Artemisinin analogue SM934 ameliorates murine experimental autoimmune encephalomyelitis through enhancing the expansion and functions of regulatory T cell.

BACKGROUND: Artemisinin analogue SM934 was previously reported to possess immunosuppressive properties. The aim of this study was to determine the effects and the underlying mechanisms of SM934 in murine experimental autoimmune encephalomyelitis (EAE). METHODS: Female C57BL/6 mice immunized with MOG35-55 were treated with or without SM934, then the clinical scores and other relevant parameters were assessed. Th1, Th17 and regulatory T (Treg) cell profiles were determined through ELISA, qRT-PCR, flow cytometry and BrdU incorporation assay. The effects of SM934 on Th1, Th17 and Treg cells differentiation were explored through intracellular staining and flow cytometry examination. RESULTS: In vivo, administration of SM934 significantly inhibited the development of EAE and suppressed the elevation of serum IL-17. Ex vivo, upon antigen-recall stimulation, IL-2, IFN-γ, IL-17 and IL-6 production were decreased, whereas IL-10 and TGF-ß production were increased from the splenocytes isolated from SM934-treated mice. Consistently, both flow cytometry and qRT-PCR results showed that SM934 treatment significantly increased the Treg, while strongly suppressed the Th17 and Th1, responses in the peripheral. Furthermore, in the spinal lesion, SM934 treatment dramatically decreased the infiltration of CD4(+) T cells, within which the Treg cells percentage was enlarged, whereas the Th17, but not Th1 percentage, was significantly decreased comparing with the vehicle-treated groups. Finally, both BrdU incorporation and in vitro Treg differentiation assays revealed that SM934 treatment could directly promote the expansion of Treg cells in vivo and in vitro. CONCLUSION: Taken together, this study demonstrated that SM934 treatment could ameliorate the murine EAE disease, which might be mediated by inducing Treg differentiation and expansion.

Astragaloside II triggers T cell activation through regulation of CD45 protein tyrosine phosphatase activity.

AIM: To investigate the immunomodulating activity of astragalosides, the active compounds from a traditional tonic herb Astragalus membranaceus Bge, and to explore the molecular mechanisms underlying the actions, focusing on CD45 protein tyrosine phosphatase (CD45 PTPase), which plays a critical role in T lymphocyte activation. METHODS: Primary splenocytes and T cells were prepared from mice. CD45 PTPase activity was assessed using a colorimetric assay. Cell proliferation was measured using a [(3)H]-thymidine incorporation assay. Cytokine proteins and mRNAs were examined with ELISA and RT-PCR, respectively. Activation markers, including CD25 and CD69, were analyzed using flow cytometry. Activation of LCK (Tyr505) was detected using Western blot analysis. Mice were injected with the immunosuppressant cyclophosphamide (CTX, 80 mg/kg), and administered astragaloside II (50 mg/kg). RESULTS: Astragaloside I, II, III, and IV concentration-dependently increased the CD45-mediated of pNPP/OMFP hydrolysis with the EC50 values ranged from 3.33 to 10.42 µg/mL. Astragaloside II (10 and 30 nmol/L) significantly enhanced the proliferation of primary splenocytes induced by ConA, alloantigen or anti-CD3. Astragaloside II (30 nmol/L) significantly increased IL-2 and IFN-γ secretion, upregulated the mRNA levels of IFN-γ and T-bet in primary splenocytes, and promoted CD25 and CD69 expression on primary CD4(+) T cells upon TCR stimulation. Furthermore, astragaloside II (100 nmol/L) promoted CD45-mediated dephosphorylation of LCK (Tyr505) in primary T cells, which could be blocked by a specific CD45 PTPase inhibitor. In CTX-induced immunosuppressed mice, oral administration of astragaloside II restored the proliferation of splenic T cells and the production of IFN-γ and IL-2. However, astragaloside II had no apparent effects on B cell proliferation. CONCLUSION: Astragaloside II enhances T cell activation by regulating the activity of CD45 PTPase, which may explain why Astragalus membranaceus Bge is used as a tonic herb in treating immunosuppressive diseases.

SM934 treated lupus-prone NZB × NZW F1 mice by enhancing macrophage interleukin-10 production and suppressing pathogenic T cell development.

BACKGROUND: Artemisinin and its derivatives were reported to possess strong regulatory effects on inflammation and autoimmune diseases. This study was designed to examine the therapeutic effects and underlying mechanisms of SM934, a water-soluble artemisinin analogue, on lupus-prone female NZB × NZW F(1) mice. METHODOLOGY/PRINCIPAL FINDINGS: NZB/W F(1) mice were treated orally with SM934 for 3 or 6 months respectively to investigate the effect on clinical manifestations and immunological correlates. To further explore the mechanisms of SM934, ovalbumin (OVA)-immunized or interferon (IFN)-γ-elicited C57BL/6 mice were used. In vivo, treatment with SM934 for 3 or 6 months significantly delayed the progression of glomerulonephritis and increased the survival rate of NZB/W F(1) mice. Clinical improvement was accompanied with decreased Th1-related anti-double-strand DNA (dsDNA) IgG2a and IgG3 Abs, serum interleukin (IL)-17, and increased Th2-related anti-dsDNA IgG1 Ab, serum IL-10 and IL-4. SM934 treatment also suppressed the accumulation of effector/memory T cells, induced the apoptosis of CD4(+) T cells, while enhancing the development of regulatory T cells in NZB/W F(1) mice. In addition, SM934 treatment promoted the IL-10 production of macrophages from NZB/W F(1) mice, OVA-immunized C57BL/6 mice and IFN-γ-elicited C57BL/6 mice. In vitro, SM934 enhanced IL-10 production from primary macrophages stimulated with IFN-γ. CONCLUSIONS/SIGNIFICANCE: The results of this study demonstrated that artemisinin analogue SM934 had therapeutic effects on lupus-prone female NZB/W F(1) mice by inhibiting the pathogenic helper T cell development and enhancing anti-inflammatory cytokine IL-10 production.

Oral administration of artemisinin analog SM934 ameliorates lupus syndromes in MRL/lpr mice by inhibiting Th1 and Th17 cell responses.

OBJECTIVE: SM934, an artemisinin derivative, possesses potent antiproliferative and antiinflammatory properties. The aim of this study was to examine the effects and explore the mechanisms of SM934 to treat autoimmune disease in lupus-prone female MRL/lpr mice. METHODS: In vitro, the effects of SM934 on the activation of polyclonal CD4+ T cells and the differentiation of naive CD4+ T cells were examined. In vivo, the preventative or therapeutic effects of SM934 in MRL/lpr mice were investigated. Ex vivo, the mechanisms of treatment were explored according to the immunologic correlates of disease. RESULTS: In vitro, SM934 inhibited interferon-γ (IFNγ) and interleukin-17 (IL-17) production from polyclonal CD4+ T cells activated by T cell receptor engagement and the differentiation of naive CD4+ T cells into Th1 and Th17 cells, but not Treg cells. In vivo, 12-week-old MRL/lpr mice treated with SM934 for 4 weeks showed significantly ameliorated proteinuria and renal lesion severity; decreased levels of blood urea nitrogen, serum IFNγ, and serum anti-double-stranded DNA antibodies; decreased spleen size; and a lower percentage of CD3+B220+CD4-CD8- T cells; 16-week-old MRL/lpr mice treated with SM934 for 8 weeks avoided severe proteinuria and survived longer. Ex vivo, SM934 treatment elevated the percentage of Treg cells, inhibited the development of Th1 and Th17 cells, and impeded the comprehensive activation of STAT-1, STAT-3, and STAT-5 proteins in splenocytes. CONCLUSION: Taken together, the results of this study demonstrated that the artemisinin analog SM934 had therapeutic effects in lupus-prone female MRL/lpr mice by inhibiting both Th1 cell and Th17 cell responses. Moreover, this study indicated that both IFNγ and IL-17 are required for the elicitation and development of murine lupus.

SM905, an artemisinin derivative, inhibited NO and pro-inflammatory cytokine production by suppressing MAPK and NF-kappaB pathways in RAW 264.7 macrophages.

AIM: To elucidate the anti-inflammatory potentials and underlying mechanisms of SM905, a novel artemisinin derivative, in lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 cells. METHODS: Nitric oxide (NO) generation, cytokine production, and the protein expression levels of inducible nitric-oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were examined using a Griess assay, an enzyme-linked immunosorbent assay (ELISA) and a Western blotting assay, respectively. The mRNA expression was measured using real-time PCR. The phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), p38, c-jun N-terminal kinase (JNK), and the degradation of IkappaBalpha were assessed by Western blotting analysis. The nuclear translocation of nuclear factor-kappaB (NF-kappaB) was observed using confocal microscopy. RESULTS: Pretreatment with SM905 (0, 0.1, 1, and 10 micromol/L) suppressed LPS-induced NO, TNF-alpha, IL-1beta, and IL-6 production, and decreased both protein and mRNA levels of iNOS and COX-2. The mRNA expression of LPS receptor Toll-like receptor 4 (TLR4) and myeloid differentiation protein-2 (MD-2) was not changed, while LPS-induced CD14 expression was slightly reduced after SM905 treatment. SM905 markedly decreased the activation of ERK1/2, p38 and JNK suppressed the degradation of IkappaBalpha, but did not modify the expression of interferon regulatory factor-1 (IRF-1), signal transducer and activator of transcription 1 (STAT1) or interferon-inducible protein-10 (IP-10). By using confocal microscopy, we further observed that NF-kappaB was correspondingly inhibited in SM905-treated cells. CONCLUSION: SM905 inhibited NO and pro-inflammatory cytokine production in LPS-stimulated RAW 264.7 cells and these effects are at least partially mediated through suppression of the MAPK and NF-kappaB signaling pathways.

SM934, a water-soluble derivative of arteminisin, exerts immunosuppressive functions in vitro and in vivo.

In the present study, we investigated the immunosuppressive effects and underlying mechanisms of beta-aminoarteether maleate (SM934), a derivative of artemisinin, against T cell activation in vitro and in vivo. In vitro, SM934 significantly inhibited the proliferation of splenocytes induced by concanavalin A (Con A), lipopolysaccharide (LPS), mixed lymphocyte reaction (MLR), and anti-CD3 plus anti-CD28 (anti-CD3/28). SM934 significantly inhibited interferon (IFN)-gamma production and CD4(+) T cell division stimulated by anti-CD3/28. SM934 also promoted apoptosis of CD69(+) population in CD4(+) T cells stimulated by anti-CD3/28. Furthermore, SM934 inhibited interleukin (IL)-2 mediated proliferation and survival through blocking Akt phosphorylation in activated T cells. In ovalbumin (OVA)-immunized mice, oral administration of SM934 suppressed OVA-specific T cell proliferation and IFN-gamma production. SM934 treatment also significantly inhibited the sheep red blood cell (SRBC)-induced delayed type hypersensitivity (DTH) reactions in mice. Taken together, SM934 showed potent immunosuppressive activities in vitro and in vivo. Our results demonstrated that SM934 might be a potential therapeutic agent for immune-related diseases.

The chemokine receptor antagonist, TAK-779, decreased experimental autoimmune encephalomyelitis by reducing inflammatory cell migration into the central nervous system, without affecting T cell function.

BACKGROUND AND PURPOSE: The C-C chemokine receptor CCR5, and the C-X-C chemokine receptor CXCR3 are involved in the regulation of T cell-mediated immune responses, and in the migration and activation of these cells. To determine whether blockade of these chemokine receptors modulated inflammatory responses in the central nervous sytem (CNS), we investigated the effect of a non-peptide chemokine receptor antagonist, TAK-779, in mice with experimental autoimmune encephalomyelitis (EAE). EXPERIMENTAL APPROACH: EAE was induced by immunization of C57BL/6 mice with myelin oligodendrocyte glycoprotein (MOG) 35-55. TAK-779 was injected s.c. once a day after immunization. Disease incidence and severity (over 3 weeks) were monitored by histopathological evaluation and FACS assay of inflammatory cells infiltrating into the spinal cord, polymerase chain reaction quantification of mRNA expression, assay of T cell proliferation, by [3H]-thymidine incorporation and cytokine production by enzyme-linked immunosorbent assay. KEY RESULTS: Treatment with TAK-779 reduced incidence and severity of EAE. It strongly inhibited migration of CXCR3/CCR5 bearing CD4+, CD8+ and CD11b+ leukocytes to the CNS. TAK-779 did not reduce proliferation of anti-MOG T cells, the production of IFN-gamma by T cells or CXCR3 expression on T cells. In addition, TAK-779 did not affect production of IL-12 by antigen-presenting cells, CCR5 induction on T cells and the potential of MOG-specific T cells to transfer EAE. CONCLUSIONS AND IMPLICATIONS: TAK-779 restricted the development of MOG-induced EAE. This effect involved reduced migration of inflammatory cells into the CNS without affecting responses of anti-MOG T cells or the ability of MOG-specific T cells to transfer EAE.

Periplocoside A, a pregnane glycoside from Periploca sepium Bge, prevents concanavalin A-induced mice hepatitis through inhibiting NKT-derived inflammatory cytokine productions.

Periploca sepium Bge, a traditional Chinese herb medicine, is widely used for treating rheumatoid arthritis in china. Periplocoside A (PSA), a pregnane glycoside, is a new nature product compound isolated from P. sepium Bge. We examined the protective effects of PSA, on concanavaline A (ConA)-induced hepatitis. Pretreatment with PSA dramatically ameliorated ConA-induced liver injury, which was characterized by reducing serum alanine transaminase (ALT), pathogenic cytokines of interleukin (IL)-4 and interferon (IFN)-gamma levels, impeding the liver necrosis, and thus elevating the survival rate. In vitro, PSA inhibited IL-4 and IFN-gamma productions of alpha-galactosylceramide (alpha-GalCer) or anti-CD3-activated Natural killer T (NKT) cells. Enzyme Linked Immunosorbent Assay (ELISA) and Reverse Transcription Polymerase Chain Reaction (RT-PCR) assays revealed PSA suppressed IL-4 transcription and IFN-gamma translation. In conclusion, PSA had significantly preventative effect on ConA-induced hepatitis, which was closely associated with inhibition of NKT-derived inflammatory cytokine productions. These findings suggested that PSA has the therapeutic potential for treatment of human autoimmune-related hepatitis.

Suppressive effect of a novel water-soluble artemisinin derivative SM905 on T cell activation and proliferation in vitro and in vivo.

Artemisinin and its derivatives exhibit potent immunosuppressive activity. The aim of this study was to investigate the suppressive effects of SM905, a new water-soluble artemisinin derivative, on T lymphocytes both in vitro and in vivo, and explore its potential mode of action. The results showed that SM905 had a high inhibitory activity in Concanavalin A (ConA)-induced splenocyte proliferation and mixed lymphocyte reaction, and a relatively low cytotoxicity in vitro. In ovalbumin-immunized mice, oral administration of SM905 dose-dependently suppressed T cell proliferative response to ovalbumin, and inhibited anti-ovalbumin interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production by T cells. Further studies showed that SM905 inhibited TCR (T cell receptor)/CD3 plus CD28-mediated primary T cell proliferation and cytokine production (IL-2 and IFN-gamma), and exerted an inhibitory action on the phosphorylation of mitogen-activated protein (MAP) kinases including extracellular signal-regulated kinase (ERK), p38 and Jun N-terminal kinase (JNK), and the activation of Ras. The results of this study provided experimental evidence that the new artemisinin derivative SM905 had immunosuppressive effects both in vitro and in vivo. SM905 suppressed T cell activation, which was associated with the inhibition of MAP kinases and Ras activation. Our results suggested a potential of SM905 to be developed as a new type agent for treating T cell-mediated immune disorder.

Alleviation of picryl chloride-induced delayed type hypersensitivity reaction by saponin fraction of Gleditsia sinensis.

The objective of the present study was to investigate the effects of saponin fraction from anomalous fruits of Gleditsia sinensis (SFGS) on picryl chloride-induced delayed type hypersensitivity (PC-DTH) and functions of T lymphocytes and macrophages in mice. SFGS (100, 200 mg/kg), orally administered during either sensitization stage or effector stage, produced remarkable inhibition of PC-DTH. In vitro, SFGS (1, 2, 4 microg/ml) concentration-dependently attenuated concanavalin A (Con A)-elicited mouse splenocyte proliferation and interleukin 2 (IL-2) production. At concentrations of 10 and 20 microg/ml, SFGS inhibited lipopolysaccharide (LPS)-induced production of nitric oxide and interleukin 1beta (IL-1beta) of mouse peritoneal macrophages. The findings indicate that SFGS attenuates PC-DTH in mice, which is probably mediated by preventing proliferation and differentiation of T cells during the sensitization stage and suppressing activation of macrophages during the effector stage.

Inhibition of immediate allergic reactions by ethanol extract from Plumbago zeylanica stems.

The antiallergic properties of the 70% ethanol extract from Plumbago zeylanica stems (EPZ) were investigated in the present study. The extract (500, 1000 mg/kg, p.o.) dose-dependently inhibited systemic anaphylactic shock induced by compound 48/80 in mice, reduced homologous passive cutaneous anaphylaxis and skin reactions induced by histamine or serotonin in rats, significant differences were observed at the dose of 1000 mg/kg. In vitro, EPZ (5, 20, 50 microg/ml) concentration-dependently reduced histamine release from rat peritoneal mast cells caused by compound 48/80 and antigen. EPZ (50 microg/ml) markedly increased intracellular cAMP content of rat mast cells. These findings demonstrate that EPZ inhibits mast cell-dependent immediate allergic reactions, which is probably mediated by reducing the release of mediators such as histamine from mast cells via elevating intracellular cAMP level and weakening the inflammatory action of mediators.