S100A9 gene silencing inhibits the release of pro‐inflammatory cytokines by blocking the IL‐17 signalling pathway in mice with acute pancreatitis

The study aimed to investigate whether S100A9 gene silencing mediating the IL‐17 pathway affected the release of pro‐inflammatory cytokines in acute pancreatitis (AP). Kunming mice were assigned to the normal, AP, AP + negative control (NC), AP + shRNA, AP + IgG and AP + anti IL‐17 groups. ELISA was applied to measure expressions of AMY, LDH, CRP, TNF‐α, IL‐6 and IL‐8. The cells were distributed into the control, blank, NC, shRNA1 and shRNA2 groups. MTT assay, flow cytometry, RT‐qPCR and Western blotting were used to evaluate cell proliferation, cell cycle and apoptosis, and expressions of S100A9, TLR4, RAGE, IL‐17, HMGB1 and S100A12 in tissues and cells. Compared with the normal group, the AP group displayed increased expressions of AMY, LDH, CRP, TNFα, IL‐6, IL‐8, S100A9, TLR4, RAGE, IL‐17, HMGB1 and S100A12. The AP + shRNA and AP + anti IL‐17 groups exhibited an opposite trend. The in vivo results: Compare with the control group, the blank, NC, shRNA1 and shRNA2 groups demonstrated increased expressions of S100A9, TLR4, RAGE, IL‐17, HMGB1 and S100A12, as well as cell apoptosis and cells at the G1 phase, with reduced proliferation. Compared with the blank and NC groups, the shRNA1 and shRNA2 groups had declined expressions of S100A9, TLR4, RAGE, IL‐17, HMGB1 and S100A12, as well as cell apoptosis and cells at the G1 phase, with elevated proliferation. The results indicated that S100A9 gene silencing suppressed the release of pro‐inflammatory cytokines through blocking of the IL‐17 pathway in AP.     

Pro‐inflammatory capacity of classically activated monocytes relates positively to muscle mass and strength

In mice, monocytes that exhibit a pro‐inflammatory profile enter muscle tissue after muscle injury and are crucial for clearance of necrotic tissue and stimulation of muscle progenitor cell proliferation and differentiation. The aim of this study was to test if pro‐inflammatory capacity of classically activated (M1) monocytes relates to muscle mass and strength in humans. This study included 191 male and 195 female subjects (mean age 64.2 years (SD 6.4) and 61.9 ± 6.4, respectively) of the Leiden Longevity Study. Pro‐inflammatory capacity of M1 monocytes was assessed by ex vivo stimulation of whole blood with Toll‐like receptor (TLR) 4 agonist lipopolysaccharide (LPS) and TLR‐2/1 agonist tripalmitoyl‐S‐glycerylcysteine (Pam₃Cys‐SK₄), both M1 phenotype activators. Cytokines that stimulate M1 monocyte response (IFN‐γ and GM‐CSF) as well as cytokines that are secreted by M1 monocytes (IL‐6, TNF‐α, IL‐12, and IL‐1β) were measured. Analyses were adjusted for age, height, and body fat mass. Upon stimulation with LPS, the cytokine production capacity of INF‐γ, GM‐CSF, and TNF‐α was significantly positively associated with lean body mass, appendicular lean mass and handgrip strength in men, but not in women. Upon stimulation with Pam₃Cys‐SK₄, IL‐6; TNF‐α; and Il‐1β were significantly positively associated with lean body mass and appendicular lean in women, but not in men. Taken together, this study shows that higher pro‐inflammatory capacity of M1 monocytes upon stimulation is associated with muscle characteristics and sex dependent.     

miR‐148a suppresses inflammation in lipopolysaccharide‐induced endometritis

Endometritis is a postnatal reproductive disorder disease, which leads to great economic losses for the modern dairy industry. Emerging evidence indicates that microRNAs (miRNAs) play a pivotal role in a variety of diseases and have been identified as critical regulators of the innate immune response. Recent miRNome profile analysis revealed an altered expression level of miR‐148a in cows with endometritis. Therefore, the present study aims to investigate the regulatory role of miR‐148a in the innate immune response involved in endometritis and estimate its potential therapeutic value. Here, we found that miR‐148a expression in lipopolysaccharide (LPS)‐stimulated endometrial epithelial cells was significantly decreased . Our results also showed that overexpression of miR‐148a using agomiR markedly reduced the production of pro‐inflammatory cytokines, such as IL‐1β and TNF‐α. Moreover, overexpression of miR‐148a also suppressed NF‐κB p65 activation by targeting the TLR4‐mediated pathway. Subsequently, we further verified that miR‐148a repressed TLR4 expression by binding to the 3′‐UTR of TLR4 mRNA. Additionally, an experimental mouse endometritis model was employed to evaluate the therapeutic value of miR‐148a. In vivo studies suggested that up‐regulation of miR‐148a alleviated the inflammatory conditions in the uterus as evidenced by H&E staining, qPCR and Western blot assays, while inhibition of miR‐148a had inverse effects. Collectively, pharmacologic stabilization of miR‐148a represents a novel therapy for endometritis and other inflammation‐related diseases.     

Toll-like receptor 4 and MYD88-dependent signaling mechanisms of the innate immune system are essential for the response to lipopolysaccharide by epithelial and stromal cells of the bovine endometrium

Infection of the bovine endometrium with Gram-negative bacteria commonly causes uterine disease. Toll-like receptor 4 (TLR4) on cells of the immune system bind Gram-negative bacterial lipopolysaccharide (LPS), stimulating the secretion of the proinflammatory cytokines interleukin 1B (IL1B) and IL6, and the chemokine IL8. Because the endometrium is the first barrier to infection of the uterus, the signaling cascade triggered by LPS and the subsequent expression of inflammatory mediators were investigated in endometrial epithelial and stromal cells, and the key pathways identified using short interfering RNA (siRNA) and biochemical inhibitors. Treatment of endometrial cells with ultrapure LPS stimulated an inflammatory response characterized by increased IL1B, IL6, and IL8 mRNA expression, and IL6 protein accumulation in epithelial cells, and by increased IL1B and IL8 mRNA expression, and IL6 and IL8 protein accumulation in stromal cells. Treatment of endometrial cells with LPS also induced the degradation of IKB and the nuclear translocation of NFKB, as well as rapid phosphorylation of mitogen-activated protein kinase 3/1 (MAPK3/1) and MAPK14. Knockdown of TLR4 or its signaling adaptor molecule, myeloid differentiation factor 88 (MYD88), using siRNA reduced the inflammatory response to LPS in epithelial and stromal cells. Biochemical inhibition of MAPK3/1, but not JNK or MAPK14, reduced LPS-induced IL1B, IL6, and IL8 expression in endometrial cells. In conclusion, epithelial and stromal cells have an intrinsic role in innate immune surveillance in the endometrium, and in the case of LPS this recognition occurs via TLR4- and MYD88-dependent cell signaling pathways.     

Decreased TLR2 signal expression in peripheral blood mononuclear cell from patients with cryptococcal meningitis

Toll‐like receptors are the most important pattern recognition receptors that can recognize conserved molecular structures shared by large groups of pathogens. Here, the aim was to determine the expression and role of TLR2 in peripheral blood mononuclear cells (PBMCs) from patients with cryptococcal meningitis and healthy controls. TLR2 expression was measured using RT‐PCR and western blotting. The role of TLR2 in cytokine production by PBMCs after Cryptococcus neoformans exposure was assessed in healthy controls prior to incubation with anti‐TLR2. TLR2 mRNA and protein expression were both weaker in patients with cryptococcal meningitis than in healthy controls. Furthermore, pre‐incubation of PBMCs from healthy donors with anti‐TLR2 led to reduced expression of IFN‐γ and IL‐12p70, but not of IL‐4 and IL‐10, following C. neoformans stimulation. Our results suggest that impaired expression of TLR2 may be involved in defective host defense to C. neoformans through an attenuated Th1 response.     

Inhibition of cytokine response to TLR stimulation and alleviation of collagen‐induced arthritis in mice by Schistosoma japonicum peptide SJMHE1

Helminth‐derived products have recently been shown to prevent the development of inflammatory diseases in mouse models. However, most identified immunomodulators from helminthes are mixtures or macromolecules with potentially immunogenic side effects. We previously identified an immunomodulatory peptide called SJMHE1 from the HSP60 protein of Schistosoma japonicum. In this study, we assessed the ability of SJMHE1 to affect murine splenocytes and human peripheral blood mononuclear cells (PBMCs) stimulated by toll‐like receptor (TLR) ligands in vitro and its treatment effect on mice with collagen‐induced arthritis (CIA). We show that SJMHE1 not only modulates the cytokine production of murine macrophage (MΦ) and dendritic cell but also affects cytokine production upon coculturing with allogeneic CD4⁺ T cell. SJMHE1 potently inhibits the cytokine response to TLR ligands lipopolysaccharide (LPS), CpG oligodeoxynucleotides (CpG) or resiquimod (R848) from mouse splenocytes, and human PBMCs stimulated by LPS. Furthermore, SJMHE1 suppressed clinical signs of CIA in mice and blocked joint erosion progression. This effect was mediated by downregulation of key cytokines involved in the pathogenesis of CIA, such as interferon‐γ (IFN‐γ), tumour necrosis factor‐α (TNF‐α), interleukin (IL)‐6, IL‐17, and IL‐22 and up‐regulation of the inhibitory cytokine IL‐10, Tgf‐β1 mRNA, and CD4⁺CD25⁺Foxp3⁺ Tregs. This study provides new evidence that the peptide from S. japonicum, which is the ‘safe’ selective generation of small molecule peptide that has evolved during host–parasite interactions, is of great value in the search for novel anti‐inflammatory agents and therapeutic targets for autoimmune diseases.     

Lactoferricin mediates anti‐inflammatory and anti‐catabolic effects via inhibition of IL‐1 and LPS activity in the intervertebral disc

The catabolic cytokine interleukin‐1 (IL‐1) and endotoxin lipopolysaccharide (LPS) are well‐known inflammatory mediators involved in degenerative disc disease, and inhibitors of IL‐1 and LPS may potentially be used to slow or prevent disc degeneration in vivo. Here, we elucidate the striking anti‐catabolic and anti‐inflammatory effects of bovine lactoferricin (LfcinB) in the intervertebral disc (IVD) via antagonism of both IL‐1 and LPS‐mediated catabolic activity using in vitro and ex vivo analyses. Specifically, we demonstrate the biological counteraction of LfcinB against IL‐1 and LPS‐mediated proteoglycan (PG) depletion, matrix‐degrading enzyme production, and enzyme activity in long‐term (alginate beads) and short‐term (monolayer) culture models using bovine and human nucleus pulposus (NP) cells. LfcinB significantly attenuates the IL‐1 and LPS‐mediated suppression of PG production and synthesis, and thus restores PG accumulation and pericellular matrix formation. Simultaneously, LfcinB antagonizes catabolic factor mediated induction of multiple cartilage‐degrading enzymes, including MMP‐1, MMP‐3, MMP‐13, ADAMTS‐4, and ADAMTS‐5, in bovine NP cells at both mRNA and protein levels. LfcinB also suppresses the catabolic factor‐induced stimulation of oxidative and inflammatory factors such as iNOS, IL‐6, and toll‐like receptor‐2 (TLR‐2) and TLR‐4. Finally, the ability of LfcinB to antagonize IL‐1 and LPS‐mediated suppression of PG is upheld in an en bloc intradiscal microinjection model followed by ex vivo organ culture using both mouse and rabbit IVD tissue, suggesting a potential therapeutic benefit of LfcinB on degenerative disc disease in the future. J. Cell. Physiol. 228: 1884–1896, 2013. © 2013 Wiley Periodicals, Inc.     

Docosahexanoic acid diet supplementation attenuates the peripheral mononuclear cell inflammatory response to exercise following LPS activation

Exercise induces changes in circulating pro- and anti-inflammatory cytokines. The aim was to investigate the effect of docosahexaenoic acid (DHA) diet supplementation on the plasma cytokine levels and on the peripheral mononuclear (PBMCs) cells cytokine production after a training season or an acute bout of exercise. Fifteen male soccer players were randomly assigned to a placebo or an experimental group. The experimental group consumed an almond-based beverage enriched with DHA, whereas the placebo group consumed the same beverage without DHA. Three blood samples were taken: in basal conditions at the beginning of the nutritional intervention and after eight weeks of training season in basal and post-exercise conditions. The DHA content increased in erythrocytes after 8 weeks of training and supplementation. Neither diet supplementation with DHA nor training season altered the basal plasma cytokines and growth factors. Only acute exercise significantly increased plasma IL6 in experimental and placebo groups. Lipopolysaccharide (LPS) activation induced the inflammatory response in PBMCs, with a significant production rate of TNFα, IL6 and IL8 mainly after acute exercise. DHA supplementation significantly reduced the rate of TNFα and IL6 production by stimulated PBMCs. Acute exercise increased the Toll-like receptor 4 (TLR4) protein levels in PBMCs, although the increase was only statistically significant in the placebo group. In conclusion, a training season does not induce significant changes in the circulating cytokine profile in well-trained soccer players. Exercise increases the PBMCs cell capabilities to produce cytokines after TLR4 stimulation with LPS and this rate of cytokine production is attenuated by diet DHA supplementation.     

Cytokine responses to eye spray adjuvants for enhancing vaccine‐induced immunity in chickens

Eye spray influenza vaccines for chickens are increasingly available; however, how to enhance cellular and antibody responses to them remains undetermined. Here, eye‐drops containing the immune‐enhancing adjuvants Pam2CSK4 or polyI:C were assessed in chickens. Application of these TLR agonists to chicken conjunctiva resulted in up‐regulation of IL‐1β, but not other cytokines, including IFN and IL‐6, in the spleen, lung and Harderian gland. Thus, responses to adjuvant applied to the conjunctival mucosa of chickens differ from those expected from the responses to intra‐nasal adjuvants in mammals. Identifying an appropriate delivery route for adjuvants is crucial for evoking immune responses in chickens.     

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.     

Age‐0 striped bass,Morone saxatilis (Walbaum, 1792), response to immunostimulation

Young‐of‐the‐year (age‐0) striped bass, Morone saxatilis, were studied to characterize their responses to inflammatory stimuli. There were two studies, with the hypotheses that (i) <5 g striped bass would respond to inflammatory insult within 6 h, and (ii) response to antigens would be maintained for >24 h in larger fish. Study I was conducted to understand the cytokine expression pattern (IL‐1β, TNF‐α, Nramp and TGF‐β) in response to lipopolysaccharide (LPS) or Freund's complete adjuvant (FCA) stimulation in age‐0 striped bass (3.44 ± 1.68 g, 70.6 ± 10.3 mm fork length) up to 24 h post injection (hpi). Study II was similar to Study I, but striped bass were sampled over a longer time frame (by 48 hpi) and larger age‐0 striped bass were used (20.6 ± 5.9 g, 129.2 ± 10.9 mm fork length). It was confirmed that immunostimulants such as LPS and FCA induce production of inflammatory cytokines and Nramp, which are important in innate immune response to bacterial infection. The responses were rapidly stimulated with LPS (IL‐1β, TNF‐α, TGF‐β >3‐fold increase, compared to PBS) or FCA (IL‐1β >3‐fold and TGF‐β >2‐fold, compared to PBS) within 6 hpi and maintained in most cases 48 hpi (spleen Nramp and TGF‐β 2‐fold >PBS, at 24 and 48 h), similar to other teleosts. Intraperitoneal injection with PBS also simulated inflammatory gene expression, but was delayed (IL‐1β, TNF‐α, TGF‐β and Nramp, FCA and LPS< at 6 h; 24 h >LPS and PBS) in comparison to LPS and FCA, suggesting that this procedure and possibly the volume used can be stimulatory and potentially harmful in age‐0 fish. Therefore, this study suggests that age‐0 striped bass are capable of strong cytokine induction in response to immunological stimulation within a very short period of time.     

Extremely low frequency electromagnetic field exposure does not modulate toll-like receptor signaling in human peripheral blood mononuclear cells

The effects of extremely low frequency electromagnetic fields (ELF-EMF) on human health remain unclear. It has been reported that ELF-EMF may modulate the innate immune response to microorganisms in animal models and mammalian cell-lines. With the recently gained insight in innate immune signaling and the discovery of pattern recognition, we aim to study whether ELF-EMF modulates innate inflammatory signaling pathways. We used human peripheral blood mononuclear cells (PBMCs), isolated from blood from healthy volunteers, which we stimulated with specific TLR2 and TLR4 ligands, and with several microorganisms. The cells were subsequently exposed in B(dc)=3 μT to a highly controlled and standardized ELF-EMF signal (20-5000Hz, B(ac)=5 μT, 30 min) and cytokine production was measured at different time points after stimulation. No significant difference in immune response, as reflected by IL-1β, IL-6, TNFα, IL-8 and IL-10 production, could be detected after stimulation with LPS (TLR4 ligand), Pam3Cys (TLR2 ligand) or a panel of heat killed microorganisms: Mycobacterium tuberculosis, Salmonella typhimurium, Candida albicans, Aspergillus fumigatus and Staphylococcus aureus (multiple TLR ligands). We therefore conclude that under our experimental conditions, ELF-EMF does not modulate the innate immune response of human primary cells after TLR stimulation in vitro.     

MNK kinases regulate multiple TLR pathways and innate proinflammatory cytokines in macrophages

The MNK kinases are downstream of both the p38 and ERK MAP kinase pathways and act to increase gene expression. MNK inhibition using the compound CGP57380 has recently been reported to inhibit tumor necrosis factor (TNF) production in macrophage cell lines stimulated with Escherichia coli lipopolysaccharide (LPS). However, the range of receptors that signal through the MNK kinases and the extent of the resultant cytokine response are not known. We found that TNF production was inhibited in RAW264.7 macrophage cells by CGP57380 in a dose-responsive manner with agonists for Toll-like receptor (TLR) 2 (HKLM), TLR4 (Salmonella LPS), TLR6/2 (FSL), TLR7 (imiquimod), and TLR9 (CpG DNA). CGP57380 also inhibited the peak of TNF mRNA production and increased the rate of TNF mRNA decay, effects not due to the destabilizing RNA binding protein tristetraprolin (TTP). Similar to its effects on TNF, CGP57380 caused dose-responsive inhibition of TTP production from stimulation with either LPS or CpG DNA. MNK inhibition also blocked IL-6 but permitted IL-10 production in response to LPS. Studies using bone marrow-derived macrophages (BMDM) isolated from a spontaneous mouse model of Crohn's disease-like ileitis (SAMP1/YitFc strain) revealed significant inhibition by CGP57380 of the proinflammatory cytokines TNF, IL-6, and monocyte chemoattractant protein-1 at 4 and 24 h after LPS stimulation. IL-10 production was higher in CGP53870-treated BMDM at 4 h but was similar to the controls by 24 h. Taken together, these data demonstrate that MNK kinases signal through a variety of TLR agonists and mediate a potent innate, proinflammatory cytokine response.