Chronic lung allograft dysfunction: clinical and experimental study

INTRODUCTION Transplantation is the only effective treatment for several end-stage lung diseases. Remarkable progress has been made in improving outcomes, although the 5-year graft survival is still less than 50% primarily because of the development of chronic lung allograft dysfunction (CLAD). CLAD has been now recognized as a heterogeneous condition that includes an obstructive form (bronchiolitis obliterans syndrome, BOS) and a restrictive allograft dysfunction (restrictive allograft syndrome, RAS). BOS, and its histological correlate obliterative bronchiolitis -OB- represents the principal form of CLAD (~75%). The specific etiology and pathogenesis of BOS/OB are not fully understood. Multiple immune mechanisms seem to contribute to the development of BOS/OB, thus it is thought to represent a final common pathway of a process triggered by both alloantigen dependent and independent mechanisms. While the role of alloimmunity has long been established more recent studies have begun to demonstrate the role of autoimmunity in the development of BOS. A few experimental and clinical studies have demonstrated that collagen V and K-α1 tubulin, modified during ischemia reperfusion injury, may trigger autoimmune response, both humoral and cell mediated. Interleukin 17 (IL17), a proinflammatory cytokine involved in autoimmune and infectious diseases, has recently been suggested to play a key role in the development of CLAD. The development of animal models, mimicking the human transplantation procedure, is of great importance to elucidate the pathogenetic mechanisms leading to BOS/OB, to identify important biomarkers of OB and finally to test the effectiveness of new target therapies. However up to today two important issues are largely discussed in rodent orthotopic models: 1) the reproducibility of the surgical procedure 2) the identification of the best genetic strain (inbred versus outbred rats) for the development of immunological lesions similar to those in humans. AIM OF THE RESEARCH The main goals of the present PhD research project were: 1) development of a reproducible orthotopic lung transplant animal model, obtaining immunological lesions, particularly CLAD, similar to those of humans; 2) evaluation of IL17/IL23 pathway, crucial in autoimmune response, through a careful investigation in preclinical models and in clinical index cases of CLAD. MATERIALS AND METHODS Two different animal models were used to perform orthotopic lung transplantation (OLT): outbred rat strain (20 CD SPF left lungs were transplanted into VAF) and inbred rat strain (32 Lewis left lung rats were transplanted into Fisher 344). Only the long term survival animals (sacrificed 30 and 90 days after LT) were subjected to a full immunological evaluation as follows: a) detection of donor-specific antibodies (DSA) testing serum samples with the flow cross match technique b) morphological and immunophenotype evaluation of acute and chronic immunological lesions developed in the graft c) immunohistochemical and molecular (RT-qPCR) analysis of IL17/IL23 pathway in the graft and bronchoalveolar lavage (BAL) of animals and in all scheduled transbronchial biopsies of two index cases that developed CLAD. RESULTS Surgical mortality and early graft failure (within 24 hours) was higher in the outbred than inbred group (only 2 of a total 20 outbred rats survived). The two survival OLT outbred rats (sacrified 13 and 14 days after OLT) developed well evident immunological disorders: one showed acute cellular rejection (ACR) with coexistent early OB and the other late OB. Immunological disorders (only minimal ACR: A1B1) were rare (only 1/11; 9%) in the first 15 days of OLT inbred rats. In this period the inbred grafts showed ischemia/reperfusion or infections. ACR (≥A2B1) developed in 2/6 (33%) inbred grafts at 30 days. Ninety days after OLT was the best time point for the development of immunological disorders: ACR (≥A2B1) and OB (both early and late) were detected in 7/15 (46%) and 8/15 (53%) animals respectively, regardless of immunosuppressive treatment. DSA IgG showed higher median levels in those with ACR or OB than those without (70% and 34%, respectively vs 13%). A strong IL17 immunostaining was detected in inbred grafts that developed ACR and OB. IL 17 was equally expressed in inflammatory cells (macrophages and lymphocytes) of inbred grafts with ACR and OB while it was more expressed in epithelial and endothelial cells of inbred grafts with OB. No staining was detected in grafts of animals without any sign of rejection. IL23 expression was high in grafts with both absence and presence of rejection. Molecular analysis of IL17 and IL23 expression in BAL fluids showed higher levels of mRNA in grafts with ACR than OB. All scheduled transbronchial biopsies of the two index cases with ACR and OB showed IL17 overexpression with the same pattern detected in the preclinical model. CONCLUSIONS Outbred rodents that could have been more similar to humans due to high genetic diversity can not be used as a reliable OLT model because of the high rate of dramatic early graft failure. A reproducible model of both ACR and OB was developed in inbred rats (Lewis to Fisher 344) and 90 days post-transplantation was the optimal endpoint established. IL17, overexpressed in ACR and overall in OB lesions, is a crucial mediator in post-transplant immunological lesions and could be considered a potential therapeutic target in clinical transplantation