Biology subjects

Keshavjee, S.

Publications and source records attributed to Keshavjee, S..

3 recordsLinked to original sources

bioRxiv2021

Pro-inflammatory alveolar macrophages associated with allograft dysfunction after lung transplantation

PurposeLung transplant (LT) recipients experience episodes of immune-mediated acute lung allograft dysfunction (ALAD). We have applied single-cell RNA sequencing (scRNAseq) to bronchoalveolar lavage (BAL) cells of stable and ALAD patients to determine key cellular elements in dysfunctional lung allografts. Our particular focus here is on studying alveolar macrophages (AMs) as scRNAseq enables us to elucidate their heterogeneity and possible association with ALAD where our knowledge from cytometry-based assays is very limited. MethodsFresh bronchoalveolar lavage (BAL) cells from 6 LT patients, 3 with stable lung function (3044 {+/-} 1519 cells) and 3 undergoing an episode of ALAD (2593 {+/-} 904 cells) were used for scRNAseq. R Bioconductor and Seurat were used to perform QC, dimensionality reduction, annotation, pathway analysis, and trajectory. Donor and recipient deconvolution was performed using single nucleotide variations. ResultsOur data revealed that AMs are highly heterogeneous (12 transcriptionally distinct subsets in stable). We identified two AM subsets uniquely represented in ALAD. Based on pathway analysis and the top differentially expressed genes in BAL we annotated them as pro-inflammatory interferon-stimulated genes (ISG) and metallothioneins-mediated inflammatory (MT). Pseudotime analysis suggested that ISG AMs represent an earlier stage of differentiation which may suggest them as monocyte drive macrophages. Our functional analysis on an independent set of BAL samples shows that ALAD samples have significantly higher expression of CXCL10, a marker of ISG AM, as we as higher secretion of pro-inflammatory cytokines. Single nucleotide variation calling algorithm has allowed us to identify macrophages of donor origin and demonstrated that donor AMs are lost with time post-transplant. ConclusionUsing scRNAseq, we observed AMs heterogeneity and identified specific subsets that may be associated with allograft dysfunction. Further exploration with scRNAseq will shed light on LT immunobiology and the role of AMs in allograft injury and dysfunction.

immunology↗

bioRxiv2021

Ex vivo delivery of regulatory T cells for control of alloimmune priming in the donor lung

Survival after lung transplantation (LTx) is hampered by uncontrolled inflammation and alloimmunity. Regulatory T cells (Tregs) are being studied for post-implantation cell therapy in solid organ transplantation. Whether these systemically administered Tregs can function at the appropriate location and time is an important concern. We hypothesized that in vitro expanded, recipient-derived Tregs can be delivered to donor lungs prior to LTx via ex vivo lung perfusion (EVLP), maintaining their immunomodulatory ability. In a rat model, Wistar Kyoto (WKy) CD4+CD25high Tregs were expanded in vitro prior to EVLP. Expanded Tregs were administered to Fisher 344 (F344) donor lungs during EVLP; left lungs were transplanted into WKy recipients. Treg localization and function post-transplant were assessed. In a proof-of-concept experiment, cryopreserved expanded human CD4+CD25+CD127low Tregs were thawed and injected into discarded human lungs during EVLP. Rat Tregs entered the lung parenchyma and retained suppressive function. Expanded Tregs had no adverse effect on donor lung physiology during EVLP; lung water as measured by wet- to-dry weight ratio was reduced by Treg therapy. The administered cells remained in the graft at 3 days post-transplant where they reduced activation of intragraft effector CD4+ T cells; these effects were diminished by day 7. Human Tregs entered the lung parenchyma during EVLP where they expressed key immunoregulatory molecules (CTLA4+, 4-1BB+, CD39+, and CD15s+). Pre-transplant Treg administration can inhibit alloimmunity within the lung allograft at early time points post- transplant. Our organ-directed approach has potential for clinical translation.

immunology↗

bioRxiv2020

Connective-Tissue Growth Factor (CTGF/CCN2) Contributes to TGF-β1-Induced Lung Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by progressive and excessive accumulation of myofibroblasts and extracellular matrix in the lung. Connective-tissue growth factor (CTGF) is known to exacerbate pulmonary fibrosis in radiation-induced lung fibrosis, and in this study, we show the upregulation of CTGF from a rat lung fibrosis model induced by adenovirus vector encoding active TGF-β1 (AdTGF-β1), and also in patients with IPF. The expression of CTGF was upregulated in vascular smooth muscle cells cultured from fibrotic lungs on days 7 or 14 as well as endothelial cells sorted from fibrotic lungs on day 14 or 28 respectively. These findings suggest the role of different cells in maintaining the fibrotic phenotype during fibrogenesis. Treatment of fibroblasts with recombinant CTGF along with TGF-β increases pro-fibrotic markers in fibroblasts, confirming the synergistic effect of recombinant CTGF with TGF-β in inducing pulmonary fibrosis. Also, fibrotic extracellular matrix upregulated the expression of CTGF, as compared to normal extracellular matrix, suggesting that not only profibrotic mediators but also a profibrotic environment contributes to fibrogenesis. We also showed that pamrevlumab, a CTGF inhibitory antibody, partially attenuates fibrosis in the model. These results suggest that pamrevlumab could be an option for the treatment of pulmonary fibrosis.Competing Interest StatementT. Yanagihara was funded by the Uehara Memorial Foundation Research Fellowship and Mitacs Canada, and the research institute of St Joseph Hospital, Hamilton, ON, Canada (Post-doctoral Fellowship Award). K. Ask reports grants and personal fees from Boehringer Ingelheim, grants from Canadian Pulmonary Fibrosis Foundation, Synairgen, Alkermes, GlaxoSmitheKline, Pharmaxis, Unity, Avalyn, Canadian Institutes of Health Research, Ceapro, Pieris, outside the submitted work. M. Kolb reports grants from the Canadian Institute for Health Research and grants/ personal fees from Roche, Boehringer Ingelheim, Prometic, Respivert, Alkermes, and Pharmaxis and personal fees from Genoa. K.E. Lipson is an employee and shareholder of FibroGen, Inc. J. Tikkanen reports personal fees from CSL Behring, outside the submitted work. S.G. Chong, M. Gholiof, Q. Zhou, C. Scallan, C. Upagupta, and S. Keshavjee report no conflict of interest.View Full Text

cell biology↗