Kaifeng Liu

The Ashwell-Morell receptor regulates hepatic thrombopoietin production via JAK2-STAT3 signaling

The hepatic Ashwell-Morell receptor (AMR) can bind and remove desialylated platelets. Here we demonstrate that platelets become desialylated as they circulate and age in blood. Binding of desialylated platelets to the AMR induces hepatic expression of thrombopoietin (TPO) mRNA and protein, thereby regulating platelet production. Endocytic AMR controls TPO expression through Janus kinase 2 (JAK2) and the acute phase response signal transducer and activator of transcription 3 (STAT3) in vivo and in vitro. Recognition of this newly identified physiological feedback mechanism illuminates the pathophysiology of platelet diseases, such as essential thrombocythemia and immune thrombocytopenia, and contributes to an understanding of the mechanisms of thrombocytopenia observed with JAK1/2 inhibition.

Long-Term Heart Transplant Survival by Targeting the Ionotropic Purinergic Receptor P2X7

Background— Heart transplantation is a lifesaving procedure for patients with end-stage heart failure. Despite much effort and advances in the field, current immunosuppressive regimens are still associated with poor long-term cardiac allograft outcomes, and with the development of complications, including infections and malignancies, as well. The development of a novel, short-term, and effective immunomodulatory protocol will thus be an important achievement. The purine ATP, released during cell damage/activation, is sensed by the ionotropic purinergic receptor P2X7 (P2X7R) on lymphocytes and regulates T-cell activation. Novel clinical-grade P2X7R inhibitors are available, rendering the targeting of P2X7R a potential therapy in cardiac transplantation. Methods and Results— We analyzed P2X7R expression in patients and mice and P2X7R targeting in murine recipients in the context of cardiac transplantation. Our data demonstrate that P2X7R is specifically upregulated in graft-infiltrating lymphocytes in cardiac-transplanted humans and mice. Short-term P2X7R targeting with periodate-oxidized ATP promotes long-term cardiac transplant survival in 80% of murine recipients of a fully mismatched allograft. Long-term survival of cardiac transplants was associated with reduced T-cell activation, T-helper cell 1/T-helper cell 17 differentiation, and inhibition of STAT3 phosphorylation in T cells, thus leading to a reduced transplant infiltrate and coronaropathy. In vitro genetic upregulation of the P2X7R pathway was also shown to stimulate T-helper cell 1/T-helper cell 17 cell generation. Finally, P2X7R targeting halted the progression of coronaropathy in a murine model of chronic rejection as well. Conclusions— P2X7R targeting is a novel clinically relevant strategy to prolong cardiac transplant survival.

A Novel Clinically Relevant Strategy to Abrogate Autoimmunity and Regulate Alloimmunity in NOD Mice

OBJECTIVE To investigate a new clinically relevant immunoregulatory strategy based on treatment with murine Thymoglobulin mATG Genzyme and CTLA4-Ig in NOD mice to prevent allo- and autoimmune activation using a stringent model of islet transplantation and diabetes reversal. RESEARCH DESIGN AND METHODS Using allogeneic islet transplantation models as well as NOD mice with recent onset type 1 diabetes, we addressed the therapeutic efficacy and immunomodulatory mechanisms associated with a new immunoregulatory protocol based on prolonged low-dose mATG plus CTLA4-Ig. RESULTS BALB/c islets transplanted into hyperglycemic NOD mice under prolonged mATG+CTLA4-Ig treatment showed a pronounced delay in allograft rejection compared with untreated mice (mean survival time: 54 vs. 8 days, P < 0.0001). Immunologic analysis of mice receiving transplants revealed a complete abrogation of autoimmune responses and severe downregulation of alloimmunity in response to treatment. The striking effect on autoimmunity was confirmed by 100% diabetes reversal in newly hyperglycemic NOD mice and 100% indefinite survival of syngeneic islet transplantation (NOD.SCID into NOD mice). CONCLUSIONS The capacity to regulate alloimmunity and to abrogate the autoimmune response in NOD mice in different settings confirmed that prolonged mATG+CTLA4-Ig treatment is a clinically relevant strategy to translate to humans with type 1 diabetes.

Reduced Cytotoxic Function of Effector CD8+ T Cells Is Responsible for Indoleamine 2,3-Dioxygenase-Dependent Immune Suppression

Indoleamine 2,3-dioxygenase (IDO), a potent immunosuppressive enzyme, contributes to tumoral escape, immune tolerance, and protection against allograft injury. In this paper, we report that inhibition of CD8+ T cell-mediated cytotoxic function is an important mechanism behind IDO’s immune-modulating property. The experimental rat lung allograft proved attractive for evaluating effector CD8+ T cells. Enhanced IDO activity achieved by using a lung-tissue-targeted nonviral human IDO gene transfer approach reduced, but did not eliminate, infiltrating CD8+ T cells. Although CD8+ T cells existed in the IDO-high lung allografts, CD8+ T cells remained viable and could proliferate for an extended period. However, cells lost their ability to attack allogeneic donor lung cells in vivo and allogeneic target cells in vitro. The impaired cytotoxic function seen in the IDO-treated CD8+ T cells was accompanied by defects in production of granule cytotoxic proteins, including perforin and granzyme A and B. Furthermore, we discovered that IDO leads to an impaired bioenergetic condition in active CD8+ T cells via selective inhibition of complex I in the mitochondrial electron transfer chain. These intriguing findings provide a base for establishing a novel mode of IDO’s immune-suppressing action. Additionally, donor lung IDO delivery, a direct and/or leukocyte passenger effect, impaired CD8+ effector cell function.

Pirfenidone Inhibits T-Cell Activation, Proliferation, Cytokine and Chemokine Production, and Host Alloresponses

Background. We previously showed that pirfenidone, an anti-fibrotic agent, reduces lung allograft injury or rejection. In this study, we tested the hypothesis that pirfenidone has immune modulating activities and evaluated its effects on the function of T-cell subsets, which play important roles in allograft rejection. Method. We first evaluated whether pirfenidone alters T-cell proliferation and cytokine release in response to T-cell receptor (TCR) activation, and whether pirfenidone alters regulatory T cells (CD4+CD25+) suppressive effects using an in vitro assay. Additionally, pirfenidone effects on alloantigen-induced T-cell proliferation in vivo were assessed by adoptive transfer of carboxyfluorescein diacetate succinimidyl ester-labeled T cells across a parent->F1 major histocompatibility complex mismatch, as well as using a murine heterotopic cardiac allograft model (BALB/c->C57BL/6). Results. Pirfenidone was found to inhibit the responder frequency of TCR-stimulated CD4+ cell total proliferation in vitro and in vivo, whereas both CD4 and CD8 proliferation index were reduced by pirfenidone. Additionally, pirfenidone inhibited TCR-induced production of multiple pro-inflammatory cytokines and chemokines. Interestingly, there was no change on transforming growth factor-&bgr; production by purified T cells, and pirfenidone had no effect on the suppressive properties of naturally occurring regulatory T cells. Pirfenidone alone showed a small but significant (P<0.05) effect on the in vivo allogeneic response, whereas the combination of pirfenidone and low dose rapamycin had more remarkable effect in reducing the alloantigen response with prolonged graft survival. Conclusion. Pirfenidone may be an important new agent in transplantation, with particular relevance to combating chronic rejection by inhibiting both fibroproliferative and alloimmune responses.

Therapeutic lymphangiogenesis ameliorates established acute lung allograft rejection

Lung transplantation is the only viable option for patients suffering from otherwise incurable end-stage pulmonary diseases such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. Despite aggressive immunosuppression, acute rejection of the lung allograft occurs in over half of transplant recipients, and the factors that promote lung acceptance are poorly understood. The contribution of lymphatic vessels to transplant pathophysiology remains controversial, and data that directly address the exact roles of lymphatic vessels in lung allograft function and survival are limited. Here, we have shown that there is a marked decline in the density of lymphatic vessels, accompanied by accumulation of low-MW hyaluronan (HA) in mouse orthotopic allografts undergoing rejection. We found that stimulation of lymphangiogenesis with VEGF-C156S, a mutant form of VEGF-C with selective VEGFR-3 binding, alleviates an established rejection response and improves clearance of HA from the lung allograft. Longitudinal analysis of transbronchial biopsies from human lung transplant recipients demonstrated an association between resolution of acute lung rejection and decreased HA in the graft tissue. Taken together, these results indicate that lymphatic vessel formation after lung transplantation mediates HA drainage and suggest that treatments to stimulate lymphangiogenesis have promise for improving graft outcomes.

Inhibitory Effects of Pirfenidone on Dendritic Cells and Lung Allograft Rejection

Background Pirfenidone (PFD) is an antifibrotic agent with beneficial effects on proinflammatory disorders. In this study, we further investigated PFD and long-acting form, “deuterated PFD,” immune-modulating properties by evaluating their effects on mouse dendritic cells (DCs). Methods The effects of PFD on DCs were examined in vivo using an orthotopic mouse lung transplant model and in vitro using isolated bone marrow–derived DCs in response to lipopolysaccharide and allogeneic stimulation. Results In mouse lung transplants, PFD and deuterated PFD treatment improved allograft lung function based on peak airway pressure, less infiltrates/consolidation on micro–computed tomography scan imaging, and reduced lung rejection/injury. DC activation from lung allografts was suppressed with PFD, and there seemed to be a greater effect of PFD on CD11c+CD11b−CD103+ lung DCs. In addition, PFD reduced the expression of several proinflammatory cytokines/chemokines from lung allografts. In vitro, DCs treated with PFD showed decreased expression of major histocompatibility complex class II and costimulatory molecules and the capacity of these DCs to stimulate T-cell activation was impaired, although antigen uptake was preserved. PFD directly inhibited the release of inflammatory cytokines from isolated DCs, was associated with a reduction of stress protein kinases, and attenuated lipopolysaccharide-dependent mitogen-activated protein kinase p38 phosphorylation. Conclusions PFD has lung allograft protective properties, and in addition to its known effects on T-cell biology, PFD immune-modulating activities encompass inhibitory effects on DC activation and function.

Inhibition of the Purinergic Pathway Prolongs Mouse Lung Allograft Survival

Lung transplantation has limited survival with current immunosuppression. ATP is released from activated T cells, which act as costimulatory molecules through binding to the purinergic receptor P2XR7. We investigated the role of blocking the ATP/purinergic pathway, primarily P2XR7, using its inhibitor oxidized ATP (oATP) in modulating rejection of mouse lung allografts. Mouse lung transplants were performed using mice with major histocompatibility complex mismatch, BALB/c to C57BL6. Recipients received suramin or oATP, and lung allografts were evaluated 15 to ≥ 60 days after transplantation. Recipients were also treated with oATP after the onset of moderate to severe rejection to determine its ability to rescue lung allografts. Outcomes measures included lung function, histology, thoracic imaging, and allo-immune responses. Blocking purinergic receptors with the nonselective inhibitor suramin or with the P2XR7-selective inhibitor oATP reduced acute rejection and prolonged lung allograft survival for ≥ 60 days with no progression in severity. There were fewer inflammatory cells within lung allografts, less rejection, and improved lung function, which was maintained over time. CD4 and CD8 T cells were reduced within lung allografts with impaired activation with prolonged impairment of CD8 responses. In vitro, oATP reduced CD8 activation of Th1 inflammatory cytokines IFN-γ and TNF-α and cytolytic machinery, granzyme B. Cotreatment with immunosuppressive agents, cyclosporine, rapamycin, or CTLA-4Ig resulted in no additive benefits, and oATP alone resulted in better outcomes than cyclosporine alone. This study illustrates a potential new pathway to target in hopes of prolonging survival of lung transplant recipients.

Identification of dedifferentiation and redevelopment phases during postpneumonectomy lung growth

Surgical resection of pulmonary tissue exerts a proregenerative stretch stimulus in the remaining lung units. Whether this regeneration process reenacts part or whole of lung morphogenesis developmental program remains unclear. To address this question, we analyzed the stretch-induced regenerating lung transcriptome in mice after left pneumonectomy (PNX) in its developmental context. We created a C57BL/6 mice lung regeneration transcriptome time course at 3, 7, 14, 28, and 56 days post-PNX, profiling the cardiac and medial lobes and whole right lung. Prominent expression at days 3 and 7 of genes related to cell proliferation (Ccnb1, Bub1, and Cdk1), extracellular matrix (Col1a1, Eln, and Tnc), and proteases (Serpinb2 and Mmp9) indicated regenerative processes that tapered off after 56 days. We projected the post-PNX transcriptomic time course into the transcriptomic principal component space of the C57BL/6 mouse developing lungs time series from embryonic day 9.5 to postnatal day 56. All post-PNX samples were localized around the late postnatal stage of developing lungs. Shortly after PNX, the temporal trajectory of regenerating lobes and right lung reversed course relative to the developing lungs in a process reminiscent of dedifferentiation. This reversal was limited to the later postnatal stage of lung development. The post-PNX temporal trajectory then moves forward in lung development time close to its pre-PNX state after days 28 to 56 in a process resembling redevelopment. A plausible interpretation is that remaining pulmonary tissue reverts to a more primitive stage of development with higher potential for growth to generate tissue in proportion to the loss.

Indoleamine 2,3-Dioxygenase and Metabolites Protect Murine Lung Allografts and Impair the Calcium Mobilization of T Cells

The enzyme indoleamine 2,3-dioxygenase (IDO) converts tryptophan into kynurenine metabolites that suppress effector T-cell function. In this study, we investigated IDO and its metabolite, 3-hydroxyanthranilic acid (3HAA), in regulating lung allograft rejection, using a murine orthotopic lung transplant model with a major mismatch (BALB/c donor and C57BL6 recipient). IDO was overexpressed in murine donor lungs, using an established nonviral (polyethylenimine carrier)-based gene transfer approach, whereas 3HAA was delivered daily via intraperitoneal injection. Increased IDO expression or its metabolite, 3HAA, resulted in a remarkable therapeutic effect with near normal lung function and little acute rejection, approximately A1, compared with A3 in untreated allografts (grading based on International Society for Heart and Lung Transplantation guidelines). We found that a high IDO environment for 7 days in lung allografts resulted in impaired T-cell activation, the production of multiple effector cytokines (IL-2, IL-4, IL-5, IL-6, IFN-γ, TNF-α, IL-12, and IL-13), and the generation of effector memory T cells (CD62L(lo)CD44(hi) phenotype). In isolated murine splenocytes, we observed that IDO/3HAA impaired T-cell receptor (TCR)-mediated T-cell activation, and more importantly, a decrease of intracellular calcium, phospholipase C-γ1 phosphorylation, and mitochondrial mass was evident. This work further illustrates the potential role of a high IDO environment in lung transplantation, and that the high IDO environment directly impairs TCR activation via the disruption of calcium signaling.