Impaired wound healing with defective expression of chemokines and recruitment of myeloid cells in TLR3-deficient mice.

Skin injury evokes both innate and adaptive immune responses to restore tissue integrity. TLRs play a critical role in host responses to injurious insults. Previous studies demonstrated that RNAs released from damaged tissues served as endogenous ligands for TLR3. In this study, we investigated the involvement of TLR3 in skin restoration after injury. Full excisional wounds were created on the skin of mice with TLR3 deficiency. We found that skin wound closure in TLR3(-/-) mice was significantly delayed compared with control littermates. Wound healing parameters, including re-epithelialization, granulation formation, and neovascularization, were decreased in TLR3(-/-) mice. Further studies revealed that the absence of TLR3 led to defective recruitment of neutrophils and macrophages, in association with decreased expression of the chemokines, MIP-2/CXCL2, MIP-1α/CCL3, and MCP-1/CCL2, in the wound. Moreover, in wild type mice, the mRNA level and protein content of TLR3 was significantly upregulated in wounded skins and silencing of TLR3 signal adaptor Toll/IL-1R domain-containing adapter inducing IFN-ß with small interfering RNA retarded wound closure. These results indicate an essential role for TLR3 and Toll/IL-1R domain-containing adapter inducing IFN-ß in wound healing by regulating chemokine production and recruitment of myeloid cells to wound for tissue repair.

HCV peptide (C5A), an amphipathic α-helical peptide of hepatitis virus C, is an activator of N-formyl peptide receptor in human phagocytes.

The hepatitis C virus (HCV) nonstructural 5A, a phosphorylated zinc metalloprotein, is an essential component of the HCV replication complex. An amphipathic α-helical peptide (HCV peptide [C5A]) derived from nonstructural 5A membrane anchor domain possesses potent anti-HCV and anti-HIV activity in vitro. In this study, we aimed to investigate the potential of HCV peptide (C5A) to regulate host immune responses. The capacity of HCV peptide (C5A) in vitro to induce migration and calcium mobilization of human phagocytes and chemoattractant receptor-transfected cells was investigated. The recruitment of phagocytes in vivo induced by HCV peptide (C5A) and its adjuvant activity were examined. The results revealed that HCV peptide (C5A) was a chemoattractant and activator of human phagocytic leukocytes by using a G-protein coupled receptor, namely formyl peptide receptor. In mice, HCV peptide (C5A) induced massive phagocyte infiltration after injection in the air pouch or the s.c. region. HCV peptide (C5A) also acted as an immune adjuvant by enhancing specific T cell responses to Ag challenge in mice. Our results suggest that HCV peptide (C5A) derived from HCV regulates innate and adaptive immunity in the host by activating the formyl peptide receptor.

High-mobility group box-1 mediates toll-like receptor 4-dependent angiogenesis.

OBJECTIVE: Inflammation is closely linked to angiogenesis, and Toll-like receptors (TLRs) are the key mediators of inflammatory responses. However, the impact of TLRs on angiogenesis is incompletely understood. In this study, we determined the involvement of TLRs in angiogenesis. METHODS AND RESULTS: In a mouse model of alkali-induced corneal neovascularization (CNV), we found that CNV was attenuated in TLR4-/- but not TLR2-/- mice. Further study revealed that the absence of TLR4 led to decreased production of proangiogenic factors in association with reduced accumulation of macrophages at the site of wounds, which was associated with reduced expression of high-mobility group box-1 (HMGB1) protein, an endogenous ligand for TLR4. Topical application of HMGB1 to the injured cornea promoted CNV with increased macrophage accumulation in wild-type mice but not in TLR4-/- mice. HMGB1 treatment in vitro also promoted the production of proangiogenic factors by mouse macrophages in a TLR4-dependent manner. Furthermore, antagonists of HMGB1 and TLR4 reduced CNV and macrophage recruitment in the injured cornea of wild-type mice. CONCLUSIONS: Our results suggest that the release of HMGB1 in the wounds initiates TLR4-dependent responses that contribute to neovascularization. Thus, targeting HMGB1-TLR4 signaling cascade may constitute a novel therapeutic approach to angiogenesis-related diseases.

Epigenetic silencing of JMJD5 promotes the proliferation of hepatocellular carcinoma cells by down-regulating the transcription of CDKN1A 686.

Proteins that contain jumonji C (JmjC) domains have recently been identified as major contributors to various malignant human cancers through epigenetic remodeling. However, the roles of these family members in the pathogenesis of hepatocellular carcinoma (HCC) are obscure. By mining public databases, we found that the HCC patients with lower JmjC domain-containing protein 5 (JMJD5) expression exhibited shorter survival time. We then confirmed that JMJD5 expression was indeed decreased in HCC specimens, which was caused by the altered epigenetic histone modifications, the decreased H3K9ac, H3K27ac and H3K4me2/3 together with the increased trimethylation of H3K27 and H3K9 on the JMJD5 promoter. Functional experiments revealed that JMJD5 knockdown promoted HCC cell proliferation and in vivo tumorigenicity by accelerating the G1/S transition of the cell cycle; in contrast, ectopic JMJD5 expression had the opposite effects. At molecular mechanism, we found that, in HCC cell lines including TP53-null Hep3B, JMJD5 knockdown led to the down-regulation of CDKN1A and ectopic expression of JMJD5 not only increased but also rescued CDKN1A transcription. Moreover, CDKN1A knockdown could abrogate the effect of JMJD5 knockdown or overexpression on cell proliferation, suggesting that JMJD5 inhibits HCC cell proliferation mainly by activating CDKN1A expression. We further revealed that JMJD5 directly enhances CDKN1A transcription by binding to CDKN1A's promoter independent of H3K36me2 demethylase activity. In short, we first prove that JMJD5 is a tumor suppressor gene in HCC pathogenesis, and the epigenetic silencing of JMJD5 promotes HCC cell proliferation by directly down-regulating CDKN1A transcription.

Formylpeptide receptor 1 mediates the tumorigenicity of human hepatocellular carcinoma cells.

G protein-coupled chemoattractant receptors (GPCRs) have been implicated in cancer progression. Formylpeptide receptor 1 (FPR1) was originally identified as a GPCR mediating anti-microbial host defense. However, the role of FPR1 in tumorigenesis remains poorly understood. The current study aims to investigate the potential of FPR1 to regulate human hepatoma growth and invasion. We found the FPR1 gene and protein expression in human intratumoral and peritumoral tissues of hepatocellular carcinoma (HCC) specimens and in human hepatoma cell lines. FPR1 activation mediated the migration, calcium mobilization and ERK-dependent IL-8 production by hepatic cancer cells. FPR1 knockdown substantially reduced the tumorigenicity of hepatoma cells in nude mice. Necrotic hepatic tumor cells released factor(s) that activated FPR1 in live tumor cells. Our results indicate a critical role of FPR1 in the progression of malignant human hepatic cancer. FPR1 thus may represent a molecular target for the development of novel anti-hepatoma therapeutics.

High glucose induces and activates Toll-like receptor 4 in endothelial cells of diabetic retinopathy.

BACKGROUND: Hyperglycemia-induced inflammation causes the dysfunction of blood vessels, and Toll-like receptor 4 (TLR4) plays a key role in inflammation-induced angiogenesis. However, the impact of TLR4 on the pathogenesis of diabetic retinopathy (DR) is poorly understood. In this study, we examined the expression of TLR4 in retinal vascular endothelial cells of patients with DR and diabetic mice, and explored the role of TLR4 in mediating inflammatory responses by human microvascular endothelial cells (HMEC-1) under high-glucose condition. METHODS: The expression of TLR4 in retinal vascular endothelial cells of patients with proliferative diabetic retinopathy and diabetic mice induced by streptozotocin was examined using immunofluorescence. HMEC-1 cells were cultured and the expression of TLR4, MyD88 and Interleukin-1ß (IL-1ß) was examined under high-glucose condition. Endothelial cells with TLR4 silencing and antagonist of TLR4 as well as endothelial cells from TLR4 deficient mice were used to study the effect of activated TLR4 on inflammation induced by high-glucose treatment. RESULTS: We observed that TLR4 was detected in CD31-labled human retinal vascular endothelia and its expression was markedly increased in fibrovascular membranes from DR patients and in retinal vascular endothelial cells of diabetic mice. The expression of TLR4, MyD88 and IL-1ß was enhanced by high glucose in cultured HMEC-1 and the expression of TLR4 and IL-1ß was inhibited by TLR4 siRNA knock-down and TLR4 antagonist. The expression of IL-1ß by endothelial cells from TLR4 deficient mice under high glucose condition was decreased. CONCLUSIONS: Our results revealed that hyperglycemia induced overexpression and activation of TLR4 in endothelial cells. This effect may lead to inflammatory responses contribute to the pathogenesis of diabetic retinopathy.

Hepatocyte-Specific Arid1a Deficiency Initiates Mouse Steatohepatitis and Hepatocellular Carcinoma.

ARID1A, encoding a subunit of chromatin remodeling SWI/SNF complexes, has recently been considered as a new type of tumor suppressor gene for its somatic mutations frequently found in various human tumors, including hepatocellular carcinoma (HCC). However, the role and mechanism of inactivated ARID1A mutations in tumorigenesis remain unclear. To investigate the role of ARID1A inactivation in HCC pathogenesis, we generated hepatocyte-specific Arid1a knockout (Arid1aLKO) mice by crossing mice carrying loxP-flanked Arid1a exon 8 alleles (Arid1af/f) with albumin promoter-Cre transgenic mice. Significantly, the hepatocyte-specific Arid1a deficiency results in mouse steatohepatitis and HCC development. In Arid1aLKO mice, we found that innate immune cells, including F4/80+ macrophages and CD11c+ neutrophil cells, infiltrate into the liver parenchyma, accompanied by the increased tumor necrosis factor (TNF)-α and interleukin (IL)-6, and activation of STAT3 and NF-κB pathways. In conclusion, hepatocyte-specific Arid1a deficiency could lead to mouse steatohepatitis and HCC development. This study provides an alternative mechanism by which Arid1a deficiency contributes to HCC tumorigenesis.

Insulin receptor tyrosine kinase substrate activates EGFR/ERK signalling pathway and promotes cell proliferation of hepatocellular carcinoma.

Insulin receptor tyrosine kinase substrate (IRTKS) is closely associated with actin remodelling and membrane protrusion, but its role in the pathogenesis of malignant tumours, including hepatocellular carcinoma (HCC), is still unknown. In this study, we showed that IRTKS was frequently upregulated in HCC samples, and its expression level was significantly associated with tumour size. Enforced expression of IRTKS in human HCC cell lines significantly promoted their proliferation and colony formation in vitro, and their capacity to develop tumour xenografts in vivo, whereas knockdown of IRTKS resulted in the opposite effects. Furthermore, the bromodeoxyuridine (BrdU) incorporation analyses and propidium iodide staining indicated that IRTKS can promote the entry into S phase of cell cycle progression. Significantly, IRTKS can interact with epidermal growth factor receptor (EGFR), results in the phosphorylation of extracellular signal-regulated kinase (ERK). By contrast, inhibition of ERK activation can attenuate the effects of IRTKS overexpression on cellular proliferation. Taken together, these data demonstrate that IRTKS promotes the proliferation of HCC cells by enhancing EGFR-ERK signalling pathway.

Production of recombinant human HMGB1 and anti-HMGB1 rabbit serum.

High-mobility group box-1 (HMGB1) plays important roles in inflammation, immune responses, and tumor progression. Since HMGB1 and its components have been shown to be mediators of a number of diseases but several sources of recombinant HMGB1 showed controversial biological activity, it is important to obtain recombinant HMGB1 with properties that resemble the native protein. For this purpose, we cloned genes coding for human HMGB1 and its active components A box and B box by PCR and inserted the cloned genes into pET28a vectors for transformation of Escherichia coli BL21. The E. coli expressed proteins were then purified with a Ni(2+)-NTA column and the endotoxin content was removed. Recombinant human HMGB1 (rhHMGB1) and its B box thus obtained stimulated, but A box inhibited, the production of the chemokine CXCL8/IL-8 by THP-1 monocytic cell line. We also used purified rhHMGB1 to immunize rabbits and generated potent anti-sera, which was capable of neutralizing the activity of rhHMGB1 in vitro and detecting the increased HMGB1 expression in inflammatory tissues in mice and humans. Thus, we have established essential means to produce biologically active rhHMGB1 that will facilitate us to study its role in diseases and to explore its potential as a therapeutic agent.

The essential roles of Toll-like receptor signaling pathways in sterile inflammatory diseases.

Toll-like receptors (TLRs) form a family of pattern recognition receptors with at least 11 members in human and 13 in mouse. TLRs recognize a wide variety of putative host-derived agonists that have emerged as key mediators of innate immunity. TLR signaling also plays an important role in the activation of the adaptive immune system by inducing pro-inflammatory cytokines and upregulating costimulatory molecules of antigen presenting cells. Inappropriate activation of TLRs by self-components generated by damaged tissues may result in sterile inflammation. This review discusses the contribution of TLR signaling to the initiation and progression of non-infectious inflammatory processes, such as ischemia and reperfusion (I/R) injury, tissue repair and regeneration and autoimmune diseases. The involvement of TLR signaling in the pathogenesis of sterile inflammation-related diseases may provide novel targets for the development of therapeutics.

The role of TLR2, TRL3, TRL4, and TRL9 signaling in the pathogenesis of autoimmune disease in a retinal autoimmunity model.

PURPOSE: Induction of tissue-specific experimental autoimmune diseases involves the use of complete Freund adjuvant containing Mycobacterium tuberculosis, whose recognition by the innate immune system depends on Toll-like receptors (TLRs) that signal through the adaptor molecule MyD88. The authors' previous study showed that MyD88(-/-) mice, but not TLR2(-/-), TLR4(-/-), or TLR9(-/-) mice, were resistant to experimental autoimmune uveitis (EAU). METHODS: The EAU induction in mice deficient in TLR3 or mice double deficient in TLR2+4, TLR2+9, and TLR4+9 was examined and the role of the TLR agonists in the adjuvant effect involved in the induction of EAU was assessed. RESULTS: TLR3-deficient and TLR2+4, TLR2+9, and TLR4+9 double-deficient mice were as susceptible to EAU as their control littermates. However, in mice immunized with a low-dose EAU regimen, TLR4 agonist lipopolysaccharide (LPS) enhanced EAU scores, delayed-type hypersensitivity responses, and antigen-specific T-cell proliferation. Antigen-specific IL-17 and IFN-gamma production by T lymphocytes was markedly increased in the LPS-treated group. The effects of LPS on EAU were abolished by treatment with an LPS deactivator polymyxin B. Inclusion of agonists for TLR2, TRL3, or TRL9 in immunization also enhanced EAU scores. CONCLUSIONS: These results suggest that signaling of TLR2, TRL3, TRL4, and TRL9 is highly redundant in the adjuvant effect needed to induce EAU and that diverse microbial infections may contribute to the pathogenesis of diseases such as uveitis.