Increased P2X7 expression in the gastrointestinal tract and skin in a humanised mouse model of graft-versus-host disease

Abstract

Background: Allogeneic haematopoietic stem cell transplantation (HSCT) is a curative therapy for blood cancers; but results in the development of graft-versus-host disease (GVHD) in up to 70% of recipients. During GVHD, tissue damage results in ATP release into the extracellular compartment activating P2X7 on antigen-presenting cells, leading to the release of pro-inflammatory cytokines and subsequent activation of donor T cells. Therefore, the aim of the present study was to examine murine (m) P2rx7 and human (h) P2RX7 gene expression in GVHD target organs of humanised mice, and further characterise disease impact in these organs. Methods: NOD-scid IL2Rγnull (NSG) mice were injected with human peripheral blood mononuclear cells (hu-PBMC-NSG mice) or phosphate-buffered saline (PBS, control). Leucocytes were assessed by flow cytometry; gene expression was measured by quantitative polymerase chain reaction (qPCR), and tissue sections examined by histology. Results: Compared with control mice, hu-PBMC-NSG mice had increased mP2rx7 and mP2rx4 expression in the duodenum, ileum and skin. hP2RX7 was expressed in all tissues examined. hu-PBMC-NSG mice also displayed increased mReg3g expression in the duodenum and ileum, despite limited histological gut GVHD. hu-PBMC-NSG mice showed histological evidence of GVHD in the skin, liver and lung. Compared with control mice, hu-PBMC-NSG mice displayed increased ear swelling. Conclusion: Combined data revealed that P2rx7 is up-regulated in gut and skin GVHD and that P2RX7 is present in target tissues of GVHD, corresponding to human leucocyte infiltration. Data also reveal increased mReg3g expression and ear swelling in hu-PBMC-NSG mice, offering new measurements of early-stage gut GVHD and skin GVHD, respectively.

P2X7 receptor antagonism increases regulatory T cells and reduces clinical and histological graft-versus-host disease in a humanised mouse model

“…Human blood was acquired and used as approved by the University of Wollongong (Wollongong, Australia) Human Ethics Research Committee. Human peripheral blood mononuclear cells (hPBMCs) were isolated as previously described (20) . Briefly, whole blood was collected from healthy human donors (n = 8, aged 23-28 y, 7 male, 1 female), with written informed consent, and separated via density centrifugation with Ficoll-Paque PLUS (GE Healthcare, Uppsala, Sweden).…”

“…All experiments using mice were carried out in the University of Wollongong Rodent Facility as approved by the University of Wollongong Animal Ethics Committee. Female NOD-scid IL2Rγ null (NSG) mice (Australian BioResources, Moss Vale, Australia) were housed and fed as described (20) . NSG mice that had been acclimatised for two weeks (6-7 weeks old) were injected intraperitoneally (i.p.)…”

Use of Humanized Mouse Models to Investigate the Roles of Purinergic Signaling in Inflammation and Immunity

“…Further complicating an immunosuppressive role for adenosine in this model, is our observation that engraftment of human PBMCs with a polymorphic variant of the ENTPD1 gene, that results in increased CD39 + T regulatory cells, worsens GVHD ( Adhikary et al, 2020 ). Finally, our studies have revealed increased expression of murine P2rx7 and P2rx4 in GVHD tissues from Hu-PBMC-NSG mice compared to those from non-engrafted NSG mice ( Cuthbertson et al, 2020 ) and the presence of functional murine P2X7 receptors in NSG mice ( Geraghty et al, 2017 ), whilst both human P2RX7 and ADORA2 are detected in Hu-PBMC-NSG mice ( Geraghty et al, 2019d ). Collectively, this data suggests Hu-PBMC-NSG mice provide a pre-clinical model of GVHD in which new therapeutics aimed at inhibiting P2X7 receptor activation can be tested, whilst the potential use of this model to test new therapeutics aimed at activating A 2A receptors remains to be established.…”

“…injection of 10 × 10 6 human (h) peripheral blood mononuclear cells (PBMCs) into NOD.Cg- Prkdc scid IL2rg tm1Wjl (NSG) mice (Day 0) results in the engraftment of human (h) CD45 + leukocytes predominately hCD4 + and hCD8 + T cells as early as Day 21. The percentages of hCD45 + leukocytes, hCD3 + T cells and hCD4 + or hCD8 + T cells represent the average percentages of these cells among total CD45 + leukocytes, hCD45 + leukocytes and hCD3 + T cells, respectively, typically observed in this model ( Cuthbertson et al, 2020 ). From Week 4, mice display signs of clinical GVHD (as indicated) corresponding with the production of circulating human interferon- γ (hIFN ( γ ), tumor necrosis factor (hTNF) and interleukins (hIL) (as indicated) ( Geraghty et al, 2017 ; Geraghty et al, 2019a ; Geraghty et al, 2019d ), increased murine P2rx7 and P2rx4 expression in GVHD tissues, and histological evidence of GVHD at endpoint (Day 70) ( Cuthbertson et al, 2020 ).…”

P2X7 Receptor in Hematological Malignancies

“…It will also be interesting to analyze isoform expression in large cohort databases to understand whether they are differentially expressed in MLL-rearranged AML and, in general, in poor outcome patients. Finally, the role of ATP and P2X7 in shaping the bone marrow niche and mediating hematopoietic precursors or leukemic stem cell localization, mobilization, and their behavior following allogeneic transplantation surely deserves further understanding (Salvestrini et al, 2017;Adamiak et al, 2018;Koldej et al, 2018;Cuthbertson et al, 2020;He et al, 2020).…”