Glucocorticoid use in acute respiratory failure from pulmonary causes and association with early changes in the systemic host immune response.

BACKGROUND: Glucocorticoids are commonly used in patients with or at-risk for acute respiratory distress syndrome (ARDS), but optimal use remains unclear despite well-conducted clinical trials. We performed a secondary analysis in patients previously enrolled in the Acute Lung Injury and Biospecimen Repository at the University of Pittsburgh. The primary aim of our study was to investigate early changes in host response biomarkers in response to real-world use of glucocorticoids in patients with acute respiratory failure due to ARDS or at-risk due to a pulmonary insult. Participants had baseline plasma samples obtained on study enrollment and on follow-up 3 to 5 days later to measure markers of innate immunity (IL-6, IL-8, IL-10, TNFr1, ST2, fractalkine), epithelial injury (sRAGE), endothelial injury (angiopoietin-2), and host response to bacterial infections (procalcitonin, pentraxin-3). In our primary analyses, we investigated the effect of receiving glucocorticoids between baseline and follow-up samples on host response biomarkers measured at follow-up by doubly robust inverse probability weighting analysis. In exploratory analyses, we examined associations between glucocorticoid use and previously characterized host response subphenotypes (hyperinflammatory and hypoinflammatory). RESULTS: 67 of 148 participants (45%) received glucocorticoids between baseline and follow-up samples. Dose and type of glucocorticoids varied. Regimens that used hydrocortisone alone were most common (37%), and median daily dose was equivalent to 40 mg methylprednisolone (interquartile range: 21, 67). Participants who received glucocorticoids were more likely to be female, to be on immunosuppressive therapy at baseline, and to have higher baseline levels of ST-2, fractalkine, IL-10, pentraxin-3, sRAGE, and TNFr1. Glucocorticoid use was associated with decreases in IL-6 and increases in fractalkine. In exploratory analyses, glucocorticoid use was more frequent in participants in the hyperinflammatory subphenotype (58% vs 40%, p = 0.05), and was not associated with subphenotype classification at the follow-up time point (p = 0.16). CONCLUSIONS: Glucocorticoid use varied in a cohort of patients with or at-risk for ARDS and was associated with early changes in the systemic host immune response.

Anti-inflammatory roles of type I interferon signaling in the lung.

Excessive or persistent inflammation may have detrimental effects on lung structure and function. Currently, our understanding of conserved host mechanisms that control the inflammatory response remains incompletely understood. In this study, we investigated the role of type I interferon signaling in the inflammatory response against diverse clinically relevant stimuli. Using mice deficient in type I interferon signaling (IFNAR1-/-), we demonstrate that the absence of interferon signaling resulted in a robust and persistent inflammatory response against Pseudomonas aeruginosa, lipopolysaccharide, and chemotherapeutic agent bleomycin. The elevated inflammatory response in IFNAR1-/- mice was manifested as elevated myeloid cells, such as macrophages and neutrophils, in the bronchoalveolar lavage. The inflammatory cell response in the IFNAR1-/- mice persisted to 14 days and there is impaired recovery and fibrotic remodeling of the lung in IFNAR1-/- mice after bleomycin injury. In the Pseudomonas infection model, the elevated inflammatory cell response led to improved bacterial clearance in IFNAR1-/- mice, although there was similar lung injury and survival. We performed RNA sequencing of lung tissue in wild-type and IFNAR1-/- mice after LPS and bleomycin injury. Our unbiased analysis identified differentially expressed genes between IFNAR1-/- and wild-type mice, including previously unknown regulation of nucleotide-binding oligomerization domain (NOD)-like receptor signaling, retinoic acid-inducible gene-I (RIG-I) signaling, and necroptosis pathway by type I interferon signaling in both models. These data provide novel insights into the conserved anti-inflammatory mechanisms of the type I interferon signaling.NEW & NOTEWORTHY Type I interferons are known for their antiviral activities. In this study, we demonstrate a conserved anti-inflammatory role of type I interferon signaling against diverse stimuli in the lung. We show that exacerbated inflammatory response in the absence of type I interferon signaling has both acute and chronic consequences in the lung including structural changes.

Lung Epithelium Releases Growth Differentiation Factor 15 in Response to Pathogen-mediated Injury.

GDF15 (growth differentiation factor 15) is a stress cytokine with several proposed roles, including support of stress erythropoiesis. Higher circulating GDF15 levels are prognostic of mortality during acute respiratory distress syndrome, but the cellular sources and downstream effects of GDF15 during pathogen-mediated lung injury are unclear. We quantified GDF15 in lower respiratory tract biospecimens and plasma from patients with acute respiratory failure. Publicly available data from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were reanalyzed. We used mouse models of hemorrhagic acute lung injury mediated by Pseudomonas aeruginosa exoproducts in wild-type mice and mice genetically deficient for Gdf15 or its putative receptor, Gfral. In critically ill humans, plasma levels of GDF15 correlated with lower respiratory tract levels and were higher in nonsurvivors. SARS-CoV-2 infection induced GDF15 expression in human lung epithelium, and lower respiratory tract GDF15 levels were higher in coronavirus disease (COVID-19) nonsurvivors. In mice, intratracheal P. aeruginosa type II secretion system exoproducts were sufficient to induce airspace and plasma release of GDF15, which was attenuated with epithelial-specific deletion of Gdf15. Mice with global Gdf15 deficiency had decreased airspace hemorrhage, an attenuated cytokine profile, and an altered lung transcriptional profile during injury induced by P. aeruginosa type II secretion system exoproducts, which was not recapitulated in mice deficient for Gfral. Airspace GDF15 reconstitution did not significantly modulate key lung cytokine levels but increased circulating erythrocyte counts. Lung epithelium releases GDF15 during pathogen injury, which is associated with plasma levels in humans and mice and can increase erythrocyte counts in mice, suggesting a novel lung-blood communication pathway.

STAT1 Employs Myeloid Cell-Extrinsic Mechanisms to Regulate the Neutrophil Response and Provide Protection against Invasive Klebsiella pneumoniae Lung Infection.

Klebsiella pneumoniae (KP) is an extracellular Gram-negative bacterium that causes infections in the lower respiratory and urinary tracts and the bloodstream. STAT1 is a master transcription factor that acts to maintain T cell quiescence under homeostatic conditions. Although STAT1 helps defend against systemic spread of acute KP intrapulmonary infection, whether STAT1 regulation of T cell homeostasis impacts pulmonary host defense during acute bacterial infection and injury is less clear. Using a clinical KP respiratory isolate and a pneumonia mouse model, we found that STAT1 deficiency led to an early neutrophil-dominant transcriptional profile and neutrophil recruitment in the lung preceding widespread bacterial dissemination and lung injury development. Yet, myeloid cell STAT1 was dispensable for control of KP proliferation and dissemination, because myeloid cell-specific STAT1-deficient (LysMCre/WT;Stat1fl/fl) mice showed bacterial burden in the lung, liver, and kidney similar to that of their wild-type littermates. Surprisingly, IL-17-producing CD4+ T cells infiltrated Stat1-/- murine lungs early during KP infection. The increase in Th17 cells in the lung was not due to preexisting immunity against KP and was consistent with circulating rather than tissue-resident CD4+ T cells. However, blocking global IL-17 signaling with anti-IL-17RC administration led to increased proliferation and dissemination of KP, suggesting that IL-17 provided by other innate immune cells is essential in defense against KP. Contrastingly, depletion of CD4+ T cells reduced Stat1-/- murine lung bacterial burden, indicating that early CD4+ T cell activation in the setting of global STAT1 deficiency is pathogenic. Altogether, our findings suggest that STAT1 employs myeloid cell-extrinsic mechanisms to regulate neutrophil responses and provides protection against invasive KP by restricting nonspecific CD4+ T cell activation and immunopathology in the lung.

BATF2 enhances proinflammatory cytokine responses in macrophages and improves early host defense against pulmonary Klebsiella pneumoniae infection.

Basic leucine zipper transcription factor ATF-like 2 (BATF2) is a transcription factor that is emerging as an important regulator of the innate immune system. BATF2 is among the top upregulated genes in human alveolar macrophages treated with LPS, but the signaling pathways that induce BATF2 expression in response to Gram-negative stimuli are incompletely understood. In addition, the role of BATF2 in the host response to pulmonary infection with a Gram-negative pathogen like Klebsiella pneumoniae (Kp) is not known. We show that induction of Batf2 gene expression in macrophages in response to Kp in vitro requires TRIF and type I interferon (IFN) signaling, but not MyD88 signaling. Analysis of the impact of BATF2 deficiency on macrophage effector functions in vitro showed that BATF2 does not directly impact macrophage phagocytic uptake and intracellular killing of Kp. However, BATF2 markedly enhanced macrophage proinflammatory gene expression and Kp-induced cytokine responses. In vivo, Batf2 gene expression was elevated in lung tissue of wild-type (WT) mice 24 h after pulmonary Kp infection, and Kp-infected BATF2-deficient (Batf2-/-) mice displayed an increase in bacterial burden in the lung, spleen, and liver compared with WT mice. WT and Batf2-/- mice showed similar recruitment of leukocytes following infection, but in line with in vitro observations, proinflammatory cytokine levels in the alveolar space were reduced in Batf2-/- mice. Altogether, these results suggest that BATF2 enhances proinflammatory cytokine responses in macrophages in response to Kp and contributes to the early host defense against pulmonary Kp infection.NEW & NOTEWORTHY This study investigates the signaling pathways that mediate induction of BATF2 expression downstream of TLR4 and also the impact of BATF2 on the host defense against pulmonary Kp infection. We demonstrate that Kp-induced upregulation of BATF2 in macrophages requires TRIF and type I IFN signaling. We also show that BATF2 enhances Kp-induced macrophage cytokine responses and that BATF2 contributes to the early host defense against pulmonary Kp infection.

Phagocytosis is a primary determinant of pulmonary clearance of clinical Klebsiella pneumoniae isolates.

Introduction: Klebsiella pneumoniae (Kp) is a common cause of hospital-acquired pneumonia. Although previous studies have suggested that evasion of phagocytic uptake is a virulence determinant of Kp, few studies have examined phagocytosis sensitivity in clinical Kp isolates. Methods: We screened 19 clinical respiratory Kp isolates that were previously assessed for mucoviscosity for their sensitivity to macrophage phagocytic uptake, and evaluated phagocytosis as a functional correlate of in vivo Kp pathogenicity. Results: The respiratory Kp isolates displayed heterogeneity in the susceptibility to macrophage phagocytic uptake, with 14 out of 19 Kp isolates displaying relative phagocytosis-sensitivity compared to the reference Kp strain ATCC 43816, and 5 out of 19 Kp isolates displaying relative phagocytosis-resistance. Intratracheal infection with the non-mucoviscous phagocytosis-sensitive isolate S17 resulted in a significantly lower bacterial burden compared to infection with the mucoviscous phagocytosis-resistant isolate W42. In addition, infection with S17 was associated with a reduced inflammatory response, including reduced bronchoalveolar lavage fluid (BAL) polymorphonuclear (PMN) cell count, and reduced BAL TNF, IL-1ß, and IL-12p40 levels. Importantly, host control of infection with the phagocytosis-sensitive S17 isolate was impaired in alveolar macrophage (AM)-depleted mice, whereas AM-depletion had no significant impact on host defense against infection with the phagocytosis-resistant W42 isolate. Conclusion: Altogether, these findings show that phagocytosis is a primary determinant of pulmonary clearance of clinical Kp isolates.

Thrombospondin-1 Restricts Interleukin-36γ-Mediated Neutrophilic Inflammation during Pseudomonas aeruginosa Pulmonary Infection.

Interleukin-36γ (IL-36γ), a member of the IL-1 cytokine superfamily, amplifies lung inflammation and impairs host defense during acute pulmonary Pseudomonas aeruginosa infection. To be fully active, IL-36γ is cleaved at its N-terminal region by proteases such as neutrophil elastase (NE) and cathepsin S (CatS). However, it remains unclear whether limiting extracellular proteolysis restrains the inflammatory cascade triggered by IL-36γ during P. aeruginosa infection. Thrombospondin-1 (TSP-1) is a matricellular protein with inhibitory activity against NE and the pathogen-secreted Pseudomonas elastase LasB-both proteases implicated in amplifying inflammation. We hypothesized that TSP-1 tempers the inflammatory response during lung P. aeruginosa infection by inhibiting the proteolytic environment required for IL-36γ activation. Compared to wild-type (WT) mice, TSP-1-deficient (Thbs1-/-) mice exhibited a hyperinflammatory response in the lungs during P. aeruginosa infection, with increased cytokine production and an unrestrained extracellular proteolytic environment characterized by higher free NE and LasB, but not CatS activity. LasB cleaved IL-36γ proximally to M19 at a cleavage site distinct from those generated by NE and CatS, which cleave IL-36γ proximally to Y16 and S18, respectively. N-terminal truncation experiments in silico predicted that the M19 and the S18 isoforms bind the IL-36R complex almost identically. IL-36γ neutralization ameliorated the hyperinflammatory response and improved lung immunity in Thbs1-/- mice during P. aeruginosa infection. Moreover, administration of cleaved IL-36γ induced cytokine production and neutrophil recruitment and activation that was accentuated in Thbs1-/- mice lungs. Collectively, our data show that TSP-1 regulates lung neutrophilic inflammation and facilitates host defense by restraining the extracellular proteolytic environment required for IL-36γ activation.IMPORTANCEPseudomonas aeruginosa pulmonary infection can lead to exaggerated neutrophilic inflammation and tissue destruction, yet host factors that regulate the neutrophilic response are not fully known. IL-36γ is a proinflammatory cytokine that dramatically increases in bioactivity following N-terminal processing by proteases. Here, we demonstrate that thrombospondin-1, a host matricellular protein, limits N-terminal processing of IL-36γ by neutrophil elastase and the Pseudomonas aeruginosa-secreted protease LasB. Thrombospondin-1-deficient mice (Thbs1-/-) exhibit a hyperinflammatory response following infection. Whereas IL-36γ neutralization reduces inflammatory cytokine production, limits neutrophil activation, and improves host defense in Thbs1-/- mice, cleaved IL-36γ administration amplifies neutrophilic inflammation in Thbs1-/- mice. Our findings indicate that thrombospondin-1 guards against feed-forward neutrophilic inflammation mediated by IL-36γ in the lung by restraining the extracellular proteolytic environment.

Recurrent deletions in the SARS-CoV-2 spike glycoprotein drive antibody escape.

Zoonotic pandemics, such as that caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can follow the spillover of animal viruses into highly susceptible human populations. The descendants of these viruses have adapted to the human host and evolved to evade immune pressure. Coronaviruses acquire substitutions more slowly than other RNA viruses. In the spike glycoprotein, we found that recurrent deletions overcome this slow substitution rate. Deletion variants arise in diverse genetic and geographic backgrounds, transmit efficiently, and are present in novel lineages, including those of current global concern. They frequently occupy recurrent deletion regions (RDRs), which map to defined antibody epitopes. Deletions in RDRs confer resistance to neutralizing antibodies. By altering stretches of amino acids, deletions appear to accelerate SARS-CoV-2 antigenic evolution and may, more generally, drive adaptive evolution.

Finding Order in the Chaos: Outstanding Questions in Klebsiella pneumoniae Pathogenesis.

Klebsiella pneumoniae are Gram-negative facultative anaerobes that are found within host-associated commensal microbiomes, but they can also cause a wide range of infections that are often difficult to treat. These infections are caused by different pathotypes of K. pneumoniae, called either classical or hypervirulent strains. These two groups are genetically distinct, inhabit nonoverlapping geographies, and cause different types of harmful infections in humans. These distinct bacterial groups have also been found to interact differently with the host immune system. Initial innate immune defenses against K. pneumoniae infection include complement, macrophages, neutrophils, and monocytes; these defenses are primary strategies employed by the host to clear infections. K. pneumoniae pathogenesis depends upon the interactions between the microbe and each of these host defenses, and it is becoming increasingly apparent that bacterial genetic diversity impacts the outcomes of these interactions. Here, we highlight recent advances in our understanding of K. pneumoniae pathogenesis, with a focus on how bacterial evolution and diversity impact K. pneumoniae interactions with mammalian innate immune host defenses. We also discuss outstanding questions regarding how K. pneumoniae can frustrate normal immune responses, capitalize upon states of immunocompromise, and cause infections with high mortality.

Intractable Coronavirus Disease 2019 (COVID-19) and Prolonged Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Replication in a Chimeric Antigen Receptor-Modified T-Cell Therapy Recipient: A Case Study.

A chimeric antigen receptor-modified T-cell therapy recipient developed severe coronavirus disease 2019, intractable RNAemia, and viral replication lasting >2 months. Premortem endotracheal aspirate contained >2 × 1010 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA copies/mL and infectious virus. Deep sequencing revealed multiple sequence variants consistent with intrahost virus evolution. SARS-CoV-2 humoral and cell-mediated immunity were minimal. Prolonged transmission from immunosuppressed patients is possible.

Low-Dose Oxygen Enhances Macrophage-Derived Bacterial Clearance following Cigarette Smoke Exposure.

Background. Chronic obstructive pulmonary disease (COPD) is a common, smoking-related lung disease. Patients with COPD frequently suffer disease exacerbations induced by bacterial respiratory infections, suggestive of impaired innate immunity. Low-dose oxygen is a mainstay of therapy during COPD exacerbations; yet we understand little about whether oxygen can modulate the effects of cigarette smoke on lung immunity. Methods. Wild-type mice were exposed to cigarette smoke for 5 weeks, followed by intratracheal instillation of Pseudomonas aeruginosa (PAO1) and 21% or 35-40% oxygen. After two days, lungs were harvested for PAO1 CFUs, and bronchoalveolar fluid was sampled for inflammatory markers. In culture, macrophages were exposed to cigarette smoke and oxygen (40%) for 24 hours and then incubated with PAO1, followed by quantification of bacterial phagocytosis and inflammatory markers. Results. Mice exposed to 35-40% oxygen after cigarette smoke and PAO1 had improved survival and reduced lung CFUs and inflammation. Macrophages from these mice expressed less TNF-α and more scavenger receptors. In culture, macrophages exposed to cigarette smoke and oxygen also demonstrated decreased TNF-α secretion and enhanced phagocytosis of PAO1 bacteria. Conclusions. Our findings demonstrate a novel, protective role for low-dose oxygen following cigarette smoke and bacteria exposure that may be mediated by enhanced macrophage phagocytosis.