The nature of chronic rejection after lung transplantation: a murine orthotopic lung transplant study.

Introduction: Chronic rejection is a major complication post-transplantation. Within lung transplantation, chronic rejection was considered as airway centred. Chronic Lung Allograft Dysfunction (CLAD), defined to cover all late chronic complications, makes it more difficult to understand chronic rejection from an immunological perspective. This study investigated the true nature, timing and location of chronic rejection as a whole, within mouse lung transplantation. Methods: 40 mice underwent an orthotopic left lung transplantation, were sacrificed at day 70 and evaluated by histology and in vivo µCT. For timing and location of rejection, extra grafts were sacrificed at day 7, 35, 56 and investigated by ex vivo µCT or single cell RNA (scRNA) profiling. Results: Chronic rejection originated as innate inflammation around small arteries evolving toward adaptive organization with subsequent end-arterial fibrosis and obliterans. Subsequently, venous and pleural infiltration appeared, followed by airway related bronchiolar folding and rarely bronchiolitis obliterans was observed. Ex vivo µCT and scRNA profiling validated the time, location and sequence of events with endothelial destruction and activation as primary onset. Conclusion: Against the current belief, chronic rejection in lung transplantation may start as an arterial response, followed by responses in venules, pleura, and, only in the late stage, bronchioles, as may be seen in some but not all patients with CLAD.

Caspase-1 and interleukin-18 in children with post infectious bronchiolitis obliterans: a case-control study.

The exact immunological mechanisms of post infectious bronchiolitis obliterans (PIBO) in childhood are not fully known. It has been shown that the inflammasome and IL-18 pathway play important roles in the pathogenesis of lung fibrosis. We aimed to investigate the role of caspase-1, IL-18, and IL-18 components in PIBO. From January to May 2020, children with PIBO, children with history of influenza infection without PIBO, and healthy children were asked to participate in the study in three pediatric pulmonology centers. Serum caspase-1, IL-18, IL-18BP, IL-18R, and INF-γ levels were measured by ELISA and compared between the 3 groups. There were 21 children in the PIBO group, 16 children in the influenza group, and 39 children in the healthy control group. No differences in terms of age and gender between the 3 groups were found. IL-18 and IL-18BP levels were higher in the healthy control group (p = 0.018, p = 0.005, respectively). IL-18R was higher in the PIBO group (p = 0.001) and caspase-1 was higher in the PIBO and influenza group than the healthy control group (p = 0.002). IFN-γ levels did not differ between the 3 groups. IL-18BP/IL-18 was higher in the influenza group than the PIBO group and the healthy control group (p = 0.003). CONCLUSIONS: Caspase-1 level was increased in patients with PIBO which suggests that inflammasome activation may have a role in fibrosis; however, IL-18 level was found to be low. Mediators other than IL-18 may be involved in the inflammatory pathway in PIBO. Further immunological studies investigating inflammasome pathway are needed for PIBO with chronic inflammation. WHAT IS KNOWN: • Post infectious bronchiolitis obliterans (PIBO) is a rare, severe chronic lung disease during childhood which is associated with inflammation and fibrosis which lead to partial or complete luminal obstruction especially in small airways. • The exact immunological mechanisms of PIBO in childhood are not fully known. WHAT IS NEW: • Inflammasome activation persists even years after acute infection and may play a role in fibrosis in PIBO. • Mediators other than IL-18 may be involved in these inflammatory pathway.

Restrictive allograft syndrome vs bronchiolitis obliterans syndrome: Immunological and molecular characterization of circulating exosomes.

BACKGROUND: Chronic lung allograft dysfunction in lung transplant recipients (LTxRs) has 2 phenotypes: obstructive bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS). Our goal was to define distinct immunologic markers of exosomes from LTxRs with BOS or RAS. METHODS: Plasma was collected from LTxRs with BOS (n = 18), RAS (n = 13), and from stable LTxRs (n = 5). Antibodies to lung self-antigens (SAgs) were determined by ELISA. Exosomes were isolated by ultracentrifugation. Donor specific antibodies to HLA were quantified using Luminex. Exosomes were characterized for lung SAgs, transcription factors, 20S proteasome, HLA class I and II, and polymeric immunoglobulin receptor protein using western blot. Exosome miRNA was analyzed using NanoString. The exosome-induced immune response was determined in mice. RESULTS: LTxRs with RAS, but not BOS, had donor specific antibodies at diagnosis. CIITA, NFkB, polymeric immunoglobulin receptor protein, 20S proteasome, HLA-DQ, and HLA-DR were significantly higher in RAS exosomes than in BOS exosomes. RAS plasma had high levels of proinflammatory cytokines and distinct exosomal miRNA. Immunization of C57BL/6 mice with RAS exosomes showed severe inflammation and peribronchial fibrosis, whereas BOS exosomes induced patchy inflammation and fibrosis. CONCLUSION: LTxRs with BOS or RAS had exosomes with distinct molecular and immunologic profiles. RAS samples had a higher concentration of proinflammatory factors, HLA class II, lung SAgs, and antibodies to HLA class II molecules, indicating severe allograft injury. Mice immunized with RAS exosomes developed lesions in airways, pleura, interlobular septum, and alveoli, whereas BOS exosomes induced mild to patchy inflammation with lung fibrosis.

Inhibition of Vascular Endothelial Growth Factor Receptors 1 and 2 Attenuates Natural Killer Cell and Innate Immune Responses in an Experimental Model for Obliterative Bronchiolitis.

Obliterative bronchiolitis (OB) after lung transplantation is a nonreversible, life-threatening complication. Herein, the role of vascular endothelial growth factor receptor (Vegfr)-1 and -2 was investigated in the development of obliterative airway disease (OAD), an experimental model for OB. The nonimmunosuppressed recipients underwent transplantation with fully major histocompatibility complex mismatched heterotopic tracheal allografts and received Vegfr1 and -2-specific monoclonal antibodies either alone or in combination, or rat IgG as a control. The treatment with Vegfr1- or -2-blocking antibody significantly decreased intragraft mRNA expression of natural killer cell activation markers early after transplantation. This was followed by reduced infiltration of Cd11b+ cells and Cd4+ T cells as well as down-regulated mRNA expression of proinflammatory chemokines and profibrotic growth factors. However, blocking of both Vegfr1 and -2 was necessary to reduce luminal occlusion. Furthermore, concomitant inhibition of the calcineurin activation pathway almost totally abolished the development of OAD. This study proposes that blocking of Vegf receptors blunted natural killer cell and innate immune responses early after transplantation and attenuated the development of OAD. The results of this study suggest that further studies on the role of Vegfr1 and -2 blocking in development of obliterative airway lesions might be rewarding.

Set Up for Failure: Pre-Existing Autoantibodies in Lung Transplant.

Lung transplant patients have the lowest long-term survival rates compared to other solid organ transplants. The complications after lung transplantation such as primary graft dysfunction (PGD) and ultimately chronic lung allograft dysfunction (CLAD) are the main reasons for this limited survival. In recent years, lung-specific autoantibodies that recognize non-HLA antigens have been hypothesized to contribute to graft injury and have been correlated with PGD, CLAD, and survival. Mounting evidence suggests that autoantibodies can develop during pulmonary disease progression before lung transplant, termed pre-existing autoantibodies, and may participate in allograft injury after transplantation. In this review, we summarize what is known about pulmonary disease autoantibodies, the relationship between pre-existing autoantibodies and lung transplantation, and potential mechanisms through which pre-existing autoantibodies contribute to graft injury and rejection.

Postdeployment Respiratory Syndrome in Soldiers With Chronic Exertional Dyspnea.

After deployment to Southwest Asia, some soldiers develop persistent respiratory symptoms, including exercise intolerance and exertional dyspnea. We identified 50 soldiers with a history of deployment to Southwest Asia who presented with unexplained dyspnea and underwent an unrevealing clinical evaluation followed by surgical lung biopsy. Lung tissue specimens from 17 age-matched, nonsmoking subjects were used as controls. Quantitative histomorphometry was performed for evaluation of inflammation and pathologic remodeling of small airways, pulmonary vasculature, alveolar tissue and visceral pleura. Compared with control subjects, lung biopsies from affected soldiers revealed a variety of pathologic changes involving their distal lungs, particularly related to bronchovascular bundles. Bronchioles from soldiers had increased thickness of the lamina propria, smooth muscle hypertrophy, and increased collagen content. In adjacent arteries, smooth muscle hypertrophy and adventitial thickening resulted in increased wall-to-lumen ratio in affected soldiers. Infiltration of CD4 and CD8 T lymphocytes was noted within airway walls, along with increased formation of lymphoid follicles. In alveolar parenchyma, collagen and elastin content were increased and capillary density was reduced in interalveolar septa from soldiers compared to control subjects. In addition, pleural involvement with inflammation and/or fibrosis was present in the majority (92%) of soldiers. Clinical follow-up of 29 soldiers (ranging from 1 to 15 y) showed persistence of exertional dyspnea in all individuals and a decline in total lung capacity. Susceptible soldiers develop a postdeployment respiratory syndrome that includes exertional dyspnea and complex pathologic changes affecting small airways, pulmonary vasculature, alveolar tissue, and visceral pleura.

Role of CMV chemokine receptor M33 in airway graft rejection in a mouse transplant model.

BACKGROUND: Cytomegalovirus (CMV) infection is a risk factor for bronchiolitis obliterans (BO), one form of chronic lung allograft dysfunction (CLAD). The viral chemokine receptor M33 is essential for successful spread of murine CMV to host salivary glands. In the present study we investigated the impact of M33 on chronic airway rejection. METHODS: MHC I-mismatched tracheas of C·B10-H2b/LilMcdJ mice were transplanted into BALB/c (H2d) recipients and infected at different dates with wild type (WT) or M33-deleted (delM33) MCMV representing clinical settings of viral recipient (R)-donor (D)-serostatus: (D-/R+) or (D+/R-). Grafts were recovered for gene expression and histological / immunofluorescence analysis, respectively. RESULTS: Evaluations showed significantly increased signs of chronic rejection in WT-infected mice compared to uninfected allografts seen in lower epithelium/lamina propria-ratio (ELR) (ELR 0.46 ± 0.07 [WT post] vs. ELR 0.66 ± 0.10 [non-inf.]; p < 0.05). The rejection in delM33-infected groups was significantly reduced vs. WT-infected groups (0.67 ± 0.04 [delM33 post]; vs. WT post p < 0.05). Furthermore, decreased rejection was observed in WT pre-infected compared to post-infected groups (0.56 ± 0.08 [WT pre]; vs. WT post p < 0.05). CD8+ T cell infiltration was significantly higher in WT-post compared to the delM33 infected or non-infected allografts. CONCLUSIONS: These data support the role of the CMV in accelerating CLAD. The deletion of chemokine receptor M33 leads to attenuated rejection.

Bronchiolitis obliterans syndrome is associated with increased senescent lymphocytes in the small airways.

BACKGROUND: Immunosuppression therapy is ineffective at preventing bronchiolitis obliterans syndrome (BOS), primarily a disease of the small airways (SAs). Our previous reports show increased senescent CD28null T and natural killer T (NKT)-like cells in the peripheral blood of patients with BOS and increased cytotoxic, proinflammatory lymphocytes in the SAs. We hypothesized that the cytotoxic, proinflammatory lymphocytes in the SAs would be steroid-resistant senescent CD28null lymphocytes. METHODS: Intracellular cytotoxic mediator granzyme B, interferon (IFN)-γ and tumor necrosis factor (TNF)-α proinflammatory cytokines, and CD28 were measured in the blood, bronchoalveolar lavage, large airway, and SA brushing T and NKT-like cells from 10 patients with BOS, 11 stable lung transplant recipients, and 10 healthy age-matched controls. SA brushings were cultured in the presence of ±1 µmol/liter prednisolone, ±5 mg/liter theophylline, and ±2.5 ng/ml cyclosporine A, and IFN-γ and TNF-α proinflammatory cytokines were assessed using flow cytometry. RESULTS: Increased SA CD28null T and NKT-like cells were identified in patients with BOS compared with that in the controls and stable transplant recipients. Loss of CD28 was associated with increased T and NKT-like cells expressing granzyme B, IFN-γ, and TNF-α. Loss of CD28 expression by CD8+ T cells was significantly associated with forced expiratory volume in 1 sec (R = 0.655, p = 0.006) and with time after transplantation (R = -0.552, p = 0.041). Treatment with prednisolone + theophylline + cyclosporin A inhibited IFN-γ and TNF-α production by SA CD28null CD8+ T and NKT-like cells additively. CONCLUSIONS: BOS is associated with the loss of CD28 in SA cytotoxic, proinflammatory senescent T and NKT-like lymphocytes. Treatment options that target the proinflammatory nature of these cells in the SAs may improve graft survival.

Decline in Club Cell Secretory Proteins, Exosomes Induction and Immune Responses to Lung Self-antigens, Kα1 Tubulin and Collagen V, Leading to Chronic Rejection After Human Lung Transplantation.

BACKGROUND: Chronic lung allograft dysfunction (CLAD), is a major hurdle for long-term lung allograft survival after lung transplant and roughly 50% of lung transplant recipients (LTxRs) develop CLAD within 5 years. The mechanisms of CLAD development remain unknown. Donor-specific immune responses to HLA and lung self-antigens (SAgs) are vital to the pathogenesis of CLAD. Reduction in Club cell secretory protein (CCSP) has been reported in bronchoalveolar lavage (BAL) fluid samples from LTxRs with bronchiolitis obliterans syndrome (BOS). CCSP levels in BAL fluid and development of antibodies to lung SAgs in plasma were determined by ELISA. Cytokines in BAL fluid were analyzed by 30-plex Luminex panel. Exosomes from BAL fluid or plasma were analyzed for SAgs, natural killer (NK) cells markers, and cytotoxic molecules. RESULTS: We demonstrate that LTxRs with BOS have lower CCSP levels up to 9 months before BOS diagnosis. LTxRs with antibodies to SAgs 1-year posttransplant also developed DSA (43%) and had lower CCSP. BOS with lower CCSP also induced Interleukin-8 and reduced vascular endothelial growth factor. Exosomes from BOS contained increased SAgs, NK cells markers, and cytotoxic molecules. CONCLUSIONS: We conclude lower CCSP leads to inflammation, pro-inflammatory cytokine production, immune responses to HLA and SAgs, and induction of exosomes. For the first time, we demonstrate that CCSP loss results in exosome release from NK cells capable of stimulating innate and adaptive immunity posttransplant. This increases the risk of BOS, suggesting a role of NK cell exosomes in CLAD development.

Self-reactive antibodies associated with bronchiolitis obliterans syndrome subtype of chronic lung allograft dysfunction.

BACKGROUND: Chronic Lung Allograft Dysfunction (CLAD) remains the major limitation in long term survival after lung transplantation. Our objective is to evaluate for the presence of autoantibodies to self-antigens, which is a pathway along with complex interplay with immune as well as non-immune mechanisms that leads to a fibroproliferative process resulting in CLAD. METHODS: Serum profiles of IgG autoantibodies were evaluated using customized proteomic microarray with 124 antigens. Output from microarray analyzed as antibody scores is correlated with bronchiolitis obliterans (BOS) subtype of CLAD using Mann-Whitney U test or Fisher exact test. Autoantibodies were evaluated for their predictive value for progressive BOS using a Cox proportional hazard model. BOS free survival and overall survival was analyzed using Kaplan-Meier survival analysis. RESULTS: Forty- two patients included in the study are grouped into "stable BOS" and "progressive BOS" for comparisons. Pulmonary fibrosis is the major indication for lung transplantation in our cohort. Progressive BOS group had significantly worse survival (p < 0.005). Sixteen IgG autoantibodies are significantly elevated at baseline in progressive BOS group. Six among them correlated with worse BOS free survival (p < 0.05). In addition, these six IgG autoantibodies remain elevated at three months and one year after lung transplantation. CONCLUSION: Pre-existing IgG autoantibodies correlate with progressive BOS and survival in a single center, small cohort of lung transplant recipients. Further validation with larger sample size, external cohort and confirmation with additional tissue, bronchoalveolar lavage samples are necessary to confirm the preliminary findings in our study.

The impact of HLA-DR mismatch status on retransplant-free survival and bronchiolitis obliterans syndrome‒free survival among sensitized lung transplant recipients.

INTRODUCTION: Donor‒recipient HLA-DR locus matching may be protective against bronchiolitis obliterans syndrome (BOS) in lung transplant recipients. It is unknown whether this benefit is more significant among sensitized (calculated panel reactive antibodies (CPRAs) of >0%) and highly sensitized (CPRAs of ≥80%) recipients who may be at a higher risk for BOS. METHODS: This was a retrospective cohort study of adults in the Scientific Registry of Transplant Recipients who underwent lung transplantation between May 5, 2005 and May 31, 2019. Retransplant-free survival and BOS-free survival were compared among recipients with 0 vs ≥1 DR mismatches, grouped according to sensitization. RESULTS: Among all 20,355 included recipients, 0 DR mismatch status was associated with improved retransplant-free survival (hazard ratio [HR] = 0.83, 95% CI = 0.74-0.93, p = 0.002) and BOS-free survival (HR = 0.86, 95% CI = 0.77-0.96, p = 0.007). Among sensitized recipients, 0 DR mismatch status was also associated with improved retransplant-free survival (HR = 0.79, 95% CI = 0.65-0.97, p = 0.02) and BOS-free survival (HR = 0.82, 95% CI = 0.67-1.00, p = 0.04). There was however no difference in retransplant-free or BOS-free survival between sensitized and non-sensitized recipients with 0 DR mismatches. Among highly sensitized recipients, 0 DR mismatch status was not associated with retransplant-free or BOS-free survival. Among sensitized and highly sensitized recipients, 0 DR mismatch status was not associated with reduced use of plasmapheresis or reduced biopsy-proven, treated acute cellular rejection compared with non-sensitized recipients. CONCLUSIONS: HLA-DR matching is associated with a similar improvement in retransplant-free and BOS-free survival among non-sensitized and sensitized lung transplant recipients. DR matching does not confer a more substantial retransplant-free or BOS-free survival benefit to highly sensitized recipients than to non-sensitized recipients.

Serum Amyloid A in lung transplantation.

BACKGROUND: Serum Amyloid A (SAA) is an acute phase protein and we analyzed its concentrations in lung transplantated patients (LTX). METHODS: 26 LTX patients (58.6 ± 11 years) and 11 healthy controls (55 ± 11.3 years). Three groups of LTX patients: acute rejection (AR, 7) bronchiolitis obliterans syndrome (BOS, 3), acute infection (INF, 9) and stable patients (NEG, 7). RESULTS: In LTX patients SAA concentrations were significantly increased, particularly in AR and INF. In LTX-AR patients were observed a correlation between SAA levels and peripheral CD4+ lymphocyte percentage (r=0.9, p<0.01) and a reverse correlation with FVC percentages (r -0.94, p=0.01). CONCLUSIONS: SAA may represent a potential biomarker of LTX acute complications, with a prognostic value in AR. (Sarcoidosis Vasc Diffuse Lung Dis 2020; 37 (1): 2-7).

The role of miRNA-155 in the immunopathogenesis of obliterative airway disease in mice induced by circulating exosomes from human lung transplant recipients with chronic lung allograft dysfunction.

Human lung transplant recipients undergoing rejection induce circulatory exosomes with lung self-antigens (SAgs), K-alpha 1 Tubulin and Collagen V, and immunization of C57BL/6 mice with exosomes induced obliterative airway disease (HEI-OAD). We analyzed whether exosomes with SAgs induced immunity in microRNA-155 knockout mice (miR-155KO), as microRNA-155 is an immune regulator. C57BL/6 and miR-155KO were immunized with exosomes from stable or chronic rejection (bronchiolitis obliterans syndrome (BOS) and on day 30, induction of exosomes, antibodies (Abs) to SAgs and cellular immunity were determined. C57BL/6 immunized with exosomes from BOS developed OAD. These immunized animals also developed Abs to SAgs and increased frequency of SAg-specific IFNγ and IL17- producing cells. In contrast, Abs to SAgs did not develop in miR-155KO and there was reduction in frequency of cells producing IL10. Upregulation of suppressor of cytokine signaling for lung inflammation was also noted resulting in abrogation of induction of exosomes with SAgs OAD.

Bronchoalveolar lavage T cell cytokine profiles and their association with lung function in children with Mycoplasma pneumoniae -associated bronchiolitis obliterans.

BACKGROUND: Mycoplasma pneumoniae (M. pneumoniae) infection may progress to bronchiolitis obliterans (BO), with an underlying chronic inflammatory process. The aim of this study was to investigate the cytokine profiles in pulmonary T-lymphocytes and their associations with lung function in patients with BO following M. pneumoniae infection. METHODS: Bronchoalveolar lavage (BAL) samples were obtained from 10 controls and 18 children with M. pneumoniae-associated BO. We analyzed the BAL T cells for interferon (IFN)-γ, interleukin (IL)-4, IL-9, IL-17, CD25, and Foxp3 by intercellular flow cytometry. The associations with T-cell subpopulations and lung function parameters were determined. RESULTS: In BAL fluid, significantly increased proportions of T-helper 1 (Th1), Th17, and Tc1 cells were found in M. pneumoniae-associated BO patients when compared with controls. The percentages of Th17 cells showed correlations with forced expiratory volume in 1 second % predicted value (r = -0585; P < .05) and forced expiratory flow at 25% to 75% (FEF25%-75% ) % predicted value (r = -.618; P < .01). Higher proportions of Tc1 (r = -.488; P < .05) and Tc17 (r = -.542; P < .05) were significantly correlated with a reduced FEF25%-75% % predicted value in M. pneumoniae-associated BO patients. CONCLUSIONS: Our comprehensive cytokines analysis of BAL T cells revealed correlations of IL-17-producing and IFN-γ-producing T cells with lung function, suggesting that increased T-cell subpopulations may play a role in M. pneumoniae-associated BO progression.

Lung Transplant Patients Show a Dissimilar Peripheral B-Cell Subset Ratio Compared With Healthy Controls.

OBJECTIVES: Lung transplant is a last treatment option for patients with end-stage pulmonary disease. Chronic lung allograft dysfunction, which generally manifests as bronchiolitis obliterans syndrome, is a major long-term survival limitation. Bronchiolitis obliterans syndrome is diagnosed when forced expiratory volume in 1 second declines > 20% in the absence of known causes. B cells can either contribute or restrain the development of bronchiolitis obliterans syndrome (eg, via induction of alloimmune antibodies, regulation of cellular immunity, and induction of tolerance). Here, we explored how peripheral B-cell subsets were altered in lung transplant recipients with bronchiolitis obliterans syndrome. MATERIALS AND METHODS: Fresh whole blood samples were analyzed from 42 lung transplant recipients, including 17 with bronchiolitis obliterans syndrome; samples from these groups were compared with 10 age-matched healthy control samples. B-cell subsets were analyzed using flow cytometry, and relative distributions of subsets were compared. Changes in forced expiratory volume in 1 second were also determined. RESULTS: Absolute B-cell count was significantly increased in transplant recipients with bronchiolitis obliterans syndrome. Transitional (CD24+CD38+) and naïve (CD27-IgD+) B cells were decreased in lung transplant patients, with transitional B cells almost absent in those with bronchiolitis obliterans syndrome. Double-negative (CD27-IgD-) memory B cells were significantly increased (P < .001). No differences were found for plasmablasts (CD38+CD24-) and switched (CD27+IgD-) and non-switched (CD27+IgD+) memory B cells. Correlation analyses showed positive correlations between lung function and naïve B cells in transplant recipients (P = .0245; r = -0.458). CONCLUSIONS: Peripheral B-cell count and subset distribution were altered in lung transplant recipients with and without bronchiolitis obliterans syndrome compared with healthy controls. Transitional and naïve B-cell decreases may be caused by differentiation toward double-negative B-cells, which were increased. The correlation between forced expiratory volume and naïve B cells during follow-up care may be clinically interesting to investigate.

Lung Transplantation Has a Strong Impact on the Distribution and Phenotype of Monocyte Subsets.

BACKGROUND: Lung transplantation (LTx) is a last treatment option for patients with an end-stage pulmonary disease. Chronic lung allograft dysfunction, which generally manifests as bronchiolitis obliterans syndrome (BOS), is a major long-term survival limitation. During injury, inflammation and BOS monocytes are recruited. We determined whether changes in count, subset distribution, and functionality by surface marker expression coincided with BOS development. METHODS: Fresh whole-blood samples were analyzed from 44 LTx patients, including 17 patients diagnosed with BOS, and compared with 10 age-matched healthy controls and 9 sarcoidosis patients as positive controls. Monocytes were quantified and analyzed using flow cytometry. Based on surface marker expression, classical, intermediate, and nonclassical subsets were determined, and functional phenotypes were investigated. RESULTS: The absolute count of monocytes was decreased in LTx and slightly increased in BOS patients. The relative count shifted toward classical monocytes at the expense of nonclassical monocytes in LTx and BOS. Surface marker expression was highest on intermediate monocytes. The expression of both CD36 and CD163 was significantly increased in the LTx and BOS cohort. The difference between the BOS cohort and the LTx cohort was only subtle, with a significant decrease in HLA-DR expression on nonclassical monocytes in BOS. CONCLUSIONS: Monocyte subsets and surface marker expression changed significantly in transplantation patients, while BOS-specific changes were understated. More research is needed to determine whether and how monocytes influence the disease process and how current immunosuppressants affect their normal function in vivo.

Nlrp3 Inflammasome Inhibitor MCC950 Ameliorates Obliterative Bronchiolitis by Inhibiting Th1/Th17 Response and Promoting Treg Response After Orthotopic Tracheal Transplantation in Mice.

BACKGROUND: Obliterative bronchiolitis (OB) remains the major complication limiting long-term survival of patients after lung transplantation. We aimed to explore the effects of the selective NACHT, LRR, and PYD domains-containing protein 3 (Nlrp3) inflammasome inhibitor MCC950 on the pathogenesis of OB. METHODS: Mouse orthotopic tracheal transplants were performed to mimic OB. MCC950 (50 mg/kg) or saline was intraperitoneally injected daily. The luminal occlusion rate and collagen deposition were evaluated by hematoxylin and eosin and Masson's trichrome staining, respectively. Infiltration of CD4+, CD8+ T cells, and neutrophils was detected with immunohistochemical staining. The frequencies of T helper 1 cell (Th1), T helper 17 cell (Th17), and regulatory T cells (Treg) were measured by flow cytometry. Cytokine levels were measured by ELISA kits. RESULTS: MCC950 treatment significantly inhibited Nlrp3 inflammasome activation after allogeneic tracheal transplant and markedly decreased the luminal occlusion rate and collagen deposition in the allograft. The numbers of infiltrating CD4+, CD8+ T cells, and neutrophils in the allograft were also significantly reduced by MCC950 treatment. MCC950 dramatically decreased the frequencies of Th1/Th17 cells and the levels of interferon gamma/interleukin (IL)-17A and increased the Treg cell frequencies and IL-10 level; however, these effects were abolished by the addition of IL-1ß and IL-18 both in vitro and in vivo. OB was also rescued by the addition of IL-1ß and/or IL-18. CONCLUSIONS: Blocking Nlrp3 inflammasome activation with MCC950 ameliorates OB lesions. The mechanistic analysis showed that MCC950 regulated the balance of Th1/Th17 and Treg cells and that this process is partially mediated by inhibition of IL-1ß and IL-18. Therefore, targeting the Nlrp3 inflammasome is a promising strategy for controlling OB after lung transplantation.

LPS-induced Airway-centered Inflammation Leading to BOS-like Airway Remodeling Distinct From RAS-like Fibrosis in Rat Lung Transplantation.

BACKGROUND: Localization of inflammatory stimuli may direct lung allografts to different phenotypes of chronic dysfunction, such as bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS). We hypothesized that airway stimulation with lipopolysaccharide (LPS) in rats leads to airway-centered inflammation similar to human BOS. METHODS: Rat left lung transplantation was conducted (donor: Brown Norway, recipient: Lewis). Allotransplant recipients received cyclosporine A (CsA) until postoperative day 56 with airway instillation of LPS (Allo-LPS, n = 8), phosphate buffered saline (Allo-PBS, n = 5) from days 35 to 46 (3 times a wk), or no further treatment (n = 4). Some allotransplant recipients received CsA until day 14 and were immunosuppression free after day 15 until day 56. Bronchial and pleural fibrosis were semiquantified; alveolar fibrosis was evaluated with a histological scale. RESULTS: The Allo-LPS group had significantly increased International Society for Heart and Lung Transplantation rejection grades (grade A, P = 0.005; grade B, P = 0.004), bronchial obstructive proportion (0.34 ± 0.04% [Allo-LPS] versus 0.11 ± 0.04% [Allo-PBS], P = 0.006), and airway resistance (3.05 ± 1.78 cm H2O·s/mL [Allo-LPS] versus 0.83 ± 0.58 cm H2O·s/mL [Allo-PBS], P = 0.007) compared with other groups. Allotransplant recipients that underwent a short course of CsA developed RAS-like fibrosis involving the airways, alveoli, and pleura. CONCLUSIONS: Airway instillation of LPS in allografts under immunosuppression resulted in BOS-like airway-centered inflammation and fibrosis distinct from RAS-like diffuse fibrosis, which was induced by a shortened course of immunosuppression. We propose novel animal models for BOS and RAS after lung transplantation.

Lung T-Cell Profile Alterations are Associated with Bronchiolitis Obliterans Syndrome in Cystic Fibrosis Lung Transplant Recipients.

The contribution of T-cells after lung transplant (LTx) remains controversial with no current consensus of their role concerning chronic lung allograft dysfunction. Using flow cytometry to assess T-cell subsets of bronchoalveolar lavage fluid (BALF) in 16 cystic fibrosis (CF) LTx recipients, we identified a decline in CD4+ T-cell frequency and an increase in CD8+ T-cell frequency in patients who developed severe bronchiolitis obliterans syndrome (BOS) (N = 10) when comparing baseline (6 months post-LTx) and follow-up (most recent bronchoscopy-clinical or surveillance per protocol). Comparing BOS to No BOS cohorts, significant differences were found in CD4+ T-cell frequency [17.4 (12.5, 28.2) vs 46.6 (44.4, 48.4), p = 0.003] and CD8+ T-cell frequency [65.6 (62.8, 75.3) vs 39.2 (32.2, 43.3), p = 0.014], respectively. The mean difference of the CD4:CD8 ratio was 0.87 units lower (95% CI - 1.44 to - 0.30, p = 0.006) than patients without BOS, while the median difference of the CD4:CD8 ratio was 0.92 units lower (95% CI - 1.83 to - 0.009, p = 0.048). Therefore, our results suggest that T-cell profiles measured through flow cytometry of BALF in the CF LTx population are associated with the development of severe BOS. Further work is needed in larger patient populations to validate our findings and to determine if this is useful for recipients who underwent LTx for other indications.