Xisheng Wang

Exposure to Cigarette Smoke Inhibits the Pulmonary T-Cell Response to Influenza Virus and Mycobacterium tuberculosis

ABSTRACT Smoking is associated with increased susceptibility to tuberculosis and influenza. However, little information is available on the mechanisms underlying this increased susceptibility. Mice were left unexposed or were exposed to cigarette smoke and then infected with Mycobacterium tuberculosis by aerosol or influenza A by intranasal infection. Some mice were given a DNA vaccine encoding an immunogenic M. tuberculosis protein. Gamma interferon (IFN-γ) production by T cells from the lungs and spleens was measured. Cigarette smoke exposure inhibited the lung T-cell production of IFN-γ during stimulation in vitro with anti-CD3, after vaccination with a construct expressing an immunogenic mycobacterial protein, and during infection with M. tuberculosis and influenza A virus in vivo. Reduced IFN-γ production was mediated through the decreased phosphorylation of transcription factors that positively regulate IFN-γ expression. Cigarette smoke exposure increased the bacterial burden in mice infected with M. tuberculosis and increased weight loss and mortality in mice infected with influenza virus. This study provides the first demonstration that cigarette smoke exposure directly inhibits the pulmonary T-cell response to M. tuberculosis and influenza virus in a physiologically relevant animal model, increasing susceptibility to both pathogens.

ESAT-6 inhibits production of IFN-γ by Mycobacterium tuberculosis-responsive human T cells

The Mycobacterium tuberculosis early secreted Ag of 6 kDa (ESAT-6) is a potent Ag for human T cells and is a putative vaccine candidate. However, ESAT-6 also contributes to virulence in animal models, mediates cellular cytolysis, and inhibits IL-12 production by mononuclear phagocytes. We evaluated the effects of ESAT-6 and its molecular chaperone, culture filtrate protein of 10 kDa (CFP10), on the capacity of human T cells to produce IFN-γ and proliferate in response to TCR activation. Recombinant ESAT-6, but not CFP10, markedly inhibited IFN-γ production by T cells stimulated with M. tuberculosis or with the combination of anti-CD3 and anti-CD28, in a dose-dependent manner. ESAT-6 also inhibited T cell production of IL-17 and TNF-α but not IL-2. Preincubation of ESAT-6 with CFP10 under conditions that favor dimer formation did not affect inhibition of IFN-γ. ESAT-6 decreased IFN-γ transcription and reduced expression of the transcription factors, ATF-2 and c-Jun, which normally bind to the IFN-γ proximal promoter and stimulate mRNA expression. ESAT-6 inhibited T cell IFN-γ secretion through mechanisms that did not involve cellular cytotoxicity or apoptosis. ESAT-6, but not CFP10, bound to T cells and inhibited expression of early activation markers without reducing activation of ZAP70. We conclude that ESAT-6 directly inhibits human T cell responses to mycobacterial Ags by affecting TCR signaling pathways downstream of ZAP70.

MprAB Regulates the espA Operon in Mycobacterium tuberculosis and Modulates ESX-1 Function and Host Cytokine Response

The ESX-1 secretion system exports the immunomodulatory protein ESAT-6 and other proteins important in the pathogenesis of Mycobacterium tuberculosis. Components and substrates of ESX-1 are encoded at several loci, but the regulation of the encoding genes is only partially understood. In this study, we investigated the role of the MprAB two-component system in the regulation of ESX-1 activity. We determined that MprAB directly regulates the espA gene cluster, a locus necessary for ESX-1 function. Transcript mapping determined that the five genes in the cluster form an operon with two transcriptional start points, and several MprA binding sites were detected in the espA promoter. Expression analyses and promoter constructs indicated that MprAB represses the espA operon. However, the MprAB mutant Rv-D981 secreted lower levels of EspA, ESAT-6, and the ESX-1 substrate EspB than control strains. Secretion of CFP10, which is normally cosecreted with ESAT-6, was similar in Rv-D981 and control strains, further demonstrating aberrant ESX-1 activity in the mutant. ESAT-6 induces proinflammatory cytokines, and macrophages infected with Rv-D981 elicited lower levels of interleukin 1β (IL-1β) and tumor necrosis factor alpha (TNF-α), consistent with the reduced levels of ESAT-6. These findings indicate that MprAB modulates ESX-1 function and reveal a new role for MprAB in host-pathogen interactions.

The Mycobacterium tuberculosis early secreted antigenic target of 6 kDa inhibits T cell interferon-γ production through the p38 mitogen-activated protein kinase pathway.

We reported previously that the early secreted antigenic target of 6 kDa (ESAT-6) from Mycobacterium tuberculosis directly inhibits human T cell IFN-γ production and proliferation in response to stimulation with anti-CD3 and anti-CD28. To determine the mechanism of this effect, we treated T cells with kinase inhibitors before stimulation with ESAT-6. Only the p38 MAPK inhibitor, SB203580, abrogated ESAT-6-mediated inhibition of IFN-γ production in a dose-dependent manner. SB203580 did not reverse ESAT-6-mediated inhibition of IL-17 and IL-10 production, suggesting a specific effect of SB203580 on IFN-γ production. SB203580 did not act through inhibition of AKT (PKB) as an AKT inhibitor did not affect ESAT-6 inhibition of T cell IFN-γ production and proliferation. ESAT-6 did not reduce IFN-γ production by expanding FoxP3+ T regulatory cells. Incubation of T cells with ESAT-6 induced phosphorylation and increased functional p38 MAPK activity, but not activation of ERK or JNK. Incubation of peripheral blood mononuclear cells with ESAT-6 induced activation of p38 MAPK, and inhibition of p38 MAPK with SB203580 reversed ESAT-6 inhibition of M. tuberculosis-stimulated IFN-γ production by peripheral blood mononuclear cells from subjects with latent tuberculosis infection. Silencing of p38α MAPK with siRNA rendered T cells resistant to ESAT-6 inhibition of IFN-γ production. Taken together, our results demonstrate that ESAT-6 inhibits T cell IFN-γ production in a p38 MAPK-dependent manner.

EspR, a regulator of the ESX-1 secretion system in Mycobacterium tuberculosis, is directly regulated by the two-component systems MprAB and PhoPR.

The regulatory mechanisms that control the ESX-1 secretion system, a key player in the pathogenesis of Mycobacterium tuberculosis, have not been fully elucidated. However, factors that regulate the ESX-1 substrate EspA usually affect ESX-1 function. Previous studies showed that espA is directly regulated by the nucleoid-associated protein EspR and the two-component system (TCS) MprAB. The PhoPR TCS also activates espA, but the direct target of PhoP was unknown. In this report, we reveal that EspR is directly regulated by MprA and PhoP-Rv, but not by PhoP-Ra. PhoP-Rv and MprA binding sites in the espR promoter were determined by gel-shift and DNase I footprinting assays, which identified a PhoP-protected region centred approximately 205 bp before the espR start codon and that encompasses MprA Region-1, one of two MprA-protected regions. MprA Region-2 is located approximately 60 bp downstream of MprA Region-1 and overlaps a known EspR binding site. Nucleotides essential for the binding of PhoP and/or MprA were identified through site-directed DNA mutagenesis. Our studies also indicate that MprA Region-2, but not MprA Region-1/PhoP region, is required for the full expression of espR. Recombinant strains carrying mutations at MprA Region-2 exhibited lower transcription levels for espR, espA and espD, and had reduced EspR and EspA levels in cell lysates. These findings indicate that EspR may mediate the regulatory effect of PhoPR and MprAB, and provide more insight into the mechanisms underlying ESX-1 control.

Early Secreted Antigenic Target of 6-kDa of Mycobacterium tuberculosis Stimulates IL-6 Production by Macrophages through Activation of STAT3

As early secreted antigenic target of 6u2009kDa (ESAT-6) of Mycobacterium tuberculosis (Mtb) is an essential virulence factor and macrophages are critical for tuberculosis infection and immunity, we studied ESAT-6 stimulated IL-6 production by macrophages. ESAT-6 stimulated significantly higher IL-6 secretion by murine bone marrow derived macrophages (BMDM) compared to culture filtrate protein 10u2009kDa (CFP10) and antigen 85A. Polymyxin B, an LPS blocker, did not affect ESAT-6 stimulated macrophage IL-6 production. ESAT-6 but not Pam3CSK4 induced IL-6 by TLR2 knockout BMDM. ESAT-6 induced phosphorylation and DNA binding of STAT3 and this was blocked by STAT3 inhibitors but not by rapamycin. STAT3 inhibitors suppressed ESAT-6-induced IL-6 transcription and secretion without affecting cell viability. This was confirmed by silencing STAT3 in macrophages. Blocking neither IL-6Rα/IL-6 nor IL-10 affected ESAT-6-induced STAT3 activation and IL-6 production. Infection of BMDM and human macrophages with Mtb with esat-6 deletion induced diminished STAT3 activation and reduced IL-6 production compared to wild type and esat-6 complemented Mtb strains. Administration of ESAT-6 but not CFP10 induced STAT3 phosphorylation and IL-6 expression in the mouse lungs, consistent with expression of ESAT-6, IL-6 and phosphorylated-STAT3 in Mtb-infected mouse lungs. We conclude that ESAT-6 stimulates macrophage IL-6 production through STAT3 activation.

The early secreted antigenic target of 6 kD of Mycobacterium tuberculosis inhibits the proliferation and differentiation of human peripheral blood CD34+ cells

Abnormalities in hematopoiesis are common in tuberculosis patients and highly prevalent in AIDS patients with tuberculosis coinfection. To explore the potential role of the early secreted antigenic target of 6-kD (ESAT-6) of Mycobacterium tuberculosis (Mtb) in abnormal hematopoiesis in tuberculosis, we studied the effect of ESAT-6 on proliferation and differentiation of inxa0vitro-expanded CD34+xa0cells isolated from the peripheral blood of the healthy donors. ESAT-6 but not control protein antigen 85A (Ag85A) of Mtb inhibited the proliferation of CD34+xa0cell derived peripheral blood stem/progenitor cells (PBSPC) in a dose dependent manner when determined by MTT-assay. ESAT-6 but not Ag85A reduced the number of colony forming cells (CFC) of PBSPC by 60-90% as determined by CFC assay by incubation of CD34+xa0cells in a semi-solid cellulose media in the presence of cytokine cocktail for two weeks. ESAT-6 but not Ag85A increased the percentages of the Annexin-V positive cells and enhanced the cleavage of caspase-3 in PBSPC in a time and dose dependent manner as determined by flow cytometry and Western blot analysis, respectively. ESAT-6 also inhibited murine bone marrow derived non-adherent cell proliferation in response to granulocyte-macrophage colony stimulating factor treatment. We conclude that ESAT-6, an essential virulence factor of Mtb, may contribute to the abnormal hematopoiesis of tuberculosis patients by inhibiting the proliferation and differentiation of hematopoietic cells via apoptosis.