The inflammatory microenvironment of the lung at the time of infection governs innate control of SARS-CoV-2 replication.

SARS-CoV-2 infection leads to vastly divergent clinical outcomes ranging from asymptomatic infection to fatal disease. Co-morbidities, sex, age, host genetics and vaccine status are known to affect disease severity. Yet, how the inflammatory milieu of the lung at the time of SARS-CoV-2 exposure impacts the control of viral replication remains poorly understood. We demonstrate here that immune events in the mouse lung closely preceding SARS-CoV-2 infection significantly impact viral control and we identify key innate immune pathways required to limit viral replication. A diverse set of pulmonary inflammatory stimuli, including resolved antecedent respiratory infections with S. aureus or influenza, ongoing pulmonary M. tuberculosis infection, ovalbumin/alum-induced asthma or airway administration of defined TLR ligands and recombinant cytokines, all establish an antiviral state in the lung that restricts SARS-CoV-2 replication upon infection. In addition to antiviral type I interferons, the broadly inducible inflammatory cytokines TNFα and IL-1 precondition the lung for enhanced viral control. Collectively, our work shows that SARS-CoV-2 may benefit from an immunologically quiescent lung microenvironment and suggests that heterogeneity in pulmonary inflammation that precedes or accompanies SARS-CoV-2 exposure may be a significant factor contributing to the population-wide variability in COVID-19 disease outcomes.

Co-infection of mice with SARS-CoV-2 and Mycobacterium tuberculosis limits early viral replication but does not affect mycobacterial loads.

Viral co-infections have been implicated in worsening tuberculosis (TB) and during the COVID-19 pandemic, the global rate of TB-related deaths has increased for the first time in over a decade. We and others have previously shown that a resolved prior or concurrent influenza A virus infection in Mycobacterium tuberculosis (Mtb)-infected mice resulted in increased pulmonary bacterial burden, partly through type I interferon (IFN-I)-dependent mechanisms. Here we investigated whether SARS-CoV-2 (SCV2) co-infection could also negatively affect bacterial control of Mtb. Importantly, we found that K18-hACE2 transgenic mice infected with SCV2 one month before, or months after aerosol Mtb exposure did not display exacerbated Mtb infection-associated pathology, weight loss, nor did they have increased pulmonary bacterial loads. However, pre-existing Mtb infection at the time of exposure to the ancestral SCV2 strain in infected K18-hACE2 transgenic mice or the beta variant (B.1.351) in WT C57Bl/6 mice significantly limited early SCV2 replication in the lung. Mtb-driven protection against SCV2 increased with higher bacterial doses and did not require IFN-I, TLR2 or TLR9 signaling. These data suggest that SCV2 co-infection does not exacerbate Mtb infection in mice, but rather the inflammatory response generated by Mtb infection in the lungs at the time of SCV2 exposure restricts viral replication.

Endogenous and Therapeutic 25-Hydroxycholesterols May Worsen Early SARS-CoV-2 Pathogenesis in Mice.

Oxysterols (i.e., oxidized cholesterol species) have complex roles in biology. 25-Hydroxycholesterol (25HC), a product of the activity of cholesterol-25-hydroxylase (CH25H) on cholesterol, has recently been shown to be broadly antiviral, suggesting therapeutic potential against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, 25HC can also amplify inflammation and be converted by CYP7B1 (cytochrome P450 family 7 subfamily B member 1) to 7α,25-dihydroxycholesterol, a lipid with chemoattractant activity, via the G protein-coupled receptor EBI2 (Epstein-Barr virus-induced gene 2)/GPR183 (G protein-coupled receptor 183). Here, using in vitro studies and two different murine models of SARS-CoV-2 infection, we investigate the effects of these two oxysterols on SARS-CoV-2 pneumonia. We show that although 25HC and enantiomeric-25HC are antiviral in vitro against human endemic coronavirus-229E, they did not inhibit SARS-CoV-2; nor did supplemental 25HC reduce pulmonary SARS-CoV-2 titers in the K18-human ACE2 (angiotensin-converting enzyme 2) mouse model in vivo. Treatment with 25HC also did not alter immune cell influx into the airway, airspace cytokines, lung pathology, weight loss, symptoms, or survival but was associated with increased airspace albumin, an indicator of microvascular injury, and increased plasma proinflammatory cytokines. Conversely, mice treated with the EBI2/GPR183 inhibitor NIBR189 displayed a modest increase in lung viral load only at late time points but no change in weight loss. Consistent with these findings, although Ch25h and 25HC were upregulated in the lungs of SARS-CoV-2-infected wild-type mice, lung viral titers and weight loss in Ch25h-/- and Gpr183-/- mice infected with the ß variant were similar to those in control animals. Taken together, endogenous 25HCs do not significantly regulate early SARS-CoV-2 replication or pathogenesis, and supplemental 25HC may have proinjury rather than therapeutic effects in SARS-CoV-2 pneumonia.

Evaluation of endogenous and therapeutic 25-hydroxycholesterols in murine models of pulmonary SARS-CoV-2 infection.

Oxysterols (i.e., oxidized cholesterol species) have complex roles in biology. 25-hydroxycholesterol (25HC), a product of activity of cholesterol-25-hydroxylase (CH25H) upon cholesterol, has recently been shown to be broadly antiviral, suggesting therapeutic potential against SARS-CoV-2. However, 25HC can also amplify inflammation and tissue injury and be converted by CYP7B1 to 7α,25HC, a lipid with chemoattractant activity via the G protein-coupled receptor, EBI2/GPR183. Here, using in vitro studies and two different murine models of SARS-CoV-2 infection, we investigate the effects of these two oxysterols on SARS-CoV-2 pneumonia. We show that while 25HC and enantiomeric-25HC are antiviral in vitro against human endemic coronavirus-229E, they did not inhibit SARS-CoV-2; nor did supplemental 25HC reduce pulmonary SARS-CoV-2 titers in the K18-human ACE2 mouse model in vivo. 25HC treatment also did not alter immune cell influx into the airway, airspace cytokines, lung pathology, weight loss, symptoms, or survival but was associated with increased airspace albumin, an indicator of microvascular injury, and increased plasma pro-inflammatory cytokines. Conversely, mice treated with the EBI2/GPR183 inhibitor NIBR189 displayed a modest increase in lung viral load only at late time points, but no change in weight loss. Consistent with these findings, although Ch25h was upregulated in the lungs of SARS-CoV-2-infected WT mice, lung viral titers and weight loss in Ch25h-/- and Gpr183-/- mice infected with the beta variant were similar to control animals. Taken together, endogenous 25-hydroxycholesterols do not significantly regulate early SARS-CoV-2 replication or pathogenesis and supplemental 25HC may have pro-injury rather than therapeutic effects in SARS-CoV-2 pneumonia.

Rapid GPR183-mediated recruitment of eosinophils to the lung after Mycobacterium tuberculosis infection.

Influx of eosinophils into the lungs is typically associated with type II responses during allergy and fungal and parasitic infections. However, we previously reported that eosinophils accumulate in lung lesions during type I inflammatory responses to Mycobacterium tuberculosis (Mtb) in humans, macaques, and mice, in which they support host resistance. Here we show eosinophils migrate into the lungs of macaques and mice as early as one week after Mtb exposure. In mice this influx is CCR3 independent and instead requires cell-intrinsic expression of the oxysterol receptor GPR183, which is highly expressed on human and macaque eosinophils. Murine eosinophils interact directly with bacilli-laden alveolar macrophages, which upregulate the oxysterol-synthesizing enzyme Ch25h, and eosinophil recruitment is impaired in Ch25h-deficient mice. Our findings show that eosinophils are among the earliest cells from circulation to sense and respond to Mtb infection of alveolar macrophages and reveal a role for GPR183 in the migration of eosinophils into lung tissue.

Intravenous administration of BCG protects mice against lethal SARS-CoV-2 challenge.

In addition to providing partial protection against pediatric tuberculosis, vaccination with bacille Calmette-Guérin (BCG) has been reported to confer nonspecific resistance to unrelated pulmonary pathogens, a phenomenon attributed to the induction of long-lasting alterations within the myeloid cell compartment. Here, we demonstrate that intravenous, but not subcutaneous, inoculation of BCG protects human-ACE2 transgenic mice against lethal challenge with SARS-CoV-2 (SCV2) and results in reduced viral loads in non-transgenic animals infected with an α variant. The observed increase in host resistance was associated with reductions in SCV2-induced tissue pathology, inflammatory cell recruitment, and cytokine production that multivariate analysis revealed as only partially related to diminished viral load. We propose that this protection stems from BCG-induced alterations in the composition and function of the pulmonary cellular compartment that impact the innate response to the virus and ensuing immunopathology. While intravenous BCG vaccination is not a clinically acceptable practice, our findings provide an experimental model for identifying mechanisms by which nonspecific stimulation of the pulmonary immune response promotes host resistance to SCV2 lethality.

Intravenous administration of BCG protects mice against lethal SARS-CoV-2 challenge.

Early events in the host response to SARS-CoV-2 are thought to play a major role in determining disease severity. During pulmonary infection, the virus encounters both myeloid and epithelioid lineage cells that can either support or restrict pathogen replication as well as respond with host protective versus detrimental mediators. In addition to providing partial protection against pediatric tuberculosis, vaccination with bacille Calmette-Guérin (BCG) has been reported to confer non-specific resistance to unrelated pulmonary pathogens, a phenomenon attributed to the induction of long-lasting alterations within the myeloid cell compartment. Here we demonstrate that prior intravenous, but not subcutaneous, administration of BCG protects human-ACE2 transgenic mice against lethal challenge with SARS-CoV-2 and results in reduced viral loads in non-transgenic animals infected with an alpha variant. The observed increase in host resistance was associated with reductions in SARS-CoV-2-induced tissue pathology, inflammatory cell recruitment and cytokine production that multivariate analysis revealed to be only partially related to diminished viral load. We propose that this protection stems from BCG-induced alterations in the composition and function of the pulmonary cellular compartment that impact the innate response to the virus and the ensuing immunopathology.

Eosinophils are part of the granulocyte response in tuberculosis and promote host resistance in mice.

Host resistance to Mycobacterium tuberculosis (Mtb) infection requires the activities of multiple leukocyte subsets, yet the roles of the different innate effector cells during tuberculosis are incompletely understood. Here we uncover an unexpected association between eosinophils and Mtb infection. In humans, eosinophils are decreased in the blood but enriched in resected human tuberculosis lung lesions and autopsy granulomas. An influx of eosinophils is also evident in infected zebrafish, mice, and nonhuman primate granulomas, where they are functionally activated and degranulate. Importantly, using complementary genetic models of eosinophil deficiency, we demonstrate that in mice, eosinophils are required for optimal pulmonary bacterial control and host survival after Mtb infection. Collectively, our findings uncover an unexpected recruitment of eosinophils to the infected lung tissue and a protective role for these cells in the control of Mtb infection in mice.

Upregulation of CD47 Is a Host Checkpoint Response to Pathogen Recognition.

It is well understood that the adaptive immune response to infectious agents includes a modulating suppressive component as well as an activating component. We now show that the very early innate response also has an immunosuppressive component. Infected cells upregulate the CD47 "don't eat me" signal, which slows the phagocytic uptake of dying and viable cells as well as downstream antigen-presenting cell (APC) functions. A CD47 mimic that acts as an essential virulence factor is encoded by all poxviruses, but CD47 expression on infected cells was found to be upregulated even by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that encode no mimic. CD47 upregulation was revealed to be a host response induced by the stimulation of both endosomal and cytosolic pathogen recognition receptors (PRRs). Furthermore, proinflammatory cytokines, including those found in the plasma of hepatitis C patients, upregulated CD47 on uninfected dendritic cells, thereby linking innate modulation with downstream adaptive immune responses. Indeed, results from antibody-mediated CD47 blockade experiments as well as CD47 knockout mice revealed an immunosuppressive role for CD47 during infections with lymphocytic choriomeningitis virus and Mycobacterium tuberculosis Since CD47 blockade operates at the level of pattern recognition receptors rather than at a pathogen or antigen-specific level, these findings identify CD47 as a novel potential immunotherapeutic target for the enhancement of immune responses to a broad range of infectious agents.IMPORTANCE Immune responses to infectious agents are initiated when a pathogen or its components bind to pattern recognition receptors (PRRs). PRR binding sets off a cascade of events that activates immune responses. We now show that, in addition to activating immune responses, PRR signaling also initiates an immunosuppressive response, probably to limit inflammation. The importance of the current findings is that blockade of immunomodulatory signaling, which is mediated by the upregulation of the CD47 molecule, can lead to enhanced immune responses to any pathogen that triggers PRR signaling. Since most or all pathogens trigger PRRs, CD47 blockade could be used to speed up and strengthen both innate and adaptive immune responses when medically indicated. Such immunotherapy could be done without a requirement for knowing the HLA type of the individual, the specific antigens of the pathogen, or, in the case of bacterial infections, the antimicrobial resistance profile.

Differential Cytokine Utilization and Tissue Tropism Results in Distinct Repopulation Kinetics of Naïve vs. Memory T Cells in Mice.

Naïve and memory T cells co-exist in the peripheral T cell pool, but the cellular mechanisms that maintain the balance and homeostasis of these two populations remain mostly unclear. To address this question, here, we assessed homeostatic proliferation and repopulation kinetics of adoptively transferred naïve and memory T cells in lymphopenic host mice. We identified distinct kinetics of proliferation and tissue-distribution between naïve and memory donor T cells, which resulted in the occupancy of the peripheral T cell pool by mostly naïve-origin T cells in short term (<1 week), but, in a dramatic reversal, by mostly memory-origin T cells in long term (>4 weeks). To explain this finding, we assessed utilization of the homeostatic cytokines IL-7 and IL-15 by naïve and memory T cells. We found different efficiencies of IL-7 signaling between naïve and memory T cells, where memory T cells expressed larger amounts of IL-7Rα but were significantly less potent in activation of STAT5 that is downstream of IL-7 signaling. Nonetheless, memory T cells were superior in long-term repopulation of the peripheral T cell pool, presumably, because they preferentially migrated into non-lymphoid tissues upon adoptive transfer and additionally utilized tissue IL-15 for rapid expansion. Consequently, co-utilization of IL-7 and IL-15 provides memory T cells a long-term survival advantage. We consider this mechanism important, as it permits the memory T cell population to be maintained in face of constant influx of naïve T cells to the peripheral T cell pool and under competing conditions for survival cytokines.

Comparing DNA enrichment of proliferating cells following administration of different stable isotopes of heavy water.

Deuterated water (2H2O) is a label commonly used for safe quantitative measurement of deuterium enrichment into DNA of proliferating cells. More recently, it has been used for labeling proteins and other biomolecules. Our in vitro - in vivo research reports important stable isotopic labeling enrichment differences into the DNA nucleosides and their isotopologues (e.g. deoxyadenosine (dA) M + 1, dA M + 2, dA M + 3), as well as tumor cell proliferation effects for various forms of commercially available stable heavy water (2H2O, H218O, and 2H218O). Using an in vitro mouse thymus tumor cell line, we determined that H218O provides superior DNA labeling enrichment quantitation, as measured by GC-positive chemical ionization (PCI)-MS/MS. In addition, at higher but physiologically relevant doses, both 2H218O and 2H2O down modulated mouse thymus tumor cell proliferation, whereas H218O water had no observable effects on cell proliferation. The in vivo labeling studies, where normal mouse bone marrow cells (i.e. high turnover) were evaluated post labeling, demonstrated DNA enrichments concordant with measurements from the in vitro studies. Our research also reports a headspace-GC-NCI-MS method, which rapidly and quantitatively measures stable heavy water levels in total body water.

In vivo kinetics and nonradioactive imaging of rapidly proliferating cells in graft-versus-host disease.

Hematopoietic stem cell transplantation (HSCT) offers a cure for cancers that are refractory to chemotherapy and radiation. Most HSCT recipients develop chronic graft-versus-host disease (cGVHD), a systemic alloimmune attack on host organs. Diagnosis is based on clinical signs and symptoms, as biopsies are risky. T cells are central to the biology of cGVHD. We found that a low Treg/CD4+ T effector memory (Tem) ratio in circulation, lymphoid, and target organs identified early and established mouse cGVHD. Using deuterated water labeling to measure multicompartment in vivo kinetics of these subsets, we show robust Tem and Treg proliferation in lymphoid and target organs, while Tregs undergo apoptosis in target organs. Since deuterium enrichment into DNA serves as a proxy for cell proliferation, we developed a whole-body clinically relevant deuterium MRI approach to nonradioactively detect cGVHD and potentially allow imaging of other diseases characterized by rapidly proliferating cells.

Distinct IL-7 signaling in recent thymic emigrants versus mature naïve T cells controls T-cell homeostasis.

IL-7 is essential for T-cell survival but its availability is limited in vivo. Consequently, all peripheral T cells, including recent thymic emigrants (RTEs) are constantly competing for IL-7 to survive. RTEs are required to replenish TCR diversity and rejuvenate the peripheral T-cell pool. However, it remains unknown how RTEs successfully compete with resident mature T cells for IL-7. Moreover, RTEs express low levels of IL-7 receptors, presumably rendering them even less competitive. Here, we show that, surprisingly, RTEs are more responsive to IL-7 than mature naïve T cells as demonstrated by markedly increased STAT5 phosphorylation upon IL-7 stimulation. Nonetheless, adoptive transfer of RTE cells into lymphopenic host mice resulted in slower IL-7-induced homeostatic proliferation and diminished expansion compared to naïve donor T cells. Mechanistically, we found that IL-7 signaling in RTEs preferentially upregulated expression of Bcl-2, which is anti-apoptotic but also anti-proliferative. In contrast, naïve T cells showed diminished Bcl-2 induction but greater proliferative response to IL-7. Collectively, these data indicate that IL-7 responsiveness in RTE is designed to maximize survival at the expense of reduced proliferation, consistent with RTE serving as a subpopulation of T cells rich in diversity but not in frequency.

Suppression of Dendritic Cell-Derived IL-12 by Endogenous Glucocorticoids Is Protective in LPS-Induced Sepsis.

Sepsis, an exaggerated systemic inflammatory response, remains a major medical challenge. Both hyperinflammation and immunosuppression are implicated as causes of morbidity and mortality. Dendritic cell (DC) loss has been observed in septic patients and in experimental sepsis models, but the role of DCs in sepsis, and the mechanisms and significance of DC loss, are poorly understood. Here, we report that mice with selective deletion of the glucocorticoid receptor (GR) in DCs (GR(CD11c-cre)) were highly susceptible to LPS-induced septic shock, evidenced by elevated inflammatory cytokine production, hypothermia, and mortality. Neutralizing anti-IL-12 antibodies prevented hypothermia and death, demonstrating that endogenous GC-mediated suppression of IL-12 is protective. In LPS-challenged GR(CD11c-cre) mice, CD8(+) DCs were identified as the major source of prolonged IL-12 production, which correlated with elevations of NK cell-derived IFN-γ. In addition, the loss of GR in CD11c(+) cells rescued LPS-induced loss of CD8(+) DCs but not other DC subsets. Unlike wild-type animals, exposure of GR(CD11c-cre) mice to low-dose LPS did not induce CD8(+) DC loss or tolerance to subsequent challenge with high dose, but neutralization of IL-12 restored the ability of low-dose LPS to tolerize. Therefore, endogenous glucocorticoids blunt LPS-induced inflammation and promote tolerance by suppressing DC IL-12 production.

Histone H3 Lysine 27 demethylases Jmjd3 and Utx are required for T-cell differentiation.

Although histone H3 lysine 27 trimethylation (H3K27Me3) is associated with gene silencing, whether H3K27Me3 demethylation affects transcription and cell differentiation in vivo has remained elusive. To investigate this, we conditionally inactivated the two H3K27Me3 demethylases, Jmjd3 and Utx, in non-dividing intrathymic CD4(+) T-cell precursors. Here we show that both enzymes redundantly promote H3K27Me3 removal at, and expression of, a specific subset of genes involved in terminal thymocyte differentiation, especially S1pr1, encoding a sphingosine-phosphate receptor required for thymocyte egress. Thymocyte expression of S1pr1 was not rescued in Jmjd3- and Utx-deficient male mice, which carry the catalytically inactive Utx homolog Uty, supporting the conclusion that it requires H3K27Me3 demethylase activity. These findings demonstrate that Jmjd3 and Utx are required for T-cell development, and point to a requirement for their H3K27Me3 demethylase activity in cell differentiation.

c-IAP ubiquitin protein ligase activity is required for 4-1BB signaling and CD8(+) memory T-cell survival.

Cellular inhibitor of apoptosis proteins (c-IAP) 1 and 2 are widely expressed ubiquitin protein ligases that regulate a variety of cellular functions, including the sensitivity of T cells to costimulation. 4-1BB is a TNF receptor family member that signals via a complex that includes TRAF family members and the c-IAPs to upregulate NF-κB and ERK, and has been implicated in memory T-cell survival. Here, we show that effector and memory T cells from mice expressing a dominant negative E3-inactive c-IAP2 (c-IAP2(H570A)) have impaired signaling downstream of 4-1BB. When infected with lymphocytic choriomeningitis virus, unlike mice in which c-IAPs were acutely downregulated by c-IAP antagonists, the primary response of c-IAP2(H570A) mice was normal. However, the number of antigen-specific CD8(+) but not CD4(+) T cells declined more rapidly and to a greater extent in c-IAP2(H570A) mice than in WT controls. Studies with T-cell adoptive transfer demonstrated that the enhanced decay of memory cells was T-cell intrinsic. Thus, c-IAP E3 activity is required for 4-1BB coreceptor signaling and maintenance of CD8(+) T-cell memory.

Thpok-independent repression of Runx3 by Gata3 during CD4+ T-cell differentiation in the thymus.

CD4(+) helper T cells are essential for immune responses and differentiate in the thymus from CD4(+) CD8(+) "double-positive" (DP) thymocytes. The transcription factor Runx3 inhibits CD4(+) T-cell differentiation by repressing Cd4 gene expression; accordingly, Runx3 is not expressed in DP thymocytes or developing CD4(+) T cells. The transcription factor Thpok is upregulated in CD4-differentiating thymocytes and required to repress Runx3. However, how Runx3 is controlled at early stages of CD4(+) T-cell differentiation, before the onset of Thpok expression, remains unknown. Here we show that Gata3, a transcription factor preferentially and transiently upregulated by CD4(+) T-cell precursors, represses Runx3 and binds the Runx3 locus in vivo. Accordingly, we show that high-level Gata3 expression and expression of Runx3 are mutually exclusive. Furthermore, whereas Runx3 represses Cd4, we show that Gata3 promotes Cd4 expression in Thpok-deficient thymocytes. Thus, in addition to its previously documented role in promoting CD4-lineage gene-expression, Gata3 represses CD8-lineage gene expression. These findings identify Gata3 as a critical pivot of CD4-CD8 lineage differentiation.

The zinc finger transcription factor Zbtb7b represses CD8-lineage gene expression in peripheral CD4+ T cells.

How CD4-CD8 differentiation is maintained in mature T cells is largely unknown. The present study has examined the role in this process of the zinc finger protein Zbtb7b, a critical factor for the commitment of MHC II-restricted thymocytes to the CD4+ lineage. We showed that Zbtb7b acted in peripheral CD4+ T cells to suppress CD8-lineage gene expression, including that of CD8 and cytotoxic effector genes perforin and Granzyme B, and was important for the proper repression of interferon-gamma (IFN-gamma) during effector differentiation. The inappropriate expression of IFN-gamma by Zbtb7b-deficient CD4+ T cells required the activities of Eomesodermin and Runx transcription factors. Runx activity was needed for Granzyme B expression, indicating that Runx proteins control expression of the cytotoxic program. We conclude that a key function of Zbtb7b in the mature CD4+ T cell compartment is to repress CD8-lineage gene expression.