Aberrant immune programming in neutrophils in cystic fibrosis.

Cystic fibrosis is a life-shortening genetic disorder, caused by mutations in the gene that encodes cystic fibrosis transmembrane-conductance regulator, a cAMP-activated chloride and bicarbonate channel. Persistent neutrophilic inflammation is a major contributor to cystic fibrosis lung disease. However, how cystic fibrosis transmembrane-conductance regulator loss of function leads to excessive inflammation and its clinical sequela remains incompletely understood. In this study, neutrophils from F508del-CF and healthy control participants were compared for gene transcription. We found that cystic fibrosis circulating neutrophils have a prematurely primed basal state with significantly higher scores for activation, chemotaxis, immune signaling, and pattern recognition. Such an irregular basal state appeared not related to the blood environment and was also observed in neutrophils derived from the F508del-CF HL-60 cell line, indicating an innate characteristic of the phenotype. Lipopolysaccharides (LPS) stimulation drastically shifted the transcriptional landscape of healthy control neutrophils toward a robust immune response; however, cystic fibrosis neutrophils were immune-exhausted, reflected by abnormal cell aging and fate determination in gene programming. Moreover, cystic fibrosis sputum neutrophils differed significantly from cystic fibrosis circulating neutrophils in gene transcription with increased inflammatory response, aging, apoptosis, and necrosis, suggesting additional environmental influences on the neutrophils in cystic fibrosis lungs. Taken together, our data indicate that loss of cystic fibrosis transmembrane-conductance regulator function has intrinsic effects on neutrophil immune programming, leading to premature priming and dysregulated response to challenge.

Mammalian Target of Rapamycin Complex 1 Activation in Macrophages Contributes to Persistent Lung Inflammation following Respiratory Tract Viral Infection.

Respiratory tract virus infections cause millions of hospitalizations worldwide each year. Severe infections lead to lung damage that coincides with persistent inflammation and a lengthy repair period. Vaccination and antiviral therapy help to mitigate severe infections before or during the acute stage of disease, but there are currently limited specific treatment options available to individuals experiencing the long-term sequelae of respiratory viral infection. Herein, C57BL/6 mice were infected with influenza A/PR/8/34 as a model for severe viral lung infection and allowed to recover for 21 days. Mice were treated with rapamycin, a well-characterized mammalian target of rapamycin complex 1 (mTORC1) inhibitor, on days 12 to 20 after infection, a time period after viral clearance. Persistent inflammation following severe influenza infection in mice was primarily driven by macrophages and T cells. Uniform manifold approximation and projection analysis of flow cytometry data revealed that lung macrophages had high activation of mTORC1, an energy-sensing kinase involved in inflammatory immune cell effector functions. Rapamycin treatment reduced lung inflammation and the frequency of exudate macrophages, T cells, and B cells in the lung, while not impacting epithelial progenitor cells or adaptive immune memory. These data highlight mTORC1's role in sustaining persistent inflammation following clearance of a viral respiratory pathogen and suggest a possible intervention for post-viral chronic lung inflammation.

Gut epithelial Interleukin-17 receptor A signaling can modulate distant tumors growth through microbial regulation.

Microbes influence cancer initiation, progression and therapy responsiveness. IL-17 signaling contributes to gut barrier immunity by regulating microbes but also drives tumor growth. A knowledge gap remains regarding the influence of enteric IL-17-IL-17RA signaling and their microbial regulation on the behavior of distant tumors. We demonstrate that gut dysbiosis induced by systemic or gut epithelial deletion of IL-17RA induces growth of pancreatic and brain tumors due to excessive development of Th17, primary source of IL-17 in human and mouse pancreatic ductal adenocarcinoma, as well as B cells that circulate to distant tumors. Microbial dependent IL-17 signaling increases DUOX2 signaling in tumor cells. Inefficacy of pharmacological inhibition of IL-17RA is overcome with targeted microbial ablation that blocks the compensatory loop. These findings demonstrate the complexities of IL-17-IL-17RA signaling in different compartments and the relevance for accounting for its homeostatic host defense function during cancer therapy.

Systems serology in cystic fibrosis: Anti-Pseudomonas IgG1 responses and reduced lung function.

Nearly one-half of patients with cystic fibrosis (CF) carry the homozygous F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene but exhibit variable lung function phenotypes. How adaptive immunity influences their lung function remains unclear, particularly the serological antibody responses to antigens from mucoid Pseudomonas in sera from patients with CF with varying lung function. Sera from patients with CF with reduced lung function show higher anti-outer membrane protein I (OprI) immunoglobulin G1 (IgG1) titers and greater antibody-mediated complement deposition. Induction of anti-OprI antibody isotypes with complement activity enhances lung inflammation in preclinical mouse models. This enhanced inflammation is absent in immunized Rag2-/- mice and is transferrable to unimmunized mice through sera. In a CF cohort undergoing treatment with elexacaftor-tezacaftor-ivacaftor, the declination in anti-OprI IgG1 titers is associated with lung function improvement and reduced hospitalizations. These findings suggest that antibody responses to specific Pseudomonas aeruginosa (PA) antigens worsen lung function in patients with CF.

Epithelial plasticity and innate immune activation promote lung tissue remodeling following respiratory viral infection.

Epithelial plasticity has been suggested in lungs of mice following genetic depletion of stem cells but is of unknown physiological relevance. Viral infection and chronic lung disease share similar pathological features of stem cell loss in alveoli, basal cell (BC) hyperplasia in small airways, and innate immune activation, that contribute to epithelial remodeling and loss of lung function. We show that a subset of distal airway secretory cells, intralobar serous (IS) cells, are activated to assume BC fates following influenza virus infection. Injury-induced hyperplastic BC (hBC) differ from pre-existing BC by high expression of IL-22Ra1 and undergo IL-22-dependent expansion for colonization of injured alveoli. Resolution of virus-elicited inflammation results in BC to IS re-differentiation in repopulated alveoli, and increased local expression of protective antimicrobial factors, but fails to restore normal alveolar epithelium responsible for gas exchange.

Deficiency of Caspase-1 Attenuates HIV-1-Associated Atherogenesis in Mice.

Within arterial plaque, HIV infection creates a state of inflammation and immune activation, triggering NLRP3/caspase-1 inflammasome, tissue damage, and monocyte/macrophage infiltration. Previously, we documented that caspase-1 activation in myeloid cells was linked with HIV-associated atherosclerosis in mice and people with HIV. Here, we mechanistically examined the direct effect of caspase-1 on HIV-associated atherosclerosis. Caspase-1-deficient (Casp-1-/-) mice were crossed with HIV-1 transgenic (Tg26+/-) mice with an atherogenic ApoE-deficient (ApoE-/-) background to create global caspase-1-deficient mice (Tg26+/-/ApoE-/-/Casp-1-/-). Caspase-1-sufficient (Tg26+/-/ApoE-/-/Casp-1+/+) mice served as the controls. Next, we created chimeric hematopoietic cell-deficient mice by reconstituting irradiated ApoE-/- mice with bone marrow cells transplanted from Tg26+/-/ApoE-/-/Casp-1-/- (BMT Casp-1-/-) or Tg26+/-/ApoE-/-/Casp-1+/+ (BMT Casp-1+/+) mice. Global caspase-1 knockout in mice suppressed plaque deposition in the thoracic aorta, serum IL-18 levels, and ex vivo foam cell formation. The deficiency of caspase-1 in hematopoietic cells resulted in reduced atherosclerotic plaque burden in the whole aorta and aortic root, which was associated with reduced macrophage infiltration. Transcriptomic analyses of peripheral mononuclear cells and splenocytes indicated that caspase-1 deficiency inhibited caspase-1 pathway-related genes. These results document the critical atherogenic role of caspase-1 in chronic HIV infection and highlight the implication of this pathway and peripheral immune activation in HIV-associated atherosclerosis.

Structural Framework for Analysis of CD4+ T-Cell Epitope Dominance in Viral Fusion Proteins.

Antigen conformation shapes CD4+ T-cell specificity through mechanisms of antigen processing, and the consequences for immunity may rival those from conformational effects on antibody specificity. CD4+ T cells initiate and control immunity to pathogens and cancer and are at least partly responsible for immunopathology associated with infection, autoimmunity, and allergy. The primary trigger for CD4+ T-cell maturation is the presentation of an epitope peptide in the MHC class II antigen-presenting protein (MHCII), most commonly on an activated dendritic cell, and then the T-cell responses are recalled by subsequent presentations of the epitope peptide by the same or other antigen-presenting cells. Peptide presentation depends on the proteolytic fragmentation of the antigen in an endosomal/lysosomal compartment and concomitant loading of the fragments into the MHCII, a multistep mechanism called antigen processing and presentation. Although the role of peptide affinity for MHCII has been well studied, the role of proteolytic fragmentation has received less attention. In this Perspective, we will briefly summarize evidence that antigen resistance to unfolding and proteolytic fragmentation shapes the specificity of the CD4+ T-cell response to selected viral envelope proteins, identify several remarkable examples in which the immunodominant CD4+ epitopes most likely depend on the interaction of processing machinery with antigen conformation, and outline how knowledge of antigen conformation can inform future efforts to design vaccines.

SARS-CoV-2 ORF8 Mediates Signals in Macrophages and Monocytes through MyD88 Independently of the IL-17 Receptor.

SARS-CoV-2 has caused an estimated 7 million deaths worldwide to date. A secreted SARS-CoV-2 accessory protein, known as open reading frame 8 (ORF8), elicits inflammatory pulmonary cytokine responses and is associated with disease severity in COVID-19 patients. Recent reports proposed that ORF8 mediates downstream signals in macrophages and monocytes through the IL-17 receptor complex (IL-17RA, IL-17RC). However, generally IL-17 signals are found to be restricted to the nonhematopoietic compartment, thought to be due to rate-limiting expression of IL-17RC. Accordingly, we revisited the capacity of IL-17 and ORF8 to induce cytokine gene expression in mouse and human macrophages and monocytes. In SARS-CoV-2-infected human and mouse lungs, IL17RC mRNA was undetectable in monocyte/macrophage populations. In cultured mouse and human monocytes and macrophages, ORF8 but not IL-17 led to elevated expression of target cytokines. ORF8-induced signaling was fully preserved in the presence of anti-IL-17RA/RC neutralizing Abs and in Il17ra-/- cells. ORF8 signaling was also operative in Il1r1-/- bone marrow-derived macrophages. However, the TLR/IL-1R family adaptor MyD88, which is dispensable for IL-17R signaling, was required for ORF8 activity yet MyD88 is not required for IL-17 signaling. Thus, we conclude that ORF8 transduces inflammatory signaling in monocytes and macrophages via MyD88 independently of the IL-17R.

EXOSOMES AND MICROVESICLES FROM ADIPOSE-DERIVED MESENCHYMAL STEM CELLS PROTECTS THE ENDOTHELIAL GLYCOCALYX FROM LPS INJURY.

ABSTRACT: Introduction: Endothelial glycocalyx damage occurs in numerous pathological conditions and results in endotheliopathy. Extracellular vesicles, including exosomes and microvesicles, isolated from adipose-derived mesenchymal stem cells (ASCs) have therapeutic potential in multiple disease states; however, their role in preventing glycocalyx shedding has not been defined. We hypothesized that ASC-derived exosomes and microvesicles would protect the endothelial glycocalyx from damage by LPS injury in cultured endothelial cells. Methods : Exosomes and microvesicles were collected from ASC conditioned media by centrifugation (10,000 g for microvesicles, 100,000 g for exosomes). Human umbilical vein endothelial cells (HUVECs) were exposed to 1 µg/mL lipopolysaccharide (LPS). LPS-injured cells (n = 578) were compared with HUVECS with concomitant LPS injury plus 1.0 µg/mL of exosomes (n = 540) or microvesicles (n = 510) for 24 hours. These two cohorts were compared with control HUVECs that received phosphate-buffered saline only (n = 786) and HUVECs exposed to exosomes (n = 505) or microvesicles (n = 500) alone. Cells were fixed and stained with FITC-labeled wheat germ agglutinin to quantify EGX. Real-time quantitative reverse-transcription polymerase chain reaction was used on HUVECs cell lystate to quantify hyaluron synthase-1 (HAS1) expression. Results: Exosomes alone decreased endothelial glycocalyx staining intensity when compared with control (4.94 vs. 6.41 AU, P < 0.001), while microvesicles did not cause a change glycocalyx staining intensity (6.39 vs. 6.41, P = 0.99). LPS injury resulted in decreased glycocalyx intensity as compared with control (5.60 vs. 6.41, P < 0.001). Exosomes (6.85 vs. 5.60, P < 0.001) and microvesicles (6.35 vs. 5.60, P < 0.001) preserved endothelial glycocalyx staining intensity after LPS injury. HAS1 levels were found to be higher in the exosome (1.14 vs. 3.67 RE, P = 0.02) and microvesicle groups (1.14 vs. 3.59 RE, P = 0.02) when compared with LPS injury. Hyaluron synthase-2 and synthase-3 expressions were not different in the various experimental groups. Conclusions: Exosomes alone can damage the endothelial glycocalyx. However, in the presence of LPS injury, both exosomes and microvesicles protect the glycocalyx layer. This effect seems to be mediated by HAS1. Level of Evidence : Basic science study.

ABERRANT IMMUNE PROGRAMMING IN NEUTROPHILS IN CYSTIC FIBROSIS.

Cystic fibrosis (CF) is a life-shortening genetic disorder, caused by mutations in the gene that encodes Cystic Fibrosis Transmembrane-conductance Regulator (CFTR), a cAMP-activated chloride and bicarbonate channel. Although multiple organ systems can be affected, CF lung disease claims the most morbidity and mortality due to chronic bacterial infection, persistent neutrophilic inflammation, and mucopurulent airway obstruction. Despite the clear predominance of neutrophils in these pathologies, how CFTR loss-of-function affects these cells per se remains incompletely understood. Here, we report the profiling and comparing of transcriptional signatures of peripheral blood neutrophils from CF participants and healthy human controls (HC) at the single-cell level. Circulating CF neutrophils had an aberrant basal state with significantly higher scores for activation, chemotaxis, immune signaling, and pattern recognition, suggesting that CF neutrophils in blood are prematurely primed. Such an abnormal basal state was also observed in neutrophils derived from an F508del-CF HL-60 cell line, indicating an innate characteristic of the phenotype. LPS stimulation drastically shifted the transcriptional landscape of HC circulating neutrophils towards a robust immune response, however, CF neutrophils were immune-exhausted. Moreover, CF blood neutrophils differed significantly from CF sputum neutrophils in gene programming with respect to neutrophil activation and aging, as well as inflammatory signaling, highlighting additional environmental influences on the neutrophils in CF lungs. Taken together, loss of CFTR function has intrinsic effects on neutrophil immune programming that leads to premature priming and dysregulated response to challenge.

Insulin-like growth factor 1 reduces coronary atherosclerosis in pigs with familial hypercholesterolemia.

Although murine models of coronary atherosclerotic disease have been used extensively to determine mechanisms, limited new therapeutic options have emerged. Pigs with familial hypercholesterolemia (FH pigs) develop complex coronary atheromas that are almost identical to human lesions. We reported previously that insulin-like growth factor 1 (IGF-1) reduced aortic atherosclerosis and promoted features of stable plaque in a murine model. We administered human recombinant IGF-1 or saline (control) in atherosclerotic FH pigs for 6 months. IGF-1 decreased relative coronary atheroma in vivo (intravascular ultrasound) and reduced lesion cross-sectional area (postmortem histology). IGF-1 increased plaque's fibrous cap thickness, and reduced necrotic core, macrophage content, and cell apoptosis, consistent with promotion of a stable plaque phenotype. IGF-1 reduced circulating triglycerides, markers of systemic oxidative stress, and CXCL12 chemokine levels. We used spatial transcriptomics (ST) to identify global transcriptome changes in advanced plaque compartments and to obtain mechanistic insights into IGF-1 effects. ST analysis showed that IGF-1 suppressed FOS/FOSB factors and gene expression of MMP9 and CXCL14 in plaque macrophages, suggesting possible involvement of these molecules in IGF-1's effect on atherosclerosis. Thus, IGF-1 reduced coronary plaque burden and promoted features of stable plaque in a pig model, providing support for consideration of clinical trials.

Multi-omic comparisons between CFBE41o- cells stably expressing wild-type CFTR and F508del-mutant CFTR.

BACKGROUND: Cystic fibrosis (CF) is characterized by chronic inflammation and excessive cytokines secretion in the lung. Isogenic human CF bronchial epithelial (CFBE41o-) cell lines stably expressing wt-CFTR (WTBE) or F508del mutant (CFBE) are widely used tools in understanding responses to stimuli or drugs and CF pathogenesis in vitro. However, the intrinsic cellular differences in culture are unknown. METHODS: We performed integrative analyses of these isogenic cells at the protein, mRNA, and chromatin levels in the submerged and air-liquid interface (ALI) conditions to determine cell intrinsic effects of mutant versus complemented CFTR expression. RESULTS: CFBE and WTBE cells displayed different cytokine secretion patterns, including IL-6, IL-8, CXCL1, CXCL10, and CCL5. The ALI culture dramatically increased cytokine secretion in both cells. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) result showed different chromatin landscapes upon polarization and CFBE cells, compared to WTBE cells, exhibited higher genome-wide chromatin accessibility under both culture methods. At the transcriptome level, differentially expressed genes identified by mRNA sequencing between two cell lines were highly concentrated in immunity-related pathways. CONCLUSIONS: This multilayered study shows that expression of wild-type CFTR has an epithelial cell intrinsic effect on the cell's epigenome and transcriptome particularly in immunity relevant activities. These data will serve as a resource for the CF community and may serve as epithelial biomarkers for CFTR mRNA therapy.

Pseudomonas Aeruginosa Lung Infection Subverts Lymphocytic Responses through IL-23 and IL-22 Post-Transcriptional Regulation.

Pseudomonas aeruginosa (P.a) is a pathogen causing significant morbidity and mortality, particularly in hospital patients undergoing ventilation and in individuals with cystic fibrosis. Although we and others have investigated mechanisms used by P.a to subvert innate immunity, relatively less is known about the potential strategies used by this bacterium to fight the adaptive immune system and, in particular, T cells. Here, using RAG KO (devoid of 'classical' αß and γδ TCR T lymphocytes) and double RAG γC KO mice (devoid of T, NK and ILC cells), we demonstrate that the lymphocytic compartment is important to combat P.a (PAO1 strain). Indeed, we show that PAO1 load was increased in double RAG γC KO mice. In addition, we show that PAO1 down-regulates IL-23 and IL-22 protein accumulation in the lungs of infected mice while up-regulating their RNA production, thereby pointing towards a specific post-transcriptional regulatory mechanism not affecting other inflammatory mediators. Finally, we demonstrate that an adenovirus-mediated over-expression of IL-1, IL-23 and IL-7 induced lung neutrophil and lymphocytic influx and rescued mice against P.a-induced lethality in all WT, RAG γC KO and RAG γC KO RAG-deficient mice, suggesting that this regimen might be of value in 'locally immunosuppressed' individuals such as cystic fibrosis patients.

Targeted NGS-Based Analysis of Pneumocystis jirovecii Reveals Novel Genotypes.

Pneumocystis jirovecii is an important etiological agent of pneumonia that is underdiagnosed due to the inability to culture the organism. The 2019 PERCH study identified Pneumocystis as the top fungal cause of pneumonia in HIV-negative children using a PCR cutoff of 104 copies of Pneumocystis per mL of sample in nasopharyngeal/oropharyngeal (NP/OP) specimens. Given that Pneumocystis consists of an environmental ascus form and a trophic from (the latter is the form that attaches to the lung epithelium), it is possible that life-form-specific molecular assays may be useful for diagnosis. However, to accomplish this goal, these assays require genotypic information, as the current fungal genomic data are largely from the US and Europe. To genotype Pneumocystis across the globe, we developed an NGS-based genotyping assay focused on genes expressed in asci as well as trophs using PERCH throat swabs from Africa, Bangladesh, and Thailand, as well as North American samples. The NGS panel reliably detected 21 fungal targets in these samples and revealed unique genotypes in genes expressed in trophs, including Meu10, an ascospore assembly gene; two in mitochondrial gene ATP8, and the intergenic region between COX1 and ATP8. This assay can be used for enhanced Pneumocystis epidemiology to study outbreaks but also permits more accurate RT-CPR- or CRISPR-based assays to be performed to improve the non-bronchoscopic diagnosis of this under-reported fungal pathogen.

IL-17RA-signaling in Lgr5+ intestinal stem cells induces expression of transcription factor ATOH1 to promote secretory cell lineage commitment.

The Th17 cell-lineage-defining cytokine IL-17A contributes to host defense and inflammatory disease by coordinating multicellular immune responses. The IL-17 receptor (IL-17RA) is expressed by diverse intestinal cell types, and therapies targeting IL-17A induce adverse intestinal events, suggesting additional tissue-specific functions. Here, we used multiple conditional deletion models to identify a role for IL-17A in secretory epithelial cell differentiation in the gut. Paneth, tuft, goblet, and enteroendocrine cell numbers were dependent on IL-17A-mediated induction of the transcription factor ATOH1 in Lgr5+ intestinal epithelial stem cells. Although dispensable at steady state, IL-17RA signaling in ATOH1+ cells was required to regenerate secretory cells following injury. Finally, IL-17A stimulation of human-derived intestinal organoids that were locked into a cystic immature state induced ATOH1 expression and rescued secretory cell differentiation. Our data suggest that the cross talk between immune cells and stem cells regulates secretory cell lineage commitment and the integrity of the mucosa.

Endothelial cell infection and dysfunction, immune activation in severe COVID-19.

Rationale: Pulmonary vascular endotheliitis, perivascular inflammation, and immune activation are observed in COVID-19 patients. While the initial SARS-CoV-2 infection mainly infects lung epithelial cells, whether it also infects endothelial cells (ECs) and to what extent SARS-CoV-2-mediated pulmonary vascular endotheliitis is associated with immune activation remain to be determined. Methods: To address these questions, we studied SARS-CoV-2-infected K18-hACE2 (K18) mice, a severe COVID-19 mouse model, as well as lung samples from SARS-CoV-2-infected nonhuman primates (NHP) and patient deceased from COVID-19. We used immunostaining, RNAscope, and electron microscopy to analyze the organs collected from animals and patient. We conducted bulk and single cell (sc) RNA-seq analyses, and cytokine profiling of lungs or serum of the severe COVID-19 mice. Results: We show that SARS-CoV-2-infected K18 mice develop severe COVID-19, including progressive body weight loss and fatality at 7 days, severe lung interstitial inflammation, edema, hemorrhage, perivascular inflammation, systemic lymphocytopenia, and eosinopenia. Body weight loss in K18 mice correlated with the severity of pneumonia, but not with brain infection. We also observed endothelial activation and dysfunction in pulmonary vessels evidenced by the up-regulation of VCAM1 and ICAM1 and the downregulation of VE-cadherin. We detected SARS-CoV-2 in capillary ECs, activation and adhesion of platelets and immune cells to the vascular wall of the alveolar septa, and increased complement deposition in the lungs, in both COVID-19-murine and NHP models. We also revealed that pathways of coagulation, complement, K-ras signaling, and genes of ICAM1 and VCAM1 related to EC dysfunction and injury were upregulated, and were associated with massive immune activation in the lung and circulation. Conclusion: Together, our results indicate that SARS-CoV-2 causes endotheliitis via both infection and infection-mediated immune activation, which may contribute to the pathogenesis of severe COVID-19 disease.