Group2 innate lymphoid cells ameliorate renal fibrosis and dysfunction associated with adenine-induced CKD.

Renal fibrosis is a common pathway of chronic kidney disease (CKD) progression involving primary kidney injury and kidney diseases. Group 2 innate lymphoid cells (ILC2s) mediate type 2 immune responses irrespective of antigen presentation and play a reno-protective role in kidney injury and disease. In the present study, we observed a decrease in kidney-resident ILC2s in CKD and found that enrichment of ILC2s in the kidney ameliorates renal fibrosis. In CKD kidney, ILC2s preferentially produced IL-13 over IL-5 in response to IL-33 stimulation, regardless of ST2L expression. Moreover, GATA3 expression was decreased in ILC2s, and T-bet+ ILC1s and RORγt+ ILC3s were increased in CKD kidney. Adoptive transfer of kidney ILC2s into adenine-induced CKD model mouse improved renal function and fibrosis. Renal fibroblasts cultured with IL33-activated kidney ILC2s suppressed myofibroblast trans-differentiation through Acta2 and Fn-1 regulation. These results suggest that kidney ILC2s prevent CKD progression via improvement of renal fibrosis. Our findings also suggest that ILC2s may contribute to the development of new therapeutic agents and strategies for tissue fibroses.

NF-κB-inducing kinase contributes to normal development of cortical thymic epithelial cells: its possible role in shaping a proper T-cell repertoire.

Nuclear factor (NF)-κB-inducing kinase (NIK) is known to be a critical regulator of multiple aspects of the immune response. Although the role of NIK in the development of medullary thymic epithelial cells (mTECs) has been well documented, the impact of NIK on the differentiation and function of cortical thymic epithelial cells (cTECs) remains ambiguous. To investigate the possible involvement of NIK in cTEC differentiation, we have compared the gene expression and function of cTECs from a NIK-mutant mouse, alymphoplasia (aly/aly) with those of cTECs from wild-type (WT) mice. Flow cytometric analyses revealed that expression levels of MHC class II, but not MHC class I or other TEC markers, were higher in aly/aly cells than in WT cells. Notably, the proportion of MHC class IIhi+ cTECs was elevated in aly/aly mice. We also demonstrated that expression of Ccl5 mRNA in the MHC class IIhi+ subset of aly/aly cTECs was decreased compared with that in WT cells, implying an abnormal pattern of gene expression in aly/aly cTECs. Analyses of bone marrow chimera using aly/aly or aly/+ mice as hosts suggested that Vß usage and CD5 expression on WT T-cells were altered when they matured in aly/aly thymi. These results collectively indicate that NIK may be involved in controlling the function of cTEC in selecting a proper T-cell repertoire.

Leucine-Rich Repeat Kinase 2 Controls Inflammatory Cytokines Production through NF-κB Phosphorylation and Antigen Presentation in Bone Marrow-Derived Dendritic Cells.

Leucine-rich repeat kinase 2 (LRRK2) is the causal molecule of familial Parkinson's disease. Although the characteristics of LRRK2 have gradually been revealed, its true physiological functions remain unknown. LRRK2 is highly expressed in immune cells such as B2 cells and macrophages, suggesting that it plays important roles in the immune system. In the present study, we investigate the roles of LRRK2 in the immune functions of dendritic cells (DCs). Bone marrow-derived DCs from both C57BL/6 wild-type (WT) and LRRK2 knockout (KO) mice were induced by culture with granulocyte/macrophage-colony stimulating factor (GM/CSF) in vitro. We observed the differentiation of DCs, the phosphorylation of the transcriptional factors NF-κB, Erk1/2, and p-38 after lipopolysaccharide (LPS) stimulation and antigen-presenting ability by flow cytometry. We also analyzed the production of inflammatory cytokines by ELISA. During the observation period, there was no difference in DC differentiation between WT and LRRK2-KO mice. After LPS stimulation, phosphorylation of NF-κB was significantly increased in DCs from the KO mice. Large amounts of inflammatory cytokines were produced by DCs from KO mice after both stimulation with LPS and infection with Leishmania. CD4+ T-cells isolated from antigen-immunized mice proliferated to a significantly greater degree upon coculture with antigen-stimulated DCs from KO mice than upon coculture with DCs from WT mice. These results suggest that LRRK2 may play important roles in signal transduction and antigen presentation by DCs.

Inhibition of microsomal prostaglandin E synthase-1 facilitates liver repair after hepatic injury in mice.

BACKGROUND & AIMS: Liver repair following hepatic ischemia/reperfusion (I/R) injury is crucial to survival. This study aims to examine the role of endogenous prostaglandin E2 (PGE2) produced by inducible microsomal PGE synthase-1 (mPGES-1), a terminal enzyme of PGE2 generation, in liver injury and repair following hepatic I/R. METHODS: mPGES-1 deficient (Ptges-/-) mice or their wild-type (WT) counterparts were subjected to partial hepatic ischemia followed by reperfusion. The role of E prostanoid receptor 4 (EP4) was then studied using a genetic knockout model and a selective antagonist. RESULTS: Compared with WT mice, Ptges-/- mice exhibited reductions in alanine aminotransferase (ALT), necrotic area, neutrophil infiltration, chemokines, and proinflammatory cytokine levels. Ptges-/- mice also showed promoted liver repair and increased Ly6Clow macrophages (Ly6Clow/CD11bhigh/F4/80high-cells) with expression of anti-inflammatory and reparative genes, while WT mice exhibited delayed liver repair and increased Ly6Chigh macrophages (Ly6Chigh/CD11bhigh/F4/80low-cells) with expression of proinflammatory genes. Bone marrow (BM)-derived mPGES-1-deficient macrophages facilitated liver repair with increases in Ly6Clow macrophages. In vitro, mPGES-1 was expressed in macrophages polarized toward the proinflammatory profile. Mice treated with the mPGES-1 inhibitor Compound III displayed increased liver protection and repair. Hepatic I/R enhanced the hepatic expression of PGE receptor subtype, EP4, in WT mice, which was reduced in Ptges-/- mice. A selective EP4 antagonist and genetic deletion of Ptger4, which codes for EP4, accelerated liver repair. The proinflammatory gene expression was upregulated by stimulation of EP4 agonist in WT macrophages but not in EP4-deficient macrophages. CONCLUSIONS: These results indicate that mPGES-1 regulates macrophage polarization as well as liver protection and repair through EP4 signaling during hepatic I/R. Inhibition of mPGES-1 could have therapeutic potential by promoting liver repair after acute liver injury. LAY SUMMARY: Hepatic ischemia/reperfusion injury is a serious complication that occurs in liver surgery. Herein, we demonstrated that inducible prostaglandin E2 synthase (mPGES-1), an enzyme involved in synthesizing prostaglandin E2, worsens the injury and delays liver repair through accumulation of proinflammatory macrophages. Inhibition of mPGES-1 offers a potential therapy for both liver protection and repair in hepatic ischemia/reperfusion injury.

Role of T-bet, the master regulator of Th1 cells, in the cytotoxicity of murine CD4+ T cells.

Although CD4+ T cells are generally regarded as helper T cells, some activated CD4+ T cells have cytotoxic properties. Given that CD4+ cytotoxic T lymphocytes (CTLs) often secrete IFN-γ, CTL activity among CD4+ T cells may be attributable to Th1 cells, where a T-box family molecule, T-bet serves as the "master regulator". However, although the essential contribution of T-bet to expression of IFN-γ has been well-documented, it remains unclear whether T-bet is involved in CD4+ T cell-mediated cytotoxicity. In this study, to investigate the ability of T-bet to confer cytolytic activity on CD4+ T cells, the T-bet gene (Tbx21) was introduced into non-cytocidal CD4+ T cell lines and their cytolytic function analyzed. Up-regulation of FasL (CD178), which provided the transfectant with cytotoxicity, was observed in Tbx21transfected CD4+ T cells but not in untransfected parental cells. In one cell line, T-bet transduction also induced perforin gene (Prf1) expression and Tbx21 transfectants efficiently killed Fas- target cells. Although T-bet was found to repress up-regulation of CD40L (CD154), which controls FasL-mediated cytolysis, the extent of CD40L up-regulation on in vitro-differentiated Th1 cells was similar to that on Th2 cells, suggesting the existence of a compensatory mechanism. These results collectively indicate that T-bet may be involved in the expression of genes, such as FasL and Prf1, which confer cytotoxicity on Th1 cells.

Cooperation of Sox4 with ß-catenin/p300 complex in transcriptional regulation of the Slug gene during divergent sarcomatous differentiation in uterine carcinosarcoma.

BACKGROUND: Uterine carcinosarcoma (UCS) represents a true example of cancer associated with epithelial-mesenchymal transition (EMT), which exhibits cancer stem cell (CSC)-like traits. Both Sox and ß-catenin signal transductions play key roles in the regulation of EMT/CSC properties, but little is known about their involvement in UCS tumorigenesis. Herein, we focused on the functional roles of the Sox/ß-catenin pathway in UCSs. METHODS: EMT/CSC tests and transfection experiments were carried out using three endometrial carcinoma (Em Ca) cell lines. Immunohistochemical investigation was also applied for a total of 32 UCSs. RESULTS: Em Ca cells cultured in STK2, a serum-free medium for mesenchymal stem cells, underwent changes in morphology toward an EMT appearance through downregulation of E-cadherin, along with upregulation of Slug, known as a target gene of ß-catenin. The cells also showed CSC properties with an increase in the aldehyde dehydrogenase (ALDH) 1(high) activity population and spheroid formation, as well as upregulation of Sox4, Sox7, and Sox9. Of these Sox factors, overexpression of Sox4 dramatically led to transactivation of the Slug promoter, and the effects were further enhanced by cotransfection of Sox7 or Sox9. Sox4 was also able to promote ß-catenin-mediated transcription of the Slug gene through formation of transcriptional complexes with ß-catenin and p300, independent of TCF4 status. In clinical samples, both nuclear ß-catenin and Slug scores were significantly higher in the sarcomatous elements as compared to carcinomatous components in UCSs, and were positively correlated with Sox4, Sox7, and Sox9 scores. CONCLUSIONS: These findings suggested that Sox4, as well as Sox7 and Sox9, may contribute to regulation of EMT/CSC properties to promote development of sarcomatous components in UCSs through transcriptional regulation of the Slug gene by cooperating with the ß-catenin/p300 signal pathway.

Thromboxane A synthase enhances blood flow recovery from hindlimb ischemia.

BACKGROUND: Thromboxane A synthase (TXAS) is the enzyme that converts the arachidonic acid derivative prostaglandin H2 to thromboxane A2 (TXA2). TXA2 induces platelet aggregation, vasoconstriction, and proliferation. TXAS and TXA2 receptors or thromboxane prostanoid (TP) receptors are elevated in numerous cardiovascular and inflammatory diseases. Platelets contain numerous angiogenesis stimulating factors. However, the involvement of TXAS on recovery from an ischemic condition is not well understood. We hypothesized that the TXAS-TXA2-TP receptor axis would induce blood flow recovery by platelet activation. MATERIAL AND METHODS: The model of hindlimb ischemia was made by the right femoral artery ligation. The blood flow was estimated by laser Doppler images. Angiogenesis was estimated by the plasma level of the vascular endothelial growth factor and the stromal cell-derived factor-1 and by immunofluorescence analysis against CD31 and P-selectin glycoprotein ligand-1 (PSGL-1). RESULTS: In wild-type mice, blood flow recovery was enhanced by treatment with murine TXAS-overexpressing fibroblasts (C57-mTXAS) compared with empty vector- (EV) treated fibroblasts (C57-EV). Compared with C57-EV-treated mice, activated platelets (P-selectin(+) platelets) and plasma levels of vascular endothelial growth factor and stromal cell-derived factor-1 were increased in C57-mTXAS-treated mice. The enhanced-blood flow recovery by C57-mTXAS treatment was suppressed in the TP knockout mice (TP(-/-)). The expression of PSGL-1 in endothelial cells around the ischemic area was enhanced by C57-mTXAS treatment in wild-type but not in TP(-/-). CONCLUSIONS: These results indicated that local administration of C57-mTXAS-induced angiogenesis by activated platelets that bind to PSGL-1 on ischemic endothelial cells.

Differential dependence on nuclear factor-κB-inducing kinase among natural killer T-cell subsets in their development.

Natural killer T cells (NKT cells) are comprised of several subsets. However, the possible differences in their developmental mechanisms have not been fully investigated. To evaluate the dependence of some NKT subpopulations on nuclear factor-κB-inducing kinase (NIK) for their generation, we analysed the differentiation of NKT cells, dividing them into subsets in various tissues of alymphoplasia (aly/aly), a mutant mouse strain that lacks functional NIK. The results indicated that the efficient differentiation of both invariant NKT (iNKT) and non-iNKT cells relied on NIK expression in non-haematopoietic cells; however, the dependence of non-iNKT cells was lower than that of iNKT cells. Especially, the differentiation of CD8(+) non-iNKT cells was markedly resistant to the aly mutation. The proportion of two other NKT cell subsets, NK1.1(+) γδ T cells and NK1.1(-) iNKT cells, was also significantly reduced in aly/aly mice, and this defect in their development was reversed in wild-type host mice given aly/aly bone marrow cells. In exerting effector functions, NIK in NKT-αß cells appeared dispensable, as NIK-deficient NKT-αß cells could secrete interleukin-4 or interferon-γ and exhibit cytolytic activity at a level comparable to that of aly/+ NKT-αß cells. Collectively, these results imply that the NIK in thymic stroma may be critically involved in the differentiation of most NKT cell subsets (although the level of NIK dependence may vary among the subsets), and also that NIK in NKT-αß cells may be dispensable for their effector function.

The role of vascular endothelial growth factor receptor-1 signaling in compensatory contralateral lung growth following unilateral pneumonectomy.

Compensatory lung growth models have been widely used to investigate alveolization because the remaining lung can be kept intact and volume loss can be controlled. Vascular endothelial growth factor (VEGF) plays an important role in blood formation during lung growth and repair, but the precise mechanisms involved are poorly understood; therefore, the aim of this study was to investigate the role of VEGF signaling in compensatory lung growth. After left pneumonectomy, the right lung weight was higher in VEGF transgenic mice than wild-type (WT) mice. Compensatory lung growth was suppressed significantly in mice injected with a VEGF neutralizing antibody and in VEGF receptor-1 tyrosine kinase-deficient mice (TK(-/-) mice). The mobilization of progenitor cells expressing VEGFR1(+) cells from bone marrow and the recruitment of these cells to lung tissue were also suppressed in the TK(-/-) mice. WT mice transplanted with bone marrow from TK(-/-)transgenic GFP(+) mice had significantly lower numbers of GFP(+)/aquaporin 5(+), GFP(+)/surfactant protein A(+), and GFP(+)/VEGFR1(+) cells than WT mice transplanted with bone marrow from WTGFP(+) mice. The GFP(+)/VEGFR1(+) cells also co-stained for aquaporin 5 and surfactant protein A. Overall, these results suggest that VEGF signaling contributes to compensatory lung growth by mobilizing VEGFR1(+) cells.

Significant involvement of nuclear factor-κB-inducing kinase in proper differentiation of αß and γδ T cells.

Nuclear factor-κB-inducing kinase (NIK) is known to play a critical role in maintaining proper immune function. This is exemplified in the spontaneous mutant mouse lacking functional NIK, alymphoplasia (aly), which is simultaneously immune-compromised and autoimmune-prone. To investigate the role of NIK in αß T-cell repertoire formation, we analysed T-cell development in aly/aly mice bearing a transgenic T-cell receptor (TCR). Although there were no apparent abnormalities in the mature αß T cells of non-transgenic aly/aly mice, the maturation efficiency of idiotype(high+) T cells in the TCR-transgenic mice was lower in aly/aly mice compared with those found in aly/+ mice, suggesting that the mature αß T-cell repertoire could be altered by the absence of functional NIK. In one strain of TCR-transgenic aly/aly mice with a negatively selecting H-2 background, the proportion of CD8(low+) idiotype(high+) cells, which are thought to potentially represent the γδ lineage of T cells, was markedly decreased. When the γδ T cells in non-transgenic aly/aly mice were investigated, the proportion of γδ T cells in the peripheral organs of aly/aly mice was found to be one-half to one-fifth of those in aly/+ mice. Analyses of bone marrow chimera mice indicated that NIK in host cells, rather than in donor cells was important for generating a normal number of peripheral γδ T cells. Collectively, these results suggest that NIK could be involved in thymic positive selection of some αß T cells and that NIK in non-haematopoietic cells is important for the optimal development and/or maintenance of γδ T cells.

Angiotensin II type 1A receptor signaling facilitates tumor metastasis formation through P-selectin-mediated interaction of tumor cells with platelets and endothelial cells.

Angiotensin II is involved in tumor growth; however, the precise mechanism is not known. Platelets also contribute to tumor growth, and angiotensin II type 1 receptor (AT1) is expressed on the platelet surface. We hypothesized that interaction of platelets with tumor cells through AT1 receptor signaling promotes tumor metastasis. B16F1 melanoma cells were intravenously injected into Agtr1a knockout mice (AT1a(-/-)) and wild-type littermates (WT); the AT1a(-/-) mice exhibited a reduction in lung colonies. Angiotensin II induced expression of P-selectin on platelets in WT but not in AT1a(-/-) mice. A selective P-selectin neutralizing antibody decreased lung colony numbers in WT but not in AT1a(-/-) mice. Levels of vascular endothelial growth factor (VEGF) and stromal cell-derived factor 1 (SDF-1) receptor in platelets at metastatic locus were lower in AT1a(-/-) mice. Treatment of neutralizing antibodies against VEGF and CXCR4 decreased lung colony numbers in WT but not in AT1a(-/-) mice. In AT1a(-/-) mice, and both mobilization of progenitor cells expressing CXCR4(+)VEGFR1(+) cells from bone marrow and their recruitment to lung tissues were suppressed. These results suggest that AT1A signaling plays a critical role in tumor metastasis through P-selectin-mediated interactions of platelets with tumor and endothelial cells and through the AT1A signaling-dependent production of VEGF and SDF-1, which may be involved in mobilization of CXCR4(+)VEGFR1(+) cells.

Insight into the significance of Foxp3 + tumor-infiltrating lymphocytes in squamous cell lung cancer.

PURPOSE: Although developing a better understanding of tumor-infiltrating Foxp3 + lymphocytes (Foxp3 + TILs) might provide essential knowledge to predict response to immunotherapy and prognosis, our current knowledge about Foxp3 + TILs is inadequate. This study investigated the prognostic significance of tumor-infiltrating Foxp3 + lymphocytes (Foxp3 + TILs) in squamous cell lung cancer (SQ-LC) objectively. METHODS: Among patients with SQ-LC surgically resected in our institution between 2011 and 2017, those with pathological stage IA3-IIIA were immunohistochemically studied to evaluate Foxp3 + TILs in their tumor stroma. The impact of Foxp3 + TILs on relapse-free survival (RFS) was analyzed with Kaplan-Meier survival analysis and multivariate analysis using a Cox proportional hazards model/Fine-Gray model. RESULTS: This study analyzed 100 patients. Multivariate analysis showed that a large number of Foxp3 + TILs in the stroma does not associate with a poor prognosis, rather that a large number of Foxp3 + TILs (≥ 64 cells) tend to be associated with a more favorable prognosis than a small number of Foxp3 + TILs (< 64 cells) (large vs small number: HR, 0.56; 95% CI, 0.17-1.83; P = 0.34). Exploratory analysis also showed that in the two populations divided by a difference in Foxp3 expression levels, similar trends to the main analysis were observed. CONCLUSION: Our results showed that a large number of Foxp3 + TILs in the stroma may not associate with a poor prognosis in SQ-LC. To use the seemingly complicated information of Foxp3 + TILs as biomarkers, better understanding the diversity and heterogeneity of Foxp3 + TILs and analyzing their subpopulations that increase in the TME may be needed.

Insight into the significance of CD8+ tumor-infiltrating lymphocytes in squamous cell lung cancer.

BACKGROUND: Although there are great expectations regarding the use of tumor-infiltrating lymphocytes (TILs) to predict effects of immunotherapies and prognosis, knowledge about TILs remains insufficient for clinical application. METHODS: We objectively investigated the prognostic significance of tumor-infiltrating CD8 + lymphocytes (CD8 + TILs) in squamous cell lung cancer (SQ-LC). Among patients who underwent surgical resection of SQ-LC in 2011-2017, 100 patients with pathological stage IA3-III were immunohistochemically studied to evaluate CD8 + TILs in the tumor stroma and parenchyma. The impact of CD8 + TILs on relapse-free survival was analyzed using a Kaplan-Meier survival analysis and multivariate analyses using Fine-Gray and Cox proportional hazards models. RESULTS: The multivariate analysis showed that large and small numbers, but not intermediate numbers, of CD8 + TILs in the tumor stroma may be related to a more favorable prognosis (small vs. intermediate: HR, 0.64; 95% CI: 0.29-1.41, p = 0.27; large vs. intermediate: HR, 0.48; 95% CI: 0.21-1.09, p = 0.08). In contrast, a large number of CD8 + TILs in the tumor parenchyma was associated with a poor prognosis (HR, 2.60; 95% CI: 0.91-7.42, p = 0.075). An exploratory analysis showed a potentially strong association between an extremely large number of CD8 + TILs in the tumor parenchyma and a poor prognosis, even with a large number of CD8 + TILs in the tumor stroma. CONCLUSION: Our study provided partial but important information on the significance of CD8 + TILs in SQ-LC. To use CD8 + TILs as biomarkers, a better understanding of CD8 + TILs as well as other important components in the tumor microenvironment and the inflammatory phenotypes they form may be needed.

The microsomal prostaglandin E synthase-1/prostaglandin E2 axis induces recovery from ischaemia via recruitment of regulatory T cells.

AIMS: Microsomal prostaglandin E synthase-1 (mPGES-1)/prostaglandin E2 (PGE2) induces angiogenesis through the prostaglandin E2 receptor (EP1-4). Among immune cells, regulatory T cells (Tregs), which inhibit immune responses, have been implicated in angiogenesis, and PGE2 is known to modulate the function and differentiation of Tregs. We hypothesized that mPGES-1/PGE2-EP signalling could contribute to recovery from ischaemic conditions by promoting the accumulation of Tregs. METHODS AND RESULTS: Wild-type (WT), mPGES-1-deficient (mPges-1-/-), and EP4 receptor-deficient (Ep4-/-) male mice, 6-8 weeks old, were used. Hindlimb ischaemia was induced by femoral artery ligation. Recovery from ischaemia was suppressed in mPges-1-/- mice and compared with WT mice. The number of accumulated forkhead box protein P3 (FoxP3)+ cells in ischaemic muscle tissue was decreased in mPges-1-/- mice compared with that in WT mice. Expression levels of transforming growth factor-ß (TGF-ß) and stromal cell derived factor-1 (SDF-1) in ischaemic tissue were also suppressed in mPges-1-/- mice. The number of accumulated FoxP3+ cells and blood flow recovery were suppressed when Tregs were depleted by injecting antibody against folate receptor 4 in WT mice but not in mPges-1-/- mice. Recovery from ischaemia was significantly suppressed in Ep4-/- mice compared with that in WT mice. Furthermore, mRNA levels of Foxp3 and Tgf-ß were suppressed in Ep4-/- mice. Moreover, the number of accumulated FoxP3+ cells in ischaemic tissue was diminished in Ep4-/- mice compared with that in Ep4+/+ mice. CONCLUSION: These findings suggested that mPGES-1/PGE2 induced neovascularization from ischaemia via EP4 by promoting the accumulation of Tregs. Highly selective EP4 agonists could be useful for the treatment of peripheral artery disease.

Thromboxane A2 receptor signaling facilitates tumor colonization through P-selectin-mediated interaction of tumor cells with platelets and endothelial cells.

Thromboxane A(2) (TXA(2) ) is a prostanoid formed by thromboxane synthase using the cyclooxygenase product, prostaglandin H(2), as the substrate. TXA(2) was shown to enhance tumor metastasis, but the underlying mechanism remains unclear. B16F1 melanoma cells were intravenously injected into TXA(2) receptor (TP) knockout mice (TP(-/-) ) and wild-type littermates (WT). TP(-/-) showed a reduction in B16F1 lung colonization and mortality rate, which were associated with a decreased number of platelets. Platelet activation as assessed by P-selectin expression was suppressed in TP(-/-) . A selective P-selectin neutralizing antibody decreased the lung colonization in WT mice, but not in TP(-/-) . The expression of P-selectin glycoprotein ligand-1 in B16F1 and HUVEC were enhanced by treatment with U46619, a thromboxane analog. The plasma levels of vascular endothelial growth factor (VEGF) and stromal-derived factor (SDF)-1 were lower in TP(-/-) . In TP(-/-) , the mobilization of progenitor cells expressing CXCR4(+) VEGFR1(+) from bone marrow and the recruitment of those cells to lung tissues were suppressed. These results suggest that TP signaling plays a critical role in tumor colonization through P-selectin-mediated interactions between platelets-tumor cells and tumor cells-endothelial cells through the TP signaling-dependent production of VEGF and SDF-1, which might be involved in the mobilization of VEGFR1(+) CXCR4(+) cells. Blockade of TP signaling might be useful in the treatment of tumor metastasis.

Gasp, a Grb2-associating protein, is critical for positive selection of thymocytes.

T cells develop in the thymus through positive and negative selection, which are responsible for shaping the T cell receptor (TCR) repertoire. To elucidate the molecular mechanisms involved in selection remains an area of intense interest. Here, we identified and characterized a gene product Gasp (Grb2-associating protein, also called Themis) that is critically required for positive selection. Gasp is a cytosolic protein with no known functional motifs that is expressed only in T cells, especially immature CD4/CD8 double positive (DP) thymocytes. In the absence of Gasp, differentiation of both CD4 and CD8 single positive cells in the thymus was severely inhibited, whereas all other TCR-induced events such as beta-selection, negative selection, peripheral activation, and homeostatic proliferation were unaffected. We found that Gasp constitutively associates with Grb2 via its N-terminal Src homology 3 domain, suggesting that Gasp acts as a thymocyte-specific adaptor for Grb2 or regulates Ras signaling in DP thymocytes. Collectively, we have described a gene called Gasp that is critical for positive selection.

Natural Killer Cell Group 7 Sequence in Cytotoxic Cells Optimizes Exocytosis of Lytic Granules Essential for the Perforin-Dependent, but Not Fas Ligand-Dependent, Cytolytic Pathway.

Cytotoxic cells, such as CD8+ T cells or NK cells, have been shown to eliminate virus-infected cells or transformed cells primarily via two pathways: the perforin/granzyme-dependent pathway and the Fas ligand-Fas pathway; however, the precise cytolytic mechanisms have not been clarified thoroughly. In our previous study, we demonstrated that a T-box transcription factor, Eomesodermin (Eomes), may play important roles in activating the perforin pathway besides inducing perforin and granzyme B mRNA expression. In this study, we identified natural killer cell group 7 sequence (Nkg7), a molecule induced by Eomes, to be found critical for perforin-dependent cytolysis. Nkg7 mRNA expression in leukocytes from normal mice was mainly restricted to cells with cytotoxicity such as NK cells, NKT cells, and activated CD8+ T cells. The cytolytic activity of NK cells or CD8+ CTLs from Nkg7-deficient mice against Fas-negative target cells was reduced significantly, whereas Fas ligand-mediated cytolysis by Nkg7-deficient CTLs was not impaired. Further, translocation of granule membrane protein CD107a to the cell surface upon CD3 stimulation was defective in CD8+ CTLs from Nkg7 knockout, whereas surface induction of another granule membrane protein, CD63, was almost normal. In addition, analyses of lytic granules in CTLs by electron microscopy revealed that the number of lytic granules with dense cores was significantly reduced in Nkg7-knockout CTLs. These results indicate that Nkg7 may specifically contribute to efficient cytolysis via the perforin/granzyme pathway by enhancing the exocytosis of a particular type of lytic granules.

Activation of iNKT Cells Facilitates Liver Repair After Hepatic Ischemia Reperfusion Injury Through Acceleration of Macrophage Polarization.

Macrophage polarization is critical for liver tissue repair following acute liver injury. However, the underlying mechanisms of macrophage phenotype switching are not well defined. Invariant natural killer T (iNKT) cells orchestrate tissue inflammation and tissue repair by regulating cytokine production. Herein, we examined whether iNKT cells played an important role in liver repair after hepatic ischemia-reperfusion (I/R) injury by affecting macrophage polarization. To this end, we subjected male C57BL/6 mice to hepatic I/R injury, and mice received an intraperitoneal (ip) injection of α-galactosylceramide (α-GalCer) or vehicle. Compared with that of the vehicle, α-GalCer administration resulted in the promotion of liver repair accompanied by acceleration of macrophage differentiation and by increases in the numbers of Ly6Chigh pro-inflammatory macrophages and Ly6Clow reparative macrophages. iNKT cells activated with α-GalCer produced interleukin (IL)-4 and interferon (IFN)-γ. Treatment with anti-IL-4 antibodies delayed liver repair, which was associated with an increased number of Ly6Chigh macrophages and a decreased number of Ly6Clow macrophages. Treatment with anti-IFN-γ antibodies promoted liver repair, associated with reduced the number of Ly6Chigh macrophages, but did not change the number of Ly6Clow macrophages. Bone marrow-derived macrophages up-regulated the expression of genes related to both a pro-inflammatory and a reparative phenotype when co-cultured with activated iNKT cells. Anti-IL-4 antibodies increased the levels of pro-inflammatory macrophage-related genes and decreased those of reparative macrophage-related genes in cultured macrophages, while anti-IFN-γ antibodies reversed the polarization of macrophages. Cd1d-deficient mice showed delayed liver repair and suppressed macrophage switching, compared with that in wild-type mice. These results suggest that the activation of iNKT cells by α-GalCer facilitated liver repair after hepatic I/R injury by both IL-4-and IFN-γ-mediated acceleration of macrophage polarization. Therefore, the activation of iNKT cells may represent a therapeutic tool for liver repair after hepatic I/R injury.

Effect of erythropoietin on angiogenesis with the increased adhesion of platelets to the microvessels in the hind-limb ischemia model in mice.

Erythropoietin (EPO) has been shown to enhance angiogenesis, but its precise mechanisms of enhancement during ischemia are not fully elucidated. We examined the effect of EPO on blood flow recovery from acute hind-limb ischemia induced by ligation of the femoral artery in male C57Bl/6 mice. The density of microvessels with platelet adhesion in ischemic tissues was assessed by intravital microscopy. Treatment with EPO (100 and 1000 IU/kg, i.p.) restored blood flow in a dose-dependent manner and increased plasma levels of soluble-P-selectin, vascular endothelial growth factor (VEGF), and stromal cell-derived factor (SDF-1). Flow cytometric analysis revealed increased P-selectin expression on platelets in EPO-treated mice compared to PBS-treated mice. Intravital microscopic studies showed that EPO increased density of microvessels with platelet adhesion selectively in the ischemic tissues. Neutralizing antibody against P-selectin reduced the density of microvessels with platelet adhesion enhanced with EPO and impaired blood flow recovery with reductions in VEGF and SDF-1 levels. These results suggest that EPO administration enhances recovery from hind-limb ischemia, and platelet adhesion to the microvessels is a key event to enhance the angiogenesis in the ischemic tissues.