Lung mucosal immunity to NTHi vaccine antigens: Antibodies in sputum of chronic obstructive pulmonary disease patients.

Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory illness in older adults. A major cause of COPD-related morbidity and mortality is acute exacerbation of COPD (AECOPD). Bacteria in the lungs play a role in exacerbation development, and the most common pathogen is non-typeable Haemophilus influenzae (NTHi). A vaccine to prevent AECOPD containing NTHi surface antigens was tested in a clinical trial. This study measured IgG and IgA against NTHi vaccine antigens in sputum. Sputum samples from 40 COPD patients vaccinated with the NTHi vaccine were collected at baseline and 30 days after the second dose. IgG and IgA antibodies against the target antigens and albumin were analyzed in the sputum. We compared antibody signals before and after vaccination, analyzed correlation with disease severity and between sputum and serum samples, and assessed transudation. Antigen-specific IgG were absent before vaccination and present with high titers after vaccination. Antigen-specific IgA before and after vaccination were low but significantly different for two antigens. IgG correlated between sputum and serum, and between sputum and disease severity. Sputum albumin was higher in patients with severe COPD than in those with moderate COPD, suggesting changes in transudation played a role. We demonstrated that immunization with the NTHi vaccine induces antigen-specific antibodies in sputum. The correlation between IgG from sputum and serum and the presence of albumin in the sputum of severe COPD patients suggested transudation of antibodies from the serum to the lungs, although local IgG production could not be excluded.Clinical Trial Registration: NCT02075541.

What is the context? Chronic obstructive pulmonary disease (COPD) is the most common chronic respiratory illness in older adults and the third leading cause of death worldwide.One bacterium in the lungs, non-typeable Haemophilus influenzae (NTHi), is responsible for acute exacerbation of the disease, characterized by an increase in airway wall inflammation and symptoms, leading to high morbidity and mortality.A vaccine targeting NTHi was previously developed but did not show efficacy in reducing exacerbations in COPD patients, probably because the vaccine did not elicit an immune response in the lung mucosae, where the bacteria are located.What is the impact? Parenteral immunization with new vaccines targeting NTHi is able to elicit immune defense at the level of lung mucosae.Now that antibodies can be measured in sputum, new vaccines against COPD exacerbations or other lung infections can be tested for efficacy in the actual target tissue.Also, lung immunity against specific pathogens can now be tested.What is new? We determined that antigen-specific antibodies were present in the lungs after vaccination; these were assessed in sputum after vaccination with NTHi surface antigens.NTHi-specific IgG were present in the lungs and appeared to have arrived there primarily by transudation, a type of leakage from the serum to the lung mucosae.Transudation appeared to be stronger in severe than in moderate COPD patients.

Lung-specific interleukin 6 mediated transglutaminase 2 activation and cardiopulmonary fibrogenesis.

Pulmonary hypertension (PH) pathogenesis is driven by inflammatory and metabolic derangements as well as glycolytic reprogramming. Induction of both interleukin 6 (IL6) and transglutaminase 2 (TG2) expression participates in human and experimental cardiovascular diseases. However, little is known about the role of TG2 in these pathologic processes. The current study aimed to investigate the molecular interactions between TG2 and IL6 in mediation of tissue remodeling in PH. A lung-specific IL6 over-expressing transgenic mouse strain showed elevated right ventricular (RV) systolic pressure as well as increased wet and dry tissue weights and tissue fibrosis in both lungs and RVs compared to age-matched wild-type littermates. In addition, IL6 over-expression induced the glycolytic and fibrogenic markers, hypoxia-inducible factor 1α, pyruvate kinase M2 (PKM2), and TG2. Consistent with these findings, IL6 induced the expression of both glycolytic and pro-fibrogenic markers in cultured lung fibroblasts. IL6 also induced TG2 activation and the accumulation of TG2 in the extracellular matrix. Pharmacologic inhibition of the glycolytic enzyme, PKM2 significantly attenuated IL6-induced TG2 activity and fibrogenesis. Thus, we conclude that IL6-induced TG2 activity and cardiopulmonary remodeling associated with tissue fibrosis are under regulatory control of the glycolytic enzyme, PKM2.

Association of IL-33 in modeling type-2 airway inflammation and pulmonary emphysema in mice.

We developed pulmonary emphysema and a type 2 airway inflammation overlap mouse model. The bronchoalveolar lavage (BAL) interleukin 13 (IL-13), IL-4, and IL-5 levels in the overlap model were higher than in the pulmonary emphysema model and lower than in the type 2 airway inflammation model, but IL-33 level in the lung was higher than in other models. IL-33 and interferon-γ (IFNγ) in lungs may control the severity of a type 2 airway inflammation in lung.

Correlating transcription and protein expression profiles of immune biomarkers following lipopolysaccharide exposure in lung epithelial cells.

Universal and early recognition of pathogens occurs through recognition of evolutionarily conserved pathogen associated molecular patterns (PAMPs) by innate immune receptors and the consequent secretion of cytokines and chemokines. The intrinsic complexity of innate immune signaling and associated signal transduction challenges our ability to obtain physiologically relevant, reproducible and accurate data from experimental systems. One of the reasons for the discrepancy in observed data is the choice of measurement strategy. Immune signaling is regulated by the interplay between pathogen-derived molecules with host cells resulting in cellular expression changes. However, these cellular processes are often studied by the independent assessment of either the transcriptome or the proteome. Correlation between transcription and protein analysis is lacking in a variety of studies. In order to methodically evaluate the correlation between transcription and protein expression profiles associated with innate immune signaling, we measured cytokine and chemokine levels following exposure of human cells to the PAMP lipopolysaccharide (LPS) from the Gram-negative pathogen Pseudomonas aeruginosa. Expression of 84 messenger RNA (mRNA) transcripts and 69 proteins, including 35 overlapping targets, were measured in human lung epithelial cells. We evaluated 50 biological replicates to determine reproducibility of outcomes. Following pairwise normalization, 16 mRNA transcripts and 6 proteins were significantly upregulated following LPS exposure, while only five (CCL2, CSF3, CXCL5, CXCL8/IL8, and IL6) were upregulated in both transcriptomic and proteomic analysis. This lack of correlation between transcription and protein expression data may contribute to the discrepancy in the immune profiles reported in various studies. The use of multiomic assessments to achieve a systems-level understanding of immune signaling processes can result in the identification of host biomarker profiles for a variety of infectious diseases and facilitate countermeasure design and development.

Regulation of pulmonary plasma cell responses during secondary infection with influenza virus.

During secondary infection with influenza virus, plasma cells (PCs) develop within the lung, providing a local source of antibodies. However, the site and mechanisms that regulate this process are poorly defined. Here, we show that while circulating memory B cells entered the lung during rechallenge and were activated within inducible bronchus-associated lymphoid tissues (iBALTs), resident memory B (BRM) cells responded earlier, and their activation occurred in a different niche: directly near infected alveoli. This process required NK cells but was largely independent of CD4 and CD8 T cells. Innate stimuli induced by virus-like particles containing ssRNA triggered BRM cell differentiation in the absence of cognate antigen, suggesting a low threshold of activation. In contrast, expansion of PCs in iBALTs took longer to develop and was critically dependent on CD4 T cells. Our work demonstrates that spatially distinct mechanisms evolved to support pulmonary secondary PC responses, and it reveals a specialized function for BRM cells as guardians of the alveoli.

Preliminary study on the protective effect of remazolam against sepsis-induced acute respiratory distress syndrome (ARDS).

Background: Sepsis can disrupt immune regulation and lead to acute respiratory distress syndrome (ARDS) frequently. Remazolam, a fast-acting hypnotic drug with superior qualities compared to other drugs, was investigated for its potential protective effects against sepsis-induced ARDS. Methods: Forty Sprague-Dawley rats were randomly divided into four groups, including the sepsis + saline group, sham operation + saline group, sham operation + remazolam group and the sepsis + remazolam group. Lung tissues of rats were extracted for HE staining to assess lung damage, and the wet weight to dry weight (W/D) ratio was calculated. The levels of proinflammatory factors, anti-inflammatory factors, CD4+ and CD8+ T cells in peripheral blood, MDA, MPO, and ATP in the lung tissue were measured by using ELISA. Western blotting was performed to determine the protein expression of HMGB1 in lung tissues. Results: In comparison to the sham operation + saline and sham operation + remazolam groups, the sepsis + saline group exhibited significantly higher values for W/D ratio, lung damage score, IL-1ß, IL-6, TNF-α, PCT, CRP, MDP and MPO, while exhibiting lower levels of CD4+ and CD8+ T lymphocytes, PaO2, PCO2, and ATP. The rats in the sepsis + saline group displayed ruptured alveolar walls and evident interstitial lung edema. However, the rats in the sepsis + remazolam group showed improved alveolar structure. Furthermore, the HMGB1 protein expression in the sepsis + remazolam group was lower than the sepsis + saline group. Conclusion: Remazolam can alleviate the inflammatory response in infected rats, thereby alleviating lung injury and improving immune function, which may be attributed to the reduction in HMGB1 protein expression.

Identification and characterization of endogenous retroviruses upon SARS-CoV-2 infection.

Endogenous retroviruses (ERVs) derived from the long terminal repeat (LTR) family of transposons constitute a significant portion of the mammalian genome, with origins tracing back to ancient viral infections. Despite comprising approximately 8% of the human genome, the specific role of ERVs in the pathogenesis of COVID-19 remains unclear. In this study, we conducted a genome-wide identification of ERVs in human peripheral blood mononuclear cells (hPBMCs) and primary lung epithelial cells from monkeys and mice, both infected and uninfected with SARS-CoV-2. We identified 405, 283, and 206 significantly up-regulated transposable elements (TEs) in hPBMCs, monkeys, and mice, respectively. This included 254, 119, 68, and 28 ERVs found in hPBMCs from severe and mild COVID-19 patients, monkeys, and transgenic mice expressing the human ACE2 receptor (hACE2) and infected with SARS-CoV-2. Furthermore, analysis using the Genomic Regions Enrichment of Annotations Tool (GREAT) revealed certain parental genomic sequences of these up-regulated ERVs in COVID-19 patients may be involved in various biological processes, including histone modification and viral replication. Of particular interest, we identified 210 ERVs specifically up-regulated in the severe COVID-19 group. The genes associated with these differentially expressed ERVs were enriched in processes such as immune response activation and histone modification. HERV1_I-int: ERV1:LTR and LTR7Y: ERV1:LTR were highlighted as potential biomarkers for evaluating the severity of COVID-19. Additionally, validation of our findings using RT-qPCR in Bone Marrow-Derived Macrophages (BMDMs) from mice infected by HSV-1 and VSV provided further support to our results. This study offers insights into the expression patterns and potential roles of ERVs following viral infection, providing a valuable resource for future studies on ERVs and their interaction with SARS-CoV-2.

Prolonged exposure to lung-derived cytokines is associated with activation of microglia in patients with COVID-19.

BACKGROUNDSurvivors of pneumonia, including SARS-CoV-2 pneumonia, are at increased risk for cognitive dysfunction and dementia. In rodent models, cognitive dysfunction following pneumonia has been linked to the systemic release of lung-derived pro-inflammatory cytokines. Microglia are poised to respond to inflammatory signals from the circulation, and their dysfunction has been linked to cognitive impairment in murine models of dementia and in humans.METHODSWe measured levels of 55 cytokines and chemokines in bronchoalveolar lavage fluid and plasma from 341 patients with respiratory failure and 13 healthy controls, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. We used flow cytometry to sort neuroimmune cells from postmortem brain tissue from 5 patients who died from COVID-19 and 3 patients who died from other causes for single-cell RNA-sequencing.RESULTSMicroglia from patients with COVID-19 exhibited a transcriptomic signature suggestive of their activation by circulating pro-inflammatory cytokines. Peak levels of pro-inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, but cumulative cytokine exposure was higher in patients with COVID-19. Treatment with corticosteroids reduced expression of COVID-19-specific cytokines.CONCLUSIONProlonged lung inflammation results in sustained elevations in circulating cytokines in patients with SARS-CoV-2 pneumonia compared with those with pneumonia secondary to other pathogens. Microglia from patients with COVID-19 exhibit transcriptional responses to inflammatory cytokines. These findings support data from rodent models causally linking systemic inflammation with cognitive dysfunction in pneumonia and support further investigation into the role of microglia in pneumonia-related cognitive dysfunction.FUNDINGSCRIPT U19AI135964, UL1TR001422, P01AG049665, P01HL154998, R01HL149883, R01LM013337, R01HL153122, R01HL147290, R01HL147575, R01HL158139, R01ES034350, R01ES027574, I01CX001777, U01TR003528, R21AG075423, T32AG020506, F31AG071225, T32HL076139.

Rapamycin improves the long-term T-cell memory and protective efficacy of tuberculosis subunit vaccine.

The formation of long-lived T-cell memory is a critical goal of vaccines against intracellular pathogens like Mycobacterium tuberculosis (M. tuberculosis). In this study, to access the adjuvant effect of rapamycin on tuberculosis subunit vaccine, we treated mice with rapamycin during the course of vaccination and then monitored the vaccine-specific long-term memory T cell recall responses and protective ability against mycobacterial organisms. Compared with the mice that received vaccine alone, rapamycin treatment enhanced the vaccine induced long-term IFN-γ and IL-2 recall responses, promoted the development of TCM (central memory) like cells and improved the long-term proliferative ability of lymphocytes. Long-duration (total 53 days) of low-dose rapamycin (75 µg/kg/day) treatment generated stronger vaccine-specific memory T cell responses than short-duration treatment (total 25 days). Moreover, rapamycin improved the vaccine's long-term protective efficacy, which resulted in a better reduction of 0.89-log10 CFU of mycobacterial organisms in the lungs compared with control without rapamycin treatment. These findings suggest that rapamycin may be considered in designing TB subunit vaccine regimens or as potential adjuvant to enhance vaccine-induced T cell memory response and to prolong the longevity of vaccine's protective efficacy.

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

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

Mucosal recombinant BCG vaccine induces lung-resident memory macrophages and enhances trained immunity via mTORC2/HK1-mediated metabolic rewiring.

Bacillus Calmette-Guérin (BCG) vaccination induces a type of immune memory known as "trained immunity", characterized by the immunometabolic and epigenetic changes in innate immune cells. However, the molecular mechanism underlying the strategies for inducing and/or boosting trained immunity in alveolar macrophages remains unknown. Here, we found that mucosal vaccination with the recombinant strain rBCGPPE27 significantly augmented the trained immune response in mice, facilitating a superior protective response against Mycobacterium tuberculosis and non-related bacterial reinfection in mice when compared to BCG. Mucosal immunization with rBCGPPE27 enhanced innate cytokine production by alveolar macrophages associated with promoted glycolytic metabolism, typical of trained immunity. Deficiency of the mammalian target of rapamycin complex 2 and hexokinase 1 abolished the immunometabolic and epigenetic rewiring in mouse alveolar macrophages after mucosal rBCGPPE27 vaccination. Most noteworthy, utilizing rBCGPPE27's higher-up trained effects: The single mucosal immunization with rBCGPPE27-adjuvanted coronavirus disease (CoV-2) vaccine raised the rapid development of virus-specific immunoglobulin G antibodies, boosted pseudovirus neutralizing antibodies, and augmented T helper type 1-biased cytokine release by vaccine-specific T cells, compared to BCG/CoV-2 vaccine. These findings revealed that mucosal recombinant BCG vaccine induces lung-resident memory macrophages and enhances trained immunity via reprogramming mTORC2- and HK-1-mediated aerobic glycolysis, providing new vaccine strategies for improving tuberculosis (TB) or coronavirus variant vaccinations, and targeting innate immunity via mucosal surfaces.

Sensory neurons promote immune homeostasis in the lung.

Cytokines employ downstream Janus kinases (JAKs) to promote chronic inflammatory diseases. JAK1-dependent type 2 cytokines drive allergic inflammation, and patients with JAK1 gain-of-function (GoF) variants develop atopic dermatitis (AD) and asthma. To explore tissue-specific functions, we inserted a human JAK1 GoF variant (JAK1GoF) into mice and observed the development of spontaneous AD-like skin disease but unexpected resistance to lung inflammation when JAK1GoF expression was restricted to the stroma. We identified a previously unrecognized role for JAK1 in vagal sensory neurons in suppressing airway inflammation. Additionally, expression of Calcb/CGRPß was dependent on JAK1 in the vagus nerve, and CGRPß suppressed group 2 innate lymphoid cell function and allergic airway inflammation. Our findings reveal evolutionarily conserved but distinct functions of JAK1 in sensory neurons across tissues. This biology raises the possibility that therapeutic JAK inhibitors may be further optimized for tissue-specific efficacy to enhance precision medicine in the future.

Mucosal boosting enhances vaccine protection against SARS-CoV-2 in macaques.

A limitation of current SARS-CoV-2 vaccines is that they provide minimal protection against infection with current Omicron subvariants1,2, although they still provide protection against severe disease. Enhanced mucosal immunity may be required to block infection and onward transmission. Intranasal administration of current vaccines has proven inconsistent3-7, suggesting that alternative immunization strategies may be required. Here we show that intratracheal boosting with a bivalent Ad26-based SARS-CoV-2 vaccine results in substantial induction of mucosal humoral and cellular immunity and near-complete protection against SARS-CoV-2 BQ.1.1 challenge. A total of 40 previously immunized rhesus macaques were boosted with a bivalent Ad26 vaccine by the intramuscular, intranasal and intratracheal routes, or with a bivalent mRNA vaccine by the intranasal route. Ad26 boosting by the intratracheal route led to a substantial expansion of mucosal neutralizing antibodies, IgG and IgA binding antibodies, and CD8+ and CD4+ T cell responses, which exceeded those induced by Ad26 boosting by the intramuscular and intranasal routes. Intratracheal Ad26 boosting also led to robust upregulation of cytokine, natural killer, and T and B cell pathways in the lungs. After challenge with a high dose of SARS-CoV-2 BQ.1.1, intratracheal Ad26 boosting provided near-complete protection, whereas the other boosting strategies proved less effective. Protective efficacy correlated best with mucosal humoral and cellular immune responses. These data demonstrate that these immunization strategies induce robust mucosal immunity, suggesting the feasibility of developing vaccines that block respiratory viral infections.

A multi-epitope/CXCL11 prime/pull coronavirus mucosal vaccine boosts the frequency and the function of lung-resident memory CD4+ and CD8+ T cells and enhanced protection against COVID-19-like symptoms and death caused by SARS-CoV-2 infection.

IMPORTANCE: Although the current rate of SARS-CoV-2 infections has decreased significantly, COVID-19 still ranks very high as a cause of death worldwide. As of October 2023, the weekly mortality rate is still at 600 deaths in the United States alone, which surpasses even the worst mortality rates recorded for influenza. Thus, the long-term outlook of COVID-19 is still a serious concern outlining the need for the next-generation vaccine. This study found that a prime/pull coronavirus vaccine strategy increased the frequency of functional SARS-CoV-2-specific CD4+ and CD8+ memory T cells in the lungs of SARS-CoV-2-infected triple transgenic HLA-DR*0101/HLA-A*0201/hACE2 mouse model, thereby resulting in low viral titer and reduced COVID-19-like symptoms.

Lung dendritic-cell metabolism underlies susceptibility to viral infection in diabetes.

People with diabetes feature a life-risking susceptibility to respiratory viral infection, including influenza and SARS-CoV-2 (ref. 1), whose mechanism remains unknown. In acquired and genetic mouse models of diabetes, induced with an acute pulmonary viral infection, we demonstrate that hyperglycaemia leads to impaired costimulatory molecule expression, antigen transport and T cell priming in distinct lung dendritic cell (DC) subsets, driving a defective antiviral adaptive immune response, delayed viral clearance and enhanced mortality. Mechanistically, hyperglycaemia induces an altered metabolic DC circuitry characterized by increased glucose-to-acetyl-CoA shunting and downstream histone acetylation, leading to global chromatin alterations. These, in turn, drive impaired expression of key DC effectors including central antigen presentation-related genes. Either glucose-lowering treatment or pharmacological modulation of histone acetylation rescues DC function and antiviral immunity. Collectively, we highlight a hyperglycaemia-driven metabolic-immune axis orchestrating DC dysfunction during pulmonary viral infection and identify metabolic checkpoints that may be therapeutically exploited in mitigating exacerbated disease in infected diabetics.

Endothelial AHR activity prevents lung barrier disruption in viral infection.

Disruption of the lung endothelial-epithelial cell barrier following respiratory virus infection causes cell and fluid accumulation in the air spaces and compromises vital gas exchange function1. Endothelial dysfunction can exacerbate tissue damage2,3, yet it is unclear whether the lung endothelium promotes host resistance against viral pathogens. Here we show that the environmental sensor aryl hydrocarbon receptor (AHR) is highly active in lung endothelial cells and protects against influenza-induced lung vascular leakage. Loss of AHR in endothelia exacerbates lung damage and promotes the infiltration of red blood cells and leukocytes into alveolar air spaces. Moreover, barrier protection is compromised and host susceptibility to secondary bacterial infections is increased when endothelial AHR is missing. AHR engages tissue-protective transcriptional networks in endothelia, including the vasoactive apelin-APJ peptide system4, to prevent a dysplastic and apoptotic response in airway epithelial cells. Finally, we show that protective AHR signalling in lung endothelial cells is dampened by the infection itself. Maintenance of protective AHR function requires a diet enriched in naturally occurring AHR ligands, which activate disease tolerance pathways in lung endothelia to prevent tissue damage. Our findings demonstrate the importance of endothelial function in lung barrier immunity. We identify a gut-lung axis that affects lung damage following encounters with viral pathogens, linking dietary composition and intake to host fitness and inter-individual variations in disease outcome.

Impact of statin treatment and exposure on the risk of chronic allograft dysfunction in Chinese lung transplant recipients.

PURPOSE: Chronic lung allograft dysfunction (CLAD) was a common complication following lung transplantation that contributed to long-term morbidity and mortality. Statin therapy had been suggested to attenuate recipient inflammation and immune response, potentially reducing the risk and severity of CLAD. This study aimed to evaluate the impact of statin use and in vivo exposure on the incidence of CLAD in lung transplant recipients (LTRs), as well as their effects on immune cells and inflammatory factors. METHODS: A retrospective cohort study was conducted on patients who underwent lung transplantation between January 2017 and December 2020. The incidence of CLAD, as per the 2019 ISHLT criteria, was assessed as the clinical outcome. The plasma concentrations of statin were measured using a validated UPLC-MS/MS method, while inflammation marker levels were determined using ELISA kits. RESULTS: The statin group exhibited a significantly lower rate of CLAD (P = 0.002). Patients receiving statin therapy showed lower CD4+ T-cell counts, total T-lymphocyte counts, and IL-6 levels (P = 0.017, P = 0.048, and P = 0.038, respectively). Among the CLAD groups, the atorvastatin level (2.51 ± 1.31 ng/ml) was significantly lower than that in the non-CLAD group (OR = 1.438, 95%CI (1.007-2.053), P = 0.046). CONCLUSION: Statin therapy significantly reduced the incidence of CLAD, as well as immune cell counts and inflammatory cytokine levels in LTRs. Although the statin exposure was significantly lower in CLAD patients, it was not associated with the incidence of CLAD.