhibits DC differentiation and maturation with preservation of an immature phenotype as evidenced by a decreased expression of MHC class II molecules, co-stimulatory molecules and IL12[43-45] (Figure 1B).
A. Effects of 1,25 Vitamin D on T cells include suppression of T cell proliferation, a shift from Th1 to a Th2 development, inhibition of Th17 cell development and facilitation of T regulatory cells. B. Effects of 1,25 Vitamin D on monocytes and dendric cells include inhibition of inflammatory cytokine production by monocytes and inhibition of dendritic cell differentiation and maturation.
Inhibition of DC differentiation and maturation is particularly important in the context of autoimmunity and the abrogation of self tolerance. Antigen presentation to a T cell by a mature DC facilitates an immune response against that antigen while antigen presentation by an immature DC facilitates tolerance. Self-antigens are abundant in the normal state from physiologic cell death and turnover. However, presentation of these self-antigens is usually by immature DCs so that tolerance to self is maintained.
Given the importance of vitamin D for a functional immune system and the profound deficiency observed in autoimmune disease, as well as the correlation of deficiency with more active disease, an important issue is whether or not the immune components in autoimmune disease are capable of responding appropriately to vitamin D. Immune cells (B cells, T cells, monocytes, DCs) from multiple autoimmune diseases appear to respond to the immunomodulatory effects of vitamin D. Examples of vitamin D responsiveness by immunologic components in different autoimmune disease follow: B cells: Abnormalities of B cells from lupus patients may be partially reversed by vitamin D. Both spontaneous and stimulated immunoglobulin production from B cells from active lupus patients are significantly decreased by pre-incubating cells with 1,25 vitamin D[46]. Additionally, preincubation with vitamin D significantly decreases spontaneous production of anti-DNA antibodies by approximately 60%[46]. T cells: T cells from patients with MS respond to vitamin D. The proliferation of stimulated CD4 cells from MS patients and controls are similarly inhibited after preincubation in increasing concentrations of vitamin D[27]. Moreover, Th17 polarized T cells from both controls and MS patients respond when incubated with vitamin D; both are downregulated with diminished production of IL-17 and gamma interferon[27]. Monocytes: Vitamin D inhibits the production of inflammatory cytokines (IL-1, TNFα) by monocyes. Cytokine production by monocytes from both normal controls and from patients with autoimmune diabetes (type 1 or latent autoimmune diabetics) is significantly diminished by vitamin D[47]. TLR 4 stimulation by LPS or LTA (leipoteichoic acid) is similarly inhibited by exposure to vitamin D[47]. DCs: Lupus DCs are susceptible to the effects of vitamin D. LPS induced DC maturation is inhibited by preincubation with vitamin D resulting in suppressed expression of HLA class II and co-stimulatory molecules. The response of lupus cells to LPS stimulation is similarly suppressed by vitamin D[48]. Furthermore, vitamin D affects the expression of the interferon (IFN) signature in SLE. Interferon is produced by plasmacytoid DCs; the IFN signature refers to the overexpression of IFN α inducible genes in peripheral blood mononuclear cells (PBMC s) of lupus patients[49]. The signature occurs in approximately 50% of patients and correlates with disease activity[50-52]. We have observed that interferon inducible genes are overexpressed in lupus patients with low serum vitamin D compared to normal serum vitamin D (Figure 2A). Expression of these interferon inducible genes may be diminished in lupus patients after receiving vitamin D supplementation (Figure 2B). In fact, we have observed that an IFN signature response, the decrease in expression of IFN inducible genes is 2.1 times more likely to occur in vitamin D supplemented lupus patients (unpublished data Ben-Zvi, I). There is currently a double-blind placebo controlled NIH sponsored trial (ClinicalTrials.gov identifier: NCT00710021) assessing the potential ability of vitamin D to suppress the interferon signature in patients with SLE.
A. Relative expression of 2 IFNα inducible genes, Mx1 and Ifit1 in SLE patients with vitamin D deficiency (≤ 20ng/ml) and sufficiency (>20ng/ml). Relative expression of these genes was determined by RTPCR on PBMCs from clinically stable SLE patients. Expression of interferon inducible genes is higher in patients with SLE with low serum vitamin D (unpublished data Ben-Zvi, I). B. Relative expression of 3 IFNα inducible genes (Mx1, Ifi1 and Ifit44) before and after (+D3) supplementation with vitamin D3 in 3 SLE patients. Vitamin D supplementation reduces expression of IFNα inducible genes (unpublished data Ben-Zvi, I).
Conclusions
Vitamin D has important functions beyond those of calcium and bone homeostasis which include modulation of the innate and adaptive immune responses. Vitamin D deficiency is prevalent in autoimmune disease. Cells of the immune system are capable of synthesizing and responding to vitamin D. Immune cells in autoimmune diseases are responsive to the ameliorative effects of vitamin D suggesting that the beneficial effects of supplementing vitamin D deficient individuals with autoimmune disease may extend beyond effects on bone and calcium homeostasis.
Footnotes
Address for reprints: same as corresponding author
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References
https://forbetterscience.com/2020/09/28/we-reached-the-stage-where-vitamin-d-is-the-cure-for-covid-19/
We arrived at Vitamin D as COVID-19 cure
It was only logical that COVID-19 will be cured with vitamin supplements. Peer-reviewed science is now catching up with the bustling Vitamin D market.
It had to happen. Vitamin supplements are promoted for every malaise, and COVID-19 is the hottest business opportunity one can get. After the initial confusion with stem cells, nanotechnology, hormones, irradiation, and of course chloroquine and its derivative hydroxychloroquine (HCQ), the world came to its senses and recognised that only a prescription-free drug can cure COVID-19, otherwise where is the fun in that.
Since most COVID-19 victims are senior citizens, anti-aging companies arrived on the spot offering their proprietary supplements as COVID-19 prevention or even therapeutic medicine. While these fancy NAD+ and resveratrol supplements sound cool and futuristic (anti-aging!) it excludes other hard-working scammer and grifters who also wish to sell something but have patented nothing. The mineral supplement Zinc is literally dirt cheap and thus has a potential, since it was prophesied by quack-messiah Vovka “Zev” Zelenko of New York as important ingredient in the HCQ cure, but then again, you still need prescription drugs HCQ and the antibiotic azithromycin for Zinc to do its magic on COVID-19. A vitamin would be perfect, pensioners always need those, vitamins are always good, the word alone is reassuring. But which vitamin?
While scratching their bottoms about whether to go for Vitamin A, Vitamin B or Vitamin C (which cures all diseases anyway, cf Nobel Prize winner Linus Pauling), with some Dutch merchants peddling Vitamin K, and surely Vitamin E is an option, too, the experts seem to have eventually arrived at Vitamin D. Why?
Well, as the coronavirus is raging uncontrolled in the United States because the President Donald Trump does not like bad news, most deaths happened among the Black and LatinX communities. Which is for sure not something making the Imperial Wizard Trump sad, but it made some biomedical scientists think. What if it is not the poverty, failed public health and blatant institutional racism which kills Black Americans in the pandemic, but something as simple and cheap as a lack of Vitamin D? In humans, the vitamin is naturally produced by exposure to sunlight, of which white skins takes up more than dark skin, so you see where this is going.
If you need scientific guidance, genetics professor Ewan Birney, director of EMBL-EBI in Edinburgh, can explain. Even a Time article highly critical of the Vitamin D theory of COVID-19 medicine wrote this:
“Indeed, many of the known risk factors for COVID-19—being Black or Hispanic; being elderly; having an underlying health condition; having a high body mass index—are also risk factors for vitamin-D deficiency.”
You see, since LatinX people are not perfectly pale white, they count as Vitamin-D deficient also, even if they live in the polar regions of California. In fact, US insurances might register being non-white as pre-existing medical condition (yes, I am aware they are eager to do exactly this as Trump dismantles Obama’s Affordable Care Act). Even The Lancet chimed in with some race medicine, via a comment article by British experts Adrian Martineau and Nita Forouhi endorsing Vitamin D supplements:
“The striking overlap between risk factors for severe COVID-19 and vitamin D deficiency, including obesity, older age, and Black or Asian ethnic origin, has led some researchers to hypothesise that vitamin D supplementation could hold promise as a preventive or therapeutic agent for COVID-19.”
Yes, we forgot Asians! Which explains such horrendous rates of COVID-19 in India, it must be the dark skin, and the lack of sun because unlike COVID-19 resistant British folks, those Indian farmers are always indoors…, ah crap, never mind. Since when are non-peer reviewed reality facts allowed to interfere with peer-reviewed science?
Vitamin D theory of COVID-19 is beautiful. It allows you to roll out any crypto-racist bunk and pretend it’s solid science, while peddling prescription-free supplements out the back. And in America, they love racial medicine, US hospital algorithms distribute (or rather deny) healthcare according to your skin colour, which bottom line is that Black people are more primitive than whites and therefore need less medical care.
Studies correlating COVID-19 to Vitamin D levels arrived quickly as the pandemic grew, and now gain traction as the HCQ hype declines. This paper from 3 September is probably the most headline-grabbing one, because it is from USA and appeared in JAMA. No wait, it’s the downstream Open Access cash-making outlet, JAMA Network Open, where it costs $3000 to publish.
David O. Meltzer, Thomas J. Best, Hui Zhang, Tamara Vokes, Vineet Arora, Julian Solway Association of Vitamin D Status and Other Clinical Characteristics With COVID-19 Test Results JAMA Network Open (2020) doi: 10.1001/jamanetworkopen.2020.19722
The authors accessed health records of COVID-19 patients and concluded that those who eventually caught the virus had low Vitamin D levels before:
“In this cohort study of 489 patients who had a vitamin D level measured in the year before COVID-19 testing, the relative risk of testing positive for COVID-19 was 1.77 times greater for patients with likely deficient vitamin D status compared with patients with likely sufficient vitamin D status, a difference that was statistically significant.”
Yes, the skin colour of patients was noted. None of the participants was actually Vitamin D deficient, so the authors set some threshold to assign them as “likely deficient”. They did notice that many had various pre-existing conditions like obesity, diabetes or cardiovascular problems, all COVID-19 risk factors. Low Vitamin D levels used to be see in the pre-COVID-19 times just an general biomarker of bad health, but now they became the main cause for getting infected with COVID-19 in the first place. Maybe confused by the fact that the majority of their analysed patients were not white, the authors conclude:
“Since African American and Hispanic populations in the US have both high rates of vitamin D deficiency and bear a disproportionate burden of morbidity and mortality from COVID-19,35,36 they may be particularly important populations to engage in studies of whether vitamin D can reduce the incidence and burden of COVID-19.“
What a lovely idea. Instead of providing public health and insurance available to everyone, why not telling Black and Hispanic Americans to pop some cheap Vitamin D pills.
As paediatrics professor Todd Alexander of University of Alberta was quoted in Folio, this Vitamin D deficiency and weakened immunity theory is not new, and has been used to explain diseases before:
“In the 1980s and ’90s, studies emerged that found a role for vitamin D in immune function. This, according to Alexander, led to a deluge of clinical studies finding reduced vitamin D levels in people affected by a host of diseases including asthma, cancer, diabetes and multiple sclerosis.”
Those cancer and asthma studies are now long forgotten, except by some unscrupulous vitamin peddlers. Correlation is not causation, but unfortunately not every scientist will agree with Alexander. Aside of rickets (which is a real Vitamin D deficiency syndrome) medical science has so far failed to convincingly connect any other disease to Vitamin D deficiency. Thanks to modern diet, there are anyway only few people with critically low Vitamin D levels in need of supplementation, while these unnecessary supplements do increase calcium uptake up to the formation of kidney stones. Doctors already noticed cases of Vitamin D poisoning in COVID-19 risk group patients who tried to “boost immunity”.
But then again, there is COVID-19 pandemic going on and their is no time for the scientific method.
The market for Vitamin D was already there, which is another beauty of the approach. Because everyone was convinced that showing yourself outside unprotected will immediately give you skin cancer, a notion supported by the extremely successful sunscreen industry (even though “melanoma rates in the U.S. have tripled since the 1970s, even as the use of sunscreen has increased“), there evolved a need to counteract the sun avoidance and the resulting potential Vitamin D deficiency with supplements. And now there is COVID-19 on, and what coincidence, Vitamin D helps also against that! The science has spoken.
Especially since the Vitamin D approach is such great business and also great fun. How about European COVID-19 death rates explained with dark-skinned Italians and lazy Spaniards doing their siesta in the shadows while the Nordic races toil in the fields, with their proud white faces turned towards the sun? Here is a paper from the (apparently COVID-19-resistant) UK on just that, its p-values for Vitamin D levels vs COVID-19 cases and mortality just happen to be exactly p=0.05, the magic value.
Petre Cristian Ilie, Simina Stefanescu & Lee Smith The role of vitamin D in the prevention of coronavirus disease 2019 infection and mortality Aging Clinical and Experimental Research (2020) doi: 10.1007/s40520-020-01570-8
“The Southern European countries have lower levels of vitamin D because of decreased exposure (prefer the shade in strong sun) [10] and also as skin pigmentation decreases vitamin D synthesis [11]. Northern part of Europe’s mean levels are better as a consequence of the consumption of cod liver oil and vitamin D supplements as well as fortification of milk and milk products (Finland) [6].”
Indeed, it must be all that Surströmming which Swedes eat, if anything it ensures social distancing. You might wonder who takes such a braindead and bigoted study seriously. Professor Helmut Schatz does, one of Germany’s top endocrinologists. Former governmental advisor Professor Hans-Konrad Biesalski of University of Hohenheim in Stuttgart analysed 30 studies, including the coronavirus effects on the “black race“, and urged Germans in June 2020 to take Vitamin D supplements for COVID-19. Elsewhere in Germany, the ultra-right xenophobes and rabid COVIDIOTS of the Alternative für Deutschland (AfD) demanded, also in June 2020 that the German Bundestag issues Vitamin D recommendation for COVID-19 prevention. Their proposal was rejected.
But also Italians and Spaniards themselves are jumping the bandwagon. Endocrinologist Luigi Gennari of University of Sienna presented his research to a closed circle at a conference, and nobody else, which should suffice. He concluded:
“Our data give strong observational support to previous suggestions that reduced vitamin D levels may favor the appearance of severe respiratory dysfunction and increase the mortality risk in patients affected with COVID-19“.
And in Spain, just look at this clinical trial from Cordoba, which used the Vitamin D analogue calcifediol. It cannot be more convincing:
Marta Entrenas Castillo, Luis Manuel Entrenas Costa, José Manuel Vaquero Barrios, Juan Francisco Alcalá Díaz, José López Miranda, Roger Bouillon, José Manuel Quesada Gomez “Effect of Calcifediol Treatment and best Available Therapy versus best Available Therapy on Intensive Care Unit Admission and Mortality Among Patients Hospitalized for COVID-19: A Pilot Randomized Clinical study” The Journal of steroid biochemistry and molecular biology (2020) doi: 10.1016/j.jsbmb.2020.105751
Only 2% of the 50 Vitamin D treated patients went to ICU as opposed to 50% of 26 patients who did not get Vitamin D!
Now, I really must commend the authors for their cleverness. The clinical trial started by dosing COVID-19 patients with HCQ, but then that chloroquine cargo cult ship has sailed, or rather ran aground (its unhinged captain Didier Raoult crying sabotage as Marseille went from HCQ religion hotspot to COVID-19 hotspot). Hence the clever Cordoba investigators quickly switched to a new magic drug, Vitamin D. The journal they chose is not really specialising on clinical trials (aside of reviews), but it does specialise on Vitamin D research. Which was good enough to find the right reviewers. Who agreed with the authors that the many COVID-19 deaths Spain has experienced are due to Spaniards not getting enough sun in the winter.
Update 17.11.2020. The Castillo et al paper seems to be extremely authoritative, maybe because not many bothered to read it properly, but here is a nice takedown by DW Science. It quotes Martin Smollich, pharmacology professor at University of Lübeck in Germany, who noticed that the placebo control arm of the clinical trial included 19% of diabetics and 57% of hypertension sufferers, who are the highest COVID-19 risk group. The Vitamin D arm, where the survival rates were so much higher, happened to have merely 6% of diabetics and 24% of hypertension patients. But it passed peer review, so how dares Smollich to criticise it now!
Now, we arrive at some really serious money making. With these authors it cannot be any more obvious that they are all unashamed Vitamin D industry shills. Especially the last character on that paper.
Harvey W. Kaufman, Justin K. Niles, Martin H. Kroll, Caixia Bi, Michael F. Holick SARS-CoV-2 positivity rates associated with circulating 25-hydroxyvitamin D levels PloS one (2020) doi: 10.1371/journal.pone.0239252
The first four authors are employees of Quest Diagnostics, which tests your blood Vitamin D levels and then sells you Vitamin D supplements. The last author is the notorious Michael Holick, described by New Yorker as “The Child-Abuse Contrarian”, who issues bizarre medical diagnoses over the phone and whose custom-curated promotional Wikipedia profile was eventually vandalised by the reality. A Controversies section was added:
“Holick has been involved in several medical controversies. While at Boston University, he was asked to leave the Division of Dermatology because of his promoting the medical benefits of sun exposure. He accepted research funding for this work from a non-profit tanning bed company, considered by many to be an important potential bias. Barbara Gilchrest, then head of the department at Boston University, called Holick’s book “shlock science” and Holick “a poster boy for the tanning industry”.[50]
Holick received nearly $163,000 from 2013 to 2017 from pharmaceutical companies, according to Medicare’s Open Payments database, which tracks payments from drug and device manufacturers. The companies paying him included Sanofi-Aventis, which markets vitamin D supplements; Shire, which makes drugs for hormonal disorders that are given with vitamin D; Amgen, which makes an osteoporosis treatment; and Roche Diagnostics and Quidel Corp., which both make vitamin D tests.[51]
Holick has also been criticized by other physicians because of his testimony, defending accused child abusers by asserting that Ehlers-Danlos Syndrome is a cause of non-traumatic fractures in infancy (rather than abuse).[52] Experts in Ehlers-Danlos syndrome, as well as pediatricians specializing in traumatic bone injury, refute Holick’s position, which is completely unsubstantiated by the medical literature. In over 300 criminal cases Holick has never concluded that the child who suffered broken bones had been abused. In one case of a child who had suffered broken bones in which Holick defended the accused parent, the child later went on to suffer severe brain injury, for which the parent has been indicted.[53]
Since May 2017, Holick has been barred from evaluating or treating children by Boston Medical Center, which subsequently reported him to the Massachusetts Board of Registration in Medicine for “health care facility discipline.” [54]
In January 2018, Robert Marvin Ray, one of the parents whom Holick worked with over child abuse suspicions, was arrested and charged with child abuse.[50][55]
His promotion of Vitamin D has been called extreme, even speculating that the extinction of dinosaurs caused by a lack of it in reduced sunlight.[56]“
Would you as a journal editor accept anything from such a “scientist”? Well, PLOS One did. Maybe they were impressed by the sheer numbers of patients:
“This study used a retrospective, observational analysis of deidentified tests performed at a national clinical laboratory to determine if circulating 25-hydroxyvitamin D (25(OH)D) levels are associated with severe acute respiratory disease coronavirus 2 (SARS-CoV-2) positivity rates. Over 190,000 patients from all 50 states with SARS-CoV-2 results performed mid-March through mid-June, 2020 and matching 25(OH)D results from the preceding 12 months were included.“
Over One Hundred Ninety Thousands of patients, who can say no to such a gigantic clinical trial dataset, especially if it promises The Cure for COVID-19? If it reminds you of the Surgisphere scandal for which both The Lancet and NEJM fell arse over tit, well, this is how scholarly publishing works. The story counts, not the author’s or their dataset’s credibility.
Holick’s Boston University of course proudly issued a press release. But PLOS One might have second thoughts:
Having tweeted their concern on 24 September, the next day PLOS One published yet another paper by Holick about curing COVID-19 with Vitamin D. It first appeared as preprint on the Elsevier’s SSRN server, which is a much fancier preprint server than others because it is named “Preprints with THE LANCET“. Now in PLOS One, what honour for the journal.
Zhila Maghbooli, Mohammad Ali Sahraian, Mehdi Ebrahimi, Marzieh Pazoki, Samira Kafan, Hedieh Moradi Tabriz, Azar Hadadi, Mahnaz Montazeri, Mehrad Nasiri, Arash Shirvani, Michael F. Holick Vitamin D sufficiency, a serum 25-hydroxyvitamin D at least 30 ng/mL reduced risk for adverse clinical outcomes in patients with COVID-19 infection PLoS ONE (2020) doi: 10.1371/journal.pone.0239799
This time, the authors saved “only” 235 patients in Iran, while PLOS One informs you: “The authors have declared that no competing interests exist“. Not even Holick, half of whose business is Vitamin D grifting (the other half being helping child abusers in court).
But now, the best Vitamin D for COVID-19 paper ever. Straight from the French Parliament, Assemblée National, and its MP, Joachim Son-Forget. This professional politician once studied medicine and did a PhD in psychology, which automatically made him expert for many things sciency, these days always ready with an educated and even peer-reviewed guess about COVID-19. Son-Forget has been promoting HCQ on Twitter and he of course is a great admirer of the greatest French scientist of all times, Didier Raoult (for whom he even raised money, it seems).
A great scientific mind himself, Son-Forget (and colleagues) postulated in Medical Hypotheses, a comedy magazine run by Elsevier, “that betathalassemic heterozygote population prevalence is correlated to immunity against COVID-19, by a regression“. Would you like to see the data they based this assumption on?
The polymath knowledge of Dr Son-Forget, MP is evident in his genius way of plotting that regression.
Incidentally, the authors list there was exactly same as on this stroke of genius, this time about the Vitamin D:
Édouard Lansiaux, Philippe P. Pébaÿ, Jean-Laurent Picard, Joachim Son-Forget Covid-19 And Vit-D: Disease Mortality Negatively Correlates With Sunlight Exposure Spatial and Spatio-temporal Epidemiology (2020) doi: 10.1016/j.sste.2020.100362
The authors (or rather only Edouard Lansiaux who has to vouch for everything) analysed COVID-19 related health data from France, except colonies and Corsica because the authors decided they have much sun but not enough hospitals there. They concluded:
“in continental metropolitan France, average annual sunlight hours are significantly (for a p-value of 1.532 × 10−32) correlated to the COVID-19 mortality rate, with a Pearson coefficient of -0.636. This correlation hints at a protective effect of sunlight exposure against COVID-19 mortality.“
And indeed, there is no COVID-19 at all in Marseille, famous for its radiant sun (who is of course Didier Raoult). Don’t believe the news.
A Letter to Editor regarding that article by Son-Forget was published in the same journal, its authors are Florian Naudet, Clara Locher, Alain Braillon and André Gillibert. They listed “Major statistical flaws”in Son-Forget’s research and concluded:
“the manuscript has no informative value at all concerning any association between “Covid-19 And Vit-D”. Therefore, we think that the article methods and conclusions are too flawed to have any value.“
But, as scholarly authorities advice, we must remain open regarding Vitamin D as COVID-19 medicine. Especially since the former CDC director Tom Frieden advised Americans already in March 2020 to take Vitamin D supplements for COVID-19 prevention.
The Vitamin D hype is working. Even Anthony Fauci himself recently advocated the supplement:
“If you are deficient in vitamin D, that does have an impact on your susceptibility to infection. So I would not mind recommending, and I do it myself taking vitamin D supplements“.
The 78 year old Fauci also recommended taking Vitamin C:
“that vitamin C is “a good antioxidant.” “So if people want to take a gram or two at the most [of] vitamin C, that would be fine”
Sure, people in Fauci’s age are in danger of vitamin deficiency and some might need supplements to support their weakened immune system. But old age is about much more than maybe lacking some Vitamin D, so the causality with COVID-19 severity is very strenuous there at best. Maybe it all is Fauci’s diplomatic way for distracting Americans from taking HCQ. Or drinking bleach.
And anyway, the only real prevention cure for COVID-19 is cabbage.
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A superior D3 rundown. You should go to the 56-minute mark on the Flux Woo video, you can find at bitchute.com. He says your blood should be at D3 90 ng/ml to deny all viruses, including the various Covids. Deny means you get nothing, or you get a case of virus ccp_xyz that can de disposed with at home. My D3 is at mid 70s.
Flux_Woo https://www.youtube.com/watch?v=Id9nEuPQ7mI&t=2s