Marwan Mounayar

TIM-3 Regulates Innate Immune Cells To Induce Fetomaternal Tolerance

TIM-3 is constitutively expressed on subsets of macrophages and dendritic cells. Its expression on other cells of the innate immune system and its role in fetomaternal tolerance has not yet been explored. In this study, we investigate the role of TIM-3–expressing innate immune cells in the regulation of tolerance at the fetomaternal interface (FMI) using an allogeneic mouse model of pregnancy. Blockade of TIM-3 results in accumulation of inflammatory granulocytes and macrophages at the uteroplacental interface and upregulation of proinflammatory cytokines. Furthermore, TIM-3 blockade inhibits the phagocytic potential of uterine macrophages resulting in a build up of apoptotic bodies at the uteroplacental interface that elicits a local immune response. In response to inflammatory cytokines, Ly-6ChiGneg monocytic myeloid–derived suppressor cells expressing inducible NO synthase and arginase 1 are induced. However, these suppressive cells fail to downregulate the inflammatory cascade induced by inflammatory granulocytes (Ly-6CintGhi) and apoptotic cells; the increased production of IFN-γ and TNF-α by inflammatory granulocytes leads to abrogation of tolerance at the FMI and fetal rejection. These data highlight the interplay between cells of the innate immune system at the FMI and their influence on successful pregnancy in mice.

Immunosuppressive Activity of Size-Controlled PEG-PLGA Nanoparticles Containing Encapsulated Cyclosporine A

We encapsulated cyclosporine A (CsA) in poly(ethylene glycol)-b-poly(d,l-lactide-co-glycolide) (PEG-PLGA) nanoparticles (NPs) by nanoprecipitation of CsA and PEG-PLGA. The resulting CsA/PEG-PLGA-NPs were <100 nm in diameter with a narrow particle size distribution. The NP size could be controlled by tuning the polymer concentration, solvent, or water/solvent ratio during formulation. The PEGylated NPs maintained non-aggregated in salt solution. Solid NPs lyoprotected with bovine serum albumin were prepared for the convenience of storage and transportation. The release kinetics of CsA (55.6% released on Day 1) showed potential for maintaining therapeutic CsA concentrations in vivo. In T-cell assays, both free CsA and CsA/PEG-PLGA-NPs suppressed T-cell proliferation and production of inflammatory cytokines dose dependently. In a mixed lymphocyte reaction assay, the IC50 values for free CsA and CsA/PEG-PLGA-NPs were found to be 30 and 35 ng/mL, respectively. This nanoparticulate CsA delivery technology constitutes a strong basis for future targeted delivery of immunosuppressive drugs with improved efficiency and potentially reduced toxicity.

The Novel Role of SERPINB9 in Cytotoxic Protection of Human Mesenchymal Stem Cells

Clinical trials using allogeneic mesenchymal stem cells (MSCs) are ongoing for the purpose of providing therapeutic benefit for a variety of human disorders. Pertinent to their clinical use are the accessibility to sufficient quantities of these cells allowing for repetitive administration, as well as a better understanding of the specific mechanisms by which allogeneic MSCs evade host immune responses that in turn influence their life span following administration. In this report, we sought to characterize and compare human peripheral blood MSCs (hPB-MSCs) with bone marrow-derived MSCs. hPB-MSCs met the established criteria to characterize this cellular lineage, including capacity for self-renewal, differentiation into tissues of mesodermal origin, and expression of phenotypic surface markers. In addition, hPB-MSCs suppressed alloreactive proliferation as well as the production of proinflammatory cytokines. Examination of the mechanisms by which allogeneic MSCs evade the host immune response, which is crucial for their therapeutic use, demonstrated that constitutive expression of serine protease inhibitor 9 (PI-9) on hPB-MSCs and bone marrow-derived MSCs is a major defense mechanism against granzyme B-mediated destruction by NK cells. Similarly, MSCs treated with small interfering RNA for PI-9 increased MSC cellular death, whereas expression of transgenic PI-9 following retroviral transduction protected MSCs. These data significantly advance our understanding of the immunomodulatory role for hPB-MSCs as well as the mechanisms by which they evade host immune responses. These findings contribute to the development of MSC-based therapies for diseases.

The Novel Therapeutic Effect of Phosphoinositide 3-Kinase-γ Inhibitor AS605240 in Autoimmune Diabetes

Type 1 diabetes (T1D) remains a major health problem worldwide, with a steadily rising incidence yet no cure. Phosphoinositide 3-kinase-γ (PI3Kγ), a member of a family of lipid kinases expressed primarily in leukocytes, has been the subject of substantial research for its role in inflammatory diseases. However, the role of PI3Kγ inhibition in suppressing autoimmune T1D remains to be explored. We tested the role of the PI3Kγ inhibitor AS605240 in preventing and reversing diabetes in NOD mice and assessed the mechanisms by which this inhibition abrogates T1D. Our data indicate that the PI3Kγ pathway is highly activated in T1D. In NOD mice, we found upregulated expression of phosphorylated Akt (PAkt) in splenocytes. Notably, T regulatory cells (Tregs) showed significantly lower expression of PAkt compared with effector T cells. Inhibition of the PI3Kγ pathway by AS605240 efficiently suppressed effector T cells and induced Treg expansion through the cAMP response element-binding pathway. AS605240 effectively prevented and reversed autoimmune diabetes in NOD mice and suppressed T-cell activation and the production of inflammatory cytokines by autoreactive T cells in vitro and in vivo. These studies demonstrate the key role of the PI3Kγ pathway in determining the balance of Tregs and autoreactive cells regulating autoimmune diabetes.

Serine Protease Inhibitor 6 Plays a Critical Role in Protecting Murine Granzyme B–Producing Regulatory T Cells

Regulatory T cells (Tregs) play a pivotal role in the maintenance of immune tolerance and hold great promise as cell therapy for a variety of immune-mediated diseases. However, the cellular mechanisms that regulate Treg maintenance and homeostasis have yet to be fully explored. Although Tregs express granzyme-B (GrB) to suppress effector T cells via direct killing, the mechanisms by which they protect themselves from GrB-mediated self-inflicted damage are unknown. To our knowledge, we show for the first time that both induced Tregs and natural Tregs (nTregs) increase their intracellular expression of GrB and its endogenous inhibitor, serine protease inhibitor 6 (Spi6) upon activation. Subcellular fractionation and measurement of GrB activity in the cytoplasm of Tregs show that activated Spi6−/− Tregs had significantly higher cytoplasmic GrB activity. We observed an increase in GrB-mediated apoptosis in Spi6−/− nTregs and impaired suppression of alloreactive T cells in vitro. Spi6−/− Tregs were rescued from apoptosis by the addition of a GrB inhibitor (Z-AAD-CMK) in vitro. Furthermore, adoptive transfer experiments showed that Spi6−/− nTregs were less effective than wild type nTregs in suppressing graft-versus-host disease because of their impaired survival, as shown in our in vivo bioluminescence imaging. Finally, Spi6-deficient recipients rejected MHC class II-mismatch heart allografts at a much faster rate and showed a higher rate of apoptosis among Tregs, as compared with wild type recipients. To our knowledge, our data demonstrate, for the first time, a novel role for Spi6 in Treg homeostasis by protecting activated Tregs from GrB-mediated injury. These data could have significant clinical implications for Treg-based therapy in immune-mediated diseases.

HCELL Expression on Murine MSC Licenses Pancreatotropism and Confers Durable Reversal of Autoimmune Diabetes in NOD Mice

Type 1 diabetes (T1D) is an immune‐mediated disease resulting in destruction of insulin‐producing pancreatic beta cells. Mesenchymal stem cells (MSCs) possess potent immunomodulatory properties, garnering increasing attention as cellular therapy for T1D and other immunologic diseases. However, MSCs generally lack homing molecules, hindering their colonization at inflammatory sites following intravenous (IV) administration. Here, we analyzed whether enforced E‐selectin ligand expression on murine MSCs could impact their effect in reversing hyperglycemia in nonobese diabetic (NOD) mice. Although murine MSCs natively do not express the E‐selectin‐binding determinant sialyl Lewisx (sLex), we found that fucosyltransferase‐mediated α(1,3)‐exofucosylation of murine MSCs resulted in sLex display uniquely on cell surface CD44 thereby creating hematopoietic cell E‐/L‐selectin ligand (HCELL), the E‐selectin‐binding glycoform of CD44. Following IV infusion into diabetic NOD mice, allogeneic HCELL+ MSCs showed threefold greater peri‐islet infiltrates compared to buffer‐treated (i.e., HCELL−) MSCs, with distribution in proximity to E‐selectin‐expressing microvessels. Exofucosylation had no effect on MSC immunosuppressive capacity in in vitro assays; however, although engraftment was temporary for both HCELL+ and HCELL− MSCs, administration of HCELL+ MSCs resulted in durable reversal of hyperglycemia, whereas only transient reversal was observed following administration of HCELL− MSCs. Notably, exofucosylation of MSCs generated from CD44−/− mice induced prominent membrane expression of sLex, but IV administration of these MSCs into hyperglycemic NOD mice showed no enhanced pancreatotropism or reversal of hyperglycemia. These findings provide evidence that glycan engineering to enforce HCELL expression boosts trafficking of infused MSCs to pancreatic islets of NOD mice and substantially improves their efficacy in reversing autoimmune diabetes. Stem Cells 2013;33:1523–1531

Impairment of Immune Systems in Diabetes

Type 1 diabetes mellitus (T1DM) is an autoimmune disease that involves the progressive destruction of the insulin-producing beta cells in the islets of langerhans. It is a complex process that results from the loss of tolerance to insulin and other beta-cell-specific antigens. Various genetic and environmental factors have been studied so far, but precise causation has yet to be established. Numerous studies in rodents and human subjects have been performed in order to elucidate the role of B and T cells, which determine the risk of development and progression of diabetes. These studies have demonstrated that while T1DM is fundamentally a T-cell-mediated autoimmune response, the development of this disease results from complex interactions between the adaptive and innate immune systems, with numerous cell types thought to contribute to pathogenesis. Like any complex disease, the variation in severity and incidence of T1DM can be attributed to a combination of genetic and environmental factors.

The mechanisms of up-regulation of dendritic cell activity by oxidative stress

Whereas DC have increasingly been recognized for their role in activating the inflammatory cascades during IRIs, the mechanisms by which oxidative stress enhances DC activation remain to be explored. We examined the role of oxidative stress on two important features of DC: T cell activation and trafficking. Bone marrow‐derived OS‐DC were compared with untreated DC. DC exposed to oxidative stress augmented allogeneic T cell proliferation and showed increased migration in a chemotaxis chamber. These results were confirmed by using hypoxanthine and xanthine oxidase as another inducer of oxidative stress. We used OT‐II and OT‐I mice to assess the effect of oxidative stress on DC activation of OVA‐specific CD4+ and CD8+ T cells, respectively. Oxidative stress increased DC capacity to promote OVA‐specific CD4+ T cell activity, demonstrated by an increase in their proliferation and production of IFN‐γ, IL‐6, and IL‐2 proinflammatory cytokines. Whereas oxidative stress increased the DC ability to stimulate IFN‐γ production by OVA‐specific CD8+ T cells, cellular proliferation and cytotoxicity were not affected. Compared with untreated DC, oxidative stress significantly reduced the capacity of DC to generate Tregs, which were restored by using anti‐IL‐6. With regard to DC trafficking, whereas oxidative stress increased DC expression of p‐Akt and p‐NF‐κB, targeting PI3Kγ and NF‐κB pathways abrogated the observed increase in DC migration. Our data propose novel insights on the activation of DC by oxidative stress and provide rationales for targeted therapies, which can potentially attenuate IRI.

Simultaneous In Vivo Monitoring of Regulatory and Effector T Lymphocytes Using Secreted Gaussia Luciferase, Firefly Luciferase, and Secreted Alkaline Phosphatase

Regulatory T cells (Tregs) are amongst the most widely studied cells in a variety of immune-mediated conditions, including transplantation and Graft Versus Host Disease (GVHD), cancer and autoimmunity; indeed, there is great interest in the tolerogenic potential of Treg-based therapy. Consequently, the need to establish the mechanisms that determine Treg survival and longevity, in addition to developing new tools to monitor these parameters, is paramount. Using both a mouse model of GVHD and a mouse model of Type 1 Diabetes (T1D), we describe herein a dual reporter system based on Gluc and multiplexed with SEAP and non-secreted Firefly luciferase (Fluc), which permits simultaneous imaging and noninvasive tracking of two different T-cell populations (CD4(+)CD25(+) Tregs and CD4(+)CD25(-) Tcon cells) in vivo by transducing the cells with different lentiviruses bearing distinct color signatures. This new technology promises to overcome the limitations of the conventional methods currently available to study lymphocyte survival in vivo. Furthermore, this novel technique has applications not only in autoimmunity and alloimmunity, but also in the wider field of immunology.

Utility of Mesenchymal Stem Cell Therapy in Type 1 Diabetes

Clinical trials are underway to test the therapeutic efficacy of mesenchymal stem cells (MSCs) in a number of diseases with no curative therapy available, including type 1 diabetes mellitus (T1D). Due to their plasticity, MSCs first gained interest in cell-based tissue engineering; however, given their low immunogenicity and profound immunodulatory effects, MSCs are now receiving much attention as a cell-based therapy for immune-mediated disorders. The use of MSCs in T1D holds potential in three different disciplines: systemic injection of MSCs to suppress autoreactive T cells; co-transplantation with islet allografts to enhance islet engraftment in an immuno-privileged microenvironment; and differentiation of MSCs into insulin-producing cells (IPC). We are focusing on the potential translation of MSCs therapy for clinical development to treat T1D.