Search

Purpose Airway inflammation after lung transplantation (LTX), resulting from infection, pollution, or aspiration, is an important risk factor for chronic lung allograft dysfunction. Classical dendritic cells (cDCs) link innate immunity and adaptive immunity, and exhibit regional and functional specialization in the lung. CD103+ cDCs reside in the airway epithelium. Their role in lung transplantation (LTX) has not been extensively investigated. We hypothesized that CD103+ cDCs augment adaptive immune responses under conditions of lipopolysaccharide (LPS)-induced airway inflammation inamouse minor alloantigen-mismatched orthotopic LTX model (C57BL/10 [B10] →C57BL/6 [B6] and vice versa). Methods BATF3-/- mice (B6 background), lacking CD103+ cDCs, were used. Three comparisons were made: 1) B10 → 6 vs. B10 → ATF3-/-, 2) B10 →B6 vs. B10 → ATF3-/- with 6 doses of intra-tracheal (i.t.) LPS (5μg in 50μlPBS)on serial postoperative days (POD), 3)B6 → 10 and BATF3-/- → 10 with 8 doses of i.t. LPS on serial PODs. The grafts were assessed on POD 28. Results Acute rejection (ISHLT criteria) did not differ significantly between B10 → 6 and B10 → ATF3-/- without LPS, or between B6 → 10 and BATF3-/- → 10 with LPS. In contrast, with LPS exposure, acute rejection scores were lower in B10àBATF3-/- compared to B10 àB6 (Figure A). Surprisingly, lung cell analysis by flow cytometry showed that monocytes and neutrophils were significantly higher in BATF3-/- recipients than wild-type recipients under repeated airway LPS exposure (Figure B). Conclusion Our data show that recipient, but not donor, CD103+ cDCs contribute to the augmentation of acute rejection after LTx in the setting of ongoing airway inflammation. As CD103+ cDCs can promote immune regulation, they may be an important therapeutic target after LTX. [ABSTRACT FROM AUTHOR]

Purpose Chronic lung allograft dysfunction (CLAD) is the primary obstacle to long-term survival after lung transplantation (LTX). Ischemia-reperfusion injury (IRI) increases the risk of CLAD in humans but the underlying mechanisms are not well understood. In a mouse minor alloantigen mismatched LTX model (C57BL/10 [B10] →C57BL/6 [B6]), we observed that IRI augments formation of B cell-rich tertiary lymphoid organs (TLOs) and chronic rejection in allografts but not isografts. We therefore hypothesized that B cells may link IRI with chronic rejection. Methods B10→B6 LTX with extended graft preservation (6 hours at 4°C followed by 45 minutes at 37°Cand 15 minutes at room temperature during anastomosis), which augments acute rejection, fibrosis, and TLO formation, was employed. Anti-CD20 antibody (aCD20: n=9) or isotype control (Iso: n=10) were given i.p. on days -2, +2, +7, +14, and +21. Peripheral blood was longitudinally assessed by flow cytometry after LTX. Grafts were assessed with histology, flow cytometry and immunofluorescence 28 days after LTX. Results aCD20 treatment effectively depleted CD19+ or B220+ B cells in peripheral blood (Figure A) and lung grafts (Figure B). Acute rejection (ISHLT A grade), peribronchial fibrosis, and airways with obliterative bronchiolitis (%OB) were lower in aCD20 mice (1.8, 1.4, and 13.1% respectively) than in Iso mice (3.1, 2.6, and 46.7% respectively) (Figure C). TLOs containing CD3+, CD19+, and peripheral node addressin (PNAd)+cells were also reduced (Figure D). Conclusion In this model of IRI-augmented chronic rejection, B cell depletion attenuated acute rejection, airway fibrosis, and lymphoid neogenesis. Our observations suggest that B cell activation and recruitment may be directly involved in the augmentation of CLAD in LTX recipients experiencing IRI. [ABSTRACT FROM AUTHOR]

Purpose Acute cellular rejection (ACR) remains the most significant risk factor for chronic lung allograft dysfunction (CLAD). While clinically-unstable and higher-grade ≥A2 ACR is generally treated with augmented immunosuppression (aIS), management of clinically-stable A1 ACR (CSA1R) remains controversial. At our center, patients with CSA1R are routinely not treated with aIS and constitute a unique population that enabled us to identify sub-phenotypes with differential risks. Our aim was to determine if bronchoalveolar lavage (BAL) biomarkers at the time of first CSA1R (fCSA1R) are predictive of subsequent CLAD or death. Methods Among all adult, first, bilateral lung transplants, performed 2010-2016, post-transplant transbronchial biopsies obtained within the first year, without concurrent infection, were categorized as clinically unstable or stable based on ≥10% concurrent drop in FEV 1. fCSA1R episodes were defined as stable biopsy-FEV 1 pairs not preceded by another ACR. 21 proteins previously associated with ACR or CLAD were measured in the BAL using a multiplex bead assay. Association between protein levels and subsequent CLAD or death was assessed using Cox PH models, adjusted for relevant peri-transplant clinical covariates. Results We identified 75 patients with fCSA1R at a median time of 98 days (range 48.5-197) post-transplant. Median time from transplant to CLAD or death was 1247 (756.5-1921.5) and 1641 days (1024.5-2326.5), respectively. In multivariable models, levels of IL6, S100A8, IL10, TNF-receptor 1, IL-1α and pentraxin 3 (PTX3) were associated with both CLAD development and death (P<0.05 for all). PTX3 remained significant for both CLAD and death after adjusting for multiple comparisons. Conclusion Our data indicate that a focused BAL protein signature, with PTX3 having the strongest association, may be useful in determining a subset of CSA1R patients who are at increased risk and may benefit from a more aggressive management strategy. [ABSTRACT FROM AUTHOR]

Purpose Regulatory T cells (Tregs) are a potential therapeutic option in lung transplantation (LTx). We have shown that expanded recipient Tregs can be seeded pre-transplant in the donor lung graft using normothermic ex vivo lung perfusion (EVLP). Here, we examined whether this therapy can alleviate lung injury early post-transplant. Methods Study design is shown in Fig A. CD4+CD25hi Tregs were isolated from Wistar Kyoto rats (WKy, RT1l) and expanded for 7d-14d. Fischer 344 (F344 RT1lv1) heart-lung blocks were flushed and placed on EVLP. Lungs were perfused with SteenTM solution (37°C, 4h). Expanded Tregs (5-120 × 106) were infused after 1h. Control lungs received no Tregs. The right lung was used for assessment at end-EVLP, and left LTx into WKy recipients was performed after 60min cold preservation. Necropsy was performed on day 3 post-LTx. Results Wet-dry ratio was lower in Treg-treated grafts compared to controls (Fig B). Transcripts for FoxP3, IL-10, CTLA-4 and GITR were elevated in Treg-treated grafts (Fig B). Tregs isolated from the right donor lung at end-EVLP suppressed CD4+CD25− T cell proliferation in vitro (Fig C). On day 3 post-LTx, ICAM1 and CD44 (adhesion molecules) expression on both CD4+ and CD8+ T cells were lower in Treg-treated grafts compared to untreated controls (Fig D). Histologically, both Treg-treated and control grafts had minor lung injury and cellular rejection on day 3 post-LTx: acute lung injury scores (4.10 ± 1.66 and 3.88 ± 1.36), ISHLT A grades (0.60 ± 0.516 and 0.625 ± 0.744), B grades (0.400 ± 0.516 and 0.313 ± 0.458). Conclusion Expanded recipient Tregs directly delivered to the lung allograft prior to transplantation exert immune regulatory effects in vivo and in vitro. Further work is needed to optimize this pre-transplant therapeutic approach. [ABSTRACT FROM AUTHOR]

The pulmonary microbiome modulates the immune milieu in the lung allograft and is in turn influenced by external factors such as microaspiration of oropharyngeal contents. A potential driver of microaspiration is gastroesophageal reflux disease (GERD), which has been associated with poor outcomes after lung transplantation. We aimed to compare the pulmonary microbiome in lung transplant recipients with and without GERD, and correlate microbial composition with concurrent lung inflammation. We selected 22 GERD patients and 49 no-GERD controls from adult lung transplant recipients with 24-hour pH-impedance testing and bronchoalveolar lavage fluid (BAL) collection at 3 months post-transplant. BAL microbial communities were characterized by 16S rRNA gene sequencing. Ten inflammatory protein markers in BAL were measured by multiplex assay. Baseline patient characteristics including age, sex, primary disease, and transplant type were comparable between GERD and no-GERD groups. Shannon diversity index was lower (MD=-0.33, p=0.04), while Berger-Parker dominance index was higher (MD=0.10, p=0.03), in GERD patients compared to no-GERD controls. Hierarchical clustering of all samples by Bray-Curtis dissimilarity identified three distinct pneumotypes (Figure 1). GERD was significantly associated with one of these pneumotypes, PT1, which was enriched in multiple anaerobic oropharyngeal taxa including Prevotella and Veillonella. In GERD patients, the presence of PT1 compared to all others was associated with higher levels of IL-8 (p=0.03). At time of GERD diagnosis, lung transplant recipients are more likely to have a pulmonary microbiome lower in diversity and enriched in oropharyngeal taxa. These perturbations are consistent with microaspiration and may contribute to allograft inflammation. Future studies are warranted to assess how the pulmonary microbiome changes over time in the context of GERD and its correlation with rejection and long-term outcomes. [ABSTRACT FROM AUTHOR]

Microaspiration induces lung inflammation which contributes to chronic lung allograft dysfunction (CLAD) and death in lung transplant (LTx) recipients. Early detection and mitigation of microaspiration may therefore improve outcomes. Bronchial wash (BW) has been underused compared to bronchoalveolar lavage (BAL) at most LTx centers. As BW likely better reflects the proximal airways, we hypothesized that the BW is a useful sample to analyze biomarkers of microaspiration and inflammation which may predict CLAD and death. Large airway BW and BAL were collected concurrently during recipient routine bronchoscopies at 3 months post-LTx (n=61, 28% of whom had gastroesophageal reflux). Mass spectrometry was performed to measure 3 bile acids, taurocholic acid (TCA), glycocholic acid (GCA), and cholic acid, as putative biomarkers of microaspiration. Ten inflammatory proteins were measured by multiplex assay. Spearman's correlation was used to assess relationships between biomarkers. Cox proportional hazards models were used for survival analysis, adjusted for age, sex, primary disease, days from LTx to BW, and acute rejection. BW bile acid levels were higher than their BAL equivalents, and correlated with inflammatory markers in both samples (Fig A). BW bile acids showed no association with CLAD, though high IL-1α and IL-6 were predictors of early CLAD (Fig B). Notably, high BW TCA and GCA, as well as IL-1β, IL-6, IL-8, IL-12, CCL2, and CCL5 were all associated with early death (Fig C). In BAL, high IL-6 predicted early CLAD, but no other biomarker was associated with CLAD or death. Bile acid and inflammatory marker levels in BW at 3 months post-LTx are significant prognostic factors for CLAD or death in LTx recipients and may be more useful predictive biomarkers than BAL levels in the context of microaspiration. Collection and analysis of BW for these biomarkers may allow for better evaluation of microaspiration and its inflammatory sequalae. [ABSTRACT FROM AUTHOR]