Anti‐arthritis activity of cationic materials

Cationic materials exhibit remarkable anti‐inflammatory activity in experimental arthritis models. Our aim was to confirm this character of cationic materials and investigate its possible mechanism. Adjuvant‐induced arthritis (AIA) models were used to test cationic materials for their anti‐inflammatory activity. Cationic dextran (C‐dextran) with different cationic degrees was used to investigate the influence of the cationic elements of materials on their anti‐inflammatory ability. Peritoneal macrophages and spleen cells were used to test the expression of cytokines stimulated by cationic materials. Interferon (IFN)‐γ receptor‐deficient mice and macrophage‐depleted rats were used to examine the possible mechanisms of the anti‐inflammatory activity of cationic materials. In AIA models, different cationic materials shared similar anti‐inflammatory characters. The anti‐inflammatory activity of C‐dextran increased with as the cationic degree increased. Cationic materials stimulated interleukin (IL)‐12 expression in peritoneal macrophages, and strong stimulation of IFN‐γ secretion was subsequently observed in spleen cells. In vivo experiments revealed that circulating IL‐12 and IFN‐γ were enhanced by the cationic materials. Using IFN‐γ receptor knockout mice and macrophage‐depleted rats, we found that IFN‐γ and macrophages played key roles in the anti‐inflammatory activity of the materials towards cells. We also found that neutrophil infiltration at inflammatory sites was reduced when AIA animals were treated with C‐dextran. We propose that cationic signals act through an unknown receptor on macrophages to induce IL‐12 secretion, and that IL‐12 promotes the expression of IFN‐γ by natural killer cells (or T cells). The resulting elevated systemic levels of IFN‐γ inhibit arthritis development by preventing neutrophil recruitment to inflammatory sites.     

CXXC finger protein 4 inhibits the CDK18‐ERK1/2 axis to suppress the immune escape of gastric cancer cells with involvement of ELK1/MIR100HG pathway

Gastric cancer, is the fourth most common tumour type yet, ranks second in terms of the prevalence of cancer‐related deaths worldwide. CXXC finger protein 4 (CXXC4) has been considered as a novel cancer suppressive factor, including gastric cancer. This study attempted to investigate the possible function of CXXC4 in gastric cancer and the underlying mechanism. The binding of the ETS domain‐containing protein‐1 (ELK1) to the long non‐coding RNA MIR100HG promoter region was identified. Then, their expression patterns in gastric cancer tissues and cells (SGC7901) were detected. A CCK‐8 assay was used to detect SGC7901 cell proliferation. Subsequently, SGC7901 cells were co‐cultured with CD3+ T cells, followed by measurement of CD3+ T cell proliferation, magnitude of IFN‐γ+ T cell population and IFN‐γ secretion. A nude mouse model was subsequently developed for in vivo validation of the in vitro results. Low CXXC4 expression was found in SGC7901 cells. Nuclear entry of ELK1 can be inhibited by suppression of the extent of ELK1 phosphorylation. Furthermore, ELK1 is able to bind the MIR100HG promoter. Overexpression of CXXC4 resulted in weakened binding of ELK1 to the MIR100HG promoter, leading to a reduced proliferative potential of SGC7901 cells, and an increase in IFN‐γ secretion from CD3+ T cells. Moreover, in vivo experiments revealed that CXXC4 inhibited immune escape of gastric cancer cells through the ERK1/2 axis. Inhibition of the CXXC4/ELK1/MIR100HG pathway suppressed the immune escape of gastric cancer cells, highlighting a possible therapeutic target for the treatment of gastric cancer.     

IL‐27 Rα+ cells promoted allorejection via enhancing STAT1/3/5 phosphorylation

Recently, emerging evidence strongly suggested that the activation of interleukin‐27 Receptor α (IL‐27Rα) could modulate different inflammatory diseases. However, whether IL‐27Rα affects allotransplantation rejection is not fully understood. Here, we investigated the role of IL‐27Rα on allorejection both in vivo and in vitro. The skin allotransplantation mice models were established, and the dynamic IL‐27Rα/IL‐27 expression was detected, and IL‐27Rα⁺ spleen cells adoptive transfer was performed. STAT1/3/5 phosphorylation, proliferation and apoptosis were investigated in mixed lymphocyte reaction (MLR) with recombinant IL‐27 (rIL‐27) stimulation. Finally, IFN‐γ/ IL‐10 in graft/serum from model mice was detected. Results showed higher IL‐27Rα/IL‐27 expression in allografted group compared that syngrafted group on day 10 (top point of allorejection). IL‐27Rα⁺ spleen cells accelerated allograft rejection in vivo. rIL‐27 significantly promoted proliferation, inhibited apoptosis and increased STAT1/3/5 phosphorylation of alloreactive splenocytes, and these effects of rIL‐27 could be almost totally blocked by JAK/ STAT inhibitor and anti‐IL‐27 p28 Ab. Finally, higher IL‐27Rα⁺IFN‐γ⁺ cells and lower IL‐27Rα⁺IL‐10⁺ cells within allografts, and high IFN‐γ/low IL‐10 in serum of allorejecting mice were detected. In conclusion, these data suggested that IL‐27Rα⁺ cells apparently promoted allograft rejection through enhancing alloreactive proliferation, inhibiting apoptosis and up‐regulating IFN‐γ via enhancing STAT pathway. Blocking IL‐27 pathway may favour to prevent allorejection, and IL‐27Rα may be as a high selective molecule for targeting diagnosis and therapy for allotransplantation rejection.     

The immune characterization of interferon‐β responses in tuberculosis patients

We aimed to assess the immunoregulatory effects of IFN‐β in patients with tuberculous pleurisy. IFN‐β, IFN‐γ and IL‐17 expression levels were detected, and correlations among these factors in different culture groups were analyzed. Pleural fluid mononuclear cells (PFMC) from tuberculous pleural effusions, but not peripheral blood mononuclear cells (PBMC) from healthy donors, spontaneously expressed IFN‐β, IL‐17 and IFN‐γ. Moreover, exogenous IFN‐β significantly inhibited the expression of IL‐17 in PFMC. By contrast, IFN‐β simultaneously enhanced the levels of IFN‐γ. To further investigate the regulation of IL‐17 and IFN‐γ by endogenous IFN‐β, an IFN‐β neutralizing antibody was simultaneously added to bacillus Calmette‐Guérin (BCG)‐stimulated PFMC. IL‐17 expression was significantly increased, but IFN‐γ production was markedly decreased in the experimental group supplemented with the IFN‐β neutralizing antibody. Simultaneously, IL‐17 production was remarkably increased in the experimental group supplemented with the IFN‐γ neutralizing antibody. Taken together, in our study, we first found that freshly isolated PFMC, but not PBMC from healthy donors, spontaneously expressed IFN‐β, IL‐17 and IFN‐γ in vivo. Moreover, IFN‐β suppressed IL‐17 expression and increased IFN‐γ production. Furthermore, both IFN‐β and IFN‐γ down‐regulated IL‐17 expression. These observations suggest that caution is required when basing anti‐tuberculosis treatment on the inhibition of IFN‐β signaling.     

Molecular properties of gp100‐reactive T‐cell receptors drive the cytokine profile and antitumor efficacy of transgenic host T cells

To study the contribution of T‐cell receptors (TCR) to resulting T‐cell responses, we studied three different human αβ TCRs, reactive to the same gp100‐derived peptide presented in the context of HLA‐A*0201. When expressed in primary CD8 T cells, all receptors elicited classic antigen‐induced IFN‐γ responses, which correlated with TCR affinity for peptide–MHC in the order T4H2 > R6C12 > SILv44. However, SILv44 elicited superior IL‐17A release. Importantly, in vivo, SILv44‐transgenic T cells mediated superior antitumor responses to 888‐A2 + human melanoma tumor cells upon adoptive transfer into tumor‐challenged mice while maintaining IL‐17 expression. Modeling of the TCR ternary complexes suggested architectural differences between SILv44 and the other complexes, providing a potential structural basis for the observed differences. Overall, the data reveal a more prominent role for the T‐cell receptor in defining host T‐cell physiology than traditionally assumed, while parameters beyond IFN‐γ secretion and TCR affinity ultimately determine the reactivity of tumor‐reactive T cells.     

T‐Cell Recruitment and Th1 Polarization in Adipose Tissue During Diet‐Induced Obesity in C57BL/6 Mice

The role of adaptive immunity in obesity‐associated adipose tissue (AT) inflammation and insulin resistance (IR) is controversial. We employed flow cytometry and quantitative PCR to assess T‐cell recruitment and activation in epididymal AT (eAT) of C57BL/6 mice during 4–22 weeks of a high‐fat diet (HFD (60% energy)). By week 6, eAT mass and stromal vascular cell (SVC) number increased threefold in mice fed HFD, coincident with onset of IR. We observed no increase in the proportion of CD3⁺ SVCs or in gene expression of CD3, interferon‐γ (IFN‐γ), or regulated upon activation, normal T‐cell expressed and secreted (RANTES) during the first 16 weeks of HFD. In contrast, CD11c⁺ macrophages (MΦ) were enriched sixfold by week 8 (P < 0.01). SVC enrichment for T cells (predominantly CD4⁺ and CD8⁺) and elevated IFN‐γ and RANTES gene expression were detected by 20–22 weeks of HFD (P < 0.01), coincident with the resolution of eAT remodeling. HFD‐induced T‐cell priming earlier in the obesity time course is suggested by (i) elevated (fivefold) interleukin‐12 (IL‐12)p40 gene expression in eAT by week 12 (P ≤ 0.01) and (ii) greater IFN‐γ secretion from phorbol myristate acetate (PMA)/ionophore‐stimulated eAT explants at week 6 (onefold, P = 0.08) and week 12 (fivefold, P < 0.001). In conclusion, T‐cell enrichment and IFN‐γ gene induction occur subsequent to AT macrophage (ATMΦ) recruitment, onset of IR and resolution of eAT remodeling. However, enhanced priming for IFN‐γ production suggests the contribution of CD4⁺ and/or CD8⁺ effectors to cell‐mediated immune responses promoting HFD‐induced AT inflammation and IR.     

Effect of heme oxygenase‐1 transduced bone marrow mesenchymal stem cells on damaged intestinal epithelial cells in vitro

In this study, we explored the effects of mesenchymal stem cells (MSCs) from bone marrow overexpressing heme oxygenase‐1 (HO‐1) on the damaged human intestinal epithelial barrier in vitro. Rat MSCs were isolated from bone marrow and transduced with rat HO‐1 recombinant adenovirus (HO‐MSCs) for stable expression of HO‐1. Colorectal adenocarinoma 2 (Caco2) cells were treated with tumor necrosis factor‐α (TNF‐α) to establish a damaged colon epithelial model. Damaged Caco2 were cocultured with MSCs, Ad‐MSCs, Ad‐HO + MSCs or HO‐MSCs. mRNA and protein expression of Zona occludens‐1 (ZO‐1) and human HO‐1 and the release of cytokines were measured. ZO‐1 and human HO‐1 in Caco2 were significantly decreased after treatment with TNF‐α; and this effect was reduced when coculture with MSCs from bone marrow. Expression of ZO‐1 was not significantly affected by Caco2 treatment with TNF‐α, Ad‐HO, and MSCs. In contrast, ZO‐1 and human HO‐1 increased significantly when the damaged Caco2 was treated with HO‐MSCs. HO‐MSCs showed the strongest effect on the expression of ZO‐1 in colon epithelial cells. Coculture with HO‐MSCs showed the most significant effects on reducing the expression of IL‐2, IL‐6, IFN‐γ and increasing the expression of IL‐10. HO‐MSCs protected the intestinal epithelial barrier, in which endogenous HO‐1 was involved. HO‐MSCs play an important role in the repair process by reducing the release of inflammatory cytokines and increasing the release of anti‐inflammatory factors. These results suggested that HO‐MSCs from bone marrow were more effective in repairing the damaged intestinal epithelial barrier, and the effectiveness of MSCs was improved by HO‐1 gene transduction, which provides favorable support for the application of stem cell therapy in the intestinal diseases.     

Modulatory effects of interferon‐γ and interleukin‐4 on cellular immune responses against Hypoderma lineatum antigens

This study investigates the in vitro modulatory effects of interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4) on both proliferative bovine T cell responses and IL‐10 production induced by different antigens [crude larval extract and the purified fractions hypodermin A, B and C (HyA, HyB, HyC)] obtained from first instars of Hypoderma lineatum (Diptera: Oestridae), alone or in the presence of the mitogen concanavalin A. Incubation with the different parasitic antigens resulted in significant inhibition of T cell proliferation and IL‐10 production, which, in general, did not revert after the addition of IFN‐γ and IL‐4. In the absence of antigens, IL‐4 induced significant inhibition of mitogen‐induced T cell responses. Exogenous IFN‐γ exhibited an inhibitory effect on cell proliferation in the presence of the purified fractions HyB and HyC. These in vitro data suggest that far from neutralizing the effects of larval antigens, the addition of IFN‐γ potentiates their anti‐proliferative activity; by contrast, IL‐4 had no consistent effects on proliferative responses to Hypoderma. IL‐4 provoked an increment of IL‐10 levels in supernatants of HyB‐stimulated cells. In conclusion, exogenous IFN‐γ and IL‐4 were unable to counteract the suppressor effects of H. lineatum antigens.     

Tim‐3 blockade promotes iNKT cell function to inhibit HBV replication

Increased expression of T cell immunoglobulin and mucin domain‐3 (Tim‐3) on invariant natural killer T (iNKT) cells is reported in chronic hepatitis B virus (HBV) infection. However, whether Tim‐3 regulates iNKT cells in chronic HBV condition remains unclear. In this study, our results showed that the expression of Tim‐3 was up‐regulated on hepatic iNKT cells from HBV‐transgenic (Tg) mice or iNKT cells stimulated with α‐galactosylceramide (α‐Galcer). Compared with Tim‐3^?iNKT cells, Tim‐3⁺iNKT cells expressed more IFN‐γ, IL‐4 and CD107a, indicating a strong relationship between Tim‐3 and iNKT cell activation. Constantly, treatment of Tim‐3 blocking antibodies significantly enhanced the production of IFN‐γ, TNF‐α, IL‐4 and CD107a in iNKT cells both in vivo and in vitro. This Tim‐3^? mediated suppression of iNKT cells was further confirmed in Tim‐3 knockout (KO) mice. Moreover, Tim‐3 blockade promoted α‐Galcer‐triggered inhibition of HBV replication, displaying as the decreased HBV DNA and HBsAg level in serum, and down‐regulated pgRNA expression in liver tissues. Collectively, our data, for the first time, demonstrated the potential role of Tim‐3 blockade in promoting iNKT cell‐mediated HBV inhibition. Therefore, combination of α‐Galcer with Tim‐3 blockade might be a promising approach in chronic hepatitis B therapy.     

Regulation of interleukin‐1β by interferon‐γ is species specific,limited by suppressor of cytokine signalling 1 and influences interleukin‐17 production

Reports describing the effect of interferon‐γ (IFNγ) on interleukin‐1β (IL‐1β) production are conflicting. We resolve this controversy by showing that IFNγ potentiates IL‐1β release from human cells, but transiently inhibits the production of IL‐1β from mouse cells. Release from this inhibition is dependent on suppressor of cytokine signalling 1. IL‐1β and Th17 cells are pathogenic in mouse models for autoimmune disease, which use Mycobacterium tuberculosis (MTB), in which IFNγ and IFNβ are anti‐inflammatory. We observed that these cytokines suppress IL‐1β production in response to MTB, resulting in a reduced number of IL‐17‐producing cells. In human cells, IFNγ increased IL‐1β production, and this might explain why IFNγ is detrimental for multiple sclerosis. In mice, IFNγ decreased IL‐1β and subsequently IL‐17, indicating that the adaptive immune response can provide a systemic, but transient, signal to limit inflammation.     

Dual specificity phosphatase16 is a negative regulator of c‐Jun NH2‐terminal kinase activity in T cells

Activation of MAPK is negatively regulated by DUSP, which dephosphorylate the phosphothreonine and phosphotyrosine residues. We have identified a novel JNK‐specific DUSP, DUSP16, from murine macrophages. Its involvement in T cells has not yet been defined. In the present study, we found expression of DUSP16 in thymocytes and activated T cells but not in naive T cells. To elucidate the roles of DUSP16 in T cells, transgenic mice expressing a dominant negative form of DUSP16 specifically in T cells were generated (dnDUSP16 Tg). JNK activity was selectively augmented in the thymocytes of these dnDUSP16 Tg mice. CD4 T cells in dnDUSP16 Tg mice showed normal levels of proliferation and IL‐2 production after TCR triggering, while they produced increased IFN‐γ but reduced Th2 cytokines compared with wild type CD4 T cells. On the other hand CD8 T cells in dnDUSP16 Tg mice produced an increased amount of IL‐2, which resulted in enhanced proliferation and IFN‐γ production. These results suggest that DUSP16 is an important regulator of JNK activity and effector functions of CD4 and CD8 T cells.     

Vaccinia virus expressing IL‐37 promotes antitumor immune responses in hepatocellular carcinoma

The aim of this study was to investigate the effect of vaccinia virus expressing IL‐37 (VV‐IL‐37) on cell proliferation, migration and invasion of hepatocellular carcinoma (HCC) and its possible underlying molecular mechanisms. In this study, we constructed a cancer‐targeted vaccinia virus carrying the IL‐37 gene knocked in the region of the viral thymidine kinase (TK) gene. Human HCC cell lines were assayed in vitro for cell proliferation, migration and invasion. Serum level, relative mRNA level and protein level of IL‐37 in HCC cell lines SMMC7721 and Bel7402 were tested by ELISA assay, qRT‐PCR and western blot, respectively. The levels of IL‐2, IFN‐γ and TNF‐α in HCC tumor tissues were also analyzed by ELISA. STAT3 and p‐STAT3 expression in tumor tissues were determined by western blot. Our results showed that VV‐IL‐37 efficiently infected and inhibited HCC cells proliferation, migration and invasion via decreasing STAT3 phosphorylation. In vivo, VV‐IL‐37 expressed IL‐37 at a high level in the transplanted tumor, reduced STAT3 activity, and eventually inhibited tumor growth. In conclusion, we demonstrate that VV‐IL‐37 promotes antitumor immune responses in HCC.     

Essential involvement of cross‐talk between IFN‐γ and TNF‐α in CXCL10 production in human THP‐1 monocytes

Interferon (IFN)‐γ‐induced protein 10 (IP‐10/CXCL10), a CXC chemokine, has been documented in several inflammatory and autoimmune disorders including atopic dermatitis and bronchial asthma. Although CXCL10 could be induced by IFN‐γ depending on cell type, the mechanisms regulating CXCL10 production following treatment with combination of IFN‐γ and TNF‐α have not been adequately elucidated in human monocytes. In this study, we showed that TNF‐α had more potential than IFN‐γ to induce CXCL10 production in THP‐1 monocytes. Furthermore, IFN‐γ synergistically enhanced the production of CXCL10 in parallel with the activation of NF‐κB in TNF‐α‐stimulated THP‐1 cells. Blockage of STAT1 or NF‐κB suppressed CXCL10 production. JAKs inhibitors suppressed IFN‐γ plus TNF‐α‐induced production of CXCL10 in parallel with activation of STAT1 and NF‐κB, while ERK inhibitor suppressed production of CXCL10 as well as activation of NF‐κB, but not that of STAT1. IFN‐γ‐induced phosphorylation of JAK1 and JAK2, whereas TNF‐α induced phosphorylation of ERK1/2. Interestingly, IFN‐γ alone had no effect on phosphorylation and degradation of IκB‐α, whereas it significantly promoted TNF‐α‐induced phosphorylation and degradation of IκB‐α. These results suggest that TNF‐α induces CXCL10 production by activating NF‐κB through ERK and that IFN‐γ induces CXCL10 production by increasing the activation of STAT1 through JAKs pathways. Of note, TNF‐α‐induced NF‐κB may be the primary pathway contributing to CXCL10 production in THP‐1 cells. IFN‐γ potentiates TNF‐α‐induced CXCL10 production in THP‐1 cells by increasing the activation of STAT1 and NF‐κB through JAK1 and JAK2. J. Cell. Physiol. 220: 690–697, 2009. © 2009 Wiley‐Liss, Inc.     

Cytokine and cellular responses to human herpesvirus‐6B in patients with B‐acute lymphoblastic leukemia

Primary infection with human herpesvirus‐6 (HHV‐6), is followed by its lifelong persistence in the host. Most T‐cell responses to HHV‐6 have been characterized using peripheral blood from healthy adults; however, the role of HHV‐6 infection in immune modulation has not been elucidated for some diseases. Therefore, in this study the immune response to HHV‐6 infection in patients with B‐acute lymphoblastic leukemia (B‐ALL) was analyzed. HHV‐6 load was quantified in blood samples taken at the time of diagnosis of leukemia and on remission. The same concentrations of anti‐ and pro‐inflammatory cytokines (IL‐4, IL‐1, IL‐6, IL‐8, IL‐12p70, IL‐17a, TNF‐α and IFN‐γ) were detected in plasma samples from 20 patients with and 20 without detectable HHV‐6 virus loads in blood. Characterization of T‐cell responses to HHV‐6 showed low specific T‐cells frequencies of 2.08% and 1.46% in patients with and without detectable viral loads, respectively. IFN‐γ‐producing T cells were detected in 0.03%–0.23% and in 0%–0.2% of CD4+T cells, respectively. Strong production of IL‐6 was detected in medium supernatants of challenged T‐cells whatever the HHV‐6 status of the patients (973.51 ± 210.06 versus 825.70 ± 210.81 pg/mL). However, concentrations of TNF‐α and IFN‐γ were low. Thus, no association between plasma concentrations of cytokines and detection of HHV‐6 in blood was identified, suggesting that HHV‐6 is not strongly associated with development of B‐ALL. The low viral loads detected may correspond with latently infected cells. Alternatively, HHV‐6B specific immune responses may be below the detection threshold of the assays used.