Chest X-ray (CXR), computed tomography (CT), and positron emission tomography-computed tomography (PET-CT) are noninvasive imaging techniques widely used in human and veterinary pulmonary research and medicine. These techniques have recently been applied in studies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-exposed non-human primates (NHPs) to complement virological assessments with meaningful translational readouts of lung disease. Our review of the literature indicates that medical imaging of SARS-CoV-2-exposed NHPs enables high-resolution qualitative and quantitative characterization of disease otherwise clinically invisible and potentially provides user-independent and unbiased evaluation of medical countermeasures (MCMs). However, we also found high variability in image acquisition and analysis protocols among studies. These findings uncover an urgent need to improve standardization and ensure direct comparability across studies.

In response to Mtb infection, the host remodels the infection foci into a dense mass of cells known as the granuloma. The key objective of the granuloma is to contain the spread of Mtb into uninfected regions of the lung. However, it appears that Mtb has evolved mechanisms to resist killing in the granuloma. Profiling granuloma transcriptome will identify key immune signaling pathways active during TB infection. Such studies are not possible in human granulomas, due to various confounding factors. Nonhuman Primates (NHPs) infected with Mtb accurately reflect human TB in clinical and pathological contexts.

The expression of indoleamine 2,3-dioxygenase (IDO), a robust immunosuppressant, is significantly induced in macaque tuberculosis (TB) granulomas, where it is expressed on IFN-responsive macrophages and myeloid-derived suppressor cells. IDO expression is also highly induced in human TB granulomas, and products of its activity are detected in patients with TB. In vivo blockade of IDO activity resulted in the reorganization of the granuloma with substantially greater T cells being recruited to the core of the lesions. This correlated with better immune control of TB and reduced lung M. tuberculosis burdens. To study if the IDO blockade strategy can be translated to a bona fide host-directed therapy in the clinical setting of TB, we studied the effect of IDO inhibitor 1-methyl-d-tryptophan adjunctive to suboptimal anti-TB chemotherapy. While two-thirds of controls and one-third of chemotherapy-treated animals progressed to active TB, inhibition of IDO adjunctive to the same therapy protected macaques from TB, as measured by clinical, radiological, and microbiological attributes. Although chemotherapy improved proliferative T cell responses, adjunctive inhibition of IDO further enhanced the recruitment of effector T cells to the lung. These results strongly suggest the possibility that IDO inhibition can be attempted adjunctive to anti-TB chemotherapy in clinical trials.

Interleukin-10 (IL-10) modulates inflammatory responses elicited in vitro and in vivo by Borrelia burgdorferi, the Lyme disease spirochete. How IL-10 modulates these inflammatory responses still remains elusive. We hypothesize that IL-10 inhibits effector functions of multiple genes induced by B. burgdorferi in macrophages to control concomitantly elicited inflammation. Because macrophages are essential in the initiation of inflammation, we used mouse J774 macrophages and live B. burgdorferi spirochetes as the model target cell and stimulant, respectively. First, we employed transcriptome profiling to identify genes that were induced by stimulation of cells with live spirochetes and that were perturbed by addition of IL-10 to spirochete cultures. Spirochetes significantly induced upregulation of 347 genes at both the 4-h and 24-h time points. IL-10 inhibited the expression levels, respectively, of 53 and 65 of the 4-h and 24-h genes, and potentiated, respectively, at 4 h and 24 h, 65 and 50 genes. Prominent among the novel identified IL-10-inhibited genes also validated by quantitative real-time PCR (qRT-PCR) were Toll-like receptor 1 (TLR1), TLR2, IRAK3, TRAF1, IRG1, PTGS2, MMP9, IFI44, IFIT1, and CD40. Proteome analysis using a multiplex enzyme-linked immunosorbent assay (ELISA) revealed the IL-10 modulation/and or potentiation of RANTES/CCL5, macrophage inflammatory protein 2 (MIP-2)/CXCL2, IP-10/CXCL10, MIP-1α/CCL3, granulocyte colony-stimulating factor (G-CSF)/CSF3, CXCL1, CXCL5, CCL2, CCL4, IL-6, tumor necrosis factor alpha (TNF-α), IL-1α, IL-1β, gamma interferon (IFN-γ), and IL-9. Similar results were obtained using sonicated spirochetes or lipoprotein as stimulants. Our data show that IL-10 alters effectors induced by B. burgdorferi in macrophages to control concomitantly elicited inflammatory responses. Moreover, for the first time, this study provides global insight into potential mechanisms used by IL-10 to control Lyme disease inflammation.

Mycobacterium tuberculosis (Mtb) executes a plethora of immune-evasive mechanisms, which contribute to its pathogenesis, limited efficacy of current therapy, and the emergence of drug-resistant strains. This has led to resurgence in attempts to develop new therapeutic strategies/targets against tuberculosis (TB). We show that Mtb down-regulates sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, in monocytes/macrophages, TB animal models, and TB patients with active disease. Activation of SIRT1 reduced intracellular growth of drug-susceptible and drug-resistant strains of Mtb and induced phagosome-lysosome fusion and autophagy in a SIRT1-dependent manner. SIRT1 activation dampened Mtb-mediated persistent inflammatory responses via deacetylation of RelA/p65, leading to impaired binding of RelA/p65 on the promoter of inflammatory genes. In Mtb-infected mice, the use of SIRT1 activators ameliorated lung pathology, reduced chronic inflammation, and enhanced efficacy of anti-TB drug. Mass cytometry-based high-dimensional analysis revealed that SIRT1 activation mediated modulation of lung myeloid cells in Mtb-infected mice. Myeloid cell-specific SIRT1 knockout mice display increased inflammatory responses and susceptibility to Mtb infection. Collectively, these results provide a link between SIRT1 activation and TB pathogenesis and indicate a potential of SIRT1 activators in designing an effective and clinically relevant host-directed therapies for TB.

Non-human primate (NHP) models of tuberculosis (TB) immunity and pathogenesis, especially rhesus and cynomolgus macaques, are particularly attractive because of the high similarity of the human and macaque immune systems. However, little is known about the MHC class II epitopes recognized in macaques, thus hindering the establishment of immune correlates of immunopathology and protective vaccination. We characterized immune responses in rhesus macaques vaccinated against and/or infected with Mycobacterium tuberculosis (Mtb), to a panel of antigens currently in human vaccine trials. We defined 54 new immunodominant CD4(+) T cell epitopes, and noted that antigens immunodominant in humans are also immunodominant in rhesus macaques, including Rv3875 (ESAT-6) and Rv3874 (CFP10). Pedigree and inferred restriction analysis demonstrated that this phenomenon was not due to common ancestry or inbreeding, but rather presentation by common alleles, as well as, promiscuous binding. Experiments using a second cohort of rhesus macaques demonstrated that a pool of epitopes defined in the previous experiments can be used to detect T cell responses in over 75% of individual monkeys. Additionally, 100% of cynomolgus macaques, irrespective of their latent or active TB status, responded to rhesus and human defined epitope pools. Thus, these findings reveal an unexpected general repertoire overlap between MHC class II epitopes recognized in both species of macaques and in humans, showing that epitope pools defined in humans can also be used to characterize macaque responses, despite differences in species and antigen exposure. The results have general implications for the evaluation of new vaccines and diagnostics in NHPs, and immediate applicability in the setting of macaque models of TB.

A major hurdle facing tuberculosis (TB) investigators who want to utilize a rapidly growing body of data from both systems biology approaches and omics technologies is the lack of a standard vocabulary for data annotation and reporting. Lacking a means to readily compare samples from different research groups, a significant quantity of potentially informative data is largely ignored by researchers. To facilitate standardizing data across studies, a simple ontology of TB terms was developed to provide a common vocabulary for annotating data sets. New terminology was developed to address animal models and experimental systems, and existing clinically focused terminology was modified and adapted. This ontology can be used to annotate host TB data in public databases and collaborations, thereby standardizing database searches and allowing researchers to more easily compare results. To demonstrate the utility of a standard TB ontology for host systems biology, a web application was developed to annotate and compare human and animal model gene expression data sets.

Mycobacterium tuberculosis can grow in the hostile intracellular environment of macrophages by actively evading macrophage-associated antibacterial activities. The stress response factor SigH contributes to this process by modulating β-chemokine and interleukin 6 (Il6) expression. Hence, Il6 is of critical importance for acquired immunity against M. tuberculosis infection. Here, we attempted to better characterize the role of Il6 in the immune response to M. tuberculosis infection.

Mycobacterium tuberculosis (Mtb) persists within lung granulomas, despite being subjected to diverse stress conditions, including hypoxia. We hypothesized that the response of host phagocytes to Mtb experiencing hypoxia is radically altered and designed in vitro experiment to study this phenomenon. Hypoxia-stressed (Mtb-H) and aerobically grown Mtb (Mtb-A) were used to infect Rhesus Macaque Bone Marrow Derived Macrophages (Rh-BMDMs) and the comparative host response to Mtb infection studied. Mechanistic insights were gained by employing RNAi. Mtb-H accumulated significantly lower bacterial burden during growth in Rh-BMDMs, concomitantly generating a drastically different host transcriptional profile (with only <2% of all genes perturbed by either infection being shared between the two groups). A key component of this signature was significantly higher TNF and apopotosis in Mtb-H- compared to Mtb-A-infected Rh-BMDMs. Silencing of TNF by RNAi reversed the significant control of Mtb replication. These results indicate a potential mechanism for the rapid clearance of hypoxia-conditioned bacilli by phagocytes. In conclusion, hypoxia-conditioned Mtb undergo significantly different interactions with host macrophages compared to Mtb grown in normoxia. These interactions result in the induction of the TNF signaling pathway, activation of apoptosis, and DNA-damage stress response. Our results show that Mtb-H bacilli are particularly susceptible to killing governed by TNF.

No abstract available

Although it is accepted that the environment within the granuloma profoundly affects Mycobacterium tuberculosis (Mtb) and infection outcome, our ability to understand Mtb gene expression in these niches has been limited. We determined intragranulomatous gene expression in human-like lung lesions derived from nonhuman primates with both active tuberculosis (ATB) and latent TB infection (LTBI). We employed a non-laser-based approach to microdissect individual lung lesions and interrogate the global transcriptome of Mtb within granulomas. Mtb genes expressed in classical granulomas with central, caseous necrosis, as well as within the caseum itself, were identified and compared with other Mtb lesions in animals with ATB (n = 7) or LTBI (n = 7). Results were validated using both an oligonucleotide approach and RT-PCR on macaque samples and by using human TB samples. We detected approximately 2,900 and 1,850 statistically significant genes in ATB and LTBI lesions, respectively (linear models for microarray analysis, Bonferroni corrected, P < 0.05). Of these genes, the expression of approximately 1,300 (ATB) and 900 (LTBI) was positively induced. We identified the induction of key regulons and compared our results to genes previously determined to be required for Mtb growth. Our results indicate pathways that Mtb uses to ensure its survival in a highly stressful environment in vivo. A large number of genes is commonly expressed in granulomas with ATB and LTBI. In addition, the enhanced expression of the dormancy survival regulon was a key feature of lesions in animals with LTBI, stressing its importance in the persistence of Mtb during the chronic phase of infection.

Primary and post-primary tuberculosis (TB) are different diseases caused by the same organism. Primary TB produces systemic immunity. Post-primary TB produces cavities to support massive proliferation of organisms for transmission of infection to new hosts from a person with sufficient immunity to prevent systemic infection. Post-primary, also known as bronchogenic, TB begins in humans as asymptomatic bronchial spread of obstructive lobular pneumonia, not as expanding granulomas. Most lesions regress spontaneously. However, some undergo caseation necrosis that is coughed out through the necrotic bronchi to form cavities. Caseous pneumonia that is not expelled through the bronchi is retained to become the focus of fibrocaseous disease. No animal reproduces this entire process. However, it appears that many mammals utilize similar mechanisms, but fail to coordinate them as do humans. Understanding this makes it possible to use human tuberculous lung sections to guide manipulation of animals to produce models of particular human lesions. For example, slowly progressive and reactivation TB in mice resemble developing human bronchogenic TB. Similarly, bronchogenic TB and cavities resembling those in humans can be induced by bronchial infection of sensitized rabbits. Granulomas in guinea pigs have characteristics of both primary and post primary TB. Mice can be induced to produce a spectrum of human like caseating granulomas. There is evidence that primates can develop bronchogenic TB. We are optimistic that such models developed by coordinated study of human and animal tissues can be used with modern technologies to finally address long-standing questions about host/parasite relationships in TB, and support development of targeted therapeutics and vaccines.

Animal models are important in understanding both the pathogenesis of and immunity to tuberculosis (TB). Unfortunately, we are beginning to understand that no animal model perfectly recapitulates the human TB syndrome, which encompasses numerous different stages. Furthermore, Mycobacterium tuberculosis infection is a very heterogeneous event at both the levels of pathogenesis and immunity. This review seeks to establish the current understanding of TB pathogenesis and immunity, as validated in the animal models of TB in active use today. We especially focus on the use of modern genomic approaches in these models to determine the mechanism and the role of specific molecular pathways. Animal models have significantly enhanced our understanding of TB. Incorporation of contemporary technologies such as single cell transcriptomics, high-parameter flow cytometric immune profiling, proteomics, proteomic flow cytometry and immunocytometry into the animal models in use will further enhance our understanding of TB and facilitate the development of treatment and vaccination strategies.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a global cause of death. Granuloma-associated lymphoid tissue (GrALT) correlates with protection during TB, but the mechanisms of protection are not understood. During TB, the transcription factor IRF4 in T cells but not B cells is required for the generation of the TH1 and TH17 subsets of helper T cells and follicular helper T (TFH)-like cellular responses. A population of IRF4+ T cells coexpress the transcription factor BCL6 during Mtb infection, and deletion of Bcl6 (Bcl6fl/fl) in CD4+ T cells (CD4cre) resulted in reduction of TFH-like cells, impaired localization within GrALT and increased Mtb burden. In contrast, the absence of germinal center B cells, MHC class II expression on B cells, antibody-producing plasma cells or interleukin-10-expressing B cells, did not increase Mtb susceptibility. Indeed, antigen-specific B cells enhance cytokine production and strategically localize TFH-like cells within GrALT via interactions between programmed cell death 1 (PD-1) and its ligand PD-L1 and mediate Mtb control in both mice and macaques.

Hypoxia promotes dormancy by causing physiologic changes to actively replicating Mycobacterium tuberculosis. DosR controls the response of M. tuberculosis to hypoxia.

The synergy between Mycobacterium tuberculosis (Mtb) and HIV in coinfected patients has profoundly impacted global mortality because of tuberculosis (TB) and AIDS. HIV significantly increases rates of reactivation of latent TB infection (LTBI) to active disease, with the decline in CD4(+) T cells believed to be the major causality. In this study, nonhuman primates were coinfected with Mtb and simian immunodeficiency virus (SIV), recapitulating human coinfection. A majority of animals exhibited rapid reactivation of Mtb replication, progressing to disseminated TB and increased SIV-associated pathology. Although a severe loss of pulmonary CD4(+) T cells was observed in all coinfected macaques, a subpopulation of the animals was still able to prevent reactivation and maintain LTBI. Investigation of pulmonary immune responses and pathology in this cohort demonstrated that increased CD8(+) memory T-cell proliferation, higher granzyme B production, and expanded B-cell follicles correlated with protection from reactivation. Our findings reveal mechanisms that control SIV- and TB-associated pathology. These CD4-independent protective immune responses warrant further studies in HIV coinfected humans able to control their TB infection. Moreover, these findings will provide insight into natural immunity to Mtb and will guide development of novel vaccine strategies and immunotherapies.

A hallmark of pulmonary tuberculosis (TB) is the formation of granulomas. However, the immune factors that drive the formation of a protective granuloma during latent TB, and the factors that drive the formation of inflammatory granulomas during active TB, are not well defined.

Antigen-specific CD4 and CD8 T cells are important components of the immune response to Mycobacterium tuberculosis, yet little information is currently known regarding how the breadth, specificity, phenotype, and function of M. tuberculosis-specific T cells correlate with M. tuberculosis infection outcome in humans. To facilitate evaluation of human M. tuberculosis-specific T cell responses targeting multiple different Ags, we sought to develop a high throughput and reproducible T cell response spectrum assay requiring low blood sample volumes. We describe here the optimization and standardization of a microtiter plate-based, diluted whole blood stimulation assay utilizing overlapping peptide pools corresponding to a functionally diverse panel of 60 M. tuberculosis Ags. Using IFN-γ production as a readout of Ag specificity, the assay can be conducted using 50 μl of blood per test condition and can be expanded to accommodate additional Ags. We evaluated the intra- and interassay variability, and implemented testing of the assay in diverse cohorts of M. tuberculosis-unexposed healthy adults, foreign-born adults with latent M. tuberculosis infection residing in the United States, and tuberculosis household contacts with latent M. tuberculosis infection in a tuberculosis-endemic setting in Kenya. The M. tuberculosis-specific T cell response spectrum assay further enhances the immunological toolkit available for evaluating M. tuberculosis-specific T cell responses across different states of M. tuberculosis infection, and can be readily implemented in resource-limited settings. Moreover, application of the assay to longitudinal cohorts will facilitate evaluation of treatment- or vaccine-induced changes in the breadth and specificity of Ag-specific T cell responses, as well as identification of M. tuberculosis-specific T cell responses associated with M. tuberculosis infection outcomes.

Failure to replace Bacille Calmette-Guerin vaccines with efficacious anti-tuberculosis (TB) vaccines have prompted outside-the-box thinking, including pulmonary vaccination to elicit local immunity. Inhalational MtbΔsigH, a stress-response-attenuated strain, protected against lethal TB in macaques. While live mycobacterial vaccines show promising efficacy, HIV co-infection and the resulting immunodeficiency prompts safety concerns about their use. We assessed the persistence and safety of MtbΔsigH, delivered directly to the lungs, in the setting of HIV co-infection. Macaques were aerosol-vaccinated with ΔsigH and subsequently challenged with SIVmac239. Bronchoalveolar lavage and tissues were sampled for mycobacterial persistence, pathology, and immune correlates. Only 35% and 3.5% of lung samples were positive for live bacilli and granulomas, respectively. Our results therefore suggest that the nonpathologic infection of macaque lungs by ΔsigH was not reactivated by simian immunodeficiency virus, despite high viral levels and massive ablation of pulmonary CD4+ T cells. Protective pulmonary responses were retained, including vaccine-induced bronchus-associated lymphoid tissue and CD8+ effector memory T cells. Despite acute simian immunodeficiency virus infection, all animals remained asymptomatic of pulmonary TB. These findings highlight the efficacy of mucosal vaccination via this attenuated strain and will guide its further development to potentially combat TB in HIV-endemic areas. Our results also suggest that a lack of pulmonary pathology is a key correlate of the safety of live mycobacterial vaccines.

TB is a catastrophic infectious disease, affecting roughly one third of the world's population. Mucosal-associated invariant T (MAIT) cells are innate-like T cells that recognize vitamin B metabolites produced by bacteria, possess effector memory phenotype, and express tissue-homing markers driving migration to sites of infection. Previous research in both Mtb and HIV infections has shown that MAIT cells are depleted in the human periphery, possibly migrating to the tissue sites of infection. We investigated this hypothesis using rhesus macaques (RMs) with active TB, latent TB (LTBI), and SIV-coinfection to explore the effects of different disease states on the MAIT cell populations in vivo. Early in infection, we observed that MAIT cells increased in the blood and bronchoalveolar lavage fluid (BAL) of all infected RMs, irrespective of clinical outcome. However, the frequency of MAIT cells rapidly normalized such that they had returned to baseline levels prior to endpoint. Furthermore, following infection, the chemokines expressed on MAIT cells reflected a strong shift towards a Th1 phenotype from a shared Th1/Th17 phenotype. In conclusion, MAIT cells with enhanced Th1 functions migrating to the site of Mtb-infection. The anti-mycobacterial effector functions of MAIT cells, particularly during the early stages of Mtb infection, had been of interest in promoting protective long-term TB immunity. Our research shows, however, that they have relatively short-acting responses in the host.