Lipid droplets-related Perilipin-3: potential immune checkpoint and oncogene in oral squamous cell carcinoma.

BACKGROUND: Lipid droplets (LDs) as major lipid storage organelles are recently reported to be innate immune hubs. Perilipin-3 (PLIN3) is indispensable for the formation and accumulation of LDs. Since cancer patients show dysregulated lipid metabolism, we aimed to elaborate the role of LDs-related PLIN3 in oral squamous cell carcinoma (OSCC). METHODS: PLIN3 expression patterns (n = 87), its immune-related landscape (n = 74) and association with B7-H2 (n = 51) were assessed by immunohistochemistry and flow cytometry. Real-time PCR, Western blot, Oil Red O assay, immunofluorescence, migration assay, spheroid-forming assay and flow cytometry were performed for function analysis. RESULTS: Spotted LDs-like PLIN3 staining was dominantly enriched in tumor cells than other cell types. PLIN3high tumor showed high proliferation index with metastasis potential, accompanied with less CD3+CD8+ T cells in peripheral blood and in situ tissue, conferring immunosuppressive microenvironment and shorter postoperative survival. Consistently, PLIN3 knockdown in tumor cells not only reduced LD deposits and tumor migration, but benefited for CD8+ T cells activation in co-culture system with decreased B7-H2. An OSCC subpopulation harbored PLIN3highB7-H2high tumor showed more T cells exhaustion, rendering higher risk of cancer-related death (95% CI 1.285-6.851). CONCLUSIONS: LDs marker PLIN3 may be a novel immunotherapeutic target in OSCC.

Annexin A5 is a novel prognostic biomarker in oral squamous cell carcinoma.

BACKGROUND: ANXA5, a notable tumor marker, displays irregular expression in diverse solid cancers, and links to local recurrence and metastasis rates. We aimed study the expression of ANXA5 in oral squamous cell carcinoma (OSCC) and its diagnostic and prognostic values. METHODS: 520 head and neck squamous cell carcinoma (HNSCC) patients in TCGA database and 124 OSCC patients in Nanjing stomatology hospital were enrolled in our study. Immunohistochemical analyses were performed using ANXA5 antibodies. Chi-square test was used to analyze the clinicopathological features. Survival rates were determined using the Kaplan-Meier method and log-rank test. RESULTS: Our results showed significantly elevated ANXA5 at the gene and protein levels in HNSCC and OSCC compared to non-tumor tissues. Histopathologically, ANXA5 was broadly present in OSCC tumor cells and fibroblast-like cells but absent in tumor-infiltrating lymphocytes, particularly at the invasive tumor front. Patients exhibiting high ANXA5 expression in these cells demonstrated poor differentiation, aggressive invasion patterns, and heightened lymph node metastasis risk, contributing to poorer postoperative outcomes. Remarkably, ANXA5 in fibroblast-like cells emerged as an independent risk factor impacting survival in OSCC patients. Gene set enrichment analysis (GSEA) highlighted ANXA5's involvement in key pathways like epithelial-mesenchymal transformation (EMT), TGF-beta signaling, and hypoxia, which correlated with adverse clinical outcomes in OSCC. CONCLUSION: ANXA5 emerges as a significant prognostic biomarker for OSCC, potentially influencing its metastasis via the EMT pathway.

Interaction between CAFs and apoptotic cancer cells promotes OSCC proliferation via STING signaling.

BACKGROUND: Apoptosis can fuel oncogenesis by the education of surrounding stromal cells. However, the function of cancer-associated fibroblasts (CAFs), which interacted with apoptotic cancer cells, in oral squamous cell carcinoma (OSCC) progression is still unknown. OBJECTIVES: This study aimed to explore the prognostic value of apoptosis and the biological effects of CAFs, interacted with apoptotic cancer cells, on OSCC. METHODS: A total of 166 samples from OSCC patients were stained via TUNEL reaction to evaluate the correlation between apoptosis and clinical characteristics. Cell viability and proliferation were assessed through flow cytometry and CCK-8 assays, respectively. Levels of mRNA and protein were examined through qRT-PCR, western blot and immunofluorescence. RESULTS: Higher percentage of apoptotic cancer cells in OSCC positively correlated with more Ki67+ cells and predicted poor clinical outcomes. Conditioned medium from CAFs exposed to apoptotic cancer cells significantly facilitated cell proliferation. Co-culture CAFs with apoptotic cancer cells dampened the phosphorylation of STING/IRF3 signaling, as well as the production of type I interferon, which was required for the inhibition of OSCC cell proliferation. CONCLUSION: These results demonstrate the interplay between apoptotic cancer cells and CAFs promotes OSCC proliferation via STING signaling, identifying a potential therapy targeted CAFs surrounded with apoptotic cancer cells for OSCC.

IL-33/ST2 signaling promotes constitutive and inductive PD-L1 expression and immune escape in oral squamous cell carcinoma.

BACKGROUND: Loss-of-function of PD-L1 induces therapy resistance of anti-PD-1/L1 therapy, and the complex regulatory mechanisms are not completely understood. We previously reported that stroma-derived interleukin-33 (IL-33) promoted the progression of oral squamous cell carcinoma (OSCC). We here focused on the immune-regulation role of IL-33 and its receptor ST2 signaling in PD-L1-positive OSCC patients. METHODS: Activated T cells in in situ and peripheral blood were analyzed by IL-33/ST3 expression. Knockdown or overexpression of ST2 combined with IL-33/IFN-γ stimulation were performed to determine PD-L1 expression and PD-L1-dependent immune escape in OSCC/human T cells co-culture system, and OSCC orthotopic model based on humanized mouse with immune reconstitution and C57BL/6 mice models. RESULTS: High IL-33/ST2 correlated with less activated T cells infiltration in situ and peripheral blood. Knockdown of ST2 down-regulated constitutive PD-L1 expression, whereas ST2 also promoted IL-33-induced PD-L1 Mechanistically, IL-33/ST2 activated JAK2/STAT3 pathway to directly promoted PD-L1 expression, and also activated MyD88/NF-κB signaling to up-regulate IFN-γ receptor (IFN-γR), which indirectly strengthen IFN-γ-induced PD-L1. Furthermore, ST2 is required for PD-L1-mediated immune tolerance in vitro and in vivo. ST2high OSCC patients have more PD-L1 and IFN-γR level in situ. CONCLUSIONS: IL-33/ST2 signaling enhanced PD-L1-mediated immune escape, ST2high OSCC patients might benefit from anti-PD-1/L1 therapy.

Effects of cancer-associated fibroblasts deletion using HSVtk suicide system in OSCC.

OBJECTIVE: To evaluate the biological characteristics of oral cancer cells co-cultured with cancer-associated fibroblasts (CAFs)-HSVtk and to assess the reliability of the CAFs-HSVtk suicide system in a co-culture model. METHODS: CAFs were lentivirus-transfected with PCDH-HSVtk. Ganciclovir (GCV) was added and the survival rates of the CAFs-HSVtk were measured. In parallel with the selective elimination of CAFs, comparison was made of the effects of CAF-HSVtk on tumor cell proliferation/migration in a CAFs-tumor co-cultural system. Cell death of co-cultured oral cancer cells was evaluated by flow cytometry. RESULTS: Q-PCR analysis showed that the expression of HSVtk in the CAFs-HSVtk group was significantly higher than in the control group (p < 0.01). The survival rates of CAFs-HSVtk with GCV were significantly reduced (p < 0.01). Following selective depletion of CAFs-HSVtk, the growth and migration rates of oral cancer cells co-cultured with CAFs-HSVtk were reduced in a mixture ratio of 1:2 (p < 0.01, p < 0.01). CONCLUSIONS: Enhanced proliferation and migration rates of oral cancer cells in co-culture were seriously impaired after deleting CAFs using the HSVtk suicide system, while oral tumor cell death was not affected. Therefore, CAFs-HSVtk can be utilized as a valid model for CAF signature identification.

SPARC promotes the proliferation and metastasis of oral squamous cell carcinoma by PI3K/AKT/PDGFB/PDGFRß axis.

Oral squamous cell carcinoma (OSCC) is a highly lethal cancer in the world, and the prognosis of OSCC is poor with a 60% 5-year survival rate in recent decades. Here, we introduced a novel secretory and acid glycoprotein with cysteine rich (secreted protein acidic and rich in cysteine, SPARC), which is correlated with the worst pattern of invasion (WPOI) and prognosis of OSCC. SPARC expression levels were measured in OSCC tissues and normal tissues using quantitative polymerase chain reaction and immunohistochemistry. The influence of SPARC on cell proliferation was examined by cell counting kit-8, colony formation, and Edu tests. Then, the effect of SPARC on the metastasis of OSCC cells was detected by wound healing and transwell migration assays. Next, the biologic characteristics of SPARC shared by STRING were analyzed. Furthermore, the underlying mechanisms were confirmed by western blot analysis. SPARC revealed higher expression in OSCC tissues than nontumor tissues. Higher SPARC expression was correlated with poorer tumor differentiation, poorer WPOI pattern, and significantly and shorter overall survival. Knockdown SPARC significantly restrained OSCC cell growth, migration, and invasion. In addition, bioinformatics analysis found SPARC had a coexpression network with the platelet-derived growth factor-B (PDGFB) and PI3K/AKT signaling pathways with minimal false discovery rate. Furthermore, SPARC promotes OSCC cells metastasis by regulating the expressions of PDGFB, PDGFRß, p-PDGFRß , and the PI3K/AKT pathway. Higher SPARC expression was positively correlated with poor WPOI and differentiation in OSCC. SPARC activates the PI3K/AKT/PDGFB/PDGFRß axis to promote proliferation and metastasis by OSCC cell lines. Therefore, SPARC may be a potential therapeutic target for patients with OSCC.

A novel stromal lncRNA signature reprograms fibroblasts to promote the growth of oral squamous cell carcinoma via LncRNA-CAF/interleukin-33.

Stromal carcinoma-related fibroblasts (CAFs) are the main type of non-immune cells in the tumor microenvironment (TME). CAFs interact with cancer cells to promote tumor proliferation. Long non-coding RNAs (lncRNAs) are known to regulate cell growth, apoptosis and metastasis of cancer cells, but their role in stromal cells is unclear. Using RNA sequencing, we identified a stromal lncRNA signature during the transformation of CAFs from normal fibroblasts (NFs) in oral squamous cell carcinoma (OSCC). We uncovered an uncharacterized lncRNA, FLJ22447, which was remarkably up-regulated in CAFs, referred to LncRNA-CAF (Lnc-CAF) hereafter. Interleukin-33 (IL-33) was mainly located in the stroma and positively co-expressed with Lnc-CAF to elevate the expression of CAF markers (α-SMA, vimentin and N-cadherin) in fibroblasts. In a co-culture system, IL-33 knockdown impaired Lnc-CAF-mediated stromal fibroblast activation, leading to decreased proliferation of tumor cells. Mechanistically, Lnc-CAF up-regulated IL-33 levels and prevented p62-dependent autophagy-lysosome degradation of IL-33, which was independent of LncRNA-protein scaffold effects. Treatment with the autophagy inducer, rapamycin, impaired the proliferative effect of Lnc-CAF/IL-33 by promoting IL-33 degradation. In turn, tumor cells further increased Lnc-CAF levels in stromal fibroblasts via exosomal Lnc-CAF. In patients with OSCC, high Lnc-CAF/IL-33 expression correlated with high TNM stage (n = 140). Moreover, high Lnc-CAF expression predicted poor prognosis. In vivo, Lnc-CAF knockdown restricted tumor growth and was associated with decreased Ki-67 expression and α-SMA+ CAF in the stroma. In conclusion, we identified a stromal lncRNA signature, which reprograms NFs to CAFs via Lnc-CAF/IL-33 and promotes OSCC development.

Exosomal miR-146a Contributes to the Enhanced Therapeutic Efficacy of Interleukin-1ß-Primed Mesenchymal Stem Cells Against Sepsis.

Improving the immunomodulatory efficacy of mesenchymal stem cells (MSCs) through pretreatment with pro-inflammatory cytokines is an evolving field of investigation. However, the underlying mechanisms have not been fully clarified. Here, we pretreated human umbilical cord-derived MSCs with interleukin-1ß (IL-1ß) and evaluated their therapeutic effects in a cecal ligation and puncture-induced sepsis model. We found that systemic administration of IL-1ß-pretreated MSCs (ßMSCs) ameliorated the symptoms of murine sepsis more effectively and increased the survival rate compared with naïve MSCs. Furthermore, ßMSCs could more effectively induce macrophage polarization toward an anti-inflammatory M2 phenotype through the paracrine activity. Mechanistically, we demonstrated that ßMSC-derived exosomes contributed to the enhanced immunomodulatory properties of ßMSCs both in vitro and in vivo. Importantly, we found that miR-146a, a well-known anti-inflammatory microRNA, was strongly upregulated by IL-1ß stimulation and selectively packaged into exosomes. This exosomal miR-146a was transferred to macrophages, resulted in M2 polarization, and finally led to increased survival in septic mice. In contrast, inhibition of miR-146a through transfection with miR-146a inhibitors partially negated the immunomodulatory properties of ßMSC-derived exosomes. Taken together, IL-1ß pretreatment effectively enhanced the immunomodulatory properties of MSCs partially through exosome-mediated transfer of miR-146a. Therefore, we believe that IL-1ß pretreatment may provide a new modality for better therapeutic application of MSCs in inflammatory disorders. Stem Cells 2017;35:1208-1221.

Frequency of circulating CD14++ CD16+ intermediate monocytes as potential biomarker for the diagnosis of oral squamous cell carcinoma.

BACKGROUND: Monocytes, which subdivided into three functional subsets (classical, intermediate, and nonclassical), play important roles in the progression of cancer. The subset composition is altered in several pathologic conditions including cancers. However, the composition and function of circulating monocyte subsets in patients with oral squamous cell carcinoma (OSCC) are still obscure. METHODS: The frequencies of monocyte subsets in peripheral blood of patients with OSCC and healthy donors are determined by flow cytometry, and their diagnostic values for OSCC were evaluated. The associations between levels of monocyte subsets and clinicopathological features of patients with OSCC were analyzed using cross-tabulation with the chi-square test. RESULTS: We demonstrated that the frequency of CD14++ CD16+ intermediate monocytes was remarkably increased (P < 0.0001) in OSCC patients compared with healthy controls (7.33% ± 2.56% of total monocytes, n = 68 versus 4.78% ± 1.50% of total monocytes, n = 57). A trend of decrease in CD14++ CD16- classical subset was observed between these two groups (P = 0.0508), whereas no significant difference was detected in CD14+ CD16++ nonclassical subset (P > 0.05). The receiver operating characteristic (ROC) curve analysis indicated that the frequency of intermediate monocytes (AUC = 0.810, P < 0.0001) could be a potential diagnostic biomarker to discriminate patients with OSCC from healthy subjects. Moreover, this parameter was significantly correlated to the worst pattern of invasion (WPOI, P < 0.05) of OSCC tissues. CONCLUSIONS: Detection of monocyte subsets in peripheral blood sheds a light on utilizing the frequency of intermediate monocytes as a potential diagnostic biomarker for OSCC.

Long Non-Coding RNA MALAT1 Promotes Proliferation, Angiogenesis, and Immunosuppressive Properties of Mesenchymal Stem Cells by Inducing VEGF and IDO.

Mesenchymal stem cells (MSCs) play an important role in regulating angiogenesis and immune balance. The abnormal MSCs in proliferation and function were reported at maternal fetal interface in patients with pre-eclampsia (PE). Long non-coding RNA MALAT1 was known to regulate the function of trophoblast cells. However, it is not clear whether MALAT1 regulates MSCs to be related to PE. In the present study, we found that the expression of MALAT1 was significantly reduced in both umbilical cord tissues and MSCs in patients with severe PE. MALAT1 did not affect the phenotype and differentiation of MSCs. Of note, transfection with MALAT1 plasmid into MSCs drove the cell cycle into G2/M phase and inhibited cell apoptosis. The supernatants from MALAT1-overexpressed MSCs promoted the migration of MSCs, invasion of HTR-8/SVneo and tube formation of HUVEC, while si-MALAT1 had the opposite effects. Moreover, we found that MALAT1-induced VEGF mediated these effects of MALAT1 on MSCs. Furthermore, we found that MALAT1-overexpressed MSCs promoted M2 macrophage polarization and this effect was mediated by MALAT1-induced IDO expression, suggesting that MALAT1 may enhance the immunosuppressive properties of MSCs in vivo. In addition, we also investigated the factors that inhibit MALAT1 expression in PE and found that peroxide was a cause for MALAT1 downregulation. Taken together, our data demonstrate that MALAT1 is an important endogenous regulator in the proliferation, angiogenesis, and immunosuppressive properties of MSCs, suggesting it may be involved in the pathogenesis of PE. J. Cell. Biochem. 118: 2780-2791, 2017. © 2017 Wiley Periodicals, Inc.

MiR-495 Promotes Senescence of Mesenchymal Stem Cells by Targeting Bmi-1.

BACKGROUND/AIMS: Mesenchymal stem cells (MSCs) play an important role in regulating angiogenesis and immune balance. Abnormal proliferation and function of MSCs were reported at maternal fetal interface in patients with pre-eclampsia (PE). Micro-RNA-495 was known to be upregulated in the MSCs derived from patients with PE. However, it is not clear whether the up-regulated miR-495 is related to the pathogenesis of PE. METHODS: We analyzed the expression of miR-495 in MSCs and umbilical cords derived from healthy pregnancies (NC) and PE, then we upregulated or downregulated the expression of miR-495 in MSCs derived from NC and tested the proliferation, apoptosis, migration, invasion, tube formation and senescence. RESULTS: In the current study, we found that the expression of miR-495 was significantly increased in both umbilical cord tissues and MSCs in patients with severe PE. Overexpressing miR-495 arrested cell cycle in S phase and promoted cell apoptosis. The supernatants from miR-495-overexpressed-MSCs inhibited the migration of MSCs and HTR-8/SVneo, invasion of HTR-8/SVneo and tube formation of HUVEC, while si-miR-495 had the opposite effects. Furthermore, we analyzed the senescence related ß-galactosidase activity and CD146 and found that miR-495 induced the senescence of MSCs. Molecular mechanism studies confirmed that Bmi-1 mediated these effects of miR-495 on MSCs. CONCLUSION: Taken together, our data demonstrated that miR-495 induced senescence of MSCs may be involved in the pathogenesis of PE.

The novel α-glucan YCP improves the survival rates and symptoms in septic mice by regulating myeloid-derived suppressor cells.

Sepsis is a life-threatening health condition that is initially characterized by uncontrolled inflammation, followed by the development of persistent immunosuppression. YCP is a novel α-glucan purified from the mycelium of the marine fungus Phoma herbarum YS4108, which has displayed strong antitumor activity via enhancing host immune responses. In this study, we investigated whether YCP could influence the development of sepsis in a mouse model. Caecal ligation and puncture (CLP)-induced sepsis was established in mice that were treated with YCP (20 mg/kg, ip or iv) 2 h before, 4 and 24 h after the CLP procedure, and then every other day. YCP administration greatly improved the survival rate (from 39% to 72% on d 10 post-CLP) and ameliorated disease symptoms in the septic mice. Furthermore, YCP administration significantly decreased the percentage of myeloid-derived suppressor cells (MDSCs) in the lungs and livers, which were dramatically elevated during sepsis. In cultured BM-derived cells, addition of YCP (30, 100 µg/mL) significantly decreased the expansion of MDSCs; YCP dose-dependently decreased the phosphorylation of STAT3 and increased the expression of interferon regulatory factor-8 (IRF-8). When BM-derived MDSCs were co-cultured with T cells, YCP dose-dependently increased the production of arginase-1 (Arg-1) and inducible nitric oxide synthase (iNOS), and activated the NF-κB pathway. In addition, the effects of YCP on MDSCs appeared to be dependent on toll-like receptor (TLR) 4. These results reveal that YCP inhibits the expansion of MDSCs via STAT3 while enhancing their immunosuppressive function, partially through NF-κB. Our findings suggest that YCP protects mice against sepsis by regulating MDSCs. Thus, YCP may be a potential therapeutic agent for sepsis.

Myeloid-derived suppressor cells contribute to systemic lupus erythaematosus by regulating differentiation of Th17 cells and Tregs.

Although major advancements have made in investigating the aetiology of SLE (systemic lupus erythaematosus), the role of MDSCs (myeloid-derived suppressor cells) in SLE progression remains confused. Recently, some studies have revealed that MDSCs play an important role in lupus mice. However, the proportion and function of MDSCs in lupus mice and SLE patients are still poorly understood. In the present study, we investigated the proportion and function of MDSCs using different stages of MRL/lpr lupus mice and specimens from SLE patients with different activity. Results showed that splenic granulocytic (G-)MDSCs were significantly expanded by increasing the expression of CCR1 (CC chemokine receptor 1) in diseased MRL/lpr lupus mice and in high-disease-activity SLE patients. However, the proportion of monocytic (M-)MDSCs remains similar in MRL/lpr lupus mice and SLE patients. G-MDSCs produce high levels of ROS (reactive oxygen species) through increasing gp91(phox) expression, and activated TLR2 (Toll-like receptor 2) and AIM2 (absent in melanoma 2) inflammasome in M-MDSCs lead to IL-1ß (interleukin 1ß) expression in diseased MRL/lpr mice and high-disease-activity SLE patients. Previous study has revealed that MDSCs could alter the plasticity of Th17 (T helper 17) cells and Tregs (regulatory T-cells) via ROS and IL-1ß. Co-culture experiments showed that G-MDSCs impaired Treg differentiation via ROS and M-MDSCs promoted Th17 cell polarization by IL-1ß in vitro Furthermore, adoptive transfer or antibody depletion of MDSCs in MRL/lpr mice confirmed that MDSCs influenced the imbalance of Tregs and Th17 cells in vivo Our results indicate that MDSCs with the capacity to regulate Th17 cell/Treg balance may be a critical pathogenic factor in SLE.

A benzenediamine derivate FC-99 attenuates lupus nephritis in MRL/lpr mice via inhibiting myeloid dendritic cell-secreted BAFF.

Myeloid dendritic cells (DCs) can produce B-cell-activating factor (BAFF) that modulates survival and differentiation of B cells and plays a pivotal role in the pathogenesis of systemic lupus erythematosus (SLE). Toll-like receptor 4 (TLR4) signaling has important functions in the process of BAFF production. Our previous study showed that a benzenediamine derivate FC-99 possesses anti-inflammation activity and directly interacts with interleukin-1 receptor-associated kinase 4 (IRAK4), which was a pivotal molecule in TLR4 signaling. In this study, we demonstrated that FC-99 attenuated lupus nephritis in the MRL/lpr mice. FC-99 also decreased the levels of total immunoglobulin G (IgG), total IgG2a and IgM in sera, as well as the activation of B cells in the spleens of MRL/lpr mice. Moreover, FC-99 inhibited abnormal activation of myeloid DCs in spleens and reduced the levels of BAFF in sera, spleens, and kidneys of MRL/lpr mice. Furthermore, upon TLR4 stimulation with lipopolysaccharide in vitro, FC-99 inhibited IRAK4 phosphorylation, as well as the activation and BAFF production in murine bone marrow-derived DCs. These data indicate that FC-99 attenuates lupus nephritis in MRL/lpr mice via inhibiting DC-secreted BAFF, suggesting that FC-99 may be a potential therapeutic candidate for the treatment of SLE.

Gene delivery with IFN-γ-expression plasmids enhances the therapeutic effects of MSCs on DSS-induced mouse colitis.

OBJECTIVE: Interferon-γ (IFN-γ) is known to enhance the immunosuppressive properties of mesenchymal stem cells (MSCs). The aim of this study was to determine whether gene modification with IFN-γ-expression plasmids could boost the therapeutic effects of MSCs on DSS-induced colitis. METHODS: We first reconstructed pcDNA3.1-IFNγ plasmids, transfected them to human umbilical cord derived MSCs, and detected the basic characters of MSCs including immune phenotype, cell vitality, proliferation, apoptosis and cell cycle progression after transfection. Subsequently, we analyzed the inhibition effect of IFN-γ-MSCs on T cell proliferation in vitro. Finally, we induced colitis in female C57BL/6 mice by 3 % DSS treatment and evaluated the therapeutic efficacy of IFN-γ-MSCs on colitis. RESULTS: Transfection with pcDNA3.1-IFNγ did not change the basic characters of MSCs. Interestingly, IFN-γ-MSCs showed more potent immunosuppressive effects on the proliferation of T cells compared to normal MSCs. Furthermore, systemic infusion with IFN-γ-MSCs more efficiently ameliorated DSS-induced mouse colitis including colitis-related ease of body weight, increase of colon length, decrease of disease activity index, and improvement of small intestine tissues structure. In addition, IFN-γ-MSCs increased the populations of Foxp3(+) Tregs and Th2 cells both in mesenteric lymph node and spleen, upregulated indoleamine 2, 3-dioxygenase expression, and suppressed inflammatory cytokine production in mouse colon. CONCLUSIONS: Gene delivery with IFN-γ-expression plasmids enhanced the therapeutic effects of MSCs on DSS-induced mouse colitis. This study provides an effective therapeutic strategy of MSCs for inflammatory diseases.

A novel benzenediamine derivate rescued mice from experimental sepsis by attenuating proinflammatory mediators via IRAK4.

We designed and synthesized a novel benzenediamine derivate, FC-99, that was tested for its ability to protect mice from experimental sepsis. Moreover, we sought to determine whether FC-99 could control a bacterial infection and to clarify the mechanism by which FC-99 inhibited LPS-activated macrophages. The effects of FC-99 on inflammation were evaluated in two experimental sepsis models and in cultured macrophages. Microarrays and docking and molecular dynamics simulations were used to determine the target of FC-99. Surface plasmon resonance and molecular detection were performed to confirm the direct interaction of FC-99 with its target. FC-99 protected mice from experimental sepsis. The mice that received FC-99 exhibited a diminished inflammatory response, had a lower local bacterial burden, and experienced a significantly improved survival rate. Genome-wide transcriptional profiling of FC-99-treated macrophages identified IRAK4 as a drug-regulated gene involved in LPS/TLR4 signaling. A computer search and calculations indicated that IRAK4 directly interacted with FC-99. Surface plasmon resonance, IRAK4-regulated signaling pathway analysis, and gene expression profiling of proinflammatory mediators confirmed the direct interaction between FC-99 and IRAK4. FC-99 is a potential therapeutic molecule for sepsis that alleviated experimental sepsis by directly inhibiting IRAK4 activation, which represents a novel target for sepsis therapy.

Calmodulin-dependent protein kinase II/cAMP response element-binding protein/Wnt/ß-catenin signaling cascade regulates angiotensin II-induced podocyte injury and albuminuria.

Angiotensin II (Ang II) plays a pivotal role in promoting podocyte dysfunction and albuminuria, however, the underlying mechanisms have not been fully delineated. In this study, we found that Ang II induced Wnt1 expression and ß-catenin nuclear translocation in cultured mouse podocytes. Blocking Wnt signaling with Dickkopf-1 (Dkk1) or ß-catenin siRNA attenuated Ang II-induced podocyte injury. Ang II could also induce the phosphorylation of calmodulin-dependent protein kinase (CaMK) II and cAMP response element-binding protein (CREB) in cultured podocytes. Blockade of this pathway with CK59 or CREB siRNA could significantly inhibit Ang II-induced Wnt/ß-catenin signaling and podocyte injury. In in vivo studies, administration of Ang II promoted Wnt/ß-catenin signaling, aggregated podocyte damage, and albuminuria in mice. CK59 could remarkably ameliorate Ang II-induced podocyte injury and albuminuria. Furthermore, ectopic expression of exogenous Dkk1 also attenuated Ang II-induced podocytopathy in mice. Taken together, this study demonstrates that the CaMK II/CREB/Wnt/ß-catenin signaling cascade plays an important role in regulating Ang II-induced podocytopathy. Targeting this signaling pathway may offer renal protection against the development of proteinuric kidney diseases.

Novel benzenediamine derivative FC99 ameliorates zymosan-induced arthritis by inhibiting RORγt expression and Th17 cell differentiation.

Increased IL-17-producing helper T (Th17) cells have been observed in patients with rheumatoid arthritis (RA). The retinoic-acid-related orphan nuclear receptor (RORγt) is the master regulator of Th17 cells. Our previous research showed that FC99 possesses anti-inflammation activity. However, to date the effects of FC99 on RORγt expression in Th17 cell differentiation have not been investigated yet. In the present study, we found that FC99 significantly attenuated arthritis-like symptoms, i.e., suppressing the development of paw edema in zymosan-induced arthritis (ZIA) mice. H&E staining showed that the infiltration of inflammatory cells in ankle synovial tissues was significantly suppressed. FC99 also reduced the mRNA levels of pro-inflammatory cytokines in ankle synovial tissues as shown by Q-PCR analysis. The protein levels of the pro-inflammatory cytokines in sera were also suppressed after FC99 treatment. Moreover, FC99 decreased the RORγt mRNA level in spleen tissues. Th17 cell percentage was significantly decreased in spleens and draining lymph nodes (dLNs). The mRNA and protein levels of IL-17A and IL-23 were reduced after FC99 treatment in ZIA mice. Furthermore, in vitro experiments showed that FC99 inhibited the expression of IL-6 in LPS-induced RAW264.7 cells and BMDCs. Moreover, FC99 significantly inhibited the RORγt expression in PMA-induced CD4(+) T cells and LPS-induced RAW264.7 cells. These data indicate that FC99 improves arthritis-like pathological symptoms in vivo and in vitro, which might be related to the inhibition of RORγt expression in Th17 cells. Our findings suggest that FC99 may be a potential therapeutic candidate for the treatment of RA and other inflammatory disorders.

Immune checkpoint CD161/LLT1-associated immunological landscape and diagnostic value in oral squamous cell carcinoma.

An active host adaptive response is characterized by the existence of programmed cell death protein 1 (PD-1)+ /IFN-γ+ cytotoxic T cells and IFN-γ-induced PD-L1+ tumor cells (TCs), which predicts high response rate to anti-PD-1/L1 therapy. Recently, CD161 and its ligand LLT1 (CLEC2D) have been identified as an emerging checkpoint for immunotherapy. Clarifying its heterogeneous clinical expression pattern and its immune landscape is a prerequisite for maximizing the response rate of CD161 blockade therapy in a specific population of oral squamous cell carcinoma (OSCC) patients. Here, we investigated the expression pattern of CD161/LLT1 and its association with major immunocytes (T cells, B cells, NK cells, and macrophages) by multiplex immunofluorescence, immunohistochemistry, and flow cytometry in 109 OSCC tissues and 102 peripheral blood samples. TCs showed higher LLT1 levels than tumor infiltrating lymphocytes (TILs), whereas CD161 was highly expressed in CD8+ T cells at the tumor front, which was decreased in paracancerous tissue. High expression of TC-derived LLT1 (LLT1TC ) conferred poor clinical outcomes, whereas higher CD161+ and LLT1+ TILs were associated with better prognosis. Meanwhile, patients with high LLT1TC showed a decreased ratio of CD8+ /Foxp3+ T cells in situ, but CD161+ TILs correlated with more peripheral CD3+ T cells. Interestingly, treatment of OSCC patients with nivolumab (anti-PD-1) could restore tumoral CD161/LLT1 signal. Furthermore, an OSCC subgroup characterized by high LLT1+ TCs and low CD161+ CD8+ T cells showed fewer peripheral T cells and a higher risk of lymph node metastasis, leading to a shorter 5-year survival time (29%). More LLT1TC at the invasive front was another risk characteristic of exhausted T cells. In conclusion, in view of this heterogeneity, the LLT1/CD161 distribution pattern should be determined before CD161-based immunotherapy.

Natural polyphenolic antibacterial bio-adhesives for infected wound healing.

Bio-adhesives used clinically, commonly have the ability to fill surgical voids and support wound healing, but which are devoid of antibacterial activity, and thus, could not meet the particular needs of the infected wound site. Herein, a series of natural polyphenolic antibacterial bio-adhesives were prepared via simple mixing and heating of polyphenols and acid anhydrides without any solvent or catalyst. Upon the acid anhydride ring opening and acylation reactions, various natural polyphenolic bio-adhesives could adhere to various substrates (i.e., tissue, wood, glass, rubber, paper, plastic, and metal) based on multi-interactions. Moreover, these bio-adhesives showed excellent antibacterial and anti-infection activity, rapid hemostatic performance and appropriate biodegradability, which could be widely used in promoting bacterial infection wound healing and hot burn infection wound repair. This work could provide a new strategy for strong adhesives using naturally occurring molecules, and provide a method for the preparation of novel multifunctional wound dressings for infected wound healing.