Helicobacter pylori ile indüklenen gastrik patogenezde kemerinin rolü

Helicobacter pylori (H. pylori) is a gram negative, spiral –shaped, microaerophilic, flagellated bacterium which colonize in the human gastric epithelium and is classified as type I carcinogen for association with the development of gastric malignancies such as chronic gastritis, gastric adenocarcinoma, peptic ulcers and gastric lymphoma. Although 50% of the world's population is infected with Helicobacter pylori, the majority of infections do not lead to symptoms or gastrointestinal disease due to the pathogen's unique ability to avoid strong innate and adaptive immune responses. The bacteria are rarely eliminated, colonization usually persists throughout life, and infection involves both innate and adaptive immune responses. Chemerin is a protein from cathelicidine/cystatin family and the major ligand of G protein coupled receptor CMKLR1 (chemokine like receptor 1). It is present in various tissues including epithelial cells, liver, spleen, lymph nodes etc. and promotes chemotaxis of leukocyte populations that express CMKLR1, such as macrophages, plasmacytoid dendritic cells and natural killer (NK) cells to site of inflammation. It is ubiquitously found in plasma as 143 amino acids prochemerin at nanomolar concentrations and becomes activated into various isoforms through proteolytic cleavage by a myriad of serine and cysteine proteases as well as carboxypeptidases. Chemerin promotes both pro- and anti-inflammatory immune response based on the stimuli provided and the disease being investigated. Inflammatory diseases, such as erythematosus, rheumatoid arthritis, psoriasis, Crohn's disease showed association of elevated chemerin in inflammation development which is also observed in several mice and human studies. CMKLR1 knockout mice showed reduced CNS inflammation in EAE and pulmonary inflammation in COPD (cigarette smoke- induced chronic obstructive pulmonary disease) model, suggesting a pro-inflammatory role of chemerin. However, anti-inflammatory response was also observed when chemerin was discarded and in CMKLR1 knockout mice in LPS-induced lung injury and acute viral pneumonia with reduced expression of pro-inflammatory markers. Macrophages are plastic and heterogenic group of cells, which can polarize according to stimuli and the local microenvironment and shape the local inflammatory status to adapt to outside stimuli. Macrophages can be polarized into the classically activated -M1 type- macrophages and the alternatively activated -M2 type- macrophage subsets. M1 type macrophages are the pro-inflammatory effector cells in innate immune response produced by granulocyte macrophage stimulating factor (GM-CSF), lipopolysaccharide (LPS) and IFN-γ and secrete pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6, IL-12/ IL-23. The antimicrobial functions of M1 macrophages are linked to up-regulation of inducible nitric oxide synthase (iNOS) that generates nitric oxide from L-arginine and substantial production of NO. M2 type macrophages work in immunosuppression and tissue repair, produced by IL-4/IL-13 or M-CSF and secrete IL-10 and IL-1RA and low expression of IL-12/ IL-23. They do not produce NO. M2 macrophages can be further divided into subsets based on their cytokine expression profiles; M2a, M2b, and M2c. There are several studies regarding effects of H. pylori on macrophages. In early studies, it has been shown that H. pylori induces the expression of inducible NO synthetase (iNOS) from macrophages along with pro- inflammatory cytokines such as IL-6, IL-8, TNF-α, IL-1-β. However, alternatively activated M2 macrophages were detected in human gastric biopsy specimens from H. pylori positive individuals. Also, upon H. pylori infection, human monocytes secreted IL-1β, IL-6, IL-10, and IL-12p40 (partially secreted as IL-23), but not IL-12p70. Furthermore, cytokines secreted from innate immune cells, antigen presentation from macrophages and dendritic cells and changes in the microenvironment, also activates the adaptive immune response against H. pylori. Macrophage mediated pro-inflammatory Th1 and Th17 response was observed as the pre-dominant adaptive response against H. pylori in humans. Chemerin/chemerin receptor signaling mediates chemotaxis and adhesion of macrophages to extracellular matrix proteins and endothelial cells through increased promotion of integrins clustering (VLA-5 and VLA-4). ChemR23-dependent anti- inflammatory and protective effects of chemerin was found in mice, whereas in humans LPS, TNF-alpha and IFN-gamma stimulation increased ChemR23 expression in monocytes and macrophages which is associated with chronic and systemic inflammation. Macrophages may also exert pro- or anti-inflammatory response to different chemerin isoforms. Chemerin was observed to suppress M2 macrophage polarization with increased pro-inflammatory cytokine profile in DSS induced colitis model in vivo and in vitro. Another study found increased chemerin expression on M1 macrophages with distinct IL-10 expression, explaining the inflammatory effect of chemerin on M1 macrophages. Marked expression of chemerin was shown to be produced by intestinal epithelial cells (IECS) which is involved in the continuous recruitment of macrophage precursors to 10-24 week fetal intestine and boosted the host defence mechanism and mucosal immunity in neonates. However, only fetal IECs but not mature IECs, produces chemerin. Many studies work on the effects of H. pylori on macrophages and gave valuable insights, there are studies that investigated the inter-relation between chemerin and macrophages as well. However, no studies have yet investigated the effect of chemerin on the polarization status of macrophages in H. pylori induced pathogenesis. It is known from the literature that H. pylori could potentially induce both M1 and M2 macrophages in human but how/whether chemerin could effect this inter-relation is not known. Therefore, in this study, we investigated the effect of both chemerin and H. pylori on the monocytic cell line THP-1 and observe the differences on its polarization status according to cytokine profiles. It was known from the literature that epithelial cells secrete chemerin. In order to observe the effect of chemerin on gastric immunopathology, it's expression was first investigated on three gastric epithelial cell lines; KATO III, AGS and MKN45. Conventional PCR results revealed expression of chemerin in different levels by these cell lines. Next we evaluated the effect of H. pylori on chemerin expression by these cell lines. In order to do that, KATO III. AGS and MKN45 cells were treated with two H. pylori sonicates, the wild type G27 strain sonicate and its ΔcagA mutant sonicate. The H. pylori virulence factor Cag A (cytotoxin- associated gene-A) was chosen since its association with development of gastric malignancies have been reported. Chemerin expression was signifantly increased in KATO III and AGS cell line when they were treated with wild type G27 and ΔcagA mutant sonicate for 6 and 24 hours. After 24 hours, the expression level of chemerin reduced. No significant expression was observed on MKN45 cell line. Therefore, the following experiments were performed on KATO III and AGS cell lines. Next, we assess if chemerin protein expression was also increased along with the mRNA expression upon H. pylori treatment. Repeated attempts of western blot optimization experiments failed to show any expected band of chemerin in any treated or untreated groups of any cell line. To clarify if the lack of detection is real or due to inefficient antibody binding to chemerin, human chemerin was cloned and transfected into HEK293T cell line. Western blot was performed using various samples from different cell lines with different treatments and chemerin-expressing HEK293T cells. Chemerin was only detected in the whole cell extract of chemerin-transfected HEK293T cell lines but not in other samples. As a conclusion, we could detect elavated chemerin in mRNA levels upon H. pylori treatment but not in protein level. Next, we investigated expression of chemerin in gastric biopsy specimens of H. pylori positive patients of acute gastritis and chronic ulcer and compared it to uninfected patients without pathology. Real-time PCR experiments revealed presence of significantly higher levels of chemerin in ulcer patients. Of note, chemerin mRNA expression in samples from patients with gastritis was not significantly different when compared to control group. These data was supported by the results obtained from immunohistochemistry analysis performed on gastric tissues of H. pylori postive patients of acute gastritis, chronic ulcer and H. pylori negative healthy individiuals. Chemerin was shown to be highly expressed in gastric tissues of ulcer patients. We investigated effects of chemerin on the polarization status of macrophages. For that, a monocytic cell line THP-1 was used and PMA (phorbol 12-myristate 13-acetate) was administered to induce differentiation of monocytes into macrophages. The differentiated macrophages were then exposed to different stimuli such as LPS and recombinant IL-4 to polarize them into M1 and M2 subtypes respectively, with or without the presence of chemerin. The presence of macrophage specific markers were used to show the differentiation of monocytes into macrophages. Also, cytokine levels were measured by conventional PCR. Two pro-inflammatory cytokines namely IL-1β and IL-6 were used as a marker for M1 macrophages. Both of them were significantly increased in LPS- treated samples. When chemerin was administered with LPS, a significantly high expression of IL-1β and low expression of IL-6 observed when compared to LPS- treated sample only in the first set. Initially it was thought chemerin might have a role in the polarization of M1 macrophages but three repeated experiments showed no difference in the expression of IL-1β and IL6 in samples treated with LPS alone or with chemerin. This disproves the possible effect of chemerin on M1 polarization. IL-10 expression was assessed to detect M2 polarization. IL-4 treatment resulted in elevated IL-10 expression compared to control group and treatment of chemerin along with IL-4 diminished this expression. This is parallel with the study performed by Lin et al. (2014) where chemerin reduced the expression of M2 associated genes. Finally, we investigated the effect of H. pylori on the polarization of macrophages with or without the presence of chemerin to check whether chemerin may alter the effect exerted by H. pylori on macrophages. The THP-1 macrophages were treated with wild type G27 and ΔcagA mutant sonicate with/without chemerin for 24 hours. The sonicate treatment polarized macrophages to M1 subtype with high profiles of pro-inflammatory IL-1β and IL6 and also increased IL-10. M1 macrophages are known to produce IL-10 in response to bacterial stimulation, this was also observed in a study on mice model stimulated by H. felis in our laboratory (unpublished data). However, no change of expression in IL-1β, IL6 and IL-10 was observed when chemerin was added with H. pylori sonicates. Therefore our results suggested chemerin may not have a role on effect of H. pylori induced M1 macrophage polarization, however M2 polarization was seen to be inhibited upon chemerin treatment which is H. pylori independent.