The Possible Role of PD-1 Protein in Ganoderma lucidum-Mediated Immunomodulation and Cancer Treatment.

Background:Ganoderma lucidum has been used in Chinese medicine for thousands years to improve health and to promote longevity. One important function of G lucidum is to modulate the immune system. However, the underlying mechanism is not well understood. Programmed cell death protein 1 (PD-1) is a cell surface protein present in certain immune cells (eg, B- and Tcells) and plays an important role in modulating the immune response. The role of PD-1 protein in G lucidum-mediated immunomodulation is unknown. Methods: Cultured human Blymphocytes and extract prepared from G lucidum spores (GLE) were used to determine PD-1 protein in G lucidum-mediated immunomodulation. Both western blotting and immunofluorescence (IF) microscopy assays were used to determine the effect of GLE treatment on PD-1 protein expression. A reverse transcription-based quantitative polymerase chain reaction (real-time PCR) assay was used to determine the effect of GLE on transcription of pdcd-1 gene. Results: Both our western blotting and IF staining results demonstrated great reduction in PD-1 protein and in proportion of PD-1+ cells in these B-lymphocytes. Our real-time PCR results indicated that this PD-1 protein reduction was not caused by a transcriptional inhibition of the gene. In addition, our western blotting study further revealed that the GLE treatment caused an increase in expression of CCL5 chemokine in the cultured B-lymphocytes. Conclusions: PD-1 protein is an important target of G lucidum-mediated immunomodulation. G lucidum and its bioactive compounds can be developed into novel immunomodulators for prevention and treatment of cancer and many other diseases.

Blocking CCL5-CXCL4 heteromerization preserves heart function after myocardial infarction by attenuating leukocyte recruitment and NETosis.

Myocardial infarction (MI) is a major cause of death in Western countries and finding new strategies for its prevention and treatment is thus of high priority. In a previous study, we have demonstrated a pathophysiologic relevance for the heterophilic interaction of CCL5 and CXCL4 in the progression of atherosclerosis. A specifically designed compound (MKEY) to block this CCL5-CXCR4 interaction is investigated as a potential therapeutic in a model of myocardial ischemia/reperfusion (I/R) damage. 8 week-old male C57BL/6 mice were intravenously treated with MKEY or scrambled control (sMKEY) from 1 day before, until up to 7 days after I/R. By using echocardiography and intraventricular pressure measurements, MKEY treatment resulted in a significant decrease in infarction size and preserved heart function as compared to sMKEY-treated animals. Moreover, MKEY treatment significantly reduced the inflammatory reaction following I/R, as revealed by specific staining for neutrophils and monocyte/macrophages. Interestingly, MKEY treatment led to a significant reduction of citrullinated histone 3 in the infarcted tissue, showing that MKEY can prevent neutrophil extracellular trap formation in vivo. Disrupting chemokine heterodimers during myocardial I/R might have clinical benefits, preserving the therapeutic benefit of blocking specific chemokines, and in addition, reducing the inflammatory side effects maintaining normal immune defence.

Radix isatidis Polysaccharides Inhibit Influenza a Virus and Influenza A Virus-Induced Inflammation via Suppression of Host TLR3 Signaling In Vitro.

Influenza remains one of the major epidemic diseases worldwide, and rapid virus replication and collateral lung tissue damage caused by excessive pro-inflammatory host immune cell responses lead to high mortality rates. Thus, novel therapeutic agents that control influenza A virus (IAV) propagation and attenuate excessive pro-inflammatory responses are needed. Polysaccharide extract from Radix isatidis, a traditional Chinese herbal medicine, exerted potent anti-IAV activity against human seasonal influenza viruses (H1N1 and H3N2) and avian influenza viruses (H6N2 and H9N2) in vitro. The polysaccharides also significantly reduced the expression of pro-inflammatory cytokines (IL-6) and chemokines (IP-10, MIG, and CCL-5) stimulated by A/PR/8/34 (H1N1) at a range of doses (7.5 mg/mL, 15 mg/mL, and 30 mg/mL); however, they were only effective against progeny virus at a high dose. Similar activity was detected against inflammation induced by avian influenza virus H9N2. The polysaccharides strongly inhibited the protein expression of TLR-3 induced by PR8, suggesting that they impair the upregulation of pro-inflammatory factors induced by IAV by inhibiting activation of the TLR-3 signaling pathway. The polysaccharide extract from Radix isatidis root therefore has the potential to be used as an adjunct to antiviral therapy for the treatment of IAV infection.

AAV-IL-22 modifies liver chemokine activity and ameliorates portal inflammation in murine autoimmune cholangitis.

There remain significant obstacles in developing biologics to treat primary biliary cholangitis (PBC). Although a number of agents have been studied both in murine models and human patients, the results have been relatively disappointing. IL-22 is a member of the IL-10 family and has multiple theoretical reasons for predicting successful usage in PBC. We have taken advantage of an IL-22 expressing adeno-associated virus (AAV-IL-22) to address the potential role of IL-22 in not only protecting mice from autoimmune cholangitis, but also in treating animals with established portal inflammation. Using our established mouse model of 2-OA-OVA immunization, including α-galactosylceramide (α-GalCer) stimulation, we treated mice both before and after the onset of clinical disease with AAV-IL-22. Firstly, AAV-IL-22 treatment given prior to 2-OA-OVA and α-GalCer exposure, i.e. before the onset of disease, significantly reduces the portal inflammatory response, production of Th1 cytokines and appearance of liver fibrosis. It also reduced the liver lymphotropic chemokines CCL5, CCL19, CXCL9, and CXCL10. Secondly, and more importantly, therapeutic use of AAV-IL-22, administered after the onset of disease, achieved a greater hurdle and significantly improved portal pathology. Further the improvements in inflammation were negatively correlated with levels of CCL5 and CXCL10 and positively correlated with levels of IL-22. In conclusion, we submit that the clinical use of IL-22 has a potential role in modulating the inflammatory portal process in patients with PBC.

Effect of salvianolic acid A and C compatibility on inflammatory cytokines in rats with unilateral ureteral obstruction.

OBJECTIVE: To investigate the effect of salvianolic acid A and C component molecules, which are involved in drug compatibility, on inflammatory cytokine expression that affects human chemokine ligand 5 (CCL5) and chemokine ligand 10 (CXCL10) levels in rats with unilateral ureteral obstruction (UUO). METHODS: Fifty Sprague Dawley rats were randomly divided into five groups: normal, model, salvianolic acid A, salvianolic acid C and salvianolic acid A and C groups. The normal group was used as the control, and the other groups of rats had a UUO model established. The control group had free access to food and water, and the other groups received the corresponding drugs for 2 weeks. After the last administration, urine ß2-microglobulin (ß 2-MG) and N-acetyl-ß-D-glucosaminidase (NAG) levels were analyzed. After 24 h, all rats were sacrificed and the serum was analyzed for creatinine (Cr) and blood urea nitrogen (BUN) levels. Rat kidneys were removed, and CCL5 and CXCL10 inflammatory cytokine mRNA expression was measured using real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR). Kidney fibrosis was observed using hematoxylin-eosin (HE) staining and Masson trichrome staining. RESULTS: In the salvianolic acid A and salvianolic acid C treatment groups, serum Cr and urine NAG levels were significantly lower than in the model group (both P < 0.05). In all treatment groups, urine ß2-MG levels were significantly lower than in the model group (all P < 0.05). Compared with model group, the pathological changes and collagen deposition improved to varying degrees (both P < 0.05). CCL5 and CXCL10 mRNA expression decreased to different degrees compared with the model group (both P < 0.05). CONCLUSION: Salvianolic acid A and C are component molecules of drug compatibility, and they may protect renal function and improve tubular function and renal pathology to a certain degree in UUO. This improvement may be related to a reduction in inflammatory cytokines CCL5 and CXCL10 secretion in the UUO rat kidney.

Chungsimyeonja-eum inhibits inflammatory responses in RAW 264.7 macrophages and HaCaT keratinocytes.

BACKGROUND: Chungsimyeonja-eum (CSYJE) is an herbal prescription used in traditional Oriental medicine for treating cerebral infarction by reducing ischemic damage. However, the effects of CSYJE on inflammation have not been verified scientifically. METHODS: Anti-inflammatory effects of CSYJE was investigated to dertermine the inhibitory effects of CSYJE against inflammation using RAW 264.7 mouse macrophages and HaCaT human keratinocytes. To measure the effects of CSYJE on inflammatory mediators and cytokines/chemokines, we used the following methods: cell viability assay, enzyme-linked immunosorbent assay (ELISA), western blotting, immunocytochemistry. RAW 264.7 cells were pretreated with CSYJE (250, 500, or 1000 µg/mL) for 4 h and treated with lipopolysaccharide (LPS) for additional 20 h. HaCaT cells were stimulated with tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) (TI), and CSYJE (125, 250, or 500 µg/mL) for 24 h. RESULTS: CSYJE suppressed the production of nitric oxide (NO, IC50 1000 µg/mL), prostaglandin E2 (PGE2, IC50 = 12.1 µg/mL), and interleukin (IL)-6 (IC50 = 248 µg/mL) in LPS-stimulated RAW 264.7 cells. CSYJE suppressed the effects of TI on the production of thymus and activation-regulated chemokine (TARC, IC50 = 330.2 µg/mL), macrophage-derived chemokine (MDC/CCL22, IC50 = 52.5 µg/mL), regulated on activation, normal T-cell expressed and secreted (RANTES/CCL5, IC50 = 372.9 µg/mL), and IL-8 (IC50 = 345.1 µg/mL) in HaCaT cells. CSYJE inhibited TI-stimulated STAT1 phosphorylation in a dose-dependent manner and nuclear translocation at 500 µg/mL in HaCaT cells. CONCLUSION: Our results suggest a possible therapeutic application of CSYJE for treating inflammatory diseases.

Anti-cellular and immunomodulatory potential of aqueous extract of Rhodiola imbricata rhizome.

In the present study, we have evaluated the anti-cellular and immunomodulatory potential of aqueous extract of Rhodiola imbricata rhizome (RAE). Rhodiola extract inhibited the proliferation of human T cell lymphoma cell line EL-4 and erythroleukemic cell line HL-60. Furthermore, treatment of human peripheral blood mononuclear cells (hPBMCs) with lipopolysaccharide (LPS) and RAE suppressed regulated upon activation, normal T cell expressed and secreted (RANTES) production. However, number of TNF-α spots was increased in RAE treated hPBMCs. The reverse transcriptase polymerase chain reaction (RT-PCR) analysis of RAE treated rat splenocytes confirmed the up regulation of TLR-4 mRNA expression. Therefore, the present study concludes that RAE has potent immune boosting activity which might be useful in immunocompromised individuals.

Antiinflammatory effects of ginger and some of its components in human bronchial epithelial (BEAS-2B) cells.

The proinflammatory chemokine interleukin-8 is increased in asthmatic patients. Traditionally, ginger is used as an antiinflammatory drug. An extract and several compounds of Zingiber officinale (ginger) were tested in human bronchial epithelial cells (BEAS-2B cells) with respect to their effect on lipopolysaccharide (LPS)-induced secretion of the proinflammatory chemokine interleukin 8 (IL-8) and RANTES (regulated upon activation, normal T-cell expressed and secreted). An oily extract of ginger rhizome with > 25% total pungent compounds, ginger volatile oil, ar-curcumene and α-pinene reduced the LPS-induced IL-8 secretion (measured by a specific enzyme-linked immunosorbent assay), whereas a spissum extract, the pungents [6]-gingerol and its metabolite [6]-shogaol, and the terpenoids citral and ß-phellandrene showed no effect. The LPS-induced slight increase of RANTES was reduced by volatile oil, ar-curcumene and α-pinene. There was no effect of LPS on TNF-α. Our results suggest that distinct ginger compounds could be used as antiinflammatory drugs in respiratory infections.

S100B protein stimulates microglia migration via RAGE-dependent up-regulation of chemokine expression and release.

The Ca(2+)-binding protein of the EF-hand type, S100B, is abundantly expressed in and secreted by astrocytes, and release of S100B from damaged astrocytes occurs during the course of acute and chronic brain disorders. Thus, the concept has emerged that S100B might act an unconventional cytokine or a damage-associated molecular pattern protein playing a role in the pathophysiology of neurodegenerative disorders and inflammatory brain diseases. S100B proinflammatory effects require relatively high concentrations of the protein, whereas at physiological concentrations S100B exerts trophic effects on neurons. Most if not all of the extracellular (trophic and toxic) effects of S100B in the brain are mediated by the engagement of RAGE (receptor for advanced glycation end products). We show here that high S100B stimulates murine microglia migration in Boyden chambers via RAGE-dependent activation of Src kinase, Ras, PI3K, MEK/ERK1/2, RhoA/ROCK, Rac1/JNK/AP-1, Rac1/NF-κB, and, to a lesser extent, p38 MAPK. Recruitment of the adaptor protein, diaphanous-1, a member of the formin protein family, is also required for S100B/RAGE-induced migration of microglia. The S100B/RAGE-dependent activation of diaphanous-1/Rac1/JNK/AP-1, Ras/Rac1/NF-κB and Src/Ras/PI3K/RhoA/diaphanous-1 results in the up-regulation of expression of the chemokines, CCL3, CCL5, and CXCL12, whose release and activity are required for S100B to stimulate microglia migration. Lastly, RAGE engagement by S100B in microglia results in up-regulation of the chemokine receptors, CCR1 and CCR5. These results suggests that S100B might participate in the pathophysiology of brain inflammatory disorders via RAGE-dependent regulation of several inflammation-related events including activation and migration of microglia.

Petasites extract Ze 339 (PET) inhibits allergen-induced Th2 responses, airway inflammation and airway hyperreactivity in mice.

BACKGROUND: The herbal Petasites hybridus (butterbur) extract (Ze 339, PET) is known to have leukotriene inhibiting properties, and therefore might inhibit allergic diseases. METHODS: The effect of PET was investigated in ovalbumin (OVA) immunized BALB/c mice given intranasally together with antigen challenge in the murine model of allergic airway disease (asthma) with the analysis of the inflammatory and immune parameters in the lung. RESULTS: PET given with the antigen challenge inhibited the allergic response. PET inhibited airway hyperresponsiveness (AHR) and eosinophil recruitment into the bronchoalveolar lavage (BAL) fluid upon allergen challenge, but had no effect in the saline control mice. Eosinophil recruitment was further assessed in the lung by eosinophil peroxidase (EPO) activity at a concentration of 100 microg PET. Microscopic investigations revealed less inflammation, eosinophil recruitment and mucus hyperproduction in the lung with 100 microg PET. Diminution of AHR and inflammation was associated with reduced IL-4, IL-5 and RANTES production in the BAL fluid with 30 microg PET, while OVA specific IgE and eotaxin serum levels remained unchanged. CONCLUSION: PET, which has been reported to inhibit leukotriene activity, reduced allergic airway inflammation and AHR by inhibiting the production of the Th2 cytokines IL-4 and IL-5, and RANTES.

Immunoregulatory role of ocular macrophages: the macrophages produce RANTES to suppress experimental autoimmune uveitis.

Murine experimental autoimmune uveitis (EAU) is a model of human uveitis. Ocular-infiltrating macrophages play a crucial role in the generation of tissue damage in EAU. In fact, several chemokines are actually produced in the inflamed eye. The aim of this study was to elucidate the role of ocular macrophage-derived chemokines in EAU. C57BL/6 mice were immunized with human interphotoreceptor retinoid binding protein peptide 1-20, and the EAU severity was scored at multiple time points based on microscopic fundus observations (retinal vascular dilatation and exudates) and histological examinations. The peak inflammatory response was observed 1 wk (day 16) after the beginning of macrophage infiltration to the eye (day 9). Ocular-infiltrating cells were enriched or depleted of macrophages by magnetic beads and analyzed by real-time RT-PCR for chemokine mRNA production. We found that only the macrophage-enriched cells from the eye produced RANTES, and thus proposed that macrophage-derived RANTES facilitated the ocular inflammations. In contrast to our postulate, neutralization of RANTES by specific Ab in vivo on days 9 and 13 exacerbated EAU. We also found that the ratio of ocular CD4/CD8 T cells was markedly increased after treatment. As a result, RANTES neutralization might exacerbate EAU by modulating the type of T cell subsets recruited to the eye. In conclusion, our data provide insight into the immunoregulatory role of macrophages and RANTES in the pathogenesis of ocular inflammation. Not all macrophage-derived chemokines cause local inflammation, since RANTES produced by ocular macrophages appears to suppress EAU.

Different chemokines are expressed in human arthritic bone biopsies: IFN-gamma and IL-6 differently modulate IL-8, MCP-1 and rantes production by arthritic osteoblasts.

In the present study we analyse chemokine expression in the remodelling of subchondral bone in arthritis patients. Trabecular bone biopsies were tested by immunohistochemistry to identify interleukin (IL)-8, GRO-alpha, MCP-1, RANTES, MIP-1alpha and MIP-1beta expression. Subsequently, we evaluated by immunoassay the effect of interferon (IFN)-gamma and IL-6 on chemokine production by osteoarthritis (OA), rheumatoid arthritis (RA) and post-traumatic (PT) patients' isolated osteoblasts (OB). OB constitutively produced in situ IL-8, GRO-alpha, MCP-1, RANTES and MIP-1alpha. MIP-1beta was positive only in mononuclear cells. In RA many of these chemokines were also produced by mononuclear cells. IFN-gamma significantly down-regulated IL-8 and up-regulated MCP-1 produced by OB from all patients tested, whereas it did not affect the other chemokines analysed. Moreover, IFN-gamma reduced IL-1beta-stimulated IL-8 production but significantly increased both MCP-1 and RANTES. Interestingly, IL-6 significantly downregulated IFN-gamma-induced MCP-1 production, that was significantly lower in OA compared to RA patients. OB expressed chemokines both in vivo and in vitro suggesting that these cells are primary effectors in the bone capable of regulating autocrine/paracrine circuits that affect bone remodelling in these diseases.

RANTES expression and contribution to monocyte chemotaxis in arthritis.

Rheumatoid arthritis (RA) is characterized by recruitment of leukocytes from the vasculature into inflamed synovial tissue (ST) and synovial fluid (SF), which depends, in part, upon the continued maintenance of chemotactic stimuli. RANTES is a potent chemoattractant for leukocytes including monocytes and CD45RO+ memory T lymphocytes. The aim of this study was to determine the production, the source, and the function of antigenic RANTES in arthritis. We detected antigenic RANTES in SFs from RA and OA patients (100 +/- 22.7 and 72 +/- 30.7 pg/ml, respectively). CM from RA ST fibroblasts stimulated with interleukin-1beta or tumor necrosis factor-alpha contained significantly more antigenic RANTES than unstimulated CM (452 +/- 181.6 and 581 +/- 200.2 pg/ml, respectively, versus 12 +/- 4.4 pg/ml, P < 0.05). PHA-stimulated RA SF mononuclear cells secreted 5- to 15-fold more antigenic RANTES than did nonstimulated mononuclear cells, while LPS induced secretion up to 4-fold. We immunolocalized antigenic RANTES to sublining macrophages (28 +/- 3.7 and 8 +/- 2.0% immunopositive cells), perivascular macrophages (56 +/- 6.9 and 19 +/- 3.4%), and synovial lining cells (37 +/- 5.8 and 60 +/- 10.4%) in RA and OA tissue, respectively. Anti-RANTES neutralized 20.2 +/- 1.3% of the RA SF chemotactic activity for normal peripheral blood monocytes (P < 0.05). These results demonstrate antigenic RANTES in RA and OA ST and SF and identify RANTES as a chemoattractant for monocytes in the RA joint.