IL-10 Impairs Local Immune Response in Lung Granulomas and Lymph Nodes during Early Mycobacterium tuberculosis Infection.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, continues to be a major global health problem. Lung granulomas are organized structures of host immune cells that function to contain the bacteria. Cytokine expression is a critical component of the protective immune response, but inappropriate cytokine expression can exacerbate TB. Although the importance of proinflammatory cytokines in controlling M. tuberculosis infection has been established, the effects of anti-inflammatory cytokines, such as IL-10, in TB are less well understood. To investigate the role of IL-10, we used an Ab to neutralize IL-10 in cynomolgus macaques during M. tuberculosis infection. Anti-IL-10-treated nonhuman primates had similar overall disease outcomes compared with untreated control nonhuman primates, but there were immunological changes in granulomas and lymph nodes from anti-IL-10-treated animals. There was less thoracic inflammation and increased cytokine production in lung granulomas and lymph nodes from IL-10-neutralized animals at 3-4 wk postinfection compared with control animals. At 8 wk postinfection, lung granulomas from IL-10-neutralized animals had reduced cytokine production but increased fibrosis relative to control animals. Although these immunological changes did not affect the overall disease burden during the first 8 wk of infection, we paired computational modeling to explore late infection dynamics. Our findings support that early changes occurring in the absence of IL-10 may lead to better bacterial control later during infection. These unique datasets provide insight into the contribution of IL-10 to the immunological balance necessary for granulomas to control bacterial burden and disease pathology in M. tuberculosis infection.

Concurrent infection with Mycobacterium tuberculosis confers robust protection against secondary infection in macaques.

For many pathogens, including most targets of effective vaccines, infection elicits an immune response that confers significant protection against reinfection. There has been significant debate as to whether natural Mycobacterium tuberculosis (Mtb) infection confers protection against reinfection. Here we experimentally assessed the protection conferred by concurrent Mtb infection in macaques, a robust experimental model of human tuberculosis (TB), using a combination of serial imaging and Mtb challenge strains differentiated by DNA identifiers. Strikingly, ongoing Mtb infection provided complete protection against establishment of secondary infection in over half of the macaques and allowed near sterilizing bacterial control for those in which a secondary infection was established. By contrast, boosted BCG vaccination reduced granuloma inflammation but had no impact on early granuloma bacterial burden. These findings are evidence of highly effective concomitant mycobacterial immunity in the lung, which may inform TB vaccine design and development.

Low Levels of T Cell Exhaustion in Tuberculous Lung Granulomas.

The hallmarks of pulmonary Mycobacterium tuberculosis infection are lung granulomas. These organized structures are composed of host immune cells whose purpose is to contain or clear infection, creating a complex hub of immune and bacterial cell activity, as well as limiting pathology in the lungs. Yet, given cellular activity and the potential for frequent interactions between host immune cells and M. tuberculosis-infected cells, we observed a surprisingly low quantity of cytokine-producing T cells (<10% of granuloma T cells) in our recent study of M. tuberculosis infection within nonhuman primate (NHP) granulomas. Various mechanisms could limit T cell function, and one hypothesis is T cell exhaustion. T cell exhaustion is proposed to result from continual antigen stimulation, inducing them to enter a state characterized by low cytokine production, low proliferation, and expression of a series of inhibitory receptors, the most common being PD-1, LAG-3, and CTLA-4. In this work, we characterized the expression of inhibitory receptors on T cells and the functionality of these cells in tuberculosis (TB) lung granulomas. We then used these experimental data to calibrate and inform an agent-based computational model that captures environmental, cellular, and bacterial dynamics within granulomas in lungs during M. tuberculosis infection. Together, the results of the modeling and the experimental work suggest that T cell exhaustion alone is not responsible for the low quantity of M. tuberculosis-responsive T cells observed within TB granulomas and that the lack of exhaustion is likely an intrinsic property of granuloma structure.

Active transforming growth factor-ß is associated with phenotypic changes in granulomas after drug treatment in pulmonary tuberculosis.

BACKGROUND: Tuberculosis (TB) chemotherapy clears bacterial burden in the lungs of patients and allows the tuberculous lesions to heal through a fibrotic process. The healing process leaves pulmonary scar tissue that can impair lung function. The goal of this study was to identify fibrotic mediators as a stepping-stone to begin exploring mechanisms of tissue repair in TB. METHODS: Hematoxylin and eosin staining and Masson's trichrome stain were utilized to determine levels of collagenization in tuberculous granulomas from non-human primates. Immunohistochemistry was then employed to further interrogate these granulomas for markers associated with fibrogenesis, including transforming growth factor-ß (TGFß), α-smooth muscle actin (αSMA), phosphorylated SMAD-2/3, and CD163. These markers were compared across states of drug treatment using one-way ANOVA, and Pearson's test was used to determine the association of these markers with one another. RESULTS: TGFß and αSMA were present in granulomas from primates with active TB disease. These molecules were reduced in abundance after TB chemotherapy. Phosphorylated SMAD-2/3, a signaling intermediate of TGFß, was observed in greater amounts after 1 month of drug treatment than in active disease, suggesting that this particular pathway is blocked in active disease. Collagen production during tissue repair is strongly associated with TGFß in this model, but not with CD163+ macrophages. CONCLUSIONS: Tissue repair and fibrosis in TB that occurs during drug treatment is associated with active TGFß that is produced during active disease. Further work will identify mechanisms of fibrosis and work towards mitigating lung impairment with treatments that target those mechanisms.

Effects of B Cell Depletion on Early Mycobacterium tuberculosis Infection in Cynomolgus Macaques.

Although recent studies in mice have shown that components of B cell and humoral immunity can modulate the immune responses against Mycobacterium tuberculosis, the roles of these components in human and nonhuman primate infections are unknown. The cynomolgus macaque (Macaca fascicularis) model of M. tuberculosis infection closely mirrors the infection outcomes and pathology in human tuberculosis (TB). The present study used rituximab, an anti-CD20 antibody, to deplete B cells in M. tuberculosis-infected macaques to examine the contribution of B cells and humoral immunity to the control of TB in nonhuman primates during the acute phase of infection. While there was no difference in the overall pathology, disease profession, and clinical outcome between the rituximab-treated and untreated macaques in acute infection, analyzing individual granulomas revealed that B cell depletion resulted in altered local T cell and cytokine responses, increased bacterial burden, and lower levels of inflammation. There were elevated frequencies of T cells producing interleukin-2 (IL-2), IL-10, and IL-17 and decreased IL-6 and IL-10 levels within granulomas from B cell-depleted animals. The effects of B cell depletion varied among granulomas in an individual animal, as well as among animals, underscoring the previously reported heterogeneity of local immunologic characteristics of tuberculous granulomas in nonhuman primates. Taken together, our data clearly showed that B cells can modulate the local granulomatous response in M. tuberculosis-infected macaques during acute infection. The impact of these alterations on disease progression and outcome in the chronic phase remains to be determined.

Construction of a tetracycline inducible expression vector and characterization of its use in Vibrio cholerae.

We report the construction of a tetracycline inducible expression vector that allows regulated gene expression in the enteric pathogen Vibrio cholerae. The expression vector, named pXB300, contains the tetracycline regulatory elements from Tn10, a multiple cloning site downstream of the tetA promoter and operator sequences, a ColE1 origin of replication, a ß-lactamase resistance gene for positive selection, and the hok/sok addiction system for selection in the absence of antibiotic. The function of the tetracycline expression system was demonstrated by cloning lacZ under control of the tetA promoter and quantifying ß-galactosidase expression in Escherichia coli and V. cholerae. The utility for pXB300 was documented by complementation of V. cholerae virulence mutants during growth under virulence inducing conditions. The results showed that pXB300 allowed high-level expression of recombinant genes with linear induction in response to the exogenous concentration of the inducer anhydrotetracycline. We further show that pXB300 was reliably maintained in V. cholerae during growth in the absence of antibiotic selection.