Interferon-Gamma Signaling in the Pathogenesis of Idiopathic Pneumonia Syndrome Following Allogeneic Bone Marrow Transplantation
Authors: Ma Hui-Hui , Sung Biin , Lu Caisheng , Song Liangsong , Gomi Rika , Wu Wenzhu , Fu Jing , Lentzsch Suzanne , Worgall Stefan , Mapara Markus 
Published: October 2019

Abstract
Idiopathic pneumonia syndrome (IPS) is a life-threatening acute noninfectious diffuse lung injury occurring after allogeneic bone marrow transplantation (BMT). We have recently shown that absence of STAT1 signaling in recipient APC leads to accelerated Graft Versus Host Disease (GVHD) and Transplant-associated mortality and morbidity after lethal irradiation in fully MHC-mismatched allogeneic or in non-irradiated P→F1 BMT models. We now addressed the impact of recipient Type II interferon signaling on the development of lung injury after allogeneic BMT using a non-irradiated P→F1 model. 1x107 BMCs plus 2.5x107 SPCs from C57BL/6 (B6) mice were i.v. injected into IFNγR-deficient CB6F1 (F1.GRKO) or CB6F1 wild type (WT) mice. F1.GRKO recipients had significantly reduced survival (MST 32 days post-BMT vs. not reached p<0.001, Log-rank test). Pulmonary function tests were performed 3 weeks post-BMT on anaesthetized mice that were tracheotomized and mechanically ventilated using a computer-controlled animal ventilator (Flexivent, SCIREQ). Static compliance was derived from plateau pressure measurements between total lung capacity and functional residual capacity, and broadband forced oscillations were used to determine airway resistance at baseline and following increasing doses of methacholine (3.1, 12.5 and 50 mg/ml). Static compliance and inspiratory capacity were reduced in F1.GRKO recipients when compared with WT mice (p<0.01). In addition, airway resistance, both at baseline and in response to methacholine, was increased in the KO recipient mice (p<0.05). Histopathology was evaluated on H&E, Trichrome and PAS stained lung sections to assess for inflammation, fibrosis and mucus hyperplasia, respectively. Lungs from the KO mice showed increased interstitial inflammation involving bronchioles, vasculature and parenchyma (pathology index 10 vs 1; p<0.001), fibrosis (fibrosis score 2.8 vs 0.15; p<0.05) and a trend for mucus hyperplasia/metaplasia. To further evaluate the cellular mediators of the observed lung injury, we assessed the cellular composition of the bronchioalveolar lavage fluid (BAL) at day 30 post-BMT. We observed a 10-fold increase in the total cell number of the BAL in F1.GRKO recipient mice compared to WT mice (4x105 vs.4x104, p<0.01); the majority of the cells in the BAL from wild type mice were macrophages/monocytes, while neutrophils predominated in the F1.GRKO mice. Importantly, more than 50% of the CD45+ BAL cells were donor derived in the F1.GRKO recipients, compared to only 10% in the wild type recipients. Furthermore, the absolute cell number of donor CD4+ and CD8+ cells in the knockout mice significantly outnumbered that in their wild type counterparts (p<0.01). Thus, our results suggest that absence of recipient IFN-γR signaling promotes accumulation and/or trafficking of donor neutrophils and T cells to the lungs. To better understand the underlying mechanisms of enhanced infiltration of donor cells in the lungs of IFNγR-/- mice, the activation, proliferation, and differentiation status of donor lymphocytes were assessed by Flowcytometry after BMT. In line with the lung infiltration, there was significantly increased donor cell engraftment in PBMC, spleen, lymph nodes, and bone marrow in F1.GRKO recipients (all with p<0.01); Moreover, there were also significantly increased activation (measured as CD44high CD62Llow cells), Th1 and Tc1 differentiation of donor CD4 and CD8 cells (p<0.01). Furthermore, enhanced Th17 differentiation, but reduced Foxp3+ Treg cell differentiation was noticed. Interestigly, we observed significantly lower PD-L1 expression on recipient APC in the F1.GRKO mice compared to wild type mice, suggesting that absence of PD-L1 expression may contribute to reduced contraction of activated donor T cells. Interestingly, in line with these findings we also observed significantly reduced PD-L1 expression when single cell suspension from lung tissues were analyzed by flow. Reduced PD-L1 expression was observed on both recipient CD45+, and CD45- cells in F1.GRKO when tested on day 22 post-BMT, meanwhile there was no difference in PD-L1 expression pattern in the cells isolated from small intestine. These results suggest that IFN-γ induced PD-L1 expression on hematopoietic and non-hematopoietic cells in the lung may be of significance in protecting recipients against acute donor-cell mediated lung injury and IPS.

Disclosures
Lentzsch: Celgene: Membership on an entity's Board of Directors or advisory committees; Axiom: Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.