Gp91phox/NADPH oxidase (NOX) 2 is the main catalytic component of NOX, which mediates the phagocytic killing of ingested pathogens via the production of reactive oxygen species (ROS). However, Mycobacterium tuberculosis (Mtb) is relatively resistant to the microbicidal effects of ROS. Thus, the exact roles of NOX2 in the innate immune control against Mtb infection are not fully resolved. In this study, we show that NOX2 is essential for TLR2-dependent inflammatory responses and 1,25-dihydroxyvitamin D3 (1,25D3)-mediated antimicrobial activity against Mtb via cathelicidin expression. NOX2-null macrophages prominently abrogated Mtb-induced ROS production and inflammatory signaling activation in a TLR2-dependent manner. Mtb triggered a physical association between NOX2 and TLR2. In addition, the knockdown of NOX2 inhibited 1,25D3-triggered antimicrobial activity against viable Mtb through the modulation of cathelicidin expression in human macrophages. Treatment of NOX2 knocked down cells with cathelicidin restored the 1,25D3-induced antimicrobial effect, suggesting that the NOX2-dependent induction of cathelicidin in macrophages is part of a defensive strategy against Mtb. Furthermore, cathelicidin expression was required for the Mtb-induced release of ROS and the production of proinflammatory cytokines/chemokines, indicating a positive circuit of inflammation in response to Mtb. Our data collectively demonstrate a novel regulatory mechanism for TLR2-dependent innate responses to Mtb involving crosstalk between NOX2 and TLR2 and the expression of cathelicidin.

SummaryMycobacterium abscessus (Mab) is an emerging and rapidly growing non-tuberculous mycobacterium (NTM). Compared with M. tuberculosis, which is responsible for tuberculosis, much less is known about NTM-induced innate immune mechanisms. Here we investigated the involvement of patternrecognition receptors and associated signalling in Mab-mediated innate immune responses. Mab activated the extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinases (MAPKs), and induced the secretion of tumour necrosis factor-a, interleukin (IL)-6 and IL-12p40 in murine macrophages via Toll-like receptor (TLR) 2. Notably, the activation of ERK1/2, but not p38, was crucial for Mab-induced pro-inflammatory cytokine production. The ITAM-like motif of dectin-1 critically contributed to Mab internalization and cytokine secretion by macrophages. In addition, dectin-1, in cooperation with TLR2, was required for the efficient phagocytosis of Mab, ERK1/2 activation and pro-inflammatory cytokine secretion. Co-immunoprecipitation and confocal analysis showed the physical interaction and colocalization of dectin-1 with TLR2 following Mab stimulation. Moreover, dectin-1-induced Syk activation was essential for the production of inflammatory cytokines and the release of reactive oxygen species by Mab-infected macrophages. Collectively, these data demonstrate that Mab actively internalizes into and robustly activates innate immune responses in macrophages through a physical and functional interaction between TLR2 and dectin-1.

Mycobacterium bovis bacillus Calmette-Gué rin (BCG) induces innate immune responses through Toll-like receptor (TLR) 2 and TLR4. We investigated the role of apoptosis-regulating signal kinase (ASK) 1 in reactive oxygen species (ROS)-mediated innate immune responses induced by BCG mycobacterial infection. In macrophages, M. bovis BCG stimulation resulted in rapid activation of mitogen-activated protein kinases (MAPKs), secretion of inflammatory cytokines, such as tumor necrosis factor (TNF)-a and interleukin (IL)-6, and ROS generation in a TLR2-and TLR4-dependent manner. M. bovis BCG-induced ROS production led to robust activation of ASK1 upstream of the c-jun-N-terminal kinase and p38 MAPK, but not extracellularregulated kinase 1/2. Blocking ASK1 activity markedly attenuated M. bovis BCG-induced TNF-a and IL-6 production by macrophages. Both TLR2 and TLR4 were required for optimal activation of ASK1 in response to M. bovis BCG. Furthermore, we present evidence that TNF receptor-associated factor (TRAF) 6 activities were essential for ROS-mediated ASK1 activation by M. bovis BCG. Finally, ASK1 activities were required for effective control of intracellular mycobacterial survival. Thus, the results of this study suggest a novel role of the TLR-ROS-TRAF6-ASK1 axis in the innate immune response to mycobacteria as a signaling intermediate. Keywords: Mycobacterium bovis bacillus Calmette-Guérin; apoptosis-regulating signal kinase 1; Toll-like receptor; reactive oxygen species; macrophages; innate immunityThe Mycobacterium bovis bacillus Calmette-Guérin (BCG) strain is a tuberculosis (TB) vaccine strain that is almost nonpathogenic, but retains the immunogenic properties of TB. 1 M. bovis BCG serves as an immune potentiator of lymphocytes through the maturation of dendritic cells. Infection of dendritic cells with M. bovis BCG or M. TB facilitates the secretion of inflammatory cytokines, including tumor necrosis factor (TNF)-a, interleukin (IL)-1b, and IL-12, and upregulation of a variety of costimulatory molecules. 2,3 Previously, it was demonstrated that higher susceptibility to TB was correlated with impaired proinflammatory responses to M. bovis BCG components in mice with respiratory infections. 4 Stimulation of Toll-like receptors (TLRs) induces the production of antimicrobial peptides and proinflammatory cytokines through nuclear factor-kB (NF-kB) and other signaling pathways, thereby playing a central role in the link between innate and adaptive immunity. 5,6 Mitogen-activated protein kinase (MAPK) pathways are crucial to macrophage signaling induced by mycobacteria through TLRs. 7,8 In MAPK signaling cascades, MAPK kinase kinase (MAPKKK) activates MAPK kinase (MAPKK), which subsequently activates MAPK. 9 Apoptosis signal-regulating kinase1 (ASK1) is a member of the MAPKKK family, which activates two different MAPK cascades, the SEK1/MKK7-JNK and MKK3/MKK6-p38 MAPK pathways. 10,11 Overexpression of wild-type (WT) or the constitutively active form of ASK1 induces apoptosis in various cell types. 10,11 In contras...

CCL5/regulated on activation, normal T expressed and secreted production (RANTES) is a principal CC chemokine, and can activate macrophages and Th1 lymphocytes, however, little is known about the CCL5 profiles associated with active tuberculosis (TB). In this study, we investigated the production of CCL5 by the peripheral blood mononuclear cells (PBMCs) of patients with active pulmonary TB after stimulation with Triton X-100 soluble proteins (TSP) or the 30-kDa antigen. The profiles of cytokines/chemokines [CXCL8/interleukin (IL)-8, IL-12 p40, and interferon (IFN)-γ] were also examined by PBMCs from TB patients, and compared with those obtained from healthy tuberculin reactors (HTR). Concordant with earlier studies, IFN-γ production was significantly depressed in the PBMCs from TB patients compared with those from HTR. In addition, the CCL5, but not CXCL8, levels in the PBMCs from TB patients were significantly depressed after stimulation for 18 hr compared to those in the PBMCs from HTRs. The CCL5 release was not significantly correlated with the release of IFN-γ in the cells from TB patients and HTRs. Further, inhibitor studies show that the 30-kDa-or TSP-induced CCL5 mRNA expression is sensitive to inhibitors of mitogen-activated protein kinase kinase (MEK) 1/2 and Janus kinase (JAK) 2, but not p38, pathway activation, suggesting a MEK1/2-or JAK2-based mechanism is responsible for modulating of the CCL5 expression in human PBMCs. Collectively, these data suggest that TB patients show depressed production of CCL5 secretion, which can be modulated by MEK-and JAK2-based transcriptional regulatory mechanisms, in response to the mycobacterial antigens.