Rapamycin modulates pulmonary pathology in a murine model of Mycobacterium tuberculosis infection.

Tuberculosis (TB) treatment regimens are lengthy, causing non-adherence to treatment. Inadequate treatment can lead to relapse and the development of drug resistance TB. Furthermore, patients often exhibit residual lung damage even after cure, increasing the risk for relapse and development of other chronic respiratory illnesses. Host-directed therapeutics are emerging as an attractive means to augment the success of TB treatment. In this study, we used C3HeB/FeJ mice as an experimental model to investigate the potential role of rapamycin, a mammalian target of rapamycin inhibitor, as an adjunctive therapy candidate during the treatment of Mycobacterium tuberculosis infection with moxifloxacin. We report that administration of rapamycin with or without moxifloxacin reduced infection-induced lung inflammation, and the number and size of caseating necrotic granulomas. Results from this study strengthen the potential use of rapamycin and its analogs as adjunct TB therapy, and importantly underscore the utility of the C3HeB/FeJ mouse model as a preclinical tool for evaluating host-directed therapy candidates for the treatment of TB.

Transmission phenotype of Mycobacterium tuberculosis strains is mechanistically linked to induction of distinct pulmonary pathology.

In a study of household contacts (HHC), households were categorized into High (HT) and Low (LT) transmission groups based on the proportion of HHC with a positive tuberculin skin test. The Mycobacterium tuberculosis (Mtb) strains from HT and LT index cases of the households were designated Mtb-HT and Mtb-LT, respectively. We found that C3HeB/FeJ mice infected with Mtb-LT strains exhibited significantly higher bacterial burden compared to Mtb-HT strains and also developed diffused inflammatory lung pathology. In stark contrast, a significant number of mice infected with Mtb-HT strains developed caseating granulomas, a lesion type with high potential to cavitate. None of the Mtb-HT infected animals developed diffused inflammatory lung pathology. A link was observed between increased in vitro replication of Mtb-LT strains and their ability to induce significantly high lipid droplet formation in macrophages. These results support that distinct early interactions of Mtb-HT and Mtb-LT strains with macrophages and subsequent differential trajectories in pathological disease may be the mechanism underlying their transmission potential.

Divergent Functions of TLR2 on Hematopoietic and Nonhematopoietic Cells during Chronic Mycobacterium tuberculosis Infection.

We have reported that TLR2 is crucial for host resistance against chronic Mycobacterium tuberculosis infection; however, which cell types are key players in this response remain unknown. This led us to decipher the relative contribution of TLR2 on nonhematopoietic and hematopoietic cells in resistance against chronic M. tuberculosis infection in mice infected with M. tuberculosis Erdman. Consistent with our previous report, at 8 wk of infection, TLR2 knockout (TLR2KO)→TLR2KO bone marrow chimeric mice exhibited increased bacterial burden, disorganized accumulation of lymphocytes and mononuclear cells, and extensive pulmonary immunopathology compared with wild-type (WT)→WT chimeric mice. Bacterial burden and pulmonary immunopathology of chimeric mice lacking TLR2 in the hematopoietic compartment (TLR2KO→WT) was comparable to TLR2KO mice. In contrast, chimeric mice deficient in TLR2 in the nonhematopoietic compartment (WT→TLR2KO) exhibited a marked attenuation in granulomatous inflammation compared with WT mice. Although the latter mice did not exhibit improved pulmonary bacterial control, significant reductions in bacterial burden in the draining lymph nodes, spleen, and liver were observed. These findings establish that the TLR2-mediated hematopoietic response promotes stable control of pulmonary bacterial burden and granuloma integrity, whereas TLR2 signaling on nonhematopoietic cells may partly facilitate granulomatous inflammation and bacterial dissemination.

1,25 (OH)2D3 treatment alters the granulomatous response in M. tuberculosis infected mice.

Induction of cathelicidin-mediated antimicrobial pathway against intracellular M. tuberculosis by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the active form of vitamin D, has been documented in vitro. However, in in vivo studies related to inflammatory disorders, 1,25(OH)2D3 has been demonstrated to induce an anti-inflammatory response. We therefore examined whether in the murine model of tuberculosis, the anti-inflammatory effects of 1,25(OH)2D3 would affect the outcome of M. tuberculosis infection. We show here that administration of 1,25(OH)2D3 to M. tuberculosis infected mice led to a change in lung granuloma architecture, characterized by a marked decrease in B cell lymphocytic aggregates. Consistent with the altered granulomas, 1,25(OH)2D3-treated mice also exhibited significantly higher bacterial burden in the lungs compared to the control group. These findings highlight the need to further investigate the effect of vitamin D on host immunity to M. tuberculosis in the context of the granulomatous response.

Disruption of an M. tuberculosis membrane protein causes a magnesium-dependent cell division defect and failure to persist in mice.

The identification of Mycobacterium tuberculosis genes necessary for persistence in vivo provides insight into bacterial biology as well as host defense strategies. We show that disruption of M. tuberculosis membrane protein PerM (Rv0955) resulted in an IFN-γ-dependent persistence defect in chronic mouse infection despite the mutant's near normal growth during acute infection. The perM mutant required increased magnesium for replication and survival; incubation in low magnesium media resulted in cell elongation and lysis. Transcriptome analysis of the perM mutant grown in reduced magnesium revealed upregulation of cell division and cell wall biosynthesis genes, and live cell imaging showed PerM accumulation at the division septa in M. smegmatis. The mutant was acutely sensitive to ß-lactam antibiotics, including specific inhibitors of cell division-associated peptidoglycan transpeptidase FtsI. Together, these data implicate PerM as a novel player in mycobacterial cell division and pathogenesis, and are consistent with the hypothesis that immune activation deprives M. tuberculosis of magnesium.

Neither primary nor memory immunity to Mycobacterium tuberculosis infection is compromised in mice with chronic enteric helminth infection.

Previously we had reported that Nippostrongylus brasiliensis, a helminth with a lung migratory phase, affected host resistance against Mycobacterium tuberculosis infection through the induction of alternatively activated (M2) macrophages. Several helminth species do not have an obligatory lung migratory phase but establish chronic infections in the host that include potent immune downregulatory effects, in part mediated through induction of a FoxP3(+) T regulatory cell (Treg) response. Treg cells exhibit duality in their functions in host defense against M. tuberculosis infection since their depletion leads to enhanced priming of T cells in the lymph nodes and attendant improved control of M. tuberculosis infection, while their presence in the lung granuloma protects against excessive inflammation. Heligmosomoides polygyrus is a strictly murine enteric nematode that induces a strong FoxP3 Treg response in the host. Therefore, in this study we investigated whether host immunity to M. tuberculosis infection would be modulated in mice with chronic H. polygyrus infection. We report that neither primary nor memory immunity conferred by Mycobacterium bovis BCG vaccination was affected in mice with chronic enteric helminth infection, despite a systemic increase in FoxP3(+) T regulatory cells. The findings indicate that anti-M. tuberculosis immunity is not similarly affected by all helminth species and highlight the need to consider this inequality in human coinfection studies.

Quest for correlates of protection against tuberculosis.

A major impediment to tuberculosis (TB) vaccine development is the lack of reliable correlates of immune protection or biomarkers that would predict vaccine efficacy. Gamma interferon (IFN-γ) produced by CD4(+) T cells and, recently, multifunctional CD4(+) T cells secreting IFN-γ, tumor necrosis factor (TNF), and interleukin-2 (IL-2) have been used in vaccine studies as a measurable immune parameter, reflecting activity of a vaccine and potentially predicting protection. However, accumulating experimental evidence suggests that host resistance against Mycobacterium tuberculosis infection is independent of IFN-γ and TNF secretion from CD4(+) T cells. Furthermore, the booster vaccine MVA85A, despite generating a high level of multifunctional CD4(+) T cell response in the host, failed to confer enhanced protection in vaccinated subjects. These findings suggest the need for identifying reliable correlates of protection to determine the efficacy of TB vaccine candidates. This article focuses on alternative pathways that mediate M. tuberculosis control and their potential for serving as markers of protection. The review also discusses the significance of investigating the natural human immune response to M. tuberculosis to identify the correlates of protection in vaccination.

Equivalent functions for B7.1 and B7.2 costimulation in mediating host resistance to Mycobacterium tuberculosis.

B7.1 and B7.2 are homologous costimulatory molecules expressed predominantly on antigen-presenting cells (APC). Interaction of these B7 molecules with CD28 and CTLA-4 expressed on T cells is a critical step in T cell activation. Previously, we reported that Mycobacterium tuberculosis infection in the combined absence of B7.1 and B7.2 resulted in impaired host resistance to the pathogen. Despite their structural similarities, the individual contribution of B7.1 and B7.2 to the development of pathogenic T cells in autoimmune diseases and protective T cells in infectious diseases is markedly distinct. In the current study, we therefore examined whether B7.1 and B7.2 have discrete, equivalent, or overlapping functions in mediating host resistance to M. tuberculosis. We found that the individual absence of either B7.1 or B7.2 had no effect on the ability of the host to contain bacterial load in the lungs, recruit immune cells to the lung, generate a Th1 response, or induce a pulmonary granulomatous response. These results indicate that B7.1 and B7.2 molecules have equal ability to mediate host resistance to M. tuberculosis, underscoring the therapeutic utility of individual B7.1 and B7.2 antagonists in treating inflammatory disorders.

Preexisting helminth infection induces inhibition of innate pulmonary anti-tuberculosis defense by engaging the IL-4 receptor pathway.

Tuberculosis and helminthic infections coexist in many parts of the world, yet the impact of helminth-elicited Th2 responses on the ability of the host to control Mycobacterium tuberculosis (Mtb) infection has not been fully explored. We show that mice infected with the intestinal helminth Nippostrongylus brasiliensis (Nb) exhibit a transitory impairment of resistance to airborne Mtb infection. Furthermore, a second dose of Nb infection substantially increases the bacterial burden in the lungs of co-infected mice. Interestingly, the Th2 response in the co-infected animals did not impair the onset and development of the protective Mtb-specific Th1 cellular immune responses. However, the helminth-induced Th2 environment resulted in the accumulation of alternatively activated macrophages (AAMs) in the lung. Co-infected mice lacking interleukin (IL) 4Rα exhibited improved ability to control Mtb infection, which was accompanied by significantly reduced accumulation of AAMs. Moreover, IL-4Rα(-/-) mice adoptively transferred with wild-type macrophages had a significantly higher Mtb load in their lungs compared with those that received IL-4Rα(-/-) macrophages, suggesting a direct contribution for the IL-4R pathway to the heightened susceptibility of co-infected animals. The Th2 response can thus enhance the intracellular persistence of Mtb, in part by mediating the alternative activation of macrophages via the IL-4Rα signaling pathway.

Development of a secondary immune response to Mycobacterium tuberculosis is independent of Toll-like receptor 2.

Published work indicates that the contribution of Toll-like receptor 2 (TLR2) to host resistance during acute Mycobacterium tuberculosis infection is marginal. However, in these studies, TLR2 participation in the memory immune response to M. tuberculosis was not determined. The substantial in vitro evidence that M. tuberculosis strongly triggers TLR2 on dendritic cells and macrophages to bring about either activation or inhibition of antigen-presenting cell (APC) functions, along with accumulating evidence that memory T cell development can be calibrated by TLR signals, led us to question the role of TLR2 in host resistance to secondary challenge with M. tuberculosis. To address this question, a memory immunity model was employed, and the response of TLR2-deficient (TLR2 knockout [TLR2KO]) mice following a secondary exposure to M. tuberculosis was compared to that of wild-type (WT) mice based on assessment of the bacterial burden, recall response, phenotype of recruited T cells, and granulomatous response. We found that upon rechallenge with M. tuberculosis, both WT and TLR2KO immune mice displayed similarly enhanced resistance to infection in comparison to their naïve counterparts. The frequencies of M. tuberculosis-specific gamma interferon (IFN-γ)-producing T cells, the phenotypes of recruited T cells, and the granulomatous responses were also similar between WT and TLR2KO immune mice. Together, the findings from this study indicate that TLR2 signaling does not influence memory immunity to M. tuberculosis.

B7 costimulation is critical for host control of chronic Mycobacterium tuberculosis infection.

Although much is understood regarding the role of B7/CD28 family of costimulatory molecules in regulating host resistance in the context of several pathogens, analogous information with Mycobacterium tuberculosis is lacking. To address the requirements of B7-mediated costimulation in host resistance against tuberculosis, mice deficient in both B7.1 and B7.2 (B7DKO) were aerosol infected with M. tuberculosis Erdman and disease progression was monitored. We report herein that B7DKO mice are initially able to contain the bacterial load in the lung, but exhibit enhanced susceptibility during chronic infection. Despite the early control of bacterial replication, B7DKO mice essentially start off with compromised Th1 immunity and slower granulomatous response in the lung, characterized by markedly reduced lymphocytic infiltration. As the infection progresses from acute phase to the chronic phase, the nascent granulomas in the B7DKO lungs never fully achieve the architecture of granulomas developing in wild-type mice. Instead, lesions spread progressively to involve much of the lung in the B7DKO mice, ultimately leading to necrosis. Thus, early control of M. tuberculosis growth in the lung can occur in the absence of B7 costimulation and is less dependent on Th1 immunity and formation of a granulomatous structure. However, B7 costimulation is critical for long-term containment of infection within lung granulomas. These findings suggest that the use of costimulation-based immunomodulators may have significant repercussions on the induction of host protective immunity against tuberculosis.

Comparative evaluation of cytokines, T-cell apoptosis, and costimulatory molecule expression in tuberculous and nontuberculous pleurisy.

In this study, we compared several immune parameters in tuberculosis (TB) and nontuberculosis (NTB) pleurisy to gain an understanding of the mechanism behind enhanced Th1 apoptosis that occurs at sites of active Myobacterium tuberculosis (M. tuberculosis) infection. An initial evaluation of the accumulated cytokines in pleural fluid (PF) demonstrated that both TB and NTB pleurisy were associated with prointflammatory cytokines, while only TB pleurisy had augmented expression of interferon (IFN)-gamma and soluble Fas ligand (sFASL). Despite enhanced expression of the apoptosis-inducing molecule in TB pleurisy, T cells derived from both types of pleurisy exhibited significant apoptosis. In both groups, T-cell apoptosis correlated with low expression of CD80 on PF-derived macrophages and elevated accumulation of TGF-beta in the PF. A causative correlation between TGF-beta and low CD80 expression in the two groups was established by in vitro studies demonstrating TGF-beta inhibition of CD80 upregulation in a macrophage cell line. Together, the findings allude to the possibility that activation in the absence of appropriate CD80 costimulation is the mechanism that leads to T-cell apoptosis at sites of active M. tuberculosis infection. Furthermore, the findings also indicate that T-cell apoptosis is perhaps a host regulatory mechanism to limit inflammation, rather than a pathogen-induced immune deviation.

Host innate immune response to Mycobacterium tuberculosis.

This review focuses on recent progress in our understanding of Mycobacterium tuberculosis survival in macrophages, the interaction of M. tuberculosis with Toll-like receptors (TLRs) and the establishment of the link between innate and adaptive immunity, and TLRs and interferon-gamma-mediated antimicrobial pathways in macrophages. We also propose a paradigm that TLR2 signaling regulates the magnitude of the host Th1 response leading to either M. tuberculosis persistence and latent infection or replication and disease.

Cutting edge: a new approach to modeling early lung immunity in murine tuberculosis.

In this study, we report a new approach that allows dissection of distinct pathways regulating induction of early adaptive immunity in response to Mycobacterium tuberculosis (Mtb). We used traceable murine dendritic cells (DCs) and macrophage populations to chart their migratory pattern in response to Mtb, and found that only DCs receiving inflammatory stimuli from Mtb up-regulated their expression of CCR7 and migrated specifically to the draining lymph nodes (LNs). Furthermore, these Mtb-modulated DCs initiated a Th1 response only in the draining LNs. Taken together, these results demonstrate that Mtb-induced modulation of DCs is critical for their migration to regional LNs and ensuing T cell priming.