Host defense responses to infection by Mycobacterium tuberculosis. Induction of IRF-1 and a serine protease inhibitor

Alveolar macrophages and newly recruited monocytes are targets of infection by Mycobacterium tuberculosis. Therefore, we examined the expression of interferon regulatory factor 1 (IRF-1), which plays an important role in host defense against M. tuberculosis, in undifferentiated and differentiated cells. Infection induced IRF-1 in both. IRF-1 from undifferentiated, uninfected monocytic cell lines was modified during extraction to produce specific species that were apparently smaller than intact IRF-1. After infection by M. tuberculosis or differentiation, intact IRF-1 was recovered. Subcellular fractions were assayed for the ability to modify IRF-1 or inhibit its modification. A serine protease on the cytoplasmic surface of an organelle or vesicle in the "lysosomal/mitochondrial" fraction from undifferentiated cells was responsible for the modification of IRF-1. Thus, the simplest explanation of the modification is cleavage of IRF-1 by the serine protease. Recovery of intact IRF-1 correlated with induction of a serine protease inhibitor that was able to significantly reduce the modification of IRF-1. The inhibitor was present in the cytoplasm of M. tuberculosis-infected or -differentiated cells. It is likely that induction of both IRF-1 and the serine protease inhibitor in response to infection by M. tuberculosis represent host defense mechanisms.     

A model to predict cell-mediated immune regulatory mechanisms during human infection with Mycobacterium tuberculosis

A key issue for the study of tuberculosis infection (TB) is to understand why individuals infected with Mycobacterium tuberculosis experience different clinical outcomes. Elaborating the immune mechanisms that determine whether an infected individual will suffer active TB or latent infection can aid in developing treatment and prevention strategies. To better understand the dynamics of M. tuberculosis infection and immunity, we have developed a virtual human model that qualitatively and quantitatively characterizes the cellular and cytokine control network operational during TB infection. Using this model, we identify key regulatory elements in the host response. In particular, factors affecting cell functions, such as macrophage activation and bactericidal capabilities, and effector T cell functions such as cytotoxicity and cytokine production can each be determinative. The model indicates, however, that even if latency is achieved, it may come at the expense of tissue damage if the response is not properly regulated. A balance in Th1 and Th2 immune responses governed by IFN-gamma, IL-10, and IL-4 facilitate this down-regulation. These results are further explored through virtual deletion and depletion experiments.     

Suppressors of cytokine signaling inhibit effector T cell responses during Mycobacterium tuberculosis infection

Protective immune responses during Mycobacterium tuberculosis (M. tuberculosis) infection are regulated at multiple levels and critically dependent on the balance in the secretion of pro-inflammatory and regulatory cytokines. A key factor that governs this balance at the cellular level is suppressors of cytokine signaling (SOCS). We recently demonstrated that toll-like receptor 2 and dendritic cell (DC)-SIGNR1 differentially regulate SOCS1 expression in DCs during M. tuberculosis infection. This consecutively regulated IL-12 production and determined M. tuberculosis survival. In this study, we characterized the role of SOCS1 in regulating effector responses from CD4(+) and CD8(+) T cells during M. tuberculosis infection. Our data indicate that T cells from M. tuberculosis-infected mice show increased and differential association of SOCS1 with CD3 and CD28, when compared with uninfected mice. While SOCS1 displays increased association with CD3 than CD28 in CD4(+) T cells; SOCS1 is associated more with CD28 than CD3 in CD8(+) T cells. Further, SOCS1 shows increased association with IL-12 and IL-2 receptors in both CD4(+) and CD8(+) T cells from infected mice when compared with naive mice. Silencing SOCS1 in T cells increased signal transduction from T cell receptor (TCR) and CD28 with enhanced activation of key signaling molecules and proliferation. Significantly, SOCS1-silenced T cells mediated enhanced clearance of M. tuberculosis inside macrophages. Finally, adoptive transfer of SOCS1-silenced T cells in M. tuberculosis-infected mice mediated significant reduction in M. tuberculosis loads in spleen. These results exemplify the negative role played by SOCS1 during T cell priming and effector functions during M. tuberculosis infection.     

Long-term control of Mycobacterium tuberculosis infection is mediated by dynamic immune responses

The primary goal of this study was to determine how chronic exposure to Ag influences the functionality of Mycobacterium tuberculosis-specific T cell responses. The frequency of IFN-gamma-producing effector CD4(+) and CD8(+) T cells dynamically changed during persistent M. tuberculosis infection. CD8(+) T cells used differential effector functions during acute and chronic phases of the immune response, where CD8(+) T cells produced negligible amounts of IFN-gamma early in infection, but switched to cytokine production during the chronic stage of infection. Using limiting dilution analysis, CD8(+) T cells isolated during the initial phase of infection demonstrated lytic potential, but this waned in the chronic stage. The apparent loss of cytotoxic activity was not associated with the lack of perforin. Ag dose could potentially govern the functional program of CD8(+) T cells. Collectively, these results depict a host immune response mounted against M. tuberculosis of a significantly more dynamic nature than previously recognized.     

Autophagy during Mycobacterium tuberculosis infection and implications for future tuberculosis medications

Autophagy is a cellular homeostasis mechanism to eliminate unwanted or excessive organelles, or for the turnover of long-life cytosolic macromolecules. During Mycobacterium tuberculosis infection, autophagy represents not only an antimicrobial mechanism for the clearance of the intracellular pathogen, but also prevents excessive inflammation, avoiding the adverse effects on host. Here we focus on the anti-tuberculosis autophagy and signal pathways involved, and attempt to depict an integrative map of the interaction between autophagy and cytokine, ROS production, vitamin D, and inflammatory response. Novel autophagy-based therapy is also summarized. This integrative insight might add some novel thoughts for better tuberculosis medications.     

Suppression of cell-mediated immunity by street rabies virus infection.

An attempt to define a severe suppression of cell-mediated immunity by street rabies virus infection was undertaken by using the mice lethally and peripherally infected with a street rabies virus (1088 strain). The cell-mediated cytotoxic (CMC) activity of the spleen cells from those mice once slightly increased until day 4 after infection but declined rapidly thereafter until their death on days 10 to 12 after infection. In parallel with a decrease of CMC response of the spleen cells from 1088-infected mice, proliferative response to Con A, IL-2 activity in the culture supernatants of Con A-induced proliferation, responsiveness to exogenously added IL-2 and to Con A to express IL-2R, of those cells became suppressed, and the marked decrease of the total number of spleen cells was observed. Selective depletion of CD4+ and CD8+ cells in the spleens, abnormalities of IL-1 and E-type prostaglandins (PGE2) production or production of inhibitory component able to block IL-2 activity by spleen cells were not observed and these factors did not appear to be associated with the suppression of proliferative response to Con A. However, an apparent association of CD8+ cells in the suppression of differentiation of pre-cytotoxic lymphocytes (CTL) into CTL was demonstrated in the co-culture experiments of the spleen cells from 1088-infected mice with spleen cells of mice infected with an attenuated rabies virus (ERA strain) which can induce higher levels of CMC response. There was no evidence of the productive replication of rabies virus in thymus and spleen of 1088-infected mice. The relationship of these observations to current theories on virus-induced immunosuppression was discussed.     

Protective immunity to Mycobacterium tuberculosis infection by chemokine and cytokine conditioned CFP-10 differentiated dendritic cells

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.     

FasL regulatory B-cells during Mycobacterium tuberculosis infection and TB disease

Tuberculosis (TB) disease remains a major health crisis. Infection with Mycobacterium tuberculosis (M.tb) cause a range of diseases ranging from latent infection to active TB disease. This active state of the disease is characterised by the formation of granulomas (a physical barrier in the lung), a structure thought to protect the host by controlling the infection through preventing the growth of the bacilli. Subsequently, the surviving bacteria become inactive and in most cases, TB reactivation is prevented by the immune response of the host. B-cells perform numerous immunological functions beyond antibody production to positively regulate the response to pathogenic assault. A subgroup of B-cells with regulatory functions express death-inducing ligands, such as Fas ligand (FasL). Expression and interaction of the Fas receptor-ligand promotes the induction of apoptosis and the induction of T-cell tolerance. Here, we focus on the significance of B-cells by addressing their FasL phenotype and regulatory functions during TB, with reference to disease in humans, non-human primates and mice.     

Highly focused T cell responses in latent human pulmonary Mycobacterium tuberculosis infection

The elucidation of the molecular and immunological mechanisms mediating maintenance of latency in human tuberculosis aids to develop more effective vaccines and to define biologically meaningful markers for immune protection. We analyzed granuloma-associated lymphocytes (GALs) from human lung biopsies of five patients with latent Mycobacterium tuberculosis (MTB) infection. MTB CD4+ and CD8+ T cell response was highly focused in the lung, distinct from PBL, as assessed by TCR-CDR3 spectratyping coupled with a quantitative analysis of TCR VB frequencies. GALs produced IFN-gamma in response to autologous macrophages infected with MTB and to defined MTB-derived HLA-A2-presented peptides Ag85a242-250, Ag85b199-207, early secreted antigenic target 6 (ESAT-6)28-36, 19-kDa Ag88-97, or the HLA-DR-presented ESAT-6(1-20) epitope. Immune recognition of naturally processed and presented MTB epitopes or the peptide ESAT-6(1-20) could be linked to specific TCR VB families, and in two patients to unique T cell clones that constituted 19 and 27%, respectively, of the CD4+ and 17% of the CD8+ GAL population. In situ examination of MTB-reactive GALs by tetramer in situ staining and confocal laser-scanning microscopy consolidates the presence of MHC class I-restricted CD8+ T cells in MTB granuloma lesions and supports the notion that clonally expanded T cells are crucial in immune surveillance against MTB.     

Key genes and pathways in tumor-educated dendritic cells by bioinformatical analysis

Specific tumor microenvironment signaling might prevent the maturation of dendritic cells (DCs) with tolerogenic and immunosuppressive potential accounting for antigen-specific unresponsiveness in the lymphoid organs and in the periphery. In the present study, dendritic cells treated with LLC lung cancer cell or 4T1 breast cancer cell culture supernatants significantly down-regulated the expression of co-stimulatory molecules MHC-II, CD40, CD80, but up-regulated the inhibitory molecule PD-L1/L2, VISTA, and increased the messengerRNA levels of interleukin (IL)-6, arginase I, and IL-10, but decreased tumor necrosis factor-α and IL-12a. RNA was isolated from the dendritic cells with or without tumor supernatant stimulation and RNA sequencing was done. Then the differential expression genes were sorted, the candidate genes were analyzed and pathway enrichment analysis was done, and the associated protein–protein interaction network (PPI) was established. After integrated bioinformatical analysis, 405 (279 up-regulated and 126 down-regulated) consistently differential expression genes were identified. Using gene ontology and pathway analysis, it was found that differential expression genes were mainly enriched in the immune response, cell–cell interaction, hemostasis, and cell surface interactions with the vascular wall. The PPI data demonstrated that 236 nodes were classified with 1072 edges, and the most remarkable three modules involved 53 central node genes associated with cell survival, cell-substrate adhesion, chemotaxis, migration, immune response, and complement receptor mediated signaling pathway. These findings revealed the immune status of dendritic cells in the tumor environment.     

Multidimensional analysis of immune responses identified biomarkers of recent Mycobacterium tuberculosis infection

The risk of tuberculosis (TB) disease is higher in individuals with recent Mycobacterium tuberculosis (M.tb) infection compared to individuals with more remote, established infection. We aimed to define blood-based biomarkers to distinguish between recent and remote infection, which would allow targeting of recently infected individuals for preventive TB treatment. We hypothesized that integration of multiple immune measurements would outperform the diagnostic performance of a single biomarker. Analysis was performed on different components of the immune system, including adaptive and innate responses to mycobacteria, measured on recently and remotely M.tb infected adolescents. The datasets were standardized using variance stabilizing scaling and missing values were imputed using a multiple factor analysis-based approach. For data integration, we compared the performance of a Multiple Tuning Parameter Elastic Net (MTP-EN) to a standard EN model, which was built to the individual adaptive and innate datasets. Biomarkers with non-zero coefficients from the optimal single data EN models were then isolated to build logistic regression models. A decision tree and random forest model were used for statistical confirmation. We found no difference in the predictive performances of the optimal MTP-EN model and the EN model [average area under the receiver operating curve (AUROC) = 0.93]. EN models built to the integrated dataset and the adaptive dataset yielded identically high AUROC values (average AUROC = 0.91), while the innate data EN model performed poorly (average AUROC = 0.62). Results also indicated that integration of adaptive and innate biomarkers did not outperform the adaptive biomarkers alone (Likelihood Ratio Test χ² = 6.09, p = 0.808). From a total of 193 variables, the level of HLA-DR on ESAT6/CFP10-specific Th1 cytokine-expressing CD4 cells was the strongest biomarker for recent M.tb infection. The discriminatory ability of this variable was confirmed in both tree-based models.A single biomarker measuring M.tb-specific T cell activation yielded excellent diagnostic potential to distinguish between recent and remote M.tb infection.     

Calcium-dependent proteolytic activity of a cysteine protease caldonopain is detected during Leishmania infection

A calcium-activated protease caldonopain in the cytosolic fraction of Leishmania donovani has been found to digest different endogenous proteins when subjected to SDS-PAGE. Gelatin-embedded gel electrophoresis confirms presence of calcium-dependent protease activity. Ca²⁺ affects proteolytic activity after 10 h. When host–parasite interaction was conducted in vitro, caldonopain was found to be active after 10 h of incubation with calcium. A 67-kDa protein is specifically digested during this time and two new proteins of 45 and 36 kDa appeared in SDS-PAGE electrophoregram. This belated action of calcium towards protease activity may be pre-requisite to facilitate invasion of host tissues and thereby mediate protein metabolism during survival of this pathogen both independently and intracellularly. It is likely that calcium metabolism in promastigotes and amastigotes does not propagate in the same manner. Involvement of calcium to initiate caldonopain activity may be critically associated with signal transduction pathways which may be responsible for the pathobiological action of this parasite. We propose that caldonopain could be a potential target to develop new chemotherapeutic approach against leishmaniasis.     

Identifying vulnerable pathways in Mycobacterium tuberculosis by using a knockdown approach

We constructed recombinant strains of Mycobacterium tuberculosis in which expression of specific genes was downregulated to identify vulnerable drug targets. Growth phenotypes in macrophages and culture were used to rank targets: the dprE1, clpP1, and fadD32 operons were the best targets and glnA1, glnE, pknL, regX3, and senX3 were poor targets.     

Florid pulmonary inflammatory responses in mice vaccinated with Antigen-85 pulsed dendritic cells and challenged by aerosol with Mycobacterium tuberculosis

Mice immunized by the intranasal route with dendritic cells harvested from the lungs and then pulsed with Ag85 (LDC-Ag85) were able to prime naive CD4(+) T cells in vivo. As a result splenic CD4(+) T cells from these immunized mice were able to produce IFNgamma following culture with Mycobacterium tuberculosis-infected antigen presenting cells. Hematoxylin and eosin stained lung sections from LDC-Ag85 immunized mice after they had been exposed to aerosol challenge with M. tuberculosis showed a florid infiltration of macrophages and lymphocytes into granulomas and parenchymal tissues when compared to lung sections from control groups implanted with dendritic cells pulsed with ovalbumin. In addition, using immunohistochemistry, these tissues appeared to have more CD4(+) and CD8(+) cells than the control groups. This was confirmed by flow cytometric analysis which showed that lung cell digests contained increased numbers of CD4 and CD8 interferongamma secreting cells. Despite this increase however, no evidence was seen that indicated that the LDC-Ag85 immunized mice were more resistant to M. tuberculosis infection than mice immunized with LDC pulsed with an irrelevant protein. Instead, the potent inflammatory response in the LDC-Ag85 resulted in serious consolidation of the lung tissue.     

Glutathione and adaptive immune responses against Mycobacterium tuberculosis infection in healthy and HIV infected individuals

Glutathione (GSH), a tripeptide antioxidant, is essential for cellular homeostasis and plays a vital role in diverse cellular functions. Individuals who are infected with Human immuno deficiency virus (HIV) are known to be susceptible to Mycobacterium tuberculosis (M. tb) infection. We report that by enhancing GSH levels, T-cells are able to inhibit the growth of M. tb inside macrophages. In addition, those GSH-replenished T cell cultures produced increased levels of Interleukin-2 (IL-2), Interleukin-12 (IL-12), and Interferon-gamma (IFN-γ), cytokines, which are known to be crucial for the control of intracellular pathogens. Our study reveals that T lymphocytes that are derived from HIV infected individuals are deficient in GSH, and that this deficiency correlates with decreased levels of Th1 cytokines and enhanced growth of M. tb inside human macrophages.     

Nramp1 expression by dendritic cells modulates inflammatory responses during Salmonella Typhimurium infection

Host resistance against Salmonella enterica serovar Typhimurium ( S . Typhimurium) is mediated by natural resistance-associated macrophage protein 1 (Nramp1/Slc11a1). Nramp1 is critical to host defence, as mice lacking Nramp1 fail to control bacterial replication and succumb to low doses of S . Typhimurium. Despite this crucial role, the mechanisms underlying Nramp1's protective effects are unclear. Dendritic cells (DCs) that sample the intestinal lumen are among the first cells encountered by S. Typhimurium following oral infection and act as a conduit for S. Typhimurium to cross the intestinal epithelial barrier. We report that DCs, including intestinal, splenic and bone marrow-derived DCs (BMDCs), express Nramp1 protein. In the small intestine, Nramp1 expression is greater in a subset of DCs (CD11c⁺CD103^-) characterized by the elevated expression of pro-inflammatory cytokines in response to bacterial products. While Nramp1 expression did not affect S. Typhimurium replication in BMDCs, infected Nramp1+/+ BMDCs and intestinal CD11c⁺CD103^- DCs secreted more inflammatory cytokines (IL-6, IL-12 and TNF-α) than Nramp1−/−, suggesting that Nramp1 expression may promote a more rapid inflammatory response following infection. Collectively, these findings reveal a new role for DCs and Nramp1 in modulating the host inflammatory response to S. Typhimurium.     

Effects of morphine during Mycobacterium tuberculosis H37Rv infection in mice

The effects of opiates in various infections are well known; however, very little is known about tuberculosis infection. Therefore, in the present study, we report for the first time, the effects of morphine during murine tuberculosis. Mice were infected intravenously with Mycobacterium tuberculosis H37Rv, administered morphine (0.1-100 mg/kg subcutaneously on day 0 and day +15) and sacrificed on day +30 for CFU enumeration in lungs and spleen. Morphine exerted maximum suppression of infection at 5 mg/kg, and sometimes completes elimination of infection; naloxone, silica and aminoguanidine blocked the protective effect of morphine. In vitro, morphine lacked direct antimycobacterial activity up to 1x10(-4) M concentration, as assessed by radiometric BACTEC method. In macrophage model of infection, morphine showed maximal killing at 1x10(-7) M concentration, the activity was blocked by naloxone and aminoguanidine. These observations suggest that morphine exerts a dose-dependent effect in murine tuberculosis, the protective effect being naloxone-reversible and may involve macrophage-mediated protective mechanisms. These results may be helpful in developing new opioid-like chemical entities against tuberculosis infection.