Comparison and subsets analysis of peripheral CD4+T cells in patients with psoriasis and psoriatic arthritis.

Psoriatic arthritis (PsA) is a disease that transformed from psoriasis (PsO), and its underlying mechanisms are still not fully understood. Overactivation of the immune system is a key factor driving inflammatory diseases. Our goal is to define the unbalanced subsets of peripheral blood CD4 +T cells between PsO and PsA patients. Blood samples from 43 patients (23 PsA and 20 PsO) and 36 healthy donors (HD) were studied. Peripheral blood mononuclear cells (PBMC) were separated from blood and underwent fluorescent staining to assess CD4+T cell subsets by flow cytometry. We found that frequencies of various CD4+T cells including Th1, Th2, Th17, and Tfh were higher in the patients with PsO or PsA than those of healthy donors, indicating the general expansion of CD4+T cells in inflammatory conditions. More importantly, we observed the significant imbalance of Th1/Th2 between patients with PsO and PsA. Pearson correlation analysis showed that Th1/Th2 ratio was positively correlated with disease activity in psoriatic arthritis (DAPSA), Tfh/Tfr ratio was positively correlated with DAPSA score and visual analogue scale (VAS) score in PsA patients. Together, our results highlight the CD4+T cell changes in the transition from PsO to PsA, may contribute to early assessment and intervention.

CX-4945 inhibits fibroblast-like synoviocytes functions through the CK2-p53 axis to reduce rheumatoid arthritis disease severity.

Fibroblast-like synoviocytes (FLS) mediate many pathological processes in rheumatoid arthritis (RA), including pannus formation, bone erosion, and inflammation. RA FLS have unique aggressive phenotypes and exhibit several tumor cell-like characteristics, including hyperproliferation, excessive migration and invasion. Casein kinase 2 (CK2) is reportedly overexpressed in numerous tumor types, and targeted inhibition of CK2 has therapeutic benefits for tumors. However, the expression level of CK2 and its functions in RA FLS remain unclear. Herein, we aimed to elucidate whether CK2 is responsible for the aggressive phenotypes of RA FLS and whether targeted therapy can alleviate the severity of RA. We found that CK2 subunits were elevated in RA FLS compared with osteoarthritis FLS, and the activity of CK2 also markedly increased in RA FLS. Targeted inhibition of CK2 using CX-4945 suppressed RA FLS proliferation through cell cycle arrest. Cell migration and invasion were also inhibited by CX-4945 treatment. Moreover, CX-4945 reduced Interleukin-6 (IL-6), CC motif chemokine ligand 2 (CCL2) and Matrix metalloproteinase-3 (MMP-3) secretion in RA FLS. Further proteomic investigation revealed that p53 signaling pathway significantly changes after CX-4945 treatment in RA FLS. The siRNA-mediated p53 knockdown partly abolished the anti-proliferation and reduced IL-6, MMP-3 secretion effects of CX-4945. Furthermore, CX-4945 administration alleviates arthritis severity in CIA mice. Collectively, our results demonstrated the abnormal elevation of CK2 and its positive association with abnormal phenotypes in RA FLS. Our novel findings suggest the possible therapeutic potential of CX-4945 for RA.

γ-Tocotrienol inhibits T helper 17 cell differentiation via the IL-6/JAK/STAT3 signaling pathway.

γ-Tocotrienol (GT3), a member of the vitamin E family, is well known for its medicinal value in clinical treatments. However, the role of GT3 in T helper 17 (Th17)/regulatory T cell (Treg) differentiation and function is not fully understood. Here, we demonstrated that GT3 suppressed Th17 differentiation in vitro by inhibiting signal transducer and activator of transcription 3 (STAT3) phosphorylation in the interleukin 6 (IL-6)/Janus kinase (JAK)/STAT3 signaling pathway. GT3 also inhibited HIF1A expression in Th17 metabolism. Additionally, we showed that GT3 treatment inhibited disease aggravation in an imiquimod (IMQ)-induced psoriasis-like mouse model by reducing the percentage of Th17 cells in the spleen in vivo. The findings of this study demonstrated the effects of GT3 on Th17 cells through the STAT3 signaling pathway.

Epigallocatechin-3 gallate regulates macrophage subtypes and immunometabolism to ameliorate experimental autoimmune encephalomyelitis.

Epigallocatechin-3 gallate (EGCG) is a polyphenolic component of tea and has potential curative effects in patients with autoimmune diseases. Multiple sclerosis (MS) is an autoimmune disease affecting the central nervous system (CNS). It remains unknown whether EGCG can regulate macrophage subtypes in MS. Here we evaluated the effects of EGCG in experimental autoimmune encephalomyelitis (EAE), MS mouse model. We found that EGCG treatment reduced EAE severity and macrophage inflammation in the CNS. Moreover, EAE severity was well correlated with the ratio of M1 to M2 macrophages, and EGCG treatment suppressed M1 macrophage-mediated inflammation in spleen. In vitro experiments showed that EGCG inhibited M1 macrophage polarization, but promoted M2 macrophage polarization. These effects were likely to be related to the inhibition of nuclear factor-κB signaling and glycolysis in macrophages by EGCG in macrophages. Overall, these findings provided important insights into the mechanisms through which EGCG may mediate MS.

T-614 Promotes Osteoblastic Cell Differentiation by Increasing Dlx5 Expression and Regulating the Activation of p38 and NF-κB.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease characterized by bone loss. Degree of inflammation has been identified as an important initiator of skeletal damage in RA. Iguratimod (T-614) is an anti-inflammatory agent which has been reported to show the inhibitory effect of bone destruction in RA. However, the role of T-614 in osteoblast differentiation is still not clear. In this study, we intended to find the effect of T-614 on the osteogenesis process. We detected osteogenesis markers and transcription factors associated with osteoblastic lineage and bone formation in the culture of mesenchymal stem cells which differentiate osteoblast. The contents and activity of alkaline phosphatase, levels of collagen type I and bone gla protein, and calcium nodule formation were increased significantly after T-614 treated. Meanwhile, the mRNAs expressions of Osterix and Dlx5 were also found to be increased significantly by real-time PCR. The changes of levels of phosphorylation of p38 and NF-κB were also detected by Western blot. The results showed that T-614 promotes osteoblastic differentiation by increasing the expression of Osterix and Dlx5 and increasing the activation of P38. T-614 could advance the ectopic expression of NF-κB to suppress inflammation, which indirectly inhibits the damage of the osteoblasts.

Berberine ameliorates TNBS induced colitis by inhibiting inflammatory responses and Th1/Th17 differentiation.

Th1 and Th17 cells, and their associated cytokines, have been associated with the pathogenesis of Crohn's disease. Berberine (BBR), a compound long used in traditional Chinese medicines, has been reported to have therapeutic effects in treating experimental colitis. In this study, we show that BBR had a protective effect on mice with TNBS-induced colitis. BBR inhibited levels of IFN-γ, IL-17, IL-6, IL-1ß and TNF-α both in the local colon and sera, and transiently increased levels of IL-22. BBR also markedly increased sIgA expression in the colon. BBR had pronounced effects on macrophage populations. Treatment with BBR adjusted the M2/M1 ratio. In addition, BBR exerted effects on adaptive immunity by suppressing numbers of Th1 and Th17 cells, as well as expression levels of their associated cytokines and transcriptional factors. BBR downregulated STAT3 and STAT1 phosphorylation, and inhibited phosphorylation of NF-kB. In vitro experiments showed that BBR inhibited the differentiation of Th17 and, to a lesser degree, Th1 cells, without affecting regulatory T cells. Therefore, we conclude that BBR plays a regulatory role in modulating the balance of immune responses in TNBS-induced colitis. Our study will help us understand the regulatory mechanisms exerted by BBR in the treatment of IBD.

OVA12, a novel tumor antigen, promotes cancer cell growth and inhibits 5-fluorouracil-induced apoptosis.

To achieve a better understanding of mechanisms that underlie carcinogenesis and to identify novel target molecules for diagnosis and therapy of carcinoma, we previously identified 24 distinct gene clones by immunoscreening of a cDNA library derived from an ovarian cancer patient through SEREX analysis. Among these genes we focused on a novel gene termed OVA12 and which putatively encodes a 114-amino-acid protein. In the present study, we found that OVA12 was ubiquitously overexpressed in diverse human tumor cell lines. Interestingly, we noticed that overexpression of OVA12 promoted proliferation of cancer cells in vitro and accelerated tumor growth in nude mice as compared to controls. Conversely, specific downregulation of OVA12 inhibited tumor cell proliferation and tumor growth both in vitro and in vivo. Furthermore, OVA12 inhibited 5-FU-induced apoptosis through specific upregulation of Mcl-1 and survivin. These results demonstrate that OVA12 is able to promote tumor growth, suggesting that this antigen might be a new potential target for development of cancer therapy.

OVA66 increases cell growth, invasion and survival via regulation of IGF-1R-MAPK signaling in human cancer cells.

Ovarian cancer-associated antigen 66 (OVA66), also known as CML66 (GenBank Accession No. AF283301), was first identified in an ovarian carcinoma complementary DNA (cDNA) expression library and was shown to play a role in tumorigenesis. Here, we find that OVA66 influences tumorigenesis by regulating the type I insulin-like growth factor receptor (IGF-1R) signaling pathway. Stable knockdown of OVA66 in cancer cells attenuated phosphorylation of IGF-1R and extracellular signal-regulated kinase 1/2 (ERK1/2)-Hsp27; similarly, a higher level of p-IGF-1R and ERK1/2-Hsp27 signaling was also detected after OVA66 overexpression in HO8910 cells. In vivo knockdown of OVA66 both reduced tumor burden in nude mice and decreased phosphorylation of IGF-1R, ERK1/2 and hsp27. We blocked IGF-1R function both by small interfering RNA (siRNA) and with the chemical inhibitor Linsitinib (OSI-906). By either method, tumorigenesis was inhibited regardless of OVA66 expression; thus, mechanistically, IGF-1R, probably, lies downstream of OVA66 in cancer cells. We also found that OVA66 regulates expression of murine double minute 2 (MDM2); this attenuates ubiquitination of IGF-1R in response to IGF-1 stimulation and promotes active ERK1/2 signaling. Thus, we propose that combined overexpression of OVA66 and MDM2 promotes oncogenesis by enhancing activation of the IGF-1R-ERK1/2 signaling pathway.

T cell vaccination inhibits Th1/Th17/Tfh frequencies and production of autoantibodies in collagen-induced arthritis.

The aim of this study is to determine whether the regulatory role of T cell vaccination (TCV) is through inhibition of Th1/Th17/Tfh and production of autoantibodies on collagen-induced arthritis (CIA). First, CIA mice were treated with TCV. After disease onset, the incidence and severity of change in joint histopathology were evaluated. Mice in the TCV-treated group showed less disease severity and less infiltration of inflammatory cells in the joint sections. TCV decreased the frequencies of Th1/Th17/Tfh cells and related cytokines. Reduction of IL-21 may be associated with both Tfh and Th17, which further influence B cell and T cell responses. In addition, inhibition of Th1/Th17/Tfh frequencies led to the reduced expression of T-bet, ROR α , ROR γ t, and Bcl6. Lastly, the proliferation of type-II-collagen-(CII-) specific T cells and the production of anti-CII antibodies were inhibited in the TCV-treated group. The results provide novel evidence that the therapeutic effects of TCV on CIA are associated with the inhibition of Th1/Th17/Tfh frequencies and autoantibodies production.

Regulatory effect of vasoactive intestinal peptide on the balance of Treg and Th17 in collagen-induced arthritis.

Vasoactive intestinal peptide (VIP) is a well-known anti-inflammatory neuropeptide. The capacity of VIP can be exhibited through inhibiting inflammatory responses, shifting the Th1/Th2 balance in favor of anti-inflammatory Th2 immunity and inducing regulatory T cells (Tregs) with suppressive activity. In addition to pro-inflammatory Th1 response, Th17 are also believed to play important roles in the pathogenesis of rheumatoid arthritis (RA). In this study, we used collagen-induced arthritis (CIA) model in Wistar rats to investigate the role of VIP in the balance of CD4(+) CD25(+) Tregs and Th17 on RA. Data presented here showed that administration of VIP decreased incidence and severity of CIA. Disease suppression was associated with the upregulation of CD4(+) CD25(+) Tregs, downregulation of Th17- and Th1-type response and influence on the RANK/RANKL/OPG system. The results provide novel evidence that the therapeutic effects of VIP on CIA rats were associated with the balance of CD4(+) CD25(+) Tregs and Th17.

Expression of IL-1alpha, IL-6, TGF-beta, FasL and ZNF265 during sertoli cell infection by ureaplasma urealyticum.

To investigate immunoregulatory mechanisms of Sertoli cells in the testis in vitro and in vivo, we utilized our well-characterized Ureaplasma Urealyticum (UU)-induced model. We investigated the expressions of IL-1alpha, IL-6, TGF-beta, FasL and ZNF265 at the first, second and third weeks post-infection. During recovery from inflammation and with the help of negative regulators TGF-beta and FasL, the high levels of IL-1alpha and IL-6 expressions were observed in the early stages of the infection, and decreased gradually in the later weeks both in vitro and in vivo. The trend of varied expression of ZNF265 was similar to those of TGF-beta and FasL in vitro and in vivo for Sertoli cells infected with UU.

[Immunoregulation of vasoactive intestinal peptide on rat Leydig cells].

AIM: To study the regulation of immune function of rat Leydig cells by vasoactive intestinal peptide (VIP). METHODS: Rat Leydig cells were seperated and infected by UU, with or without the incubation with VIP. The expression of FasL on Leydig cells in different group was analysed by flow cytometry(FCM). The mRNA expression of IL-1, IL-6, TGF-beta and FasL of Leydig cells in different group was identified by RT-PCR. SD rats were infected by UU with or without the injection of VIP, and the testis tissnes of each group were har vested and observed under transmission electron microscope. RESULTS: When testis was infected by UU, VIP up-regulated the mRNA expression of IL-1, IL-6, and TGF-beta, and down-regulated the expression of FasL in vitro. In addition, there was significant difference in testis morphous of rats from different group. CONCLUSION: VIP could regulate the expression pattern of IL-1, IL-6, TGF-beta and FasL by Leydig cells, which may contribute to maintain immune privilege of the testis. VIP could regulate rat Leydig cell immune function.

[Influence of FasL overexpression in transgenic mice on the immune regulative function of Sertoli cell].

AIM: To study the influence of FasL overexpression in transgenic mice on the Sertoli cell immune response to testicular infection. METHODS: Ureaplasma urealyticum (UU) was directly injected into bladders of FasL transgenic mice and wild-type mice respectively, to mimic an ascending infection pathway. At week 1, 2 and 3 after injection, respectively, the mice were put to death to observe the pathological alterations in testis section. And at the same time the differences of FasL, TGF-beta, IL-1alpha and IL-6 expressions on Sertoli cells were compared by immunohistochemical staining between wild mice and transgenic mice before and after infection, respectively. The high-purified Sertoli cells were isolated from the testis tissue of wild-type mice, comparing apoptotic capability of Fas(+) Jurkat cells mediated by FasL(+) Sertoli cells of wild control and wild UU-infected groups. RESULTS: The pathological changes of testis tissue from transgenic mice were more serious as compared with wild-type mice and the model of cytokines secreted by sertoli cells was distinctive between the two kinds of mice. The UU-infected Sertoli cells increased Fas(+) Jurkat cell apoptosis. CONCLUSION: FasL overexpression can influence the cytokine's secretion in the process of anti-infection immunity and further affects the immune balance of testis locality. Not all FasL over expression is benefit to body's anti-infection immune response.