Helicobacter felis'in makrofaj polarizasyonu üzerine etkisi

Helicobacter pylori (H. pylori) has been identified and classified as type I carcinogen for gastric malignancies such as chronic gastritis, peptic ulcer and gastric adenocarcinoma. Even though more than half of the world's population is infected with the H. pylori, only a minority develops gastric complications and/or remain asymptomatic. The bacteria are rarely eliminated, colonization usually persists throughout life, and infection involves both innate and adaptive immune responses. Helicobacter felis (H. felis) is a gram-negative, spiral- shaped bacterium which was first isolated from the stomach of a domestic cat. It is more immunogenic on mice and zoonotic species of H. pylori. Hence, it is widely used in murine Helicobacter studies because it causes similar pathogenic effect on mice as H. pylori on humans. However, H. felis lacks Vacuolating cytotoxin A (VacA) and Cytotoxin-associated gene A (CagA) virulence factors. Despite lacking important H. pylori virulence factors, it causes more severe gastric inflammation than H. pylori does on mice. Neutrophils, dendritic cells and macrophages mediate innate immune response against H. pylori. Macrophages are plastic and heterogenic group of cells, which can polarize to different types under different stimuli. Polarization status of macrophages changes according to stimuli and the local microenvironment, allowing them to shape the local inflammatory status to adapt to outside stimuli. However, different stimuli do not exist alone in tissues and macrophages may not form clear-cut activated subsets or expand clonally. The various macrophage functions are associated with the stimuli variety, receptor recognition on the macrophage upon stimuli and the presence of cytokines. There are two distinct states of polarized activation for macrophages: the classically activated -M1 type- macrophages and the alternatively activated -M2 type- macrophage subsets. Cell markers alone do not fully define the many subpopulations of macrophages. Therefore, macrophages should be defined based on their specific functional activities. M1 type macrophages are the pro-inflammatory effector cells in innate immune response. Granulocyte macrophage stimulating factor (GM-CSF), lipopolysaccharide (LPS) and IFN- polarize macrophages towards the M1 phenotype which induces the macrophage to produce large amounts of pro-inflammatory cytokines, such as TNF-, IL-1, 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. M1 type macrophages are professional antigen presenting cells and phagocytes, they express high levels of MHC I and class II antigens, CD40, CD80 (B7.1), CD86 (B7.2) co-stimulatory molecules and secrete complement factors to facilitate complement-mediated phagocytosis. Even though cell markers alone are not enough to fully define the subpopulations of macrophages, it has been shown that M1 type macrophages express CD11c surface marker along with the specific macrophage identification markers such as CD11b and F4/80. M2 type macrophages function in immunosuppression and tissue repair. Exposure to IL-4 or treatment with M-CSF produces an "anti-inflammatory" meaning, alternatively activated M2 macrophages. M2 type macrophages are generally characterized by production of high levels of IL-10 and IL-1RA and low expression of IL-12/ IL-23, combined with high levels of scavenger, mannose (CD206). They repurpose arginine metabolism to ornithine and polyamine by arginase, which promotes growth. Meaning that, they do not produce NO. However, M2 macrophages can be further divided into subsets. M2a, M2b, and M2c based on their cytokine expression profiles. The M2a subtype is induced by IL-4 or IL-13. The M2b is elicited by IL-1R ligands or exposure to immune complexes plus LPS. The M2c subtype is characterized by production of IL-10, TGF- and glucocorticoid hormones. The fourth type of macrophage M2d (or TAMs) is characterized by an IL-10high IL-12low M2 profile. M2d's have phenotypic and functional attributes distinct from M2a-c. Their functions are immunosuppression, low tumoricidal activity and promotion of tissue remodeling and angiogenesis. 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, in recent studies, it has been shown that, in human gastric biopsy specimens from H. pylori positive individuals, CD163+ (alternatively activated; M2) macrophages were detected. Also, upon H. pylori infection, human monocytes secreted IL-1, IL-6, IL-10, and IL-12p40 (partially secreted as IL-23), but not IL-12p70, meaning that, M2 macrophages were up-regulated and secreted IL-10 but produced less of the pro-inflammatory cytokines than M1 macrophages. 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. Pro-inflammatory Th1 and Th17 CD4+T cells mediate the pre-dominant adaptive response against H. pylori, the latter being induced by IL-23, IL-1, and IL-6. In spite of evidence for CD4+ T-cell-mediated protection against H. pylori infection in mouse models, Th1 and Th17 responses in humans are also present in chronic H. pylori infection as is IL-17. In mice, the induction of regulatory T (Treg) cells with the simultaneous suppression of Th17 cells may contribute to bacterial persistence. Also, recent findings revealed that murine splenic B cells produce and secrete IL-10 upon Helicobacter-infection in vitro as well as in vivo. IL-10 producing regulatory B cells restrain excessive Th1-type pro-inflammatory immune response and gastric immunopathology of C57BL/6 mice via suppression of CD4+ effector T cells. The interaction between regulatory B cells and T cells were also denoted as required for the function of regulatory B cells (Bregs). Bregs were shown to be able to convert CD4+ T cells into IL-10-producing T regulatory 1 (Tr-1) cell through direct interaction. Tr-1 cells and Bregs work in harmony in order to restore the immune balance in Helicobacter-infection by ameliorating excessive gastric immunopathology while preventing bacterial clearance in the gastric mucosa. Furthermore, in humoral response, H. pylori- specific serum IgM antibodies were present in H. pylori infected humans. Also, Serum IgA and IgG antibodies were directed toward many different H. pylori antigens. Above information shows that there is some information about the effects of H. pylori on macrophages but not definite characterization of H. pylori or H. felis on murine or human studies. Therefore, in this study, we investigated the effect of H. felis on polarization of two types of macrophages: bone marrow- derived macrophages and peritoneal macrophages to show the polarization status and their differences according to their surface receptor expressions and cytokine profiles. For that purpose, firstly bone marrow cells were isolated from leg bones of C57BL/6 mice and were differentiated to bone marrow- derived macrophages in the presence of M-CSF derived from L929 cell line. Peritoneal macrophages were collected from peritoneal cavity of thioglycollate induced C56BL/6 mice. After determining the percentage of differentiation and purity of isolation, cells were treated with LPS, H. felis sonicate, or left untreated as an internal control for 24 hours. Small portion of cell pellets were used for surface marker stainings of activation markers (CD40, CD80 and CD86) and of M1 and M2- type specific surface markers (CD11c and CD206, respectively). Also, their supernatants were collected for ELISA to measure IL-12/IL-23 (p40), TNF-, IL-1, IL-10, and NO secretion levels via griess reagent protocol. In addition to that, their pellets were collected for relative gene expression analysis of IL-12/IL-23 (p40), TNF-, IL-1, IL-10, IL-6 and iNOS using real-time PCR assay. The results of this study indicated that both LPS and H. felis sonicate- treated cells express high levels of activation markers of CD40 and CD80. CD86, which also is an activation marker, was the determining marker in identifying the activation status. CD86 marker expression suggesting the activation status, did not affect the polarization status of both bone marrow- derived and peritoneal macrophages. LPS treated bone marrow- derived macrophages were categorized as M1 type macrophages with the high CD11c and low CD206 expression, and H. felis sonicate- treated bone marrow -derived macrophages were categorized as M2 type macrophages with the high CD206 and low CD11c expression. However, high levels of CD11c surface marker expression was detected in LPS and H. felis sonicate- treated, thioglycollate induced peritoneal macrophages. Despite the high levels of CD11c expression, there was higher CD206 expression among H. felis sonicate- treated peritoneal macrophages when compared to LPS treated group. Therefore, as a result of surface marker expressions, there seems to be no correlation between the activation status and polarization status of peritoneal macrophages. After that, cytokine profiles of activated macrophages were also examined. Firstly, activation status of bone marrow- derived and peritoneal macrophages were assessed via CD86 marker expression. After that, cytokine profiles of activated macrophages were identified. As a result, LPS- treated BM- derived and peritoneal macrophages were polarized to M1 phenotype with high IL-12/IL-23 (p40), TNF-, IL-1, IL-6 cytokine expression and secretion, NO production and iNOS expression. Furthermore, H. felis sonicate- treated bone marrow -derived and peritoneal macrophages categorized as M2b phenotype with the high anti-inflammatory IL-10 production along with TNF-, IL-1, and IL-6 production. Also, IL-10 secretion observed from LPS- treated bone marrow- derived and peritoneal macrophages was thought to be the protective effect of IL-10 against LPS toxicity. However, production of pro-inflammatory IL-12/IL23 (p40) cytokine lead us to the conclusion that H. felis does not drive macrophages to polarize into only one phenotype of M2b, but there is probably M1 type macrophages mixed in the population. In conclusion, this study has contributed to the literature through providing definitive characterization of H. felis infected bone marrow- derived and peritoneal macrophages polarization, describing the surface marker and cytokine profiles for the first time.