Src homology 3-interacting domain of Rv1917c of Mycobacterium tuberculosis induces selective maturation of human dendritic cells by regulating PI3K-MAPK-NF-kappaB signaling and drives Th2 immune responses.
ABSTRACT: Mycobacterium tuberculosis, an etiological agent of pulmonary tuberculosis, causes significant morbidity and mortality worldwide. Pathogenic mycobacteria survive in the host by subverting host innate immunity. Dendritic cells (DCs) are professional antigen-presenting cells that are vital for eliciting immune responses to infectious agents, including pathogenic mycobacteria. DCs orchestrate distinct Th responses based on the signals they receive. In this perspective, deciphering the interactions of the proline-glutamic acid/proline-proline-glutamic acid (PE/PPE) family of proteins of M. tuberculosis with DCs assumes significant pathophysiological attributes. In this study, we demonstrate that Rv1917c (PPE34), a representative member of the proline-proline-glutamic-major polymorphic tandem repeat family, interacts with TLR2 and triggers functional maturation of human DCs. Signaling perturbations implicated a critical role for integrated cross-talk among PI3K-MAPK and NF-?B signaling cascades in Rv1917c-induced maturation of DCs. However, this maturation of DCs was associated with a secretion of high amounts of anti-inflammatory cytokine IL-10, whereas Th1-polarizing cytokine IL-12 was not induced. Consistent with these results, Rv1917c-matured DCs favored secretion of IL-4, IL-5, and IL-10 from CD4(+) T cells and contributed to Th2-skewed cytokine balance ex vivo in healthy individuals and in patients with pulmonary tuberculosis. Interestingly, the Rv1917c-skewed Th2 immune response involved induced expression of cyclooxygenase-2 (COX-2) in DCs. Taken together, these results indicate that Rv1917c facilitates a shift in the ensuing immunity toward the Th2 phenotype and could aid in immune evasion by mycobacteria.
Project description:The role of the unique proline-glutamic acid (PE)/proline-proline-glutamic acid (PPE) family of proteins in the pathophysiology and virulence of Mycobacterium tuberculosis is not clearly understood. One of the PE family proteins, PE11 (LipX or Rv1169c), specific to pathogenic mycobacteria is found to be over-expressed during infection of macrophages and in active TB patients. In this study, we report that M. smegmatis expressing PE11 (Msmeg-PE11) exhibited altered colony morphology and cell wall lipid composition leading to a marked increase in resistance against various environmental stressors and antibiotics. The cell envelope of Msmeg-PE11 also had greater amount of glycolipids and polar lipids. Msmeg-PE11 was found to have better survival rate in infected macrophages. Mice infected with Msmeg-PE11 had higher bacterial load, showed exacerbated organ pathology and mortality. The liver and lung of Msmeg-PE11-infected mice also had higher levels of IL-10, IL-4 and TNF-? cytokines, indicating a potential role of this protein in mycobacterial virulence.
Project description:Background:The malaria causing parasite Plasmodium subverts host immune responses by several strategies including the modulation of dendritic cells (DCs). Methods:In this study, we show that Plasmodium falciparum skewed CD16+ DC cytokine responses towards interleukin (IL)-10 production in vitro, distinct to the cytokine profile induced by Toll-like receptor ligation. To determine CD16+ DC responsiveness in vivo, we assessed their function after induced P falciparum infection in malaria-naive volunteers. Results:CD16+ DCs underwent distinctive activation, with increased expression of maturation markers human leukocyte antigen (HLA)-DR and CD86, enhanced tumor necrosis factor (TNF) production, and coproduction of TNF/IL-10. In vitro restimulation with P falciparum further increased IL-10 production. In contrast, during naturally acquired malaria episode, CD16+ DCs showed diminished maturation, suggesting increased parasite burden and previous exposure influence DC subset function. Conclusions:These findings identify CD16+ DCs as the only DC subset activated during primary blood-stage human Plasmodium infection. As dual cytokine producers, CD16+ DCs contribute to inflammatory as well as regulatory innate immune processes.
Project description:Mycobacterium tuberculosis is a highly successful human pathogen that primarily resides in host phagocytes, such as macrophages and dendritic cells (DCs), and interferes with their functions. Although multiple strategies used by M. tuberculosis to modulate macrophage responses have been discovered, interactions between M. tuberculosis and DCs are less well understood. DCs are the primary APCs of the immune system and play a central role in linking innate and adaptive immune responses to microbial pathogens. In this study, we show that M. tuberculosis impairs DC cytokine secretion, maturation, and Ag presentation through the cell envelope-associated serine hydrolase, Hip1. Compared to wild-type, a hip1 mutant strain of M. tuberculosis induced enhanced levels of the key Th1-inducing cytokine IL-12, as well as other proinflammatory cytokines (IL-23, IL-6, TNF-?, IL-1?, and IL-18) in DCs via MyD88- and TLR2/9-dependent pathways, indicating that Hip1 restricts optimal DC inflammatory responses. Infection with the hip1 mutant also induced higher levels of MHC class II and costimulatory molecules CD40 and CD86, indicating that M. tuberculosis impairs DC maturation through Hip1. Further, we show that M. tuberculosis promotes suboptimal Ag presentation, as DCs infected with the hip1 mutant showed increased capacity to present Ag to OT-II- and early secreted antigenic target 6-specific transgenic CD4 T cells and enhanced Th1 and Th17 polarization. Overall, these data show that M. tuberculosis impairs DC functions and modulates the nature of Ag-specific T cell responses, with important implications for vaccination strategies.
Project description:Tuberculosis (TB) is a major health problem of global concern. The control of this disease requires appropriate preventive measures, including vaccines. In TB, T helper (Th)1 cytokines provide protection whereas Th2 and T regulatory (Treg) cytokines contribute to the pathogenesis and Th17 cytokines play a role in both protection and pathogenesis. Previous studies with Mycobacterium tuberculosis-specific proteins have identified seven low molecular weight proteins, PE35, ESXA, ESXB, Rv2346c, Rv2347c, Rv3619c, and Rv3620c, as immunodominant antigens inducing Th1-cell responses in humans following natural infection with M. tuberculosis. The aim of this study was to characterize the cytokine responses induced in mice immunized with these proteins, using various adjuvants and delivery systems, i.e. chemical adjuvants (Alum and IFA), non-pathogenic mycobacteria (M. smegmatis and M. vaccae) and a DNA vaccine plasmid (pUMVC6). The immune responses were monitored by quantifying the marker cytokines secreted by Th1 (IFN-?), Th2 (IL-5), Treg (IL-10), and Th17 (IL-17A) cells. DNA corresponding to pe35, esxa, esxb, rv2346c, rv2347c, rv3619c, and rv3620c genes were cloned into the expression vectors pGES-TH-1, pDE22 and pUMVC6 for expression in Escherichia coli, mycobacteria and eukaryotic cells, respectively. Mice were immunized with the recombinants using different adjuvants and delivery systems, and spleen cells were stimulated in vitro with peptides of immunizing proteins to investigate antigen-specific secretion of Th1 (IFN-?), Th2 (IL-5), Treg (IL-10), and Th17 (IL-17A) cytokines. The results showed that spleen cells, from mice immunized with all antigens, secreted the protective Th1 cytokine IFN-?, except ESXB, with one or more adjuvants and delivery systems. However, only Rv3619c consistently induced Th1-biased responses, without the secretion of significant concentrations of Th2, Th17 and Treg cytokines, with all adjuvants and delivery systems. Rv3619c also induced antigen-specific IgG antibodies in immunized mice.
Project description:Recent work demonstrated the importance of macroautophagy in dendritic cell (DC) maturation and innate cytokine production upon viral infection through delivery of cytoplasmic viral components to intracellular TLRs. To study the functional consequences of impaired autophagosome formation during a respiratory syncytial virus (RSV) infection, mice harboring significant autophagy defects due to Beclin-1 haploinsufficiency (Beclin-1(+/-)) were used. Upon RSV infection in vivo, lungs of Beclin-1(+/-) mice showed increased Th2 cytokine production, mucus secretion, and lung infiltration of eosinophils and inflammatory DCs. Although isolated airway epithelial cells from Beclin-1(+/-) mice demonstrated little change compared with wild-type mice, Beclin-1(+/-) pulmonary and bone marrow-derived DCs showed decreased expression of MHC class II and innate cytokine production upon RSV infection. Further examination indicated that Beclin-1(+/-) DCs stimulated less IFN-? and IL-17 production by cocultured CD4(+) T cells and increased Th2 cytokine production in comparison with wild-type controls. Finally, adoptive transfer of RSV-infected Beclin-1(+/-) DCs into the airways of wild-type mice produced severe lung pathology and increased Th2 cytokine production upon subsequent RSV challenge compared with wild-type DC transfer controls. These results indicate a critical role for autophagy in DCs during pulmonary viral infection, facilitating appropriate antiviral adaptive immune responses.
Project description:STAT3 is a master regulator of the immune responses. Here we show that M. tuberculosis-infected stat3fl/fl lysm cre mice, defective for STAT3 in myeloid cells, contained lower bacterial load in lungs and spleens, reduced granuloma extension but higher levels of pulmonary neutrophils. STAT3-deficient macrophages showed no improved control of intracellular mycobacterial growth. Instead, protection associated to elevated ability of stat3fl/fl lysm cre antigen-presenting cells (APCs) to release IL-6 and IL-23 and to stimulate IL-17 secretion by mycobacteria-specific T cells. The increased IL-17 secretion accounted for the improved control of infection since neutralization of IL-17 receptor A in stat3fl/fl lysm cre mice hampered bacterial control. APCs lacking SOCS3, which inhibits STAT3 activation via several cytokine receptors, were poor inducers of priming and of the IL-17 production by mycobacteria-specific T cells. In agreement, socs3fl/fl cd11c cre mice deficient of SOCS3 in DCs showed increased susceptibility to M. tuberculosis infection. While STAT3 in APCs hampered IL-17 responses, STAT3 in mycobacteria-specific T cells was critical for IL-17 secretion, while SOCS3 in T cells impeded IL-17 secretion. Altogether, STAT3 signalling in myeloid cells is deleterious in the control of infection with M. tuberculosis.
Project description:The role played by lung dendritic cells (DCs) which are influenced by external antigens and by their redox state in controlling inflammation is unclear. We studied the role played by nitric oxide (NO) in DC maturation and function. Human DCs were stimulated with a long-acting NO donor, DPTA NONOate, prior to exposure to lipopolysaccharide (LPS). Dose-and time-dependent experiments were performed with DCs with the aim of measuring the release and gene expression of inflammatory cytokines capable of modifying T-cell differentiation, towardsTh1, Th2 and Th17 cells. NO changed the pattern of cytokine release by LPS-matured DCs, dependent on the concentration of NO, as well as on the timing of its addition to the cells during maturation. Addition of NO before LPS-induced maturation strongly inhibited the release of IL-12, while increasing the expression and release of IL-23, IL-1? and IL-6, which are all involved in Th17 polarization. Indeed, DCs treated with NO efficiently induced the release of IL-17 by T-cells through IL-1?. Our work highlights the important role that NO may play in sustaining inflammation during an infection through the preferential differentiation of the Th17 lineage.
Project description:BACKGROUND:Dendritic cells (DCs) play major roles in mediating immune responses to mycobacteria. A crucial aspect of this is the priming of T cells via chemokines and cytokines. In this study we investigated the roles of chemokines RANTES and IP-10 in regulating protective responses from Mycobacterium tuberculosis (M. tb) 10 kDa Culture Filtrate Protein-10 (CFP-10) differentiated DCs (CFP10-DCs). METHODS AND FINDINGS:Infection of CFP10-DCs with mycobacteria down-modulated RANTES and IP-10 levels. Pathway specific microarray analyses showed that in addition to RANTES and IP-10, mycobacteria infected CFP10-DCs showed reduced expression of many Th1 promoting chemokines and chemokine receptors. Importantly, T cells co-cultured with RANTES and IP-10 conditioned CFP10-DCs mediated killing of mycobacteria from infected macrophages. Similarly, T cells recruited by RANTES and IP-10 conditioned CFP10-DCs mediated significant killing of mycobacteria from infected macrophages. IFN-gamma treatment of CFP10-DCs restored RANTES and IP-10 levels and T cells activated by these DCs mediated significant killing of virulent M. tb inside macrophages. Adoptive transfer of either RANTES and IP-10 or IL-12 and IFN-gamma conditioned CFP10-DCs cleared an established M. tb infection in mice. The extent of clearance was similar to that obtained with drug treatment. CONCLUSIONS:These results indicate that chemokine and cytokine secretion by DCs differentiated by M. tb antigens such as CFP-10 play major roles in regulating protective immune responses at sites of infection.
Project description:Understanding the early immunologic events accompanying reactivated tuberculosis (TB) in HIV-infected individuals may yield insight into causes of reactivation and improve treatment modalities. We used the cynomolgus macaque (Macaca fascicularis) model of HIV-Mycobacterium tuberculosis coinfection to investigate the dynamics of multifunctional T cell responses and granuloma T cell phenotypes in reactivated TB. CD4(+) and CD8(+) T cells expressing Th1 cytokines (IFN-?, IL-2, TNF) and Th2 cytokines (IL-4 and IL-10) were followed from latent M. tuberculosis infection to reactivation after coinfection with a pathogenic SIV. Coinfected animals experienced increased Th1 cytokine responses to M. tuberculosis Ags above the latent-response baseline 3-5 wk post-SIV infection that corresponded with peak plasma viremia. Th2 cytokine expression was not Ag specific, but strong, transient IL-4 expression was noted 4-7 wk post-SIV infection. Animals reactivating <17 wk post-SIV infection had significantly more multifunctional CD4(+) T cells 3-5 wk post-SIV infection and more Th2-polarized and fewer Th0-, Th1-polarized CD8(+) T cells during weeks 1-10 post-SIV infection than animals reactivating >26 wk post-SIV infection. Granuloma T cells included Th0-, Th1-, and Th2-polarized phenotypes but were particularly rich in cytolytic (CD107(+)) T cells. When combined with the changes in peripheral blood T cells, these factors indicate that events during acute HIV infection are likely to include distortions in proinflammatory and anti-inflammatory T cell responses within the granuloma that have significant effects on reactivation of latent TB. Moreover, it appears that mycobacteria-specific multifunctional T cells are better correlates of Ag load (i.e., disease status) than of protection.
Project description:Atopic asthma is an inflammatory pulmonary disease associated with Th2 adaptive immune responses triggered by innocuous antigens. While dendritic cells (DCs) are known to shape the adaptive immune response, the mechanisms by which DCs promote Th2 differentiation remain elusive. Herein we demonstrate that Th2-promoting stimuli induce DC expression of IRF4. Mice with conditional deletion of Irf4 in DCs show a dramatic defect in Th2-type lung inflammation, yet retain the ability to elicit pulmonary Th1 antiviral responses. Using loss- and gain-of-function analysis, we demonstrate that Th2 differentiation is dependent on IRF4 expression in DCs. Finally, IRF4 directly targets and activates the Il-10 and Il-33 genes in DCs. Reconstitution with exogenous IL-10 and IL-33 recovers the ability of Irf4-deficient DCs to promote Th2 differentiation. These findings reveal a regulatory module in DCs by which IRF4 modulates IL-10 and IL-33 cytokine production to specifically promote Th2 differentiation and inflammation.