Acute cellular rejection (ACR) is a common complication after lung transplantation (LTX) and it is considered a risk factor for chronic lung allograft dysfunction (CLAD). Lung transbronchial biopsy is still the gold standard for a correct diagnosis of ACR. The aim of the present study was to evaluate the predictive role of bronchoalveolar lavage (BAL) cellular composition in combination with CT scan features for the diagnosis of ACR. We retrospectively evaluated all LTX recipients who underwent transbronchial biopsies combined with BAL procedures and CT scan at a single Institution between January 2019 and October 2024 (n = 169). ACR histological diagnosis was made according to current guidelines, BAL analysis included percentage of cellular composition, lymphocytes' typing and microbiology. A qualitative analysis of specific CT was conducted by an expert thoracic radiologist. Among the 169 biopsies analyzed, 34% showed acute cellular rejection (ACR), predominantly grade A1 (68%). Patients with ACR exhibited significantly higher lymphocyte percentages in BAL (p = 0.025), and the cutoff of 25% showed 22% sensibility and 92% specificity for the diagnosis of ACR. Combing BAL findings with CT features, patients with lymphocyte ≥ 25% in BAL and concomitant pleural effusion showed 95.7% specificity of ACR. Infections were associated with elevated neutrophil levels in BAL (p = 0.026); eosinophil levels were significantly higher in patients with significant ACR (grade ≥ 2) and concomitant infection than those with infection only (p = 0.0014). BAL cellular composition proved to be a strong predictive tool for the diagnosis of ACR. The lymphocyte threshold of 25% was able to distinguish patients with ACR, while the combination of increased BAL lymphocytes with ACR associated CT scan abnormalities especially pleural effusion significantly enhanced diagnostic accuracy. Elevated eosinophil levels were associated to more severe rejection and concomitant infection, highlighting their crucial role in the alloreactive immune response. These findings suggest the role of BAL and CT scan in combination as a valuable diagnostic tool in ACR diagnosis, although histological confirmation remains the gold standard.

BackgroundOzone exposure is a major risk factor for chronic obstructive pulmonary disease (COPD). In this study, we investigated the potential role of targeting mTOR signaling in the treatment of COPD induced by ozone exposure.MethodsThe public database was chosen to explore the expression of mTOR mRNA, S6K1 mRNA, and LC3B mRNA in COPD patients, and potential correlations with FEV1(%pred). In an ozone-exposed mouse model, large airway and small airway function were evaluated by spirometry. After intraperitoneal injection of a mTOR inhibitor known as rapamycin, the emphysema index, and inflammation scores in lung tissue were measured. Inflammatory cell infiltration in bronchoalveolar lavage fluid (BALF) and levels of cytokines in the lung tissue were also observed. Airway remodeling in the lung tissue was detected using Masson’s trichrome stains and immunohistochemical staining. Mucus hypersecretion was evaluated by PAS staining. The protein expression of the mTOR pathway and autophagy marker LC3B in the lung tissue was determined through Western blot.ResultsmTOR mRNA and S6K1 mRNA were upregulated in patients with COPD compared to the control subjects, whereas LC3B mRNA showed a downward shift in patients with COPD. Mice that received mTOR inhibitor treatment displayed higher FEV50/FVC, FEF25, FEF50, FEF75, and MMEF. The mTOR inhibitor rapamycin improved the emphysema index and inflammation scores in mice lung tissue. Moreover, it significantly inhibited inflammatory cell infiltration in BALF, IL-1β, TNF-α, and NF-κb in the lung tissue of ozone-exposed mice. The mTOR inhibitor significantly suppressed mucus hypersecretion in large and small airways and decreased the protein expression of collagen I and α-SMA in the lung tissue of ozone-exposed mice. Notably, mTOR repression also decreased the protein expression of S6K1 and increased LC3B expression in the lung tissue.ConclusionThe mTOR inhibitor rapamycin ameliorates ozone-induced airway inflammation and emphysema in a LC3B-dependent manner. mTOR inhibition may offer a promising therapeutic approach for preventing ozone-induced COPD by mitigating airway inflammation, reducing airway remodeling, and alleviating mucus hypersecretion.

This study aimed to assess the diagnostic and prognostic potential of adhesion molecules ICAM-1, ICAM-2, and VCAM-1 by analyzing their levels in serum and bronchoalveolar lavage (BAL) samples from patients with lung cancer. This prospective, single-center, cross-sectional study was conducted at the chest diseases clinic of Atatürk University Faculty of Medicine from March 2024 to May 2025. Patients diagnosed with malignant or benign lung disease by bronchoscopy, along with a control group of healthy volunteers, were included in the study. Serum samples were collected from all participants, and BAL samples were obtained from the patient groups. ICAM-1, ICAM-2, and VCAM-1 levels were measured by ELISA. Statistical analyses were performed using the SPSS 20.0 software package. Diagnostic accuracy was evaluated with ROC analysis, and 1-year survival was assessed using Kaplan-Meier curves. In the lung cancer group, ICAM-1, ICAM-2, and VCAM-1 levels in serum and BAL samples were significantly higher compared to the benign and control groups (p < 0.001 for all). BAL ICAM-2 level showed the highest diagnostic performance (AUC: 0.990; sensitivity and specificity: 96.4%). Serum ICAM-2 and ICAM-1 also showed high diagnostic performance. BAL ICAM-1 levels were significantly higher in non-small cell lung cancer (NSCLC) than in small cell lung cancer (SCLC) (p = 0.036). High serum ICAM-1 and BAL ICAM-2 levels were associated with mortality (p = 0.048 and p = 0.015, respectively). Serum and BAL levels of ICAM-1, ICAM-2, and VCAM-1 show promise as potential diagnostic biomarkers for lung cancer. Among these, BAL ICAM-2 levels are especially notable due to their high diagnostic accuracy and link to survival.

BackgroundDetecting Histoplasma (HP) and Blastomyces (BM) antigen (Ag) in urine (U) and serum (S) is an essential diagnostic tool for these pathogens. Labs have added bronchoalveolar lavage (BAL) and cerebrospinal fluid (CSF) as additional HP and BM Ag testing sources. We evaluated the sensitivity of Ag detection in U and S in patients with detected HP or BM Ag in BAL or CSF by enzyme immunoassay (EIA).Methods14 patients with HP or BM Ag-positive results made up the CSF cohort. The Ag concentrations were below the limit of quantification (< LoQ, N=3), above the limit of quantification ( > LoQ, N=1), and 0.3 to 9.5 ng/mL (x̄ = 2.3 ng/mL, N=10).The BM Ag-positive in BAL group included 24 patients, with concentrations of < LoQ (N=4), >LoQ (N=5), and 0.3 to 11.3 ng/mL (x̄ = 3.5 ng/mL, N=15).HP Ag-positive in BAL comprised 36 patients, with 3 < LoQ, 10 >LoQ, and 23 ranging from 0.8 to 16.9 ng/mL (x̄ = 4.7 ng/mL).16 patients in the BAL cohorts were Ag-positive for HP and BM, demonstrating the inability to discriminate between these dimorphic fungi through Ag testing.All patients were checked for routine clinical HP and BM EIA Ag test results on U and S. HP BAL patients were also investigated for cross-reacting galactomannan (GM; Aspergillus EIA in S or BAL) and (1→3)-β-d-glucan (BDG; Fungitell EIA in S) antigen test orders. All additional test results over 14 days before or after CSF or BAL Ag testing were excluded.Results100% (14/14), 96% (22/23), and 83% (30/36) of HP/BM CSF, BM BAL, and HP BAL Ag-positive patients were also positive in U and/or S, when both additional sources were ordered, respectively. Of the 6 HP BAL patients without positive U or S results, 3 had GM and BDG testing performed. All 3 had high signal GM and/or BDG results in S and/or BAL. In conjunction with low HP Ag levels in BAL (1.0 to 1.53), these findings suggest cross-reactivity in the HP BAL Ag test. Classifying these 3 patients as HP BAL false-positive shows a positive agreement of 91% (30/33) between HP BAL and HP U/S.ConclusionHP and BM CSF Ag testing showed no increase in sensitivity when U and S were tested. Minimal increases in sensitivity were observed in HP and BM BAL versus U and S. However, false-positive results in BAL due to cross-reactivity with GM and BDG cannot be ruled out, suggesting limited utility of the invasive CSF and BAL sources for HP and BM EIA Ag testing.DisclosuresAll Authors: No reported disclosures

ABSTRACT Graphene oxide (GO) is a nanomaterial (NM) derived from graphite associated with oxygenated functional groups which confer catalytic and hydrophilic properties, enhancing its potential for industrial and therapeutic applications. This study aimed to integrate physicochemical characterization utilizing in vitro and in vivo toxicological assessments of GO developed by CTNANO-UFMG and CENPES/Petrobras for enhanced oil recovery. ATR–FTIR and TGA were used to characterize the chemical functional group profile. GO exhibited hydrodynamic diameters (HD) below 1 µm at different times. SEM analysis revealed mean equivalent circular diameters (ECD) of 11.37 (water) and 11.78 µm (DMEM). Concentrations of 5, 10, 50, 100, 250 or 500 µg/ml did not significantly affect HepG2 cell viability. Intraperitoneal GO administration (5, 12.5, 25 or 50 mg/kg) to BALB/c mice transiently reduced food and water intake and body weight. In pleural cavity lavage (PCL), number of leukocytes was decreased accompanied by lower levels of mononuclear cells and eosinophils but elevated neutrophils. In bronchoalveolar lavage (BAL), neutrophils were also increased. No significant changes were observed in IFN-γ and TNF levels in plasma, PCL, or BAL, although IL-6 was decreased in PCL. Serum AST levels were increased, and GO accumulated preferentially in liver, with additional deposits in adipose tissue adjacent to spleen and kidney. Histological changes and inflammatory infiltrates were observed in liver, spleen, and lungs. These findings indicate that GO induces mild local inflammatory responses and dose-dependent hepatic accumulation, indicating need for further studies with prolonged exposure periods to assess potential adverse effects and reversibility.

BackgroundVeno-venous (V–V) extracorporeal membrane oxygenation (ECMO) is widely used in critical care but remains associated with high mortality rates (22–68%). In septic shock, increased pulmonary inflammation and impaired intestinal and hepatic microcirculation have been observed during ECMO therapy. To explore the impact of ECMO-induced inflammation, this study used a rat model with varying ECMO blood flows to assess intestinal and hepatic microcirculation and lung inflammation.MethodsThirty male Lewis rats were randomised into three groups: sham, low-flow ECMO (60 mL/kg/min), and high-flow ECMO (90 mL/kg/min). V–V ECMO was established via femoral drainage and jugular return. Microcirculation in the intestine and liver was measured using micro-light guide spectrophotometry after laparotomy. Systemic and pulmonary inflammation were evaluated through cytokine levels in plasma and bronchoalveolar lavage (BAL), focusing on tumour necrosis factor-alpha (TNF-α), interleukins 6 (IL6) and 10 (IL10), and C–X–C motif chemokine ligands 2 (CXCL2) and 5 (CXCL5). Hemodynamic data were obtained using a left ventricular pressure–volume catheter.ResultsIntestinal oxygenation was significantly impaired only during low-flow ECMO therapy (65% [62–70%]) compared to sham therapy (76% [72–79%], p = 0.003), while hepatic microcirculation was reduced during both low-flow (21% [14–26%]) and high-flow (19% [16–21%]) ECMO therapy compared to sham therapy (43% [38–48%], all p < 0.001). Serum TNF-α levels were only significantly elevated during high-flow ECMO therapy (1 h: 14 [12–22] pg/mL; 2 h: 18 [15–38] pg/mL) compared to the sham procedure (1 h: 10 [9–11] pg/mL; 2 h: 10 [9–11] pg/mL; p = 0.033). In contrast, BAL IL6 levels were significantly lower during both high- and low-flow ECMO therapy (32 pg/mL) than sham therapy (81 pg/mL, p ≤ 0.001). IL10, CXCL2, and CXCL5 levels did not differ significantly between the low- and high-flow ECMO and sham therapies.ConclusionsECMO-induced inflammation is blood flow dependent. In healthy rats, high-flow ECMO did not impair intestinal microcirculation and was associated with reduced pulmonary inflammation, likely due to lung-protective ventilation.

Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase inhibitor. ADMA concentrations are augmented in lung, plasma samples of asthmatics. It remains unclear whether the increased concentration of ADMA in airway inflammation contributes to the pathophysiology or acts as a protective mechanism. The objective of this study was to evaluate the effects of exogenous ADMA on airway inflammation in mice. Ex vivo/in vivo airway inflammation models were used. Tracheal tissues, isolated from female Balb/c mice were incubated with lipopolysaccharide (LPS), ADMA, or LPS + ADMA for 4 days in tissue culture. Tracheal reactivity was evaluated afterward by agonist-induced contraction responses. Female Balb/c mice were applied i.n. LPS (0,1mg/ml) to induce in vivo airway inflammation. ADMA (30mg/kg) was administered by i.n. route 1 h before and 24h after LPS application. I.n. ADMA application augmented lung ADMA levels, reduced nitrite levels in bronchoalveolar lavage (BAL) fluid, prevented bronchial hyperreactivity and airway inflammation. ADMA also prevented bronchial hyperreactivity when applied 15min before methacholine nebulization during the assessment of airway function. Exogenously applied ADMA may have protective effects in airway inflammation by limiting excessive nitric oxide (NO) production. Targeting the DDAH/ADMA/NOS pathway may provide a therapeutic approach for airway inflammation.

Lung surfactant is a lipid-protein complex essential for maintaining alveolar stability by reducing surface tension. In surfactant-deficient states such as pneumonia or acute respiratory distress syndrome, alveolar collapse and impaired gas exchange may occur. Exogenous supplementation of surfactant phospholipids has been proposed to improve lung function. In this work, we performed LC-MS/MS analysis of phosphatidylcholines from a commercially available Essentiale phospholipid preparation in bronchoalveolar lavage (BAL) fluid. The dynamics of BAL phosphatidylcholine levels in mice following intravenous and aerosol administration of Essentiale were investigated for the first time, revealing the time course and extent of pulmonary accumulation for each route. A 20-min aerosol inhalation of Essentiale (≈3 mg/kg phospholipids) achieved BAL phosphatidylcholine concentrations comparable to those obtained with a 500 mg/kg intravenous dose. Repeated daily intravenous dosing caused a gradual increase in BAL phosphatidylcholine over 3-4 days, whereas prolonged aerosol exposure beyond 20 min did not further elevate BAL levels, likely due to uptake by alveolar cells. These findings highlight aerosol inhalation as a more efficient delivery method for surfactant phospholipids and suggest an optimal dosing regimen (≈ 3 mg/kg, 20 min) for adjunctive therapy in conditions associated with surfactant deficiency.

Background/Objectives: Obesity and hyperglycemia predispose patients to respiratory infections. Although the lung is a major organ to utilize glucose, pulmonary glucose homeostasis in type 2 diabetic (T2Dx) subjects remains poorly characterized. We hypothesized that pulmonary glucose transport would be altered during T2Dx, which would be rescued with long-term metformin treatment. Methods: T2Dx was induced by feeding mice a high-fat diet for 16 weeks, with metformin treatment administered during the final 8 weeks. Results: Glucose transporter (GLUT) protein expression and trafficking was quantified by Western blotting and the biotinylated photolabeling assay, respectively. T2Dx mice exhibited obesity, and increased glucose levels in blood and bronchoalveolar lavage (BAL) fluid. T2Dx also significantly decreased protein expression of GLUTs from Class I (i.e., GLUT-2 and -4) and class III (i.e., GLUT-10 and -12) isoforms in lung. Metformin treatment restored the protein expression of GLUT-2, -4, and -10, but not GLUT-12. Pulmonary cell surface expression of GLUT-4 and -8 was also significantly reduced in T2Dx mice and rescued by metformin. Conclusions: These findings suggest that alterations in pulmonary GLUT expression and trafficking during diabetes could contribute to the elevated airway glucose levels and severity of respiratory infections. Metformin treatment restored pulmonary glucose transport during T2Dx.

Role of IL-10 signaling in the circadian control of host response to influenza infection.

Excessive mucus in the airways is an underlying pathological feature of many airway diseases, including asthma. Therapeutic options to reduce mucus production in the airways remain limited. One possible therapeutic target is the airway sympathetic nerves. Although lung sympathetic innervation has been considered sparse, sympathetic nerves secrete neurotransmitters that act on adrenergic receptors, including β2-adrenergic receptor (β2AR). Interestingly, in experimental models, chronic use of β2AR agonists can augment mucus secretion. Thus, in the present study, we tested the hypothesis that airway sympathetic nerves regulate mucus production in the airway in response to the type 2 cytokine interleukin 13 (IL-13). We performed airway sympathectomy using intranasal instillation of the synthetic neurotoxin 6-hydroxydopamine (6-OHDA). Airway sympathectomy attenuated multiple IL-13-mediated airway deficits, including density of goblet cells containing neutral mucins, transcriptional ratio of mucin 5ac (Muc5ac) to mucin 5b (Muc5b), and airway elastance and tissue damping. Although total Muc5ac and Muc5b transcript levels and Muc5ac and Muc5b protein levels in bronchoalveolar lavage were not significantly altered, these changes suggest that airway sympathectomy modifies goblet cell phenotype and mucin composition. Airway sympathectomy also dampened IL-13-mediated increases in total lung transcripts important for regulating allergic responses, including interleukin 6, complement component 3, and colony-stimulating factor. This study reveals that airway sympathetic nerves regulate physiologic, molecular, and inflammatory responses to type 2 (IL-13-mediated) airway inflammation and raises the possibility that they may serve as potential targets for therapeutic intervention.NEW & NOTEWORTHY The role of airway sympathetic nerves in regulating airway responses remains largely undefined. We demonstrated that chemical depletion of airway sympathetic nerves attenuates specific IL-13-induced airway deficits at the molecular, cellular, and functional level. Our data suggest that airway sympathetic nerves may represent novel therapeutic targets to alleviate some pathologic features due to type 2 (IL-13-mediated) airway inflammation.

IntroductionResistive breathing (RB) is the hallmark of diseases of airway obstruction, especially during exacerbations, resulting in significant mechanical stress on the lung. RB induces pulmonary inflammation and injury in previously healthy animals. Whether RB exerts additional injurious effects when superimposed on cigarette smoke (CS) exposure is unknown.MethodsAdult mice were exposed to CS for 1 or 6 months, followed by RB for 24 h, induced through tracheal banding. Subsequently, respiratory system mechanics were assessed, bronchoalveolar lavage (BAL) was performed and cytokine levels were measured by ELISA in lung tissue samples. Surfactant protein D (SP-D) was measured in blood, and BAL and histology were performed. Emphysema was quantified by the mean linear intercept (Lm) and the destructive index (DI).ResultsCS exposure for 1 and 6 months increased BAL cellularity (∼8-fold and ∼1.9-fold to air, respectively, p<0.01). RB aggravated BAL cellularity at both time points (p=0.025 and p=0.002 to CS) and increased MCP-1 levels at 1 month (p=0.002 to control). Histology revealed augmented focal membrane thickening at 1 month following combination of RB with CS exposure (p=0.011), while at 6 months emphysema was more severe after RB in CS-exposed mice (p=0.001 to CS for both Lm and DI). CS increased Sp-D levels in BAL (p<0.01 at both time points), while RB caused increased levels in blood (p<0.01 to CS). RB decreased static compliance at 1 month of air exposure (p=0.007).ConclusionCombining resistive breathing with cigarette smoke exposure results in augmented inflammatory responses, increased lung injury and augmented emphysema.

Asthma is a debilitating lung disease characterized by airway inflammation and airflow obstruction. Immune cells, particularly T helper 2 (Th2) lymphocytes, are central players in the pathogenesis of asthma and mesenchymal stem cells (MSCs) have shown the capability of softening pathological inflammatory responses in asthma. Hence, we researched the immunopathologic effects MSCs cocultured with interferon (IFN)-γ, the main Th1 cytokine, in asthmatic mice. After isolation, MSCs were cocultured with IFN-γ and administered to asthmatic mice. Subsequent analyses included enumeration of broncho-alveolar lavage (BAL) fluid's inflammatory cells, determination of the levels of immunoglobulin E (IgE), leukotrienes (LTs), cytokines, chemokines, and histopathology assessment. The administration of IFN-γ-cocultured MSCs reduced the percentage of eosinophils in the BAL fluid and levels of IgE, LTs, cytokines, and chemokines. Also, there was a decrease in the eosinophilic infiltration of perivascular areas and periairways. IFN-γ cocultured MSCs could modulate immune responses and harness pathological events in allergic asthma.

Obesity is associated with metabolic inflammation, which includes changes to innate immune responses relevant to acute lung injury. NOD1 is a cytosolic pattern recognition receptor involved in sensing bacterial peptidoglycan and has been linked to metabolic inflammation. However, its role in obesity‐associated acute lung injury, especially in females, remains unclear. Here, we investigated the impact of NOD1 deficiency on pulmonary inflammation in female mice subjected to a high‐fat diet and lipopolysaccharide‐induced acute lung injury. Compared to wild‐type controls, obese Nod1−/− mice showed reduced leukocyte and neutrophil numbers in the bronchoalveolar lavage (BAL), but increased BAL levels of TNF‐α, IL‐1β, IL‐6, IL‐17A, and IL‐22, suggesting impaired neutrophil clearance. In the lung tissue, NOD1 deficiency during obesity led to elevated neutrophil accumulation, increased myeloperoxidase activity, reduced CD163+ macrophages, and enhanced β‐galactosidase activity. Gene expression analysis revealed upregulation of chemokines, adhesion molecules, and inflammasome components, alongside downregulation of M2 polarization markers. Additionally, obese Nod1−/− mice showed higher NF‐κB and ERK1/2 activation and lower p38 phosphorylation. These findings indicate that NOD1 regulates leukocyte dynamics, inflammation, and macrophage function in the obese lung. We identify NOD1 as a key protective modulator of pulmonary immune responses during acute lung injury under metabolic stress.

Platelet membrane-cloaked biomimetic nanoparticles for targeted acute lung injury therapy.

Abstract RATIONALE: Interstitial lung diseases, particularly those that manifest a progressive fibrotic phenotype, are poorly understood with little known about factors driving the progression of fibrosis in some individuals and not others. The P2 purinergic receptors are known to play a role in pulmonary fibrosis although the exact nature of this role remains unclear. We hypothesize that adenosine triphosphate (ATP) acting via the G protein-coupled (P2Y) and ATP-gated ion channel (P2X) receptors, alters the early inflammatory response of the lung to insult, initiating fibrogenesis. Thus, our aim was to determine P2X and P2Y receptor expression, levels of ATP activator, and inflammatory cytokines in airways and blood samples of patients with progressive pulmonary fibrosis (pwPPF). METHODS: Blood samples and bronchoalveolar lavage (BAL) were collected from patients and healthy controls. ATP levels in plasma and BAL were measured via bioluminescence. Gene expression was assessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Interleukin-1 β (IL-1β) and tumour necrosis factor α (TNF-α) were quantified by enzyme-linked immunosorbent assay (ELISA). RESULTS: Elevated ATP levels were recorded in plasma (p&amp;lt;.0001) and BAL (p=0.0015) of pwPPF (n=12). Increased mRNA expression of P2X4R (p=0.0001), P2X7R (p=0.016), P2Y4R (p=0.03), P2Y6R (p=0.0008), P2Y12R (p=0.0005) and P2Y13R (p=0.004) were detected in PPF monocytes. Increased expression of P2X3 (p=0.004), P2X7 (p=0.04), P2Y6 (p=0.01), P2Y12 (p=0.02) and P2Y13 (p=0.004) were detected in BAL immune cells of pwPPF. Increased IL-1β and TNF-α correlated significantly with ATP levels in PPF plasma (p=0.02 and p=0.001, respectively) and BAL (p=0.005 and p=0.001, respectively). In turn, ATP, TNF-α and IL-1β correlated negatively with patient FVC (%) (p=0.001, p=0.002 and p=0.007, respectively). Mechanistically, ATP and TNF-α stimulated PPF monocyte NLR family pyrin domain containing 3 (NLRP-3) inflammasome activation, resulting in increased IL-1β release, an effect reduced by inclusion of specific P2X4 (5-BDBD) and P2X7 (A438079) inhibitors. CONCLUSION: P2 purinergic signalling is implicated in pulmonary fibrosis, with purinergic receptor remodelling of circulating monocytes and resident bronchial immune cells. ATP and TNF-α act synergistically on P2X4 and P2X7 receptors to drive inflammation in monocytes. These findings provide insights into the role of circulating immune cells in the pathogenesis of pulmonary fibrosis and suggest novel targets for therapeutic intervention.

Abstract Background: Idiopathic pulmonary fibrosis (IPF) is interstitial lung disease characterized by dysregulated response to injury, increased oxidative stress and progressive lung scarring.Oxidation-specific epitopes (OSEs) are neoantigens that form during oxidative stress and can trigger immune response and further inflammation. OSEs have been described in animal models of lung injury, but their role in IPF is unknown. Antibodies against OSEs can limit inflammation, and we previously reported that anti-OSE IgA against oxidized cholesterol is elevated in interstitial lung diseases including IPF. In the current study, we identified unique B-cell phenotypes associated with anti-OSE IgA in IPF and further explored anti-OSE IgA role in IPF. Methods: We performed deep phenotyping of circulating B-cells in a cohort of 20 patients with IPF and 9 healthy controls utilizing mass cytometry time of flight (CyTOF). We utilized a bleomycin murine model of pulmonary fibrosis to determine anti-OSE IgA production in vivo and performed in vitro studies to elucidate effects of cytokines on anti-OSE IgA production. To determine whether anti-OSE IgA forms in other types of lung injury, we analyzed anti-OSE IgA and B-Cells in 49 patients with COVID-19. Results: In patients with IPF and age-matched controls, we identified an atypical, switched IgA+ B-cell phenotype which significantly correlated with anti-OSE IgA serum antibody levels (p&amp;lt;0.05) (Figure). This B-cell cluster was characterized by IgD-CD27-CD21-CD11c+ phenotype. Similar B-cell populations were previously reported in various autoimmune diseases under the term “atypical memory cells” or “double negative B-cells”. IgD-CD27- B-cells were expanded in IPF compared to controls (p&amp;lt;0.05), and early plasma-cell marker CD43 was significantly increased in IgA+IgD-CD27- B-cell population (p&amp;lt;0.05), suggesting increased terminal differentiation into antibody-secreting cells. In a murine model of pulmonary fibrosis, anti-OSE IgA levels in bronchoalveolar lavage were significantly elevated post bleomycin treatment (p&amp;lt;0.05). TGF beta, a major pro-fibrotic cytokine, increased anti-OSE IgA antibody production in peripheral blood mononuclear cells of normal donors (p&amp;lt;0.05). Anti-OSE IgA antibodies were also significantly elevated in inpatients with COVID-19 compared to outpatients (p&amp;lt;0.05) and correlated with circulating IgD-CD27-CD11c+ B-cell frequency (p&amp;lt;0.01). Conclusions: In patients with IPF and COVID-19, anti-OSE IgA antibodies were elevated and associated with an atypical IgD-CD27-CD11c+ B-cell phenotype. Anti-OSE IgA antibodies accumulated in lungs of mice with experimental pulmonary fibrosis, and TGF-beta, a major pro-fibrotic mediator, promoted OSE-IgA production in vitro. Together, these findings suggest that atypical CD11c+ B-cells produce anti-OSE IgA antibodies as a response to oxidative stress in both acute and chronic lung injury.

We have previously demonstrated that the circadian clock regulates the host response to influenza A virus (IAV) infection, conferring a time-of-day-specific protection -infection at dawn resulted in a threefold increase in survival and reduced immunopathology compared to infection at dusk. While IL10 is well-known for its immunoregulatory function, its role in IAV remains unclear, with studies reporting both protective and detrimental effects. Given the diurnal rhythmicity of IL-10 receptor ( Il10ra ) expression in the lung, we investigated the contribution of IL-10 signaling to time-of-day-specific IAV protection. We found that blocking IL10 signaling abrogated the time of day protection, leading to increased immunopathology characterized by enhanced lymphocyte infiltration and global immune activation (transcriptomic analysis). Interestingly, while later, IL-10R blockade also eliminated the time-of-day difference in IAV outcomes, it improved the outcome of dusk-infected mice. Furthermore, natural killer (NK) cell depletion suppressed IL-10 levels in bronchoalveolar lavage, suggesting a role for NK cells in regulating IL-10 signaling. In conclusion, incorporating the circadian context has not only clarified the IL-10 role in IAV infection but also underscored the pivotal influence of circadian regulation on immune responses.

Mucosal secretory IgA (SIgA) produced by subepithelial plasma cells in the lamina propria is the major antigen-specific defense mechanism against mucosal infections. We investigated if a retinoic acid (RA)-containing adjuvant in parenteral immunization, can induce vaccine-specific SIgA in the jejunal lumen in a dose-dependent manner in neonatal pigs immunized with a Chlamydia hybrid antigen. To accurately quantify SIgA responses in mucosal secretions, an antigen-specific ELISA method with secondary detection of porcine secretory component rather than IgA was developed. RA facilitated a stronger (or faster) IgG, IgA, IgM and SIgA response in serum after primary immunization, and a more than 10-fold significantly increased level of vaccine-specific SIgA in jejunum at termination 2weeks after the secondary boost, whereas IgA or SIgA responses in bronchoalveolar lavage (BAL) were not significantly increased after immunization with RA. Analyses of different isotype responses to vaccination and different sampling sites, revealed significant correlations between IgG and IgA responses in serum, and between IgG in serum and jejunum, while IgA in jejunum was neither correlated with IgA in serum nor with IgG in jejunum. This is indicative of IgG in jejunum being primarily a transudate from serum, while IgA is not. Jejunum SIgA correlated significantly with jejunum IgA and with both serum SIgA and IgA. Our results thus support the use of SC-specific reagents for mucosal SIgA responses, although IgA reagents to a lesser extent also reflects local antibodies. Although the IgA and SIgA levels in BAL were not significantly different with or without RA, we observed a significant correlation of vaccine-specific SIgA in jejunum and BAL, indicating a level of commonality in the regulation of mucosal antibodies in gut and respiratory system. In conclusion, an adjuvant with high concentration of RA was shown to increase the local intestinal mucosal antibody response after parenteral immunization in pigs.

S-Denitrosylation counteracts local inflammation and improves survival in mice infected with K. pneumoniae.