Interleukin‐6 maintains bone marrow‐derived mesenchymal stem cell stemness by an ERK1/2‐dependent mechanism

Adult human mesenchymal stem cells (MSCs) hold promise for an increasing list of therapeutic uses due to their ease of isolation, expansion, and multi‐lineage differentiation potential. To maximize the clinical potential of MSCs, the underlying mechanisms by which MSC functionality is controlled must be understood. We have taken a deconstructive approach to understand the individual components in vitro, namely the role of candidate “stemness” genes. Our recent microarray gene expression profiling data suggest that interleukin‐6 (IL‐6) may contribute to the maintenance of MSCs in their undifferentiated state. In this study, we showed that IL‐6 gene expression is significantly higher in undifferentiated MSCs as compared to their chondrogenic, osteogenic, and adipogenic derivatives. Moreover, we found that MSCs secrete copious amounts of IL‐6 protein, which decreases dramatically during osteogenic differentiation. We further evaluated the role of IL‐6 for maintenance of MSC “stemness,” using a series of functional assays. The data showed that IL‐6 is both necessary and sufficient for enhanced MSC proliferation, protects MSCs from apoptosis, inhibits adipogenic and chondrogenic differentiation of MSCs, and increases the rate of in vitro wound healing of MSCs. We further identified ERK1/2 activation as the key pathway through which IL‐6 regulates both MSC proliferation and inhibition of differentiation. Taken together, these findings show for the first time that IL‐6 maintains the proliferative and undifferentiated state of bone marrow‐derived MSCs, an important parameter for the optimization of both in vitro and in vivo manipulation of MSCs. J. Cell. Biochem. 108: 577–588, 2009. Published 2009 Wiley‐Liss, Inc.     

Interleukin‐17 family cytokines in protective immunity against infections: role of hematopoietic cell‐derived and non‐hematopoietic cell‐derived interleukin‐17s

Interleukin‐17 family cytokines, consisting of six members, participate in immune response in infections and autoimmune and inflammatory diseases. The prototype cytokine of the family, IL‐17A, was originally identified from CD4+ T cells which are now termed Th17 cells. Later, IL‐17A‐producing cells were expanded to include various hematopoietic cells, namely CD8+ T cells (Tc17), invariant NKT cells, γδ T cells, non‐T non‐B lymphocytes (termed type 3 innate lymphoid cells) and neutrophils. Some IL‐17 family cytokines other than IL‐17A are also expressed by CD4+ T cells: IL‐17E by Th2 cells and IL‐17F by Th17 cells. IL‐17A and IL‐17F induce expression of pro‐inflammatory cytokines to induce inflammation and anti‐microbial peptides to kill pathogens, whereas IL‐17E induces allergic inflammation. However, the functions of other IL‐17 family cytokines have been unclear. Recent studies have shown that IL‐17B and IL‐17C are expressed by epithelial rather than hematopoietic cells. Interestingly, expression of IL‐17E and IL‐17F by epithelial cells has also been reported and epithelial cell‐derived IL‐17 family cytokines shown to play important roles in immune responses to infections at epithelial sites. In this review, we summarize current information on hematopoietic cell‐derived IL‐17A and non‐hematopoietic cell‐derived IL‐17B, IL‐17C, IL‐17D, IL‐17E and IL‐17F in infections and propose functional differences between these two categories of IL‐17 family cytokines.     

Interleukin‐6 contributes to chemoresistance in MDA‐MB‐231 cells via targeting HIF‐1α

Chemoresistance is a critical challenge in the clinical treatment of triple‐negative breast cancer (TNBC). It has been well documented that inflammatory mediators from tumor microenvironment are involved in the pathogenesis of TNBC and might be related to chemoresistance of cancer cells. In this study, the contribution of interleukin‐6 (IL‐6), one of the principal oncogenic molecules, in chemoresistance of a TNBC cell line MDA‐MB‐231 was first investigated. The results showed that IL‐6 treatment could induce upregulation of HIF‐1α via the activation of STAT3 in MDA‐MB‐231 cells, which consequently contributed to its effect against chemotherapeutic drug‐induced cytotoxicity and cell apoptosis. However, knockdown of HIF‐1α attenuated such effect via affecting the expressions of apoptosis‐related molecules as Bax and Bcl‐2 and drug transporters as P‐gp and MRP1. This study indicated that targeting at IL‐6/HIF‐1α signaling pathway might be an effective strategy to overcome chemoresistance in TNBC therapy.     

Large numbers of interleukins‐22‐ and ‐17A‐producing T helper cells in cholangiocarcinoma related to liver fluke infection

Cholangiocarcinoma (CCA) associated with liver fluke infection involves inflammatory and immune processes; however, whether these involve the proinflammatory cytokine IL‐17A and proliferative cytokine IL‐22 remains unclear. Here, numbers of IL‐22‐ and IL‐17A‐producing Th cells and cytokine concentrations in 30 patients with CCA and long‐term liver fluke infection, 40 patients with liver‐fluke infection but not CCA, and 16 healthy controls were compared. Analyses were performed using immunohistochemistry, flow cytometry, ELISA and RT‐PCR. Immunohistochemical staining showed weaker expression of IL‐22 and IL‐17A in patients with CCA with than in those without liver fluke infection (P < 0.01). Flow cytometry revealed significantly greater median proportions of IL‐22‐producing T helper cells in patients with CCA (2.2%) than in those without it (0.69%) or controls (0.4%, P < 0.001). Similar results were obtained for IL‐17A‐producing T helper cells. ELISA revealed plasma concentrations of IL‐22 were 1.3‐fold higher in patients with CCA than in those without it and 4.6‐fold higher than in controls (P < 0.001). Plasma concentrations of IL‐17A were 2.5‐fold higher in patients with CCA than in those without it, and 21‐fold higher than in controls (P < 0.001). Amounts of IL‐22 and IL‐17A mRNAs in blood were significantly higher in patients with CCA than in the other two groups. Proportions of CD4⁺CD45RO⁺ T cells producing IL‐22 correlated with proportions producing IL‐17A (r = 0.759; P < 0.001), and plasma concentrations of IL‐22 correlated with those of IL‐17A (r = 0.726; P < 0.001). These results suggest that both IL‐17A and IL‐22 affect development of CCA related to liver fluke infection.

     

Correlation of interleukin‐6 and monocyte chemotactic protein‐1 concentrations with crescent formation and myeloperoxidase‐specific anti‐neutrophil cytoplasmic antibody titer in SCG/Kj mice by treatment with anti‐interleukin‐6 receptor antibody or mizoribine

Myeloperoxidase‐specific anti‐neutrophil cytoplasmic antibody (MPO–ANCA) is associated with rapidly progressive glomerulonephritis (RPGN) and glomerular crescent formation. Pathogenic factors in RPGN were analyzed by using SCG/Kj mice, which spontaneously develop MPO–ANCA‐associated RPGN. The serum concentration of soluble IL‐6R was determined by using ELISA and those of another 23 cytokines and chemokines by Bio‐Plex analysis. Sections of frozen kidney tissue were examined by fluorescence microscopy and the CD3⁺B220⁺ T cell subset in the spleen determined by a flow cytometry. Concentrations of IL‐6 and monocyte chemotactic protein‐1 were significantly correlated with the percentages of crescent formation. Anti‐IL‐6R antibody, which has been effective in patients with rheumatoid arthritis, was administered to SCG/Kj mice to elucidate the role of IL‐6 in the development of RPGN. MPO–ANCA titers decreased after administration of anti‐IL‐6R antibody, but not titers of mizoribine, which is effective in Kawasaki disease model mice. These results suggest that IL‐6‐mediated signaling is involved in the production of MPO–ANCA.     

H2S transiently blocks IL‐6 expression in rheumatoid arthritic fibroblast‐like synoviocytes and deactivates p44/42 mitogen‐activated protein kinase

Sulfur bath therapy represents the oldest form of treatment for patients with different types of rheumatic disorders. However, scientific reports about the beneficial effects of this form of therapy are controversial, rare and of poor scientific quality. Also, little is known about the role and underlying molecular mechanisms of H₂S. Therefore, this topic encouraged us to investigate the influence of H₂S on fibroblasts isolated from the synovial membrane of RA (rheumatoid arthritis) patients. FLSs (fibroblast‐like synoviocytes) were treated with different concentrations of an exogenous H₂S donor (NaHS). At defined time points, secretion of IL‐6 was quantified by ELISA. Activation/deactivation of MAPKs (mitogen‐activated protein kinases), p38 and p44/42 MAPK (ERK1/2) were confirmed by Western blot experiments. FLSs constitutively express and secrete large quantities of IL‐6 and IL‐8. Data provided prove that, in FLSs, constitutive as well as IL‐1β‐induced expression of IL‐6 is transiently and partially down‐regulated by the short treatment of cells with low concentrations of NaHS. Another key finding is that H₂S deactivates p44/42 MAPK (ERK1/2). Long‐term exposure of FLSs to H₂S provides stimulatory effects, leading to reinforced activation of p38 MAPK and ERK1/2 accompanied by upregulation of IL‐6 expression. Presented data seem of importance for studying (patho‐) physiological functions of H₂S and also for re‐evaluating sulfur spa therapy as one of the oldest forms of therapy for rheumatic disorders.     

The classic signalling and trans‐signalling of interleukin‐6 are both injurious in podocyte under high glucose exposure

Interleukin‐6 (IL‐6) is a multifunctional cytokine that employs IL‐6 classic and trans‐signalling pathways, and these two signal channels execute different or even opposite effects in certain diseases. As a cardinal event of diabetic kidney disease (DKD), whether the podocyte abnormalities are associated with IL‐6 signalling, especially classic or trans‐signalling respectively, remains unclear. In this study, we identified that the circulatory IL‐6, soluble IL‐6R (sIL‐6R) and soluble glycoprotein 130 (sgp130) levels are elevated in patients with DKD. The expressions of membrane‐bound IL‐6R (mIL‐6R), sIL‐6R and gp130 are enhanced in kidney cortex of diabetic mice accompanying with activated STAT3 by tyrosine 705 residue phosphorylation, while not serine 727. Above data infer both classic signalling and trans‐signalling of IL‐6 are activated during DKD. In cultured podocyte, high glucose (HG) up‐regulates the expression of mIL‐6R and gp130, as well as STAT3 tyrosine 705 phosphorylation, in a time‐dependent manner. Entirely blocking IL‐6 signalling by gp130 shRNA, gp130 or IL‐6 neutralizing antibodies attenuates HG‐induced podocyte injury. Interestingly, either inhibiting IL‐6 classic signalling by mIL‐6R shRNA or suppressing its trans‐signalling using sgp130 protein dramatically alleviates HG‐induced podocyte injury, suggesting both IL‐6 classic signalling and trans‐signalling play a detrimental role in HG‐induced podocyte injury. Additionally, activation of IL‐6 classic or trans‐signalling aggravates podocyte damage in vitro. In summary, our observations demonstrate that the activation of either IL‐6 classic or trans‐signalling advances podocyte harming under hyperglycaemia. Thus, suppressing IL‐6 classic and trans‐signalling simultaneously may be more beneficial in podocyte protection and presents a novel therapeutic target for DKD.     

Raised Interleukin‐6 Levels in Obese Patients

Objective: Obese patients demonstrate a variety of biochemical, metabolic, and pulmonary abnormalities. Inflammatory mediators such as tumor necrosis factor‐α and interleukin‐6 (IL‐6) may have a direct effect on glucose and lipid metabolism. Hypoxemia in itself induces release of IL‐6. The aim of this study was to examine the relationship between IL‐6 levels in healthy volunteers (control group) and three different groups of obese patients: patients without obstructive sleep apnea syndrome (OSAS), patients with OSAS, and patients with obesity hypoventilation syndrome (OHS) (daytime baseline oxygen saturation of <93%). Research Methods and Procedures: We measured serum IL‐6 levels in 25 obese patients (body mass index of >35 kg/m²) and 12 healthy women. Results: The results demonstrate statistically significant differences in serum IL‐6 levels between the control group (1.28 ± 0.85 pg/mL) and obese patients without OSAS (7.69 ± 5.06 pg/mL, p < 0.05) and with OSAS (5.58 ± 0.37 pg/mL, p < 0.0005). In the patients with OHS, IL‐6 concentrations were highest (43.13 ± 24.27 pg/mL). Discussion: We conclude that serum IL‐6 is increased in obese patients. The highest IL‐6 levels were found in the patients with OHS.     

Crystal structure of norcoclaurine‐6‐O‐methyltransferase,a key rate‐limiting step in the synthesis of benzylisoquinoline alkaloids

Growing pharmaceutical interest in benzylisoquinoline alkaloids (BIA) coupled with their chemical complexity make metabolic engineering of microbes to create alternative platforms of production an increasingly attractive proposition. However, precise knowledge of rate‐limiting enzymes and negative feedback inhibition by end‐products of BIA metabolism is of paramount importance for this emerging field of synthetic biology. In this work we report the structural characterization of (S)‐norcoclaurine‐6‐O‐methyltransferase (6OMT), a key rate‐limiting step enzyme involved in the synthesis of reticuline, the final intermediate to be shared between the different end‐products of BIA metabolism, such as morphine, papaverine, berberine and sanguinarine. Four different crystal structures of the enzyme from Thalictrum flavum (Tf 6OMT) were solved: the apoenzyme, the complex with S‐adenosyl‐l ‐homocysteine (SAH), the complexe with SAH and the substrate and the complex with SAH and a feedback inhibitor, sanguinarine. The Tf 6OMT structural study provides a molecular understanding of its substrate specificity, active site structure and reaction mechanism. This study also clarifies the inhibition of Tf 6OMT by previously suggested feedback inhibitors. It reveals its high and time‐dependent sensitivity toward sanguinarine.     

Interferon‐α curbs production of interleukin‐22 by human peripheral blood mononuclear cells exposed to live Borrelia burgdorferi

Cytokine networks initiated by means of innate immunity are regarded as a major determinant of host defence in response to acute infection by bacteria including Borrelia burgdorferi. Herein, we demonstrate that interferon (IFN)‐α, either endogenously produced after exposure of cells to toll‐like receptor‐9‐activating CpG oligonucleotides or provided as recombinant cytokine, weakens activation of the anti‐bacterial interleukin (IL)‐1/IL‐22 axis in human peripheral blood mononuclear cells exposed to viable B. burgdorferi. As IFN‐α has been related to pathological dissemination of the spirochaete, data suggest an immunoregulatory role of type I IFN in this context that is able to significantly modify cytokine profiles thereby possibly determining early course of B. burgdorferi infection.     

WNT‐3A and WNT‐5A counteract lipopolysaccharide‐induced pro‐inflammatory changes in mouse primary microglia

Surveying microglia, the resident macrophage‐like cells in the central nervous system, continuously screen their surroundings to sense imbalance in tissue homeostasis. Their activity is tightly regulated in both a pro‐ and anti‐inflammatory manner. We have previously shown that the lipoglycoproteins WNT‐3A and WNT‐5A drive pro‐inflammatory transformation in primary mouse microglia cells, arguing that WNTs have a role in the modulation of the central nervous system immune response. In this study, we address the effects of recombinant WNT‐3A and WNT‐5A on lipopolysaccharide (LPS)‐activated mouse primary microglia to investigate the putative anti‐inflammatory modulation of microglia by WNTs. While both WNT‐3A and WNT‐5A alone induce an up‐regulation of cyclooxygenase 2 (COX2), a generic pro‐inflammatory microglia marker, LPS exceeds these effects dramatically. However, combination of LPS and WNTs results in a dose‐dependent decrease in LPS‐induced cyclooxygenase 2 protein and mRNA expression. In conclusion, our data suggest that WNTs have a dual and context‐dependent effect on microglia acting in a homeostatic pro‐ and anti‐inflammatory manner.     

Crosstalk between Akt and NF‐κB pathway mediates inhibitory effect of gas6 on monocytes‐endothelial cells interactions stimulated by P. gingivalis‐LPS

Correlation between periodontitis and atherosclerosis is well established, and the inherent mechanisms responsible for this relationship remain unclear. The biological function of growth arrest‐specific 6 (gas6) has been discovered in both atherosclerosis and inflammation. Inhibitory effects of gas6 on the expression of inflammatory factors in human umbilical vein endothelial cells (HUVECs) stimulated by Porphyromonas gingivalis lipopolysaccharide (P. gingivalis‐LPS) were reported in our previous research. Herein, the effects of gas6 on monocytes‐endothelial cells interactions in vitro and their probable mechanisms were further investigated. Gas6 protein in HUVECs was knocked down with siRNA or overexpressed with plasmids. Transwell inserts and co‐culturing system were introduced to observe chemotaxis and adhering affinity between monocytes and endothelial cells in vitro. Expression of gas6 was decreased in inflammatory periodontal tissues and HUVECs challenged with P. gingivalis‐LPS. The inhibitory effect of gas6 on chemotaxis and adhesion affinity between monocytes and endothelial cells was observed, and gas6 promoted Akt phosphorylation and inhibited NF‐κB phosphorylation. To our best knowledge, we are first to report that gas6 inhibit monocytes‐endothelial cells interactions in vitro induced by P. gingivalis‐LPS via Akt/NF‐κB pathway. Additionally, inflammation‐mediated inhibition of gas6 expression is through LncRNA GAS6‐AS2, rather than GAS6‐AS1, which is also newly reported.