Catharine M. Bosio

Intracellular pathogens manipulate host metabolic systems to establish a replicative niche and evade immune responses. However, mechanisms by which metabolism can be altered for successful infection remain poorly understood. Here, we identify a metabolism-based strategy used by the intracellular pathogen Francisella tularensis subsp. tularensis (FTT) to condition its host cell for optimal replication. FTT, through its associated O-antigen capsule, augmented lactate oxidation in macrophages, thereby improving mitochondrial bioenergetics, reducing redox imbalances and production of pro-inflammatory cytokines, and maintaining cellular viability. Heavy isotope tracing and extracellular flux analysis confirmed lactate incorporation into the TCA cycle with increased mitochondrial ATP production, and these outcomes were associated with upregulation of lactate import and conversion systems, specifically monocarboxylate transporter 4 (MCT4) and LDHB, respectively. Targeting MCT4 using the small molecule inhibitor MSC-4381, controlled FTT infection-mediated manipulation of mitochondrial function and increased cell death in infected macrophages, thereby controlling infection. MSC-4381 also directly controlled FTT infection in defined medium implicating a critical role of short-chain carbon intermediates among the bacteria. Ultimately, these data demonstrate that the lactate oxidation metabolic node, including MCT4 activity, is integral for successful FTT infection and contributes to the pleiotropic effects of the FTT capsule on immune evasion. IMPORTANCE: Francisella tularensis subsp. tularensis (FTT) is an extremely virulent pathogen for which antibiotic intervention must be delivered early, often before proper diagnosis and effective, durable vaccines do not exist. Part of what makes FTT so problematic is that it is proficient at manipulating host metabolic pathways that govern immune activation. However, specific mechanisms by which this manipulation occurs have remained elusive. Discovering new ways in which FTT modulates host cell metabolism will reveal new therapeutic targets to help treat this difficult-to-control infection. We show here that FTT, through its O-antigen polysaccharide capsule, increased lactate oxidation, which in turn reduced inflammatory cytokine production, redox imbalance, and cell death. Targeting proteins responsible for lactate transport controlled FTT infection and blocked the capsule’s ability to manipulate the host cell. These data are the first to show lactate oxidation as a potential target for pathogen success and may be a therapeutic target for FTT infection.

Pulmonary infections often fail to produce long-lived immune memory and the underlying mechanism(s) for this are unclear. Given the complex interactions between cells within the lung, we predicted intrinsic and extrinsic factors contribute to development of poor memory immune responses. To identify these factors, we used a multiomics approach to determine host-driven responses that undermine or support development of effective immune responses in two mouse models of pulmonary bacterial infections. Single cell RNA analysis and spatial imaging of the lung revealed that, in contrast to Bordetella pertussis driven immunity, subpar responses following Francisella tularensis infection were associated with the inability of T cells to readily proliferate upon re-challenge and absence of formation of iBALT. Further, we also identified that these features were partially a consequence of IFN-γ driven reprogramming of endothelial cells resulting in expression of IDO1 and dysregulated tryptophan metabolism. Interestingly, IDO1 expression and imbalanced tryptophan persisted even after clearance of the primary infection. The importance of expression of IDO1 was confirmed using IDO1 knock out mice. Specifically, these animals could withstand higher doses of the initial infection and developed significantly larger pools of functional T cells compared to wild type controls. Together, these results demonstrate critical crosstalk among cells in the lung that influences spatial organization of immune cells which affects the ability to develop effective memory immune responses against secondary bacterial infection. Our data also underscores the challenge of utilizing a live vaccine strategy against tularemia and the necessity for identifying novel, acellular vaccine candidates.

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection involves an initial viral infection phase followed by a host‐response phase that includes an eicosanoid and cytokine storm, lung inflammation and respiratory failure. While vaccination and early anti‐viral therapies are effective in preventing or limiting the pathogenic host response, this latter phase is poorly understood with no highly effective treatment options. Inhibitors of soluble epoxide hydrolase (sEH) increase levels of anti‐inflammatory molecules called epoxyeicosatrienoic acids (EETs). This study aimed to investigate the impact of sEH inhibition on the host response to SARS‐CoV‐2 infection in a mouse model with human angiotensin‐converting enzyme 2 (ACE2) expression. Mice were infected with SARS‐CoV‐2 and treated with either vehicle or the sEH inhibitor 1‐trifluoromethoxyphenyl‐3‐(1‐propionylpiperidin‐4‐yl) urea (TPPU). At day 5 post‐infection, SARS‐CoV‐2 induced weight loss, clinical signs, a cytokine storm, an eicosanoid storm, and severe lung inflammation with ~50% mortality on days 6–8 post‐infection. SARS‐CoV‐2 infection induced lung expression of phospholipase A ₂ (PLA ₂ ), cyclooxygenase (COX) and lipoxygenase (LOX) pathway genes, while suppressing expression of most cytochrome P450 genes. Treatment with the sEH inhibitor TPPU delayed weight loss but did not alter clinical signs, lung cytokine expression or overall survival of infected mice. Interestingly, TPPU treatment significantly reversed the eicosanoid storm and attenuated viral‐induced elevation of 39 fatty acids and oxylipins from COX, LOX and P450 pathways, which suggests the effects at the level of PLA ₂ activation. The suppression of the eicosanoid storm by TPPU without corresponding changes in lung cytokines, lung inflammation or mortality reveals a surprising dissociation between systemic oxylipin and cytokine signaling pathways during SARS‐CoV‐2 infection and suggests that the cytokine storm is primarily responsible for morbidity and mortality in this animal model.

Route of exposure to pathogens can inform divergent disease pathogenesis and mortality rates. However, the features that contribute to these differences are not well established. Host metabolism has emerged as a critical element governing susceptibility and the metabolism of tissue exposure sites are unique. Therefore, specific metabolic niches may contribute to the course and outcome of infection depending on route of infection. In the current study, we utilized a combination of imaging and systems metabolomics to map the spatiotemporal dynamics of the host response to intranasal (i.n.) or intradermal (i.d.) infection of mice using the bacterium Francisella tularensis subsp tularensis (FTT). FTT causes lethal disease through these infection routes with similar inoculation doses and replication kinetics, which allowed for isolation of host outcomes independent of bacterial burden. We observed metabolic modifications that were both route dependent and independent. Specifically, i.d. infection resulted in early metabolic reprogramming at the site of infection and draining lymph nodes, whereas the lungs and associated draining lymph nodes were refractory to metabolic reprogramming following i.n. infection. Irrespective of exposure route, FTT promoted metabolic changes in systemic organs prior to colonization, and caused massive dysregulation of host metabolism in these tissues prior to onset of morbidity. Preconditioning infection sites towards a more glycolytic and pro-inflammatory state prior to infection exacerbated FTT replication within the lungs but not intradermal tissue. This enhancement of replication in the lungs was associated with the ability of FTT to limit redox imbalance and alter the pentose phosphate pathway. Together, these studies identify central metabolic features of the lung and dermal compartments that contribute to disease progression and identify potential tissue specific targets that may be exploited for novel therapeutic approaches.

Dysregulation of host metabolism is a feature of lethal SARS-CoV-2 infection. Perturbations in α-ketoglutarate levels can elicit metabolic reprogramming through 2-oxoglutarate–dependent dioxygenases (2-ODDGs), leading to stabilization of the transcription factor HIF-1α. HIF1-α activation has been reported to promote antiviral mechanisms against SARS-CoV-2 through direct regulation of ACE2 expression (a receptor required for viral entry). However, given the numerous pathways HIF-1α serves to regulate it is possible that there are other undefined metabolic mechanisms contributing to the pathogenesis of SARS-CoV-2 independent of ACE2 downregulation. In this study, we used in vitro and in vivo models in which HIF-1α modulation of ACE2 expression was negated, allowing for isolated characterization of the host metabolic response within SARS-CoV-2 disease pathogenesis. We demonstrated that SARS-CoV-2 infection limited stabilization of HIF-1α and associated mitochondrial metabolic reprogramming by maintaining activity of the 2-ODDG prolyl hydroxylases. Inhibition of 2-ODDGs with dimethyloxalylglycine promoted HIF-1α stabilization following SARS-CoV-2 infection, and significantly increased survival among SARS-CoV-2–infected mice compared with vehicle controls. However, unlike previous reports, the mechanism by which activation of HIF-1α responses contributed to survival was not through impairment of viral replication. Rather, dimethyloxalylglycine treatment facilitated direct effects on host metabolism including increased glycolysis and resolution of dysregulated pools of metabolites, which correlated with reduced morbidity. Taken together, these data identify (to our knowledge) a novel function of α-ketoglutarate–sensing platforms, including those responsible for HIF-1α stabilization, in the resolution of SARS-CoV-2 infection and support targeting these metabolic nodes as a viable therapeutic strategy to limit disease severity during infection.

Obesity is considered an important comorbidity for a range of noninfectious and infectious disease states including those that originate in the lung, yet the mechanisms that contribute to this susceptibility are not well defined. In this study, we used the diet-induced obesity (DIO) mouse model and two models of acute pulmonary infection, Francisella tularensis subspecies tularensis strain SchuS4 and SARS-CoV-2, to uncover the contribution of obesity in bacterial and viral disease. Whereas DIO mice were more resistant to infection with SchuS4, DIO animals were more susceptible to SARS-CoV-2 infection compared with regular weight mice. In both models, neither survival nor morbidity correlated with differences in pathogen load, overall cellularity, or influx of inflammatory cells in target organs of DIO and regular weight animals. Increased susceptibility was also not associated with exacerbated production of cytokines and chemokines in either model. Rather, we observed pathogen-specific dysregulation of the host lipidome that was associated with vulnerability to infection. Inhibition of specific pathways required for generation of lipid mediators reversed resistance to both bacterial and viral infection. Taken together, our data demonstrate disparity among obese individuals for control of lethal bacterial and viral infection and suggest that dysregulation of the host lipidome contributes to increased susceptibility to viral infection in the obese host.

Pulmonary infections elicit a combination of tissue-resident and circulating T cell responses. Understanding the contribution of these anatomically distinct cellular pools in protective immune responses is critical for vaccine development. Francisella tularensis is a highly virulent bacterium capable of causing lethal systemic disease following pulmonary infection for which there is no currently licensed vaccine. Although T cells are required for survival of F. tularensis infection, the relative contribution of tissue-resident and circulating T cells is not completely understood, hampering design of effective, long-lasting vaccines directed against this bacterium. We have previously shown that resident T cells were not sufficient to protect against F. tularensis, suggesting circulating cells may serve a critical role in host defense. To elucidate the role of circulating T cells, we used a model of vaccination and challenge of parabiotic mice. Intranasally infected naive mice conjoined to immune animals had increased numbers of circulating memory T cells and similar splenic bacterial burdens as vaccinated–vaccinated pairs. However, bacterial loads in the lungs of naive parabionts were significantly greater than those observed in vaccinated–vaccinated pairs, but despite early control of F. tularensis replication, all naive–vaccinated pairs succumbed to infection. Together, these data define the specific roles of circulating and resident T cells in defense against infection that is initiated in the pulmonary compartment but ultimately causes disseminated disease. These data also provide evidence for employing vaccination strategies that elicit both pools of T cells for immunity against F. tularensis and may be a common theme for other disseminating bacterial infections.

Cellular and molecular mechanisms driving morbidity following SARS-CoV-2 infection have not been well defined. The receptor for advanced glycation end products (RAGE) is a central mediator of tissue injury and contributes to SARS-CoV-2 disease pathogenesis. In this study, we temporally delineated key cell and molecular events leading to lung injury in mice following SARS-CoV-2 infection and assessed efficacy of therapeutically targeting RAGE to improve survival. Early following infection, SARS-CoV-2 replicated to high titers within the lungs and evaded triggering inflammation and cell death. However, a significant necrotic cell death event in CD45 ^– populations, corresponding with peak viral loads, was observed on day 2 after infection. Metabolic reprogramming and inflammation were initiated following this cell death event and corresponded with increased lung interstitial pneumonia, perivascular inflammation, and endothelial hyperplasia together with decreased oxygen saturation. Therapeutic treatment with the RAGE antagonist FPS-ZM1 improved survival in infected mice and limited inflammation and associated perivascular pathology. Together, these results provide critical characterization of disease pathogenesis in the mouse model and implicate a role for RAGE signaling as a therapeutic target to improve outcomes following SARS-CoV-2 infection.

Pre-existing comorbidities such as obesity or metabolic diseases can adversely affect the clinical outcome of COVID-19. Chronic metabolic disorders are globally on the rise and often a consequence of an unhealthy diet, referred to as a Western Diet. For the first time in the Syrian hamster model, we demonstrate the detrimental impact of a continuous high-fat high-sugar diet on COVID-19 outcome. We observed increased weight loss and lung pathology, such as exudate, vasculitis, hemorrhage, fibrin, and edema, delayed viral clearance and functional lung recovery, and prolonged viral shedding. This was accompanied by an altered, but not significantly different, systemic IL-10 and IL-6 profile, as well as a dysregulated serum lipid response dominated by polyunsaturated fatty acid-containing phosphatidylethanolamine, partially recapitulating cytokine and lipid responses associated with severe human COVID-19. Our data support the hamster model for testing restrictive or targeted diets and immunomodulatory therapies to mediate the adverse effects of metabolic disease on COVID-19.