Sahana Holla

NOD2-Nitric Oxide-responsive MicroRNA-146a Activates Sonic Hedgehog Signaling to Orchestrate Inflammatory Responses in Murine Model of Inflammatory Bowel Disease

Background: Genetic variants of NOD2 are linked to inflammatory bowel disease (IBD) etiology. Results: DSS model of colitis in wild-type and inducible nitric-oxide synthase (iNOS) null mice revealed that NOD2-iNOS/NO-responsive microRNA-146a targets NUMB gene facilitating Sonic hedgehog (SHH) signaling. Conclusion: miR-146a-mediated NOD2-SHH signaling regulates gut inflammation. Significance: Identification of novel regulators of IBD provides new insights into pathophysiology and development of new therapy concepts. Inflammatory bowel disease (IBD) is a debilitating chronic inflammatory disorder of the intestine. The interactions between enteric bacteria and genetic susceptibilities are major contributors of IBD etiology. Although genetic variants with loss or gain of NOD2 functions have been linked to IBD susceptibility, the mechanisms coordinating NOD2 downstream signaling, especially in macrophages, during IBD pathogenesis are not precisely identified. Here, studies utilizing the murine dextran sodium sulfate model of colitis revealed the crucial roles for inducible nitric-oxide synthase (iNOS) in regulating pathophysiology of IBDs. Importantly, stimulation of NOD2 failed to activate Sonic hedgehog (SHH) signaling in iNOS null macrophages, implicating NO mediated cross-talk between NOD2 and SHH signaling. NOD2 signaling up-regulated the expression of a NO-responsive microRNA, miR-146a, that targeted NUMB gene and alleviated the suppression of SHH signaling. In vivo and ex vivo studies confirmed the important roles for miR-146a in amplifying inflammatory responses. Collectively, we have identified new roles for miR-146a that established novel cross-talk between NOD2-SHH signaling during gut inflammation. Potential implications of these observations in therapeutics could increase the possibility of defining and developing better regimes to treat IBD pathophysiology.

Sonic hedgehog-Dependent Induction of MicroRNA 31 and MicroRNA 150 Regulates Mycobacterium bovis BCG-Driven Toll-Like Receptor 2 Signaling

ABSTRACT Hedgehog (HH) signaling is a significant regulator of cell fate decisions during embryogenesis, development, and perpetuation of various disease conditions. Testing whether pathogen-specific HH signaling promotes unique innate recognition of intracellular bacteria, we demonstrate that among diverse Gram-positive or Gram-negative microbes, Mycobacterium bovis BCG, a vaccine strain, elicits a robust activation of Sonic HH (SHH) signaling in macrophages. Interestingly, sustained tumor necrosis factor alpha (TNF-α) secretion by macrophages was essential for robust SHH activation, as TNF-α−/− macrophages exhibited compromised ability to activate SHH signaling. Neutralization of TNF-α or blockade of TNF-α receptor signaling significantly reduced the infection-induced SHH signaling activation both in vitro and in vivo. Intriguingly, activated SHH signaling downregulated M. bovis BCG-mediated Toll-like receptor 2 (TLR2) signaling events to regulate a battery of genes associated with divergent functions of M1/M2 macrophages. Genome-wide expression profiling as well as conventional gain-of-function or loss-of-function analysis showed that SHH signaling-responsive microRNA 31 (miR-31) and miR-150 target MyD88, an adaptor protein of TLR2 signaling, thus leading to suppression of TLR2 responses. SHH signaling signatures could be detected in vivo in tuberculosis patients and M. bovis BCG-challenged mice. Collectively, these investigations identify SHH signaling to be what we believe is one of the significant regulators of host-pathogen interactions.

Selective inhibition of IFNG-induced autophagy by Mir155- and Mir31-responsive WNT5A and SHH signaling

Autophagy is one of the major immune mechanisms engaged to clear intracellular infectious agents. However, several pathogens have evolved strategies to evade autophagy. Here, we demonstrated that Mycobacteria, Shigella, and Listeria but not Klebsiella, Staphylococcus, and Escherichia inhibit IFNG-induced autophagy in macrophages by evoking selective and robust activation of WNT and SHH pathways via MTOR. Utilization of gain- or loss-of-function analyses as well as mir155-null macrophages emphasized the role of MTOR-responsive epigenetic modifications in the induction of Mir155 and Mir31. Importantly, cellular levels of PP2A, a phosphatase, were regulated by Mir155 and Mir31 to fine-tune autophagy. Diminished expression of PP2A led to inhibition of GSK3B, thus facilitating the prolonged activation of WNT and SHH signaling pathways. Sustained WNT and SHH signaling effectuated the expression of anti-inflammatory lipoxygenases, which in tandem inhibited IFNG-induced JAK-STAT signaling and contributed to evasion of autophagy. Altogether, these results established a role for new host factors and inhibitory mechanisms employed by the pathogens to limit autophagy, which could be targeted for therapeutic interventions.

The WNT Signaling Pathway Contributes to Dectin-1-Dependent Inhibition of Toll-Like Receptor-Induced Inflammatory Signature

ABSTRACT Macrophages regulate cell fate decisions during microbial challenges by carefully titrating signaling events activated by innate receptors such as dectin-1 or Toll-like receptors (TLRs). Here, we demonstrate that dectin-1 activation robustly dampens TLR-induced proinflammatory signature in macrophages. Dectin-1 induced the stabilization of β-catenin via spleen tyrosine kinase (Syk)-reactive oxygen species (ROS) signals, contributing to the expression of WNT5A. Subsequently, WNT5A-responsive protein inhibitors of activated STAT (PIAS-1) and suppressor of cytokine signaling 1 (SOCS-1) mediate the downregulation of IRAK-1, IRAK-4, and MyD88, resulting in decreased expression of interleukin 12 (IL-12), IL-1β, and tumor necrosis factor alpha (TNF-α). In vivo activation of dectin-1 with pathogenic fungi or ligand resulted in an increased bacterial burden of Mycobacteria, Klebsiella, Staphylococcus, or Escherichia, with a concomitant decrease in TLR-triggered proinflammatory cytokines. All together, our study establishes a new role for dectin-1-responsive inhibitory mechanisms employed by virulent fungi to limit the proinflammatory environment of the host.

Mycobacteria-responsive sonic hedgehog signaling mediates programmed death-ligand 1-and prostaglandin E 2 -induced regulatory T cell expansion

CD4+CD25+FoxP3+ regulatory T cells (Tregs) are exploited by mycobacteria to subvert the protective host immune responses. The Treg expansion in the periphery requires signaling by professional antigen presenting cells and in particularly dendritic cells (DC). However, precise molecular mechanisms by which mycobacteria instruct Treg expansion via DCs are not established. Here we demonstrate that mycobacteria-responsive sonic hedgehog (SHH) signaling in human DCs leads to programmed death ligand-1 (PD-L1) expression and cyclooxygenase (COX)-2-catalyzed prostaglandin E2 (PGE2) that orchestrate mycobacterial infection-induced expansion of Tregs. While SHH-responsive transcription factor GLI1 directly arbitrated COX-2 transcription, specific microRNAs, miR-324-5p and miR-338-5p, which target PD-L1 were downregulated by SHH signaling. Further, counter-regulatory roles of SHH and NOTCH1 signaling during mycobacterial-infection of human DCs was also evident. Together, our results establish that Mycobacterium directs a fine-balance of host signaling pathways and molecular regulators in human DCs to expand Tregs that favour immune evasion of the pathogen.

Nitric oxide and KLF4 protein epigenetically modify class II transactivator to repress Major histocompatibility complex II expression during Mycobacterium bovis Bacillus Calmette-Guerin infection

Background: Mycobacteria down-regulates class II transactivator (CIITA)/MHC-II expression and antigen presentation. Results: During Mycobacterium bovis BCG infection, iNOS/NO responsive KLF4 induces EZH2 and miR-150 functions to regulate CIITA expression and thus antigen presentation. Conclusion: CIITA/MHC-II down-regulation by mycobacteria involves NOTCH/iNOS/NO/KLF4 signaling cross-talk and functions. Significance: Identification of novel regulators of host-mycobacteria interactions provides promising therapeutic potential. Pathogenic mycobacteria employ several immune evasion strategies such as inhibition of class II transactivator (CIITA) and MHC-II expression, to survive and persist in host macrophages. However, precise roles for specific signaling components executing down-regulation of CIITA/MHC-II have not been adequately addressed. Here, we demonstrate that Mycobacterium bovis bacillus Calmette-Guérin (BCG)-mediated TLR2 signaling-induced iNOS/NO expression is obligatory for the suppression of IFN-γ-induced CIITA/MHC-II functions. Significantly, NOTCH/PKC/MAPK-triggered signaling cross-talk was found critical for iNOS/NO production. NO responsive recruitment of a bifunctional transcription factor, KLF4, to the promoter of CIITA during M. bovis BCG infection of macrophages was essential to orchestrate the epigenetic modifications mediated by histone methyltransferase EZH2 or miR-150 and thus calibrate CIITA/MHC-II expression. NO-dependent KLF4 regulated the processing and presentation of ovalbumin by infected macrophages to reactive T cells. Altogether, our study delineates a novel role for iNOS/NO/KLF4 in dictating the mycobacterial capacity to inhibit CIITA/MHC-II-mediated antigen presentation by infected macrophages and thereby elude immune surveillance.

Mycobacterium bovis BCG promotes tumor cell survival from tumor necrosis factor-α-induced apoptosis

BackgroundIncreased incidence of lung cancer among pulmonary tuberculosis patients suggests mycobacteria-induced tumorigenic response in the host. The alveolar epithelial cells, candidate cells that form lung adenocarcinoma, constitute a niche for mycobacterial replication and infection. We thus explored the possible mechanism of M. bovis Bacillus Calmette-Guérin (BCG)-assisted tumorigenicity in type II epithelial cells, human lung adenocarcinoma A549 and other cancer cells.MethodsCancer cell lines originating from lung, colon, bladder, liver, breast, skin and cervix were treated with tumor necrosis factor (TNF)-α in presence or absence of BCG infection. p53, COP1 and sonic hedgehog (SHH) signaling markers were determined by immunoblotting and luciferase assays, and quantitative real time PCR was done for p53-responsive pro-apoptotic genes and SHH signaling markers. MTT assays and Annexin V staining were utilized to study apoptosis. Gain- and loss-of-function approaches were used to investigate the role for SHH and COP1 signaling during apoptosis. A549 xenografted mice were used to validate the contribution of BCG during TNF-α treatment.ResultsHere, we show that BCG inhibits TNF-α-mediated apoptosis in A549 cells via downregulation of p53 expression. Substantiating this observation, BCG rescued A549 xenografts from TNF-α-mediated tumor clearance in nude mice. Furthermore, activation of SHH signaling by BCG induced the expression of an E3 ubiquitin ligase, COP1. SHH-driven COP1 targeted p53, thereby facilitating downregulation of p53-responsive pro-apoptotic genes and inhibition of apoptosis. Similar effects of BCG could be shown for HCT116, T24, MNT-1, HepG2 and HELA cells but not for HCT116 p53-/- and MDA-MB-231 cells.ConclusionOur results not only highlight possible explanations for the coexistence of pulmonary tuberculosis and lung cancer but also address probable reasons for failure of BCG immunotherapy of cancers.

MUSASHI-Mediated Expression of JMJD3, a H3K27me3 Demethylase, Is Involved in Foamy Macrophage Generation during Mycobacterial Infection

Foamy macrophages (FM)s harbor lipid bodies that not only assist mycobacterial persistence within the granulomas but also are sites for intracellular signaling and inflammatory mediators which are essential for mycobacterial pathogenesis. However, molecular mechanisms that regulate intracellular lipid accumulation in FMs during mycobacterial infection are not clear. Here, we report for the first time that jumonji domain containing protein (JMJD)3, a demethylase of the repressive H3K27me3 mark, orchestrates the expression of M. tuberculosis H37Rv-, MDR-JAL2287-, H37Ra- and M. bovis BCG-induced genes essential for FM generation in a TLR2-dependent manner. Further, NOTCH1-responsive RNA-binding protein MUSASHI (MSI), targets a transcriptional repressor of JMJD3, Msx2-interacting nuclear target protein, to positively regulate infection-induced JMJD3 expression, FM generation and M2 phenotype. Investigations in in vivo murine models further substantiated these observations. Together, our study has attributed novel roles for JMJD3 and its regulators during mycobacterial infection that assist FM generation and fine-tune associated host immunity.

Inhibition of Programmed Death 1 Ligand 1 on Dendritic Cells Enhances Mycobacterium-Mediated Interferon γ (IFN-γ) Production Without Modulating the Frequencies of IFN-γ–Producing CD4+ T Cells

TO THE EDITOR—Mycobacterium tuberculosis, the causative agent of tuberculosis, uses several strategies to evade the immune system, which include inhibition of phagosomal maturation and antigen presentation, blockade of apoptosis and autophagy of infected cells, suppression of T-helper type 1 (Th1) and interferon γ (IFN-γ) responses, and expansion of CD4CD25FoxP3 regulatory T cells (Tregs) [1–3]. Recently Singh et al reported that M. tuberculosis exploits the programmed death 1 (PD-1) pathway to inhibit IFN-γ responses [4]. Conversely, blockade of the PD-1 pathway either by blocking PD-1 on CD3 T cells or blocking PD-1 ligand 1 (PD-L1) on monocytes in vitro rescued IFN-γ–producing T cells from undergoing apoptosis. However, 2 issues remain unanswered: (1) the specific role of PD-L1 on CD4 T cells and (2) the contribution of PD-L1 on dendritic cells (DCs), the professional antigen-presenting cells, in polarizing Mycobacterium-mediated IFN-γ responses from naive CD4 T cells. Human CD4 T cells, when activated, were reported to express PD-L1 [5]. Therefore, it is likely that interaction of PD-L1–expressing CD4 T cells with PD-1–positive T cells might modulate IFN-γ responses. We found thatMycobacterium induced only a marginal increase in PD-L1 expression on CD4 T cells (Figure 1A). Our results thus indicate that the relatively high expression of PD-L1 on CD3 T cells (up to 25%) observed by Singh et al [4] upon stimulation with mycobacterial antigens might reflect modulation of PD-L1 expression on CD8 T cells, rather than CD4 T cells. PD-L2 expression, however, remained negative on these activated CD4 T cells (data not shown). In accordance with data on low-level expression of PD-L1 on CD4 T cells, blockade of this molecule by using monoclonal antibodies (mAbs) did not significantly modulate either the frequency of IFN-γCD4 T cells (Figure 1B and 1C) or the quantities of IFN-γ secreted from these cells (Figure 1D). Thus, our results suggest that PD-L1 on CD4 T cells plays only a marginal role in mediating impaired IFN-γ responses byMycobacterium. Dendritic cells (DCs) are sentinels of the immune system that orchestrate primary immune responses to Mycobacterium by polarizing distinct CD4 T-cell responses from naive T cells [1]. Therefore, we next examined the role of PD-L1 on DCs in regulating IFN-γ polarizing responses from naive CD4 T cells. DCs were generated from circulating monocytes as previously described [6]. Similar to the results obtained with monocytes [4], stimulation of DCs with gamma-irradiated M. tuberculosis H37Rv or bacillus Calmette–Guérin induced significant upregulation of PD-L1 (Figure 1E and 1F). Live Mycobacterium bacilli were more efficient in inducing PD-L1 than killed bacilli, implying that, in addition to cell-wall pathogen-associated molecular patterns, secretory antigens and signals associated with replication of bacteria provide stimuli for the induction of PD-L1. However, we could not detect PD-L2 on DCs (data not shown). Analysis of polarization of T-cell responses from naive CD4 T cells revealed that so-called Mycobacterium-educated DCs significantly enhanced the frequency of IFN-γ Th1 cells (Figure 1G and 1H). However, it was not associated with the increased quantities of IFN-γ secretion from these CD4 T cells (Figure 1I), possibly because of negative signaling by PDL1 on DCs. Therefore, we attempted to confirm this proposition by blocking PD-L1 on DCs. We confirm that blocking mAbs to PD-L1 were functional, as these antibodies quenched even the basal expression of PD-L1 (Figure 1E ). Further, in contrast to the results obtained with monocytes [4], blocking PD-L1 on DCs did not significantly alter the frequency of IFNγCD4 T cells (Figure 1G and 1H). However, PD-L1 blockade led to significant increase in the quantity of IFN-γ produced by CD4 T cells (Figure 1I). It should be noted that to analyze the expression of surface molecules and intracellular T-cell cytokines, Singh et al stimulated peripheral blood mononuclear cells with M. tuberculosis antigens for 48 hours in the presence of brefeldin A, a Golgi transport blocker [4]. For blocking experiments involving PD ligands or receptors, monocyte–T-cell cultures were treated with brefeldin A for 72 hours [4]. However, because brefeldin A is highly toxic to cells if they are treated for longer periods, short-period treatment (duration, typically 4–6 hours) is recommended. Hence, we suggest that the results reported by Singh et al on Mycobacterium-mediated IFN-γ responses need to be judged with caution because of the possible toxic effects of brefeldin A. Together, these results provide insight on how PD-L1 on innate cells regulates IFN-γ responses to Mycobacterium. However, the functional repercussion of

WNT-Inflammasome Signaling Mediates NOD2-Induced Development of Acute Arthritis in Mice

In addition to its role in innate immunity, the intracellular pathogen sensor nucleotide-binding oligomerization domain 2 (NOD2) has been implicated in various inflammatory disorders, including the development of acute arthritis. However, the molecular mechanisms involved in the development of NOD2-responsive acute arthritis are not clear. In this study, we demonstrate that NOD2 signals to a cellular protein, Ly6/PLAUR domain–containing protein 6, in a receptor-interacting protein kinase 2–TGF-β–activated kinase 1–independent manner to activate the WNT signaling cascade. Gain- or loss-of-function of the WNT signaling pathway in an in vivo experimental mouse arthritis model or in vitro systems established the role for WNT-responsive X-linked inhibitor of apoptosis during the development of acute arthritis. Importantly, WNT-stimulated X-linked inhibitor of apoptosis mediates the activation of inflammasomes. The subsequent caspase-1 activation and IL-1β secretion together contribute to the phenotypic character of the inflammatory condition of acute arthritis. Thus, identification of a role for WNT-mediated inflammasome activation during NOD2 stimulation serves as a paradigm to understand NOD2-associated inflammatory disorders and develop novel therapeutics.