The mechanisms by which TLR2/1 activation of monocytes triggers a vitamin D-dependent induction of antimicrobial activity are central to host defense against intracellular M. tuberculosis 
. Here we demonstrate that convergence of IL-1β and vitamin D transcriptional activation induced expression of the antimicrobial peptide DEFB4. Importantly, TLR2/1L 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. A synergy between NF-κB and VDR activation was sufficient to upregulate expression of DEFB4. Finally, knockdown of either DEFB4 or cathelicidin resulted in the loss of TLR2/1-induced antimicrobial activity against intracellular mycobacteria. Therefore, these data identify a novel mechanism of host defense requiring the induction of two distinct signaling pathways, involving IL-1βin synergy with vitamin D activation, for TLR-induced antimicrobial activity against an intracellular pathogen.
Upon triggering TLR2/1 with lipopeptide, both human and murine macrophages activate antimicrobial pathways against intracellular mycobacteria 
. In comparing the human and mouse TLR-induced antimicrobial pathways, it is noteworthy that the human antimicrobial response requires induction of multiple antimicrobial peptides, both cathelicidin and DEFB4, as opposed to the murine antimicrobial response which is dependent upon generation of nitric oxide 
. It remains to be determined if the cathelicidin- and DEFB4-mediated antimicrobial activity is bactericidal or bacteriostatic. In humans, both the cathelicidin and DEFB4 promoters contain VDREs 
, VDR activation was required for their induction, as well as TLR-induced antimicrobial activity 
. In contrast, there are no VDRE sites present in the mouse cathelicidin promoter region 
, and to our knowledge there are no other known vitamin D-dependent antimicrobial peptides in the murine genome. Interestingly, 1,25D induces nitric oxide generation in murine macrophages 
, although the role for vitamin D in this murine TLR-induced antimicrobial response remains to be determined. Although our data indicate that the human innate immune system has evolved a powerful antimicrobial mechanism against intracellular mycobacterial infection through the vitamin D dependent production of antimicrobial peptides, it does not preclude a role for nitric oxide 
and other mechanisms such as superoxide generation and autophagy 
Although VDR activation was required for induction of both DEFB4 and cathelicidin, neutralization of IL-1β prior to TLR-activation resulted in the loss of DEFB4 but not cathelicidin. Analysis of the cathelicidin and DEFB4 promoter regions revealed two NF-κB response elements present in the DEFB4 promoter region, and none in the cathelicidin promoter. In contrast, both genes contained vitamin D response elements, one in DEFB4 promoter and three in cathelicidin. These data suggested a potential mechanism by which IL-1β could uniquely influence the TLR-induced expression of DEFB4 and not cathelicidin while both genes shared a requirement for vitamin D. Transfection of NF-κB p65, the predominant IL-1β induced NF-κB subunit 
, into monocytes resulted in a significant expression of DEFB4 only with VDR activation. In contrast, NF-κB p65 transfection decreased 1,25D3 induced cathelicidin, further highlighting the differential regulation of these two antimicrobial genes. However, the mechanism by which NF-κB p65 transfection inhibits cathelicidin is currently unclear and warrants further exploration. Taken together these data demonstrate a novel role for IL-1β in the antimicrobial activity against intracellular pathogens, through the induction of DEFB4 in synergy with vitamin D. Furthermore, this suggests that there are two distinct vitamin D-dependent TLR-induced antimicrobial mechanisms, with DEFB4 expression also dependent on induction of IL-1β.
In addition to induction of IL-1β expression and VDR activation, triggering of TLR2/1 was found to modulate IL-1β activity by increasing the cell's responsiveness to the secreted IL-1β. The monocyte response to IL-1β was upregulated by TLR2/1 activation through the simultaneous secretion of IL-1β, upregulation of cell surface IL-1R1 and downregulation of baseline IL-1RA. Addition of IL-1β to TLR2/1L stimulated monocytes resulted in synergistic upregulation of DEFB4, which reflects the increased IL-1β responsiveness. On the other hand, IL-1β plus 1,25D3 did not induce expression of DEFB4, presumably because the IL-1R1 was not upregulated and/or the IL-1RA was not downregulated. These findings provide a potential molecular mechanism for the previously known association of a high IL-1β/low IL-1RA producing haplotype with pleural tuberculosis, a form of the disease that generally resolves without chemotherapy 
. In addition, there is also an elevated level of 1,25D in the pleural effusion fluid compared to serum of the same individual, resulting in an optimal local microenvironment for the induction of antimicrobial responses, including cathelicidin and DEFB4 
. Furthermore, IL-1R1 has been demonstrated to be required for host defense against M. tuberculosis
in a murine model 
. Given that excessive activation of IL-1β results in tissue injury and sepsis 
, IL-1β activity is subject to negative feedback by the IL-1RA 
. While both IL-1β and IL-1RA are produced during inflammatory conditions, IL-1RA is also induced by anti-inflammatory signals 
. As such, the balance between IL-1β and IL-1RA during an innate immune response plays a major role the pathogenesis of inflammatory diseases such as diabetes, arthritis, inflammatory bowel disease 
, and as presented here, TLR-mediated host defense mechanisms.
Although TLR activation induces DEFB4 in both monocytes and epithelial cells 
, IL-1β alone was not sufficient to induce expression of DEFB4 in monocytes, yet it is a potent inducer of DEFB4 in epithelial cells 
. The differential regulation of antimicrobial peptide expression in monocytes vs. epithelial cells reflects the location of these cell types and the immunomodulatory properties of antimicrobial peptides. Epithelial cells form the barrier at which pathogens are initially encountered, and they efficiently utilize the antimicrobial peptide family of genes, for host defense at body surfaces interacting with the outside environment 
. However, in addition to their antimicrobial activity, β-defensins and cathelicidin trigger inflammation, by their chemotactic activity for innate and adaptive immune cells such as monocytes, neutrophils, mast cells, dendritic cells and T cells 
. At body surfaces the antimicrobial peptides are rapidly shed, such that this inflammatory activity is transient, but when released in tissues, the resulting inflammatory cell infiltrate contributes to immune-mediated injury, a complication of host defense in chronic infectious diseases including mycobacterial infections such as leprosy, tuberculosis and Buruli ulcer. Therefore, the more stringent regulation for monocytic expression of cathelicidin and DEFB4 could potentially represent a mechanism to prevent tissue injury in confined spaces.
IL-1β is clearly pivotal to host defense against microbial pathogens, required for cell recruitment and induction of inflammation, but also augmenting acquired lymphocyte responses 
. In murine models, a key role for IL-1 has been shown in immunity to a variety of pathogens, including Listeria monocytogenes
, Staphylococcus aureus
, and Helicobacter pylori 
. Here, we have identified a novel mechanism by which IL-1β contributes to human host defense, requiring convergence of the TLR-induced IL-1β and VDR pathways, to trigger expression DEFB4. Furthermore, the data establish that the antimicrobial peptides, DEFB4 and cathelicidin, are required effector molecules in the TLR-induced antimicrobial response against intracellular mycobacteria in macrophages. Elucidation of the immune defense mechanisms utilized by human macrophages to combat pathogens provides possible targets for the development of new therapeutic strategies, potentially useful given the emergence of multidrug resistant strains of M. tuberculosis
and other lethal microbes.