Systems genetics uncover new loci containing functional gene candidates in Mycobacterium tuberculosis-infected Diversity Outbred mice.

Mycobacterium tuberculosis infects two billion people across the globe, and results in 8-9 million new tuberculosis (TB) cases and 1-1.5 million deaths each year. Most patients have no known genetic basis that predisposes them to disease. Here, we investigate the complex genetic basis of pulmonary TB by modelling human genetic diversity with the Diversity Outbred mouse population. When infected with M. tuberculosis, one-third develop early onset, rapidly progressive, necrotizing granulomas and succumb within 60 days. The remaining develop non-necrotizing granulomas and survive longer than 60 days. Genetic mapping using immune and inflammatory mediators; and clinical, microbiological, and granuloma correlates of disease identified five new loci on mouse chromosomes 1, 2, 4, 16; and three known loci on chromosomes 3 and 17. Further, multiple positively correlated traits shared loci on chromosomes 1, 16, and 17 and had similar patterns of allele effects, suggesting these loci contain critical genetic regulators of inflammatory responses to M. tuberculosis. To narrow the list of candidate genes, we used a machine learning strategy that integrated gene expression signatures from lungs of M. tuberculosis-infected Diversity Outbred mice with gene interaction networks to generate scores representing functional relationships. The scores were used to rank candidates for each mapped trait, resulting in 11 candidate genes: Ncf2, Fam20b, S100a8, S100a9, Itgb5, Fstl1, Zbtb20, Ddr1, Ier3, Vegfa, and Zfp318. Although all candidates have roles in infection, inflammation, cell migration, extracellular matrix remodeling, or intracellular signaling, and all contain single nucleotide polymorphisms (SNPs), SNPs in only four genes (S100a8, Itgb5, Fstl1, Zfp318) are predicted to have deleterious effects on protein functions. We performed methodological and candidate validations to (i) assess biological relevance of predicted allele effects by showing that Diversity Outbred mice carrying PWK/PhJ alleles at the H-2 locus on chromosome 17 QTL have shorter survival; (ii) confirm accuracy of predicted allele effects by quantifying S100A8 protein in inbred founder strains; and (iii) infection of C57BL/6 mice deficient for the S100a8 gene. Overall, this body of work demonstrates that systems genetics using Diversity Outbred mice can identify new (and known) QTLs and functionally relevant gene candidates that may be major regulators of complex host-pathogens interactions contributing to granuloma necrosis and acute inflammation in pulmonary TB.

B cells in perivascular and peribronchiolar granuloma-associated lymphoid tissue and B-cell signatures identify asymptomatic Mycobacterium tuberculosis lung infection in Diversity Outbred mice.

Because most humans resist Mycobacterium tuberculosis infection, there is a paucity of lung samples to study. To address this gap, we infected Diversity Outbred mice with M. tuberculosis and studied the lungs of mice in different disease states. After a low-dose aerosol infection, progressors succumbed to acute, inflammatory lung disease within 60 days, while controllers maintained asymptomatic infection for at least 60 days, and then developed chronic pulmonary tuberculosis (TB) lasting months to more than 1 year. Here, we identified features of asymptomatic M. tuberculosis infection by applying computational and statistical approaches to multimodal data sets. Cytokines and anti-M. tuberculosis cell wall antibodies discriminated progressors vs controllers with chronic pulmonary TB but could not classify mice with asymptomatic infection. However, a novel deep-learning neural network trained on lung granuloma images was able to accurately classify asymptomatically infected lungs vs acute pulmonary TB in progressors vs chronic pulmonary TB in controllers, and discrimination was based on perivascular and peribronchiolar lymphocytes. Because the discriminatory lesion was rich in lymphocytes and CD4 T cell-mediated immunity is required for resistance, we expected CD4 T-cell genes would be elevated in asymptomatic infection. However, the significantly different, highly expressed genes were from B-cell pathways (e.g., Bank1, Cd19, Cd79, Fcmr, Ms4a1, Pax5, and H2-Ob), and CD20+ B cells were enriched in the perivascular and peribronchiolar regions of mice with asymptomatic M. tuberculosis infection. Together, these results indicate that genetically controlled B-cell responses are important for establishing asymptomatic M. tuberculosis lung infection.

Preclinical murine models for the testing of antimicrobials against Mycobacterium abscessus pulmonary infections: Current practices and recommendations.

Mycobacterium abscessus, a rapidly growing nontuberculous mycobacterium, is increasingly recognized as an important pathogen of the human lung, disproportionally affecting people with cystic fibrosis (CF) and other susceptible individuals with non-CF bronchiectasis and compromised immune functions. M. abscessus infections are extremely difficult to treat due to intrinsic resistance to many antibiotics, including most anti-tuberculous drugs. Current standard-of-care chemotherapy is long, includes multiple oral and parenteral repurposed drugs, and is associated with significant toxicity. The development of more effective oral antibiotics to treat M. abscessus infections has thus emerged as a high priority. While murine models have proven instrumental in predicting the efficacy of therapeutic treatments for M. tuberculosis infections, the preclinical evaluation of drugs against M. abscessus infections has proven more challenging due to the difficulty of establishing a progressive, sustained, pulmonary infection with this pathogen in mice. To address this issue, a series of three workshops were hosted in 2023 by the Cystic Fibrosis Foundation (CFF) and the National Institute of Allergy and Infectious Diseases (NIAID) to review the current murine models of M. abscessus infections, discuss current challenges and identify priorities toward establishing validated and globally harmonized preclinical models. This paper summarizes the key points from these workshops. The hope is that the recommendations that emerged from this exercise will facilitate the implementation of informative murine models of therapeutic efficacy testing across laboratories, improve reproducibility from lab-to-lab and accelerate preclinical-to-clinical translation.

Myc Dysregulation in Activated Macrophages Initiates Iron-Mediated Lipid Peroxidation that Fuels Type I Interferon and Compromises TB Resistance.

A quarter of human population is infected with Mycobacterium tuberculosis, but less than 10% of those infected develop clinical, mostly pulmonary, TB. To dissect mechanisms of susceptibility in immunocompetent individuals, we developed a genetically defined sst1-susceptible mouse model that uniquely reproduces a defining feature of human TB: development of necrotic lung lesions after infection with virulent Mtb. In this study, we explored the connectivity of the sst1-regulated pathways during prolonged macrophage activation with TNF. We determined that the aberrant response of the sst1-susceptible macrophages to TNF was primarily driven by conflicting Myc and antioxidant response pathways that resulted in a coordinated failure to properly sequester intracellular iron and activate ferroptosis inhibitor enzymes. Consequently, iron-mediated lipid peroxidation fueled IFNß superinduction and sustained the Type I Interferon (IFN-I) pathway hyperactivity that locked the sst1-susceptible macrophages in a state of unresolving stress and compromised their resistance to Mtb. The accumulation of the aberrantly activated, stressed, macrophages within granuloma microenvironment led to the local failure of anti-tuberculosis immunity and tissue necrosis. Our findings suggest a novel link between metabolic dysregulation in macrophages and susceptibility to TB, offering insights into potential therapeutic targets aimed at modulating macrophage function and improving TB control.

BACH1 promotes tissue necrosis and Mycobacterium tuberculosis susceptibility.

Oxidative stress triggers ferroptosis, a form of cellular necrosis characterized by iron-dependent lipid peroxidation, and has been implicated in Mycobacterium tuberculosis (Mtb) pathogenesis. We investigated whether Bach1, a transcription factor that represses multiple antioxidant genes, regulates host resistance to Mtb. We found that BACH1 expression is associated clinically with active pulmonary tuberculosis. Bach1 deletion in Mtb-infected mice increased glutathione levels and Gpx4 expression that inhibit lipid peroxidation. Bach1-/- macrophages exhibited increased resistance to Mtb-induced cell death, while Mtb-infected Bach1-deficient mice displayed reduced bacterial loads, pulmonary necrosis and lipid peroxidation concurrent with increased survival. Single-cell RNA-seq analysis of lungs from Mtb-infected Bach1-/- mice revealed an enrichment of genes associated with ferroptosis suppression. Bach1 depletion in Mtb-infected B6.Sst1S mice that display human-like necrotic lung pathology also markedly reduced necrosis and increased host resistance. These findings identify Bach1 as a key regulator of cellular and tissue necrosis and host resistance in Mtb infection.

Cell state transition analysis identifies interventions that improve control of Mycobacterium tuberculosis infection by susceptible macrophages.

Understanding cell state transitions and purposefully controlling them to improve therapies is a longstanding challenge in biological research and medicine. Here, we identify a transcriptional signature that distinguishes activated macrophages from the tuberculosis (TB) susceptible and resistant mice. We then apply the cSTAR (cell state transition assessment and regulation) approach to data from screening-by-RNA sequencing to identify chemical perturbations that shift the transcriptional state of tumor necrosis factor (TNF)-activated TB-susceptible macrophages toward that of TB-resistant cells, i.e., prevents their aberrant activation without suppressing beneficial TNF responses. Last, we demonstrate that the compounds identified with this approach enhance the resistance of the TB-susceptible mouse macrophages to virulent Mycobacterium tuberculosis.

The Many Hosts of Mycobacteria 9 (MHM9): A conference report.

The Many Hosts of Mycobacteria (MHM) meeting series brings together basic scientists, clinicians and veterinarians to promote robust discussion and dissemination of recent advances in our knowledge of numerous mycobacterial diseases, including human and bovine tuberculosis (TB), nontuberculous mycobacteria (NTM) infection, Hansen's disease (leprosy), Buruli ulcer and Johne's disease. The 9th MHM conference (MHM9) was held in July 2022 at The Ohio State University (OSU) and centered around the theme of "Confounders of Mycobacterial Disease." Confounders can and often do drive the transmission of mycobacterial diseases, as well as impact surveillance and treatment outcomes. Various confounders were presented and discussed at MHM9 including those that originate from the host (comorbidities and coinfections) as well as those arising from the environment (e.g., zoonotic exposures), economic inequality (e.g. healthcare disparities), stigma (a confounder of leprosy and TB for millennia), and historical neglect (a confounder in Native American Nations). This conference report summarizes select talks given at MHM9 highlighting recent research advances, as well as talks regarding the historic and ongoing impact of TB and other infectious diseases on Native American Nations, including those in Southwestern Alaska where the regional TB incidence rate is among the highest in the Western hemisphere.

Adjunctive Integrated Stress Response Inhibition Accelerates Tuberculosis Clearance in Mice.

Despite numerous advances in tuberculosis (TB) drug development, long treatment durations have led to the emergence of multidrug resistance, which poses a major hurdle to global TB control. Shortening treatment time therefore remains a top priority. Host-directed therapies that promote bacterial clearance and/or lung health may improve the efficacy and treatment duration of tuberculosis antibiotics. We recently discovered that inhibition of the integrated stress response, which is abnormally activated in tuberculosis and associated with necrotic granuloma formation, reduced bacterial numbers and lung inflammation in mice. Here, we evaluated the impact of the integrated stress response (ISR) inhibitor ISRIB, administered as an adjunct to standard tuberculosis antibiotics, on bacterial clearance, relapse, and lung pathology in a mouse model of tuberculosis. Throughout the course of treatment, ISRIB robustly lowered bacterial burdens compared to the burdens with standard TB therapy alone and accelerated the time to sterility in mice, as demonstrated by significantly reduced relapse rates after 4 months of treatment. In addition, mice receiving adjunctive ISRIB tended to have reduced lung necrosis and inflammation. Together, our findings identify the ISR pathway as a promising therapeutic target with the potential to shorten TB treatment durations and improve lung health. IMPORTANCE Necrosis of lung lesions is a hallmark of tuberculosis (TB) that promotes bacterial growth, dissemination, and transmission. This process is driven by the persistent hyperactivation of the integrated stress response (ISR) pathway. Here, we show that adjunctive ISR inhibition during standard antibiotic therapy accelerates bacterial clearance and reduces immunopathology in a clinically relevant mouse model of TB, suggesting that host-directed therapies that de-escalate these pathological stress responses may shorten TB treatment durations. Our findings present an important conceptual advance toward overcoming the challenge of improving TB therapy and lowering the global burden of disease.

Cell state transition analysis identifies interventions that improve control of M. tuberculosis infection by susceptible macrophages.

Understanding cell state transitions and purposefully controlling them to improve therapies is a longstanding challenge in biological research and medicine. Here, we identify a transcriptional signature that distinguishes activated macrophages from TB-susceptible and TB-resistant mice. We then apply the cSTAR (cell State Transition Assessment and Regulation) approach to data from screening-by-RNA sequencing to identify chemical perturbations that shift the. transcriptional state of the TB-susceptible macrophages towards that of TB-resistant cells. Finally, we demonstrate that the compounds identified with this approach enhance resistance of the TB-susceptible mouse macrophages to virulent M. tuberculosis .

Progression and Dissemination of Pulmonary Mycobacterium Avium Infection in a Susceptible Immunocompetent Mouse Model.

Pulmonary infections caused by the group of nontuberculosis mycobacteria (NTM), Mycobacterium avium complex (MAC), are a growing public health concern with incidence and mortality steadily increasing globally. Granulomatous inflammation is the hallmark of MAC lung infection, yet reliable correlates of disease progression, susceptibility, and resolution are poorly defined. Unlike widely used inbred mouse strains, mice that carry the mutant allele at the genetic locus sst1 develop human-like pulmonary tuberculosis featuring well-organized caseating granulomas. We characterized pulmonary temporospatial outcomes of intranasal and left intrabronchial M. avium spp. hominissuis (M.av) induced pneumonia in B6.Sst1S mice, which carries the sst1 mutant allele. We utilized traditional semi-quantitative histomorphological evaluation, in combination with fluorescent multiplex immunohistochemistry (fmIHC), whole slide imaging, and quantitative digital image analysis. Followingintrabronchiolar infection with the laboratory M.av strain 101, the B6.Sst1S pulmonary lesions progressed 12-16 weeks post infection (wpi), with plateauing and/or resolving disease by 21 wpi. Caseating granulomas were not observed during the study. Disease progression from 12-16 wpi was associated with increased acid-fast bacilli, area of secondary granulomatous pneumonia lesions, and Arg1+ and double positive iNOS+/Arg1+ macrophages. Compared to B6 WT, at 16 wpi, B6.Sst1S lungs exhibited an increased area of acid-fast bacilli, larger secondary lesions with greater Arg1+ and double positive iNOS+/Arg1+ macrophages, and reduced T cell density. This morphomolecular analysis of histologic correlates of disease progression in B6.Sst1S could serve as a platform for assessment of medical countermeasures against NTM infection.

Medium throughput protocol for genome-based quantification of intracellular mycobacterial loads and macrophage survival during in vitro infection.

Here, we present a streamlined protocol for assessing intracellular Mycobacterium tuberculosis (Mtb) loads in macrophages. This protocol describes the simultaneous assessment of macrophage viability using automated microscopy. Further, we detail the quantification of mycobacterial loads using a rapid, inexpensive, and accurate approach for mycobacterial DNA isolation from paraformaldehyde-fixed macrophages. Simultaneous assessment of the bacterial loads using internal standard and macrophage viability allows for precise quantification of the effects of perturbations on Mtb and host cells while accounting for technical artifacts. For complete details on the use and execution of this protocol, please refer to Chatterjee et al. (2021).

Channeling macrophage polarization by rocaglates increases macrophage resistance to Mycobacterium tuberculosis.

Macrophages contribute to host immunity and tissue homeostasis via alternative activation programs. M1-like macrophages control intracellular bacterial pathogens and tumor progression. In contrast, M2-like macrophages shape reparative microenvironments that can be conducive for pathogen survival or tumor growth. An imbalance of these macrophages phenotypes may perpetuate sites of chronic unresolved inflammation, such as infectious granulomas and solid tumors. We have found that plant-derived and synthetic rocaglates sensitize macrophages to low concentrations of the M1-inducing cytokine IFN-gamma and inhibit their responsiveness to IL-4, a prototypical activator of the M2-like phenotype. Treatment of primary macrophages with rocaglates enhanced phagosome-lysosome fusion and control of intracellular mycobacteria. Thus, rocaglates represent a novel class of immunomodulators that can direct macrophage polarization toward the M1-like phenotype in complex microenvironments associated with hypofunction of type 1 and/or hyperactivation of type 2 immunity, e.g., chronic bacterial infections, allergies, and, possibly, certain tumors.

CXCL1: A new diagnostic biomarker for human tuberculosis discovered using Diversity Outbred mice.

More humans have died of tuberculosis (TB) than any other infectious disease and millions still die each year. Experts advocate for blood-based, serum protein biomarkers to help diagnose TB, which afflicts millions of people in high-burden countries. However, the protein biomarker pipeline is small. Here, we used the Diversity Outbred (DO) mouse population to address this gap, identifying five protein biomarker candidates. One protein biomarker, serum CXCL1, met the World Health Organization's Targeted Product Profile for a triage test to diagnose active TB from latent M.tb infection (LTBI), non-TB lung disease, and normal sera in HIV-negative, adults from South Africa and Vietnam. To find the biomarker candidates, we quantified seven immune cytokines and four inflammatory proteins corresponding to highly expressed genes unique to progressor DO mice. Next, we applied statistical and machine learning methods to the data, i.e., 11 proteins in lungs from 453 infected and 29 non-infected mice. After searching all combinations of five algorithms and 239 protein subsets, validating, and testing the findings on independent data, two combinations accurately diagnosed progressor DO mice: Logistic Regression using MMP8; and Gradient Tree Boosting using a panel of 4: CXCL1, CXCL2, TNF, IL-10. Of those five protein biomarker candidates, two (MMP8 and CXCL1) were crucial for classifying DO mice; were above the limit of detection in most human serum samples; and had not been widely assessed for diagnostic performance in humans before. In patient sera, CXCL1 exceeded the triage diagnostic test criteria (>90% sensitivity; >70% specificity), while MMP8 did not. Using Area Under the Curve analyses, CXCL1 averaged 94.5% sensitivity and 88.8% specificity for active pulmonary TB (ATB) vs LTBI; 90.9% sensitivity and 71.4% specificity for ATB vs non-TB; and 100.0% sensitivity and 98.4% specificity for ATB vs normal sera. Our findings overall show that the DO mouse population can discover diagnostic-quality, serum protein biomarkers of human TB.

Role of the transcriptional regulator SP140 in resistance to bacterial infections via repression of type I interferons.

Type I interferons (IFNs) are essential for anti-viral immunity, but often impair protective immune responses during bacterial infections. An important question is how type I IFNs are strongly induced during viral infections, and yet are appropriately restrained during bacterial infections. The Super susceptibility to tuberculosis 1 (Sst1) locus in mice confers resistance to diverse bacterial infections. Here we provide evidence that Sp140 is a gene encoded within the Sst1 locus that represses type I IFN transcription during bacterial infections. We generated Sp140-/- mice and found that they are susceptible to infection by Legionella pneumophila and Mycobacterium tuberculosis. Susceptibility of Sp140-/- mice to bacterial infection was rescued by crosses to mice lacking the type I IFN receptor (Ifnar-/-). Our results implicate Sp140 as an important negative regulator of type I IFNs that is essential for resistance to bacterial infections.

Visualizing the dynamics of tuberculosis pathology using molecular imaging.

Nearly 140 years after Robert Koch discovered Mycobacterium tuberculosis, tuberculosis (TB) remains a global threat and a deadly human pathogen. M. tuberculosis is notable for complex host-pathogen interactions that lead to poorly understood disease states ranging from latent infection to active disease. Additionally, multiple pathologies with a distinct local milieu (bacterial burden, antibiotic exposure, and host response) can coexist simultaneously within the same subject and change independently over time. Current tools cannot optimally measure these distinct pathologies or the spatiotemporal changes. Next-generation molecular imaging affords unparalleled opportunities to visualize infection by providing holistic, 3D spatial characterization and noninvasive, temporal monitoring within the same subject. This rapidly evolving technology could powerfully augment TB research by advancing fundamental knowledge and accelerating the development of novel diagnostics, biomarkers, and therapeutics.

The integrated stress response mediates necrosis in murine Mycobacterium tuberculosis granulomas.

The mechanism by which only some individuals infected with Mycobacterium tuberculosis develop necrotic granulomas with progressive disease while others form controlled granulomas that contain the infection remains poorly defined. Mice carrying the sst1-suscepible (sst1S) genotype develop necrotic inflammatory lung lesions, similar to human tuberculosis (TB) granulomas, which are linked to macrophage dysfunction, while their congenic counterpart (B6) mice do not. In this study we report that (a) sst1S macrophages developed aberrant, biphasic responses to TNF characterized by superinduction of stress and type I interferon pathways after prolonged TNF stimulation; (b) the late-stage TNF response was driven via a JNK/IFN-ß/protein kinase R (PKR) circuit; and (c) induced the integrated stress response (ISR) via PKR-mediated eIF2α phosphorylation and the subsequent hyperinduction of ATF3 and ISR-target genes Chac1, Trib3, and Ddit4. The administration of ISRIB, a small-molecule inhibitor of the ISR, blocked the development of necrosis in lung granulomas of M. tuberculosis-infected sst1S mice and concomitantly reduced the bacterial burden. Hence, induction of the ISR and the locked-in state of escalating stress driven by the type I IFN pathway in sst1S macrophages play a causal role in the development of necrosis in TB granulomas. Interruption of the aberrant stress response with inhibitors such as ISRIB may offer novel host-directed therapy strategies.

SON DNA-binding protein mediates macrophage autophagy and responses to intracellular infection.

Intracellular pathogens affect diverse host cellular defence and metabolic pathways. Here, we used infection with Francisella tularensis to identify SON DNA-binding protein as a central determinant of macrophage activities. RNAi knockdown of SON increases survival of human macrophages following F. tularensis infection or inflammasome stimulation. SON is required for macrophage autophagy, interferon response factor 3 expression, type I interferon response and inflammasome-associated readouts. SON knockdown has gene- and stimulus-specific effects on inflammatory gene expression. SON is required for accurate splicing and expression of GBF1, a key mediator of cis-Golgi structure and function. Chemical GBF1 inhibition has similar effects to SON knockdown, suggesting that SON controls macrophage functions at least in part by controlling Golgi-associated processes.

Publisher Correction: Type I interferon-driven susceptibility to Mycobacterium tuberculosis is mediated by IL-1Ra.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Diversity Outbred Mouse Population Is an Improved Animal Model of Vaccination against Tuberculosis That Reflects Heterogeneity of Protection.

Many studies of Mycobacterium tuberculosis infection and immunity have used mouse models. However, outcomes of vaccination and challenge with M. tuberculosis in inbred mouse strains do not reflect the full range of outcomes seen in people. Previous studies indicated that the novel Diversity Outbred (DO) mouse population exhibited a spectrum of outcomes after primary aerosol infection with M. tuberculosis Here, we demonstrate the value of this novel mouse population for studies of vaccination against M. tuberculosis aerosol challenge. Using the only currently licensed tuberculosis vaccine, we found that the DO population readily controlled systemic Mycobacterium bovis BCG bacterial burdens and that BCG vaccination significantly improved survival across the DO population upon challenge with M. tuberculosis Many individual DO mice that were vaccinated with BCG and then challenged with M. tuberculosis exhibited low bacterial burdens, low or even no systemic dissemination, little weight loss, and only minor lung pathology. In contrast, some BCG-vaccinated DO mice progressed quickly to fulminant disease upon M. tuberculosis challenge. Across the population, most of these disease parameters were at most modestly correlated with each other and were often discordant. This result suggests the need for a multiparameter metric to better characterize "disease" and "protection," with closer similarity to the complex case definitions used in people. Taken together, these results demonstrate that DO mice provide a novel small-animal model of vaccination against tuberculosis that better reflects the wide spectrum of outcomes seen in people.IMPORTANCE We vaccinated the Diversity Outbred (DO) population of mice with BCG, the only vaccine currently used to protect against tuberculosis, and then challenged them with M. tuberculosis by aerosol. We found that the BCG-vaccinated DO mouse population exhibited a wide range of outcomes, in which outcomes in individual mice ranged from minimal respiratory or systemic disease to fulminant disease and death. The breadth of these outcomes appears similar to the range seen in people, indicating that DO mice may serve as an improved small-animal model to study tuberculosis infection and immunity. Moreover, sophisticated tools are available for the use of these mice to map genes contributing to control of vaccination. Thus, the present studies provided an important new tool in the fight against tuberculosis.