Lori Phillips

Blocking angiotensin-converting enzyme induces potent regulatory T cells and modulates TH1- and TH17-mediated autoimmunity

The renin-angiotensin-aldosterone system (RAAS) is a major regulator of blood pressure. The octapeptide angiotensin II (AII) is proteolytically processed from the decapeptide AI by angiotensin-converting enzyme (ACE), and then acts via angiotensin type 1 and type 2 receptors (AT1R and AT2R). Inhibitors of ACE and antagonists of the AT1R are used in the treatment of hypertension, myocardial infarction, and stroke. We now show that the RAAS also plays a major role in autoimmunity, exemplified by multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Using proteomics, we observed that RAAS is up-regulated in brain lesions of MS. AT1R was induced in myelin-specific CD4+ T cells and monocytes during autoimmune neuroinflammation. Blocking AII production with ACE inhibitors or inhibiting AII signaling with AT1R blockers suppressed autoreactive TH1 and TH17 cells and promoted antigen-specific CD4+FoxP3+ regulatory T cells (Treg cells) with inhibition of the canonical NF-κB1 transcription factor complex and activation of the alternative NF-κB2 pathway. Treatment with ACE inhibitors induces abundant CD4+FoxP3+ T cells with sufficient potency to reverse paralytic EAE. Modulation of the RAAS with inexpensive, safe pharmaceuticals used by millions worldwide is an attractive therapeutic strategy for application to human autoimmune diseases.

Differential Expression of MicroRNAs During Allograft Rejection

MicrorRNA are small noncoding RNA molecules that regulate the posttranscriptional expression of target genes. In addition to being involved in many biologic processes, microRNAs are important regulators in innate and adaptive immune responses. Distinct sets of expressed microRNAs are found in different cell types and tissues and aberrant expression of microRNAs is associated with many disease states. MicroRNA expression was examined in a model of heterotopic heart transplantation by microarray analyses and a unique profile was detected in rejecting allogeneic transplants (BALB/c → C57BL/6) as compared to syngeneic transplants (C57BL/6 → C57BL/6). The microRNA miR‐182 was significantly increased in rejecting cardiac allografts and in mononuclear cells that infiltrate the grafts. Forkhead box (FOX) proteins are a family of important transcription factors and FOXO1 is a target of miR‐182. As miR‐182 increases after transplant, there is a concomitant posttranscriptional decrease in FOXO1 expression in heart allografts that is localized to both the cardiomyocytes and CD3+ T cells. The microRNA miR‐182 is significantly increased in both peripheral blood mononuclear cells and plasma during graft rejection suggesting potential as a biomarker of graft status. Our results identify microRNAs that may regulate alloimmune responses and graft outcomes.

Syk Activation of Phosphatidylinositol 3-Kinase/Akt Prevents HtrA2-dependent Loss of X-linked Inhibitor of Apoptosis Protein (XIAP) to Promote Survival of Epstein-Barr Virus+ (EBV+) B Cell Lymphomas

Background: Syk activation is required for B cell survival. EBV can induce B cell lymphomas. Results: Syk, PI3K/Akt inhibition induces apoptosis of EBV+ B cell lymphomas. Syk PI3K/Akt inhibition results in HtrA2-dependent loss of XIAP protein. Conclusion: Syk activates PI3K/Akt to promote survival by preventing HtrA2-dependent loss of XIAP. Significance: Syk, PI3K/Akt, and XIAP are new therapeutic targets for EBV+ B cell lymphomas. B cell lymphoma survival requires tonic or ligand-independent signals through activation of Syk by the B cell receptor. The Epstein-Barr virus (EBV) protein latent membrane 2a (LMP2a), a mimic of the B cell receptor, provides constitutive survival signals for latently infected cells through Syk activation; however, the precise downstream mechanisms coordinating this survival response in EBV+ B cell lymphomas remain to be elucidated. Herein, we assess the mechanism of Syk survival signaling in EBV+ B cell lymphomas from post-transplant lymphoproliferative disorder (PTLD) to discover virally controlled therapeutic targets involved in lymphomagenesis and tumor progression. Using small molecule inhibition and siRNA strategies, we show that Syk inhibition reduces proliferation and induces apoptosis of PTLD-derived EBV+ B cell lines. Syk inhibition also reduces autocrine IL-10 production. Although Syk inhibition attenuates signaling through both the PI3K/Akt and Erk pathways, only PI3K/Akt inhibition causes apoptosis of PTLD-derived cell lines. Loss of the endogenous caspase inhibitor XIAP is observed after Syk or PI3K/Akt inhibition. The loss of XIAP and apoptosis that results from Syk or PI3K/Akt inhibition is reversed by inhibition of the mitochondrial protease HtrA2. Thus, Syk drives EBV+ B cell lymphoma survival through PI3K/Akt activation, which prevents the HtrA2-dependent loss of XIAP. Syk, Akt, and XIAP antagonists may present potential new therapeutic strategies for PTLD through targeting of EBV-driven survival signals.

MHC mismatch inhibits neurogenesis and neuron maturation in stem cell allografts.

Background The role of histocompatibility and immune recognition in stem cell transplant therapy has been controversial, with many reports arguing that undifferentiated stem cells are protected from immune recognition due to the absence of major histocompatibility complex (MHC) markers. This argument is even more persuasive in transplantation into the central nervous system (CNS) where the graft rejection response is minimal. Methodology/Principal Findings In this study, we evaluate graft survival and neuron production in perfectly matched vs. strongly mismatched neural stem cells transplanted into the hippocampus in mice. Although allogeneic cells survive, we observe that MHC-mismatch decreases surviving cell numbers and strongly inhibits the differentiation and retention of graft-derived as well as endogenously produced new neurons. Immune suppression with cyclosporine-A did not improve outcome but non-steroidal anti-inflammatory drugs, indomethacin or rosiglitazone, were able to restore allogeneic neuron production, integration and retention to the level of syngeneic grafts. Conclusions/Significance These results suggest an important but unsuspected role for innate, rather than adaptive, immunity in the survival and function of MHC-mismatched cellular grafts in the CNS.

Toll-like receptor 4 contributes to small intestine allograft rejection.

Background. Although outcomes for small intestine transplantation (SIT) have improved in recent years, allograft rejection rates remain among the highest of solid organ grafts. The high load of commensal bacteria in the small intestine may contribute through activation of the toll-like receptor (TLR) pathway. In this study, we examine the participation of TLR4 in acute allograft rejection in an orthotopic mouse model of SIT. Methods. Wild-type C57Bl/6 (H-2b) or TLR4−/− (H-2b) mice were transplanted with syngeneic (C57Bl/6), allogeneic (BALB/c; H-2d), or F1 (BALB/c×C57Bl/6; H-2d/b) vascularized, orthotopic small intestine grafts. Graft recipients were killed on days 2 to 6 posttransplant. Serum cytokines were measured by Luminex, and tissue was obtained for histology and quantitative real-time polymerase chain reaction. Results. BALB/c grafts transplanted into C57Bl/6 recipients exhibited mixed inflammatory infiltrates, destruction of the mucosa, and significant apoptosis. TLR2 and TLR4 transcripts were modestly increased in syngeneic grafts on days 2 and 6 compared with native bowel, whereas TLR2 and TLR4 were significantly increased on days 2 and 6 in allogeneic grafts. Although fully mismatched and F1 grafts were rejected by C57Bl/6 recipients (mean survival time=8.2 and 9.3 days, respectively), graft survival was significantly prolonged in TLR4−/− recipients (mean survival time=10.6 and 14.3 days, respectively). Proinflammatory cytokines were markedly reduced in TLR4−/− graft recipients. Conclusions. Small intestine graft survival is prolonged in the absence of TLR4, suggesting that gut flora associated with the graft may augment alloimmune responses through TLR4. Thus, the TLR pathway may be a novel therapeutic target for improving SIT allograft survival.

Syk‐Induced Phosphatidylinositol‐3‐Kinase Activation in Epstein–Barr Virus Posttransplant Lymphoproliferative Disorder

Posttransplant lymphoproliferative disorder (PTLD)‐associated Epstein–Barr virus (EBV)+ B cell lymphomas are serious complications of solid organ and bone marrow transplantation. The EBV protein LMP2a, a B cell receptor (BCR) mimic, provides survival signals to virally infected cells through Syk tyrosine kinase. Therefore, we explored whether Syk inhibition is a viable therapeutic strategy for EBV‐associated PTLD. We have shown that R406, the active metabolite of the Syk inhibitor fostamatinib, induces apoptosis and cell cycle arrest while decreasing downstream phosphatidylinositol‐3′‐kinase (PI3K)/Akt signaling in EBV+ B cell lymphoma PTLD lines in vitro. However, Syk inhibition did not inhibit or delay the in vivo growth of solid tumors established from EBV‐infected B cell lines. Instead, we observed tumor growth in adjacent inguinal lymph nodes exclusively in fostamatinib‐treated animals. In contrast, direct inhibition of PI3K/Akt significantly reduced tumor burden in a xenogeneic mouse model of PTLD without evidence of tumor growth in adjacent inguinal lymph nodes. Taken together, our data indicate that Syk activates PI3K/Akt signaling which is required for survival of EBV+ B cell lymphomas. PI3K/Akt signaling may be a promising therapeutic target for PTLD, and other EBV‐associated malignancies.

Natural Killer Cell-activating receptor NKG2D mediates innate immune targeting of allogeneic neural progenitor cell grafts

Cell replacement therapy holds promise for a number of untreatable neurological or psychiatric diseases but the immunogenicity of cellular grafts remains controversial. Emerging stem cell and reprogramming technologies can be used to generate autologous grafts that minimize immunological concerns but autologous grafts may carry an underlying genetic vulnerability that reduces graft efficacy or survival. Healthy allogeneic grafts are an attractive and commercially scalable alternative if immunological variables can be controlled. Stem cells and immature neural progenitor cells (NPC) do not express major histocompatibility complex (MHC) antigens and can evade adaptive immune surveillance. Nevertheless, in an experimental murine model, allogeneic NPCs do not survive and differentiate as well as syngeneic grafts, even when traditional immunosuppressive treatments are used. In this study, we show that natural killer (NK) cells recognize the lack of self‐MHC antigens on NPCs and pose a barrier to NPC transplantation. NK cells readily target both syngeneic and allogeneic NPC, and killing is modulated primarily by NK‐inhibiting “self” class I MHC and NK‐activating NKG2D‐ligand expression. The absence of NKG2D signaling in NK cells significantly improves NPC‐derived neuron survival and differentiation. These data illustrate the importance of innate immune mechanisms in graft outcome and the potential value of identifying and targeting NK cell‐activating ligands that may be expressed by stem cell derived grafts. STEM Cells 2013;31:2024‐2030