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

Philip T Liu,Xiaoran Zhang,Genhong Cheng,Mirjam Schenk,Aria Vazirnia,Bruce W Hollis,Paul W Dempsey,Ulrich Zügel,Valencia P Walker,Melissa Kanchanapoomi,Andreas Steinmeyer,Matthew Wheelwright,Robert L Modlin

doi:10.1371/journal.pone.0005810

Abstract

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.

Highlights

The innate immune system rapidly responds to infectious pathogens, through recognition of microbial ligands and subsequent triggering of an antimicrobial response
Optimal induction of cathelicidin by TLR2/1L was performed at 24 hours based on previous time course experiments. 1,25D3 was purchased (BioMol, Plymouth Meeting, PA, USA) and resuspended in sterile filtered ethanol at 1022 M in amber tubes and stored at 280uC in small aliquots. 1,25D3 was added to culture at a concentration of 1027 M to 1029 M, or 1028 M in the absence of a titration, the concentration previously determined to be optimal for provocation of primary human monocytes
The induction of cathelicidin by 1,25D3 in human monocytes, and as previously shown the downstream target CYP24A1 suggested that the vitamin D receptor (VDR) is functional [4], yet its activation is not sufficient to induce defensin beta 4 gene (DEFB4)

Summary

Introduction

The innate immune system rapidly responds to infectious pathogens, through recognition of microbial ligands and subsequent triggering of an antimicrobial response. For the intracellular pathogen Mycobacterium tuberculosis, a key antimicrobial mechanism involves recognition of bacterial lipoproteins by Toll-like receptors (TLRs), induction of the 25-hydroxyvitamin D3-1a-hydroxylase (CYP27B1), which converts the vitamin D prohormone (25D) into the active 1,25D form, upregulation and activation of the vitamin D receptor (VDR) [1,2,3,4,5]. It has long been known that activation of the VDR alone in human monocytes induces an antimicrobial activity against M. tuberculosis, first demonstrated in the laboratories of Crowle and Rook by addition of the VDR agonist [7,8]. We reasoned that the VDR-induced antimicrobial pathway involves a set of regulated genes that contribute to host defense against M. tuberculosis infection. We investigated the mechanism by which activation of the innate system could induce DEFB4 expression in human monocytes

Methods

Results

Conclusion