Platelets Induce Cell Apoptosis of Cardiac Cells via FasL after Acute Myocardial Infarction.

Acute myocardial infarction (AMI) is one of the leading causes of death worldwide. Cell apoptosis in the myocardium plays an important role in ischemia and reperfusion (I/R) injury, leading to cardiac damage and dysfunction. Platelets are major players in hemostasis and play a crucial role in vessel occlusion, inflammation, and cardiac remodeling after I/R. Here, we studied the impact of platelets on cell apoptosis in the myocardium using a close-chest mouse model of AMI. We found caspase-3-positive resident cardiac cells, while leukocytes were negative for caspase-3. Using two different mouse models of thrombocytopenia, we detected a significant reduction in caspase-3 positive cells in the infarct border zone after I/R injury. Further, we identified platelet FasL to induce cell apoptosis via the extrinsic pathway of Fas receptor activation of target cells. Mechanistically, hypoxia triggers platelet adhesion to FasR, suggesting that platelet-induced apoptosis is elevated after I/R. Platelet-specific FasL knock-out mice showed reduced Bax and Bcl2 expression, suggesting that platelets modulate the intrinsic and extrinsic pathways of apoptosis, leading to reduced infarct size after myocardial I/R injury. Thus, a new mechanism for how platelets contribute to tissue homeostasis after AMI was identified that should be validated in patients soon.

Intrinsic factors and CD1d1 but not CD1d2 expression levels control invariant natural killer T cell subset differentiation.

Invariant natural killer T (NKT) cell subsets are defined based on their cytokine-production profiles and transcription factors. Their distribution is different in C57BL/6 (B6) and BALB/c mice, with a bias for NKT1 and NKT2/NKT17 subsets, respectively. Here, we show that the non-classical class I-like major histocompatibility complex CD1 molecules CD1d2, expressed in BALB/c and not in B6 mice, could not account for this difference. We find however that NKT cell subset distribution is intrinsic to bone marrow derived NKT cells, regardless of syngeneic CD1d-ligand recognition, and that multiple intrinsic factors are likely involved. Finally, we find that CD1d expression levels in combination with T cell antigen receptor signal strength could also influence NKT cell distribution and function. Overall, this study indicates that CD1d-mediated TCR signals and other intrinsic signals integrate to influence strain-specific NKT cell differentiation programs and subset distributions.

Ambivalent role of FasL in murine acute graft-versus-host-disease.

Fas ligand is increased in several immune-mediated diseases, including acute graft-versus-host disease, a donor cell-mediated disorder post-hematopoietic stem cell transplantation. In this disease, Fas ligand is involved in T-cell-mediated damage to host tissues. However, the role of its expression on donor non-T cells has, so far, never been addressed. Using a well-established CD4- and CD8-mediated graft-versus-host disease murine model, we found that precocious gut damage and mice mortality are increased with a graft of donor T- and B-depleted bone marrow cells devoid of Fas ligand as compared with their wild-type counterparts. Interestingly, serum levels of both soluble Fas ligand and IL-18 are drastically reduced in the recipients of Fas ligand-deficient grafts, indicating that soluble Fas ligand stems from donor bone marrow-derived cells. In addition, the correlation between the concentrations of these 2 cytokines suggests that IL-18 production arises through a soluble Fas ligand-driven mechanism. These data highlight the importance of Fas ligand-dependent production in IL-18 production and in mitigating acute graft-versus-host disease. Overall, our data reveal the functional duality of Fas ligand according to its source.

CpG-Activated Regulatory B-Cell Progenitors Alleviate Murine Graft-Versus-Host-Disease.

Development of Graft Versus Host Disease (GVHD) represents a major impediment in allogeneic hematopoietic stem cell transplantation (HSCT). The observation that the presence of bone marrow and circulating hematogones correlated with reduced GVHD risks prompted us to evaluate whether B-cell progenitors, which provide protection in various autoimmune disease models following activation with the TLR-9 agonist CpG (CpG-proBs), could likewise reduce this allogeneic disorder. In a murine model of GVHD that recapitulates an initial phase of acute GVHD followed by a phase of chronic sclerodermatous GVHD, we found that CpG-proBs, adoptively transferred during the initial phase of disease, reduced the diarrhea score and mostly prevented cutaneous fibrosis. Progenitors migrated to the draining lymph nodes and to the skin where they mainly differentiated into follicular B cells. CpG activation and IFN-γ expression were required for the protective effect, which resulted in reduced CD4+ T-cell-derived production of critical cytokines such as TGF-ß, IL-13 and IL-21. Adoptive transfer of CpG-proBs increased the T follicular regulatory to T follicular helper (Tfr/Tfh) ratio. Moreover, CpG-proBs privileged the accumulation of IL-10-positive CD8+ T cells, B cells and dendritic cells in the skin. However, CpG-proBs did not improve survival. Altogether, our findings support the notion that adoptively transferred CpG-proBs exert immunomodulating effect that alleviates symptoms of GVHD but require additional anti-inflammatory strategy to improve survival.

Characterization of the developmental landscape of murine RORγt+ iNKT cells.

Invariant natural killer T (iNKT) cells expressing the retinoic acid receptor-related orphan receptor γt (RORγt) and producing IL-17 represent a minor subset of CD1d-restricted iNKT cells (iNKT17) in C57BL/6J (B6) mice. We aimed in this study to define the reasons for their low distribution and the sequence of events accompanying their normal thymic development. We found that RORγt+ iNKT cells have higher proliferation potential and a greater propensity to apoptosis than RORγt- iNKT cells. These cells do not likely reside in the thymus indicating that thymus emigration, and higher apoptosis potential, could contribute to RORγt+ iNKT cell reduced thymic distribution. Ontogeny studies suggest that mature HSAlow RORγt+ iNKT cells might develop through developmental stages defined by a differential expression of CCR6 and CD138 during which RORγt expression and IL-17 production capabilities are progressively acquired. Finally, we found that RORγt+ iNKT cells perceive a strong TCR signal that could contribute to their entry into a specific 'Th17 like' developmental program influencing their survival and migration. Overall, our study proposes a hypothetical thymic developmental sequence for iNKT17 cells, which could be of great use to study molecular mechanisms regulating this developmental program.

Fas Ligand Deficiency Impairs Tumor Immunity by Promoting an Accumulation of Monocytic Myeloid-Derived Suppressor Cells.

The Fas receptor ligand FasL regulates immune cell levels by inducing apoptosis of Fas receptor-positive cells. Here, we studied the impact of host FasL on tumor development in mice. Genetically targeting FasL in naïve mice increased myeloid cell populations, but, in marked contrast, it reduced the levels of myeloid-derived suppressor cells (MDSC) in mice bearing Lewis lung carcinoma tumors. Analysis of the MDSC subset distribution revealed that FasL deficiency skewed cell populations toward the M-MDSC subset, which displays a highly immunosuppressive activity. Furthermore, tumor-bearing mice that were FasL-deficient displayed an enhanced proportion of tumor-associated macrophages and regulatory T cells. Overall, the immunosuppressive environment produced by FasL targeting correlated with reduced survival of tumor-bearing mice. These results disclose a new role for FasL in modulating immunosuppressive cells.

Infiltration of circulating myeloid cells through CD95L contributes to neurodegeneration in mice.

Neuroinflammation is increasingly recognized as a hallmark of neurodegeneration. Activated central nervous system-resident microglia and infiltrating immune cells contribute to the degeneration of dopaminergic neurons (DNs). However, how the inflammatory process leads to neuron loss and whether blocking this response would be beneficial to disease progression remains largely unknown. CD95 is a mediator of inflammation that has also been proposed as an apoptosis inducer in DNs, but previous studies using ubiquitous deletion of CD95 or CD95L in mouse models of neurodegeneration have generated conflicting results. Here we examine the role of CD95 in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin (MPTP)-induced neurodegeneration using tissue-specific deletion of CD95 or CD95L. We show that DN death is not mediated by CD95-induced apoptosis because deletion of CD95 in DNs does not influence MPTP-induced neurodegeneration. In contrast, deletion of CD95L in peripheral myeloid cells significantly protects against MPTP neurotoxicity and preserves striatal dopamine levels. Systemic pharmacological inhibition of CD95L dampens the peripheral innate response, reduces the accumulation of infiltrating myeloid cells, and efficiently prevents MPTP-induced DN death. Altogether, this study emphasizes the role of the peripheral innate immune response in neurodegeneration and identifies CD95 as potential pharmacological target for neurodegenerative disease.

PD-L1 is a novel direct target of HIF-1α, and its blockade under hypoxia enhanced MDSC-mediated T cell activation.

Tumor-infiltrating myeloid cells such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) form an important component of the hypoxic tumor microenvironment. Here, we investigated the influence of hypoxia on immune checkpoint receptors (programmed death [PD]-1 and CTLA-4) and their respective ligands (PD-1 ligand 1 [PD-L1], PD-L2, CD80, and CD86) on MDSCs. We demonstrate that MDSCs at the tumor site show a differential expression of PD-L1 as compared with MDSCs from peripheral lymphoid organ (spleen). Hypoxia caused a rapid, dramatic, and selective up-regulation of PD-L1 on splenic MDSCs in tumor-bearing mice. This was not limited to MDSCs, as hypoxia also significantly increased the expression of PD-L1 on macrophages, dendritic cells, and tumor cells. Furthermore, PD-L1 up-regulation under hypoxia was dependent on hypoxia-inducible factor-1α (HIF-1α) but not HIF-2α. Chromatin immunoprecipitation and luciferase reporter assay revealed direct binding of HIF-1α to a transcriptionally active hypoxia-response element (HRE) in the PD-L1 proximal promoter. Blockade of PD-L1 under hypoxia enhanced MDSC-mediated T cell activation and was accompanied by the down-regulation of MDSCs IL-6 and IL-10. Finally, neutralizing antibodies against IL-10 under hypoxia significantly abrogated the suppressive activity of MDSCs. Simultaneous blockade of PD-L1 along with inhibition of HIF-1α may thus represent a novel approach for cancer immunotherapy.

Astrocytic Fas ligand expression is required to induce T-cell apoptosis and recovery from experimental autoimmune encephalomyelitis.

In T-cell-mediated autoimmune diseases of the CNS, apoptosis of Fas(+) T cells by FasL contributes to resolution of disease. However, the apoptosis-inducing cell population still remains to be identified. To address the role of astrocytic FasL in the regulation of T-cell apoptosis in experimental autoimmune encephalomyelitis, we immunized C57BL/6 glial fibrillary acid protein (GFAP)-Cre FasL(fl/fl) mice selectively lacking FasL in astrocytes with MOG(35-55) peptide. GFAP-Cre FasL(fl/fl) mice were unable to resolve EAE and suffered from persisting demyelination and paralysis, while FasL(fl/fl) control mice recovered. In contrast to FasL(fl/fl) mice, GFAP-Cre FasL(fl/fl) mice failed to induce apoptosis of Fas(+) activated CD4(+) T cells and to increase numbers of Foxp3(+) Treg cells beyond day 15 post immunization, the time point of maximal clinical disease in control mice. The persistence of activated and GM-CSF-producing CD4(+) T cells in GFAP-Cre FasL(fl/fl) mice also resulted in an increased IL-17, IFN-γ, TNF, and GM-CSF mRNA expression in the CNS. In vitro, FasL(+) but not FasL(-) astrocytes induced caspase-3 expression and apoptosis of activated T cells. In conclusion, FasL expression of astrocytes plays an important role in the control and elimination of autoimmune T cells from the CNS, thereby determining recovery from EAE.

Opposing roles for membrane bound and soluble Fas ligand in glaucoma-associated retinal ganglion cell death.

Glaucoma, the most frequent optic neuropathy, is a leading cause of blindness worldwide. Death of retinal ganglion cells (RGCs) occurs in all forms of glaucoma and accounts for the loss of vision, however the molecular mechanisms that cause RGC loss remain unclear. The pro-apoptotic molecule, Fas ligand, is a transmembrane protein that can be cleaved from the cell surface by metalloproteinases to release a soluble protein with antagonistic activity. Previous studies documented that constitutive ocular expression of FasL maintained immune privilege and prevented neoangeogenesis. We now show that FasL also plays a major role in retinal neurotoxicity. Importantly, in both TNFα triggered RGC death and a spontaneous model of glaucoma, gene-targeted mice that express only full-length FasL exhibit accelerated RGC death. By contrast, FasL-deficiency, or administration of soluble FasL, protected RGCs from cell death. These data identify membrane-bound FasL as a critical effector molecule and potential therapeutic target in glaucoma.

CD95-ligand on peripheral myeloid cells activates Syk kinase to trigger their recruitment to the inflammatory site.

Injury to the central nervous system initiates an uncontrolled inflammatory response that results in both tissue repair and destruction. Here, we showed that, in rodents and humans, injury to the spinal cord triggered surface expression of CD95 ligand (CD95L, FasL) on peripheral blood myeloid cells. CD95L stimulation of CD95 on these cells activated phosphoinositide 3-kinase (PI3K) and metalloproteinase-9 (MMP-9) via recruitment and activation of Syk kinase, ultimately leading to increased migration. Exclusive CD95L deletion in myeloid cells greatly decreased the number of neutrophils and macrophages infiltrating the injured spinal cord or the inflamed peritoneum after thioglycollate injection. Importantly, deletion of myeloid CD95L, but not of CD95 on neural cells, led to functional recovery of spinal injured animals. Our results indicate that CD95L acts on peripheral myeloid cells to induce tissue damage. Thus, neutralization of CD95L should be considered as a means to create a controlled beneficial inflammatory response.

Prevention of autoimmunity and control of recall response to exogenous antigen by Fas death receptor ligand expression on T cells.

Mice with mutations in the gene encoding Fas ligand (FasL) develop lymphoproliferation and systemic autoimmune diseases. However, the cellular subset responsible for the prevention of autoimmunity in FasL-deficient mice remains undetermined. Here, we show that mice with FasL loss on either B or T cells had identical life span as littermates, and both genotypes developed signs of autoimmunity. In addition, we show that T cell-dependent death was vital for the elimination of aberrant T cells and for controlling the numbers of B cells and dendritic cells that dampen autoimmune responses. Furthermore, we show that the loss of FasL on T cells affected the follicular dentritic cell network in the germinal centers, leading to an impaired recall response to exogenous antigen. These results disclose the distinct roles of cellular subsets in FasL-dependent control of autoimmunity and provide further insight into the role of FasL in humoral immunity.

Cutting edge: two distinct motifs within the Fas ligand tail regulate Fas ligand-mediated costimulation.

The cytoplasmic domain of Fas ligand is sufficient to costimulate CD8(+) T cells by driving Fas ligand recruitment into lipid rafts and association with select Src homology 3-containing proteins, activating PI3K and MAPK pathways, mediating nuclear translocation of the transcription factors NFAT and AP-1, and enhancing IFN-gamma production and Ag-specific CD8(+) T cell proliferation. We now show that Fas ligand molecules lacking amino acids 45-54 in the proline-rich region of the cytoplasmic domain fail to costimulate but serve as effective death inducers. Death induction and costimulation by Fas ligand are therefore clearly separable functions. Further, upon Fas ligand-mediated costimulation, casein kinase I phosphorylates Fas ligand, in which two conserved casein kinase I binding sites regulate NFAT activation and costimulation. These results help resolve how one molecule can serve as a double-edged immunomodulator by directing discrete biological consequences.

Complete loss of Fas ligand gene causes massive lymphoproliferation and early death, indicating a residual activity of gld allele.

To investigate the in vivo function of Fas ligand (FasL), we produced a mouse strain with a FasL gene flanked by loxP sequences. Mice with homozygous floxed FasL gene showed no obvious abnormalities. However, germline deletion of the FasL gene, obtained after mating with mice expressing ubiquitous Cre recombinase, resulted in an unexpectedly severe phenotype. FasL(-/-) mice exhibited an extreme splenomegaly and lymphadenopathy associated with lymphocytic infiltration into multiple organs and autoimmune disease. This severe phenotype led to the premature death at 4 mo of age of >50% of the homozygous mice. It stands in sharp contrast with the milder disease observed in gld (generalized lymphoproliferative disease) mice, indicating that the FasL allele of these mice encodes a protein still able to bind, albeit at a very low level, the Fas receptor.

Unexpected sensitivity of nonobese diabetic mice with a disrupted poly(ADP-Ribose) polymerase-1 gene to streptozotocin-induced and spontaneous diabetes.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that consumes NAD in response to DNA strand breaks. Its excessive activation seems particularly deleterious to pancreatic beta-cells, as exemplified by the complete resistance of PARP-1-deficient mice to the toxic diabetes induced by streptozotocin. Because of the possible implication of this enzyme in type 1 diabetes, many human trials using nicotinamide, an inhibitor of PARP-1, have been conducted either in patients recently diagnosed or in subjects highly predisposed to this disease. To analyze the role of this enzyme in murine type 1 diabetes, we introgressed a disrupted PARP-1 allele onto the autoimmune diabetes-prone nonobese diabetic (NOD) mouse strain. We showed that these mice were protected neither from spontaneous nor from cyclophosphamide-accelerated diabetes. Surprisingly they were also highly sensitive to the diabetes induced by a single high dose of streptozotocin, standing in sharp contrast with C57BL/6 mice that bear the same inactivated PARP-1 allele. Our results suggest that NOD mice are characterized not only by their immune dysfunction but also by a peculiarity of their islets leading to a PARP-1-independent mechanism of streptozotocin-induced beta-cell death.