Contribution of Lipid Mediators in Divergent Outcomes following Acute Bacterial and Viral Lung Infections in the Obese Host.

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.

Impairing RAGE signaling promotes survival and limits disease pathogenesis following SARS-CoV-2 infection in mice.

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.

Age-related differences in immune dynamics during SARS-CoV-2 infection in rhesus macaques.

Advanced age is a key predictor of severe COVID-19. To gain insight into this relationship, we used the rhesus macaque model of SARS-CoV-2 infection. Eight older and eight younger macaques were inoculated with SARS-CoV-2. Animals were evaluated using viral RNA quantification, clinical observations, thoracic radiographs, single-cell transcriptomics, multiparameter flow cytometry, multiplex immunohistochemistry, cytokine detection, and lipidomics analysis at predefined time points in various tissues. Differences in clinical signs, pulmonary infiltrates, and virus replication were limited. Transcriptional signatures of inflammation-associated genes in bronchoalveolar lavage fluid at 3 dpi revealed efficient mounting of innate immune defenses in both cohorts. However, age-specific divergence of immune responses emerged during the post-acute phase. Older animals exhibited sustained local inflammatory innate responses, whereas local effector T-cell responses were induced earlier in the younger animals. Circulating lipid mediator and cytokine levels highlighted increased repair-associated signals in the younger animals, and persistent pro-inflammatory responses in the older animals. In summary, despite similar disease outcomes, multi-omics profiling suggests that age may delay or impair antiviral cellular immune responses and delay efficient return to immune homeostasis.

Validation and Application of a Benchtop Cell Sorter in a Biosafety Level 3 Containment Setting.

Introduction: Fluorescent-activated cell sorting (FACS) is often the most appropriate technique to obtain pure populations of a cell type of interest for downstream analysis. However, aerosol droplets can be generated during the sort, which poses a biosafety risk when working with samples containing risk group 3 pathogens such as Francisella tularensis, Mycobacterium tuberculosis, Yersinia pestis, and severe acute respiratory syndrome coronavirus 2. For many researchers, placing the equipment required for FACS at biosafety level 3 (BSL-3) is often not possible due to expense, space, or expertise available. Methods: We performed aerosol testing as part of the biosafety evaluation of the MACSQuant Tyto, a completely closed, cartridge-based cell sorter. We also established quality control procedures to routinely evaluate instrument performance. Results: The MACSQuant Tyto does not produce aerosols as part of the sort procedure. Discussion: These data serve as guidance for other facilities with containment laboratories wishing to use the MACSQuant Tyto for cell sorting. Potential users should consult with their Institutional Biosafety Committees to perform in-house risk assessments of this equipment. Conclusion: The MACSQuant Tyto can safely be used on the benchtop to sort samples at BSL-3.

Cutting Edge: Lung-Resident T Cells Elicited by SARS-CoV-2 Do Not Mediate Protection against Secondary Infection.

Immunity to pulmonary infection typically requires elicitation of lung-resident T cells that subsequently confer protection against secondary infection. The presence of tissue-resident T cells in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) convalescent patients is unknown. Using a sublethal mouse model of coronavirus disease 2019, we determined if SARS-CoV-2 infection potentiated Ag-specific pulmonary resident CD4+ and CD8+ T cell responses and if these cells mediated protection against secondary infection. S protein-specific T cells were present in resident and circulating populations. However, M and N protein-specific T cells were detected only in the resident T cell pool. Using an adoptive transfer strategy, we found that T cells from SARS-CoV-2 immune animals did not protect naive mice. These data indicate that resident T cells are elicited by SARS-CoV-2 infection but are not sufficient for protective immunity.

Pulmonary infection induces persistent, pathogen-specific lipidomic changes influencing trained immunity.

Resolution of infection results in development of trained innate immunity which is typically beneficial for defense against unrelated secondary infection. Epigenetic changes including modification of histones via binding of various polar metabolites underlie the establishment of trained innate immunity. Therefore, host metabolism and this response are intimately linked. However, little is known regarding the influence of lipids on the development and function of trained immunity. Utilizing two models of pulmonary bacterial infection combined with multi-omic approaches, we identified persistent, pathogen-specific changes to the lung lipidome that correlated with differences in the trained immune response against a third unrelated pathogen. Further, we establish the specific cellular populations in the lung that contribute to this altered lipidome. Together these results expand our understanding of the pulmonary trained innate immune response and the contributions of host lipids in informing that response.

Validation and Application of a Bench Top Cell Sorter in a BSL-3 Containment Setting.

Rigorous assessment of the cellular and molecular changes during infection typically requires isolation of specific immune cell subsets for downstream application. While there are numerous options for enrichment/isolation of cells from tissues, fluorescent activated cell sorting (FACS) is accepted as a method that results in superior purification of a wide variety of cell types. Flow cytometry requires extensive fluidics and aerosol droplets can be generated during collection of target cells. Pathogens such as Francisella tularensis, Mycobacterium tuberculosis, Yersinia pestis, and SARS-CoV-2 require manipulation at biosafety level-3 (BSL-3). Due to the concern of potential aerosolization of these pathogens, use of flow cytometric-based cell sorting in these laboratory settings requires placement of the equipment in dedicated biosafety cabinets within the BSL-3. For many researchers, this is often not possible due to expense, space, or expertise available. Here we describe the safety validation and utility of a completely closed cell sorter that results in gentle, rapid, high purity, and safe sorting of cells on the benchtop at BSL-3. We also provide data demonstrating the need for cell sorting versus bead purification and the applicability of this technology for BSL-3 and potentially BSL-4 related infectious disease projects. Adoption of this technology will significantly expand our ability to uncover important features of the most dangerous infectious diseases leading to faster development of novel vaccines and therapeutics.