Vaccination with Klebsiella pneumoniae OmpX and the Th17 adjuvant LTA1 elicits lung CD4+ tissue-resident memory (TRM) Th17 cells that provide serotype-independent protection against K. pneumoniae, but vaccine efficacy declines as TRM numbers wane. To define the mechanisms sustaining these cells, we developed an ovalbumin (OVA) model with tetramer enrichment to track antigen-specific CD4+ TRM cells over time. Serial single-cell RNA sequencing on days 30, 90, and 184 after LTA1/OVA immunization revealed persistent glycolytic signatures across all time points, while ornithine decarboxylase 1 (Odc1), although required for Th17 differentiation in vitro, was dispensable for TRM maintenance in vivo. Inhibition of glycolysis with 2-deoxy-D-glucose impaired IL-17A production both in vivo and in precision-cut lung slice (PCLS) cultures which preserve functional TRM cells ex vivo. These findings identify glycolysis as a key metabolic pathway sustaining lung CD4+ Th17 TRM effector function and highlight PCLS as an ex vivo model.

OBJECTIVE:Maternal exposure during pregnancy is a strong determinant of offspring health outcomes. Such exposure induces changes in the offspring epigenome resulting in gene expression and functional changes. In this study, we investigated the effect of maternal Western hypercaloric diet (HCD) programming during the perinatal period on neuronal plasticity and cardiometabolic health in adult offspring. METHODS:C57BL/6J dams were fed HCD for 1 month prior to mating with regular diet (RD) sires and kept on the same diet throughout pregnancy and lactation. At weaning, offspring were maintained on either HCD or RD for 3 months resulting in 4 treatment groups that underwent cardiometabolic assessments. DNA and RNA were extracted from the hypothalamus to perform whole genome methylation, mRNA, and miRNA sequencing followed by bioinformatic analyses. RESULTS:Maternal programming resulted in male-specific hypertension and hyperglycemia, with both males and females showing increased sympathetic tone to the vasculature. Surprisingly, programmed male offspring fed HCD in adulthood exhibited lower glucose levels, less insulin resistance, and leptin levels compared to non-programmed HCD-fed male mice. Hypothalamic genes involved in inflammation and type 2 diabetes were targeted by differentially expressed miRNA, while genes involved in glial and astrocytic differentiation were differentially methylated in programmed male offspring. These data were supported by our findings of astrogliosis, microgliosis and increased microglial activation in programmed males in the paraventricular nucleus (PVN). Programming induced a protective effect in male mice fed HCD in adulthood, resulting in lower protein levels of hypothalamic TGFβ2, NF-κB2, NF-κBp65, Ser-pIRS1, and GLP1R compared to non-programmed HCD-fed males. Although TGFβ2 was upregulated in male mice exposed to HCD pre- or post-natally, only blockade of the brain TGFβ receptor in RD-HCD mice improved glucose tolerance and a trend to weight loss. CONCLUSIONS:Our study shows that maternal HCD programs neuronal plasticity in the offspring and results in male-specific hypertension and hyperglycemia associated with hypothalamic inflammation in mechanisms and pathways distinct from post-natal HCD exposure. Together, our data unmask a compensatory role of HCD programming, likely via priming of metabolic pathways to handle excess nutrients in a more efficient way.

Excessive formation of macrophage extracellular trap (MET) has been implicated in several autoimmune disease pathogeneses; however, its impact on type 1 diabetes (T1D) and related mechanisms remains enigmatic. We demonstrated the pivotal role of peptidyl arginine deiminase 4 (PAD4) in driving profuse MET formation and macrophage M1 polarization in intestinal inflammation in NOD mice. Genetic knockout of PAD4 or adoptive transfer of METs altered the proportion of proinflammatory T cells in the intestine, subsequently influencing their migration to the pancreas. Combining RNA sequencing and CUT&Tag analysis, we found activated PAD4 transcriptionally regulated CXCL10 expression. This study comprehensively investigated how excessive PAD4-mediated MET formation in the colon increases the aggravation of intestinal inflammation and proinflammatory T-cell migration and finally is involved in T1D progression, suggesting that inhibition of MET formation may be a potential therapeutic target in T1D. ARTICLE HIGHLIGHTS:

BackgroundNeutrophil extracellular traps (NETs) play an important role in the development and progression of ulcerative colitis (UC). Peptidyl arginine deiminase 4 (PAD4) is essential for the formation of NETs via catalyzing histone citrullination. This study mainly to explore the role of PAD4-mediated NETs in intestinal inflammation of dextran sulfate sodium (DSS)-induced UC.MethodsAcute and chronic colitis mouse models were established by supplementing DSS in drinking water. Colon tissues from colitis mice were analyzed for the level of PAD4 expression, citrullinated histone H3(Cit-H3), intestinal histopathology, and inflammatory cytokines secretion. Serum samples were tested for systemic neutrophil activation biomarkers. Colitis mice administered with Cl-amidine, a PAD4 inhibitor, and PAD4 knockout mice were investigated to detect NETs formation, intestinal inflammation, and barrier function.ResultWe found the formation of NETs significantly increased in DSS-induced colitis mice and was correlated with disease markers. Blocking NETs formation by Cl-amidine or PAD4 genetic knockout could alleviate clinical colitis index, intestinal inflammation, and barrier dysfunction.ConclusionThis study provided a research basis for the role of PAD4-mediated NETs formation in the pathogenesis of UC and suggested that inhibition of PAD4 activity and the formation of NETs may be helpful for the prevention and treatment of UC.

supported by the identification of ionocytes lacking detectable CFTR protein in sectioned airways from people with asthma (a high-IL-13 condition).Conclusions: Our findings suggest that IL-13 decreases CFTR expression in ionocytes while increasing CFTR-mediated short-circuit current.Other cell types, such as secretory cells, likely contribute to the increase in CFTRmediated short-circuit current after IL-13 treatment.

Aims: Extra virgin olive oil (EVOO) is the highest quality olive oil available and has been shown to regulate postprandial blood glucose in patients with type 1 diabetes (T1D). However, it remains uncertain whether EVOO can prevent the onset of T1D. In this study, we investigated the potential preventive effect of orally administered EVOO on T1D in non-obese diabetic (NOD) mice. Main methods: We analyzed changes in fecal microbes using 16 s rDNA sequencing and serum metabolites using Ultra High-Performance Liquid Chromatography and Quadrupole Time-of-Flight Mass Spectrometry (Q-TOFKey findings: Our findings showed that EVOO supplementation in NOD mice slowed gastric emptying, reduced insulitis, and delayed T1D onset. Moreover, EVOO altered the composition of fecal microbes, increasing the Bacteroidetes/Firmicutes ratio, and promoting the growth of short-chain fatty acids (SCFAs)-producing bacteria, such as Lachnoclostridium and Ruminococcaceae_UCG-005. Moreover, it also increased beneficial serum metabolites, including unsaturated fatty acid and triterpenoid, which positively correlated with the increased SCFAproducing bacteria and negatively correlated with the disease indicators. Conversely, most decreased serum lipid metabolites, such as Oleamide, showed the opposite trend. Significance: Our study demonstrates that EVOO may ameliorate pancreas inflammation and prevent T1D onset in NOD mice by modulating gut microbiota and serum metabolites.

PurposeGlioma has been demonstrated as one of the most malignant intracranial tumors and currently there is no effective treatment. Based on our previous RNA-sequencing data for oxidative phosphorylation (OXPHOS)-inhibition resistant and OXPHOS-inhibition sensitive cancer cells, we found that vimentin (VIM) is highly expressed in the OXPHOS-inhibition resistant cancer cells, especially in glioma cancer cells. Further study of VIM in the literature indicates that it plays important roles in cancer progression, immunotherapy suppression, cancer stemness and drug resistance. However, its role in glioma remains elusive. This study aims to decipher the role of VIM in glioma, especially its role in OXPHOS-inhibition sensitivity, which may provide a promising therapeutic target for glioma treatment.MethodsThe expression of VIM in glioma and the normal tissue has been obtained from The Cancer Genome Atlas (TCGA) database, and further validated in Human Protein Atlas (HPA) and Chinese Glioma Genome Atlas (CGGA). And the single-cell sequencing data was obtained from TISCH2. The immune infiltration was calculated via Tumor Immune Estimation Resource (TIMER), Estimation of Stromal and Immune Cells in Malignant Tumors using Expression Data (ESTIMATE) and ssGSEA, and the Immunophenoscore (IPS) was calculated via R package. The differentiated expressed genes were analyzed including GO/KEGG and Gene Set Enrichment Analysis (GSEA) between the VIM-high and -low groups. The methylation of VIM was checked at the EWAS and Methsurv. The correlation between VIM expression and cancer stemness was obtained from SangerBox. We also employed DepMap data and verified the role of VIM by knocking down it in VIM-high glioma cell and over-expressing it in VIM-low glioma cells to check the cell viability.ResultsVim is highly expressed in the glioma patients compared to normal samples and its high expression negatively correlates with patients' survival. The DNA methylation in VIM promoters in glioma patients is lower than that in the normal samples. High VIM expression positively correlates with the immune infiltration and tumor progression. Furthermore, Vim is expressed high in the OXPHOS-inhibition glioma cancer cells and low in the OXPHOS-inhibition sensitive ones and its expression maintains the OXPHOS-inhibition resistance.ConclusionsIn conclusion, we comprehensively deciphered the role of VIM in the progression of glioma and its clinical outcomes. Thus provide new insights into targeting VIM in glioma cancer immunotherapy in combination with the current treatment.

Background: In humans and other large mammals, airway submucosal glands (SMGs) secrete mucus involved in bacterial killing and mucociliary transport.Regulating the function and secretion of SMGs is key to respiratory host defense, but in CF, SMG hypertrophy and hypersecretion generate large amounts of elastic mucus that obstructs the airway.Understanding the pathophysiology of SMGs is critical to treating mucus obstruction in CF, but little is known about the cellular and molecular mechanisms of SMG mucus secretion and SMG hypertrophy in normal and CF lungs.Methods: We used single-cell RNA sequencing, single-molecule fluorescence in situ hybridization, and immunofluorescence staining to build a single-cell atlas of SMGs in newborn normal and CF pigs.We investigated how SMGs sense and respond to airway surface inflammatory signals by SMG live imaging, Ca2+ imaging, and primary SMG cell culture.Results: Single-cell atlas identified cellular and molecular features of newborn pig SMGs.Cell types and gene expression were the same in normal and CF SMGs, suggesting no developmental defects of CF SMGs at birth.Mucous and serous cells expressed the same ion transporters and neurohumoral receptors, suggesting the importance of balancing mucin and liquid secretion to produce optimal mucus properties.We discovered that SMGs contain pulmonary neuroendocrine cells (PNECs), a rare chemosensory cell type previously detected only on the airway surface.PNECs express SUCNR1 to detect succinate, a stress signal that accumulates on the airway surface in response to inflammation and infection.Succinate activates SUCNR1 in PNECs to trigger ATP release, which stimulates P2Y1 purinergic receptors in myoepithelial cells to facilitate SMG contraction.This process is disrupted in CF when mucus plugs the gland ducts, impairing access from the airway surface.In CF SMGs with lung disease, PNEC hyperplasia released calcitonin gene-related peptide, which might trigger inflammation and SMG hypertrophy.Conclusions: These findings increase our understanding of SMGs at singlecell resolution and reveal a local circuit in which rare PNECs within SMGs sense an environmental cue to modulate the function of airway SMGs.They also shed light on the mechanisms of SMG hypertrophy in CF with lung disease.

Ferroptosis is a form of regulated cell death induced by iron-dependent lipid peroxidation, and it has been studied extensively since its discovery in 2012. Induced by iron overload and ROS accumulation, ferroptosis is modulated by various cellular metabolic and signaling pathways. The GSH-GPX4 pathway, the FSP1-CoQ10 pathway, the GCH1-BH4 pathway, the DHODH-CoQH2 system and the sex hormones suppress ferroptosis. Mitochondrial iron metabolism regulates ferroptosis and mitochondria also undergo a morphological change during ferroptosis, these changes include increased membrane density and reduced mitochondrial cristae. Moreover, mitochondrial energy metabolism changes during ferroptosis, the increased oxidative phosphorylation and ATP production rates lead to a decrease in the glycolysis rate. In addition, excessive oxidative stress induces irreversible damage to mitochondria, diminishing organelle integrity. ROS production, mitochondrial membrane potential, mitochondrial fusion and fission, and mitophagy also function in ferroptosis. Notably, some ferroptosis inhibitors target mitochondria. Ferroptosis is a major mechanism for cell death associated with the progression of cancer. Metastasis-prone or metastatic cancer cells are more susceptible to ferroptosis. Inducing ferroptosis in tumor cells shows very promising potential for treating drug-resistant cancers. In this review, we present a brief retrospect of the discovery and the characteristics of ferroptosis, then we discuss the regulation of ferroptosis and highlight the unique role played by mitochondria in the ferroptosis of cancer cells. Furthermore, we explain how ferroptosis functions as a double-edged sword as well as novel therapies aimed at selectively manipulating cell death for cancer eradication.

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.

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.

BackgroundCystic 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.MethodsWe 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.ResultsCFBE 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.ConclusionsThis 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.

Background: Classical forced oscillation technique (FOT) parameters, resistance (Rrs), and reactance have the disadvantage of not being corrected for the lung volume at which they are measured.A novel FOT index, specific respiratory system conductance (sGrs), might overcome this limitation [1,2].The aim of the study was to determine sGrs in patients with cystic fibrosis (CF) and relate it to severity of lung disease.Methods: Forty-four people with CF (aged 16.3 ± 6, range 6.9-27; 45.5% male) underwent spirometry, multiple-breath nitrogen washout, and FOT measurements (Resmon Pro Full).Outcomes included forced expiratory volume in 1 second (FEV 1 ) z-score, lung clearance index (LCI), functional residual capacity (FRC), Scond, Sacin, M1/M0, M2/M0, and sGrs (calculated as 1/[R rs at 5 Hz × FRC]).Spearman correlation and linear regression after adjustment for age and FEV 1 z-score were used to assess relationships between the outcome parameters.Results: sGrs was strongly correlated with LCI (r = -0.788),Scond (r = -0.723),M1/M0 (r = -0.792),and M2/M0 (r = -0.793)and moderately correlated with Sacin (r = -0.544),FEV 1 z-score (r = 0.439), and FEV 1 /forced vital capacity (FVC) (r = 0.584).sGrs was a significant predictor of LCI (beta = -0.603,p < 0.001), Scond (beta = -0.615,p < 0.001), Sacin (beta = -0.585,p = 0.002), M1/M0 (beta = -0.601,p < 0.001), and M2/M0 (beta = -0.547,p < 0.001), independent of FEV 1 z-score and age.Conclusions: In people with CF, sGrs is consistent with ventilation heterogeneity and relates closely to severity of lung disease.This novel, easy-to-obtain FOT index may thus improve CF monitoring in the outpatient setting.

The highly complex and variable genotype-phenotype relationships observed in cystic fibrosis (CF) have been an area of growing interest since the discovery of the CF transmembrane conductance regulator (CFTR) gene >30 y ago. The consistently observed excessive, yet ineffective, activation of both the innate and adaptive host immune systems and the establishment of chronic infections within the lung, leading to destruction and functional decline, remain the primary causes of morbidity and mortality in CF. The fact that both inflammation and pathogenic bacteria persist despite the introduction of modulator therapies targeting the defective protein, CFTR, highlights that we still have much to discover regarding mucosal immunity determinants in CF. Gene modifier studies have overwhelmingly implicated immune genes in the pulmonary phenotype of the disease. In this context, we aim to review recent advances in our understanding of the innate and adaptive immune systems in CF lung disease.

Cystic fibrosis (CF) is caused by mutations in CFTR (cystic fibrosis transmembrane conductance regulator), which results in defects in ion transport. The leading causes of morbidity and mortality are respiratory symptoms and progressive pulmonary failure. Three hallmarks of this pathogenesis identified to date are abnormal mucus accumulation, mucus tethering, chronic sinopulmonary inflammation, and recurrent infections (1). However, there has been continued debate about what comes first: infection or inflammation, akin to the chicken or the egg argument. Evidence of intrinsic airway inflammation has been described in fetal lungs homozygous for the DF508del-CFTRmutation. This study found that even in fetal lung tissue, which was presumably sterile, there was overexpression of proinflammatory proteins and evidence of nuclear factor-kB activation in the airway (2). Meanwhile, studies from AREST-CF (the Australian Respiratory Early Surveillance Team for Cystic Fibrosis) showed that bacterial infections can exacerbate airway inflammation and worsen other clinical outcomes in early CF lung disease as well (3, 4). Newborn screening allows us to diagnose CF and apply early interventions before clinical presentation. Early childhoodmay represent a critical time point to delay or prevent the onset of lung damage and may impact the future clinical trajectory (5). In this issue of the Journal, Bouzek and colleagues (pp. 692–702) used the CF pig model to study bacterial-dependent and -independent inflammatory responses and mucus accumulation in newborn pigs (6). The authors chose the CF pig model because this model develops spontaneous lung disease. Commonly used rodent models are not ideal in the CF lung field as they fail to develop spontaneous lung disease as observed in humans (7). Investigators have developed newer animal models for CF, including pigs (8), ferrets (9), and rabbits (10), that to some extent overcome this limitation. The newborn pigs in this paper have similar anatomical, physiological, and biochemical features to those of early events in humans with CF compared with other models, such as 1) pigs contain submucosal glands throughout the cartilaginous airways, whereas those in rodents are limited in trachea; 2) the major type of secretory cell is the goblet cell instead of the club cell in mice; and 3) newborn pigs with the CFTRmutation have acidic airway surface liquid pH (11) and mucous tethering (12), leading to lower bacterial killing efficiency and abnormal mucociliary clearance. Within months, they develop spontaneous sinopulmonary diseases with hallmarks of CF such as infection, inflammation, mucus accumulation, airway remodeling, and lobar pathological heterogeneity (13). To determine the effects on airway bacterial burden as well as airway inflammation, the authors applied early-onset continuous broad-spectrum antibiotics to newborn CF pigs. Before the initiation of antibiotics, CF pigs at 3 weeks of age had a greater absolute number of bacteria as well as a greater number of bacterial species in lung tissue than non-CF pigs. Continuous antibiotic treatment (a combination of ceftiofur hydrochloride intramuscularly from birth as well as oral cephalexin, ciprofloxacin, and trimethoprim-sulfamethoxazole till study completion) reduced the bacterial burden as well as the bacterial species abundance in lung tissue. Furthermore, antibiotic treatment reduced certain aspects of CF lung pathology, including less mucus accumulation, measured by periodic acid–Schiff staining, and less lung parenchymal heterogeneity, measured by computed tomography (CT) lung scanning, compared with control CF pigs. However, some lung abnormalities persisted. Antibiotic-treated CF pigs still showed similar inflammatory histopathologic scores in hematoxylin and eosin staining and air trapping abnormalities on CT imaging. Notably, this extensive antibiotic regimen did not affect sinusitis, with no reduction in bacterial burdens in this anatomic niche. However, the authors did find that antibiotics altered the dominant bacterial species from Streptococcus spp. to Enterococcus spp. and Pseudomonas spp., which may suggest an active selection mechanism. This shift in bacterial species induced by antibiotics was also observed in the lungs and had a strong correlation with lung heterogeneity on CT imaging. This raises concerns for the use of routine clinical antibiotics as the main option in treating or preventing frequent infections in CF (14), given the potential risks of antibiotic resistance, disruption of gastrointestinal tract microbiome, and bacterial selection. In turn, this shift in bacterial populations may further contribute to the persistent sinopulmonary disease. Stutman and colleagues demonstrated higher colonization of P. aeruginosa and no improvement on the major health outcomes after continuous antistaphylococcal antibiotics prophylaxis (15). This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0. For commercial usage and reprints, please e-mail Diane Gern.

Tissue-resident memory (TRM) cells are thought to play a role in lung mucosal immunity to pathogens, but strategies to elicit TRM by mucosal vaccines have not yet been fully realized. Here, we formulated a vaccine composed of outer membrane protein (Omp) X from Klebsiella pneumoniae and LTA1 adjuvant that was administered by the intrapulmonary route. This vaccine elicited both TH1 and TH17 cells that shared transcriptional features with cells elicited by heat-killed K. pneumoniae. Antibody responses were required to prevent bacterial dissemination but dispensable for lung-specific immunity. In contrast, lung immunity required CD4+ T cells, STAT3 expression, and IL-17R signaling in fibroblasts. Lung-specific CD4+ T cells from OmpX+LTA1–immunized mice were observed homing to the lung and could mediate protection against infection in an adoptive transfer model. Vaccine-elicited TH17 cells showed reduced plasticity and were resistant to the immunosuppressant FK506 compared with TH1 cells, and TH17 cells conferred protection under conditions of transplant immunosuppression. These data demonstrate a promising vaccine strategy that elicits lung TRM cells and promotes serotype-independent immunity to K. pneumoniae.

Cystic fibrosis (CF) is an inherited lethal disease caused by mutations in CFTR. Approximately 60% of patients have homozygous F508del mutation, but their lung function is quite variable. Gene modifier studies identified I MHC II alleles are associated with the age of first Pseudomonas aeruginosa (PA) infection and lung function. Thus, we assessed serological responses to PA among CF subjects with poor lung function (FEV1<70%) versus good lung function (FEV1>90%). Immunoprecipitation-proteomics using outer membrane proteins (OMPs) from PA and human serum identified OprI as a potential important B cell antigen. We found increased anti-mucoid PA antibodies in CF and higher titers against OMPs and recombinant OprI in patients with poor lung function. Mice that were immunized with either recombinant OprI or inactivated whole PA subcutaneously developed high IgG titers in serum. Immunized mice that were challenged with mucoid PA had more weight loss and more severe lung pathology compared to unimmunized controls. There was minimal weight loss in PA immunized Rag2KO mice (impaired T and B cells), suggesting a role of adaptive immunity in the pathology. We observed more sensitive responses in FABP-hCFTR mice (CFTR knockout except GI tract) that OprI-immunized group exhibited the highest anti-OprI IgG titer and the most significant weight loss after challenge. These data suggest that one mechanism by which class II MHC is a modifier gene in CF is through antigen presentation and determining the types of anti-PA antibodies are generated in CF. Anti-PA antibodies may contribute to CF lung disease that we are conducting systems serology studies to understand which antibody functions are exacerbating disease.

Cystic fibrosis (CF) is an inherited life-threatening disease caused by mutated chlorine channel cystic fibrosis transmembrane conductance regulator(CFTR). Around 60% of CF patients have homozygous F508del mutation. Along with chronic bacterial infection, the integrity and function of pulmonary epithelium were compromised. Pseudomonas aeruginosa (P. a) is the most predominant pathogen, leading to decreased lung function and increased mortality in CF patients. So, prevention/eradication of airway P.a colonization is crucial to maintain lung function. Genome-wide association studies revealed human leukocyte antigen class II(HLAII) within the F508del population are associated with the age of P.a colonization and lung disease severity. However, the role of HLA class II in CF progress is not fully defined. We hypothesized that there may exist differences in antibody isotypes, titer, specificity or affinity that influence/determine lung function of CF patients. Therefore, we recruited a cohort of non-progressive F508del CF patients with confirmed P. a colonization but different lung function. Our results showed sera of CF patients have significantly higher specific IgG and IgG1 titers and stronger bacterial surface binding against mucoid P.a compared of non-CF controls. To assess antigen specificity, we performed immunoprecipitation followed by proteomics, that antibodies from CF sera have a common target, outer membrane protein OprI. Besides, CF sera inhibited bacterial biofilm formation time-dependently compared to non-CF serum. Taken together these studies may shed light on the role of HLA class II as a gene modifier in CF.