A zebrafish model of CLN2 disease is deficient in tripeptidyl peptidase 1 and displays progressive neurodegeneration accompanied by a reduction in proliferation.

Tripeptidyl peptidase 1 (TPP1) deficiency causes CLN2 disease, late infantile (or classic late infantile neuronal ceroid lipofuscinosis), a paediatric neurodegenerative disease of autosomal recessive inheritance. Patients suffer from blindness, ataxia, epilepsy and cognitive defects, with MRI indicating widespread brain atrophy, and profound neuron loss is evident within the retina and brain. Currently there are no effective therapies for this disease, which causes premature death in adolescence. Zebrafish have been successfully used to model a range of neurological and behavioural abnormalities. The aim of this study was to characterize the pathological and functional consequences of Tpp1 deficiency in zebrafish and to correlate these with human CLN2 disease, thereby providing a platform for drug discovery. Our data show that homozygous tpp1(sa0011) mutant (tpp1(sa0011)(-/-)) zebrafish display a severe, progressive, early onset neurodegenerative phenotype, characterized by a significantly small retina, a small head and curved body. The mutant zebrafish have significantly reduced median survival with death occurring 5 days post-fertilization. As in human patients with CLN2 disease, mutant zebrafish display storage of subunit c of mitochondrial ATP-synthase, hypertrophic lysosomes as well as localized apoptotic cell death in the retina, optic tectum and cerebellum. Further neuropathological phenotypes of these mutants provide novel insights into mechanisms of pathogenesis in CLN2 disease. Secondary neurogenesis in the retina, optic tectum and cerebellum is impaired and axon tracts within the spinal cord, optic nerve and the posterior commissure are disorganized, with the optic nerve failing to reach its target. This severe neurodegenerative phenotype eventually results in functional motor impairment, but this is preceded by a phase of hyperactivity that is consistent with seizures. Importantly, both of these locomotion phenotypes can be assayed in an automated manner suitable for high-throughput studies. Our study provides proof-of-principle that tpp1(sa0011)(-/-) mutants can utilize the advantages of zebrafish for understanding pathogenesis and drug discovery in CLN2 disease and other epilepsies.

Cutting edge: cell-extrinsic immune regulation by CTLA-4 expressed on conventional T cells.

The CTLA-4 pathway is a key regulator of T cell activation and a critical failsafe against autoimmunity. Although early models postulated that CTLA-4 transduced a negative signal, in vivo evidence suggests that CTLA-4 functions in a cell-extrinsic manner. That multiple cell-intrinsic mechanisms have been attributed to CTLA-4, yet its function in vivo appears to be cell-extrinsic, has been an ongoing paradox in the field. Although CTLA-4 expressed on conventional T cells (Tconv) can mediate inhibitory function, it is unclear why this fails to manifest as an intrinsic effect. In this study, we show that Tconv-expressed CTLA-4 can function in a cell-extrinsic manner in vivo. CTLA-4(+/+) T cells, from DO11/rag(-/-) mice that lack regulatory T cells, were able to regulate the response of CTLA-4(-/-) T cells in cotransfer experiments. This observation provides a potential resolution to the above paradox and suggests CTLA-4 function on both Tconv and regulatory T cells can be achieved through cell-extrinsic mechanisms.

Cutting edge: CTLA-4 on effector T cells inhibits in trans.

CTLA-4 is thought to inhibit effector T cells both intrinsically, by competing with CD28 for B7 ligands, and extrinsically, through the action of regulatory T cells (Tregs). We studied in vivo responses of normal and CTLA-4-deficient Ag-specific murine effector CD4(+) T cells. We directly demonstrate that effector T cell-restricted CTLA-4 inhibits T cell responses in a cell-extrinsic manner. Cotransfer experiments show that CTLA-4 on normal effector CD4(+) T cells completely abrogates the dramatically increased expansion normally experienced by their CTLA-4-deficient counterparts. Neither the wild-type nor the CTLA-4-deficient T cells express the Treg transcription factor Foxp3 when transferred alone or together. Thus, cell-extrinsic inhibition of T cell responses by CTLA-4 is not limited to Tregs but is also a function of effector T cells.

Protein kinase C-θ inhibits inducible regulatory T cell differentiation via an AKT-Foxo1/3a-dependent pathway.

Protein kinase C (PKC)-θ has been shown to be a critical TCR signaling molecule that promotes the activation and differentiation of naive T cells into inflammatory effector T cells. In this study, we demonstrate that PKC-θ-mediated signals inhibit inducible regulatory T cell (iTreg) differentiation via an AKT-Foxo1/3A pathway. TGF-ß-induced iTreg differentiation was enhanced in PKC-θ(-/-) T cells or wild-type cells treated with a specific PKC-θ inhibitor, but was inhibited by the PKC-θ activator PMA, or by CD28 crosslinking, which enhances PKC-θ activation. PKC-θ(-/-) T cells had reduced activity of the AKT kinase, and the expression of a constitutively active form of AKT in PKC-θ(-/-) T cells restored the ability to inhibit iTreg differentiation. Furthermore, knockdown or overexpression of the AKT downstream targets Foxo1 and Foxo3a was found to inhibit or promote iTreg differentiation in PKC-θ(-/-) T cells accordingly, indicating that the AKT-Foxo1/3A pathway is responsible for the inhibition of iTreg differentiation of iTregs downstream of PKC-θ. We conclude that PKC-θ is able to control T cell-mediated immune responses by shifting the balance between the differentiation of effector T cells and inhibitory Tregs.

IFN-γ promotes muscle damage in the mdx mouse model of Duchenne muscular dystrophy by suppressing M2 macrophage activation and inhibiting muscle cell proliferation.

Duchenne muscular dystrophy is a degenerative disorder that leads to death by the third decade of life. Previous investigations have shown that macrophages that invade dystrophic muscle are a heterogeneous population consisting of M1 and M2 macrophages that promote injury and repair, respectively. In the present investigation, we tested whether IFN-γ worsens the severity of mdx dystrophy by activating macrophages to a cytolytic M1 phenotype and by suppressing the activation of proregenerative macrophages to an M2 phenotype. IFN-γ is a strong inducer of the M1 phenotype and is elevated in mdx dystrophy. Contrary to our expectations, null mutation of IFN-γ caused no reduction of cytotoxicity of macrophages isolated from mdx muscle and did not reduce muscle fiber damage in vivo or improve gross motor function of mdx mice at the early, acute peak of pathology. In contrast, ablation of IFN-γ reduced muscle damage in vivo during the regenerative stage of the disease and increased activation of the M2 phenotype and improved motor function of mdx mice at that later stage of the disease. IFN-γ also inhibited muscle cell proliferation and differentiation in vitro, and IFN-γ mutation increased MyoD expression in mdx muscle in vivo, showing that IFN-γ can have direct effects on muscle cells that could impair repair. Taken together, the findings show that suppression of IFN-γ signaling in muscular dystrophy reduces muscle damage and improves motor performance by promoting the M2 macrophage phenotype and by direct actions on muscle cells.

The NF-κB transcription factor c-Rel is required for Th17 effector cell development in experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease involving effector Th subsets such as Th1 and Th17. In this study, we demonstrate that mice lacking the NF-κB transcription factor family member c-Rel (rel(-/-)), which are known to be resistant to EAE, show impaired Th17 development. Mixed bone marrow chimeras and EAE adoptive transfer experiments show that the deficiency of effector Th17 cells in rel(-/-) mice is T cell intrinsic. Consistent with this finding, c-Rel was activated in response to TCR signaling in the early stages of Th17 development and controlled the expression of Rorc, which encodes the Th17 transcription factor retinoic acid-related orphan receptor γt. CD28, but not IL-2, repression of Th17 development was dependent on c-Rel, implicating a dual role for c-Rel in modulating Th17 development. Adoptive transfer experiments also suggested that c-Rel control of regulatory T cell differentiation and homeostasis influences EAE development and severity by influencing the balance between Th17 and regulatory T cells. Collectively, our findings indicate that in addition to promoting Th1 differentiation, c-Rel regulates the development and severity of EAE via multiple mechanisms that impact on the generation of Th17 cells.

Apolipoprotein A-II suppressed concanavalin A-induced hepatitis via the inhibition of CD4 T cell function.

Con A-induced hepatitis has been used as a model of human autoimmune or viral hepatitis. During the process of identifying immunologically bioactive proteins in human plasma, we found that apolipoprotein A-II (ApoA-II), the second major apolipoprotein of high-density lipoprotein, inhibited the production of IFN-γ by Con A-stimulated mouse and human CD4 T cells. Con A-induced hepatitis was attenuated by the administration of ApoA-II. The beneficial effect of ApoA-II was associated with reduced leukocyte infiltration and decreased production of T cell-related cytokines and chemokines in the liver. ApoA-II inhibited the Con A-induced activation of ERK-MAPK and nuclear translocation of NFAT in CD4 T cells. Interestingly, exacerbated hepatitis was observed in ApoA-II-deficient mice, indicating that ApoA-II plays a suppressive role in Con A-induced hepatitis under physiological conditions. Moreover, the administration of ApoA-II after the onset of Con A-induced hepatitis was sufficient to suppress disease. Thus, the therapeutic effect of ApoA-II could be useful for patients with CD4 T cell-related autoimmune and viral hepatitis.

GCN5 regulates the superoxide-generating system in leukocytes via controlling gp91-phox gene expression.

The superoxide anion (O(2)(-))-generating system is an important mechanism of innate immune response against microbial infection in phagocytes and is involved in signal transduction mediated by various physiological and pathological signals in phagocytes and other cells, including B lymphocytes. The O(2)(-)-generating system is composed of five specific proteins: p22-phox, gp91-phox, p40-phox, p47-phox, p67-phox, and a small G protein, Rac. Little is known regarding epigenetic regulation of the genes constituting the O(2)(-)-generating system. In this study, by analyzing the GCN5 (one of most important histone acetyltransferases)-deficient DT40 cell line, we show that GCN5 deficiency causes loss of the O(2)(-)-generating activity. Interestingly, transcription of the gp91-phox gene was drastically downregulated (to ∼4%) in GCN5-deficient cells. To further study the involvement of GCN5 in transcriptional regulation of gp91-phox, we used in vitro differentiation system of U937 cells. When human monoblastic U937 cells were cultured in the presence of IFN-γ, transcription of gp91-phox was remarkably upregulated, and the cells were differentiated to macrophage-like cells that can produce O(2)(-). Chromatin immunoprecipitation assay using the U937 cells during cultivation with IFN-γ revealed not only that association of GCN5 with the gp91-phox gene promoter was significantly accelerated, but also that GCN5 preferentially elevated acetylation levels of H2BK16 and H3K9 surrounding the promoter. These results suggested that GCN5 regulates the O(2)(-)-generating system in leukocytes via controlling the gp91-phox gene expression as a supervisor. Our findings obtained in this study should be useful in understanding the molecular mechanisms involved in epigenetic regulation of the O(2)(-)-generating system in leukocytes.

DAP12 promotes IRAK-M expression and IL-10 production by liver myeloid dendritic cells and restrains their T cell allostimulatory ability.

Freshly isolated hepatic dendritic cells (DC) are comparatively immature, relatively resistant to maturation, and can downmodulate effector T cell responses. Molecular mechanisms that underlie these properties are ill defined. DNAX-activating protein of 12 kDa (DAP12) is an ITAM-bearing transmembrane adaptor protein that integrates signals through several receptors, including triggering receptor expressed on myeloid cells-1, -2, and CD200R. Notably, DC propagated from DAP12-deficient mice exhibit enhanced maturation in response to TLR ligation. Given the constitutive exposure of liver DC to endotoxin draining from the gut, we hypothesized that DAP12 might regulate liver DC maturation. We show that DAP12 is expressed by freshly isolated liver, spleen, kidney, and lung myeloid DC. Moreover, inhibition of DAP12 expression by liver DC using small interfering RNA promotes their phenotypic and functional maturation, resulting in enhanced TNF-α, IL-6, and IL-12p70 production, reduced secretion of IL-10, and enhanced CD4(+) and CD8(+) T cell proliferation. Furthermore, DAP12 silencing correlates with decreased STAT3 phosphorylation in mature liver DC and with diminished expression of the IL-1R-associated kinase-M, a negative regulator of TLR signaling. These findings highlight a regulatory role for DAP12 in hepatic DC maturation, and suggest a mechanism whereby this function may be induced/maintained.

The Src-like adaptor protein regulates GM-CSFR signaling and monocytic dendritic cell maturation.

GM-CSF is an important cytokine involved in myeloid differentiation and inflammatory processes. Signaling through the GM-CSFR also plays a critical role in the generation of monocyte-derived dendritic cells (DC). In this article, we report that the Src-like adaptor protein (SLAP) functions as a negative regulator of the GM-CSFR. In bone marrow-derived DC (BM-DC) lacking SLAP and the closely related SLAP2, downregulation of GM-CSFRß is impaired, leading to enhanced phosphorylation of Jak2 and prolonged activation of Akt and Erk1/2 in response to GM-CSF stimulation. Compared with wild-type bone marrow, SLAP/SLAP2(-/-) bone marrow gave rise to similar numbers of CD11c(+) and CD11b(+) DC, but SLAP/SLAP2(-/-) BM-DC failed to acquire high levels of MHC class II, CD80, and CD86, indicating an impairment in maturation. Furthermore, MHC class II expression in SLAP/SLAP2(-/-) BM-DC was rescued by decreasing GM-CSF concentration, suggesting that enhanced GM-CSF signaling mediates the block in maturation. In addition, SLAP/SLAP2(-/-) BM-DC produced less IL-12 and TNF-α in response to LPS compared with controls and failed to stimulate T cells in an MLR. Ag-specific T cell activation assays showed that SLAP/SLAP2(-/-) BM-DC were less robust at inducing IFN-γ secretion by DO11.10 T cells. These results indicated that SLAP-mediated GM-CSFR regulation is important for the generation of functionally mature monocytic DC.

Cutting edge: TIGIT has T cell-intrinsic inhibitory functions.

Costimulatory molecules regulate the functional outcome of T cell activation, and disturbance of the balance between activating and inhibitory signals results in increased susceptibility to infection or the induction of autoimmunity. Similar to the well-characterized CD28/CTLA-4 costimulatory pathway, a newly emerging pathway consisting of CD226 and T cell Ig and ITIM domain (TIGIT) has been associated with susceptibility to multiple autoimmune diseases. In this study, we examined the role of the putative coinhibitory molecule TIGIT and show that loss of TIGIT in mice results in hyperproliferative T cell responses and increased susceptibility to autoimmunity. TIGIT is thought to indirectly inhibit T cell responses by the induction of tolerogenic dendritic cells. By generating an agonistic anti-TIGIT Ab, we demonstrate that TIGIT can inhibit T cell responses directly independent of APCs. Microarray analysis of T cells stimulated with agonistic anti-TIGIT Ab revealed that TIGIT can act directly on T cells by attenuating TCR-driven activation signals.

A critical role for mast cells and mast cell-derived IL-6 in TLR2-mediated inhibition of tumor growth.

Several TLR agonists are effective in tumor immunotherapy, but their early innate mechanisms of action, particularly those of TLR2 agonists, are unclear. Mast cells are abundant surrounding solid tumors where they are often protumorigenic and enhance tumor angiogenesis. However, antitumor roles for mast cells have also been documented. The impact of mast cells may be dependent on their activation status and mediator release in different tumors. Using an orthotopic melanoma model in wild-type C57BL/6 and mast cell-deficient Kit(W-sh/W-sh) mice and a complementary Matrigel-tumor model in C57BL/6 mice, mast cells were shown to be crucial for TLR2 agonist (Pam(3)CSK(4))-induced tumor inhibition. Activation of TLR2 on mast cells reversed their well-documented protumorigenic role. Tumor growth inhibition after peritumoral administration of Pam(3)CSK(4) was restored in Kit(W-sh/W-sh) mice by local reconstitution with wild-type, but not TLR2-deficient, mast cells. Mast cells secrete multiple mediators after Pam(3)CSK(4) activation, and in vivo mast cell reconstitution studies also revealed that tumor growth inhibition required mast cell-derived IL-6, but not TNF. Mast cell-mediated anticancer properties were multifaceted. Direct antitumor effects in vitro and decreased angiogenesis and recruitment of NK and T cells in vivo were observed. TLR2-activated mast cells also inhibited the growth of lung cancer cells in vivo. Unlike other immune cells, mast cells are relatively radioresistant making them attractive candidates for combined treatment modalities. This study has important implications for the design of immunotherapeutic strategies and reveals, to our knowledge, a novel mechanism of action for TLR2 agonists in vivo.

SHP-1 in T cells limits the production of CD8 effector cells without impacting the formation of long-lived central memory cells.

During responses against viruses and malignancies, naive CD8 T lymphocytes expand to form both short-lived effector cells and a population containing cells with the potential to be long-lived and participate in memory responses (memory precursor effector cells). The strength of antigenic, costimulatory, and cytokine signals during responses impacts the magnitude and type of CD8 populations formed. In vitro studies have revealed that the tyrosine phosphatase Src homology region 2 domain-containing phosphatase-1 (SHP-1) regulates signal transduction from receptors on T cells including the TCR, helping set the activation threshold, and therefore may shape responses of mature CD8 T cells in vivo. Analysis of CD8 T cells from motheaten mice, which are globally deficient in SHP-1, proved problematic due to cell-extrinsic effects of SHP-1 deficiency in non-T cells on CD8 T cells. Therefore, a conditional knockout of SHP-1 in mature single-positive T cells was developed to analyze cell-intrinsic consequences of complete and partial SHP-1 deficiency on CD8 T cell responses to acute viral infection. The results demonstrated that SHP-1 has disparate effects on subpopulations of responding cells, limiting the magnitude and quality of primary and secondary responses by reducing the number of short-lived effector cells generated without affecting the size of the memory precursor effector cell pool that leads to formation of long-term memory.

IL-10 restricts memory T cell inflation during cytomegalovirus infection.

The beta-herpesvirus CMV induces a substantial and progressive expansion of virus-specific memory CD8 T cells, which protect the host against viral reactivation from latency. In this paper, we report that this expansion, or "inflation," of memory T cells is amplified dramatically during mouse CMV infection of IL-10 knockout (IL-10(-/-)) mice. T cells from IL-10(-/-) mice were oligoclonal, exhibited a highly activated phenotype, expressed antiviral cytokines, and degranulated in response to cognate Ag encounter ex vivo. Moreover, latent viral load was reduced in IL-10(-/-) mice. Importantly, these results were recapitulated by IL-10R blockade during chronic/latent infection of wild-type mice. These data demonstrate that regulatory immune mechanisms can influence CMV-specific T cell memory and suggest a possible rationale for the acquisition of functional IL-10 orthologs by herpesviruses.

Targeted knockdown of SEPT9_v1 inhibits tumor growth and angiogenesis of human prostate cancer cells concomitant with disruption of hypoxia-inducible factor-1 pathway.

Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor in the hypoxic response pathway. We recently identified a novel interaction between HIF-1alpha and the mammalian septin family member, septin 9 protein, isoform 1 (SEPT9_i1), a protein product of septin 9 transcript variant 1 (SEPT9_v1). Septins are a highly conserved family of GTP-binding cytoskeletal proteins that are implicated in multiple cellular functions, including oncogenesis. SEPT9_i1 binds and stabilizes HIF-1alpha protein and stimulates HIF-1 transcriptional activity by preventing its RACK1-mediated ubiquitination and degradation. SEPT9_i1-HIF-1 activation promotes tumor growth and angiogenesis. The effect of SEPT9_v1 silencing in prostate cancer cells was studied. SEPT9_v1 stable knockdown was generated in PC-3 cells using a specific shRNA. SEPT9_v1 silencing reduced HIF-1alpha protein expression and inhibited HIF-1 transcriptional activity. SEPT9_v1 knockdown affected cell morphology, deregulated cell cycle, and decreased migration. The antiproliferative effect of shSEPT9_v1 was abolished in HIF-1alpha knockout colon cancer cells. In vivo, SEPT9_i1 depletion reduced HIF-1alpha protein expression, cellular proliferation, tumor growth, and angiogenesis. These results provide new insights and validation for applying SEPT9_v1 as a potential target for antitumor therapy by interrupting the HIF-1 pathway.

Estradiol and G1 reduce infarct size and improve immunosuppression after experimental stroke.

Reduced risk and severity of stroke in adult females is thought to depend on normal endogenous levels of estrogen, a well-known neuroprotectant and immunomodulator. In male mice, experimental stroke induces immunosuppression of the peripheral immune system, characterized by a reduction in spleen size and cell numbers and decreased cytokine and chemokine expression. However, stroke-induced immunosuppression has not been evaluated in female mice. To test the hypothesis that estradiol (E2) deficiency exacerbates immunosuppression after focal stroke in females, we evaluated the effect of middle cerebral artery occlusion on infarct size and peripheral and CNS immune responses in ovariectomized mice with or without sustained, controlled levels of 17-beta-E2 administered by s.c. implant or the putative membrane estrogen receptor agonist, G1. Both E2- and G1-replacement decreased infarct volume and partially restored splenocyte numbers. Moreover, E2-replacement increased splenocyte proliferation in response to stimulation with anti-CD3/CD28 Abs and normalized aberrant mRNA expression for cytokines, chemokines, and chemokine receptors and percentage of CD4(+)CD25(+)FoxP3(+) T regulatory cells observed in E2-deficient animals. These beneficial changes in peripheral immunity after E2 replacement were accompanied by a profound reduction in expression of the chemokine, MIP-2, and a 40-fold increased expression of CCR7 in the lesioned brain hemisphere. These results demonstrate for the first time that E2 replacement in ovariectomized female mice improves stroke-induced peripheral immunosuppression.

Notch2 signaling is required for potent antitumor immunity in vivo.

CD8(+) T cells play a central role in cancer immunosurveillance, and the efficient induction of CTLs against tumor Ags is required for successful immunotherapy for cancer patients. Notch signaling directly regulates the transcription of effector molecules in CTLs. However, it remains unclear whether Notch signaling in CD8(+) T cells is required for antitumor CTL responses and whether modulation of Notch signaling can augment antitumor CTL responses. In this study, we demonstrate that signaling by Notch2 but not Notch1 in CD8(+) T cells is required for antitumor CTL responses. Notch2(flox/flox) mice crossed with E8I-cre transgenic (N2F/F-E8I) mice, in which the Notch2 gene is absent only in CD8(+) T cells, die earlier than control mice after inoculation with OVA-expressing EG7 thymoma cells. In contrast, Notch1(flox/flox) mice crossed with E8I-cre transgenic mice inoculated with EG7 cells die comparable to control mice, indicating that Notch2 is crucial for exerting antitumor CTL responses. Injection of anti-Notch2 agonistic Ab or delta-like 1-overexpressing dendritic cells augmented the antitumor response in C57BL/6 mice inoculated with EG7 cells. These findings indicate that Notch2 signaling in CD8(+) T cells is required for generating potent antitumor CTLs, thus providing a crucial target for augmenting tumor immune responses.

Adenosine A1 receptor activation as a brake on the microglial response after experimental traumatic brain injury in mice.

We reported that adenosine A(1) receptor (A(1)AR) knockout (KO) mice develop lethal status epilepticus after experimental traumatic brain injury (TBI), which is not seen in wild-type (WT) mice. Studies in epilepsy, multiple sclerosis, and neuro-oncology suggest enhanced neuro-inflammation and/or neuronal death in A(1)AR KO. We hypothesized that A(1)AR deficiency exacerbates the microglial response and neuronal damage after TBI. A(1)AR KO and WT littermates were subjected to mild controlled cortical impact (3 m/sec; 0.5 mm depth) to left parietal cortex, an injury level below the acute seizure threshold in the KO. At 24 h or 7 days, mice were sacrificed and serial sections prepared. Iba-1 immunostaining was used to quantify microglia at 7 days. To assess neuronal injury, sections were stained with Fluoro-Jade C (FJC) at 24 h to evaluate neuronal death in the hippocampus and cresyl violet staining at 7 days to analyze cortical lesion volumes. We also studied the effects of adenosine receptor agonists and antagonists on (3)H-thymidine uptake (proliferation index) by BV-2 cells (immortalized mouse microglial). There was no neuronal death in CA1 or CA3 quantified by FJC. A(1)AR KO mice exhibited enhanced microglial response; specifically, Iba-1 + microglia were increased 20-50% more in A(1)AR KO versus WT in ipsilateral cortex, CA3, and thalamus, and contralateral cortex, CA1, and thalamus (p < 0.05). However, contusion and cortical volumes did not differ between KO and WT. Pharmacological studies in cultured BV-2 cells indicated that A(1)AR activation inhibits microglial proliferation. A(1)AR activation is an endogenous inhibitor of the microglial response to TBI, likely via inhibition of proliferation, and this may represent a therapeutic avenue to modulate microglia after TBI.

SHIP is required for dendritic cell maturation.

Although several groups have investigated the role of SHIP in macrophage (M) development and function, SHIP's contribution to the generation, maturation, and innate immune activation of dendritic cells (DCs) is poorly understood. We show herein that SHIP negatively regulates the generation of DCs from bone marrow precursors in vitro and in vivo, as illustrated by the enhanced expansion of DCs from SHIP(-/-) GM-CSF cultures, as well as increased numbers of DCs in the spleens of SHIP-deficient mice. Interestingly, however, these SHIP(-/-) DCs display a relatively immature phenotype and secrete substantially lower levels of IL-12 after TLR ligand stimulation than wild type DCs. This, in turn, leads to a dramatically reduced stimulation of Ag-specific T cell proliferation and Th1 cell responses in vitro and in vivo. This immature phenotype of SHIP(-/-) DCs could be reversed with the PI3K inhibitors LY294002 and wortmannin, suggesting that SHIP promotes DC maturation by reducing the levels of the PI3K second messenger phosphatidylinositol-3,4,5-trisphosphate. These results are consistent with SHIP being a negative regulator of GM-CSF-derived DC generation but a positive regulator of GM-CSF-derived DC maturation and function.

IL-12 suppresses vascular endothelial growth factor receptor 3 expression on tumor vessels by two distinct IFN-gamma-dependent mechanisms.

IL-12 has been shown to be effective in enhancing antitumor responses. However, how IL-12 exerts its antiangiogenic effect is largely unknown. In this study, we elucidate this mechanism using B16 transfected to express IL-12 (B16/IL-12), a system that provides constant, local production of IL-12 within the tumor microenvironment. Intratumoral IL-12 resulted in a significant delay in tumor growth and phenotypic changes in the vasculature. Vessels found within B16 tumors are chaotic and poorly formed and express vascular endothelial growth factor receptor 3 (VEGFR3), a growth factor receptor not expressed on normal adult vessels. However, the vessels within B16/IL-12 tumors have a more normal morphology and do not express VEGFR3. We have shown that IFN-gamma is required for IL-12 to suppress the aberrant expression of VEGFR3. Indeed, the presence of intratumoral IL-12 stimulates the immune system resulting in more IFN-gamma-producing tumor-infiltrating lymphocytes per tumor when compared with parental B16 tumors, which may have a marked effect on control of tumor growth. Interestingly, within B16/IL-12 tumors, T cells are necessary to suppress VEGFR3 expression on tumor vessels. Finally, using IFN-gamma receptor knockout mice in a bone marrow chimera system, we show that the IFN-gamma produced within the tumor suppresses VEGFR3 expression in two ways: 1) acting directly on tumor vessel endothelial cells, and 2) acting on the tumor-infiltrating lymphocytes to indirectly alter endothelial cells' VEGFR3 expression. Our data indicate a mechanism in which tumor-infiltrating immune cells regulate tumor vessel phenotype.