BACKGROUNDChronic pulmonary graft-versus-host disease (cpGVHD) after hematopoietic cell transplant (HCT) manifests as progressive airway and parenchymal lung fibrosis. On the basis of our prior data, mice that undergo allogeneic HCT with Tbet-knockout donors (AlloTbet) have increased lung Th17 cells and IL-17A and develop fibrosis resembling human cpGVHD. The role of IL-17A in posttransplant pulmonary fibrosis remains incompletely understood. We hypothesized that IL-17A is necessary for development of murine cpGVHD in this model.METHODSAlloTbet mice received weekly intraperitoneal anti-IL-17A or IgG (200 μg/mouse) starting 2 weeks post-HCT and were sacrificed after week 5. Histologic airway and parenchymal fibrosis were semiquantitatively graded in a blinded fashion. Lung cells and proteins were measured by flow cytometry, ELISA, and multicytokine assays.RESULTSAnti-IL-17A modestly decreased airway and parenchymal lung fibrosis, along with a striking reduction in pulmonary neutrophilia, IL-6, MIP-1α, MIP-1β, CXCL1, and CXCL5 in AlloTbet mice. Additionally, anti-IL-17A decreased CCL2, inflammatory monocytes and macrophages, and Th17 cells.CONCLUSIONSIn the setting of murine AlloHCT with Tbet donors, IL-17A blockade decreases fibrotic features of cpGVHD. This may be mediated by the observed reduction in neutrophils or specific lung monocyte and macrophage populations or alternatively via a direct effect on fibroblasts. Collectively, our results further suggest that anti-IL-17A strategies could prove useful in preventing alloimmune-driven fibrotic lung diseases.

PurposeChronic lung allograft dysfunction (CLAD) after lung transplant and chronic pulmonary graft-versus-host disease (cpGVHD) after bone marrow transplant (BMT) manifest as progressive airway and parenchymal lung fibrosis. Based on our prior data, mice that undergo allogeneic BMT (AlloBMT) with Tbet-/- donors have increased lung Th17 cells and IL17 and develop fibrosis that resembles human cpGVHD and CLAD. While IL-17 has been shown to play a role in neutrophil recruitment and fibrosis in other models, its relative importance in promoting post-transplant pulmonary fibrosis remains unclear. We hypothesized that IL-17 is necessary for the development of fibrosis in our model of cpGVHD.MethodsB10.BR mice underwent AlloBMT with Tbet-/- donors (on C57BL/6 background). Recipients received weekly intraperitoneal injections of anti-IL-17A or control IgG (200μg/mouse) starting 2 weeks after BMT and were sacrificed 5 weeks after BMT. Collagen was stained with Masson-Trichrome and histologic airway and parenchymal lung fibrosis was semi-quantitatively graded in a blinded fashion. Lung proteins were assessed by ELISA and multi-cytokine assay. Lung cell subsets were analyzed by flow cytometry.ResultsAnti-IL-17 significantly decreases qualitative measures of airway and parenchymal lung fibrosis in AlloBMT mice transplanted with Tbet-/- donors. IL-17 neutralization leads to reduced lung neutrophils and neutrophil-recruiting proteins IL-6, MIP-1α, MIP-2, KC, and CXCL5. In addition, anti-IL-17 decreases CCL2, inflammatory monocytes, and antigen presenting cells (APC), as well as the number of overall T cells and Th17 cells.ConclusionIn the setting of murine AlloBMT with Tbet-/- donors, IL-17 blockade decreases fibrotic features of cpGVHD. This decrement in fibrosis may be mediated by the observed reduction in lung neutrophils and/or specific lung APC populations. Collectively, our results suggest that anti-IL-17 strategies could prove useful in the treatment of alloimmune-driven chronic fibrotic lung diseases. PurposeChronic lung allograft dysfunction (CLAD) after lung transplant and chronic pulmonary graft-versus-host disease (cpGVHD) after bone marrow transplant (BMT) manifest as progressive airway and parenchymal lung fibrosis. Based on our prior data, mice that undergo allogeneic BMT (AlloBMT) with Tbet-/- donors have increased lung Th17 cells and IL17 and develop fibrosis that resembles human cpGVHD and CLAD. While IL-17 has been shown to play a role in neutrophil recruitment and fibrosis in other models, its relative importance in promoting post-transplant pulmonary fibrosis remains unclear. We hypothesized that IL-17 is necessary for the development of fibrosis in our model of cpGVHD. Chronic lung allograft dysfunction (CLAD) after lung transplant and chronic pulmonary graft-versus-host disease (cpGVHD) after bone marrow transplant (BMT) manifest as progressive airway and parenchymal lung fibrosis. Based on our prior data, mice that undergo allogeneic BMT (AlloBMT) with Tbet-/- donors have increased lung Th17 cells and IL17 and develop fibrosis that resembles human cpGVHD and CLAD. While IL-17 has been shown to play a role in neutrophil recruitment and fibrosis in other models, its relative importance in promoting post-transplant pulmonary fibrosis remains unclear. We hypothesized that IL-17 is necessary for the development of fibrosis in our model of cpGVHD. MethodsB10.BR mice underwent AlloBMT with Tbet-/- donors (on C57BL/6 background). Recipients received weekly intraperitoneal injections of anti-IL-17A or control IgG (200μg/mouse) starting 2 weeks after BMT and were sacrificed 5 weeks after BMT. Collagen was stained with Masson-Trichrome and histologic airway and parenchymal lung fibrosis was semi-quantitatively graded in a blinded fashion. Lung proteins were assessed by ELISA and multi-cytokine assay. Lung cell subsets were analyzed by flow cytometry. B10.BR mice underwent AlloBMT with Tbet-/- donors (on C57BL/6 background). Recipients received weekly intraperitoneal injections of anti-IL-17A or control IgG (200μg/mouse) starting 2 weeks after BMT and were sacrificed 5 weeks after BMT. Collagen was stained with Masson-Trichrome and histologic airway and parenchymal lung fibrosis was semi-quantitatively graded in a blinded fashion. Lung proteins were assessed by ELISA and multi-cytokine assay. Lung cell subsets were analyzed by flow cytometry. ResultsAnti-IL-17 significantly decreases qualitative measures of airway and parenchymal lung fibrosis in AlloBMT mice transplanted with Tbet-/- donors. IL-17 neutralization leads to reduced lung neutrophils and neutrophil-recruiting proteins IL-6, MIP-1α, MIP-2, KC, and CXCL5. In addition, anti-IL-17 decreases CCL2, inflammatory monocytes, and antigen presenting cells (APC), as well as the number of overall T cells and Th17 cells. Anti-IL-17 significantly decreases qualitative measures of airway and parenchymal lung fibrosis in AlloBMT mice transplanted with Tbet-/- donors. IL-17 neutralization leads to reduced lung neutrophils and neutrophil-recruiting proteins IL-6, MIP-1α, MIP-2, KC, and CXCL5. In addition, anti-IL-17 decreases CCL2, inflammatory monocytes, and antigen presenting cells (APC), as well as the number of overall T cells and Th17 cells. ConclusionIn the setting of murine AlloBMT with Tbet-/- donors, IL-17 blockade decreases fibrotic features of cpGVHD. This decrement in fibrosis may be mediated by the observed reduction in lung neutrophils and/or specific lung APC populations. Collectively, our results suggest that anti-IL-17 strategies could prove useful in the treatment of alloimmune-driven chronic fibrotic lung diseases. In the setting of murine AlloBMT with Tbet-/- donors, IL-17 blockade decreases fibrotic features of cpGVHD. This decrement in fibrosis may be mediated by the observed reduction in lung neutrophils and/or specific lung APC populations. Collectively, our results suggest that anti-IL-17 strategies could prove useful in the treatment of alloimmune-driven chronic fibrotic lung diseases.

Background Pulmonary GVHD (pGVHD) is an important complication of hematopoietic cell transplant (HCT) and is thought to be a consequence of the HCT conditioning regimen, allogeneic donor cells, and posttransplant lung exposures. We have previously demonstrated that serial inhaled lipopolysaccharide (LPS) exposures potentiate the development of pGVHD after murine allogeneic HCT. In the current study we hypothesized that allogeneic lymphocytes and environmental exposures alone, in the absence of a pre-conditioning regimen, would cause features of pGVHD and would lead to a different T cell expansion pattern compared to syngeneic cells. Methods Recipient Rag1−/− mice received a transfer of allogeneic (Allo) or syngeneic (Syn) spleen cells. After 1 week of immune reconstitution, mice received 5 daily inhaled LPS exposures and were sacrificed 72 hours after the last LPS exposure. Lung physiology, histology, and protein levels in bronchoalveolar lavage (BAL) were assessed. Lung cells were analyzed by flow cytometry. Results Both Allo and Syn mice that undergo LPS exposures (AlloLPS and SynLPS) have prominent lymphocytic inflammation in their lungs, resembling pGVHD pathology, not seen in LPS-unexposed or non-transplanted controls. Compared to SynLPS, however, AlloLPS have significantly increased levels of BAL protein and enhancement of airway hyperreactivity, consistent with more severe lung injury. This injury in AlloLPS mice is associated with an increase in CD8 T cells and effector CD4 T cells, as well as a decrease in regulatory to effector CD4 T cell ratio. Additionally, cytokine analysis is consistent with a preferential Th1 differentiation and upregulation of pulmonary CCL5 and granzyme B. Conclusions Allogeneic lymphocyte transfer into lymphocyte-deficient mice, followed by LPS exposures, causes features of pGVHD and lung injury in the absence of a pre-conditioning HCT regimen. This lung disease associated with an expansion of allogeneic effector T cells provides a novel model to dissect mechanisms of pGVHD independent of conditioning.

Environmental exposures are a potential trigger of chronic pulmonary graft-versus-host disease (pGVHD) after successful recovery from hematopoietic cell transplant (HCT). We hypothesized that inhalations of LPS, a prototypic environmental stimulus, trigger pGVHD via increased pulmonary recruitment of donor-derived antigen-presenting cells (APCs) through the C-C motif ligand 2 (CCL2)-C-C motif receptor 2 (CCR2) chemokine axis. B10.BR(H2(k)) and C57BL/6(H2(b)) mice underwent allogeneic (Allo) or syngeneic (Syn) HCT with wild-type (WT) C57BL/6, CCL2(-/-), or CCR2(-/-) donors. After 4 weeks, recipient mice received daily inhaled LPS for 5 days and were killed at multiple time points. Allo mice exposed to repeated inhaled LPS developed prominent lymphocytic bronchiolitis, similar to human pGVHD. The increase in pulmonary T cells in Allo mice after LPS exposures was accompanied by increased CCL2, CCR2, and Type-1 T-helper cytokines as well as by monocytes and monocyte-derived dendritic cells (moDCs) compared with Syn and nontransplanted controls. Using CCL2(-/-) donors leads to a significant decrease in lung DCs but to only mildly reduced CD4 T cells. Using CCR2(-/-) donors significantly reduces lung DCs and moDCs but does not change T cells. CCL2 or CCR2 deficiency does not alter pGVHD pathology but increases airway hyperreactivity and IL-5 or IL-13 cytokines. Our results show that hematopoietic donor-derived CCL2 and CCR2 regulate recruitment of APCs to the Allo lung after LPS exposure. Although they do not alter pathologic pGVHD, their absence is associated with increased airway hyperreactivity and IL-5 and IL-13 cytokines. These results suggest that the APC changes that result from CCL2-CCR2 blockade may have unexpected effects on T cell differentiation and physiologic outcomes in HCT.