Participation of the Cytoskeletal and Lysosomal Compartments in Campylobacter jejuni Invasion of Caco-2 cells, the Cellular Response by Morphometric Analysis and the Presence of Cytokine and Chemokine Transcripts

This study aimed to evaluate the participation of actin and tubulin in the process of internalisation, the interaction of bacterial phagosomes with lysosomes, the morphometric changes and the expression of inflammatory cytokines in Caco-2 cells infected with Campylobacter jejuni. Both actin and tubulin participated in the process of internalisation. Inside the cells, lysosomes fuse with phagosomes, which may lead to bacterial death because after 2 h, the bacteria were not detected by Transmission electron microscopy (TEM). There is increased expression of TGF-β3 during the early stages, and IL-8 was expressed after 60 min p.i. This work showed that C. jejuni invades and causes major morphometric changes in epithelial cells. In response, the cells increase their expression of cytokines that can lead to inflammation. The mechanisms of invasion are dependent on actin and tubulin, and once internalised, lysosomes fuse with phagosomes.     

Cytokine and cytokine receptor gene polymorphisms and their functionality

Cytokines, signaling proteins produced by a variety of cell types, are essential for the development and functioning of both innate and adaptive immune response. Cytokine gene expression is tightly regulated, and aberrant expression from environmental and genetic polymorphism has been implicated in a range of diseases, susceptibility to infections, and responses to treatment. This review concentrates on the functionality of cytokine and cytokine receptor gene polymorphisms; it is through these variants that genuine disease-associations are based. Several mechanisms for single nucleotide polymorphism (SNP) functionality are present within cytokine genes including: amino acid changes (IL-6R, IL-13, IL-1α), exon skipping (IL-7Rα), proximal promoter variants (IL-1β, IL-Ra, IL-2, IL-6, IL-10, IL-12, IL-13, IL-16, TNF, IFN-γ, TGF-β), distal promoter variants (IL-6, IL-18) and intronic enhancer variants (IL-8).     

Contrasting effect of transforming growth factor type beta 1 (TGF-β1) on proliferation and interleukin-2 receptor expression in activated and rapidly cycling immature (CD3−CD4−CD8−) thymocytes

Transforming growth factor β (TGF-β) is a cytokine with immunoregulatory properties that acts negatively on T lymphocyte proliferation. However, with the EL 4–6.1 variant of the murine thymoma EL 4 activated with phorbol ester and/or interleukin-1 (IL-1), we recently found that it up-regulates interleukin-2-receptor (IL-2R) expression. Since EL 4–6.1 cells share phenotypic and functional characteristics with the immature thymic subset lacking CD4 and CD8 accessory molecules (DN), we investigated the effect of TGF-β1 on the IL-2R 55kD α chain expression and proliferation of activated DN cells and especially in DN cells that do not express CD3. We observed that TGF-β1 was able to increase both the percentage of CD3^?DN cells expressing IL-2Rα chains and the expression of IL-2Rα chain in these cells. This stimulatory effect of TGF-β1 was distal from early transduction events. In addition, TGF-β1 was found to modulate CD3^?DN cell proliferation. During differentiation in the thymus, CD3^?DN cells transiently express the IL-2Rα chain of the IL-2R and these IL-2R⁺ CD3^?DN cells are preprogrammed to down-regulate the IL-2Rα chain and up-regulate the CD4 and CD8 accessory molecule. We thus also tested the effect of TGF-β1 on IL-2Rα chain expression in these in vitro differentiating CD3^?DN cells. We found that TGF-β1 neither significantly affected IL-2R expression nor changed CD4 or CD8 expression. Hence, in CD3^?DN cells, the effect of TGF-β1 on IL-2R expression seems to be restricted to proliferating cells. © 1993 Wiley-Liss, Inc.     

Altered expression of inflammatory cytokine receptors in response to LPS challenge through interaction between intestinal epithelial cells and lymphocytes of Peyer's patch

The intense innate immunological activities occurring at the enteric mucosal surface involve interactions between intestinal epithelial cells and immune cells. Our previous studies have indicated that Peyer's patch lymphocytes may modulate intestinal epithelial barrier and ion transport function in homeostasis and host defense via cell-cell contact as well as cytokine signaling. The present study was undertaken using the established co-culture system of Caco-2 epithelial cells with lymphocytes of Peyer's patch to investigate the expression of IL-8 and IL-6 cytokines and cytokine receptors in the co-culture system after challenge with Shigella F2a-12 lipopolysaccharide (LPS). The human colonic epithelial cell line Caco-2 was co-cultured with freshly isolated lymphocytes from the murine Peyer's patch either in the mixed or separated (isolated but permeable compartments) co-culture configuration, and was challenged with Shigella F2a-12 LPS for 8 h. The level of mRNA expressions of human interleukin-8 (hIL-8), human interleukin-8 receptor (hIL-8R), mouse interleukin-8 receptor (mIL-8R), mouse interleukin-6 (mIL-6), mouse interleukin-6 receptor (mIL-6R) and human interleukin-6 receptor (hIL-6R) was examined by semi-quantitative PCR. In both co-culture groups, hIL-8 expression of Caco-2 cells was decreased, and hIL-8R expression was increased compared to the Caco-2 alone group. Upon LPS challenge, hIL-8 expression from the Caco-2 cells of both co-culture groups was higher than in the Caco-2 control group. The increased hIL-8 expression of Caco-2 cells in the separated co-culture group is correlated with a decreased hIL-8R expression and an increased mIL-8R expression. In the mixed co-culture group, the increased expression of hIL-8 was associated with the upregulated hIL-8R expression on Caco-2 cells and downregulated mIL-8R on murine Peyer's patch lymphocytes (PPL). mIL-6 expression from mouse PPL was also upregulated by LPS in mixed co-culture. However, upon the treatment with LPS, hIL-6R expression of Caco-2 cells was decreased in the mixed co-culture, but increased in separated co-culture. The data suggest that release of hIL-8 from epithelial cells may act on lymphocytes through a paracrine pathway, but it may also act on the epithelial cells themselves via an autocrine pathway. The data also suggest that the release of mIL-6 from Peyer's patch lymphocytes affects epithelial cells in a paracrine fashion.     

Lactoferrin downregulates pro-inflammatory cytokines upexpressed in intestinal epithelial cells infected with invasive or noninvasive Escherichia coli strains.

Intestinal epithelial cells are able to differentially interact with commensal or pathogenic microorganisms, triggering a physiological or destructive inflammation, respectively. To mimic commensal-enteroinvasive bacteria-host cell interaction, we infected Caco-2 cells with noninvasive Escherichia coli HB101 and with recombinant invasive E. coli HB101(pRI203). Using DNA microarray mRNA profiling and ELISA assays, we studied the expression of several cytokine and cytokine-related genes in infected Caco-2 cells in the absence or presence of bovine lactoferrin (bLf). Infection of Caco-2 cells with the noninvasive strain induced a slight increase in the expression of interleukin 8 (IL-8), whereas infection with invasive E. coli HB101(pRI203) induced a significant increase in the expression of IL-8 as well as other pro-inflammatory cytokines. The addition of bLf, in native- or holo-form, did not influence expression of cytokine genes by uninfected Caco-2 cells, but it decreased expression of IL-8 by cells infected with E.coli HB101. Moreover, except for IL-8, bLfs dramatically downregulated pro-inflammatory cytokines upexpressed by Caco-2 cells infected with the invasive strain. Although IL-8 was decreased by bLfs, it remained upregulated, suggesting that it could be a signal of persistence of intracellular bacteria. The bLf ability to reduce expression of some pro-inflammatory cytokines, which appears independent of its iron saturation, might represent an important natural mechanism in regulating epithelial cell responses to pathogenic bacteria and in limiting cell damage and the spread of infections.     

Interleukin-26 in host defense and inflammatory disorders of the airways

The dimeric cytokine interleukin (IL)-26 belongs to the IL-10 family. Whereas it was originally perceived as a T-helper (Th)17 cytokine, subsequent studies have shown that IL-26 is produced by several populations of leukocytes and structural cells. This cytokine binds to a heterodimeric receptor complex including IL-10R2 and -20R1 (IL-26R) and signals through STAT 1 and 3 to induce the release of chemokines and growth factors. Remarkably, IL-26 directly kills bacteria and inhibits viral replication. The most recent studies on human airways confirm multiple cellular sources in this critical interphase of host defense and demonstrate that stimulation of toll-like receptors (TLR) trigger the release of IL-26. Once released, it exerts a dualistic effect on cytokine production and up-regulates gene expression of IL-26R. It also potentiates chemotaxis and inhibits chemokinesis for neutrophils, thereby facilitating the accumulation of innate effector cells at the site of bacterial stimulation. The high levels of IL-26 in human airways are altered in inflammatory airway disorders such as asthma and chronic obstructive pulmonary disease. Thus, IL-26 emerges as an important mediator, providing direct and indirect actions on microbes, actions that are essential for host defense and inflammation and bears potential as a biomarker of disease.     

Influence of seminal plasma, spermatozoa and semen extender on cytokine expression in the porcine endometrium after insemination

The effects of semen components or extender alone on the expression of selected cytokines [interleukine (IL)-1β, IL-6, granulocyte-macrophage colony stimulating factor (GM-CSF), IL-10 and transforming growth factor (TGF)-β1] on the porcine endometrium were studied, as well as the presence of polymorphonuclear neutrophilic granulocytes (PMNs). In experiment (Exp) I, groups of gilts were sampled at 5-6h after insemination with fresh semen in extender (Beltsville thawing solution, BTS), spermatozoa in extender (Spz), seminal plasma (SP), or only BTS (control). In Exp II, gilts were sampled 35-40h after insemination with Spz, SP, BTS or only catheter inserted (as control). Immunohistochemical (IHC) labelling of IL-6, IL-10 and TGF-β1 was evident, especially in surface and glandular epithelia of the porcine endometrium. There were no consistent differences in IHC-labelling of the cytokines in relation to different treatments. However, the scores for IL-6 and IL-10 in surface epithelium and sub-epithelial connective tissue compartments were higher at 35-40h than shortly (5-6h) after treatment. Cytoplasmic labelling in the sub-epithelial connective tissue was observed in scattered individual cells but not in PMNs. Shortly (5-6h) after insemination, there were no differences between animals inseminated with BTS (control) and the semen components for any of the cytokine mRNAs. Later however, at 35-40h, lower endometrial expression of TGF-β1 mRNA was observed in the Spz and BTS groups compared with the control (catheter only). The same pattern was found for IL-10 (NS). The mRNA expression of IL-6 in the BTS inseminated group was higher compared to the control group. Insemination with SP resulted in significantly lower PMN cell infiltration in the sub-epithelial connective tissue compared with Spz or BTS groups shortly (5-6h) after insemination. Later (35-40h), a significant difference was found between SP (lower) and the control group (only catheter). To conclude, our results show that insemination and/or inseminated components modulated cytokine expression in the gilt endometrium. The semen extender BTS stimulated immune reactivity, as shown by down-regulation of the suppressive cytokine TGF-β1. Insemination with solely SP clearly decreased PMN cell infiltration of the gilt endometrium. However, no clear relation between the cytokines studied and PMN cell presence was found.     

Oncostatin M (OSM) primes IL-13- and IL-4-induced eotaxin responses in fibroblasts: Regulation of the type-II IL-4 receptor chains IL-4Rα and IL-13Rα1

Oncostatin M (OSM), a pleiotropic cytokine and a member of the gp130/IL-6 cytokine family, has been implicated in regulation of various chronic inflammatory processes. Previous work has shown that OSM induces eosinophil accumulation in mouse lungs in vivo and stimulates the eosinophil-selective chemokine eotaxin-1 synergistically with IL-4 in vitro. To examine the role of receptor regulation by OSM in synergistic eotaxin-1 responses, we here examine the modulation of the type-II IL-4 receptor (IL-4Rα and IL-13Rα1) by OSM and other gp130/IL-6 cytokine family members using NIH3T3 fibroblasts and primary mouse lung fibroblasts. We first show that OSM with either IL-13 or IL-4 synergistically induces eotaxin-1 expression in a dose-dependent fashion. Analysis of IL-4Rα expression at the protein (Western blot and FACS) and RNA (TAQMAN) levels showed that OSM markedly elevates expression by 3 h. OSM enhanced IL-13Rα1 mRNA and induced a smaller but detectable increase in total IL-13Rα1 protein. Priming fibroblasts with OSM for 6 h markedly enhanced subsequent IL-13 and IL-4-induced eotaxin-1 responses and STAT6 tyrosine-641 phosphorylation. Regulation of IL-4Rα by OSM was sensitive to inhibition of the PI3′K pathway by LY294002. These studies provide novel mechanistic insights in OSM role in regulation of synergistic eotaxin-1 responses and IL-4Rα expression in fibroblasts.     

Modulation of the catabolic effects of interleukin-1β on human articular chondrocytes by transforming growth factor-β

The effects of IL-1β and TGF-β on the biosynthesis of extracellular matrix structural components relative to the metalloproteinases and their inhibitor TIMP1 in human articular chondrocytes were investigated. It has been proposed that TGF-β, acting as a positive regulator of matrix accretion, can counteract the increased loss of cartilage matrix induced by IL-1β. To allow a comparison of their effects on mRNA levels for these different components, quantitation by competitive RT/PCR was employed. This method was found to give reproducible estimates of mRNA levels and the observed effects of IL-1β and TGF-β on individual components of this system agree with qualitative data obtained by northern blotting. IL-1β had a more pronounced effect on aggrecan mRNA levels than on those for type II collagen. Similar quantitative differences were observed between collagenase and stromelysin mRNA levels. TGF-β generally counteracted the effects of IL-1β, and new steady state levels were attained within 24 h. However, the reversal of IL-1β induced suppression of matrix protein mRNA levels appeared more effective than its suppression of the increase in stromelysin and collagenase mRNA levels. Similarly TGF-β did not reduce the extent of IL-1β induced secretion of stromelysin at the protein level. TIMP1 mRNA levels were only slightly reduced by IL-1β; however this cytokine effectively surpressed its induction by TGF-β. The higher concentrations of TGF-β and longer exposure times required to overcome the surpressive effects of IL-1β suggest that the interaction between IL-1β and TGF-β in the regulation of TIMP1 expression follows a different mechanism to that operating for the metalloproteinases and matrix proteins. Thus the overall potential of TGF-β to inhibit proteolysis is attenuated by its much slower effect on TIMP1 mRNA levels. © 1996 Wiley-Liss, Inc.     

Impaired IL-17 signaling pathway contributes to the increased collagen expression in scleroderma fibroblasts

Among IL-17 families, IL-17A and IL-17F share amino acid sequence similarity and bind to IL-17R type A. IL-17 signaling is implicated in the pathogenesis of various autoimmune diseases, but its role in the regulatory mechanism of extracellular matrix expression and its contribution to the phenotype of systemic sclerosis (SSc) both remain to be elucidated. This study revealed that IL-17A expression was significantly increased in the involved skin and sera of SSc patients, whereas the IL-17F levels did not increase. In contrast, the expression of IL-17R type A in SSc fibroblasts significantly decreased in comparison with that in normal fibroblasts, due to the intrinsic TGF-β1 activation in these cell types. Moreover, IL-17A, not IL-17F, reduced the protein expression of α1(I) collagen and connective tissue growth factor. miR-129-5p, one of the downregulated microRNAs in SSc fibroblasts, increased due to IL-17A and mediated the α1(I) collagen reduction. These results suggest that IL-17A signaling, not IL-17F, has an antifibrogenic effect via the upregulation of miR-129-5p and the downregulation of connective tissue growth factor and α1(I) collagen. IL-17A signaling is suppressed due to the downregulation of the receptor by the intrinsic activation of TGF-β1 in SSc fibroblasts, which may amplify the increased collagen accumulation and fibrosis characteristic of SSc. Increased IL-17A levels in the sera and involved skin of SSc may be due to negative feedback. Clarifying the novel regulatory mechanisms of fibrosis by the cytokine network consisting of TGF-β and IL-17A may lead to a new therapeutic approach for this disease.     

Brucella invasion of human intestinal epithelial cells elicits a weak proinflammatory response but a significant CCL20 secretion

In spite of the frequent acquisition of Brucella infection by the oral route in humans, the interaction of the bacterium with cells of the intestinal mucosa has been poorly studied. Here, we show that different Brucella species can invade human colonic epithelial cell lines (Caco-2 and HT-29), in which only smooth species can replicate efficiently. Infection with smooth strains did not produce a significant cytotoxicity, while the rough strain RB51 was more cytotoxic. Infection of Caco-2 cells or HT-29 cells with either smooth or rough strains of Brucella did not result in an increased secretion of TNF-α, IL-1β, MCP-1, IL-10 or TGF-β as compared with uninfected controls, whereas all the infections induced the secretion of IL-8 and CCL20 by both cell types. The MCP-1 response to flagellin from Salmonella typhimurium was similar in Brucella-infected or uninfected cells, ruling out a bacterial inhibitory mechanism as a reason for the weak proinflammatory response. Infection did not modify ICAM-1 expression levels in Caco-2 cells, but increased them in HT-29 cells. These results suggest that Brucella induces only a weak proinflammatory response in gut epithelial cells, but produces a significant CCL20 secretion. The latter may be important for bacterial dissemination given the known ability of Brucella to survive in dendritic cells.     

TGF-β mediates proinflammatory seminal fluid signaling in human cervical epithelial cells

The cervix is central to the female genital tract immune response to pathogens and foreign male Ags introduced at coitus. Seminal fluid profoundly influences cervical immune function, inducing proinflammatory cytokine synthesis and leukocyte recruitment. In this study, human Ect1 cervical epithelial cells and primary cervical cells were used to investigate agents in human seminal plasma that induce a proinflammatory response. TGF-β1, TGF-β2, and TGF-β3 are abundant in seminal plasma, and Affymetrix microarray revealed that TGF-β3 elicits changes in Ect1 cell expression of several proinflammatory cytokine and chemokine genes, replicating principal aspects of the Ect1 response to seminal plasma. The differentially expressed genes included several induced in the physiological response of the cervix to seminal fluid in vivo. Notably, all three TGF-β isoforms showed comparable ability to induce Ect1 cell expression of mRNA and protein for GM-CSF and IL-6, and TGF-β induced a similar IL-6 and GM-CSF response in primary cervical epithelial cells. TGF-β neutralizing Abs, receptor antagonists, and signaling inhibitors ablated seminal plasma induction of GM-CSF and IL-6, but did not alter IL-8, CCL2 (MCP-1), CCL20 (MIP-3α), or IL-1α production. Several other cytokines present in seminal plasma did not elicit Ect1 cell responses. These data identify all three TGF-β isoforms as key agents in seminal plasma that signal induction of proinflammatory cytokine synthesis in cervical cells. Our findings suggest that TGF-β in the male partner's seminal fluid may influence cervical immune function after coitus in women, and potentially be a determinant of fertility, as well as defense from infection.     

TGF-β1 suppresses IL-6-induced STAT3 activation through regulation of Jak2 expression in prostate epithelial cells

Chronic inflammation plays an important role in the initiation and progression of various human diseases including benign prostatic hyperplasia or prostate cancer. Here we show that the proinflammatory cytokine interleukin-6 (IL-6) has prosurvival effects and chronically activates the Jak2/STAT3 signalling pathway in a model of benign prostatic hyperplasia (BPH-1). We demonstrate that the antiinflammatory cytokine transforming growth factor-β1 (TGF-β1), which also permanently activates its canonical signalling pathway through SMAD proteins in BPH-1 cells, modifies the effects of IL-6 on cell proliferation. Importantly, TGF-β1 inhibits IL-6 signal transduction by decreasing the phosphorylation levels of STAT3. This effect is associated with decreased expression of Jak2 at both mRNA and protein levels. Moreover, we showed that TGF-β1 inhibits IL-6-induced expression of the cancer-associated gene MUC1. These observations demonstrated a novel interaction between TGF-β1 and IL-6 signalling and suggested another mechanism of how defects in TGF-β signalling, frequently associated with prostate pathologies, can contribute to the disruption of tissue homeostasis.     

IL-10 is required for polarization of macrophages to M2-like phenotype by mycobacterial DnaK (heat shock protein 70)

Macrophages are key cells in the innate immune system. They phagocytose pathogens and cellular debris, promote inflammation, and have important roles in tumor immunity. Depending on the microenvironment, macrophages can polarize to M1 (inflammatory) or M2 (anti-inflammatory) phenotypes. Extracellular DnaK (the bacterial ortholog of the mammalian Hsp70) from Mycobacterium tuberculosis (Mtb) was described to exert immune modulatory roles in an IL-10 dependent manner. We have previously observed that endotoxin-free DnaK can polarize macrophages to an M2-like phenotype. However, the mechanisms that underlie this polarization need to be further investigated. IL-10 has been described to promote macrophage polarization, so we investigated the involvement of this cytokine in macrophages stimulated with extracellular DnaK. IL-10 was required to induce the expression of M2 markers - Ym1 and Fizz, when macrophages were treated with DnaK. Blockade of IL-10R also impaired DnaK induced polarization, demonstrating the requirement of the IL-10/IL-10R signaling pathway in this polarization. DnaK was able to induce TGF-β mRNA in treated macrophages in an IL-10 dependent manner. However, protein TGF-β could not be detected in culture supernatants. Finally, using an in vivo allogeneic melanoma model, we observed that DnaK-treated macrophages can promote tumor growth in an IL-10-dependent manner. Our results indicate that the IL-10/IL-10R axis is required for DnaK-induced M2-like polarization in murine macrophages.     

Adhesion of lactic acid bacteria to caco-2 cells and their effect on cytokine secretion

Cytokines secreted by human enterocytes play a critical role in mucosal and systemic immunity. Intestinal microorganisms can influence this secretion. In the present study, 30 strains of lactic acid bacteria were characterized for their adhesion to Caco-2 cells and their potential to stimulate proinflammatory cytokine secretion by this cell line. The bacteria adhered in a strain-dependent manner to Caco-2 cells. Contact with lactobacilli did not result in the production of IL-6 or IL-8. A slight IL-6 and IL-8 production by a Caco-2 cell was detected after exposure to 8 of the tested Bifidobacterium strains. No correlation was found between adhesion and cytokine induction among the bacteria tested. This indicates that lactic acid bacteria, even those with strong adhesive properties, are not very likely to trigger an inflammatory response in human enterocytes.