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.

We investigated the mechanisms by which T-cell cytokines are able to influence the Toll-like receptor (TLR)-induced, vitamin Ddependent antimicrobial pathway in human monocytes. T-cell cytokines differentially influenced TLR2/1-induced expression of the antimicrobial peptides cathelicidin and DEFB4, being upregulated by IFN-γ, down-regulated by IL-4, and unaffected by IL-17. The Th1 cytokine IFN-γ up-regulated TLR2/1 induction of 25-hydroxyvitamin D-1α-hydroxylase (i.e., CYP27B1), leading to enhanced bioconversion of 25-hydroxyvitamin D 3 (25D 3 ) to its active metabolite 1,25D 3 . In contrast, the Th2 cytokine IL-4, by itself and in combination with the TLR2/1 ligand, induced catabolism of 25D 3 to the inactive metabolite 24,25D 3 , and was dependent on expression of vitamin D-24-hydroxylase (i.e., CYP24A1). Therefore, the ability of T-cell cytokines to differentially control monocyte vitamin D metabolism represents a mechanism by which cell-mediated immune responses can regulate innate immune mechanisms to defend against microbial pathogens. Mycobacterium tuberculosisT he ability of Toll-like receptors (TLRs) to trigger a direct antimicrobial activity is a key aspect of their role in innate immunity. In mouse monocytes, activation of the TLR2/1 heterodimer by microbial lipoproteins (1-3), induces an antimicrobial activity against Mycobacterium tuberculosis that is nitric oxide (NO)-dependent, but in human monocytes is NO-independent (4). Instead, a key antimicrobial mechanism for TLR-activated human monocytes involves induction of the 25-hydroxyvitamin D-1α-hydroxylase (i.e., CYP27B1), which enzymatically converts the major circulating form of vitamin D, 25-hydroxyvitamin D3 (25D 3 ) into the active form of vitamin D, 1,25D 3 . Parallel TLRmediated up-regulation of the vitamin D receptor (VDR) and activation of this receptor by 1,25D 3 leads to downstream induction of the genes encoding the antimicrobial peptides cathelicidin and DEFB4 (5-10). Here, we tested the hypothesis that adaptive T-cell cytokines, including key cytokines of the Th1, Th2, and Th17 pattern, regulate the TLR2/1-induced, vitamin Ddependent antimicrobial pathway. ResultsEffect of T-Cell Cytokines on TLR2/1 Induction of Cathelicidin and DEFB4. To determine the role of individual cytokines on the TLRtriggered vitamin D-dependent induction of antimicrobial peptides, monocytes were treated with TLR2/1L with or without a specific T-cell cytokine, and cathelicidin and DEFB4 mRNAs measured at 24 h. IFN-γ by itself up-regulated cathelicidin and DEFB4 mRNA levels by twofold ( Fig. 1A; P < 0.05 and P < 0.001). Consistent with previous findings, TLR2/1L induced both cathelicidin and DEFB4 mRNAs (8, 10). However, whereas IFN-γ augmented TLR2/1L-triggered induction of cathelicidin by 4.1-fold (P < 0.01), it had no effect on TLR2/1L-mediated induction of DEFB4 (Fig. 1A). The addition of IL-17 had no effect on induction of antimicrobial peptide gene expression in the presence or absence of TLR2/1L (Fig. 1B).Whereas IFN-γ augmented TLR2/1 induction of cath...

Leprosy provides a model to investigate mechanisms of immune regulation in humans, given that the disease forms a clinical-immunological spectrum. Here, we identified 13 miRNAs that were differentially expressed in the lesions of subjects with progressive lepromatous (L-lep) vs. the self-limited tuberculoid (T-lep) disease. Bioinformatic analysis revealed a significant enrichment of L-lep-specific miRNAs that preferentially target key immune genes downregulated in L-lep vs. T-lep lesions. The most differentially expressed miRNA in L-lep lesions, hsa-mir-21, was upregulated in M. leprae-infected monocytes. Hsa-mir-21, by downregulating toll-like receptor 2/1 (TLR2/1)-induced CYP27B1 and IL1B as well as upregulating IL-10, inhibited gene expression of the vitamin D-dependent antimicrobial peptides, CAMP and DEFB4A. Conversely, knockdown of hsa-mir-21 in M. leprae-infected monocytes enhanced expression of CAMP and DEFB4A and restored TLR2/1-mediated antimicrobial activity against M. leprae. Therefore, the ability of M. leprae to upregulate hsa-mir-21 targets multiple genes associated with the immunologically localized disease form, providing an effective mechanism to escape from the vitamin D-dependent antimicrobial pathway.

Human heart failure due to myocardial infarction is a major health concern. The paucity of organs for transplantation limits curative approaches for the diseased and failing adult heart. Human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) have the potential to provide a long-term, viable, regenerative-medicine alternative. Significant progress has been made with regard to efficient cardiac myocyte generation from hiPSCs. However, directing hiPSC-CMs to acquire the physiological structure, gene expression profile and function akin to mature cardiac tissue remains a major obstacle. Thus, hiPSC-CMs have several hurdles to overcome before they find their way into translational medicine. In this review, we address the progress that has been made, the void in knowledge and the challenges that remain.

A role for vitamin A in host defense against Mycobacterium tuberculosis has been suggested through epidemiological and in vitro studies; however, the mechanism is unclear. Here, we demonstrate that vitamin A-triggered antimicrobial activity against M. tuberculosis requires expression of Niemann-Pick disease type C2 (NPC2). Comparison of monocytes stimulated with all-trans retinoic acid (ATRA) or 1,25-dihydroxyvitamin D3 (1,25D3), the biologically active forms of vitamin A and vitamin D, respectively, indicates that ATRA and 1,25D3 induce mechanistically distinct antimicrobial activities. Stimulation of primary human monocytes with ATRA did not result in expression of the antimicrobial peptide cathelicidin, which is required for 1,25D3 antimicrobial activity. In contrast, ATRA triggers a reduction in the total cellular cholesterol concentration, whereas 1,25D3 did not. Blocking ATRA-induced cellular cholesterol reduction inhibits antimicrobial activity as well. Bioinformatic analysis of ATRA and 1,25D3 induced gene profiles suggests Niemann-Pick disease type C2 (NPC2) is a key gene in ATRA-induced cholesterol regulation. Knockdown experiments demonstrate that ATRA-mediated decrease of total cellular cholesterol content and increase in lysosomal acidification are both dependent upon expression of NPC2. Expression of NPC2 was lower in caseous tuberculosis granulomas and M. tuberculosis-infected monocytes compared to normal lung and uninfected cells, respectively. Loss of NPC2 expression ablated ATRA-induced antimicrobial activity. Taken together, these results suggest that the vitamin A-mediated antimicrobial mechanism against M. tuberculosis requires NPC2-dependent expression and function, indicating a key role for cellular cholesterol regulation in the innate immune response.