SARS-CoV-2 viral clearance and evolution varies by type and severity of immunodeficiency.

Despite vaccination and antiviral therapies, immunocompromised individuals are at risk for prolonged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but the immune defects that predispose an individual to persistent coronavirus disease 2019 (COVID-19) remain incompletely understood. In this study, we performed detailed viro-immunologic analyses of a prospective cohort of participants with COVID-19. The median times to nasal viral RNA and culture clearance in individuals with severe immunosuppression due to hematologic malignancy or transplant (S-HT) were 72 and 40 days, respectively, both of which were significantly longer than clearance rates in individuals with severe immunosuppression due to autoimmunity or B cell deficiency (S-A), individuals with nonsevere immunodeficiency, and nonimmunocompromised groups (P < 0.01). Participants who were severely immunocompromised had greater SARS-CoV-2 evolution and a higher risk of developing resistance against therapeutic monoclonal antibodies. Both S-HT and S-A participants had diminished SARS-CoV-2-specific humoral responses, whereas only the S-HT group had reduced T cell-mediated responses. This highlights the varied risk of persistent COVID-19 across distinct immunosuppressive conditions and suggests that suppression of both B and T cell responses results in the highest contributing risk of persistent infection.

SARS-CoV-2 Viral Clearance and Evolution Varies by Extent of Immunodeficiency.

Despite vaccination and antiviral therapies, immunocompromised individuals are at risk for prolonged SARS-CoV-2 infection, but the immune defects that predispose to persistent COVID-19 remain incompletely understood. In this study, we performed detailed viro-immunologic analyses of a prospective cohort of participants with COVID-19. The median time to nasal viral RNA and culture clearance in the severe hematologic malignancy/transplant group (S-HT) were 72 and 40 days, respectively, which were significantly longer than clearance rates in the severe autoimmune/B-cell deficient (S-A), non-severe, and non-immunocompromised groups (P<0.001). Participants who were severely immunocompromised had greater SARS-CoV-2 evolution and a higher risk of developing antiviral treatment resistance. Both S-HT and S-A participants had diminished SARS-CoV-2-specific humoral, while only the S-HT group had reduced T cell-mediated responses. This highlights the varied risk of persistent COVID-19 across immunosuppressive conditions and suggests that suppression of both B and T cell responses results in the highest contributing risk of persistent infection.

Surfactant Protein D Influences Mortality During Abdominal Sepsis by Facilitating Escherichia coli Colonization in the Gut.

Determine the role of surfactant protein D (SPD) in sepsis. DESIGN: Murine in vivo study. SETTING: Research laboratory at an academic medical center. PATIENTS: SPD knockout (SPD-/-) and wild-type (SPD+/+) mice. INTERVENTIONS: SPD-/- and SPD+/+ mice were subjected to cecal ligation and puncture (CLP). After CLP, Escherichia coli bacteremia was assessed in both groups. Cecal contents from both groups were cultured to assess for colonization by E. coli. To control for parental effects on the microbiome, SPD-/- and SPD+/+ mice were bred from heterozygous parents, and levels of E. coli in their ceca were measured. Gut segments were harvested from mice, and SPD protein expression was measured by Western blot. SPD-/- mice were gavaged with green fluorescent protein, expressing E. coli and recombinant SPD (rSPD). MEASUREMENTS AND MAIN RESULTS: SPD-/- mice had decreased mortality and decreased E. coli bacteremia compared with SPD+/+ mice following CLP. At baseline, SPD-/- mice had decreased E. coli in their cecal flora. When SPD-/- and SPD+/+ mice were bred from heterozygous parents and then separated after weaning, less E. coli was cultured from the ceca of SPD-/- mice. E. coli gut colonization was increased by gavage of rSPD in SPD-/- mice. The source of enteric SPD in SPD+/+ mice was the gallbladder. CONCLUSIONS: Enteral SPD exacerbates mortality after CLP by facilitating colonization of the mouse gut with E. coli.

The effect of elexacaftor/tezacaftor/ivacaftor (ETI) on glycemia in adults with cystic fibrosis.

BACKGROUND: Cystic fibrosis related diabetes (CFRD) is associated with pulmonary decline and compromised nutritional status. Emerging data suggest that CFTR dysfunction may play a direct role in the pathogenesis of CFRD; however, studies investigating the effect of CFTR modulators on glycemic outcomes in patients with cystic fibrosis (CF) have shown mixed results. The impact of elexacaftor-tezacaftor-ivacaftor (ETI) on glycemic control is currently unknown. Our objective was to investigate the effect of ETI initiation on glycemia in adults with CF using continuous glucose monitoring (CGM). METHODS: In this prospective observational study, 34 adults with CF and at least one F508del CFTR mutation wore CGM sensors for 14 days prior to starting ETI and again 3-12 months after ETI initiation. Hypoglycemia symptoms were queried at each visit, and most recent anthropometric measures and spirometry data were obtained by chart review. RESULTS: Twenty-three participants completed the study. Compared to baseline, average glucose (AG), standard deviation (SD), % time >200 mg/dL, and peak sensor glucose decreased with ETI treatment, and % time in target range 70-180 mg/dL increased. Improvements in glycemic parameters were most notable in individuals with CFRD. There was no significant change in CGM-measured or self-reported hypoglycemia before and after ETI initiation. CONCLUSION: Initiation of ETI in adults with CF was associated with improvement CGM-derived measures of hyperglycemia and glycemic variability with no effect on hypoglycemia. Further studies are needed to investigate underlying etiology of these changes and the long-term impact of ETI on glycemic control in patients with CF.

CFTR regulates B cell activation and lymphoid follicle development.

BACKGROUND: Cystic fibrosis (CF) is an inherited disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that promotes persistent lung infection and inflammation and progressive loss of lung function. Patients with CF have increased lung lymphoid follicles (LFs) and B cell-activating factor of tumor necrosis factor family (BAFF) that regulates B cell survival and maturation. A direct role for CFTR in B cell activation and disease pathogenesis in CF remains unclear. METHODS: The number of LFs, BAFF+, TLR4+ and proliferation marker Ki67+ B cells in lung explants or resections from subjects with CF and normal controls was quantified by immunostaining. The role of CFTR in B cell activation and LF development was then examined in two independent cohorts of uninfected CFTR-deficient mice (Cftr -/-) and wild type controls. The number of lung LFs, B cells and BAFF+, CXCR4+, immunoglobulin G+ B cells was examined by immunostaining. Lung and splenocyte B cell activation marker and major histocompatibility complex class II (MHC class II) expression was quantified by flow cytometry. Inflammatory cytokine levels were measured in supernatants from isolated B cells from Cftr -/- and wild type mice stimulated in vitro with Pseudomonas aeruginosa lipopolysaccharide (LPS). RESULTS: There was a significant increase in well-formed LFs in subjects with CF compared to normal controls. Increased B cell activation and proliferation was observed in lung LFs from CF subjects as was quantified by a significant increase in B cell BAFF, TLR4 and Ki67 expression. Uninfected Cftr -/- mice had increased lung LFs and BAFF+ and CXCR4+ B cells compared to wild type controls. Lung B cells isolated from uninfected Cftr -/- mice demonstrated increased MHC class II expression. In vitro, isolated B cells from Cftr -/- mice produced increased IL-6 when stimulated with LPS compared to wild type controls. CONCLUSIONS: These data support a direct role for CFTR in B cell activation, proliferation and inflammatory cytokine production that promotes lung LF follicle development in cystic fibrosis.

Macrophage FABP4 is required for neutrophil recruitment and bacterial clearance in Pseudomonas aeruginosa pneumonia.

Fatty acid binding protein 4 (FABP4), an intracellular lipid chaperone and adipokine, is expressed by lung macrophages, but the function of macrophage-FABP4 remains elusive. We investigated the role of FABP4 in host defense in a murine model of Pseudomonas aeruginosa pneumonia. Compared with wild-type (WT) mice, FABP4-deficient (FABP4-/-) mice exhibited decreased bacterial clearance and increased mortality when challenged intranasally with P. aeruginosa. These findings in FABP4-/- mice were associated with a delayed neutrophil recruitment into the lungs and were followed by greater acute lung injury and inflammation. Among leukocytes, only macrophages expressed FABP4 in WT mice with P. aeruginosa pneumonia. Chimeric FABP4-/- mice with WT bone marrow were protected from increased mortality seen in chimeric WT mice with FABP4-/- bone marrow during P. aeruginosa pneumonia, thus confirming the role of macrophages as the main source of protective FABP4 against that infection. There was less production of C-X-C motif chemokine ligand 1 (CXCL1) in FABP4-/- alveolar macrophages and lower airway CXCL1 levels in FABP4-/- mice. Delivering recombinant CXCL1 to the airways protected FABP4-/- mice from increased susceptibility to P. aeruginosa pneumonia. Thus, macrophage-FABP4 has a novel role in pulmonary host defense against P. aeruginosa infection by facilitating crosstalk between macrophages and neutrophils via regulation of macrophage CXCL1 production.-Liang, X., Gupta, K., Rojas Quintero, J., Cernadas, M., Kobzik, L., Christou, H., Pier, G. B., Owen, C. A., Çataltepe, S. Macrophage FABP4 is required for neutrophil recruitment and bacterial clearance in Pseudomonas aeruginosa pneumonia.

Neutrophil cytoplasts induce TH17 differentiation and skew inflammation toward neutrophilia in severe asthma.

Severe asthma is a debilitating and treatment refractory disease. As many as half of these patients have complex neutrophil-predominant lung inflammation that is distinct from milder asthma with type 2 eosinophilic inflammation. New insights into severe asthma pathogenesis are needed. Concomitant exposure of mice to an aeroallergen and endotoxin during sensitization resulted in complex neutrophilic immune responses to allergen alone during later airway challenge. Unlike allergen alone, sensitization with allergen and endotoxin led to NETosis. In addition to neutrophil extracellular traps (NETs), enucleated neutrophil cytoplasts were evident in the lungs. Surprisingly, allergen-driven airway neutrophilia was decreased in peptidyl arginine deiminase 4-deficient mice with defective NETosis but not by deoxyribonuclease treatment, implicating the cytoplasts for the non-type 2 immune responses to allergen. Neutrophil cytoplasts were also present in mediastinal lymph nodes, and the cytoplasts activated lung dendritic cells in vitro to trigger antigen-specific interleukin-17 (IL-17) production from naïve CD4+ T cells. Bronchoalveolar lavage fluid from patients with severe asthma and high neutrophil counts had detectable NETs and cytoplasts that were positively correlated with IL-17 levels. Together, these translational findings have identified neutrophil cytoplast formation in asthmatic lung inflammation and linked the cytoplasts to T helper 17-mediated neutrophilic inflammation in severe asthma.

Natural killer cell-mediated inflammation resolution is disabled in severe asthma.

Severe asthma is typically characterized by chronic airway inflammation that is refractory to corticosteroids and associated with excess morbidity. Patients were recruited into the National Heart, Lung, and Blood Institute-sponsored Severe Asthma Research Program and comprehensively phenotyped by bronchoscopy. Bronchoalveolar lavage (BAL) cells were analyzed by flow cytometry. Compared with healthy individuals (n = 21), patients with asthma (n = 53) had fewer BAL natural killer (NK) cells. Patients with severe asthma (n = 29) had a marked increase in the ratios of CD4+ T cells to NK cells and neutrophils to NK cells. BAL NK cells in severe asthma were skewed toward the cytotoxic CD56dim subset, with significantly increased BAL fluid levels of the cytotoxic mediator granzyme A. The numbers of BAL CD56dim NK cells and CCR6-CCR4- T helper 1-enriched CD4+ T cells correlated inversely with lung function [forced expiratory volume in 1 s (FEV1) % predicted] in asthma. Relative to cells from healthy controls, peripheral blood NK cells from asthmatic patients had impaired killing of K562 myeloid target cells despite releasing more cytotoxic mediators. Ex vivo exposure to dexamethasone markedly decreased blood NK cell lysis of target cells and cytotoxic mediator release. NK cells expressed airway lipoxin A4/formyl peptide receptor 2 receptors, and in contrast to dexamethasone, lipoxin A4-exposed NK cells had preserved functional responses. Together, our findings indicate that the immunology of the severe asthma airway is characterized by decreased NK cell cytotoxicity with increased numbers of target leukocytes, which is exacerbated by corticosteroids that further disable NK cell function. These failed resolution mechanisms likely contribute to persistent airway inflammation in severe asthma.

ALX receptor ligands define a biochemical endotype for severe asthma.

BACKGROUND: In health, inflammation resolution is an active process governed by specialized proresolving mediators and receptors. ALX/FPR2 receptors (ALX) are targeted by both proresolving and proinflammatory ligands for opposing signaling events, suggesting pivotal roles for ALX in the fate of inflammatory responses. Here, we determined if ALX expression and ligands were linked to severe asthma (SA). METHODS: ALX expression and levels of proresolving ligands (lipoxin A4 [LXA4], 15-epi-LXA4, and annexin A1 [ANXA1]), and a proinflammatory ligand (serum amyloid A [SAA]) were measured in bronchoscopy samples collected in Severe Asthma Research Program-3 (SA [n = 69], non-SA [NSA, n = 51] or healthy donors [HDs, n = 47]). RESULTS: Bronchoalveolar lavage (BAL) fluid LXA4 and 15-epi-LXA4 were decreased and SAA was increased in SA relative to NSA. BAL macrophage ALX expression was increased in SA. Subjects with LXA4loSAAhi levels had increased BAL neutrophils, more asthma symptoms, lower lung function, increased relative risk for asthma exacerbation, sinusitis, and gastroesophageal reflux disease, and were assigned more frequently to SA clinical clusters. SAA and aliquots of LXA4loSAAhi BAL fluid induced IL-8 production by lung epithelial cells expressing ALX receptors, which was inhibited by coincubation with 15-epi-LXA4. CONCLUSIONS: Together, these findings have established an association between select ALX receptor ligands and asthma severity that define a potentially new biochemical endotype for asthma and support a pivotal functional role for ALX signaling in the fate of lung inflammation. TRIAL REGISTRATION: Severe Asthma Research Program-3 (SARP-3; ClinicalTrials.gov NCT01606826)FUNDING Sources. National Heart, Lung and Blood Institute, the NIH, and the German Society of Pediatric Pneumology.

Alternative Macrophage Activation Is Increased in Asthma.

The immune responses of type 2 T helper cells (Th2) play an important role in asthma and promote the differentiation of alternatively activated (M2) macrophages. M2 macrophages have been increasingly understood to contribute to Th2 immunity. We hypothesized that M2 macrophages are altered in asthma and modulate Th2 responses. The aim of this study was to characterize the phenotype and function of human monocyte-derived M2 and bronchoalveolar lavage fluid (BALF) macrophages from healthy control subjects and subjects with asthma. Phenotypic characteristics and effector function of M2 macrophages were examined using monocyte-derived and BALF macrophages obtained from subjects with asthma (n = 28) and healthy volunteers (n = 9) by flow cytometry and quantitative PCR. Resting monocyte-derived (M0) and M2 macrophages were generated by the addition of macrophage colony-stimulating factor or macrophage colony-stimulating factor plus IL-4, respectively. M2 macrophage cytokine expression and their impact on dendritic and CD4+ T cell activation were examined in vitro. High levels of CD206 and major histocompatibility complex class II expression identify macrophages with an M2 phenotype that are increased 2.9-fold in the BALF of subjects with asthma compared with control subjects. M2 macrophages have elevated IL-6, IL-10, and IL-12p40 production compared with conventional macrophages and modulate dendritic and CD4+ T cell interactions. Histamine receptor 1 and E-cadherin expression identify M2 macrophage subsets associated with increased airflow obstruction. M2 macrophages have a distinct cell surface and effector phenotype and are found in increased numbers in subjects with asthma. These findings suggest that M2 macrophages may play an important role in allergic asthma through their bidirectional interactions with immune and structural cells, and inflammatory mediators.

Circadian rhythm reprogramming during lung inflammation.

Circadian rhythms are known to regulate immune responses in healthy animals, but it is unclear whether they persist during acute illnesses where clock gene expression is disrupted by systemic inflammation. Here we use a genome-wide approach to investigate circadian gene and metabolite expression in the lungs of endotoxemic mice and find that novel cellular and molecular circadian rhythms are elicited in this setting. The endotoxin-specific circadian programme exhibits unique features, including a divergent group of rhythmic genes and metabolites compared with the basal state and a distinct periodicity and phase distribution. At the cellular level, endotoxin treatment also alters circadian rhythms of leukocyte counts within the lung in a bmal1-dependent manner, such that granulocytes rather than lymphocytes become the dominant oscillating cell type. Our results show that inflammation produces a complex re-organization of cellular and molecular circadian rhythms that are relevant to early events in lung injury.

Adam8 limits the development of allergic airway inflammation in mice.

To determine whether a disintegrin and metalloproteinase-8 (Adam8) regulates allergic airway inflammation (AAI) and airway hyperresponsiveness (AHR), we compared AAI and AHR in wild-type (WT) versus Adam8(-/-) mice in different genetic backgrounds sensitized and challenged with OVA or house dust mite protein extract. OVA- and house dust mite-treated Adam8(-/-) mice had higher lung leukocyte counts, more airway mucus metaplasia, greater lung levels of some Th2 cytokines, and higher methacholine-induced increases in central airway resistance than allergen-treated WT mice. Studies of OVA-treated Adam8 bone marrow chimeric mice confirmed that leukocyte-derived Adam8 predominantly mediated Adam8's anti-inflammatory activities in murine airways. Airway eosinophils and macrophages both expressed Adam8 in WT mice with AAI. Adam8 limited AAI and AHR in mice by reducing leukocyte survival because: 1) Adam8(-/-) mice with AAI had fewer apoptotic eosinophils and macrophages in their airways than WT mice with AAI; and 2) Adam8(-/-) macrophages and eosinophils had reduced rates of apoptosis compared with WT leukocytes when the intrinsic (but not the extrinsic) apoptosis pathway was triggered in the cells in vitro. ADAM8 was robustly expressed by airway granulocytes in lung sections from human asthma patients, but, surprisingly, airway macrophages had less ADAM8 staining than airway eosinophils. Thus, ADAM8 has anti-inflammatory activities during AAI in mice by activating the intrinsic apoptosis pathway in myeloid leukocytes. Strategies that increase ADAM8 levels in myeloid leukocytes may have therapeutic efficacy in asthma.

Integrating murine gene expression studies to understand obstructive lung disease due to chronic inhaled endotoxin.

RATIONALE: Endotoxin is a near ubiquitous environmental exposure that that has been associated with both asthma and chronic obstructive pulmonary disease (COPD). These obstructive lung diseases have a complex pathophysiology, making them difficult to study comprehensively in the context of endotoxin. Genome-wide gene expression studies have been used to identify a molecular snapshot of the response to environmental exposures. Identification of differentially expressed genes shared across all published murine models of chronic inhaled endotoxin will provide insight into the biology underlying endotoxin-associated lung disease. METHODS: We identified three published murine models with gene expression profiling after repeated low-dose inhaled endotoxin. All array data from these experiments were re-analyzed, annotated consistently, and tested for shared genes found to be differentially expressed. Additional functional comparison was conducted by testing for significant enrichment of differentially expressed genes in known pathways. The importance of this gene signature in smoking-related lung disease was assessed using hierarchical clustering in an independent experiment where mice were exposed to endotoxin, smoke, and endotoxin plus smoke. RESULTS: A 101-gene signature was detected in three murine models, more than expected by chance. The three model systems exhibit additional similarity beyond shared genes when compared at the pathway level, with increasing enrichment of inflammatory pathways associated with longer duration of endotoxin exposure. Genes and pathways important in both asthma and COPD were shared across all endotoxin models. Mice exposed to endotoxin, smoke, and smoke plus endotoxin were accurately classified with the endotoxin gene signature. CONCLUSIONS: Despite the differences in laboratory, duration of exposure, and strain of mouse used in three experimental models of chronic inhaled endotoxin, surprising similarities in gene expression were observed. The endotoxin component of tobacco smoke may play an important role in disease development.

Lipoxin A4 regulates natural killer cell and type 2 innate lymphoid cell activation in asthma.

Asthma is a prevalent disease of chronic inflammation in which endogenous counterregulatory signaling pathways are dysregulated. Recent evidence suggests that innate lymphoid cells (ILCs), including natural killer (NK) cells and type 2 ILCs (ILC2s), can participate in the regulation of allergic airway responses, in particular airway mucosal inflammation. We have identified both NK cells and ILC2s in human lung and peripheral blood in healthy and asthmatic subjects. NK cells were highly activated in severe asthma, were linked to eosinophilia, and interacted with autologous eosinophils to promote their apoptosis. ILC2s generated antigen-independent interleukin-13 (IL-13) in response to the mast cell product prostaglandin D2 alone and in a synergistic manner with the airway epithelial cytokines IL-25 and IL-33. Both NK cells and ILC2s expressed the pro-resolving ALX/FPR2 receptors. Lipoxin A4, a natural pro-resolving ligand for ALX/FPR2 receptors, significantly increased NK cell-mediated eosinophil apoptosis and decreased IL-13 release by ILC2s. Together, these findings indicate that ILCs are targets for lipoxin A4 to decrease airway inflammation and mediate the catabasis of eosinophilic inflammation. Because lipoxin A4 generation is decreased in severe asthma, these findings also implicate unrestrained ILC activation in asthma pathobiology.

Endothelial cell-fatty acid binding protein 4 promotes angiogenesis: role of stem cell factor/c-kit pathway.

Fatty acid binding protein 4 (FABP4) plays an important role in regulation of glucose and lipid homeostasis as well as inflammation through its actions in adipocytes and macrophages. FABP4 is also expressed in a subset of endothelial cells, but its role in this cell type is not known. We found that FABP4-deficient human umbilical vein endothelial cells (HUVECs) demonstrate a markedly increased susceptibility to apoptosis as well as decreased migration and capillary network formation. Aortic rings from FABP4(-/-) mice demonstrated decreased angiogenic sprouting, which was recovered by reconstitution of FABP4. FABP4 was strongly regulated by mTORC1 and inhibited by Rapamycin. FABP4 modulated activation of several important signaling pathways in HUVECs, including downregulation of P38, eNOS, and stem cell factor (SCF)/c-kit signaling. Of these, the SCF/c-kit pathway was found to have a major role in attenuated angiogenic activity of FABP4-deficient ECs as provision of exogenous SCF resulted in a significant recovery in cell proliferation, survival, morphogenesis, and aortic ring sprouting. These data unravel a novel pro-angiogenic role for endothelial cell-FABP4 and suggest that it could be exploited as a potential target for diseases associated with pathological angiogenesis.

Chronic endotoxin exposure produces airflow obstruction and lung dendritic cell expansion.

Little is known about the mechanisms of persistent airflow obstruction that result from chronic occupational endotoxin exposure. We sought to analyze the inflammatory response underlying persistent airflow obstruction as a result of chronic occupational endotoxin exposure. We developed a murine model of daily inhaled endotoxin for periods of 5 days to 8 weeks. We analyzed physiologic lung dysfunction, lung histology, bronchoalveolar lavage fluid and total lung homogenate inflammatory cell and cytokine profiles, and pulmonary gene expression profiles. We observed an increase in airway hyperresponsiveness as a result of chronic endotoxin exposure. After 8 weeks, the mice exhibited an increase in bronchoalveolar lavage and lung neutrophils that correlated with an increase in proinflammatory cytokines. Detailed analyses of inflammatory cell subsets revealed an expansion of dendritic cells (DCs), and in particular, proinflammatory DCs, with a reduced percentage of macrophages. Gene expression profiling revealed the up-regulation of a panel of genes that was consistent with DC recruitment, and lung histology revealed an accumulation of DCs in inflammatory aggregates around the airways in 8-week-exposed animals. Repeated, low-dose LPS inhalation, which mirrors occupational exposure, resulted in airway hyperresponsiveness, associated with a failure to resolve the proinflammatory response, an inverted macrophage to DC ratio, and a significant rise in the inflammatory DC population. These findings point to a novel underlying mechanism of airflow obstruction as a result of occupational LPS exposure, and suggest molecular and cellular targets for therapeutic development.