# IMMUNE-MODULATORY EFFECTS OF VITAMIN D

EDITED BY : Susu M. Zughaier, Erik Lubberts and Abdulbari Bener PUBLISHED IN : Frontiers in Immunology

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ISSN 1664-8714 ISBN 978-2-88966-174-9 DOI 10.3389/978-2-88966-174-9

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# IMMUNE-MODULATORY EFFECTS OF VITAMIN D

Topic Editors: Susu M. Zughaier, Qatar University, Qatar Erik Lubberts, Erasmus University Rotterdam, Netherlands Abdulbari Bener, Istanbul University Cerrahpasa Faculty of Medicine, Turkey

Citation: Zughaier, S. M., Lubberts, E., Bener, A., eds. (2020). Immune-Modulatory Effects of Vitamin D. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88966-174-9

# Table of Contents

Soraya Mousavi, Fábia Daniela Lobo de Sá, Jörg-Dieter Schulzke, Roland Bücker, Stefan Bereswill and Markus M. Heimesaat

*37 Vitamin D Signaling in the Context of Innate Immunity: Focus on Human Monocytes*

Carsten Carlberg

*45 Vitamin D Receptor Gene Polymorphisms are Associated With Leprosy in Southern Brazil*

Afonso Carrasco Pepineli, Hugo Vicentin Alves, Bruna Tiaki Tiyo, Luciana Conci Macedo, Lorena Visentainer, Quirino Alves de Lima Neto, Joana Maira Valentini Zacarias, Ana Maria Sell and Jeane Eliete Laguila Visentainer

*51 Regulation of the Immune Balance During Allogeneic Hematopoietic Stem Cell Transplantation by Vitamin D*

Cindy Flamann, Katrin Peter, Marina Kreutz and Heiko Bruns

Peter Crauwels, Elena Bank, Bianca Walber, Ulf Alexander Wenzel, Birgitta Agerberth, Menberework Chanyalew, Markos Abebe, Renate König, Uwe Ritter, Norbert Reiling and Ger van Zandbergen

*91 Vitamin D Receptor Inhibits NLRP3 Activation by Impeding Its BRCC3-Mediated Deubiquitination*

Zebing Rao, Xin Chen, Junxian Wu, Mengjun Xiao, Jing Zhang, Binghao Wang, Lei Fang, Hongjie Zhang, Xiaoming Wang, Shuo Yang and Yunzi Chen

#### *106 Polarization of Human Monocyte-Derived Cells With Vitamin D Promotes Control of* Mycobacterium tuberculosis *Infection*

Jagadeeswara Rao Muvva, Venkata Ramanarao Parasa, Maria Lerm, Mattias Svensson and Susanna Brighenti

*119 Vitamin D Induces Differential Effects on Inflammatory Responses During Bacterial and/or Viral Stimulation of Human Peripheral Blood Mononuclear Cells*

Jeremy Anderson, Lien Anh Ha Do, Zheng Quan Toh, Edwin Hoe, Andrea Reitsma, Kim Mulholland and Paul V. Licciardi

Md Asiful Islam, Shahad Saif Khandker, Przemysław J. Kotyla and Rosline Hassan

# Editorial: Immune-Modulatory Effects of Vitamin D

#### Susu M. Zughaier 1 \*, Erik Lubberts 2 and Abdulbari Bener 3,4

*1 Department of Basic Medical Sciences, College of Medicine, QU Health, Qatar University, Doha, Qatar, 2 Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands, 3 Faculty of Medicine, Istanbul University Cerrahpasa, Istanbul, Turkey, 4 International School of Medicine, Istanbul Medipol University, Istanbul, Turkey*

Keywords: vitamin D, immune modulation, monocytes, T cells, infection

**Editorial on the Research Topic**

#### **Immune-Modulatory Effects of Vitamin D**

Vitamin D plays an essential role in bone development. However, recent studies are beginning to uncover its role as a modulator of the immune system (1). Several reports have shown associations between vitamin D deficiency (2) and the incidence as well as the severity of chronic inflammatory diseases such as cardiovascular disease (3, 4), inflammatory bowel disease (5), asthma (6), and chronic obstructive pulmonary disease (COPD) (7). Consistent with this, vitamin D supplementations have been shown to reduce the severity of and inflammation markers in chronic inflammatory diseases (8). At the molecular level, the hormonally active form of vitamin D (α1,25 dihydroxyvitamin D3) regulates the expression of vitamin D responsive genes that can lead to differential regulation of signaling pathways in immune cells. For example, vitamin D positively regulates iron homeostasis and erythropoiesis via the iron-hepcidin-ferroportin axis (9). Vitamin D deficiency is highly prevalent world-wide including countries with abundance of sunshine (10). Singh et al. reviewed the causes of vitamin D deficiency where they dissected the complex impact of genetic predisposition, gut microbiota, and immune system. In this review, authors examined GWAS database (11) and listed genes variants with SNPs that associate with risk of vitamin D deficiency. These alleles are common in vitamin D receptor (VDR) and vitamin D binding protein (VDPB). Since gut microbiota plays a crucial role in nutrients and vitamins production, absorption and degradation, authors also highlighted the role of vitamin D metabolism and VDR is regulating host-gut microbiota interactions.

At the cellular level, vitamin D exerts anti-inflammatory effects on immune cells that express the vitamin D receptor (VDR) such as monocytes, macrophages, and T lymphocytes, which in turn shapes the immune response during the onset of inflammation and infection and following vaccination. Vitamin D exerts anti-inflammatory effects by reducing pro-inflammatory cytokines production from macrophages and T cells (12, 13). Carlberg eloquently demonstrated that vitamin D/VDR signaling impacts chromatin modeling leading to significant modification of human monocytes epigenome during perturbation, consequently reducing cytokine release and modulating trained innate immunity. The immune modulatory effects of vitamin D include reduction in inflammatory cytokines release such as IL-1β, which is induced via inflammasome activation. Rao et al. showed that VDR inhibited NALP3-inflammasome activation leading to reduction in IL-1β release. To confirm the inhibitory effect of VDR on NLP3 inflammasome activation, authors used VDR-deficient mice and showed that IL-1β release was significantly reduced in vivo confirming that VDR inhibited NALP3 inflammasome activation. Vitamin D deficiency is associated with bone pain in particular and chronic disease pain in general (12, 14). The proposed mechanisms by which Vitamin D/VDR signaling modulate pain sensation is reviewed by Habib et al.. Authors delineate vitamin D/VDR interactions with pain sensing

Edited and reviewed by: *Francesca Granucci, University of Milano-Bicocca, Italy*

> \*Correspondence: *Susu M. Zughaier<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology*

Received: *19 August 2020* Accepted: *28 August 2020* Published: *29 September 2020*

#### Citation:

*Zughaier SM, Lubberts E and Bener A (2020) Editorial: Immune-Modulatory Effects of Vitamin D. Front. Immunol. 11:596611. doi: 10.3389/fimmu.2020.596611* pathways including dorsal root ganglion (DRG) neurons, nerve growth factor (NGF), epidermal growth factor receptor (EGFR), glial-derived neurotrophic factor (GDNF), and opioid receptors.

Sufficient levels of vitamin D have been shown to regulate T cell proliferation by controlling T cell antigen receptor and T cell activation as well as enhancing the phagocytic activity of macrophages (12, 14). The immune suppressive effects of regulatory T cells (T-reg) in controlling inflammation and preventing autoimmunity is well-established. Maternal immune tolerance to fetus is also integral to successful pregnancy. Vitamin D deficiency is quite common in pregnancy which has been associated with adverse outcomes. Cyprian et al. reviewed the consequences of vitamin D deficiency during pregnancy and detailed the role of T-regs in mitigating pregnancy loss and adverse events such as preeclampsia. Further, Vitamin D is shown to play an important role in modulating graft-vs-host disease (GvHD) by balancing immune responses. Flamann et al. described the role of vitamin D in inducing immune suppressive T regs and balancing immune responses in allogeneic hematopoietic stem cell transplantation. The immune modulatory effects of vitamin D increased immune tolerance and enhanced anti-tumor activity.

In cases of autoimmune diseases, low vitamin D levels are associated with increased B cell proliferation and autoantibody production. Low vitamin D levels have also been associated with the incidence of autoimmune diseases such as Rheumatoid Arthritis (RA), Multiple Sclerosis (MS), and Systemic Lupus Erythematosus (SLE) (15). A systematic review by Islam et al. on the immunomodulatory effects of diet and nutrients in Systemic Lupus Erythematosus (SLE) presented the beneficial effect of vitamin D supplementation such as alleviating fatigue. Metaanalysis studies showed that SLE patients have significantly lower vitamin D levels compared to healthy participants. In human and animal studies of SLE, vitamin D is shown to inhibit cytokines such as IL-10, IL-17, IFN-γ, and modulate cellular proliferation of B-cells, Th1, Th17, CD4+ T cells. Therefore, vitamin D exerts various immune modulatory effects during autoimmune diseases. Another example of autoimmune diseases is Type 1 diabetes mellitus (T1DM) where insulin producing pancreatic β-cells are attacked by T cell-mediated autoimmunity (12, 16). Stem cell transplantation has emerged as regenerative treatment for T1DM as it possess immune suppressive potential. A clinical trial (NCT03920397) conducted by Araujo et al. on the effect of vitamin D treatment with allogenic adipose tissue-derived stromal/stem cells (ASCs) in 13 patients with recent onset of T1DM. One group received ASC and vitamin D (cholecalciferol 2,000 UI/day) for 3 months while the second group received standard insulin therapy. They observed that allogenic ASC and vitamin D therapy lead to reduced insulin requirement and more stable C peptide.

Low vitamin D status has been shown to be a risk factor for infectious diseases (17, 18). During infections such as Tuberculosis (TB), vitamin D induces the expression of cathelicidin (LL-37), a host defense peptide that enhances the bactericidal activity of immune cells like macrophages, thereby limiting the growth of mycobacteria that causes TB (18). Furthermore, LL-37 exerts anti-inflammatory effects via its ability to neutralize bacterial molecules like endotoxins and capsular polysaccharides that activate TLR signaling pathways, consequently inhibiting the release of pro-inflammatory mediators from macrophages. In this special topic several research papers investigated the role of vitamin D in infection outcomes. Muvva et al. investigated the effect of vitamin D on controlling intracellular Mycobacterium tuberculosis (Mtb) infection in human monocytes-derived macrophages. They showed that vitamin D treatment resulted in an appropriate macrophage polarization M1/M2 phenotype with enhanced expression of antimicrobial LL-37 and antiinflammatory cytokine IL-10 but reduced expression of the immune suppressive enzyme IDO. This in vitro study provides evidence to the immune modulatory effects of vitamin D in controlling Mtb infection. Similarly, LL-37 is shown to control Leishmania parasitic infections in human. Crauwels et al. observed increased expression of LL-37 in skin biopsies from cutaneous leishmaniasis. They demonstrated that recombinant LL-37 reduced Leishmania parasite viability in a dose-dependent manner. Accordingly, they investigated the effect of vitamin D treatment on human monocytic-derived macrophages and documented that LL-37 mediated Leishmania restriction in macrophages. Vitamin D role in controlling infection is also demonstrated in acute enterocolitis. Mousavi et al. used an animal model to investigate the beneficial effects of vitamin D on preserving intestinal barrier and exerting anti-pathogenic effects during Campylobacter jejuni infection. They reported that preclinical administration of vitamin D ameliorated acute campylobacteriosis in mice model by reducing bacterial colonization and translocation, restoring intestinal epithelial cells regeneration and dampening inflammatory responses.

Vitamin D deficiency increases the risk of bacterial and viral infections (17). The immune modulatory effects of vitamin D during bacterial infections seem to vary from those effects during viral infections. In this regards, Anderson et al. observed the differential effects of vitamin D on induced inflammatory responses in primary human peripheral mononuclear cells (PBMC) during bacterial pneumococcal infection alone, respiratory syncytial viral (RSV) infection or coinfection. They showed that vitamin D exerted anti-inflammatory effects by reducing Th17 inflammatory cell expression and cytokines during pneumococcal infection alone and RSV alone but not during co-infection with RSV.

Genetic predisposition plays a role in host susceptibility to infections (19). Similarly, polymorphism in genes involved in vitamin D metabolism and function such as VDR impacts host susceptibility to infections. Pepineli et al. evaluated four genetic variants of VDR in the Leprosy immune pathogenesis in Brazil. They investigated a cohort of 404 leprosy patients in comparison to 432 control individuals without disease. Although no association is identified in VDR polymorphism genetic frequency between patients with active clinical leprosy and controls, they observed bAt haplotype to confer protection from leprosy. Taken together, data from original research paper, clinical trial, systematic analysis, and reviews included herein provide compelling evidence to the immune modulatory effects of vitamin D. Therefore, vitamin D deficiency should be treated to gain the extra skeletal beneficial effects on the immune system in health and disease.

#### REFERENCES

#### AUTHOR CONTRIBUTIONS

SZ wrote the editorial. EL and AB reveiwed the editorial. All authors contributed to the article and approved the submitted version.

**Conflict of Interest:** The 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.

Copyright © 2020 Zughaier, Lubberts and Bener. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Vitamin D Deficiency in the Gulf Cooperation Council: Exploring the Triad of Genetic Predisposition, the Gut Microbiome and the Immune System

#### Parul Singh, Manoj Kumar and Souhaila Al Khodor\*

*Research Department, Sidra Medicine, Doha, Qatar*

#### Edited by:

*Susu M. Zughaier, Qatar University, Qatar*

#### Reviewed by:

*Michael F. Holick, Boston Medical Center, United States Jun Sun, University of Illinois at Chicago, United States*

> \*Correspondence: *Souhaila Al Khodor<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology*

Received: *30 December 2018* Accepted: *24 April 2019* Published: *10 May 2019*

#### Citation:

*Singh P, Kumar M and Al Khodor S (2019) Vitamin D Deficiency in the Gulf Cooperation Council: Exploring the Triad of Genetic Predisposition, the Gut Microbiome and the Immune System. Front.  Vitamin D is essential for the growth and development of bones and helps in reducing inflammation by strengthening muscles and the immune system. Despite the endless supply of sunlight in the Gulf Cooperation Council (GCC) countries which includes United Arab Emirates, Qatar, Kuwait, Bahrain, Saudi Arabia, and Oman, Vitamin D deficiency in the (GCC) general population at various age groups remains alarmingly high. In parallel runs the increasing prevalence of acute and chronic illnesses including, autoimmune diseases, cancer, type 1 diabetes mellitus, cardiovascular disease and Inflammatory bowel disease in the adult as well as the pediatric population of these countries. The exact association between Vitamin D deficiency and chronic disease conditions remains unclear; however, studies have focused on the mechanism of Vitamin D regulation by assessing the role of the Vitamin D associated genes/proteins such as VDR (Vitamin D receptor), VDBP (Vitamin D Binding protein), CYP27B1 as these are integral parts of the Vitamin D signaling pathway. VDR is known to regulate the expression of more than 200 genes across a wide array of tissues in the human body and may play a role in controlling the Vitamin D levels. Moreover, reduced Vitamin D level and downregulation of VDR have been linked to gut dysbiosis, highlighting an intriguing role for the gut microbiome in the Vitamin D metabolism. However, this role is not fully described yet. In this review, we aim to expand our understanding of the causes of Vitamin D deficiency in the GCC countries and explore the potential relationship between the genetic predisposition, Vitamin D levels, immune system and the gut microbiome composition. Trying to unravel this complex interaction may aid in understanding the mechanism by which Vitamin D contributes to various disease conditions and will pave the way toward new therapeutics treatments for Vitamin D deficiency and its associated outcomes.

Keywords: hypovitaminosis D, microbial dysbiosis, VDR, VDBP, CYP27B1, GCC

# INTRODUCTION

Vitamin D is well-known for its many health benefits. The role of Vitamin D in the elimination of rickets remains one of the most notable discoveries in medicine (1). Calcium and phosphate are two minerals that are required for normal bone formation. Upon demand, Vitamin D stimulates the intestines, bones, and kidneys to maintain calcium and phosphorus levels in the blood, and thus promotes the mineralization of the bone matrix and osteoclasts differentiation (2). Hypovitaminosis D or severe Vitamin D deficiency results in poor mineralization and bone loss, leading to osteoporosis, fractures, muscle weakness, and frank hypocalcemia (3). Vitamin D mediates its biological function via metabolizing into its active steroid form 1α,25 dihydroxyvitamin D3 [1α,25(OH)2D3 or simply 1,25(OH)2D], often referred to as a hormone, influencing many genes across various tissues in the human body, such as kidneys (4–6), intestines (7), bones (8–11), as well as cancer and immune cells (12–14).

Vitamin D is also known to interact with cells from both the innate and adaptive immune system, where it plays an important role in antigen presentation, immune regulation and antibacterial response (15). Historical Vitamin D supplementation has been used to treat lupus and mycobacterial infections such as tuberculosis and leprosy (16, 17). Studies examining the immunomodulatory properties of Vitamin D have linked its deficiency to a higher incidence of autoimmune diseases, such as type I diabetes (T1D), multiple sclerosis (MS), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and inflammatory bowel disease (IBD) and also to an increased risk of developing various types of cancers including breast, ovarian, colon, and prostate cancer (18–21). Vitamin D is also a potent effector of vascular endothelial cells and thus has a role in cardio-protection (22), as it regulates blood pressure by stimulating the renin angiotensin system, vascular calcification and smooth muscle cell proliferation (23). Hypovitaminosis D is directly linked to the development of hypertension, and a reduction in blood pressure has been noted to be a consequence of Vitamin D supplementation (24, 25). Evidence also supports the role of Vitamin D in neuroprotection, brain development and maintenance of cognitive functions via immunomodulation, neuronal calcium regulation, antioxidative mechanisms, enhanced nerve conduction, and detoxification mechanisms (26). There have been some reports linking decreased Vitamin D levels with depression (27), cognitive delays and an increased risk of Alzheimer's disease (28). Cerebrovascular events such as the risk of vessel thrombosis, cerebrovascular infracts and strokes have also been associated with deficiency of Vitamin D (29).

Insufficient levels of Vitamin D may also be a contributing factor for other abnormalities such as poor diet, short stature, liver disease, and diabetes (30–32). The role of Vitamin D during pregnancy is also of great consideration since maternal nutritional status determines the health of fetus and newborn. Maternal Vitamin D deficiency has been associated with increased risk of preeclampsia, calcium malabsorption, bone loss, and other myopathies (33). It is suggested that the development of the fetus in a state of hypovitaminosis D can have significant impact on innate immune functions. In vitro study conducted with Monocytes cultured in Vitamin D deficient plasma, showed significantly decreased in a TLR-dependent expression of cathelicidin compared to the control (34) Vitamin D status in the cord blood was found to be associated with the risk of lower respiratory tract infection in the first year of life consistent with the in vitro study results (35). Severe deficiency can also contribute toward abnormalities such as small stature for gestational age, neonatal hypocalcemia, hypocalcemic seizures, infantile heart failure, enamel defects, large fontanelle, congenital rickets, among others (36, 37). The role of Vitamin D goes above and beyond the traditionally ascribed ones and its significance in human physiology is undeniable. In the most recent years, several papers have addressed the importance of Vitamin D and its intracellular receptor VDR in regulation of gut hemostasis and immune response (38–41), here we aim to go further and present a comprehensive review examining the epidemic of Vitamin D deficiency in Gulf Cooperation Council (GCC) which is an alliance of six Middle Eastern countries— Saudi Arabia, Qatar, Kuwait, the United Arab Emirates, Bahrain, and Oman. We would also like to study the possible role of specific genes in predisposing the gulf population to Vitamin D deficiency, and how this increasing epidemic leads to disturbed microbial balance in the intestines and manifestation of various immune mediated diseases such as IBD.

# METHODS USED TO REVIEW THE LITERATURE IN THE FIELD

A comprehensive literature search was carried out in PubMed, ScienceDirect, Google Scholar and SpringerLink databases using keywords like "Vitamin D," "Human microbiome," "Vitamin D metabolism," "Vitamin D and Gut microbiome," "Vitamin D deficiency in Gulf countries," "VDR and Immune regulation," "Immune related disease in Gulf countries," "25-hydroxy vitamin D." Only articles published in English and related to the study topic were included in this review. The search was not restricted to the type of study i.e., species, meta-analysis, case-control, randomized control trials, cohort studies, reviews, sample size, or year of publication. Bibliographies and citation of the included reviews were scanned for additional studies that may have been missed by the database searches. The exclusion criteria comprised the following: unpublished data, conference publications, articles available only in the abstract form, and doctoral or master's thesis. Endnote software (Thomas Reuters, Philadelphia, PA) was used to create library and manage the findings of the search as recognized by the above-mentioned strategies. The selected articles were read and organized under the following headings: (1) Vitamin D, importance, metabolism, status and supplementation (2) Prevalence of Vitamin D deficiency or insufficiency and immune related disorders in GCC countries (3). Possible relationship between disturbances of the gut microbiota and/or Vitamin D deficiency, VDR dysfunction, and role of Vitamin D in immune system and related disorders.

# VITAMIN D METABOLISM

In humans, Vitamin D originates from three potential sources: Ultraviolet B (UVB) dependent exogenous synthesis, nutritional sources and supplements (18, 42, 43). However, it is primarily synthesized in the skin by the action of sunlight (UV dependent) as very few naturally occurring food sources have adequate amounts of Vitamin D (18, 42, 43). Dietary sources such as fish, milk, orange juice, and cereals contain one of the two forms, cholecalciferol (Vitamin D3) or ergocalciferol (Vitamin D2) (18, 42, 43). Regardless of the source, the Vitamin D synthesis pathway follows several common steps as detailed in **Figure 1**.

Dermal synthesis starts with the conversion of cutaneously derived cholesterol precursor 7-dehydroxycholestrol to previtamin D3 by the action of UV sunlight (44, 47). Previtamin D undergoes a temperature dependent isomerization to Vitamin D3. Vitamin D-binding protein (VDBP) carries the Vitamin D3 synthesized in the skin and Vitamin D2/Vitamin D3 absorbed via intestine from the dietary sources to the liver (45). In the liver, Vitamin D2/Vitamin D3 is hydrolyzed to 25-hydroxyvitamin D[25(OH)D] by the action of enzyme 25-hydroxylase (CYP2R1) (44). The final step of activation occurs in the kidney with the conversion of 25-hydroxyvitamin D[25(OH)D] to 1,25(OH)2D by the 1α-hydroxylase enzyme (CYP27B1) (48). Finally, 1,25(OH)2D binds to its principal receptor VDR through which it regulates the expression of a large number of genes across various tissues in the human body (46). VDR plays a central role in mediating the biological functions of Vitamin D, via both genomic and non-genomic pathways as elucidated in **Figure 2**.

# VITAMIN D STATUS AND DOSAGE

Levels of Vitamin D are measured in the serum as 25(OH)D, reflecting the Vitamin D status. The results of this blood test help with the clinical decision as to whether to take Vitamin D supplement or expose the skin to the sun. However, there is no international guidelines for reading the Vitamin D levels, as different organizations interpret the levels differently. Levels indicated as normal by one method may be interpreted differently by the other. The Vitamin D Council guidelines recommends 40–80 ng/ml as the ideal level, with levels of 0– 30 ng/ml being considered deficient and 31–39 ng/ml considered insufficient (50). According to the Endocrine Society (51) the recommended preferred range falls between 40 and 60 ng/ml, levels below 20 ng/ml should indicate deficiency, 20–29 ng/ml defines insufficiency and ≥30 ng/ml is considered sufficient, all these guidelines along with the ones from the Food and Nutrition Board (52) are summarized in **Figure 3**.

Measurement of 25(OH)D can be performed with a number of different analytical techniques. Automated immunoassay's is used by most pathology lab to measure total serum 25(OH) D (25(OH) D2 + 25(OH) D3). Because of the clinical importance of Vitamin D testing it is important to note the existing

FIGURE 2 | Molecular actions of the Vitamin D/VDR axis. Inactive Vitamin D (25(OH)D) circulates in the blood stream bound to Vitamin D binding protein (VDBP) and undergoes hydroxylation by the renal mitochondrial 1-hydroxylase (CYP27B1) enzyme to convert into active form (1α,25(OH)2D). Active Vitamin D binds to its primary receptor VDR to modulate the expression of more than 200 genes in human body. Some notable ones are grouped into various biological processes including (1) bone metabolism (8–11) (2) immune cell regulation (14, 49) (3) cancer (12) (4) cell cycle (8) (5) metabolism (4–7) demonstrating the wide range of VDR dependent genomic actions. Non-genomic actions include the activation of one or more intracellular signaling molecules, such as G protein-coupled receptors (G-protein), Protein kinase C (PKC), phosphatidylinositol-3′ -kinase (PI3K), and Phospholipase C (PLC) resulting in opening of the voltage-gated channels, generation of the specific second messengers and cross talk with genomic pathways (8).

discrepancies in Vitamin D total immunoassays as reported by several studies (53, 54). Various causes of the discrepancies have been noted (55) including the cross-reactivities of various Vitamin D metabolites (56). Therefore, the assays for Vitamin D testing need to be standardized so there is less variability in the results, the Vitamin D Standardization Program (VDSP), was launched in 2010 in collaboration with the National Institutes of Health, the Centers for Disease Control and Prevention (CDC), the National Institute for Standards and Technology (NIST), and Ghent University in Belgium to correct the disparity and ensure reliable Vitamin D measurement. However, we are still a long way to go to attain the goal of standardization of assay and results, till then we have to rely on the judgement of physicians and experts in the field. The researchers who publish in this area will likely continue to point toward such disparities.

To overcome the deficiency, Vitamin D supplementation is usually advised, however there is no consensus on the optimal dose to be prescribed, as many factors such as age and serum levels of 25(OH)D must be taken into account before recommending the best possible dose for instance for obese people (BMI > 30 kg/m2 ), a daily Vitamin D dose was set as "three times" greater than the recommended dose for subjects with normal body weight (51). The most common form of Vitamin D supplementation is Cholecalciferol and Ergocalciferol with both considered highly effective and safe (57). Vitamin D toxicity generally results from having serum levels of 25(OH)D > 150 ng/ml, which in most cases is attributed to prolonged and unintended daily intakes of >40,000 IU of supplementation (58). **Table 1** details the group-wise recommendations for daily dose of Vitamin D supplementation from different organizations.

*Disparity in Vitamin D dosage recommendations by the Vitamin D council (1), Endocrine society (2), IOM food and nutrition board (3) are shown. Generally, however, pregnant or breastfeeding women and the elderly as well as children and adults lacking enough exposure to sunlight are in need of Vitamin D supplementation (50–52).*

# VITAMIN D DEFICIENCY: A HEALTH PROBLEM WORLDWIDE AND IN THE GCC COUNTRIES

Vitamin D deficiency is a major public health problem worldwide, affecting more than a billion people across the globe from both the developing and industrialized countries (59, 60). Studies have suggested that more than 70% of USA and 50% of UK adults may have insufficient Vitamin D levels (61, 62). Among other European populations, 1 in 8 adults have low circulating levels of 25-hydroxyvitamin D (63) and a similar pattern has also been reported in India, Australia and New Zealand (64, 65).

Despite the ample amount of year-round sunlight in the GCC region, pandemic levels of Vitamin D deficiency have been observed in recent years as represented in **Figure 4**. The 2016–2017 annual report from the Qatar Biobank highlights that close to 86% of the total Qatar biobank population (comprising of adults above 18–85 year-old, 80% of them Qatari national and the rest were long-term residents) suffers from Vitamin D inadequacy and more interestingly, 14% (more women 65% than men 35%) remained deficient despite taking supplementation, 70% of the participants were also obese (71). A review examining mixed population in Saudi Arabia (e.g., pregnant/ lactating women, children, adults) found that in 81% of all the groups the levels of 25(OH) D was <20 ng/mL (<50 nmol/L) (67). The numbers run high in UAE as well, a study conducted with a large cohort of patients (60,979) including UAE nationals and visitors of other nationalities, showed that up to 82.5% of the population suffers from deficiency or insufficiency of Vitamin D. Around 86.1% UAE nationals and 78.9% visitors had serum levels of 25(OH)D < 75 nmol/L. The study also showed that the extreme cases of deficiency were higher in females (26.4%) than males (18.4%) (68). Comparable percentages have also been reported in different study cohorts' in other countries in the gulf peninsula (69, 70).

Recently many studies have associated high levels of Vitamin D deficiency with immune-mediated and inflammatory diseases. IBD is the term used to describe disorders that involve chronic inflammation of the GI tract. The main types of IBD include: ulcerative colitis and Crohn's disease. Recently the Epi-IBD study reported high prevalence of low Vitamin D levels in treatment-naive European IBD populations, with 79% of the patients showing either insufficient or deficient levels of Vitamin D (72). Vitamin D supplementation was associated with reduced intestinal inflammation in patients with active UC (73) and also in controlling the relapse rate of IBD (74).

Systemic lupus erythematosus (SLE) is a chronic autoimmune inflammatory disease which involves the connective tissue effecting multiple organs such as the brain, lungs, kidneys, heart, blood vessels, muscles, skin etc., and is more common among women (75). Vitamin D deficiency was significantly higher in SLE patients when compared to healthy controls in a study conducted with a cohort of Bahraini patients (76). Similar results were observed in a Saudi cohort (71) with high prevalence of Vitamin D inadequacy was observed in Saudi patients with SLE.

T1DM prevalence/incidence is increasing worldwide (77) the rise seems much steeper in gulf countries. A study conducted to determine the association between Vitamin D status and T1DM along with other factors in the young population of the State of Qatar found out that the incidences of severe Vitamin D deficiency was considerably higher in T1DM (28.8%) compared with healthy children (17.1%) (78), similarly in the Saudi cohort 84% of the T1DM children, and 59% of the healthy children were Vitamin D deficient (79). Low serum Vitamin D status was found to be associated with high prevalence and early onset of type-1 diabetes mellitus in Kuwaiti children as well (80). In a case control study done with Multiple sclerosis patient in Kuwait, VDR variants TaqI and BsmI were found to be associated with MS risk (81).The therapeutic effect of Vitamin D in immune diseases should be further assessed in interventional studies.

A comparative associative study between Vitamin D deficiency and IBS (Irritable bowel syndrome) was conducted with patients visiting the gastroenterology clinic in Saudi Arabia, Vitamin D deficiency was detected in 82% of the patients in the IBS group and 31% in the control group (82).

The potential limitation to the findings of this review should be considered, relatively small number of studies were available to be included in the review and also there is limited data to prove causal relationship between Vitamin D deficiency and risk of Immune system related illness. The studies included employ various definitions of hypovitaminosis D, and these variations may result in the over or underestimation of Vitamin D deficiency in GCC population. Thus, development of global or local standard and guidelines will help in better screening to define the candidates and treating those who are at most risk for Vitamin D deficiency.

As we previously discussed, synthesis of Vitamin D occurs in the skin and it depends on several factors such as time of sun exposure, season, latitude, altitude, clothing, veiling, use of sunscreen, old age, and skin color (83). It was previously shown that limited exposure to sunlight results in poor Vitamin D synthesis (84), thus even in sunny climates like the GCC countries, high rates of Vitamin D deficiency is found because of cultural and social habits that limit exposure to sunlight (85). The association between reduced 25D concentrations and obesity is also well-established with several large-scale studies

that found obesity to be associated with lower 25D, 1,25D concentrations (86, 87). A bi-directional genetic study revealed that though the effect of lower 25D on BMI may not be significant but higher BMI leads to lower 25D (88) various explanations have been proposed to define this association, such as reduced cutaneous synthesis (89) and altered metabolism (90). Dietary sources make up for <10% of the total Vitamin D synthesized in the body (91), so even with a varied balanced diet, individuals will not be able to achieve the recommended Vitamin D levels.

However, the insufficiency of Vitamin D may not be solely due to diet or lack of sunlight. Several studies have suggested that various genes can define the Vitamin D status, thus largescale genome-wide association studies have identified selected genes mainly those involved in the synthesis, metabolism or transport of Vitamin D to be associated with a variation in the Vitamin D status (92, 93). The SNPs associated with these genes were initially singled out in a European population (92) and were later replicated in the African American (94) and Asian populations (95). We utilized the GWAS catalog to generate the list of 49 gene variants or risk allele associated with Vitamin D Levels as shown in **Table 2** below. After literature review we identified two gene-association studies conducted in the GCC region, one in the Saudi populations (104) and the other in the Kuwaiti population (105). The study performed in the Saudi population identified a significant risk of Vitamin D deficiency and insufficiency associated with the SNP rs2228570 [Chromosome: 12, Position: 47879112, Gene: Vitamin D Receptor (VDR)], rs4588 [Chromosome: 4, Position: 71752606, Gene: GC] and rs10741657[Chromosome: 11, Position: 14,893,332, Gene: Vitamin D 25-hydroxylase (CYP2R1)] (104), while the study conducted in Kuwait showed that the polymorphism in the GC gene coding for the Vitamin D Binding protein (VDBP) may play a major role in determining Vitamin D levels in this population (105).

# VITAMIN D AND THE GUT MICROBIOME: IS IT A BIDIRECTIONAL RELATIONSHIP

The human gut microbiota (the microbial taxa associated with humans) is home to an estimated 1014 microorganisms, with around 500–1,000 species of bacteria (106). The vastness of the human microbiome (the catalog of human microbiota and their genes) can be imagined in terms that it contains 10 times more cells and 100 times more genes than the human (107). These microorganisms create a "mini-ecosystem" inside our bodies, and they work together as biochemical factories performing a wide array of activities such as acquisition of nutrients, Vitamins production, degradation of toxins and enhancing host immune responses by functioning as a barrier from pathogenic microorganisms (108).

Gut microbiota exists in a symbiotic relationship with the host. The composition of the gut microbiome plays a crucial role in maintaining host homeostasis, as the wrong combination of microbes contribute to an array of chronic digestive or immune disorders such as IBD, obesity, diabetes mellitus, metabolic syndrome, atherosclerosis, alcoholic liver disease, nonalcoholic fatty liver disease, cirrhosis, and hepatocellular carcinoma (109, 110).

The compositions of the gut microbiota is determined by several factors as summarized in **Figure 5**, which include age, host genomics, immune health, exercise, intake of medication specifically antibiotics, and dietary habits (111). This imbalance or maladaptation of microbial communities is often referred to as Dysbiosis (also called dysbacteriosis) which has been categorized into three different types: (1) loss of beneficial organisms, (2) excessive growth of potentially harmful organisms, and (3) loss of overall microbial diversity. These types can occur in combination and are not mutually exclusive (112, 113). In such cases, the normally dominant species become underrepresented and the resulting void is TABLE 2 | List of key variants or risk allele associated with Vitamin D level as reported by the GWAS catalog.

*(Continued)*

#### TABLE 2 | Continued

\**GC gene codes for the Vitamin D binding protein (VDBP).*

filled by increasing numbers of opportunistic or pathogenic species resulting in overall decrease in overall bacterial diversity (112). Dysbiosis is commonly reported in the gastrointestinal tract (112) and is associated with a loss of the integrity of the intestinal mucosa, which may result in gut impairment and inflammation (114–116).

Several studies have demonstrated the role of Vitamin D and its receptor VDR in regulating host-microbial interactions. The active and inactive forms of Vitamin D circulate in the bloodstream bound to VDBP. The active form known as Calcitriol 1,25(OH)2D is known to bind to the calcitriol receptor, commonly known as the VDR (117). Calcitriol binds to the VDR, which then forms a heterodimer with the retinoid-X receptor and other co-activators. This transcriptional complex then binds to discrete sites on DNA known as Vitamin D responsive elements (VDREs) resulting in the expression or repression of specific gene products (summarized in **Figure 6**) (131). It is also known that Vitamin D plays a role in various microRNA-directed post-transcriptional mechanisms (132).

The Interplay between Vitamin D, VDBP, Cyp27B1, and VDR not only regulate the transcriptional and post-transcriptional responses, but a growing evidence supports the concept that Vitamin D metabolism impacts the intestinal microbial balance

and gut homeostasis (132, 133). As previously mentioned, VDR is expressed in a variety of cell types such as kidney, muscles, prostate, immune cells; and high levels of expression in the cells of the gastrointestinal tract (GI) have been shown (134). Moreover, the Vitamin D activating enzyme Cyp27B1 is also expressed in various immune cells, as well as intestinal epithelial cells (135, 136). These two vital players of Vitamin D metabolism have also been colocalized in different cells of the GI tract suggesting the need for active Vitamin D in those cells (137).

the individual along with their genetic makeup (111).

In addition, the type of bacterial communities in the gut have been shown to regulate the expression of both VDR and Cyp27B1 (138, 139). VDR expression is inversely related to the presence of pathogenic bacteria, and correlate with probiotic bacteria with the former decreasing (138) and the latter increasing VDR expression (139). Animal studies done with germ-free and antibiotics-treated mice have reported reduced levels of Cyp27B1 expression in those animal models (140). This suggests that the intestinal bacteria regulate Vitamin D metabolism and, as a result, they regulate the innate immune response. The synthesis of active Vitamin D is modulated according to the local needs by Cyp27B1 and VDR enzymes whose expression is dependent on the "nature of local microbiota."

The VDR, Cyp27B1, and VDBP genes have also been studied for their association with autoimmune diseases such as IBD and its forms Crohn's disease (CD) and Ulcerative colitis (UC) (72, 141, 142). Several VDR polymorphisms such as ApaI, TaqI, and FokI have been associated with the development of UC (143, 144). VDR and Cyp27B1 gene knockout mice have shown increased susceptibility to the development of intestinal colitis, elevated expression of proinflammatory cytokines, and microbial dysbiosis, with more Proteobacteria and less firmicutes, a condition commonly observed in patients with IBD (145). Vitamin D supplementation in human and 1,25D treatment of CYP27B1 knockout mice decreased Proteobacteria and increased beneficial organism including members of the Firmicutes phyla (132, 146).

Gut mucosal integrity is a crucial barrier, playing a vital role in protection against pathogenic microorganisms, as disruption in mucosal barrier function and hyperpermeability can predispose to various diseases such as asthma, tuberculosis, cystic fibrosis and intestinal lung diseases (147–149). Vitamin D and its associated molecules provide protection for the epithelial barriers in various tissues including the gut mucosa, by increasing the expression of several tight and adherent junction proteins (150– 152). VDR signaling specifically has been known to regulate the gut mucosal cell inflammation by suppressing intestinal epithelial cell apoptosis (153). The increasing awareness of the role of Vitamin D as an immunomodulator was prompted by the discovery of VDR and production of active Vitamin D (1,25(OH)2 D) in almost all immune cells, including activated CD4+ and CD8+ T cells, B cells, neutrophils and antigenpresenting cells (APCs) such as macrophages and dendritic cells. The details of this regulation are described in the section below.

## VITAMIN D IN INNATE AND ADAPTIVE IMMUNE SYSTEM

The innate immune system is the first and immediate line of the defense against invading pathogens and is an alliance of components both from the host and resident microbiota. The host defense comprises of diverse components such as physical defense (such as epithelial cells of the skin, mucous membrane, and microbiome), cellular defense (as mast cells, dendritic cells, macrophages, neutrophils, natural killer cells etc.), cell receptors that recognize pathogens (as Toll-like Receptors), antimicrobial peptides and proteins (as defensins, cathelicidins).

Vitamin D is able to modulate many of the above listed components of the innate immune system. It is known to reinforce the physical barrier function of epithelial cells. The active Vitamin D and VDR are important in regulating the genes of proteins required for the tight, adheres, and gap junctions such as zonulin occluden-1, zonulin occluden-2 through the up regulation of claudin 2 and 12 (154). Studies in transgenic mice demonstrated that over-expression of VDR in the intestinal epithelium decreases mucosal inflammation suppressing epithelial cell apoptosis and boosting the function of tight junction (119, 155). The importance of active Vitamin D in maintaining healthy gut microbiota has been discussed in the earlier section.

Monocytes and macrophages are crucial members of the innate immunity, Vitamin D stimulates the differentiation of precursor monocytes to mature phagocytic macrophage, the high expression of VDR by monocytes ensures sensitivity of these cells to the differentiating effects of active Vitamin D (120). Mature macrophages sense pathogen-associated molecular patterns (PAMPs) by means of pattern-recognition receptors, such as Toll-like receptors (TLRs). The presence of CYP27B1 (Vitamin D activating enzyme) in macrophages is important for the physiological action of host defense against infection, activation of 25(OH) D to 1, 25(OH)2 D via CYP27B1 in macrophages leads to regulation of TLR2 (121), enhanced production of defensin β2, cathelicidin antimicrobial peptide (CAMP) (49) leading to induction of autophagy (122). The ability of 1,25(OH)2D to increase the production of other antimicrobial peptides, has been demonstrated both in vitro by monocytes stimulation (156) and in vivo in pediatric patients' blood (157).

At the same time, the active Vitamin D can inhibit the dendritic cell (DCs) differentiation and maturation. In human and murine DC cultures, the activation of VDR signaling pathways inhibited DC-maturation as shown by downregulation of DC markers, MHC-class II, co-stimulatory molecules (CD40, CD80, and CD86), and other maturation molecules (e.g., CD1a, CD83), chemokine (CXCL10) which is involved in the recruitment of T helper 1 (Th1) cells. Furthermore, active Vitamin D also modulates DC-derived cytokine and chemokine expression, by inhibiting the production of pro-inflammatory cytokine (IL-12 and IL-23) and enhancing the release of antiinflammatory cytokine (IL-10) and the chemokine [CCL22, involved in the recruitment regulatory T cells (Tregs)] (123–125).

However, this interaction between active Vitamin D and cells from innate immunity also have downstream effects on cells from the adaptive immune system. The adaptive immune system or acquired immune system is the second line of defense against infection. It comes into action upon exposure to pathogens and uses immunological memory to learn about the pathogens and enhance the immune response accordingly. The adaptive immune system is composed of T and B cells and is also responsible for autoimmune reaction**.** 1,25(OH)2 D suppresses the immune responses mediated by Type 1 T helper (Th1) cells, by inhibiting the inflammatory cytokines IL-2 and interferon gamma (IFNγ) (125, 126) On the other hand, it has been reported to enhance cytokines associated with Th2 cells (127). The 1,25(OH)2 D also regulate Treg cells (known for their role in the inhibition of inflammation) by induction of Foxp3 (the transcription factor involved in the development and function of Treg cells) (128). In addition to Th1, Th2, and Treg cells (subsets from the CD4 + T cell lineage) there are distinct subset termed Th17 cells that produce IL-17, which has been implicated in the pathogenesis of a number of autoimmune diseases. Th17 is inhibited by 1,25(OH)2 D (129). 1,25(OH)2 D prevents the activation of nuclear factor kappa-B (NF-κB), a component of proinflammatory signaling pathway at the level of transcription [VDR/RXR has been reported to bind to NF-κB promotor (IL-12p20) thus preventing its activation] (158).

The proliferation of VDR-expressing B-cells is also suppressed by active Vitamin D negatively impacting Ig production and also inhibiting the differentiation of plasma cells and class-switched memory cells highlighting a potential role for Vitamin D in B-cell-related disorders such as SLE (130). **Figure 6** details the proposed model of the overall impact of Vitamin D in the regulation of the epithelial barrier function, gut microbiota, and the immune system.

The above data elucidate the role of Vitamin D/VDR signaling in not only maintaining the microbial landscape in the gut in check and keeping the gut mucosal integrity intact but also as a potent immunomodulator. However, to fully understand the extent to which Vitamin D impact these complex systems, the multidirectional interaction between them needs to be considered. The importance of the interplay presents an encouraging field for further research with potential clinical implications.

## DISCUSSION

Vitamin D deficiency is a major health problem in GCC countries including Qatar, indicating its importance as a regional and national health problem (67–71). Vitamin D deficiency affects various age groups and threatens the GCC populations with increased prevalence for chronic diseases such as IBD, cancer, diabetes among others. The reasons for the deficiency include limited exposure to sunlight, full body covering and low consumption of food rich in Vitamin D (83–85). The deficiency is treated with Vitamin D supplementation or intramuscular injections, however, despite being treated in a similar manner; there are interracial and interindividual variations in response to Vitamin D intake (159). The differences in serum Vitamin D levels among diverse ethnic groups have been previously reported in GWAS studies that have identified SNPs associated with genes from the Vitamin D pathway (92–95). These studies have indicated that certain ethnic groups may be predisposed to lower serum Vitamin D levels. We believe the same may be true for the

#### REFERENCES

1. Steenbock H. The induction of growth promoting and calcifying properties in a ration by exposure to light. Science. (1924) 60:224– 5. doi: 10.1126/science.60.1549.224

population residing in GCC countries. These genetic variations may also impact the response to Vitamin D supplementation in these populations.

Originally known to cure rickets, our understanding on how Vitamin D impacts human health has broadened. The discovery of VDRs and Vitamin D associated enzymes in a variety of cells including cells of the innate and adaptive immune system was crucial in appreciating its role as a potent immunomodulator. Immune cells are not only a target for Vitamin D, but are also able to activate the hormone locally, thus postulating for an autocrine and paracrine role for the active Vitamin D (160). Inadequate amounts of Vitamin D could be linked to defective functioning of the associated autocrine and paracrine circuits eventually leading to various immune abnormalities.

Among other extra-skeletal effects, we have also come to realize its role in maintaining a healthy gut microbiota. The GI tract is home to a plethora of microorganisms that program the immune system preventing colonization by harmful bacteria and viruses. Several studies referenced in this review have suggested that Vitamin D signaling influences the microbial load and composition and ensures adequate innate and adaptive immune responses to pathogenic threats; though the questions still linger about the bidirectional and multidirectional interaction between these intricate systems. Vitamin D deficiency may result in abnormal immune functions causing inflammation, which, when unresolved, may lead to chronic conditions.

In conclusion, Vitamin D signaling mediated by key players, such as VDR, VDBP, and Cyp27B1, has a diverse physiological impact, enhancing the intestinal barrier function and contributing to enteric homeostasis. Studies have shown that low serum Vitamin D levels might be linked to several diseases and given the fact that the Vitamin D status is a modifiable factor, the potential therapeutic benefits from Vitamin D supplementation in the prevention of various diseases and maintenance of a healthy microbiota are undeniable. Therefore, the widespread Vitamin D deficiency in the GCC countries demands discussion about including Vitamin D testing as part of routine clinical care practices.

# AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

#### ACKNOWLEDGMENTS

This work has been supported by Sidra Internal Research Funds SDR200006. The authors thank Dr. Bernice Lo for her assistance in proofreading the review.

effects on cardiovascular diseases: a comprehensive review. Korean J Intern Med. (2016) 31:1018–29. doi: 10.3904/kjim.2015.224

randomization analysis of multiple cohorts. PLoS Med. (2013) 10:e1001383. doi: 10.1371/journal.pmed.1001383

**Conflict of Interest Statement:** The 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.

Copyright © 2019 Singh, Kumar and Al Khodor. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Vitamin D in Acute Campylobacteriosis–Results From an Intervention Study Applying a Clinical *Campylobacter jejuni* Induced Enterocolitis Model

#### Soraya Mousavi 1 , Fábia Daniela Lobo de Sá2 , Jörg-Dieter Schulzke2 , Roland Bücker 2 , Stefan Bereswill 1† and Markus M. Heimesaat 1 \* †

#### *Edited by:*

Susu M. Zughaier, Qatar University, Qatar

#### *Reviewed by:*

Chad Porter, Naval Medical Research Center, United States Derrick Richard Samuelson, LSU Health Sciences Center New Orleans, Louisiana State University, United States

#### *\*Correspondence:*

Markus M. Heimesaat<EMAIL_ADDRESS>
†These authors have contributed equally to this work

#### *Specialty section:*

This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology

*Received:* 21 June 2019 *Accepted:* 20 August 2019 *Published:* 03 September 2019

#### *Citation:*

Mousavi S, Lobo de Sá FD, Schulzke J-D, Bücker R, Bereswill S and Heimesaat MM (2019) Vitamin D in Acute Campylobacteriosis–Results From an Intervention Study Applying a Clinical Campylobacter jejuni Induced Enterocolitis Model. Front. Immunol. 10:2094. doi: 10.3389/fimmu.2019.02094 1 Institute of Microbiology, Infectious Diseases and Immunology, Charité - University Medicine Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany, 2 Institute of Clinical Physiology, Department of Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany

Human Campylobacter infections are progressively rising and of high socioeconomic impact. In the present preclinical intervention study we investigated anti-pathogenic, immuno-modulatory, and intestinal epithelial barrier preserving properties of vitamin D applying an acute campylobacteriosis model. Therefore, secondary abiotic IL-10−/− mice were perorally treated with synthetic 25-OH-cholecalciferol starting 4 days before peroral Campylobacter jejuni infection. Whereas, 25-OH-cholecalciferol application did not affect gastrointestinal pathogen loads, 25-OH-cholecalciferol treated mice suffered less frequently from diarrhea in the midst of infection as compared to placebo control mice. Moreover, 25-OH-cholecalciferol application dampened C. jejuni induced apoptotic cell responses in colonic epithelia and promoted cell-regenerative measures. At day 6 post-infection, 25-OH-cholecalciferol treated mice displayed lower numbers of colonic innate and adaptive immune cell populations as compared to placebo controls that were accompanied by lower intestinal concentrations of pro-inflammatory mediators including IL-6, MCP1, and IFN-γ. Remarkably, as compared to placebo application synthetic 25-OH-cholecalciferol treatment of C. jejuni infected mice resulted in lower cumulative translocation rates of viable pathogens from the inflamed intestines to extra-intestinal including systemic compartments such as the kidneys and spleen, respectively, which was accompanied by less compromised colonic epithelial barrier function in the 25-OH-cholecalciferol as compared to the placebo cohort. In conclusion, our preclinical intervention study provides evidence that peroral synthetic 25-OH-cholecalciferol application exerts inflammation-dampening effects during acute campylobacteriosis.

Keywords: vitamin D, *Campylobacter jejuni*, campylobacteriosis model, intervention study, host-pathogen interaction, acute enterocolitis, intestinal epithelial barrier function

# INTRODUCTION

Campylobacter jejuni constitute major infectious bacterial agents of zoonotic enteric morbidities with increasing prevalences worldwide (1). Humans become infected via the food chain by consumption of raw or undercooked meat derived from contaminated livestock animals or by ingestion of C. jejuni containing surface water (2–4). Infected individuals present with symptoms of varying degree depending on the virulence of the acquired bacterial strain on one side and the host immune status on the other (1, 5–7). Some patients display rather mild symptoms including watery diarrhea, whereas others develop acute campylobacteriosis (8, 9).  During infection intestinal tissues are destroyed by innate immune responses and display profound histopathological inflammatory changes such as ulcerations, crypt abscesses, and increased numbers of innate and adaptive immune cells in the colonic mucosa and lamina propria (5, 8, 10, 11). The vast majority of human infections are usually self-limiting and treated (if at all) symptomatically. Only severely compromised patients with immuno-suppressive comorbidities, for instance, require hospitalization and receive antimicrobial treatment (6, 8, 9). In rare cases, however, postinfectious sequelae such as Guillain-Barré syndrome, Miller Fisher syndrome, Reiter's syndrome, and reactive arthritis might arise with a latency of weeks to months (8, 9, 12).

Despite the progressively increasing prevalences of human campylobacteriosis, cellular, and molecular events that are involved in disease development are not yet fully understood. However, previous clinical studies revealed that in humans acute C. jejuni induced disease courses and post-infectious sequelae such as Guillain-Barré syndrome are triggered by the pathogenic surface molecule lipooligosaccharide (LOS) causing hyper-activation of the innate immune system in the sialylated form (13). For quite a long time in vivo studies have been hampered by the scarcity of appropriate animal models. This is mainly because the gastrointestinal microbiota of mice mediates a strong colconization resistance to C. jejuni and mice are per se about 10,000-fold more resistant to LOS and lipopolysaccharide (LPS) as compared to humans (14). Our group has recently shown that secondary abiotic IL-10−/− mice in which the gut microbiota had been depleted by broad-spectrum antibiotic treatment can not only be effectively colonized by the pathogen upon peroral infection, but also develop key features of acute campylobacteriosis such as wasting and bloody diarrhea within 1 week (15). One major reason for these severe immunopathological responses mounting in acute ulcerative enterocolitis is the absence of colonization resistance and the lack of interleukin-10 (IL-10) providing murine resistance to C. jejuni LOS (16, 17). In consequence, C. jejuni infected IL-10−/− mice display pronounced LOS induced and Toll-like receptor-4 (TLR-4) dependent innate and adaptive immune responses that are not restricted to the intestinal tract, but can also be observed in extra-intestinal including systemic compartments (15, 18–25).

Vitamin D has primarily been known for its regulatory properties in bone metabolism due to the tight control of calcium reabsortion in the intestinal tract and in bone remodeling (26). After exposure to ultraviolet (UV) B light the steroid hormone is produced in the skin from 7-dehydroxy-cholesterol followed by hydroxylation steps in the liver and the kidneys to the biologically active forms 25-hydroxy-vitamin D and 1,25-dihydroxy-vitamin D, respectively (27). After ingestion of food or supplements, circulating 25-hydroxy-vitamin D can be utilized by many cells including immune cells and intestinal intraepithelial cells expressing the 1α-hydroxylase enzyme CYP27B, whereas 24-hydroxylase CYP24A exerts counter-regulatory properties subsequently providing local 1,25-dihydroxy-vitamin D sources in a well-balanced fashion (27).

The identification of the vitamin D receptor (VDR) on peripheral blood mononuclear cells in the 1980s first pointed to immune-related functions of vitamin D (28, 29). In fact, vitamin D has been shown to be involved in modulating both, innate and adaptive immune responses (30–33) and to exert antiinflammatory effects (34). Furthermore, several reports underline the anti-microbial properties of vitamin D (33). For instance, vitamin D could effectively inhibit the growth of Gram-positive bacterial strains such as Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus mutans, but also of Gram-negative species including Klebsiella pneumoniae and Escherichia coli (35– 37). In addition, the production of antimicrobial peptides such as cathelicidin and defensins are stimulated by vitamin D (38– 40). Both, immune-modulatory and antimicrobial effects might be responsible for the beneficial effects of exogenous vitamin D observed in infectious morbidities caused by Helicobacter pylori (41) and in respiratory tract infections (42). Moreover, vitamin D has been shown to be involved in maintenance of the intestinal epithelial barrier integrity (43).

This prompted us in our present preclinical intervention study to investigate potential pathogen-lowering, immunomodulatory, intestinal epithelial barrier preserving and hence, disease-alleviating effects of synthetic 25-OH-cholecalciferol applying a clincial model of acute campylobacterosis.

# MATERIALS AND METHODS

#### Ethics Statement

All animal experiments were conducted in accordance with the European Guidelines for animal welfare (2010/63/EU) following approval by the commission for animal experiments headed by the "Landesamt für Gesundheit und Soziales" (LaGeSo, Berlin, registration numbers G0172/16 and G0247/16). Twice a day clinical conditions of mice were assessed.

#### Generation of Secondary Abiotic Mice

Female and male IL-10−/− mice (all in C57BL/6j background) were bred and reared under specific pathogen free (SPF) conditions in the same unit of the Forschungseinrichtungen

**Abbreviations:** CBA, Cytometric Bead Array; CFU, colony-forming units; HPF, High power fields; IFN, interferon; IL, interleukin; LOS, Lipo-oligosaccharide; LPS, Lipo-polysaccharide; MCP-1, monocyte chemoattractant protein 1; MLN, mesenteric lymph nodes; PBS, phosphate buffered saline; p.i., post-infection; PLC, Placebo; Rt, Transmural electrical resistance; SPF, specific pathogen free; Th, T helper cell; TLR, toll-like receptor; TNF, tumor necrosis factor; Treg, regulatory T cells; UV, ultraviolet; VDR, vitamin D receptor; VitD, Vitamin D.

für Experimentelle Medizin (FEM, Charité–University Medicine Berlin). Three to five mice were maintained in one cage including filter tops within an experimental semi-barrier (accessible only with lab coat, overshoes, caps, and sterile gloves) under standard conditions (22–24◦C room temperature, 55 ± 15% humidity, 12 h light/12 dark cycle) and had free access to autoclaved standard chow (food pellets: ssniff R/M-H, V1534-300, Sniff, Soest, Germany).

In order to assure stable gastrointestinal C. jejuni colonization and to override physiological colonization resistance (44), microbiota-depleted (i.e., secondary abiotic) mice were generated (44, 45). In brief, immediately post-weaning 3-week old mice were subjected to a 10-week course of broad-spectrum antibiotic treatment by adding ampicillin plus sulbactam (1 g/L; Ratiopharm, Germany), vancomycin (500 mg/L; Cell Pharm, Germany), ciprofloxacin (200 mg/L; Bayer Vital, Germany), imipenem (250 mg/L; MSD, Germany) and metronidazole (1 g/L; Fresenius, Germany) to the autoclaved drinking water (ad libitum) as described elsewhere (45). To assure antibiotic washout, the antibiotic cocktail was withdrawn 4 days prior infection and thus immediately before start of the vitamin D treatment.

#### Vitamin D Treatment

Vitamin D treatment started 4 days before C. jejuni infection. Therefore, synthetic 25-OH-cholecalciferol (purchased from Sigma-Aldrich, München, Germany) was dissolved in Tween 80 (0.2% v/v) and administered to mice via the autoclaved tap water (ad libitum). Considering a body weight of ∼25 g per mouse and a daily drinking volume of ∼5 mL, the final concentration of the synthetic 25-OH-cholecalciferol solution was 2.5µg/mL resulting in a daily treatment dosage of 500 µg per kg body weight (equivalent to 20,000 IU per kg) (46). Hence, the applied daily vitamin D dose was far beyond the toxic doses defined for rodents (i.e., 42 mg/kg/day) (47, 48) and humans (i.e., 150 mg/kg/day) (49). Age and sex matched placebo (PLC) control mice received vehicle (i.e., Tween 80) via the drinking water (ad libitum).

# *C. jejuni* Infection, Gastrointestinal Colonization, and Translocation

For infection, a stock solution of C. jejuni 81-176 strain that had been stored at −80◦C was thawed, aliquots streaked onto karmali agar (Oxoid, Wesel, Germany) and incubated in a microaerophilic atmosphere at 37◦C for 48 h. Immediately before peroral infection of mice, bacteria were harvested in sterile PBS (Oxoid) to a final inoculum of 109 bacterial cells. Mice (3 months of age) were perorally infected on two consecutive days (i.e., days 0 and 1). Animals were continuously maintained in a sterile environment (autoclaved food and drinking water) and handled under strict aseptic conditions to prevent from contaminations.

In order to assess gastrointestinal colonization and translocation, C. jejuni were quantitatively assessed in fecal samples over time post-infection (p.i.) and furthermore, in luminal samples derived from distinct parts of the gastrointestinal tract (i.e., from the stomach, duodenum, ileum, and colon) and in organ homogenates at day 6 p.i. by culture as stated elsewhere (44, 50). The detection limit of viable pathogens was ≈100 CFU per g (CFU/g). To assess C. jejuni bacteremia, thioglycollate enrichment broths (BD Bioscience, Germany) were inoculated with ∼200 µL cardiac blood of individual mice, incubated for 7 days at 37◦C, and streaked onto respective media for further identification as described (44).

#### Clinical Conditions

Before and after C. jejuni infection the clinical conditions of mice were assessed on a daily basis by using a standardized cumulative clinical score (maximum 12 points), addressing the clinical aspect/wasting (0: normal; 1: ruffled fur; 2: less locomotion; 3: isolation; 4: severely compromised locomotion, pre-final aspect), the abundance of blood in feces (0: no blood; 2: microscopic detection of blood by the Guajac method using Haemoccult, Beckman Coulter/PCD, Germany; 4: macroscopic blood visible), and diarrhea (0: formed feces; 2: pasty feces; 4: liquid feces) as described earlier (19).

#### Sampling Procedures

At day 6 p.i., mice were sacrificed by isofluran inhalation (Abbott, Germany). Luminal gastrointestinal samples (from stomach, duodenum, ileum, and colon) and ex vivo biopsies from colon, ileum, mesenteric lymph nodes (MLN), spleen, liver, kidneys, and lungs were taken under sterile conditions. For serum cytokine measurements cardiac blood was taken. Colonic and extraintestinal samples were collected from each mouse in parallel for microbiological, immunohistopathological, and immunological analyses. The absolute colonic and small intestinal lengths were measured with a ruler (in cm).

#### Immunohistochemistry

In situ immunohistochemical analyses were performed in colonic ex vivo biopsies that had been immediately fixed in 5% formalin and embedded in paraffin as described earlier (51–54). In brief, in order to detect apoptotic epithelial cells, proliferation epithelial cells, macrophages/monocytes, T lymphocytes, and regulatory T cells (Tregs), 5µm thin paraffin sections of ex vivo biopsies were stained with primary antibodies directed against cleaved caspase 3 (Asp175, Cell Signaling, Beverly, MA, USA, 1:200), Ki67 (TEC3, Dako, Denmark, 1:100), F4/80 (# 14- 4801, clone BM8, eBioscience, San Diego, CA, USA, 1:50), CD3 (#N1580, Dako, 1:10), and FOXP3 (clone FJK-165, #14-5773, eBioscience, 1:100), respectively. Positively stained cells were then examined by light microscopy (magnification 100× and 400×), and for each mouse the average number of respective positively stained cells was determined within at least six high power fields (HPF, 0.287 mm2 , 400× magnification) by a blinded independent investigator.

#### Inflammatory Mediator Detection in Supernatants of Intestinal and Extra-Intestinal *ex vivo* Biopsies

Colonic ex vivo biopsies were cut longitudinally, washed in phosphate buffered saline (PBS; Gibco, Life Technologies, UK), and strips of ∼1 cm2 tissue and ex vivo biopsies derived from MLN (3–4 lymph nodes), liver, and spleen (one half) were placed in 24-flat-bottom well-culture plates (Nunc, Germany) containing 500 µL serum-free RPMI 1640 medium (Gibco, life technologies, UK) supplemented with penicillin (100 U/mL) and streptomycin (100µg/mL; PAA Laboratories, Germany). After 18 h at 37◦C, respective culture supernatants as well as serum samples were tested for IL-6, monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor (TNF), and interferonγ (IFN-γ) by the Mouse Inflammation Cytometric Bead Assay (CBA; BD Biosciences, Germany) on a BD FACSCanto II flow cytometer (BD Biosciences). Systemic pro-inflammatory cytokine concentrations were measured in serum samples.

#### Electrophysiological Measurements

Distal colonic ex vivo biopsies were mounted unstripped in Ussing chambers (0.049 cm2 area). Transmural electrical resistance (Rt) was recorded under voltage clamp conditions by an automatic clamp device (CVC6, Fiebig Hard and Software, Berlin, Germany) at 37◦C over 1 h. The bathing solution was composed of NaCl (113.6 mmol/L), NaHCO3 (21.0 mmol/L), KCl (5.4 mmol/L), Na2HPO4 (2.4 mmol/L), MgCl2 (1.2 mmol/L), CaCl2 (1.2 mmol/L), NaH2PO4 (0.6 mmol/L), D(+)-glucose (10.0 mmol/L), D(+)-mannose (10.0 mmol/L), beta-hydroxybutyric acid (0.5 mmol/L), and L-glutamine (2.5 mmol/L) equilibrated with carbogen gas (pH 7.4).

#### Statistical Analysis

Medians and levels of significance were determined using Mann-Whitney test (GraphPad Prism v7, USA) for pairwise comparisons of not normally distributed data, and using the one-sided ANOVA with Tukey post-correction or the Kruskal-Wallis test with Dunn's post-correction for multiple comparisons as indicated. Two-sided probability (p) values ≤ 0.05 were considered significant. Experiments were performed in a blinded fashion and reproduced three times.

#### RESULTS

#### Intestinal Pathogen Loads Over Time Following Vitamin D Treatment of *C. jejuni* Infected Mice With Acute Enterocolitis

Secondary abiotic IL-10−/− mice were subjected to synthetic 25-OH-cholecalciferol treatment via the drinking water starting 4 days before C. jejuni infection. On two consecutive days, namely days 0 and 1, mice were then perorally challenged with 109 viable pathogens by gavage. Daily cultural analyses of fecal samples revealed that 25-OH-cholecalciferol application did not affect pathogenic intestinal colonization properties as indicated by stable median fecal C. jejuni loads of 109 CFU/g over time p.i. that did not differ between both cohorts at respective time points (n.s.; **Figure S1**). Upon necropsy, luminal gastrointestinal C. jejuni densities did not differ between 25-OH-cholecalciferol and placebo treated mice as determined in stomach, duodenum, ileum and colon at day 6 post-infection (n.s.; **Figure 1**). Hence, synthetic 25-OH-cholecalciferol treatment did not affect gastrointestinal C. jejuni loads.

FIGURE 1 | Gastrointestinal C. jejuni loads following vitamin D treatment of infected mice. Secondary abiotic IL-10−/− mice were treated with synthetic 25-OH-cholecalciferol (vitamin D, VitD, open circles) or placebo (PLC, closed circles) via the drinking water starting 4 days before peroral C. jejuni 81-176 strain infection on days 0 and 1. At necropsy (i.e., day 6 post-infection), luminal C. jejuni loads were quantitatively assessed from each mouse in distinct gastrointestinal compartments as indicated by culture and expressed in colony forming units per g (CFU/g). Medians (black bars) and numbers of analyzed animals (in parentheses) are indicated. Data were pooled from four independent experiments.

# Comprehensive Survey of Clinical Conditions Over Time Following Vitamin D Treatment of *C. jejuni* Infected Mice With Acute Enterocolitis

Within 6 days following C. jejuni infection mice from either cohort developed comparably severe symptoms of acute enterocolitis as daily quantitated applying a standardized cumulative clinical scoring system (**Figure S2**) assessing wasting symptoms, abundance of fecal blood, and the severity of diarrhea. Whereas overall pathogen-induced clinical symptoms were comparable between the two cohorts over time (n.s.; **Figure S2**), cumulate relative frequencies of diarrhea were lower in 25-OHcholecalciferol treated mice as compared to placebo controls as early as 24 h following the latest infection (i.e., day 2 p.i.) until 4 p.i. (**Figure 2**). Hence, synthetic 25-OH-cholecalciferol treatment results in less frequent C. jejuni induced diarrhea in the midst of infection.

#### Macroscopic and Microscopic Inflammatory Sequelae Following Vitamin D Treatment of *C. jejuni* Infected Mice With Acute Enterocolitis

Given that intestinal inflammation is association with a significant shortening of the affected part of the intestinal tract (15, 45), we measured the lengths of both, the small and large intestines upon necropsy. In fact, C. jejuni infection was accompanied with shorter colons of placebo as well as of 25-OHcholecalciferol treated mice (p < 0.001; **Figure S3A**), whereas the small intestinal lengths were virtually unaffected at day 6 p.i. (n.s.;

(see Materials and Methods). Bars indicate the cumulative frequencies of diarrhea (in %). Numbers of diarrheal mice out of the total number of analyzed animals are given in parentheses. Data were pooled from four independent experiments.

**Figure S3B**). Hence, synthetic 25-OH-cholecalciferol treatment does not ameliorate C. jejuni induced macroscopic disease.

Since apoptosis is regarded a reliable parameter for the grading of intestinal inflammation (44), we further quantitatively assessed caspase3+ apoptotic epithelial cells in large intestinal ex vivo biopsies applying in situ immunohistochemistry. At day 6 p.i., C. jejuni infected mice exhibited multifold increased numbers of apoptotic cells in their colonic epithelia (p < 0.001), that were, however, more than 60% lower in 25-OHcholecalciferol as compared to placebo treated mice (p < 0.05; **Figure 3A**, **Figure S4A**). Conversely, numbers of Ki67+ colonic epithelial cells indicative for cell proliferation and regeneration increased upon C. jejuni infection (p < 0.001), but more distinctly following 25-OH-cholecalciferol as compared to placebo treatment (p < 0.05; **Figure 3B**, **Figure S4B**). Hence, synthetic 25-OH-cholecalciferol treatment dampens C. jejuni induced apoptotic cell responses and promotes cell regenerative measures counteracting intestinal cell damage upon pathogenic exposure.

# Intestinal Immune Cell Responses Following Vitamin D Treatment of *C. jejuni* Infected Mice With Acute Enterocolitis

We further quantitatively surveyed both, innate and adaptive immune cell responses in the large intestinal tract following synthetic 25-OH-cholecalciferol treatment of C. jejuni infected mice by immunohistochemical staining of colonic paraffin sections. As early as 6 days upon C. jejuni infection, numbers of F4/80+ innate immune cell subsets including macrophages and monocytes had increased in the large intestinal mucosa and lamina propria (p < 0.001), but less distinctly in 25-OHcholecalciferol as compared to placebo challenged mice (p < 0.01; **Figure 4A**, **Figure S4C**). Similarly, C. jejuni induced increases in adaptive immune cells such as CD3+ lymphocytes, were less pronounced in the 25-OH-cholecalciferol vs. placebo cohort at day 6 p.i. (p < 0.05, VitD vs. PLC; **Figure 4B**, **Figure S4D**). Interestingly, numbers of FOXP3+ regulatory T cells (Treg) were slightly higher following vitamin D as

(B) proliferating (Ki67+) cells were assessed microscopically from six high power fields (HPF, 400× magnification) per animal in immunohistochemically stained colonic paraffin sections. Uninfected and untreated mice (none, open diamonds) served as negative control animals. Medians (black bars), levels of significance (p-values) assessed by the Kruskal-Wallis test and Dunn's post-correction or the one-sided ANOVA test with Tukey post-correction and numbers of analyzed animals (in parentheses) are indicated. Data were pooled from four independent experiments.

compared to placebo treated C. jejuni infected mice (p < 0.01; **Figure 4C**, **Figure S4E**). Hence, synthetic 25-OH-cholecalciferol treatment results in less pronounced C. jejuni induced intestinal responses of distinct innate and adaptive immune cell populations.

# Intestinal Pro-inflammatory Mediator Secretion Following Vitamin D Treatment of *C. jejuni* Infected Mice With Acute Enterocolitis

We next measured pro-inflammatory mediators in intestinal ex vivo biopsies. At day 6 following C. jejuni infection increased IL-6 and MCP-1 concentrations could be assessed in the colon of placebo (p < 0.01 and p < 0.05, respectively), but not 25-OHcholecalciferol treated mice (**Figures 5A,B**). C. jejuni induced increases in large intestinal TNF and IFN-γ concentrations (p < 0.05–0.001 vs. none), however, were unaffected by 25-OHcholecalciferol challenge (n.s. vs. PLC; **Figures 5C,D**). In line, ileal IL-6 and MCP-1 as well as IFN-γ levels were elevated upon C. jejuni infection of mice from the placebo (p < 0.05–0.01), but not from the 25-OH-cholecalciferol cohort (**Figures 6A,B,D**), whereas like in the colon, ileal TNF concentrations were comparably elevated at day 6 post-infection of either cohort (p < 0.001; **Figure 6C**). Hence, synthetic 25-OH-cholecalciferol treatment of C. jejuni infected mice results in less pronounced secretion of distinct pro-inflammatory mediators in the intestinal tract.

# Extra-Intestinal Inflammatory Immune Responses Following Vitamin D Treatment of *C. jejuni* Infected Mice

We further asked whether the 25-OH-cholecalciferol mediated anti-inflammatory effects were restricted to the intestinal tract or also effective in extra-intestinal compartments. In fact, IFNγ concentrations were lower in MLN and liver of 25-OHcholecalciferol as compared to placebo treated mice at day 6 p.i. (p < 0.05; **Figures 7A,B**). Interestingly, C. jejuni infection resulted in decreased IFN-γ secretion in splenic ex vivo biopsies irrespective of the treatment regimen (p < 0.001; **Figure 7C**). Hence, synthetic 25-OH-cholecalciferol treatment of C. jejuni infected mice resulted in less distinct IFN-γ secretion in MLN and liver.

## Systemic Pro-inflammatory Mediator Secretion Following Vitamin D Treatment of *C. jejuni* Infected Mice With Acute Enterocolitis

We next addressed whether synthetic 25-OH-cholecalciferol treatment might alleviate systemic C. jejuni induced proinflammatory immune responses. At day 6 p.i., mice from either cohort exhibited comparably elevated IL-6, MCP1, TNF, and IFN-γ serum concentrations (p < 0.001 vs. none; **Figure S5**). Hence, synthetic 25-OH-cholecalciferol treatment does not affect C. jejuni induced systemic pro-inflammatory mediator secretion.

FIGURE 4 | Colonic immune cell responses following vitamin D treatment of C. jejuni infected mice. Secondary abiotic IL-10−/− mice were treated with synthetic 25-OH-cholecalciferol (vitamin D, VitD, open circles) or placebo (PLC, closed circles) via the drinking water starting 4 days before peroral C. jejuni 81-176 strain infection on days 0 and 1. At necropsy (i.e., day 6 post-infection), the average numbers of (A) macrophages and monocytes (F4/80+), (B) T lymphocytes (CD3+) and (C) regulatory T cells (FOXP3+) were assessed microscopically from six high power fields (HPF, 400× magnification) per animal in immunohistochemically stained colonic paraffin sections. Uninfected and untreated mice (none, open diamonds) served as negative control animals. Medians (black bars), levels of significance (p-values) assessed by the one-sided ANOVA test with Tukey post-correction and numbers of analyzed animals (in parentheses) are indicated. Data were pooled from four independent experiments.

# Bacterial Translocation Following Vitamin D Treatment of *C. jejuni* Infected Mice With Acute Enterocolitis

We further asked whether synthetic 25-OH-cholecalciferol treatment had an impact of the translocation rates of viable pathogens from the infected intestines to extra-intestinal including systemic tissue sites. Whereas, C. jejuni could be cultured at similar frequencies from MLN, liver and lungs derived from 25-OH-cholecalciferol and placebo treated mice (**Figures 8A–C**), cumulative pathogenic translocation rates were lower in the kidneys (12.5 vs. 31.6%) and the spleen (12.5 vs. 26.3%) taken from the former as compared to the latter at day 6 p.i. (**Figures 8D,E**). Notably, all blood cultures remained C. jejuni negative (**Figure 8F**). Hence, synthetic 25-OH-cholecalciferol treatment was associated with lower cumulative translocation rates of C. jejuni originating from the inflamed intestines to the kidneys and the spleen.

## Colonic Epithelial Barrier Changes Following Vitamin D Treatment of *C. jejuni* Infected Mice With Acute Enterocolitis

Given the lower cumulative pathogenic translocation rates we assessed whether synthetic 25-OH-cholecalciferol treatment resulted in a less compromised colonic epithelial barrier function in C. jejuni infected mice. Therefore, we performed electrophysiological resistance measurements of colonic ex vivo biopsies in the Ussing chamber. In fact, transmural resistances were lower in the large intestines derived from placebo, but not 25-OH-cholecalciferol treated mice at day 6 p.i. as compared to uninfected and untreated control animals (p < 0.05; **Figure 9**). Hence, synthetic 25-OH-cholecalciferol treatment results in uncompromised colonic epithelial barrier function following C. jejuni infection.

# DISCUSSION

Due to the pleiotropic beneficial effects of vitamin D in health and disease, the application of vitamin D as safe dietary supplement is currently discussed as promising option for the adjunct treatment and prophylaxis of various immunopathological morbidities including infectious diseases, intestinal inflammatory conditions, and cancer, for instance (33, 55). In our present vitamin D intervention study applying a clinical acute campylobacteriosis model, prophylactic synthetic 25-OH-cholecalciferol application starting 4 days prior murine infection resulted in dampened C. jejuni induced intestinal and extra-intestinal inflammatory sequalae, but could not lower the high intestinal pathogen loads of more than 109 viable C. jejuni per g feces. In support, recent reports revealed that the beneficial effects of vitamin D during gastrointestinal infection with distinct bacterial species such as Salmonella (56) or Listeria monocytogenes (57) are rather due to the pleiotropic immuno-modulatory than direct antimicrobial properties of the steroid hormone. In addition, one needs to take into consideration, that, in contrary to humans, the expression of the antimicrobial peptide cathelicidin in mice is not regulated by vitamin D, given that in the murine cathelicidin gene promoter the vitamin D response element is missing (58, 59). This could explain our observation that external 25-OHcholecalciferol application, even in high doses, did not reduce intestinal C. jejuni burdens. However, it is tempting to speculate that this could be the case in humans.

Despite the high intestinal pathogenic burdens, 25-OHcholecalciferol treated mice suffered less frequently from diarrhea

supernatants derived from colonic ex vivo biopsies. Uninfected and untreated mice (none, open diamonds) served as negative control animals. Medians (black bars), levels of significance (p-values) assessed by the Kruskal-Wallis test and Dunn's post-correction and numbers of analyzed animals (in parentheses) are indicated. Data were pooled from four independent experiments.

in the midst of campylobacteriosis development as compared to placebo controls, but exhibited comparable macroscopic disease at the end of the observation period. Notably, the macroscopic outcome particularly in such a non-selflimiting detrimental intestinal infection and inflammation model is due to the sum effect of many different intestinal, extra-intestinal and systemic events within this hyper-inflammatory scenario (24). It is therefore remarkable, that less distinct C. jejuni induced apoptosis of colonic epithelial cells, whereas, conversely, large intestinal cell regenerative properties counteracting pathogeninduced cell damage were promoted upon 25-OH-cholecalciferol application in mice suffering from acute enterocolitis. In support, the intestinal epithelial vitamin D receptor has been shown to regulate mucosal inflammation by suppressing intestinal epithelial cell apoptosis (60). Less severe colonic apoptosis upon 25-OH-cholecalciferol treatment was accompanied by less distinct immune cell responses upon C. jejuni infection, which is supported by several studies showing that vitamin D regulates both, innate and adaptive immunity (61–63). In our study, lower numbers of innate immune cell populations such as macrophages and monocytes could be assessed in the colonic mucosa and lamina propria of C. jejuni infected mice that had been pretreated with synthetic 25-OH-cholecalciferol. In line, recent reports revealed that vitamin D stimulation of antigen presenting cells including macrophages and dendritic cells resulted in decreased pro-inflammatory mediator secretion

(59, 64). In addition, colonic mucosal numbers of T lymphocytes were lower in 25-OH-cholecalciferol as compared to placebo treated mice with C. jejuni induced enterocolitis. In fact, T cells have been shown to be direct and indirect targets of vitamin D (65, 66). Previous in vitro, ex vivo, and in vivo studies revealed that vitamin D treatment of T cells and of mice resulted in less distinct T cell proliferation and in decreased T helper cell (Th)−1 dependent secretion of pro-inflammatory cytokines and subsequently in ameliorated inflammation (66, 67). In our present study, the colonic concentrations of proinflammatory mediators including IL-6 and MPC-1 measured in 25-OH-cholecalciferol pretreated, C. jejuni infected mice were

comparable to those obtained from naive controls. In support, vitamin D was shown to reduce recruitment of innate immune cells such as monocytes and to decrease IL-6 and MCP-1 releases upon in vitro stimulation (68). Notably, the 25-OHcholecalciferol associated decreased pro-inflammatry mediator secretion was not restricted to the large intestines, the major predilection site of C. jejuni induced enterocolitis (15, 69). In fact, C. jejuni induced increased secretion of IL-6, MCP-1, and additionally of IFN-γ could be observed in the terminal ileum of mice from the placebo, but not from the 25-OHcholecalciferol treatment cohort. Interestingly, as opposed to 25- OH-cholecalciferol related decreases in large intestinal T cell

pooled from four independent experiments.

FIGURE 7 | Extra-intestinal IFN-γ secretion following vitamin D treatment of C. jejuni infected mice. Secondary abiotic IL-10−/− mice were treated with synthetic 25-OH-cholecalciferol (vitamin D, VitD, open circles) or placebo (PLC, closed circles) via the drinking water starting 4 days before peroral C. jejuni 81-176 strain infection on days 0 and 1. At necropsy (i.e., day 6 post-infection), IFN-γ concentrations were determined in supernatants of ex vivo biopsies derived from (A) mesenteric lymph nodes (MLN), (B) liver, and (C) spleen. Uninfected and untreated mice (none, open diamonds) served as negative control animals. Medians (black bars), levels of significance (p-values) assessed by the Kruskal-Wallis test and Dunn's post-correction and numbers of analyzed animals (in parentheses) are indicated. Data were pooled from four independent experiments.

treated with synthetic 25-OH-cholecalciferol (vitamin D, VitD, open circles) or placebo (PLC, closed circles) via the drinking water starting 4 days before peroral C. jejuni 81-176 strain infection on days 0 and 1. At necropsy (i.e., day 6 post-infection), the transmural electrical resistance of distal colon was measured in Ussing chambers as described in Materials and Methods. Uninfected and untreated mice (none, open diamonds) served as negative control animals. Medians (black bars), levels of significance (p-values) assessed by the Kruskal-Wallis test and Dunn's post-correction and numbers of analyzed animals (in parentheses) are indicated. Data were pooled from two independent experiments.

numbers, higher numbers of (potentially anti-inflammatory) FOXP3+ regulatory T cells could be assessed in the colonic mucosa and lamina propria of 25-OH-cholecalciferol vs. placebo treated mice with enterocolitis. In support, recent studies reported that vitamin D results in enhanced recruitment of regulatory T cells to inflamed tissue sites (70–72). Given that we did not perform co-staining analyses in our present study, however, we can not answer which specific immune cell subset was expressing FOXP3.

Remarkably, the pro-inflammatory immune responsedampening effects of exogenous 25-OH-cholecalciferol were not restricted to the intestinal tract, but were also effective in extra-intestinal compartments given that C. jejuni induced IFN-γ secretion was less pronounced in MLN draining the inflamed intestines and in the liver upon 25-OH-cholecalciferol treatment. In line, previous studies provide evidence that vitamin D application or even skin exposure to UV light could ameliorate or prevent from liver inflammation due to vitamin D mediated dampening of immune cellular responses and inhibition of liver apoptosis, for instance (73, 74).

At the first glance unexpectedly, C. jejuni infection was associated with decreases in splenic IFN-γ concentrations in either cohort. One possible explanation might be that upon pathogenic infection leukocytes were recruited from the spleen to the site of infection in order to limit pathogenic spread. One could have expected an even more prominent effect following synthetic 25-OH-cholecalciferol application due to the known immune cell recruiting properties of vitamin D (75).

C. jejuni infection results in impaired epithelial barrier function in vitro (76) and campylobacteriosis is characterized by a leaky gut syndrome facilitating pathogenic translocation from the inflamed intestines to extra-intestinal including systemic compartments (15, 69). Given that vitamin D has been shown to preserve epithelial barrier function (75), we assessed potential 25- OH-cholecalciferol mediated effects on pathogenic translocation frequencies in our preclinical survey. In fact, when taking results of the four independent experiment together, C. jejuni could be cultured less frequently from the kidneys and the spleen of infected mice following 25-OH-cholecalciferol as compared to placebo treatment, whereas cumulative relative C. jejuni translocation rates to MLN, liver and lungs were comparable. Of note, all blood cultures remained C. jejuni negative, irrespective of the treatment regimen. One needs to take into consideration, however, that soluble bacterial molecules including LOS and others might have been transported via the circulation contributing to the observed extra-intestinal collateral damages of C. jejuni infection. Nevertheless, the observed inflammation-alleviating effects upon 25-OH-cholecalciferol application were further accompanied by a less compromised colonic epithelial barrier function in 25-OH-cholecalciferol as compared to placebo treated, C. jejuni infected mice. This in turn very likely reduced the risk of spread of both, viable bacteria and soluble bacterial molecules in the former vs. the latter. We therefore hypothesize that the 25-OH-cholecalciferol associated anti-inflammatory effects in particular prevent from further bacteria-induced damages in this acute C. jejuni induced inflammation model.

#### CONCLUSION

Our preclinical intervention study provides evidence that prophylactic peroral synthetic 25-OH-cholecalciferol application dampens intestinal and extra-intestinal inflammatory responses during acute campylobacteriosis in the clinical mouse model applied here. Further studies are needed in order to define appropriate vitamin D doses for the prevention and combat of distinct gastrointestinal infectious morbidities in humans.

#### DATA AVAILABILITY

All datasets generated for this study are included in the manuscript/**Supplementary Files**.

#### AUTHOR CONTRIBUTIONS

SM performed experiments, analyzed data, and co-wrote paper. FL and RB performed experiments, analyzed data, and co-edited paper. J-DS and SB provided advice in experimental design, critically discussed results, and co-edited paper. MH designed and performed experiments, analyzed data, and wrote paper.

# FUNDING

This work was supported from the German Federal Ministries of Education and Research (BMBF) in frame of the zoonoses research consortium PAC-Campylobacter to SM, SB, and MH (IP7/01KI1725D) and to RB and J-DS (IP8/01KI1725D) as part of the Research Network Zoonotic Infectious Diseases and from the German Federal Ministries of Economy and Energy to SB and MH (ZIM; ZF4117904 AJ8). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

#### ACKNOWLEDGMENTS

We thank Alexandra Bittroff-Leben, Ines Puschendorf, Ulrike Fiebiger, Ulrike Escher, Anna-Maria Schmidt, Gernot Reifenberger, and the staff of the animal research facility at Charité–University Medicine Berlin for excellent technical assistance and animal breeding.

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.02094/full#supplementary-material

Figure S1 | Fecal C. jejuni loads over time following vitamin D treatment of infected mice. Secondary abiotic IL-10−/− mice were treated with (A) placebo (closed circles) or (B) synthetic 25-OH-cholecalciferol (vitamin D, open circles) via the drinking water starting four days before peroral C. jejuni 81-176 strain infection on day (d) 0 and d1. Fecal C. jejuni loads were quantitatively assessed from each mouse on a daily basis post-infection (p.i.) by culture and expressed in colony forming units per g (CFU/g). Medians (black bars) and numbers of analyzed animals (in parentheses) are indicated. Data were pooled from four independent experiments.

Figure S2 | Kinetic survey of overall clinical conditions following vitamin D treatment of C. jejuni infected mice. Secondary abiotic IL-10−/− mice were treated with (A) placebo (closed circles) or (B) synthetic 25-OH-cholecalciferol (vitamin D, open circles) via the drinking water starting four days before peroral C.

#### REFERENCES

jejuni 81-176 strain infection on days 0 and 1. Clinical symptoms were quantitatively assessed applying a standardized clinical scoring system from d0 until d6 post-infection (see Materials and Methods). Uninfected and untreated mice (none, open diamonds) served as negative control animals. Medians (black bars) and numbers of analyzed animals (in parentheses) are indicated. Data were pooled from four independent experiments.

Figure S3 | Intestinal lengths following vitamin D treatment of C. jejuni infected mice. Secondary abiotic IL-10−/− mice were treated with synthetic 25-OH-cholecalciferol (vitamin D, VitD, open circles) or placebo (PLC, closed circles) via the drinking water starting 4 days before peroral C. jejuni 81-176 strain infection on days 0 and 1. At necropsy (i.e., day 6 post-infection), the absolute lengths of the (A) colon and (B) small intestines were measured with a ruler. Uninfected and untreated mice (none, open diamonds) served as negative control animals. Medians (black bars), levels of significance (p-values) assessed by one-sided ANOVA test with Tukey post-correction and numbers of analyzed animals (in parentheses) are indicated. Data were pooled from four independent experiments.

Figure S4 | Representative photomicrographs illustrating apoptotic and proliferating epithelial as well as immune cells responses in large intestines following vitamin D treatment of C. jejuni infected mice. Secondary abiotic IL-10−/− mice were treated with synthetic 25-OH-cholecalciferol (vitamin D) or placebo via the drinking water starting 4 days before peroral C. jejuni 81-176 strain infection on days 0 and 1. Naive mice served as uninfected and untreated controls. Photomicrographs reepresentative for four independent experiments illustrate the average numbers of (A) apoptotic epithelial cells (Casp3+), (B) proliferating epithelial cells, (C) macrophages and monocytes (F4/80+), (D) T lymphocytes (CD3+) and (E) regulatory T cell (Treg, FOXP3+) in at least six high power fields (HPF) as quantitatively assessed in ileal paraffin sections applying in situ immunohistochemistry at day 6 post-infection (100× magnification, scale bar 100µm).

Figure S5 | Systemic secretion of pro-inflammatory mediators following vitamin D treatment of C. jejuni infected mice. Secondary abiotic IL-10−/− mice were treated with synthetic 25-OH-cholecalciferol (vitamin D, VitD, open circles) or placebo (PLC, closed circles) via the drinking water starting 4 days before peroral C. jejuni 81-176 strain infection on days 0 and 1. At necropsy (i.e., day 6 post-infection), (A) IL-6, (B) MCP1, (C) TNF, and (D) IFN-γ concentrations were determined in serum samples. Uninfected and untreated mice (none, open diamonds) served as negative control animals. Medians (black bars), levels of significance (p-values) assessed by the Kruskal-Wallis test and Dunn's post-correction and numbers of analyzed animals (in parentheses) are indicated. Data were pooled from four independent experiments.

mice via Toll-like-receptor-2 and−4 signaling. PLoS ONE. (2012) 7:e40761. doi: 10.1371/journal.pone.0040761

**Conflict of Interest Statement:** The 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.

Copyright © 2019 Mousavi, Lobo de Sá, Schulzke, Bücker, Bereswill and Heimesaat. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Vitamin D Signaling in the Context of Innate Immunity: Focus on Human Monocytes

Carsten Carlberg\*

*School of Medicine, Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland*

The vitamin D3 metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) activates at sub-nanomolar concentrations the transcription factor vitamin D receptor (VDR). VDR is primarily involved in the control of cellular metabolism but in addition modulates processes important for immunity, such as anti-microbial defense and the induction of T cell tolerance. Monocytes and their differentiated phenotypes, macrophages and dendritic cells, are key cell types of the innate immune system, in which vitamin D signaling was most comprehensively investigated *via* the use of next generation sequencing technologies. These investigations provided genome-wide maps illustrating significant effects of 1,25(OH)2D3 on the binding of VDR, the pioneer transcription factors purine-rich box 1 (PU.1) and CCAAT/enhancer binding protein α (CEBPA) and the chromatin modifier CCCTC-binding factor (CTCF) as well as on chromatin accessibility and histone markers of promoter and enhancer regions, H3K4me3 and H3K27ac. Thus, the epigenome of human monocytes is at multiple levels sensitive to vitamin D. These data served as the basis for the chromatin model of vitamin D signaling, which mechanistically explains the activation of a few hundred primary vitamin D target genes. Comparable epigenome- and transcriptome-wide effects of vitamin D were also described in peripheral blood mononuclear cells isolated from individuals before and after supplementation with a vitamin D3 bolus. This review will conclude with the hypothesis that vitamin D modulates the epigenome of immune cells during perturbations by antigens and other immunological challenges suggesting that an optimal vitamin D status may be essential for an effective epigenetic learning process, in particular of the innate immune system.

Keywords: vitamin D, VDR, epigenome, transcriptome, gene regulation, vitamin D target genes, monocytes, PBMCs

#### INTRODUCTION

Vitamin D3 is an evolutionary very old molecule that is produced from the direct cholesterol precursor 7-dehydrocholesterol in a non-enzymatic reaction using energy provided by the UV-B component of sunlight (1). Thus, every species that exerts cholesterol biosynthesis and is exposed to sunlight should be able to synthesize vitamin D3. The molecule itself is biologically inert, but when it is converted to 25-hydroxyvitamin D3 (25(OH)D3) and then to 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), it acts as a nuclear hormone. The jawless fish lamprey is the oldest known species that some 550 million years ago evolved with the transcription factor VDR a nuclear receptor

#### Edited by:

*Susu M. Zughaier, Qatar University, Qatar*

#### Reviewed by:

*Anthony George Tsolaki, Brunel University London, United Kingdom Lakshna Mahajan, Swami Shraddhanand College, University of Delhi, India*

\*Correspondence:

*Carsten Carlberg<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology*

Received: *17 May 2019* Accepted: *02 September 2019* Published: *13 September 2019*

#### Citation:

*Carlberg C (2019) Vitamin D Signaling in the Context of Innate Immunity: Focus on Human Monocytes. Front. Immunol. 10:2211. doi: 10.3389/fimmu.2019.02211*

**37**

changes in markers of active TSS regions (H3K4me3) and active chromatin (H3K27ac). The genome-wide binding of VDR, CTCF, PU.1, CEBPα, and histone markers were determined by ChIP-seq in three biological repeats. The time of 1,25(OH)2D3 stimulation is indicated for each dataset.

that gets activated by 1,25(OH)2D3 at sub-nanomolar concentrations (2). After the manifestation of VDR, vitamin D turned from a product of UV-B absorption, i.e., the output of a radiation protection pathway as found in plankton, to an endocrine molecule in higher species (3). Thus, vitamin D has via its metabolite 1,25(OH)2D3 direct effects on gene regulation (**Figure 1**). In human, the main sites of 1,25(OH)2D3 production for endocrine purposes are proximal tubule cells of the kidneys, but for para- and autocrine use also monocytes, macrophages, and dendritic cells of the innate immune system and other cell types in skin and bone are able to produce the nuclear hormone (4).

Since vitamin D3 can be synthesized endogenously in human skin (5), the term "vitamin" seems not to be appropriate. However, compared to the past, humans spend far more time indoors, largely cover their skin by textile when being outdoors and often live at latitudes where during winter UV-B radiation is too low for many months, there is insufficient endogenous vitamin D3 production, i.e., under these conditions vitamin D3 is an essential micronutrient (6). Average human diet is low in vitamin D, so that dietary products, such as milk, margarine and juices, are fortified and direct vitamin D supplementation via pills is recommended in winter months (7). Interestingly, already more than 100 years ago cod-liver oil as well as UV-B exposure had been proposed for the protection against rickets (an infant bone malformation disease) as well as for the treatment of tuberculosis (an infectious disease caused by intra-cellular bacteria) (8, 9). Thus, vitamin D deficiency causes not only bone disorders (10) but also affects the protective roles of the molecule against a large number of other diseases (11). The autoimmune disease multiple sclerosis is the most prominent example, which may be largely preventable by a sufficient vitamin D status (12). This status is defined via the serum concentrations of the most stable vitamin D metabolite, 25(OH)D3, which for

**Abbreviations:** 1,25(OH)2D3, 1α,25-dihydroxyvitamin D3; 25(OH)D3, 25 hydroxyvitamin D3; BRD7, bromodomain containing 7; CAMP, cathelicidin antimicrobial peptide; CD14, CD14 molecule; CDKN1C, cyclin dependent kinase inhibitor 1C; CEBP, CCAAT/enhancer binding protein; ChIP-seq, chromatin immunoprecipitation sequencing; CTCF, CCCTC-binding factor; CYP, cytochrome P450; DENND6B, DENN domain containing 6B; FAIREseq, formaldehyde-assisted isolation of regulatory elements sequencing; FBP1, fructose-bisphosphatase 1; GABPA, GA binding protein transcription factor α; HLA, human leukocyte antigen; KDM6B, lysine demethylase 6B; MYO1G, myosin IG; NF-AT, nuclear factor activated T cells; NF-κB, nuclear factor κ-lightchain-enhancer of activated B cells; PBMCs, peripheral blood mononuclear cells; PFKFB4, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4; PU.1, purinerich box 1, Spi-1 proto-oncogene (official gene symbol: SPI1); qPCR, quantitative polymerase chain reaction; RNA-seq, RNA sequencing; TAD, topologically associated domain; TNFSF11, TNF superfamily member 11; TSS, transcription start site; VDR, vitamin D receptor; ZMIZ1, zinc finger MIZ-type containing 1.

good bone health should be 50 nM (13), but also levels of 75 nM or more are suggested (14). Accordingly, instructions for daily supplementation with vitamin D3 range from 10 to 50 µg (400– 2,000 IU). However, these population-wide recommendations do not take inter-individual variations into account, such as a different molecular response to vitamin D, which are expressed by the vitamin D response index (15). As discussed below in more detail, this index can be determined via the genome-wide response of peripheral blood mononuclear cells (PBMCs) to an in vivo challenge with vitamin D3 (16).

In extension to a recent summary on the nutrigenomic role of vitamin D (17), the aim of this short review is to present the epigenome-wide impact of the nuclear hormone in relation to immunity. Special attention is given to human monocytes and PBMCs serving as in vitro and in vivo model systems for vitamin D signaling.

#### VITAMIN D AND THE EPIGENOME

Chromatin is a complex of histone proteins and genomic DNA (18, 19) that by default largely restricts the access of RNA polymerases to promoter regions and of transcription factors to enhancer regions. Therefore, in a differentiated cell only some 200,000 genomic loci are accessible (20). The epigenome comprises genome-wide information represented by covalent and structural modifications of chromatin, such as cytosine methylation, post-translational modifications of histone proteins and 3D structure of the nucleus, that do not involve any alterations in the sequence of genomic DNA (21). Epigenetic programming is a memory creating event that during embryogenesis and cellular differentiation, such as of monocytes after immune challenges (**Figure 2C**), determines the specialized roles of terminally differentiated cells via changes of their epigenome (25). In these cases epigenetic programming is irreversible and leads to static outcomes, in order to keep the identity of tissues and cell types. Thus, the epigenome largely determines gene expression and the functional profile of a cell; i.e., alternations of the epigenome precede those of the transcriptome.

A number of diet-derived metabolites, such as resveratrol, genistein, curcumin and polyphenols from fruits, vegetables, spices, teas and medicinal herbs, can affect the activity of chromatin modifiers and transcription factors (17, 26). Chromatin modifiers are nuclear enzymes that catalyze epigenetic modifications, such as DNA methylation as well as histone acetylation and methylation, while chromatin remodelers are another class of nuclear enzymes that change the position and composition of nucleosomes. VDR communicates in a liganddependent fashion both with chromatin modifiers, such as lysine demethylase 6B (KDM6B) (27), as well as with chromatin remodelers, such as bromodomain containing 7 (BRD7) (28). This explains how vitamin D can significantly change the intensity of histone markers for active chromatin, H3K27ac, as well as those for active transcription start sites (TSSs), H3K4me3, as observed by chromatin immunoprecipitation sequencing (ChIP-seq) in THP-1 human monocytic leukemia cells (29, 30) (**Figure 1**). The epigenome of these cells responds to a stimulation with 1,25(OH)2D3 at the loci of more than 500 promoters and 2,500 enhancers. Moreover, the method of formaldehyde-assisted isolation of regulatory elements sequencing (FAIRE-seq) monitored in the same cellular system that vitamin D changes the accessibility of some 4,500 chromatin loci (out of some 100,000 in total) at a given time point (22). Thus, a stimulation with vitamin D represents a cellular perturbation that results in changes of the epigenome and in this way affects the epigenetic memory of the cell (24). However, in contrast to epigenetic programming during cellular differentiation, many of these epigenetic memorizing events are dynamic; i.e., they persist only for a shorter time period and are reversible.

In human cells, the cistrome of VDR, i.e., the genome-wide binding pattern of the transcription factor, was determined by the ChIP-seq in lymphocytes (31), colorectal cancer cells (32), hepatic stellate cells (33), prostate cells (34), macrophage-like cells (35) and most comprehensively in monocytes (36, 37). In all these in vitro cell culture models stimulation with ligand resulted in a 2- to 10-fold increase in VDR binding sites; i.e., the significantly enhanced VDR cistrome represents the most eminent response of the human epigenome to a perturbation with vitamin D. In monocytes the VDR cistrome comprises more than 10,000 loci, of which a subgroup of a few hundred persistent sites is always occupied (37). These sites seem to be the primary contact points of the human genome with vitamin D and may coordinate the genome's spatio-temporal response to the nuclear hormone.

In THP-1 cells, statistically significant epigenome-wide effects of vitamin D were also described for the binding of the pioneer factors PU.1 (38), CEBPA (30) and GA binding protein transcription factor α (GABPA) (39) as well as for the chromatin organizer CTCF (40). The pioneer factors contribute to the increase in VDR loci after ligand stimulation by helping the receptor accessing its genomic binding sites (**Figure 1**). Since CTCF majorly contributes to DNA loop formation of the genome into topologically associated domains (TADs) (41), the vitamin D sensitivity of the protein implies that some 500 TADs are triggered by VDR and its ligand. Thus, vitamin D affects the epigenome on multiple levels, such as the binding of VDR and pioneer factors, histone markers, chromatin accessibility, and 3D organization of the nucleus.

## CHROMATIN MODEL OF VITAMIN D SIGNALING

The chromatin model of vitamin D signaling (24, 42) (**Figure 1**) was developed on the basis of above described epigenomic data, which had been primarily obtained in THP-1 cells after a stimulation with 1,25(OH)2D3 for 24 h. The model suggests that a primary vitamin D target gene is modulated in its expression, when the TAD, in which the gene is localized, contains a prominent VDR binding site. This applies to 425 vitamin D sensitive TADs comprising 90% of all target genes in THP-1 cells (40). An additional condition for effective gene regulation is, that

*(Continued)*

FIGURE 2 | individual was challenged with a vitamin D3 bolus (2,000 µg) and PBMCs were isolated before (day 0) and at days 1 (24 h) and 2 (48 h) (23). The peak tracks represent merger of each three biological repeats. Gene structures are shown in blue. Different types of immune challenges program the epigenome of the pool of human monocytes, which "memorize" these encounters in form of differently programmed epigenomes leading to subtype differentiation (bottom, differently colored dots of in nuclei of monocyte subpopulations, C). The recently discovered epigenome modulating effect of vitamin D [via the VDR (24)] modulates on multiple levels this epigenetic programming process. The stabilization of the epigenomes of the subtypes of monocytes, macrophages and dendritic cells by vitamin D can prevent or delay the onset of common age-related diseases.

the TSS of the target gene as well as the vitamin D-sensitive enhancer have to be located within accessible chromatin (43). DNA looping between the enhancer binding ligand-activated VDR supported by pioneer factors, such as CEBPA and PU.1, and the TSS of a vitamin D target genes changes at both genomic regions H3K27ac and H3K4me3 histone marks as well as chromatin accessibility (**Figure 1**). Thus, many epigenetic events are required before RNA polymerase II on the TSSs is activated and mRNA synthesis can start.

A meta-analysis of four independent transcriptome-wide datasets of 1,25(OH)2D3-stimulated undifferentiated THP-1 cells (22, 30, 36, 44) revealed 126 common genes, 72% of which are primary vitamin D targets (45). Nearly all (97%) of these vitamin D target genes are up-regulated and primarily encode for enzymes, receptors and transporters, half of which are located in membranes. Gene ontology analysis indicated the modulation of innate immunity as the most prominent common function of these genes, although this covers <25% of all. Four classes of gene regulatory scenarios, which are based on differential VDR, PU.1, and CEBPA binding to promoter and enhancer regions, can explain the regulation of most (85%) primary vitamin D target genes (45). Interestingly, immune system-related genes are often prominently up-regulated by vitamin D, while genes involved in cellular metabolism are less sensitive to the nuclear hormone. This was confirmed by an independent analysis of vitamin D-triggered TAD classes, where genes that are important for immune function are regulated in a tightly controlled "on/off " modus (37).

#### VITAMIN D AND IMMUNITY

Based on the evolutionary history of nuclear receptors (46, 47), VDR's original function was the regulation of cellular metabolism. This role specialized into the control of calcium homeostasis, when some 400 million years ago species left the ocean and had to improve their calcium-based skeleton, in order to resist to gravitation (48). Although there are no direct effects of vitamin D on bone mineralization, bone-resorbing osteoclasts derive from monocytes, the differentiation of which is controlled by the vitamin D target gene TNF superfamily member 11 (TNFSF11, encoding the cytokine RANKL) (49). VDR's tasks in the control of metabolism involves regulating genes mediating energy metabolism, like the glycolytic enzymes fructose-bisphosphatase 1 (FBP1) (36) and 6-phosphofructo-2 kinase/fructose-2,6-biphosphatase 4 (PFKFB4) (50), as well as in the catabolism of lipophilic intra-cellular molecules, like those encoding for the cytochrome P450 (CYP) enzymes CYP26B1, CYP19A1, and CYP24A1 (51). Both functions supported and enhanced the expansion of the energy demanding immune system as suggested by the concept of immuno-metabolism (52). Moreover, VDR became a critical transcription factor in regulating the expression of genes involved in inflammation and anti-bacterial defense, such as CD14 (53) and cathelicidin antimicrobial peptide (CAMP) (54). Thus, the immune-modulating function of vitamin D is probably evolutionary older than its role in calcium homeostasis.

During hematopoiesis VDR acts together with the pioneer transcription factors PU.1 and CEBPA as a key regulator of myeloid differentiation toward key cells in innate immunity, such as monocytes and granulocytes (55). Furthermore, vitamin D can inhibit the maturation, differentiation and the stimulatory capacity of dendritic cells, which derive from monocytes (56). A profile change of dendritic cells induces the production of regulatory T cells and leads to immunological tolerance. In parallel, vitamin D and its receptor are able to antagonize the pro-inflammatory actions of the transcription factors nuclear factor activated T cells (NF-AT) and nuclear factor κ-lightchain-enhancer of activated B cells (NF-κB) in T cells (57). In this way, vitamin D reduces autoimmunity, such as the onset and progression of multiple sclerosis (58), as well as chronic inflammation, such as in inflammatory bowel disease (59).

Most cells of the immune system have a rapid turnover, which enables them to respond more flexible to environmental changes than other cell types of the human body. For example, monocytes coordinate not only inflammatory pathways, but also control via in their differentiated forms, macrophages and dendritic cells, metabolic pathways, and general stress responses. Cellular perturbations, such as an encounter of immune cells with an antigen, affect via signal transduction cascades the epigenome. For example, most inflammatory lesions are initiated by monocyte-derived macrophages, the altered gene expression profile of which is based on changes of their epigenome in response to extra-cellular signals (**Figure 2C**). Moreover, the differentiation process of monocytes to macrophages (or dendritic cells) is based on epigenome changes in response to contacts with antigens. Such a subtype specification is also referred to as trained immunity, as demonstrated by studies of the BLUEPRINT consortium (www.blueprint-epigenome.eu) (60, 61). This rather short-term epigenetic memory monitors the close relationship between immune challenges and effects on chromatin. Epigenetic memory prepares innate immune cells for a possible next microbe encounter (62). In the context of these immunological processes, high affinity receptors for lipophilic signaling molecules, such as VDR and other members of the nuclear receptor superfamily, are in a prime position sensing environmental changes and other signals with a potential of creating cellular stress. Thus, VDR and its ligand are predestined for modulating the process of recording epigenetic memory in innate immunity (63) (**Figure 2C**).

#### IN VIVO RESPONSE OF IMMUNE CELLS TO VITAMIN D