SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues.

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.

Multimodal profiling of lung granulomas in macaques reveals cellular correlates of tuberculosis control.

Mycobacterium tuberculosis lung infection results in a complex multicellular structure: the granuloma. In some granulomas, immune activity promotes bacterial clearance, but in others, bacteria persist and grow. We identified correlates of bacterial control in cynomolgus macaque lung granulomas by co-registering longitudinal positron emission tomography and computed tomography imaging, single-cell RNA sequencing, and measures of bacterial clearance. Bacterial persistence occurred in granulomas enriched for mast, endothelial, fibroblast, and plasma cells, signaling amongst themselves via type 2 immunity and wound-healing pathways. Granulomas that drove bacterial control were characterized by cellular ecosystems enriched for type 1-type 17, stem-like, and cytotoxic T cells engaged in pro-inflammatory signaling networks involving diverse cell populations. Granulomas that arose later in infection displayed functional characteristics of restrictive granulomas and were more capable of killing Mtb. Our results define the complex multicellular ecosystems underlying (lack of) granuloma resolution and highlight host immune targets that can be leveraged to develop new vaccine and therapeutic strategies for TB.

A computational model tracks whole-lung Mycobacterium tuberculosis infection and predicts factors that inhibit dissemination.

Mycobacterium tuberculosis (Mtb), the causative infectious agent of tuberculosis (TB), kills more individuals per year than any other infectious agent. Granulomas, the hallmark of Mtb infection, are complex structures that form in lungs, composed of immune cells surrounding bacteria, infected cells, and a caseous necrotic core. While granulomas serve to physically contain and immunologically restrain bacteria growth, some granulomas are unable to control Mtb growth, leading to bacteria and infected cells leaving the granuloma and disseminating, either resulting in additional granuloma formation (local or non-local) or spread to airways or lymph nodes. Dissemination is associated with development of active TB. It is challenging to experimentally address specific mechanisms driving dissemination from TB lung granulomas. Herein, we develop a novel hybrid multi-scale computational model, MultiGran, that tracks Mtb infection within multiple granulomas in an entire lung. MultiGran follows cells, cytokines, and bacterial populations within each lung granuloma throughout the course of infection and is calibrated to multiple non-human primate (NHP) cellular, granuloma, and whole-lung datasets. We show that MultiGran can recapitulate patterns of in vivo local and non-local dissemination, predict likelihood of dissemination, and predict a crucial role for multifunctional CD8+ T cells and macrophage dynamics for preventing dissemination.

Lymph nodes are sites of prolonged bacterial persistence during Mycobacterium tuberculosis infection in macaques.

Tuberculosis is commonly considered a chronic lung disease, however, extrapulmonary infection can occur in any organ. Even though lymph nodes (LN) are among the most common sites of extrapulmonary Mycobacterium tuberculosis (Mtb) infection, and thoracic LNs are frequently infected in humans, bacterial dynamics and the effect of Mtb infection in LN structure and function is relatively unstudied. We surveyed thoracic LNs from Mtb-infected cynomolgus and rhesus macaques analyzing PET CT scans, bacterial burden, LN structure and immune function. FDG avidity correlated with the presence of live bacteria in LNs at necropsy. Lymph nodes have different trajectories (increasing, maintaining, decreasing in PET activity over time) even within the same animal. Rhesus macaques are more susceptible to Mtb infection than cynomolgus macaques and this is in part due to more extensive LN pathology. Here, we show that Mtb grows to the same level in cynomolgus and rhesus macaque LNs, however, cynomolgus macaques control Mtb at later time points post-infection while rhesus macaques do not. Notably, compared to lung granulomas, LNs are generally poor at killing Mtb, even with drug treatment. Granulomas that form in LNs lack B cell-rich tertiary lymphoid structures, disrupt LN structure by pushing out T cells and B cells, introduce large numbers of macrophages that can serve as niches for Mtb, and destroy normal vasculature. Our data support that LNs are not only sites of antigen presentation and immune activation during infection, but also serve as important sites for persistence of significant numbers of Mtb bacilli.

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.

Immunology studies in non-human primate models of tuberculosis.

Non-human primates, primarily macaques, have been used to study tuberculosis for decades. However, in the last 15 years, this model has been refined substantially to allow careful investigations of the immune response and host-pathogen interactions in Mycobacterium tuberculosis infection. Low-dose challenge with fully virulent strains in cynomolgus macaques result in the full clinical spectrum seen in humans, including latent and active infection. Reagents from humans are usually cross-reactive with macaques, further facilitating the use of this model system to study tuberculosis. Finally, macaques develop the spectrum of granuloma types seen in humans, providing a unique opportunity to investigate bacterial and host factors at the local (lung and lymph node) level. Here, we review the past decade of immunology and pathology studies in macaque models of tuberculosis.

Prospective Discrimination of Controllers From Progressors Early After Low-Dose Mycobacterium tuberculosis Infection of Cynomolgus Macaques using Blood RNA Signatures.

The cynomolgus macaque model of low-dose Mycobacterium tuberculosis infection recapitulates clinical aspects of human tuberculosis pathology, but it is unknown whether the 2 systems are sufficiently similar that host-based signatures of tuberculosis will be predictive across species. By blind prediction, we demonstrate that a subset of genes comprising a human signature for tuberculosis risk is simultaneously predictive in humans and macaques and prospectively discriminates progressor from controller animals 3-6 weeks after infection. Further analysis yielded a 3-gene signature involving PRDX2 that predicts tuberculosis progression in macaques 10 days after challenge, suggesting novel pathways that define protective responses to M. tuberculosis.

PET CT Identifies Reactivation Risk in Cynomolgus Macaques with Latent M. tuberculosis.

Mycobacterium tuberculosis infection presents across a spectrum in humans, from latent infection to active tuberculosis. Among those with latent tuberculosis, it is now recognized that there is also a spectrum of infection and this likely contributes to the variable risk of reactivation tuberculosis. Here, functional imaging with 18F-fluorodeoxygluose positron emission tomography and computed tomography (PET CT) of cynomolgus macaques with latent M. tuberculosis infection was used to characterize the features of reactivation after tumor necrosis factor (TNF) neutralization and determine which imaging characteristics before TNF neutralization distinguish reactivation risk. PET CT was performed on latently infected macaques (n = 26) before and during the course of TNF neutralization and a separate set of latently infected controls (n = 25). Reactivation occurred in 50% of the latently infected animals receiving TNF neutralizing antibody defined as development of at least one new granuloma in adjacent or distant locations including extrapulmonary sites. Increased lung inflammation measured by PET and the presence of extrapulmonary involvement before TNF neutralization predicted reactivation with 92% sensitivity and specificity. To define the biologic features associated with risk of reactivation, we used these PET CT parameters to identify latently infected animals at high risk for reactivation. High risk animals had higher cumulative lung bacterial burden and higher maximum lesional bacterial burdens, and more T cells producing IL-2, IL-10 and IL-17 in lung granulomas as compared to low risk macaques. In total, these data support that risk of reactivation is associated with lung inflammation and higher bacterial burden in macaques with latent Mtb infection.

Early Whole Blood Transcriptional Signatures Are Associated with Severity of Lung Inflammation in Cynomolgus Macaques with Mycobacterium tuberculosis Infection.

Whole blood transcriptional profiling offers great diagnostic and prognostic potential. Although studies identified signatures for pulmonary tuberculosis (TB) and transcripts that predict the risk for developing active TB in humans, the early transcriptional changes immediately following Mycobacterium tuberculosis infection have not been evaluated. We evaluated the gene expression changes in the cynomolgus macaque model of TB, which recapitulates all clinical aspects of human M. tuberculosis infection, using a human microarray and analytics platform. We performed genome-wide blood transcriptional analysis on 38 macaques at 11 postinfection time points during the first 6 mo of M. tuberculosis infection. Of 6371 differentially expressed transcripts between preinfection and postinfection, the greatest change in transcriptional activity occurred 20-56 d postinfection, during which fluctuation of innate and adaptive immune response-related transcripts was observed. Modest transcriptional differences between active TB and latent infection were observed over the time course with substantial overlap. The pattern of module activity previously published for human active TB was similar in macaques with active disease. Blood transcript activity was highly correlated with lung inflammation (lung [18F]fluorodeoxyglucose [FDG] avidity) measured by positron emission tomography and computed tomography at early time points postinfection. The differential signatures between animals with high and low lung FDG were stronger than between clinical outcomes. Analysis of preinfection signatures of macaques revealed that IFN signatures could influence eventual clinical outcomes and lung FDG avidity, even before infection. Our data support that transcriptional changes in the macaque model are translatable to human M. tuberculosis infection and offer important insights into early events of M. tuberculosis infection.

Computational and Empirical Studies Predict Mycobacterium tuberculosis-Specific T Cells as a Biomarker for Infection Outcome.

Identifying biomarkers for tuberculosis (TB) is an ongoing challenge in developing immunological correlates of infection outcome and protection. Biomarker discovery is also necessary for aiding design and testing of new treatments and vaccines. To effectively predict biomarkers for infection progression in any disease, including TB, large amounts of experimental data are required to reach statistical power and make accurate predictions. We took a two-pronged approach using both experimental and computational modeling to address this problem. We first collected 200 blood samples over a 2- year period from 28 non-human primates (NHP) infected with a low dose of Mycobacterium tuberculosis. We identified T cells and the cytokines that they were producing (single and multiple) from each sample along with monkey status and infection progression data. Machine learning techniques were used to interrogate the experimental NHP datasets without identifying any potential TB biomarker. In parallel, we used our extensive novel NHP datasets to build and calibrate a multi-organ computational model that combines what is occurring at the site of infection (e.g., lung) at a single granuloma scale with blood level readouts that can be tracked in monkeys and humans. We then generated a large in silico repository of in silico granulomas coupled to lymph node and blood dynamics and developed an in silico tool to scale granuloma level results to a full host scale to identify what best predicts Mycobacterium tuberculosis (Mtb) infection outcomes. The analysis of in silico blood measures identifies Mtb-specific frequencies of effector T cell phenotypes at various time points post infection as promising indicators of infection outcome. We emphasize that pairing wetlab and computational approaches holds great promise to accelerate TB biomarker discovery.

Computational modeling predicts IL-10 control of lesion sterilization by balancing early host immunity-mediated antimicrobial responses with caseation during mycobacterium tuberculosis infection.

Although almost a third of the world's population is infected with the bacterial pathogen Mycobacterium tuberculosis, our understanding of the functions of many immune factors involved in fighting infection is limited. Determining the role of the immunosuppressive cytokine IL-10 at the level of the granuloma has proven difficult because of lesional heterogeneity and the limitations of animal models. In this study, we take an in silico approach and, through a series of virtual experiments, we predict several novel roles for IL-10 in tuberculosis granulomas: 1) decreased levels of IL-10 lead to increased numbers of sterile lesions, but at the cost of early increased caseation; 2) small increases in early antimicrobial activity cause this increased lesion sterility; 3) IL-10 produced by activated macrophages is a major mediator of early antimicrobial activity and early host-induced caseation; and 4) increasing levels of infected macrophage derived IL-10 promotes bacterial persistence by limiting the early antimicrobial response and preventing lesion sterilization. Our findings, currently only accessible using an in silico approach, suggest that IL-10 at the individual granuloma scale is a critical regulator of lesion outcome. These predictions suggest IL-10-related mechanisms that could be used as adjunctive therapies during tuberculosis.

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.

Bioinformatic and empirical analysis of novel hypoxia-inducible targets of the human antituberculosis T cell response.

We analyzed whole genome-based transcriptional profiles of Mycobacterium tuberculosis subjected to prolonged hypoxia to guide the discovery of novel potential Ags, by a combined bioinformatic and empirical approach. We analyzed the fold induction of the 100 most highly induced genes at 7 d of hypoxia, as well as transcript abundance, peptide-binding prediction (ProPred) adjusted for population-specific MHC class II allele frequency, and by literature search. Twenty-six candidate genes were selected by this bioinformatic approach and evaluated empirically using IFN-γ and IL-2 ELISPOT using immunodominant Ags (Acr-1, CFP-10, ESAT-6) as references. Twenty-three of twenty-six proteins induced an IFN-γ response in PBMCs of persons with active or latent tuberculosis. Five novel immunodominant proteins-Rv1957, Rv1954c, Rv1955, Rv2022c, and Rv1471-were identified that induced responses similar to CFP-10 and ESAT-6 in both magnitude and frequency. IL-2 responses were of lower magnitude than were those of IFN-γ. Only moderate evidence of infection stage-specific recognition of Ags was observed. Reconciliation of bioinformatic and empirical hierarchies of immunodominance revealed that Ags could be predicted, providing transcriptomic data were combined with peptide-binding prediction adjusted by population-specific MHC class II allele frequency.

CD4+ T cells are homeostatic regulators during Mtb reinfection.

Immunological priming - either in the context of prior infection or vaccination - elicits protective responses against subsequent Mycobacterium tuberculosis (Mtb) infection. However, the changes that occur in the lung cellular milieu post-primary Mtb infection and their contributions to protection upon reinfection remain poorly understood. Here, using clinical and microbiological endpoints in a non-human primate reinfection model, we demonstrate that prior Mtb infection elicits a long-lasting protective response against subsequent Mtb exposure and that the depletion of CD4+ T cells prior to Mtb rechallenge significantly abrogates this protection. Leveraging microbiologic, PET-CT, flow cytometric, and single-cell RNA-seq data from primary infection, reinfection, and reinfection-CD4+ T cell depleted granulomas, we identify differential cellular and microbial features of control. The data collectively demonstrate that the presence of CD4+ T cells in the setting of reinfection results in a reduced inflammatory lung milieu characterized by reprogrammed CD8+ T cell activity, reduced neutrophilia, and blunted type-1 immune signaling among myeloid cells, mitigating Mtb disease severity. These results open avenues for developing vaccines and therapeutics that not only target CD4+ and CD8+ T cells, but also modulate innate immune cells to limit Mtb disease.

CD8+ lymphocytes are critical for early control of tuberculosis in macaques.

The functional role of CD8+ lymphocytes in tuberculosis remains poorly understood. We depleted innate and/or adaptive CD8+ lymphocytes in macaques and showed that loss of all CD8α+ cells (using anti-CD8α antibody) significantly impaired early control of Mycobacterium tuberculosis (Mtb) infection, leading to increased granulomas, lung inflammation, and bacterial burden. Analysis of barcoded Mtb from infected macaques demonstrated that depletion of all CD8+ lymphocytes allowed increased establishment of Mtb in lungs and dissemination within lungs and to lymph nodes, while depletion of only adaptive CD8+ T cells (with anti-CD8ß antibody) worsened bacterial control in lymph nodes. Flow cytometry and single-cell RNA sequencing revealed polyfunctional cytotoxic CD8+ lymphocytes in control granulomas, while CD8-depleted animals were unexpectedly enriched in CD4 and γδ T cells adopting incomplete cytotoxic signatures. Ligand-receptor analyses identified IL-15 signaling in granulomas as a driver of cytotoxic T cells. These data support that CD8+ lymphocytes are required for early protection against Mtb and suggest polyfunctional cytotoxic responses as a vaccine target.

In silico identification and synthesis of a multi-drug loaded MOF for treating tuberculosis.

Conventional drug delivery systems have been applied to a myriad of active ingredients but may be difficult to tailor for a given drug. Herein, we put forth a new strategy, which designs and selects the drug delivery material by considering the properties of encapsulated drugs (even multiple drugs, simultaneously). Specifically, through an in-silico screening process of 5109 MOFs using grand canonical Monte Carlo simulations, a customized MOF (referred as BIO-MOF-100) was selected and experimentally verified to be biologically stable, and capable of loading 3 anti-Tuberculosis drugs Rifampicin+Isoniazid+Pyrazinamide at 10% + 28% + 23% wt/wt (total > 50% by weight). Notably, the customized BIO-MOF-100 delivery system cleared naturally Pyrazinamide-resistant Bacillus Calmette-Guérin, reduced growth of virulent Erdman infection in macaque macrophages 10-100-fold compared to soluble drugs in vitro and was also significantly reduced Erdman growth in mice. These data suggest that the methodology of identifying-synthesizing materials can be used to generate solutions for challenging applications such as simultaneous delivery of multiple, small hydrophilic and hydrophobic molecules in the same molecular framework.

High prevalence of subclinical tuberculosis in HIV-1-infected persons without advanced immunodeficiency: implications for TB screening.

BACKGROUND: The prevalence of asymptomatic tuberculosis (TB) in recently diagnosed HIV-1-infected persons attending pre-antiretroviral therapy (ART) clinics is not well described. In addition, it is unclear if the detection of Mycobacterium tuberculosis in these patients clearly represents an early asymptomatic phase leading to progressive disease or transient excretion of bacilli. OBJECTIVE: To describe the prevalence and outcome of subclinical TB disease in HIV-1-infected persons not eligible for ART. METHODS: The study was conducted in 274 asymptomatic ART-naïve HIV-1-infected persons in Khayelitsha Day Hospital, Cape Town, South Africa. All participants were screened for TB using a symptom screen and spoligotyping was performed to determine genotypes. RESULTS: The prevalence of subclinical TB disease was 8.5% (95% CI 5.1% to 13.0%) (n=18; median days to culture positivity 17 days), with 22% of patients being smear-positive. Spoligotyping showed a diverse variety of genotypes with all paired isolates being of the same spoligotype, effectively excluding cross-contamination. 56% of patients followed up developed symptoms 3 days to 2 months later. All were well and still in care 6-12 months after TB diagnosis; 60% were started on ART. A positive tuberculin skin test (OR 4.96, p=0.064), low CD4 count (OR 0.996, p=0.06) and number of years since HIV diagnosis (OR 1.006, p=0.056) showed trends towards predicting TB disease. Conclusion This study found a high prevalence but good outcome (retained in care) of subclinical TB disease in HIV-1-infected persons. The results suggest that, in high HIV/TB endemic settings, a positive HIV-1 test should prompt TB screening by sputum culture irrespective of symptoms, particularly in those with a positive tuberculin skin test, longer history of HIV infection and low CD4 count. Operational difficulties in resource-constrained settings with respect to screening with TB culture highlight the need for rapid and affordable point-of-care tests to identify persons with clinical and subclinical TB disease.

Neutrophil Dynamics Affect Mycobacterium tuberculosis Granuloma Outcomes and Dissemination.

Neutrophil infiltration into tuberculous granulomas is often associated with higher bacteria loads and severe disease but the basis for this relationship is not well understood. To better elucidate the connection between neutrophils and pathology in primate systems, we paired data from experimental studies with our next generation computational model GranSim to identify neutrophil-related factors, including neutrophil recruitment, lifespan, and intracellular bacteria numbers, that drive granuloma-level outcomes. We predict mechanisms underlying spatial organization of neutrophils within granulomas and identify how neutrophils contribute to granuloma dissemination. We also performed virtual deletion and depletion of neutrophils within granulomas and found that neutrophils play a nuanced role in determining granuloma outcome, promoting uncontrolled bacterial growth in some and working to contain bacterial growth in others. Here, we present three key results: We show that neutrophils can facilitate local dissemination of granulomas and thereby enable the spread of infection. We suggest that neutrophils influence CFU burden during both innate and adaptive immune responses, implying that they may be targets for therapeutic interventions during later stages of infection. Further, through the use of uncertainty and sensitivity analyses, we predict which neutrophil processes drive granuloma severity and structure.

Measurement of leukocyte trafficking kinetics in macaques by serial intravascular staining.

Leukocyte trafficking enables detection of pathogens, immune responses, and immune memory. Dysregulation of leukocyte trafficking is often found in disease, highlighting its important role in homeostasis and the immune response. Whereas some of the molecular mechanisms mediating leukocyte trafficking are understood, little is known about the regulation of trafficking, including trafficking kinetics and its impact on immune homeostasis. We developed a method of serial intravascular staining (SIVS) to measure trafficking kinetics in nonhuman primates using infusions of fluorescently labeled antibodies to label circulating leukocytes. Because antibody infusions labeled only leukocytes in the blood, cells were "barcoded" according to their location at the time of each infusion, providing positional histories that could be used to infer trafficking kinetics. We used SIVS and multiparameter flow cytometry to quantitate cellular trafficking into lymphoid tissues of healthy animals at homeostasis and to identify perivascular cells that could be unique to nonlymphoid organs. To investigate how these parameters could be influenced during disease, SIVS was used to quantify lymphocyte trafficking in macaques infected with the bacterial pathogen Mycobacterium tuberculosis and to enumerate intravascular leukocytes in lung granulomas. We showed that whereas most cells in lung granulomas were localized there for more than 24 hours, granulomas were dynamic with a slow continual cellular influx, the rate of which predicted clearance of M. tuberculosis from the granulomas. SIVS, in combination with intracellular staining and multiparametric flow cytometry, is a powerful method to quantify the kinetics of leukocyte trafficking in nonhuman primates in vivo.