Ved Prakash Dwivedi

Mycobacterium tuberculosis Controls MicroRNA-99b (miR-99b) Expression in Infected Murine Dendritic Cells to Modulate Host Immunity

Background: Modulation of host miRNAs coincides with increased pathogenicity in various infectious diseases. Results: miR-99b is up-regulated in M. tuberculosis-infected dendritic cells, which inhibits production of proinflammatory cytokines. Conclusion: Our findings unfold a novel immune evasion strategy of M. tuberculosis by modulating miRNAs. Significance: Our study opens up the possibility to design vaccines and immunotherapies for tuberculosis by targeting specific miRNAs. Mycobacterium tuberculosis resides and replicates within host phagocytes by modulating host microbicidal responses. In addition, it suppresses the production of host protective cytokines to prevent activation of and antigen presentation by M. tuberculosis-infected cells, causing dysregulation of host protective adaptive immune responses. Many cytokines are regulated by microRNAs (miRNAs), a newly discovered class of small noncoding RNAs, which have been implicated in modulating host immune responses in many bacterial and viral diseases. Here, we show that miRNA-99b (miR-99b), an orphan miRNA, plays a key role in the pathogenesis of M. tuberculosis infection. We found that miR-99b expression was highly up-regulated in M. tuberculosis strain H37Rv-infected dendritic cells (DCs) and macrophages. Blockade of miR-99b expression by antagomirs resulted in significantly reduced bacterial growth in DCs. Interestingly, knockdown of miR-99b in DCs significantly up-regulated proinflammatory cytokines such as IL-6, IL-12, and IL-1β. Furthermore, mRNA and membrane-bound protein data indicated that inhibition of miR-99b augments TNF-α and TNFRSF-4 production. Thus, miR-99b targets TNF-α and TNFRSF-4 receptor genes. Treatment of anti-miR-99b-transfected DCs with anti-TNF-α antibody resulted in increased bacterial burden. Thus, our findings unveil a novel host evasion mechanism adopted by M. tuberculosis via miR-99b, which may open up new avenues for designing miRNA-based vaccines and therapies.

Early secreted antigen ESAT-6 of Mycobacterium tuberculosis promotes protective T helper 17 cell responses in a toll-like receptor-2-dependent manner.

Despite its relatively poor efficacy, Bacillus Calmette-Guérin (BCG) has been used as a tuberculosis (TB) vaccine since its development in 1921. BCG induces robust T helper 1 (Th1) immune responses but, for many individuals, this is not sufficient for host resistance against Mycobacterium tuberculosis (M. tb) infection. Here we provide evidence that early secreted antigenic target protein 6 (ESAT-6), expressed by the virulent M. tb strain H37Rv but not by BCG, promotes vaccine-enhancing Th17 cell responses. These activities of ESAT-6 were dependent on TLR-2/MyD88 signalling and involved IL-6 and TGF-β production by dendritic cells. Thus, animals that were previously infected with H37Rv or recombinant BCG containing the RD1 region (BCG::RD1) exhibited improved protection upon re-challenge with virulent H37Rv compared with mice previously infected with BCG or RD1-deficient H37Rv (H37RvΔRD1). However, TLR-2 knockout (TLR-2-/-) animals neither showed Th17 responses nor exhibited improved protection in response to immunization with H37Rv. Furthermore, H37Rv and BCG::RD1 infection had little effect on the expression of the anti-inflammatory microRNA-146a (miR146a) in dendritic cells (DCs), whereas BCG and H37RvΔRD1 profoundly induced its expression in DCs. Consistent with these findings, ESAT-6 had no effect on miR146a expression in uninfected DCs, but dramatically inhibited its upregulation in BCG-infected or LPS-treated DCs. Collectively, our findings indicate that, in addition to Th1 immunity induced by BCG, RD1/ESAT-6-induced Th17 immune responses are essential for optimal vaccine efficacy.

Malaria parasite tyrosyl-tRNA synthetase secretion triggers pro-inflammatory responses

Malaria infection triggers pro-inflammatory responses in humans that are detrimental to host health. Parasite-induced enhancement in cytokine levels correlate with malaria-associated pathologies. Here we show that parasite tyrosyl-tRNA synthetase (PfTyrRS), a housekeeping protein translation enzyme, induces pro-inflammatory responses from host immune cells. PfTyrRS exits from the parasite cytoplasm into the infected red blood cell (iRBC) cytoplasm, from where it is released into the extracellular medium on iRBC lysis. Using its ELR peptide motif, PfTyrRS specifically binds to and internalizes into host macrophages, leading to enhanced secretion of the pro-inflammatory cytokines TNF-α and IL-6. PfTyrRS-macrophage interaction also augments expression of adherence-linked host endothelial receptors ICAM-1 and VCAM-1. Our description of PfTyrRS as a parasite-secreted protein that triggers pro-inflammatory host responses, along with its atomic resolution crystal structure in complex with tyrosyl-adenylate, provides a novel platform for targeting PfTyrRS in anti-parasitic strategies.

Mycobacterium tuberculosis directs T helper 2 cell differentiation by inducing interleukin-1β production in dendritic cells

Background: Mycobacterium tuberculosis survives within the host by modulating host immune responses. Results: RD-1/ESAT-6 from M. tuberculosis induces IL-1β in dendritic cells to direct Th2 differentiation, which facilitates disease progression by inhibiting host protective Th1 responses. Conclusion: Cytokines produced by M. tuberculosis-infected cells play a role in promoting Th2 responses to subvert host immunity. Significance: These findings contribute to understanding the immune evasion mechanisms of M. tuberculosis. Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), resides and replicates within phagocytes and persists in susceptible hosts by modulating protective innate immune responses. Furthermore, M. tuberculosis promotes T helper 2 (Th2) immune responses by altering the balance of T cell polarizing cytokines in infected cells. However, cytokines that regulate Th2 cell differentiation during TB infection remain unknown. Here we show that IL-1β, produced by phagocytes infected by virulent M. tuberculosis strain H37Rv, directs Th2 cell differentiation. In sharp contrast, the vaccine strain bacille Calmette-Guérin as well as RD-1 and ESAT-6 mutants of H37Rv failed to induce IL-1β and promote Th2 cell differentiation. Furthermore, ESAT-6 induced IL-1β production in dendritic cells (DCs), and CD4+ T cells co-cultured with infected DCs differentiated into Th2 cells. Taken together, our findings indicate that IL-1β induced by RD-1/ESAT-6 plays an important role in the differentiation of Th2 cells, which in turn facilitates progression of TB by inhibiting host protective Th1 responses.

Small molecule-directed immunotherapy against recurrent infection by Mycobacterium tuberculosis.

Background: M. tuberculosis evades host-immune-responses by polarizing T helper (Th)2 and regulatory T cell (Treg) responses, which diminish protective Th1 responses. Results: Mice that are unable to generate Th2 cells and Tregs are resistant to M. tuberculosis infection. Simultaneous inhibition of these T cell subsets by therapeutic compounds dramatically reduced bacterial burden. Conclusion: Inhibition of Th2 and Treg cells increases Th1 responses that protect against M. tuberculosis infection. Significance: As therapeutic agents employed here do not directly act on harbored pathogens, they should avoid generation of drug-resistant M. tuberculosis variants. Tuberculosis remains the biggest infectious threat to humanity with one-third of the population infected and 1.4 million deaths and 8.7 million new cases annually. Current tuberculosis therapy is lengthy and consists of multiple antimicrobials, which causes poor compliance and high treatment dropout, resulting in the development of drug-resistant variants of tuberculosis. Therefore, alternate methods to treat tuberculosis are urgently needed. Mycobacterium tuberculosis evades host immune responses by inducing T helper (Th)2 and regulatory T (Treg) cell responses, which diminish protective Th1 responses. Here, we show that animals (Stat-6−/−CD4-TGFβRIIDN mice) that are unable to generate both Th2 cells and Tregs are highly resistant to M. tuberculosis infection. Furthermore, simultaneous inhibition of these two subsets of Th cells by therapeutic compounds dramatically reduced bacterial burden in different organs. This treatment was associated with the generation of protective Th1 immune responses. As these therapeutic agents are not directed to the harbored organisms, they should avoid the risk of promoting the development of drug-resistant M. tuberculosis variants.

Host ICAMs play a role in cell invasion by Mycobacterium tuberculosis and Plasmodium falciparum

Intercellular adhesion molecules (ICAMs) belong to the immunoglobulin superfamily and participate in diverse cellular processes including host-pathogen interactions. ICAM-1 is expressed on various cell types including macrophages, whereas ICAM-4 is restricted to red blood cells. Here we report the identification of an 11-kDa synthetic protein, M5, that binds to human ICAM-1 and ICAM-4, as shown by in vitro interaction studies, surface plasmon resonance and immunolocalization. M5 greatly inhibits the invasion of macrophages and erythrocytes by Mycobacterium tuberculosis and Plasmodium falciparum, respectively. Pharmacological and siRNA-mediated inhibition of ICAM-1 expression also results in reduced M. tuberculosis invasion of macrophages. ICAM-4 binds to P. falciparum merozoites, and the addition of recombinant ICAM-4 to parasite cultures blocks invasion of erythrocytes by newly released merozoites. Our results indicate that ICAM-1 and ICAM-4 play roles in host cell invasion by M. tuberculosis and P. falciparum, respectively, either as receptors or as crucial accessory molecules.

Simultaneous Inhibition of T Helper 2 and T Regulatory Cell Differentiation by Small Molecules Enhances Bacillus Calmette-Guerin Vaccine Efficacy against Tuberculosis

Background: Immunological parameters induced by BCG and the requirement of immunologic responses for optimal vaccine efficacy is incompletely understood. Results: Small-molecule inhibitors of Th2 and Treg cells promote BCG vaccine efficacy. Conclusion: Immunomodulators enhance the capacity of the BCG vaccine to protect against tuberculosis. Significance: Our studies reveal a simple and cost-effective approach to improve BCG vaccine efficacy. Tuberculosis affects nine million individuals and kills almost two million people every year. The only vaccine available, Bacillus Calmette-Guerin (BCG), has been used since its inception in 1921. Although BCG induces host-protective T helper 1 (Th1) cell immune responses, which play a central role in host protection, its efficacy is unsatisfactory, suggesting that additional methods to enhance protective immune responses are needed. Recently we have shown that simultaneous inhibition of Th2 cells and Tregs by using the pharmacological inhibitors suplatast tosylate and D4476, respectively, dramatically enhances Mycobacterium tuberculosis clearance and induces superior Th1 responses. Here we show that treatment with these two drugs during BCG vaccination dramatically improves vaccine efficacy. Furthermore, we demonstrate that these drugs induce a shift in the development of T cell memory, favoring central memory T (Tcm) cell responses over effector memory T (Tem) cell responses. Collectively, our findings provide evidence that simultaneous inhibition of Th2 cells and Tregs during BCG vaccination promotes vaccine efficacy.

Transforming growth factor-β protein inversely regulates in vivo differentiation of interleukin-17 (IL-17)-producing CD4+ and CD8+ T cells.

Background: In vitro TGF-β differentiates Th17 and Tc17 cells, but TGF-βRIIDN mice display multiorgan autoimmune disorders. Results: CD4+T cells from TGF-βRIIDN mice are resistant to Th17 cell differentiation, whereas CD8+T cells acquire IL-17-producing phenotype, and IL-17 neutralization or depletion inhibited inflammation in TGF-βRIIDN mice. Conclusion: Tc17 cell differentiations in vivo are distinct from Tc17 cell differentiations in vitro. Significance: The Tc17 cell differentiation program is unique to Th17. TGF-β is a pleiotropic cytokine that predominantly exerts inhibitory functions in the immune system. Unexpectedly, the in vitro differentiation of both Th17 and Tc17 cells requires TGF-β. However, animals that are impaired in TGF-β signaling (TGF-βRIIDN mice) display multiorgan autoimmune disorders. Here we show that CD4+ T cells from TGF-βRIIDN mice are resistant to Th17 cell differentiation and, paradoxically, that CD8+ T cells from these animals spontaneously acquire an IL-17-producing phenotype. Neutralization of IL-17 or depletion of CD8+ T cells dramatically inhibited inflammation in TGF-βRIIDN mice. Therefore, the absence of TGF-β triggers spontaneous differentiation of IL-17-producing CD8+ T cells, suggesting that the in vivo and in vitro conditions that promote the differentiation of IL-17-producing CD8+ T cells are distinct.

CD4+ T Cell-derived Novel Peptide Thp5 Induces Interleukin-4 Production in CD4+ T Cells to Direct T Helper 2 Cell Differentiation

Background: CD4+ T cells produce IL-4 that drives Th2 cell differentiation. Early production of IL-4 in naïve T cells leads to Th2 cell differentiation. Results: Thp5, a novel peptide, regulates IL-4 production in early activated CD4+ T cells. Conclusion: Early activated CD4+ T cells produce Thp5, which plays a critical role in the differentiation of Th2 cells. Significance: Thp5 acts as an important determinant of Th2 cell differentiation during early T cell activation. The differentiation of naïve CD4+ T cells into T helper 2 (Th2) cells requires production of the cytokine IL-4 in the local microenvironment. It is evident that naïve/quiescently activated CD4+ T cells produce the IL-4 that drives Th2 cell differentiation. Because early production of IL-4 in naïve T cells leads to preferential Th2 cell differentiation, this process needs to be tightly regulated so as to avoid catastrophic and misdirected Th2 cell differentiation. Here, we show that Thp5, a novel peptide with structural similarity to vasoactive intestinal peptide, regulates production of early IL-4 in newly activated CD4+ T cells. Induction of IL-4 in CD4+ T cells by Thp5 is independent of the transcription factor STAT6 but dependent on ERK1/2 signaling. Furthermore, cytokines (IL-12 and TGF-β) that promote the differentiation of Th1 or Th17 cells inhibit Thp5 induction, thus suppressing Th2 cell differentiation. We further showed that Thp5 enhances Th2 responses and exacerbates allergic airway inflammation in mice. Taken together, our findings reveal that early activated CD4+ T cells produce Thp5, which plays a critical role as a molecular switch in the differentiation of Th cells, biasing the response toward the Th2 cell phenotype.

Early secretory antigenic target-6 of Mycobacterium tuberculosis: enigmatic factor in pathogen-host interactions.

T helper (Th) 1 and 17 cells play important roles in host protective responses against tuberculosis. Early Secretory Antigenic Target 6; a Region of Difference 1 (RD1) encoded protein, mounts Th17-responses in the lung. Therefore, lack of RD-1 region makes Bacillus Calmette-Guérin (BCG) less vaccine efficacious than parent strains.