Induced IL-6 Secretion Research Articles

Heat shock protein 60 stimulates the migration of vascular smooth muscle cells via Toll-like receptor 4 and ERK MAPK activation.

Accumulating evidence indicates that heat shock protein (HSP) 60 is strongly associated with the pathology of atherosclerosis (AS). However, the precise mechanisms by which HSP60 promotes atherosclerosis remain unclear. In the present study, we found that HSP60 mRNA and protein expression levels in the thoracic aorta are enhanced not only in a mouse model of AS but also in high-fat diet (HFD) mice. HSP60 expression and secretion was activated by platelet-derived growth factor-BB (PDGF-BB) and interleukin (IL)-8 in both human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (VSMCs). HSP60 was found to induce VSMC migration, and exposure to HSP60 activated ERK MAPK signaling. U0126, an inhibitor of ERK, reduced VSMC migration. The HSP60-stimulated VSMCs were found to express TLR4 mRNA but not TLR2 mRNA. Knockdown of TLR4 by siRNA reduced HSP60-induced VSMC migration and HSP60-induced ERK activation. Finally, HSP60 induced IL-8 secretion in VSMCs. Together these results suggest that HSP60 is involved in the stimulation of VSMC migration, via TLR4 and ERK MAPK activation. Meanwhile, activation of HSP60 is one of the most powerful methods of sending a ‘danger signal’ to the immune system to generate IL-8, which assists in the management of an infection or disease.

PDGF upregulates CLEC-2 to induce T regulatory cells.

The effect of platelet derived growth factor (PDGF) on immune cells is not elucidated. Here, we demonstrate PDGF inhibited the maturation of human DCs and induced IL-10 secretion. Culture of PDGF-DCs with T cells induced the polarization of T cells towards FoxP3 expressing T regulatory cells that secreted IL-10. Gene expression studies revealed that PDGF induced the expression of C-type lectin like receptor member 2, (CLEC-2) receptor on DCs. Furthermore, DCs transfected with CLEC-2 induced T regulatory cells in DC-T cell co-culture. CLEC-2 is naturally expressed on platelets. Therefore, to confirm whether CLEC-2 is responsible for inducing the T regulatory cells, T cells were cultured with either CLEC-2 expressing platelets or soluble CLEC-2. Both conditions resulted in the induction of regulatory T cells. The generation of T regulatory cells was probably due to the binding of CLEC-2 with its ligand podoplanin on T cells, since crosslinking of podoplanin on the T cells also resulted in the induction of T regulatory cells. These data demonstrate that PDGF upregulates the expression of CLEC-2 on cells to induce T regulatory cells.

Abstract 2139: Wild type N-Ras, overexpressed in basal-like breast cancer, promotes tumor formation by inducing IL8 secretion via JAK2 activation

Abstract “Basal-like” breast cancer (BLBC) is the most aggressive breast cancer subtype, with a very poor prognosis _ median time to distant recurrence is just 2.6 years vs. 5 years overall, and survival time from diagnosis of distant metastatic disease is 9 months vs. 22 months. BLBC tumors usually do not express ER, Her2, or progesterone receptor. As such, they cannot be treated by the current targeted therapies, which target these molecules. What drive the formation and progression of BLBCs is largely unclear. Ras GTPases are best known for mediating growth factor signaling, and oncogenic mutations in the RAS genes, and K-RAS in particular, are found in more than 30% of human tumors. Surprisingly, oncogenic RAS mutations are rare in breast cancer. However, we found that wild-type N-RAS is overexpressed in BLBCs, partly via promoter demethylation, but not in other breast cancer subtypes. Repressing N-RAS inhibits transformation and tumor growth, while overexpression enhances these processes even in preinvasive BLBC cells. In contrast, repressing N-RAS expression does not affect growth and transforming activities of breast cancer cells of other subtypes. We identified N-Ras-responsive genes, most of which encode chemokines, e.g., IL8. High expression levels of these N-Ras-responsive genes as well as of N-RAS itself in tumors correlate with poor patient outcome. N-Ras, but not K-Ras, induces IL8 by binding and activating the cytoplasmic pool of JAK2; IL8 then acts on both the cancer cells and stromal fibroblasts. Thus N-Ras drives BLBC by promoting transformation in epithelial cells, which in turn remodels the tumor microenvironment to create a proinvasive state. These results illustrate an N-Ras-dependent convergence between the growth factor and cytokine pathways to enable efficient transformation of epithelial cells, which then orchestrate the remodeling of the tumor microenvironment. Although oncogenic mutations affecting RAS are common in many other human cancers, tumorigenesis in an important subset of breast cancers is driven instead by increasing activity of wild-type N-Ras. Thus, to fully assess the impact of Ras on tumorigenesis, the role of wild-type as well as mutant Ras proteins must be carefully examined. Citation Format: Ze-Yi Zheng, Wen Bu, Lin Tian, Cheng Fan, Xia Gao, Cuijuan Yu, Liu Jun, Xiaomei Zhang, Yi-Hua Liao, Yi Li, Michael T. Lewis, Dean Edwards, Susan G. Hilsenbeck, Daniel Medina, Thomas P. Zwaka, Charles M. Perou, Chad J. Creighton, Xiang Zhang, Eric C. Chang. Wild type N-Ras, overexpressed in basal-like breast cancer, promotes tumor formation by inducing IL8 secretion via JAK2 activation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2139. doi:10.1158/1538-7445.AM2015-2139

Preservation of Helicobacter pylori CagA Translocation and Host Cell Proinflammatory Responses in the Face of CagL Hypervariability at Amino Acid Residues 58/59.

Carriage of the CagA oncoprotein by the human gastric cancer-associated pathogen Helicobacter pylori is significantly associated with this typically benign chronic infection advancing to a potentially fatal outcome. However it remains to be elucidated why only a small subset of individuals infected with H. pylori CagA-positive strains develops gastric cancer. H. pylori translocates CagA into host cells using a type IV secretion apparatus that interacts with host integrin receptors via a three-amino-acid-residue RGD motif on the H. pylori protein CagL. The RGD motif of CagL also plays a major role in the induction of proinflammatory responses. Upstream of this motif is a conserved glycine flanked by four hypervariable amino acid residues (residues 58, 59, 61 and 62). Certain amino acid polymorphisms at 58 and 59 are significantly prevalent in strains from gastric cancer patients in particular geographic regions; Y58E59 is seen in Taiwan and D58K59 in India. In light of the seemingly contradictory findings of recent CagL mutagenesis studies, we have examined the contribution of sequence promiscuity specifically at CagL residues 58 and 59 to CagA translocation and H. pylori-mediated proinflammatory responses of gastric epithelial cells. Using isogenic mutants of H. pylori strains P12 and 26695 with amino acid substitutions at CagL residues 58 and 59, we determined that carriage of the polymorphisms Y58E59, D58K59, D58E59, N58E59 or N58K59 did not significantly alter the capacity of H. pylori to translocate CagA into, or induce IL-8 secretion in, host cells. Our findings, together with other recently published data, suggest that the variation at CagL residues 58 and 59 does not influence type IV secretion system function in isolation, but rather may work in concert with particular polymorphisms elsewhere in CagL to modulate disease progression.

Galectin-3 Modulates Experimental Colitis

Background: We recently identified galectin-3 (gal-3) as a new and strong fibroblast activator produced by colonic epithelial cells. Very little is known about the influence of gal-3 in inflammatory bowel disease. We, therefore, investigated the impact of gal-3 on dextran sodium sulfate (DSS)-induced colitis in a mouse model. Methods: Colonic lamina propria fibroblasts of healthy controls were used for co-incubation studies of gal-3 with gal-1, TGF-β1, IFNγ, IL-4 and IL-10. Acute and chronic DSS colitis was induced by 3% DSS in drinking water in female Balb/c mice weighing 20-22 g. Recombinant gal-3 was expressed by the pET vector system and used for a 5-day treatment in different concentrations intraperitoneally. The distal third of the colon was used for histologic analysis. Colonic cytokine expression was determined by quantitative RT-PCR. Results: In vitro, gal-3 induced IL-8 secretion was significantly reduced by co-incubation with IL-10 (5 ng/ml) and IL-4 (10 ng/ml). Acute DSS-induced colitis was ameliorated by gal-3 treatment as indicated by increased colonic length and reduced weight loss compared to that of controls. In acute and chronic colitis, gal-3 treatment resulted in a significant suppression of colonic IL-6. Conclusion: Gal-3 significantly reduces inflammation in acute and chronic DSS colitis in mice indicating a potential role in intestinal inflammation.

B10 cells induced by Schistosoma japonicum soluble egg antigens modulated regulatory T cells and cytokine production of T cells.

A distinct subset of B cells, also known as regulatory B cells, can negatively regulate T cell immune responses, but the role of these cells in schistosomiasis has not been clarified. Soluble egg antigen (SEA) and soluble adult worm antigen preparation (SWAP), which are two important antigen sources during Schistosoma japonicum infection, both can induce Th1, Th2, Th17, and Treg cells and the corresponding cytokines. However, whether they can induce the production of regulatory B cells and the regulatory function of schistosome-induced regulatory B cells remains unclear. In our studies, we first analyzed the production of regulatory B cells stimulated by SEA or SWAP using flow cytometry and enzyme-linked immunosorbent assay, and observed these cells in mice immunized by SEA or SWAP. Then, B10 cells sorted by MicroBeads were co-cultured with CD4(+) T cells, and the proportion of Treg cells were detected. At the same time, the IFN-γ, IL-4, and IL-17 levels in the culture supernatant were measured. The results showed that B10 cells were preferentially induced by SEA in vitro, and B10 could also be induced in mice immunized by SEA. SEA-induced B10 cells promoted the expansion of regulatory T cells and induced IL-4 secretion, but inhibited IL-17 production. These findings reveal that the generation of B10 cells is determined by parasitic antigen, and suggest the function of B10 cell induced by SEA. This study significantly contributes to the understanding of the immune regulatory role in schistosomiasis and may help protect hosts from infection.

In Vitro Evidence for Immune-Modulatory Properties of Non-Digestible Oligosaccharides: Direct Effect on Human Monocyte Derived Dendritic Cells.

Infant formulas containing non-digestible oligosaccharides (NDO) similar to the composition in breast milk or a combination of lactic acid bacteria (LAB) and NDO have been shown to harbor preventive effects towards immune-regulatory disorders. The aim of this study was to investigate the immune-modulatory potential of non-digestible short chain galacto- and long chain fructo-oligosaccharides (scGOS/lcFOS) mimicking the natural distribution of oligosaccharides in human breast milk in presence or absence of certain LAB strains in human monocyte derived dendritic cells (MoDC). Immature human MoDC prepared from peripheral blood of healthy non-atopic volunteers were screened in vitro after stimulation with specific scGOS/lcFOS in presence or absence of LAB. IL-10 and IL-12p70 release was analyzed after 24 hours in cell-free supernatants by enzyme-linked immunosorbent assay (ELISA). A luminex-based assay was conducted to assess further cytokine and chemokine release by MoDC. To investigate the resulting T cell response, stimulated MoDC were co-incubated with naïve T cells in allogeneic stimulation assays and intracellular Foxp3 expression, as well as immune-suppressive capacity was determined. Oligosaccharides did not induce relevant amounts of IL-12p70 production, but did promote IL-10 release by MoDC. Furthermore, scGOS/lcFOS mixtures exerted a significant enhancing effect on LAB induced IL-10 secretion by MoDC while no increase in IL-12p70 production was observed. Blocking toll like receptor (TLR)4 abrogated the increase in IL-10 in both the direct stimulation and the LAB stimulation of MoDC, suggesting that scGOS/lcFOS act via TLR4. Finally, scGOS/lcFOS-treated MoDC were shown to upregulate the number of functional suppressive Foxp3 positive T cells following allogeneic stimulation. Our results indicate anti-inflammatory and direct, microbiota independent, immune-modulatory properties of scGOS/lcFOS mixtures on human MoDC suggesting a possible induction of regulatory T cells (Tregs). The tested combinations of LAB and scGOS/lcFOS might represent a useful dietary ingredient for the maintenance of intestinal homeostasis via the induction of Tregs.

Wild-Type N-Ras, Overexpressed in Basal-like Breast Cancer, Promotes Tumor Formation by Inducing IL-8 Secretion via JAK2 Activation.

Basal-like breast cancers (BLBCs) are aggressive, and their drivers are unclear. We have found that wild-type N-RAS is overexpressed in BLBCs but not in other breast cancer subtypes. Repressing N-RAS inhibits transformation and tumor growth, whereas overexpression enhances these processes even in preinvasive BLBC cells. We identified N-Ras-responsive genes, most of which encode chemokines; e.g., IL8. Expression levels of these chemokines and N-RAS in tumors correlate with outcome. N-Ras, but not K-Ras, induces IL-8 by binding and activating the cytoplasmic pool of JAK2; IL-8 then acts on both the cancer cells and stromal fibroblasts. Thus, BLBC progression is promoted by increasing activities of wild-type N-Ras, which mediates autocrine/paracrine signaling that can influence both cancer and stroma cells.

A Humanized Monoclonal Antibody against Heat Shock Protein 60 Suppresses Murine Arthritis and Colitis and Skews the Cytokine Balance toward an Anti-Inflammatory Response.

We have previously shown that naturally occurring as well as acquired Abs against the Mycobacterium tuberculosis heat shock protein (HSP)65 protect against the induction of murine autoimmune inflammatory arthritis. In the present work, we have studied the anti-inflammatory effect of prozumab, a humanized anti-HSP mAb in murine inflammatory arthritis and colitis, and its effects on cytokine secretion. Prozumab was shown to bind to HSP60, the highly conserved mammalian homolog of the bacterial protein, and it was found to be effective in protecting and suppressing autoimmune arthritis in the models of adjuvant arthritis and collagen-induced arthritis in rats and mice, respectively, as well as in acute hapten-mediated colitis and chronic, spontaneous colitis models. Mechanistically, prozumab induces IL-10 secretion from naive human PBMCs and suppresses the secretion of IFN-γ and IL-6 from anti-CD3-activated human PBMCs. These findings make prozumab a promising potential drug for treating human rheumatoid arthritis and inflammatory bowel disease, as well as a wide range of autoimmune inflammatory diseases.

Analysis of the relationship between invasive capability of Helicobacter pylori and gastroduodenal diseases.

Helicobacter pylori (H. pylori) may enter into host cells, maybe as a facultative intracellular pathogen. This study aims to reveal the roles of internalized H. pylori in the bacterial pathopoiesis. Transmission electron microscopy was used to observe the invasion of H. pylori. Invasion rates of H. pylori (two standard strains and 43 clinical strains) were examined by gentamicin invasion assay. The cagA, cagE and vacA genes of H. pylori were detected by PCR. The cagA 3'region (cagA-EPIYA) of each strain was sequenced. The secretion of IL-8 from AGS cells and activity of NF-κB induced by intracellular H. pylori were tested by ELISA and the dual-luciferase reporter assay system, respectively. It was found that H. pylori could adhere to and invade AGS cells, then continue to survive and multiply in the cytoplasm. The average invasion rate of H. pylori gastric cancer plants and that of ulcer plants were both higher than that of gastritis plants (P ≈ 0.0001). In the clinical strains, cagA, vacA and cagE were all positive; cagA-EPIYA genotypes included ABD 90.7% (39/43) and ABBD 9.3% (4/43), all without comparability. Notably, the average invasion rate of H. pylori vacA s1c-i1-m1b plants was higher than that of vacA s1c-i1-m2 plants (P=0.0445). In addition, the intracellular H. pylori all could induce IL-8 secretion, which was decreased after cells were pretreated with anti-β1-integrin antibody or SN-50 (an NF-κB inhibitor). The intracellular H. pylori all activated NF-κB, which would be inhibited after cells were pretreated with anti-β1-integrin antibody. These results demonstrate that H. pylori invasive ability and disease severity have a positive correlation, and this intension of invasive ability is associated with the vacA mid-region, not with cagA, cagA-EPIYA or cagE. It is possible that cagA and cagE are essential for the bacterial invasion. Internalized H. pylori can activate NF-κB signal pathway and induce IL-8 secretion, which suggests that H. pylori invasion may be an important strategy to play a role in the development of H. pylori associated diseases.

The C-terminal disulfide bonds of Helicobacter pylori GroES are critical for IL-8 secretion via the TLR4-dependent pathway in gastric epithelial cells.

Helicobacter pylori GroES (HpGroES), a potent immunogen, is a secreted virulence factor that stimulates production of proinflammatory cytokines and may contribute to gastric carcinogenesis. HpGroES is larger than other bacterial orthologs because of an additional C-terminal region, known as domain B. We found that the HpGroES-induced IL-8 release by human gastric epithelial cells was dependent on activation of the MAPK and NF-κB pathways. HpGroES lacking domain B was unable to induce IL-8 release. Additionally, a TLR4 inhibitor significantly inhibited IL-8 secretion and reduced HpGroES-induced activation of MAPKs. Furthermore, HpGroES-induced IL-8 release by primary gastric epithelial cells from TLR4(-/-) mice was significantly lower than from wild-type mice. We also found that HpGroES bound to TLR4 in cell lysates and colocalized with TLR4 on the cell membrane only when domain B was present. We then constructed two deletion mutants lacking C-terminal regions and mutants with point mutations of two of the four cysteine residues, C111 and C112, in domain B and found that the deletion mutants and a double mutant lacking the C94-C111 and C95-C112 disulfide bonds were unable to interact with TLR4 or induce IL-8 release. We conclude that HpGroES, in which a unique conformational structure, domain B, is generated by these two disulfide bonds, induces IL-8 secretion via a TLR4-dependent mechanism.

Functional analysis of free fatty acid receptor GPR120 in human eosinophils: implications in metabolic homeostasis.

Recent evidence has shown that eosinophils play an important role in metabolic homeostasis through Th2 cytokine production. GPR120 (FFA4) is a G protein-coupled receptor (GPCR) for long-chain fatty acids that functions as a regulator of physiological energy metabolism. In the present study, we aimed to investigate whether human eosinophils express GPR120 and, if present, whether it possesses a functional capacity on eosinophils. Eosinophils isolated from peripheral venous blood expressed GPR120 at both the mRNA and protein levels. Stimulation with a synthetic GPR120 agonist, GW9508, induced rapid down-regulation of cell surface expression of GPR120, suggesting ligand-dependent receptor internalization. Although GPR120 activation did not induce eosinophil chemotactic response and degranulation, we found that GW9508 inhibited eosinophil spontaneous apoptosis and Fas receptor expression. The anti-apoptotic effect was attenuated by phosphoinositide 3-kinase (PI3K) inhibitors and was associated with inhibition of caspase-3 activity. Eosinophil response investigated using ELISpot assay indicated that stimulation with a GPR120 agonist induced IL-4 secretion. These findings demonstrate the novel functional properties of fatty acid sensor GPR120 on human eosinophils and indicate the previously unrecognized link between nutrient metabolism and the immune system.

A1.4 Inflammatory microenvironment of rheumatoid and osteoarthritic joint affects immunomodulatory activity of adipose-derived mesenchymal stem cells

Background and objectives Adipose-derived mesenchymal stem cells (ASCs) have immunomodulatory properties, but their activity is dependent on signals provided by local microenvironment. ASCs administration in rheumatoid arthritis (RA) did not bring consistent results, while in osteoarthritis (OA) it has beneficial effects. It is likely that proinflammatory milieu of rheumatoid joint affects ASCs activity. To test this hypothesis, the function of rheumatoid ASCs (RA-ASCs) and osteoarthritic ASCs (OA-ASCs) isolated from infrapatellar fat pad of the knee joint have been analysed. Materials and methods Infrapatellar fat pads were obtained from 29 RA and 12 OA patients undergoing total knee joint replacement surgery. Then, ASCs were isolated accordingly to routinely applied procedure. ASCs were treated or not with IFNγ or TNF. The expression of indoleamine 2,3-dioxygenase and heme oxygenase 1 mRNA was measured using quantitative PCR, while ASCs secretory potential was assessed by specific ELISAs. Rheumatoid synovial fibroblasts (RA-FLS) or peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured in ASC-conditioned media or co-cultured with ASCs. Next, cytokines secretion and cells proliferation were measured using ELISAs and bromodeoxyuridine/ 3 H-thymidine proliferation assays, respectively. Results RA- and OA-ASCs activity in vitro is comparable, nonetheless some differences in spontaneous secretory activity and heme oxygenase 1 mRNA expression have been observed. Unstimulated RA- and OA-ASCs slightly inhibited PBMCs proliferation and induced IL-10 secretion but increased IL-17A production and failed to suppress release of other proinflammatory factors (TNF, IFNγ, RANTES) by PBMCs. Under TNF stimulation, RA- and OA-ASCs up-regulated IL-6 and MMP-3 secretion in RA-FLS cultures and IL-17A release by PBMCs. ASCs treatment with IFNγ or TNF did not intensify their anti-inflammatory activity. Conclusions Our study demonstrates that immunosuppressive function of RA- and OA-ASCs derived from infrapatellar fat pad is impaired. Possibly, it is due to the localisation of ASCs in the site of inflammation. Moreover, TNF triggers proinflammatory properties of ASCs which indicates that when administered to the rheumatoid joint, ASCs may shift from an anti-inflammatory to proinflammatory phenotype. Acknowledgements This work was supported by the Polish National Science Centre (grant no. 2011/01/N/NZ5/00932) and by the European Union funds (European Social Fund, Human Capital Programme).

Wild-Type N-Ras, Overexpressed in Basal-like Breast Cancer, Promotes Tumor Formation by Inducing IL-8 Secretion via JAK2 Activation

Wild-Type N-Ras, Overexpressed in Basal-like Breast Cancer, Promotes Tumor Formation by Inducing IL-8 Secretion via JAK2 Activation

Interferon-tau attenuates uptake of nanoparticles and secretion of interleukin-1β in macrophages.

BackgroundType I interferons (IFNs), including IFN-alpha (IFNA) and IFN-beta (IFNB), have anti-inflammatory properties and are used to treat patients with autoimmune and inflammatory disorders. However, little is known of the role of IFN-tau (IFNT), a type I IFN produced by ruminant animals for inflammation. Because IFNB has recently been shown to inhibit nucleotide-binding oligomerization domain-like receptor, pyrin domain-containing 3 (NLRP3) inflammasome activation and subsequent secretion of the potent inflammatory cytokine interleukin (IL)-1β, we examined the effects of ruminant IFNT on NLRP3 inflammasome-mediated IL-1β secretion in human THP-1 macrophages.Methods and ResultsIFNT dose-dependently inhibited IL-1β secretion induced by nano-silica, a well-known activators of NLRP3 inflammasomes, in human macrophages primed with lipopolysaccharide (LPS, TLR4 agonist) and Pam3CSK4 (TLR1/2 agonist). IFNT also suppressed phagocytosis of nano-silica and reactive oxygen species (ROS) generation. Western blot analysis showed that IFNT inhibited both pro-IL-1β and mature IL-1β. In addition, real-time RT-PCR analysis showed that IFNT suppressed IL-1β mRNA expression induced by LPS and Pam3CSK4. Although nano-silica particles did not induce IL-10 secretion, IFNT induced IL-10 secretion in a dose-dependent manner. Furthermore, IFNT-suppressed IL-1β secretion was restored by anti-IL-10 neutralizing antibody.ConclusionsRuminant IFNT inhibits NLRP3 inflammasome-driven IL-1β secretion in human macrophages via multiple pathways, including the uptake of nano-silica particles, generation of ROS, and IL-10-mediated inhibition of pro-IL-1β induction. It may be a therapeutic alternative to IFNA and IFNB.

Antioxidative stress effect of phosphoserine dimers is mediated via activation of the Nrf2 signaling pathway.

Phosphoserine-containing peptides have been shown to exert antioxidative stress effects, by lowering lipid peroxidation, increasing intracellular glutathione, and increasing the expression of antioxidant enzymes in human intestinal epithelial cells. However, the role of phosphoserine residues in antioxidative stress activity, and their mechanism of action, remains unknown. The antioxidative stress activity of phosphoserine and phosphoserine peptides was examined using an in vitro model of hydrogen peroxide (H2 O2 )-induced oxidative stress in Caco-2 cells. Phosphoserine dimers (2PS) reduced IL-8 secretion in H2 O2 -treated Caco-2 cells, and reduced H2 O2 -induced expression of genes involved in inflammation and generation of reactive oxygen species (ROS), including chemokine (C-C motif) ligand 5 (CCL5), lactoperoxidase (LPO), myeloperoxidase (MPO), neutrophil cytosolic factor 1/2 (NCF1/2), and nitric oxide synthase 2A (NOS2), and upregulated metallothionein 3 (MT3), peroxiredoxin 3 (PRDX3), and surfactant, pulmonary-associated protein D (SFTPD), which are involved in protection against oxidative stress and activation of the Nrf2 signaling pathway. At the protein level, 2PS reduced H2 O2 -induced phosphorylation of the ERK1/2 and JNK MAPKs, and increased Nrf2 expression. Moreover, the ability of 2PS to reduce H2 O2 -induced IL-8 secretion, a marker of inflammation and oxidative stress, was abrogated in Nrf2 knockdown cells. These results suggest that 2PS reduce H2 O2 -induced oxidative stress via the Nrf2 signaling pathway, and reveal a potential mechanism for the antioxidative stress activity of phosphoserine-containing peptides.

Reaction of human macrophages on protein corona covered TiO2 nanoparticles

The cytokine secretion of primary cells of human macrophages during the interaction of TiO2 nanoparticles (with an average primary size of 100-120nm) is investigated down to concentration levels suggested to be relevant for in vivo conditions. We find that high TiO2 concentrations induce the cytokines Interleukin IL-1β, IL-6, and IL-10 secretion, while at low concentrations only IL-6 secretion is observed. To obtain further evidence on in vivo conditions we investigated the development and structure of the protein corona of the nanoparticles. We demonstrated that the surface of TiO2 particles attract preferably secondary modified proteins which then induce cytokine secretion of macrophages. Our results indicate that concentration of corona covered TiO2 particles below 1μg/ml induce IL-6 secretion which is reported to be responsible for the development of autoimmune diseases as well as for the secretion of acute phase proteins. From the Clinical EditorThis study investigates the effects of protein corona covered titanium dioxide nanoparticles on human macrophages, concluding that concentration of such particles below 1 μg/ml induces IL-6 secretion, which may be responsible for the development of autoimmune diseases as well as for the secretion of acute phase proteins. This finding has important implications on future applications of such nanoparticles.

Secretion of biologically active human interleukin 22 (IL-22) by Lactococcus lactis.

Interleukin-22 (IL-22) participates in the modulation of innate immunity and inflammation. This cytokine has important therapeutic potential, such as with ulcerative colitis, liver and lung injury, and infection, in different animal models. We generated a Lactococcus lactis strain that secretes human IL-22 under the regulation of the nisin-inducible promoter. Identification and secretion of this cytokine was demonstrated using western blots of culture supernatants from IL-22-expressing bacteria. The recombinant IL-22 protein produced by L. lactis was biologically active as determined by its ability to induce IL-10 secretion when co-cultured with a colon epithelial cell line in vitro. We consider this novel strain a promising live vaccine for various therapeutic applications.

Gastroprotection as an example: Antiadhesion against Helicobacter pylori, anti-inflammatory and antioxidant activities of aqueous extracts from the aerial parts of Lippia integrifolia Hieron

Ethnopharmacological relevanceThe aerial parts of Lippia integrifolia (Gris.) Hieronymus (Verbenaceae), known as incayuyo, are used by the peasant population of Argentina for treatment of diseases related to a gastrointestinal system, mainly for inflammation of the stomach and have also been included into the Argentina Food Code. This study aimed to investigate the phytochemical profile of hydrophilic extracts from the herbal material by LC−MS and to evaluate potential pharmacological mechanisms rationalizing the traditional use of incayuyo aqueous extracts potential anti-inflammatory treatment of gastrointestinal disorders. Materials and methodsPhytochemical profiling: LC−MS of an aqueous decoction. Antiadhesive effects against Helicobacter pylori: in vitro FACS assay using FITC-labeled bacteria and AGS human stomach cells. Influence of extracts on stomach cells and RAW 264.7 macrophages: MTT viability assay and BrdU proliferation ELISA. Influence of extracts on IL-6 and IL-8 secretion from stomach cells was quantitated by ELISA after infection of the cells with Helicobacter pylori. Influence of test extracts on macrophages: phagocytosis of FITC-labeled Zymosan particles and NO production. Antioxidative capacity: DPPH assay and O2-induced caroten oxidation. ResultsLC−MS profiling indicated the presence of compounds 1−20 with flavonoid hexosides, phenylethanoides (acteoside, isoacteoside) and sesquiterpenes [(epi)lippidulcine, peroxylippidulcine] in the decoction extract and subfraction PhF. The extract exhibits strong in vitro antioxidative capacity and inhibited adhesion of Helicobacter pylori to stomach cells up to 40%, while an EtOH-soluble fraction showed inhibition rates of up to 60%. Decoction increased the cellular viability of AGS cells significantly at >10μg/mL, while proliferation rate was not influenced. Helicobacter pylori induced IL-8 secretion was significantly reduced by coincubation of AGS cells with the extracts. Aqueous extracts stimulated phagocytosis rate of macrophages and inhibited the LPS-induced NO-secretion. ConclusionsThe traditional use of aqueous extracts from Lippia integrifolia for gastric inflammation seems to be rationalized: besides anti-inflammatory effects on stomach cells antiadhesive properties of the extracts against the main bacterial inductor of gastritis Helicobacter pylori were identified. Additional effects for stimulation of innate immunity and potential radical scavenging effects may additionally contribute to the activity of the extracts.

IL-8 secretion in primary cultures of prostate cells is associated with prostate cancer aggressiveness.

BackgroundChronic inflammation is believed to be a major factor in prostate cancer initiation and promotion and has been studied using prostate cancer cells and immortalized cell lines. However, little is known about the contribution of normal cells to the prostatic microenvironment and inflammation. We aim to study the contribution of normal prostate epithelial cells to prostate inflammation and to link the inflammatory status of normal cells to prostate cancer aggressiveness.Materials and methodsShort-term primary cell cultures of normal epithelial prostate cells were derived from prostate biopsies from 25 men undergoing radical prostatectomy, cystoprostatectomy, or organ donation. Cells were treated with polyinosinic:polycytidylic acid, a mimic of double-stranded viral RNA and a potent inducer of the inflammatory response. Secretion of interleukin (IL)-8 in the cell culture medium by untreated and treated cells was measured and we determined the association between IL-8 levels in these primary cell cultures and prostate cancer characteristics. The Fligner–Policello test was used to compare the groups.ResultsBaseline and induced IL-8 secretion were highly variable between cultured cells from different patients. This variation was not related to drug use, past medical history, age, or preoperative prostate-specific antigen value. Nonetheless, an elevated secretion of IL-8 from normal cultured epithelial cells was associated with prostate cancer aggressiveness (P=0.0005).ConclusionThe baseline secretion of IL-8 from normal prostate epithelial cells in culture is strongly correlated with cancer aggressiveness and may drive prostate cancer carcinogenesis. A better characterization of individual prostate microenvironment may provide a basis for personalized treatment and for monitoring the effects of strategies aimed at preventing aggressive prostate cancer.