Granulocyte recruitment to the pulmonary compartment is a hallmark of progressive tuberculosis (TB). This process is well-documented to promote immunopathology, but can also enhance the replication of the pathogen. Both the specific granulocytes responsible for increasing mycobacterial burden and the underlying mechanisms remain obscure. We report that the known immunomodulatory effects of these cells, such as suppression of protective T-cell responses, play a limited role in altering host control of mycobacterial replication in susceptible mice. Instead, we find that the adaptive immune response preferentially restricts the burden of bacteria within monocytes and macrophages compared to granulocytes. Specifically, mycobacteria within inflammatory lesions are preferentially found within long-lived granulocytes that express intermediate levels of the Ly6G marker and low levels of antimicrobial genes. These cells progressively accumulate in the lung and correlate with bacterial load and disease severity, and the ablation of Ly6G-expressing cells lowers mycobacterial burden. These observations suggest a model in which dysregulated granulocytic influx promotes disease by creating a permissive intracellular niche for mycobacterial growth and persistence.

The outcome of an encounter with Mycobacterium tuberculosis (Mtb) depends on the pathogen’s ability to adapt to the heterogeneous immune response of the host. Understanding this interplay has proven difficult, largely because experimentally tractable small animal models do not recapitulate the heterogenous disease observed in natural infections. We leveraged the genetically diverse Collaborative Cross (CC) mouse panel in conjunction with a library of Mtb mutants to associate bacterial genetic requirements with host genetics and immunity. We report that CC strains vary dramatically in their susceptibility to infection and represent reproducible models of qualitatively distinct immune states. Global analysis of Mtb mutant fitness across the CC panel revealed that a large fraction of the pathogen’s genome is necessary for adaptation to specific host microenvironments. Both immunological and bacterial traits were associated with genetic variants distributed across the mouse genome, elucidating the complex genetic landscape that underlies host-pathogen interactions in a diverse population.

In 2017 over 550,000 estimated new cases of multi-drug/rifampicin resistant tuberculosis (MDR/RR-TB) occurred, emphasizing a need for new treatment strategies. Linezolid (LZD) is a potent antibiotic for drug-resistant Gram-positive infections and is an effective treatment for TB. However, extended LZD use can lead to LZD-associated host toxicities, most commonly bone marrow suppression. LZD toxicities may be mediated by IL-1, an inflammatory pathway important for early immunity during M. tuberculosis infection. However, IL-1 can contribute to pathology and disease severity late in TB progression. Since IL-1 may contribute to LZD toxicity and does influence TB pathology, we targeted this pathway with a potential host-directed therapy (HDT). We hypothesized LZD efficacy could be enhanced by modulation of IL-1 pathway to reduce bone marrow toxicity and TB associated-inflammation. We used two animal models of TB to test our hypothesis, a TB-susceptible mouse model and clinically relevant cynomolgus macaques. Antagonizing IL-1 in mice with established infection reduced lung neutrophil numbers and partially restored the erythroid progenitor populations that are depleted by LZD. In macaques, we found no conclusive evidence of bone marrow suppression associated with LZD, indicating our treatment time may have been short enough to avoid the toxicities observed in humans. Though treatment was only 4 weeks (the FDA approved regimen at the time of study), we observed sterilization of the majority of granulomas regardless of co-administration of the FDA-approved IL-1 receptor antagonist (IL-1Rn), also known as Anakinra. However, total lung inflammation was significantly reduced in macaques treated with IL-1Rn and LZD compared to LZD alone. Importantly, IL-1Rn administration did not impair the host response against Mtb or LZD efficacy in either animal model. Together, our data support that inhibition of IL-1 in combination with LZD has potential to be an effective HDT for TB and the need for further research in this area.

In 2017, there were over 550,000 estimated new cases of multi-drug/rifampicin resistant tuberculosis (MDR/RR-TB), emphasizing a need for new treatment strategies. Linezolid (LZD) is a potent antibiotic for antibiotic-resistant Gram-positive infections and is an effective treatment for TB. However, extended LZD use can lead to LZD-associated host toxicities, most commonly bone marrow suppression. LZD toxicities may be mediated by IL-1, a pathway important for early immunity during M. tuberculosis infection that later contributes to pathology. We hypothesized LZD efficacy could be enhanced by modulation of IL-1 pathway to reduce BM toxicity and TB associated-inflammation. We used two animal models of TB to test our hypothesis, mice and cynomolgus macaques. Antagonizing IL-1 in chronically-infected mice reduced lung neutrophil numbers and partially restored the erythroid progenitor populations that are depleted by LZD. In macaques, we found no conclusive evidence of BM suppression associated with LZD, indicating our treatment time may have been short enough to avoid the toxicities observed in humans. Though treatment was only 1 month, the majority of granulomas were sterilized with reduced inflammation (assessed by PET/CT) in animals treated with both LZD and IL-1 receptor antagonist (IL-1Rn). However, overall lung inflammation was significantly reduced in macaques treated with both IL-1Rn and LZD, compared to LZD alone. Importantly, IL-1Rn administration did not noticeably impair the host response against Mtb or LZD efficacy in either animal model. Together, our data support that inhibition of IL-1 in combination with LZD has potential to be an effective HDT for TB.

Nitric oxide contributes to protection from tuberculosis. It is generally assumed that this protection is due to direct inhibition of Mycobacterium tuberculosis growth, which prevents subsequent pathological inflammation. In contrast, we report that nitric oxide primarily protects mice by repressing an interleukin-1- and 12/15-lipoxygenase-dependent neutrophil recruitment cascade that promotes bacterial replication. Using M. tuberculosis mutants as indicators of the pathogen's environment, we inferred that granulocytic inflammation generates a nutrient-replete niche that supports M. tuberculosis growth. Parallel clinical studies indicate that a similar inflammatory pathway promotes tuberculosis in patients. The human 12/15-lipoxygenase orthologue, ALOX12, is expressed in cavitary tuberculosis lesions; the abundance of its products correlates with the number of airway neutrophils and bacterial burden and a genetic polymorphism that increases ALOX12 expression is associated with tuberculosis risk. These data suggest that M. tuberculosis exploits neutrophilic inflammation to preferentially replicate at sites of tissue damage that promote contagion.

The outcome of Mycobacterium tuberculosis infection and the immunological response to the bacillus Calmette-Guerin (BCG) vaccine are highly variable in humans. Deciphering the relative importance of host genetics, environment, and vaccine preparation for the efficacy of BCG has proven difficult in natural populations. We developed a model system that captures the breadth of immunological responses observed in outbred individual mice, which can be used to understand the contribution of host genetics to vaccine efficacy. This system employs a panel of highly diverse inbred mouse strains, consisting of the founders and recombinant progeny of the "Collaborative Cross" project. Unlike natural populations, the structure of this panel allows the serial evaluation of genetically identical individuals and the quantification of genotype-specific effects of interventions such as vaccination. When analyzed in the aggregate, our panel resembled natural populations in several important respects: the animals displayed a broad range of susceptibility to M. tuberculosis, differed in their immunological responses to infection, and were not durably protected by BCG vaccination. However, when analyzed at the genotype level, we found that these phenotypic differences were heritable. M. tuberculosis susceptibility varied between lines, from extreme sensitivity to progressive M. tuberculosis clearance. Similarly, only a minority of the genotypes was protected by vaccination. The efficacy of BCG was genetically separable from susceptibility to M. tuberculosis, and the lack of efficacy in the aggregate analysis was driven by nonresponsive lines that mounted a qualitatively distinct response to infection. These observations support an important role for host genetic diversity in determining BCG efficacy and provide a new resource to rationally develop more broadly efficacious vaccines. IMPORTANCE: Tuberculosis (TB) remains an urgent global health crisis, and the efficacy of the currently used TB vaccine, M. bovis BCG, is highly variable. The design of more broadly efficacious vaccines depends on understanding the factors that limit the protection imparted by BCG. While these complex factors are difficult to disentangle in natural populations, we used a model population of mice to understand the role of host genetic composition in BCG efficacy. We found that the ability of BCG to protect mice with different genotypes was remarkably variable. The efficacy of BCG did not depend on the intrinsic susceptibility of the animal but, instead, correlated with qualitative differences in the immune responses to the pathogen. These studies suggest that host genetic polymorphism is a critical determinant of vaccine efficacy and provide a model system to develop interventions that will be useful in genetically diverse populations.

Interleukin 1 (IL-1) is an important mediator of innate immunity but can also promote inflammatory tissue damage. During chronic infections such as tuberculosis, the beneficial antimicrobial role of IL-1 must be balanced with the need to prevent immunopathology. By exogenously controlling the replication of Mycobacterium tuberculosis in vivo, we obviated the requirement for antimicrobial immunity and discovered that both IL-1 production and infection-induced immunopathology were suppressed by lymphocyte-derived interferon-gamma (IFN-gamma). This effect was mediated by nitric oxide (NO), which we found specifically inhibited assembly of the NLRP3 inflammasome via thiol nitrosylation. Our data indicate that the NO produced as a result of adaptive immunity is indispensable in modulating the destructive innate inflammatory responses elicited during persistent infections.