Andreas Steinmeyer

Injury enhances TLR2 function and antimicrobial peptide expression through a vitamin D–dependent mechanism

An essential element of the innate immune response to injury is the capacity to recognize microbial invasion and stimulate production of antimicrobial peptides. We investigated how this process is controlled in the epidermis. Keratinocytes surrounding a wound increased expression of the genes coding for the microbial pattern recognition receptors CD14 and TLR2, complementing an increase in cathelicidin antimicrobial peptide expression. These genes were induced by 1,25(OH)2 vitamin D3 (1,25D3; its active form), suggesting a role for vitamin D3 in this process. How 1,25D3 could participate in the injury response was explained by findings that the levels of CYP27B1, which converts 25OH vitamin D3 (25D3) to active 1,25D3, were increased in wounds and induced in keratinocytes in response to TGF-beta1. Blocking the vitamin D receptor, inhibiting CYP27B1, or limiting 25D3 availability prevented TGF-beta1 from inducing cathelicidin, CD14, or TLR2 in human keratinocytes, while CYP27B1-deficient mice failed to increase CD14 expression following wounding. The functional consequence of these observations was confirmed by demonstrating that 1,25D3 enabled keratinocytes to recognize microbial components through TLR2 and respond by cathelicidin production. Thus, we demonstrate what we believe to be a previously unexpected role for vitamin D3 in innate immunity, enabling keratinocytes to recognize and respond to microbes and to protect wounds against infection.

Vitamin D3 down-regulates monocyte TLR expression and triggers hyporesponsiveness to pathogen-associated molecular patterns.

Toll‐like receptors (TLR) represent an ancient front‐line defence system that enables the host organism to sense the presence of microbial components within minutes. As inducers of inflammation, TLR act as important triggers of distinct entities such as sepsis or autoimmune disease exacerbation. We report here that vitamin D3 [1α,25‐dihydroxycholecalciferol, 1,25(OH)2D3] suppresses the expression of TLR2 and TLR4 protein and mRNA in human monocytes in a time‐ and dose‐dependent fashion. Despite 1,25(OH)2D3‐induced up‐regulation of CD14, challenge of human monocytes with either LPS or lipoteichoic acid resulted in impaired TNF‐α and procoagulatory tissue factor (CD142) production, emphasizing the critical role of TLR in the induction of inflammation. Moreover, reduced TLR levels in 1,25(OH)2D3‐treated phagocytes were accompanied by impaired NF‐κB/RelA translocation to the nucleus and by reduced p38 and p42/44 (extracellular signal‐regulated kinase 1/2) phosphorylation upon TLR‐ligand engagement. Both TLR down‐regulation and CD14 up‐regulation were substantially inhibited by the vitamin D receptor (VDR) antagonist ZK 159222, indicating that the immunomodulatory effect of 1,25(OH)2D3 on innate immunity receptors requires VDR transcription factor activation. Our data provide strong evidence that 1,25(OH)2D3 primes monocytes to respond less effectively to bacterial cell wall components in a VDR‐dependent mechanism, most likely due to decreased levels of TLR2 and TLR4.

Vitamin D is required for IFN-gamma-mediated antimicrobial activity of human macrophages.

Vitamin D is required for both innate and adaptive immunity to tuberculosis. The Sunny Side of Antimicrobial Response Nearly one-third of the world’s population is thought to be infected with Mycobacterium tuberculosis, which causes a potentially fatal lung disease in untreated patients. Although most M. tuberculosis infections can be treated by antibiotic therapy, the burden of infection is especially high in immunodeficient (HIV+) patients and individuals from developing nations. Moreover, drug-resistant M. tuberculosis is increasingly prevalent. Yet, most humans with M. tuberculosis infection are asymptomatic, perhaps because of successful immunological control. Understanding the mechanisms behind immune control of M. tuberculosis infection may pinpoint potential new therapeutic avenues. Now, Fabri et al. examine the antimicrobial function of M. tuberculosis–infected human macrophages. The authors found that cells from the adaptive immune system—T cells—governed bacterial control by releasing the cytokine interferon-γ (IFN-γ), which then activated infected macrophages, inciting the cells to attack the invading M. tuberculosis. This activation depended on the presence of vitamin D, a fat-soluble prohormone thought to be beneficial for everything from bone health to cancer therapy. Indeed, this antimicrobial response was not seen with macrophages maintained in human sera from subjects with insufficient vitamin D levels. Vitamin D3 has been used historically to treat M. tuberculosis infection, but its effects have not been thoroughly tested in clinical trials. This study suggests that increasing serum levels of vitamin D, whether through supplementation or increased sun exposure, should improve the human immune response to M. tuberculosis and supports further testing of vitamin D in the clinic. Control of tuberculosis worldwide depends on our understanding of human immune mechanisms, which combat the infection. Acquired T cell responses are critical for host defense against microbial pathogens, yet the mechanisms by which they act in humans remain unclear. We report that T cells, by the release of interferon-γ (IFN-γ), induce autophagy, phagosomal maturation, the production of antimicrobial peptides such as cathelicidin, and antimicrobial activity against Mycobacterium tuberculosis in human macrophages via a vitamin D–dependent pathway. IFN-γ induced the antimicrobial pathway in human macrophages cultured in vitamin D–sufficient sera, but not in sera from African-Americans that have lower amounts of vitamin D and who are more susceptible to tuberculosis. In vitro supplementation of vitamin D–deficient serum with 25-hydroxyvitamin D3 restored IFN-γ–induced antimicrobial peptide expression, autophagy, phagosome-lysosome fusion, and antimicrobial activity. These results suggest a mechanism in which vitamin D is required for acquired immunity to overcome the ability of intracellular pathogens to evade macrophage-mediated antimicrobial responses. The present findings underscore the importance of adequate amounts of vitamin D in all human populations for sustaining both innate and acquired immunity against infection.

Convergence of IL-1β and VDR Activation Pathways in Human TLR2/1-Induced Antimicrobial Responses

Antimicrobial effector mechanisms are central to the function of the innate immune response in host defense against microbial pathogens. In humans, activation of Toll-like receptor 2/1 (TLR2/1) on monocytes induces a vitamin D dependent antimicrobial activity against intracellular mycobacteria. Here, we report that TLR activation of monocytes triggers induction of the defensin beta 4 gene (DEFB4), requiring convergence of the IL-1β and vitamin D receptor (VDR) pathways. TLR2/1 activation triggered IL-1β activity, involving the upregulation of both IL-1β and IL-1 receptor, and downregulation of the IL-1 receptor antagonist. TLR2/1L induction of IL-1β was required for upregulation of DEFB4, but not cathelicidin, whereas VDR activation was required for expression of both antimicrobial genes. The differential requirements for induction of DEFB4 and cathelicidin were reflected by differences in their respective promoter regions; the DEFB4 promoter had one vitamin D response element (VDRE) and two NF-κB sites, whereas the cathelicidin promoter had three VDREs and no NF-κB sites. Transfection of NF-κB into primary monocytes synergized with 1,25D3 in the induction of DEFB4 expression. Knockdown of either DEFB4 or cathelicidin in primary monocytes resulted in the loss of TLR2/1-mediated antimicrobial activity against intracellular mycobacteria. Therefore, these data identify a novel mechanism of host defense requiring the induction of IL-1β in synergy with vitamin D activation, for the TLR-induced antimicrobial pathway against an intracellular pathogen.

1,25-dihydroxyvitamin D3 promotes IL-10 production in human B cells

1,25‐dihydroxyvitamin D3 (calcitriol) regulates immune responses, e.g., inhibits expression of IgE by B cells and enhances expression of IL‐10 by dendritic cells and T cells. We report here that activation of human B cells by B cell receptor, CD40 and IL‐4 signals induces expression of the gene for 25‐hydroxyvitamin‐D3–1α‐hydroxylase (CYP1α). Accordingly, these B cells generate and secrete significant amounts of calcitriol. In activated B cells calcitriol induces expression of the genes Cyp24, encoding a vitamin D hydroxylase, and Trpv6, encoding a calcium selective channel protein. Calcitriol enhances IL‐10 expression of activated B cells more than threefold, both by recruiting the vitamin D receptor to the promoter of Il‐10, and to lesser extent by modulation of calcium‐dependent signaling. The molecular link in activated B cells between vitamin D signaling, expression of IgE and IL‐10, and their ability to produce calcitriol from its precursor, suggest that pro‐vitamin D (25‐hydroxyvitamin D3) can be used as a modulator of allergic immune responses.

1,25-Dihydroxyvitamin D3 Is an autonomous regulator of the transcriptional changes leading to a tolerogenic dendritic cell phenotype

Activation of vitamin D receptor (VDR) by 1,25-dihydroxyvitamin D3 (1,25-vitD) reprograms dendritic cells (DC) to become tolerogenic. Previous studies suggested that 1,25-vitD could inhibit the changes brought about by differentiation and maturation of DCs. Underpinning the described phenotypic and functional alterations, there must be 1,25-vitD-coordinated transcriptional events. However, this transcriptional program has not been systematically investigated, particularly not in a developmental context. Hence, it has not been explored how 1,25-vitD-regulated genes, particularly the ones bringing about the tolerogenic phenotype, are connected to differentiation. We conducted global gene expression analysis followed by comprehensive quantitative PCR validation to clarify the interrelationship between 1,25-vitD and differentiation-driven gene expression patterns in developing human monocyte-derived and blood myeloid DCs. In this study we show that 1,25-vitD regulates a large set of genes that are not affected by differentiation. Interestingly, several genes, impacted both by the ligand and by differentiation, appear to be regulated by 1,25-vitD independently of the developmental context. We have also characterized the kinetics of generation of 1,25-vitD by using three early and robustly regulated genes, the chemokine CCL22, the inhibitory receptors CD300LF and CYP24A1. We found that monocyte-derived DCs are able to turn on 1,25-vitD sensitive genes in early phases of differentiation if the precursor is present. Our data collectively suggest that exogenous or endogenously generated 1,25-vitD regulates a large set of its targets autonomously and not via inhibition of differentiation and maturation, leading to the previously characterized tolerogenic state.

Vitamin D inhibition of pro-fibrotic effects of transforming growth factor β1 in lung fibroblasts and epithelial cells

The mechanisms that control fibroproliferation and matrix deposition in lung fibrosis remain unclear. We speculate that vitamin D deficiency may contribute to pulmonary fibrosis since vitamin D deficiency has been implicated in several diseases. First, we confirmed the presence of vitamin D receptors (VDRs) in cultured NIH/3T3 and lung fibroblasts. Fibroblasts transfected with a vitamin D response element-reporter construct and exposed to the active vitamin D metabolite, 1,25(OH)(2)D(3), showed increased promoter activity indicating VDR functionality in these cells. Testing the effects of 1,25(OH)(2)D(3) on fibroblasts treated with transforming growth factor beta1 (TGFbeta1), considered a driver of many fibrotic disorders, we found that 1,25(OH)(2)D(3) inhibited TGFbeta1-induced fibroblast proliferation in a dose-dependent fashion. 1,25(OH)(2)D(3) also inhibited TGFbeta1 stimulation of alpha-smooth muscle actin expression and polymerization and prevented the upregulation of fibronectin and collagen in TGFbeta1-treated fibroblasts. Finally, we examined how 1,25(OH)(2)D(3) affects epithelial-mesenchymal transformation of lung epithelial cells upon exposure to TGFbeta1. We showed that the TGFbeta1-induced upregulation of mesenchymal cell markers and abnormal expression of epithelial cell markers were blunted by 1,25(OH)(2)D(3). These observations suggest that under TGFbeta1 stimulation, 1,25(OH)(2)D(3) inhibits the pro-fibrotic phenotype of lung fibroblasts and epithelial cells.

Vitamin D Analogs Differentially Control Antimicrobial Peptide/“Alarmin”Expression in Psoriasis

Antimicrobial peptides (AMPs) are strongly expressed in lesional skin in psoriasis and play an important role as proinflammatory “alarmins” in this chronic skin disease. Vitamin D analogs like calcipotriol have antipsoriatic effects and might mediate this effect by changing AMP expression. In this study, keratinocytes in lesional psoriatic plaques showed decreased expression of the AMPs β-defensin (HBD) 2 and HBD3 after topical treatment with calcipotriol. At the same time, calcipotriol normalized the proinflammatory cytokine milieu and decreased interleukin (IL)-17A, IL-17F and IL-8 transcript abundance in lesional psoriatic skin. In contrast, cathelicidin antimicrobial peptide expression was increased by calcipotriol while psoriasin expression remained unchanged. In cultured human epidermal keratinocytes the effect of different vitamin D analogs on the expression of AMPs was further analyzed. All vitamin D analogs tested blocked IL-17A induced HBD2 expression by increasing IκB-α protein and inhibition of NF-κB signaling. At the same time vitamin D analogs induced cathelicidin through activation of the vitamin D receptor and MEK/ERK signaling. These studies suggest that vitamin D analogs differentially alter AMP expression in lesional psoriatic skin and cultured keratinocytes. Balancing AMP “alarmin” expression might be a novel goal in treatment of chronic inflammatory skin diseases.

Osteopontin: A Fibrosis-Related Marker Molecule in Cardiac Remodeling of Enterovirus Myocarditis in the Susceptible Host

The characteristics of dilated cardiomyopathy (DCM) resulting from chronic viral myocarditis are remodeling processes of the extracellular matrix. Based on our findings of enhanced osteopontin (OPN) expression in inflamed human hearts, we further investigated in the murine model of acute and chronic coxsackievirus (CV)B3-myocarditis the role of OPN regarding its involvement in resolution of cardiac virus infection and fibrosis. In hearts of A.BY/SnJ mice susceptible to chronic CVB3-myocarditis, a pronounced increase of OPN expression levels was detected by microarray analysis and quantitative RT-PCR during acute stages of myocarditis. Combined immunohistochemistry and in situ hybridization identified infiltrating macrophages as main OPN producers. In contrast to resistant C57BL/6 and OPN gene–deficient mice, transcription levels of matrix metalloproteinase-3, TIMP1 (tissue inhibitor of metalloproteinases-1), uPA (urokinase-type plasminogen activator), and transforming growth factor β1 were elevated in susceptible mice, and as a consequence, procollagen-1α mRNA expression and fibrosis was considerably enhanced. Treatment of infected susceptible mice with the vitamin D analog ZK 191784 led to decreased myocardial expression levels of OPN, metalloproteinase-3, TIMP1, uPA, and procollagen-1α and subsequently to reduced fibrosis. Concurrently, the fibrosis-relevant signaling molecules pERK (phosphorylated extracellular signal-regulated kinase) and pAkt (phosphorylated Akt), increased in A.BY/SnJ mice, were diminished in ZK 191784–treated mice. Here, we show that high expression levels of OPN in acute myocarditis are associated with consecutive development of extensive fibrosis that can be reduced by treatment with a vitamin D analog. Thus, OPN may serve as a diagnostic tool as well as a potential therapeutic target to limit cardiac remodeling in chronic myocarditis.

The New Low Calcemic Vitamin D Analog 22-Ene-25-Oxa-Vitamin D Prominently Ameliorates T Helper Cell Type 1-Mediated Colitis in Mice

In addition to its well defined role as a key regulator of calcium and bone metabolism, 1,25-dihydroxyvitamin D3 (calcitriol) has been established as a potent modulator of immune cell function. Still, because of the hypercalcemic toxicity occurring after systemic application of the parent compound, its clinical application as an immunosuppressant has been hampered. Recently, we described 22-ene-25-oxa-vitamin D (ZK156979) as a representative of a novel class of low calcemic vitamin D analogs with well preserved immunosuppressive activity in vitro. Here, in vivo colitis was induced by applying a rectal enema of 2,4,6-trinitrobenzene sulfonic acid (TNBS) to male BALB/c mice, and calcitriol (0.2 μg/kg) or ZK156979 (0.1–2.0 μg/kg) was given i.p. from days 0 to 3 or 3 to 5. Body mass and clinical activity score of colitis were recorded daily. Colon tissue was analyzed macroscopically and microscopically, myeloperoxidase activity and cytokine levels [tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-10, and IL-4] were determined by enzyme-linked immunosorbent assay, and T-box transcription factor (T-bet) expression was determined by immunoblot analysis. We found that treatment with ZK156979 clearly reduced the severity of TNBS-induced colitis without exhibiting calcemic effects. Both early and late treatment abrogated body weight loss, diarrhea, and macroscopic intestinal inflammation with a potency comparable with that of calcitriol. The therapeutic effect of ZK156979 was accompanied by a down-regulation of myeloperoxidase activity, TNF-α, IFN-γ, and T-bet expression decreased, whereas local tissue IL-10 and IL-4 protein levels increased. To conclude, our data provide the first clear evidence that ZK156979 exhibits a beneficial prophylactic as well as therapeutic profile in T helper cell type 1-like experimental colitis, offering new therapeutic options for the treatment of human inflammatory bowel diseases.