Theses supervised by Doç. Dr. Ayça Sayı Yazgan
10 theses · İstanbul Technical University
Helicobacter-aktive regülatör B hücrelerinde gen ifadesinin belirlenmesi
The immune system provides the primary defense against the invasion of foreign organisms including; bacteria, viruses, fungi or any antigen recognized as a foreign antigen by immune cells. Every moment inside our bodies, the immune system encounters many antigens around it. For that reason, the immune system should discriminate the non-self pathogens from self-antigens and respond appropriately. Broadly, the immune system is divided into innate and adaptive arms. The innate immune cells are the early line of defense in front of any pathogen in a nonspecific manner and they proceed to an inflammation. In addition, the most important innate immune cells are neutrophils and macrophages, which are responding quickly to local infection. On the other hand, the adaptive immune cells are T- and B- lymphocytes, both have specific cell surface markers, and mainly take time to recognize the specific foreign antigen. The innate and adaptive immune system often work in harmony to remove pathogens or foreign invaders. Helicobacter pylori (H. pylori) is a gram-negative bacterium that selectively colonizes the gastric epithelium and is the most common bacterial infection worldwide. More than 50% of the world's population are H. pylori infected. Approximately, 60% of adults and 30% of children. Clear evidence indicates that once the Helicobacter infection is acquired, it usually persists for lifelong unless it is eradicated by antimicrobial therapy. H. pylori is located in gastric mucosal cells. All of the H. pylori-infected patients mainly develop asymptomatic chronic gastric inflammation. The presence of genes such as Cag pathogenicity island (cag-PAI) and vacuolating cytotoxin A (vacA) that increases the risk for gastric atrophy, gastric adenocarcinoma development, recruitment and activation of immune cells in the underlying mucosal host tissue. The present study reported that the relative risk of gastric cancer is 1.7-5.3 times higher in H. pylori - infected individuals compared to uninfected individuals. The mechanism of interaction between H. pylori and host lymphocytes as well as antigen presenting cells is complicated. Therefore, the regulatory B and T cells have a pivotal role in the balance between immune pathogenicity and protection. Recently, it has been shown that the regulatory T cells could reduce H. pylori-induced gastritis in mice. At the same time, the regulatory T cells allow the bacterium to colonize the mucosa at higher densities. In addition, H. pylori-specific CD4+ CD25+ regulatory T cells suppress memory T-cell responses against H. pylori in infected humans. In addition to that, the role of regulatory B cells in Helicobacter - driven immune response was investigated. It was reported that IL-10- producing B cells were activated upon Helicobacter infection. The IL-10- producing B cells were activated through Toll-Like Receptor 2 (TLR-2)-myeloid differentiation primary-response protein 88 (MyD88) signaling. In fact, the IL-10- producing B cells lead to differentiation of T regulatory-1 (Tr-1) cells from naive T cells. The interaction xxvi between Tr-1 and IL-10-producing B cells may prevent the gastric precancerous lesions and serve as a good immune modulator against Helicobacter. While phenotype of H. felis stimulated (Hfstim) B cells were investigated two H. felis induced Breg subsets were determined; Hfstim-IL-10+ B and Hfstim-IL-10– B cells. Both subsets were identified with various surface markers and cytokine profiles. Hfstim-IL-10+ B cells (CD1d+CD5+ CD21+CD23+) and produce IL-10, and Hfstim-IL-10– B cells (CD21+CD23lo) and produce IgM, IgG2b and TGF-β. Furthermore, we assessed the level of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) in Hfstim-IL-10+ B and in Hfstim-IL-10– B cells. Both the m RNA and protein levels of TNF-α and IL-6 cytokines are expressed significantly higher in Hfstim-IL-10– B cells compared to Hfstim-IL-10+ B. Based on ours and others findings, we aimed to identify the functional role of Hfstim-Bregs upon interaction with naive CD4+ T cells. Previous studies showed the transcriptome profile of B10 cells by using RNA-Seq and microarray analysis. In our study, we aimed to investigate the expression profile of Helicobacter - stimulated regulatory B cells (Hfstim-Bregs) by microarray analysis. Our samples include four groups; unstimulated B cells, Hfstim-total B cells, Hfstim-IL-10+ B and Hfstim-IL-10– B cells. Our microarray data indicated that 1615 genes are up-regulated and 794 genes are down–regulated in Hf stim- IL-10+ B cells compared to Hf stim- IL-10– B cells. CD9 and PDCD1 were shown to be highly expressed in IL-10+ B cells by a recent publication. Based on our microarray and real -time PCR data, we found differential expression of CD9, PDCD1, and NRP2 together with two other candidate genes (S100A8 and Trib1) in Hfstim-IL-10+ B cells in comparing to Hfstim-IL-10– B cells. Afterward, the surface expression of PD-1 and CD9 was investigated by flow cytometry analysis in Hfstim-IL-10+ B and Hfstim-IL-10– B cells. The PD-1 and CD9 protein levels are significantly high in Hfstim-IL-10+ B cells compared to Hfstim-IL-10– B cells. Of note Of noteOf note , both q-RT-PCR results and flow analysis confirmed the expression of PD-1 in Hfstim-IL-10+ B cells. Accordingly, we pointed out to identify the functional implication of a PDCD1 in Helicobacter stimulated Bregs. The flow cytometric analysis is applied to assess CD9, PD-1, CD19, CD4, IL-10, PD-L1, and PD-L2 expression on B cell groups. In line with the previous reports, we found high expression of PD-1 in mRNA and protein level in Hf stim-IL-10+ B cells. While, there was a distinct surface expression of PD-L1 in Hf stim-IL-10– B cells compared to Hf stim-IL-10+ B cells. Besides the m RNA level of PD-L1 was high in Hf stim-IL-10+ B cells compared to Hf stim-IL-10– B cells. In this regard, we blocked PD-1 or PD-L1 in Hfstim-IL-10+ B or in Hfstim-IL-10– B cells prior to co-culturing B cells with naive CD4+ T cells. The anti PD-1 or anti PD-L1was used to block the PD-1-PD-L1 axis pathway. Remarkably, blockade of PD-1 or PD-L1 in Hfstim-IL-10+ B cells led to a pronounced down regulation of IL-10+ CD4+ Tr1 cell differentiation. Interestingly, blockade of PD-1 or PD-L1 on CD4+ T cell prior to co-culturing with Hfstim-IL-10+ B / Hfstim-IL-10– B cells also decrease the percentage of Tr1 differentiated cells. Therefore, PD-1/ PD-L1 interaction between B and T cells is important in inducing Tr-1 cell differentiation. Also, the Hfstim-PD-1+ Bregs have a potential role in inducing Tr1 cells. Furthermore, the PD-1+ Bregs could be used, in future, as a therapeutic target to provoke the immune response and prevent the progression of gastric cancer. Currently, it was reported that PD-1 expression was high in CD4+ T cells of H. pylori infected patients. Notably, they found a significant correlation between H. pylori xxvii density with CD4+ PD-1+ T cell frequency or CD4+ CD25hi Tregs. Therefore, we studied PD-1 mRNA expression in Helicobacter-infected gastric tissue. H. pylori infection status of gastric tissue in this study was confirmed previously using ure A and ure B qPCR. Here, we determined the expression levels of PD-1 in gastric tissue of H. pylori negative individuals and in H. pylori infected gastritis and ulcer patients. The PD-1 mRNA is expressed significantly higher in ulcers compared to gastritis. Further studies would be performed to investigate the correlation between the human PD-1 in H. pylori infected gastric tissue with the IL-10- producing B cells or IL-10- producing T cells.
Examination of the correlation between Helicobacter pylori and T cell response with the determination of PD-1 expression level in gastric pathologies
Helicobacter pylori (H. pylori) is a gram negative microaerobic bacterium that coccoid or sphiral shaped bacterium that colonizes in stomach. Bacteria is around 2.5-3 μm length. Each H. pylori has around 6 flagella. This flagellas enables the bacterium movement and survival under harsh environmental conditions by escaping. More than 50% of people all around the world are infected by this bacterium and world health organization accepts H. pylori as primary cancerous. Most of the infected people by H. pylori do not show any clinical disease evidence, it maintains its existence sub clinically. Generally, infection starts at childhood and maintain lifelong. However, if bacterium could not detect early and treated properly, it caused several gastric pathologies. These pathologies are ulcer, gastritis, gastric carcinoma and MALT. Gastric Cancer has two main subtypes; intestinal type gastric cancer and diffuse type gastric cancer. Intestinal type gastric cancer is more frequently diagnosed than diffuse type. The prevalence of intestinal type is around 60% and there is a stepwise progression of disease. When the healthy gastric epithelium infected with H. pylori, host immune system responds it. Inflammation starts and if bacterium is not treated or eliminated by immune system, inflammation becomes a chronic phase; chronic gastritis. If bacterium does not eradicated atrophic changes in gastric epithelium takes place. One of the most important atrophic changes is degeneracy of glandular structure of gastric epithelium. After the degradation of gastric epithelium structure, disease could be progressed to intestinal metaplasia stage. Intestinal metaplasia is a pathological discrimination of gastric disease, which is mainly the differentiation of gastric epithelial cells to small intestine or colon epithelial cells. After the formation of these epithelial changes, treatments against H. pylori could not efficient to preventation of gastric cancer. Therefore, bacterium detection must be take place in early stages and this detection must be accurate. Detection of H. pylori could be happened by invasive or non-invasive methods. Non-invasive and invasive methods could be used at the same time for more accurate diagnosis. All those H. pylori related diseases are directly related with the host immune responses, genetic susceptibility and virulence factors of bacterium. Especially the resistance of bacterium to the treatment and increasing the survival period in host stomach is directly associated with the virulence factors. Even the genetic strain of H. pylori shows geographical differences in between countries, most important and prevalent virulence factors are urease subunit A (ureA), urease subunit B (ureB) Cytotoxin-associated gene A (cagA), vacuolating cytotoxin gene A (vacA), outer inflammatory protein A (oipA), blood group antigen-binding adhesion (BabA), putative neuraminyllactose-binding hemaggluttinin homolog A (hpaA), neutrophil activating protein A (NapA), duodenal ulcer promoting gene A (dupA). Not every Helicobacter pylori strain has to carry all of those virulence factors but they have to carry one of the urease subunit; ureA or ureB. These virulence factors are importants for bacterial infection, adhesion, colonization and survival periods. When the H. pylori infected, host immune system starts an immediate response against it by innate or adaptive immunity. Innate immunity gives immediate but non-specific responds. However, adaptive immune response is highly specific to pathogenic antigens. Especially, as an adaptive immune response, T cells have an importance on determining the clinical outcome of the infection. One of the recently discovered subtypes of T helper cell is Th17. T helper 17 (Th17) cells are functional in defending the host against the extracellular microorganism infections and has role in induction and propagation of autoimmunity. The master regulatory transcription factor of Th17 is Retineic-acid-receptor-related orphan nuclear receptor gamma t (RORγT). RORγT mainly expresses in thymus and controls the lineage of Th17. Th17 has been secreted 8 cytokines known. IL-17 is main cytokine groups of Th17. PD-1 is programmed death protein that act as immune checkpoint protein. It is expressing by the effector T cells as a surface receptor. Normally, absent on resting T cells. This surface receptor protein is responsible in controlling the T cell response of the body. When it binds its ligand PD-L1, it turns active T cells to inactive T cells. This feature of receptor is important in progression of many diseases. The effect of H. pylori on T cells and gastric epithelium is known but, effect on PD-1 is not clearly explained yet. In this thesis, we worked with the gastric biopsy and resection samples. In total of 60 patients were selected from 5 different pathological conditions. With in this 5 pathological groups; there is 15 normal, 20 active chronic gastritis, 3 inactive chronic gastritis, 9 intestinal metaplasia and 14 gastric cancer patient exists. Normal group was used as uninfected and unaffected control group for gastric malignecies. Patients were selected from patients who admitted to Acıbadem Hospital Gastroenterology Department who undergo endoscopies and Acıbadem Hospital General Surgery Department who undergo resection in between 18 to 80 years old admitted in this study. Each patient informed about the study, and his or her permissions taken. Endoscopic biopsy samples were collected from antrum of patients and resection samples were from tumor sites. Tumor site was detected by macroscopic examination and verified by hematoxylin and eosin staining. First step of this study, we detected the each virulence factors by PCR assay. H. pylori G27 strain used as positive control. G27 strain expresses all of those 9 virulence factors. We correlated the virulence factors expression profiles in between active chronic gastritis and intestinal metaplasia patients. Then verification of datas were confirmed with multiplex PCR assay. Then, we compared our PCR results with pathology reports of patients, which were prepared, based on histological appearance of H. pylori. According to this result, our PCR assay shows almost perfect agreement. Secondly, we examined the gene expression profiles of IL-17, RORγT and PD-1 by RT-PCR. We tried to correlate expression of these three genes with H. pylori infection. We observed that only PD-1 showed significant increase expression profile in infected patiens. The expressions of IL-17 and RORγT did not show significant varience between infected and uninfected patients. Then, we examined and tried to correlate expression profiles of those genes in between normal (uninfected and unaffected), precancerous lesions (inactive chronic gastritis, active chronic gastritis and intestinal metaplasia) and gastric cancer. For, IL-17 and RORγT, highest expression found in normal (uninfected and unaffected) patients. PD-1 had highest expression in active chronic gastritis. Consequently, there was not any stepwise increase or decrease of IL-17 and RORγT expression in between progression steps of gastric cancer but PD-1 showed increasing expression profile.
Investigation of the relation between Helicobacter pylori and T cell response with examination of PD-L1 expression level in gastric pathogenesis
Gastric cancer is the third most common cancer in the world. The reason of high death rate in this type of cancer is late diagnosis and bad prognosis. Chronic inflammation due to Helicobacter pylori (H. pylori) infection is a key factor in triggering the development of gastric cancer, as well as many factors such as genetic susceptibility, environmental factors, diet and age. Helicobacter pylori (H. pylori) is a gram-negative, microaerophilic, spiral-shaped bacterium that colonizes the stomach of more than half of the world's population during their life span. The bacteria appear around 3μm in length and possess a unipolar bundle of two to six sheathed flagella, about 0,5μm in size, that enable the bacteria to move in the acidic environment of stomach. H. pylori infection is usually acquired in early childhood and persists throughout the life. While most infected individuals remain asymptomatic, H. pylori infection can cause peptic ulcer, chronic gastritis, mucosa-associated lymphoid tissue (MALT) lymphomas and gastric cancer in 20% of the infected ones.H. pylori has several virulence factors that may be associated with diseases. The presence of this virulence factor increases the risk of stomach cancer by causing a higher inflammation in the stomach mucosa. Most studied virulence factor is vacA, which forms vacuoles in cells. H. pylori strains that contains the vacA virulence factor are more likely to develop gastritis. In addition, it increases mucosal inflammation and prognosis of the disease by increasing the colonization of babA and napA virulence factors of bacteria, which are responsible for bacterial adhesion to the gastric epithelium. Another protein found in the outer membrane of bacteria is oipA, which causes IL-8 release together with cagA and it is effective in progressing duodenal ulceration. Also, hpaA, which is found in the outer membrane is effective in colonization of the bacterium. The dupA gene is located in the plasticity zone of the H. pylori genome and has been associated with increased risk of duodenal ulcer. In addition, the ureA and ureB genes are required for the urease activity that protect the bacteria to stomach microenvironment. The stages of development of gastric cancer are classified as chronic gastritis, atrophic gastritis, intestinal metaplasia and dysplasia. In Turkish population, there are patients with gastric precancerous lesions at different levels resulting from a high incidence of H. pylori infection. Some of these patients have potential to develop stomach cancer in the future. However, the identification of whether these patients will develop gastric cancer is not possible because the pathogenesis of the stomach is not fully known from the genetic and immunological aspects. At the same time, the relationship between the characteristics of virulence factors of H. pylori and the response of the infected site is unknown. H. pylori in the gastric mucosa can survive at high degreess for a long time. This suggests that the immune response of the host failed to remove the pathogen. This may be due to a change in the expression of the cytokines that involved in the immune response. H. pylori infection leads to Th1 cells mediated immune response; In contrast, regulatory T cells (Tregs) prevent host inflammation against H. pylori and allow the bacteria to maintain in the stomach for long periods of time. In addition, the link between immune check proteins, programmed death-1 (PD-1) and its ligand-programmed death-ligand-1 (PD-L1), which are found on the surface of active T and B cells leads to negative regulation of T cell response. Therefore, in this study, the correlation between Th1 associated cytokine IFN-γ, Treg transcription factor FOXP3 and PD-L1 expression levels together with H.pylori infection was investigated in patients with gastric precancerous lesions and gastric cancer patients. This study included in total 60 patients; 20 with active chronic gastritis, 3 with inactive chronic gastritis, 9 with intestinal metaplasia and 14 with gastric cancer. In addition, 15 individuals with uneffected and uninfected normal gastric pathology were included as negative control group. These patients were applied to Acıbadem Hospitals with gastrointestinal symptoms and underwent endoscopy gastric cancer surgery.Three tissue samples were taken from the antrum region of patients' stomach who underwent endoscopy and resection. DNA was isolated from one of the received tissue samples and RNA was isolated from the other; the third biopsy specimen was used for hematoxylin and eosin staining to detect stomach pathogenesis and the satatus of Helicobacter pylori (H.pylori). Isolated DNA samples were used for Conventional PCR studies to identify ten different virulence factors of H.pylori. In the characterization of virulence factors, G27 H.pylori strain, which known to carry all virulence factors, was used as a positive control. cDNA synthesized from RNA samples and the expression of Treg-specific transcription factor Forkhead box P3 (FOXP3), Th1 derived cytokine interferon-g (IFN-ɣ) and programmed death ligand-1 (PD-L1) were examined by quantitative Real Time PCR (qRT-PCR) assays . Patients who had high inflammation were selected for optimization of quantitative Real Time PCR (qRT-PCR) assays. Selected patients were examined by conventional PCR assay to generate standard curve for mRNA level expression analysis of FOXP3, IFN-ɣ and PD-L1 by quantitavive Real Time PCR. Characterization of 10 different virulence factors by conventional PCR studies was carried out in patients who known to be H. pylori positive according to the pathology reports. The presence of ureA and ureB virulence factors which required for diagnosis of H. pylori was detected by conventional urease PCR. Conventional urease PCR results was compared with results of histopathological staining of H.pylori which was performed routinely for every patient. Comparison studies have shown a strong correlation between conventional urease PCR and pathological evaluation methods. Virulence factors determined as a result of conventional PCR studies were evaluated in two different groups of patients. The most prevalent virulence factor in patients with active chronic gastritis were; napA, hpaA, and cagA. The most common virulence factor in intestinal metaplasia were; cagA, vacA and hpaA. Expressions of FOXP3, IFN-ɣ, and PD-L1 were evaluated in precancerous and gastric cancer patient groups and expression levels were compared with uneffected and uninfected control individuals. The comparison results were analyzed by student's t test. According to the results of comparison with control group, expression levels of IFN-ɣ and FOXP3 were found to be decreased in patients with gastric precancerous lesions and gastric cancer patients. Otherwise, the level of expression of PD-L1 was higher in gastric cancer and gastric precancerous patients. When compared patients with precancerous lesions and gastric cancer, it was determined that only the expression level of FOXP3 was higher in patients with precancerous lesions. When the patients who included in this study were grouped according to H. pylori infection, only the expression level of IFN-ɣ was found to be higher in H. pylori-infected patients. In this study, the characterization of H. pylori virulence factors, the status of immune responses, and PD-L1 levels were investigated in staircase precancerous lesions to stomach cancer stages progressively for the first time.
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.
Cellular characterization of immune cells in non-small cell lung cancer and mesothelioma
Lung cancer is the leading cause of cancer deaths worldwide for both women and men. Mesothelioma, form of cancer concerning membrane surrounding the lungs, is under-examined especially in terms of immune cells. Tumor microenvironment mainly affects immunological responses via either suppressing effector cells or converting them to the regulatory cell types. Secretion of immune suppressor cytokines by regulatory cell types contributes to suppression of immune responses within cancer microenvironment. Regulatory cell types including regulatory T and B cells contribute to the inhibitory responses with their ability to secrete anti-inflammatory cytokines such as Interleukin-10 (IL-10). This study aims to characterize immune cells in non-small cell lung cancer and mesothelioma according to cell surface molecules and cytokine profiles of the cells. Cellular characterization of effector and regulatory immune cells in blood and tumor tissue samples of non-small cell lung cancer and mesothelioma patients is aimed to be performed. Our second aim was to investigate the effects of NSCLC microenvironment on healthy T and B cells. Overall, this study especially aims to identify types of peripheral and tumor infiltrating regulatory B cells that are not identified before in lung cancer and mesothelioma. Most of the studies in the literature regarding regulatory B cells are restricted to the studies of peripheral blood, which reflects only a systemic immune responses for NSCLC. For that reason, investigating regulatory B cells within its microenviroment is necessary. To investigate the relationship among CD4+ T cells, CD19+ B cells and their subsets, T cell specific (CD4 and CD8), regulatory B cell specific (CD19, CD24 ve CD38), and PD-1 and PD-L1 cell surface molecules were examined along with anti-inflammatory (IL-10) and pro-inflammatory (IFNγ and IL-17) cytokine profiles. In this study, we performed flow cytometry analysis of peripheral blood and tumor tissue samples collected from patients with NSCLC and mesothelioma from Istanbul University, Cerrahpaşa Medical School, Thoracic Surgery Department. Peripheral Mononuclear Blood Cells (PBMC) were isolated from both healthy and patient samples. Tumor infiltrating lymphocytes were obtained from tumor tissues that were resected during surgery. PBMC and TILs were stimulated with CpG ODN K3 (0,7 μg/ml) for 24 hours and PMA/ionomycin before staining of IL-10 and regulatory B cell surface markers. Immune cell characterization was performed by flow cytometry using anti-CD4, anti-CD8, anti-CD19, anti-CD24, anti-CD38, anti-PD1, anti-IL10, anti-IFNγ and anti-IL-17 antibodies. To study the effects of NSCLC microenvironment, CD4+ T cells and B cells were purified from healthy PBMCs by magnetic cell sorting with a column free commercial reagent kit (Stemcell). Then, B and T cells were cultured in either tumor or normal tissue conditioned medium which were obtained by culturing cells of tumor and normal tissue for 2 days. After that, IL10, IFNγ and PD-L1 expression were analyzed. Proliferation assay was also performed for T cells. 7 healty controls (3 females and 4 males, median age was 36 years; range, 25 to 52 years), 16 non-small lung cancer (3 females and 13 males, median age was 59 years; range, 42 to 69) and 3 mesothelioma (3 males, median age was 66 years; range 55 to 78) patients were analyzed. IL-10 expression of CD4+ and CD19+ (0.82-24.1%; 0.85-17.3%) cells were increased in bloods and also in TILs (0-23.7%; 0-20%) of patients with NSCLC compared with healthy individuals. CD4+ IL10+ (0-35.7%) cell frequencies were significantly increased in blood samples of mesothelioma patients. In addition, CD19+IL10+ cells were significantly increased in their tumors. IL-10 expression by CD19+CD24+CD38+ B cells (0-75%) from lung cancer patients' blood were greater than CD19+CD24+CD38+ B cells of healthy controls. PD-1 and IL-10 double positive B cells were significantly increased in PBMCs of LC patients (0,68-5,45%) in comparison to healthy individuals (0-0,94%). We observed increased IL-10 expression by both CD4+ T cells (0.82-24.1%) and CD19+ B cells (0.85-17.3%) for blood samples of the patients with lung cancer. Moreover, there was a high incidence of IL-10 expression in their tumor infiltrating CD4+ T cells (0-23,7%) and CD19+ B cells (0-20%). In addition, tumor infiltrating CD19+IL-10+ B cells (1,15-10%) were significantly increased in mesothelioma patients. Our results were also showed that IFNγ+ cytotoxic T cells were significantly decreased in TIL of lung cancer patients to the healthy control level compared with their blood samples (20,3-94,9% to 3,03-53,2%). In addition, IFNγ producing CD4+ T cell frequencies were significantly decreased in bloods of LC patients (10,4-38,3% to 1,82-21,4%). In turn, increased CD4+IL-10+ T cell frequencies suggests that CD4+ T cell response shift to the anti-inflammatory responses for both systemic and local responses. Moreover, our in vitro studies showed that IFNγ expression by CD4+ T cells decreased (15,2 to 10,7%) in tumor microenvironment compared to normal one. Also, IL-10 production was significantly elevated in healthy control B cells when they were cultured with supernatant of primary NSCLC tumor cells. Even though, we have low numbers of samples, the frequency of CD19+ CD24+ CD38+ IL-10+ B cells (0-75%) were highly increased in for NSCLC. PD-1 expression on B cells were significatly increased in both blood (2,14-11,1%) and tumor tissue (3,52-28,6%) samples of NSCLC patients compared to healthy individuals (0,79-3,9%). Moreover, PD-1 and IL-10 double positive B cells were significantly increased in both blood (0,68-5,45%) and tumor tissue samples (1,58-7,04%) of patients with NSCLC compared to healthy individuals (0-0,94%). Altogether, regulatory B cels were significantly elevated in both blood and TILs of patients with NSCLC while they were significantly elevated in TILs of patients with mesothelioma. We also observed that IL-10 production levels of PD-L1+ B cells in tumor microenviroment were increased. In conclusion, these results indicate an anti-inflammatory environment within tumor region both in lung cancer and mesothelioma.
Functional interaction between helicobacter-stimulated B (Hstim-B) cells and dendritic cells
Helicobacter pylori (H. pylori) is a microaerophilic gram negative and spiral-shaped bacterium which is firstly identified by Robbin Warren and Barry Marshall. About more than 50% of the people in the world have been infected with this bacterium; however, the bacteria cause serious gastrointestinal diseases only in 10-20% of infected people, whereas the remaining infected people has only asymptomatic gastritis. H. pylori can lead to development of gastric cancer and categorized as a group-I carcinogen by World Health Organization. Due to lack of efficient immune response against H.pylori in murines; mice has been infected with Helicobacter felis (H. felis) to create mouse model of Helicobacter infection. Dendritic cells (DCs) which are firstly identified by Steinmann, are the member of antigen presenting cells (APCs) and provide a connection between innate and adaptive immune response due to having a crucial role for activation of specific immune response against foreign antigens. Regulatory DCs express low levels of IL-12 and co-stimulatory molecules such as CD40, CD80 and CD86. Instead, expression of inhibitory molecules such as IL-10, PD-L1, CD95L and indoleamine 2,3 deoxygenase (IDO) are upregulated in regulatory DCs. Moreover, in addition to immature DCs, also semi-mature DCs characterized with MHC-IIhiCD80/CD86hiIL-12-TNF-α-IL-10+ phentotype are thought as tolerogenic DCs and inducer of type-1 regulatory T (Tr1) cell-mediated immune suppression. B cells have an important role for regulation of immune response in addition to their effector roles such as antibody production and antigen presentation. Regulatory role of B cells was shown with the use of mice lacking B cells which was not able to overcome autoimmune encephalitis. Helicobacter felis activates B cells via TLR2 and Myd88 and suppress immune responses in vivo and in vitro by triggering IL-10-producing Tr1-like cells. This suppression results in persistency of bacteria in gastric mucosa and decrease in pathology score. Therefore, balance between regulatory and effector response could determine the fate of infection. As demonstrated by our previous laboratory studies, H. felis-mediated activation of B cells induce two different B cell subgroups: IL-10-producing IL-10+ B cells and TGF-β-producing IL-10- B cells. Our first aim was to understand the effects of H. felis on bone marrow-derived dendritic cells (BM-DCs). Second aim was to determine effects of Helicobacter-stimulated B (Hfstim-B) cells on bone marrow-derived dendritic cells (BM-DCs). To perform these experiments; C57BL/6 mouse bone marrow cells were differentiated into immature dendritic cells in the presence of IL-4 and GM-CSF. B cells were magnetically isolated from spleens of C57BL/6 mice and treated with H. felis sonicate in order to obtain Helicobacter-stimulated IL-10+ B and IL-10- B cells through magnetic separation. Afterwards, these B cell subgroups were treated with H.felis sonicate, their supernatant were collected and applied onto immature BM-DCs. Following incubation and washing of the cells, BM-DCs were treated with LPS or H. felis sonicate. Cytokine profile and activation status of DCs were determined on protein and expression level. Moreover; for functional analysis, these DCs were co-cultured with naive CD4+ T cells at a ratio of 1:2 (DC:T) in the presence of 10 ng/ml IL-2 and 1 µg/ml anti-CD3Ԑ. Then, frequency of IL-10 secreting CD4+ T cells in different groups were analyzed by flow cytometry. H. felis-induced BM-DCs secrete IL-12/IL-23 (p40), IL-1β, TNF- α and IL-10 into the culture medium. Consistently, expression of IL-12/IL-23 (p40), TNF-α and IL-10 in addition to IL-6 was also induced. Flow cytometric analysis showed that H. felis activates DCs and causes upregulation of CD86 expression. All Hfstim-B cell groups including IL-10+ B and IL-10- B cells, induced DCs to express CD86 molecules in the absence of additional antigenic stimulation. However, there was no difference between DC groups stimulated with supernatant of IL-10- B and IL-10+ B in terms of expression of CD86 in untreated and LPS- or H. felis-treated groups. Moreover, IL-10 secretion was not induced upon stimulation of DCs with supernatant of unstimulated B, Helicobacter felis-stimulated B (Hfstim) or IL-10+ B cell followed by LPS or H. felis sonicate treatment. Nevertheless, DCs secreted significantly more IL-10 upon stimulation with IL-10+ B cell followed by LPS or H.felis treatment compared to IL-10- B cell supernatant stimulation. IL-12 secretion from DCs was induced by supernatant of all Helicobacter-stimulated B cell types. Strikingly, TNF-α secretion from DCs was suppressed upon stimulation of DCs with Hfstim-B cell supernatant prior to LPS or H. felis treatment. Interaction of BM-DCs with supernatant of Hfstim-IL-10+ B cells in the absence of subsequent LPS or H. felis treatment retained the frequency of IL-10-producing CD4+ T cells generated by immature DCs. Moreover, LPS treatment of DCs which had been previously stimulated with supernatant of unstimulated B cells, Hfstim-B cells or Hfstim-IL-10- B cells, but not Hfstim-IL-10+ B cells, resulted in the decreased frequency of IL-10-producing CD4+ T cells compared to LPS-treated control group. However, H. felis treatment of DCs following stimulation with supernatant of B cells have no effect on DCs to induce IL-10-producing CD4+ population. Therefore, it is possible that in addition to IL-10, also some other factors may be involved in differentiation of IL-10-producing CD4+ T cells by DCs stimulated with supernatant of different Hfstim-B cells subgroups. Furthermore, immature BM-DCs were co-cocultured with Hfstim-B cells at a ratio of 1:1 and 1:2 (DC:B) to evaluate the role of cell-to cell contact on this interaction. Accordingly, direct contact between Hfstim-B cells and immature DCs activates DCs and upregulates CD86 expression. B cells alone did not induce any IL-10 and TNF-α secretion regardless of the ratio between DC and B cell. However LPS or H. felis treatment of DCs after interaction with B cells resulted in decreased IL-10 production when the ratio between these cells was 1:1. Importantly, interaction of immature BM-DCs with Hfstim-B cells prior to LPS treatment induced production of IL-10 by DCs; whereas, unstimulated B cells exhibited inhibitory effect on IL-10 production when the ratio between these cells was 1:2 (DC:B). Moreover, TNF-α production by DCs was inhibited efficiently by Hfstim-B cells and unstimulated B cells when BM-DCs were co-cultured with these B cells at a ratio of 1:2 (DC:B) prior to LPS or H. felis treatment. It seems that the number of B cells co-cultured with immature DCs could be a critical factor to exhibit their regulatory function on DCs. Interestingly, Hfstim-IL-10+ B cells and Hfstim-IL-10- B cells did not affect the production of TNF-α by DCs. Collectively; some molecules secreted from Hfstim-B cells in addition to IL-10 synergistically with cell-to-cell contact have a regulatory role on activation and function of DCs. Consequently; first, DCs were activated and secreted both pro-inflammatory and anti-infammatory cytokines in response to H. felis antigens in vitro. Second, Helicobacter felis-stimulated B cells converts immature BM-DCs into IL-10-secreting tolerogenic form. Therefore, this study includes comprehensive analysis for the effects of H. felis on mouse BM-DCs and the first investigation that clarifies the ex vivo interaction of Hfstim-B cells subgroups with BM-DCs.
Investigating the behavior of endothelial cells and monocytes under disturbed flow conditions within a microfluidic system
Cells are capable of sensing and responding to the mechanical forces exerted by their environment. Reception of physical stimuli is followed by transmission and conversion of the signal into biological activity in a process defined as mechanotransduction. Mechanical signals combine with biochemical signals to produce multiple responses within a cell. Cumulative regulation in response to mechanical cues by a population of cells in close proximity can maintain important tissue functions. Disturbance in physiological patterns of mechanical forces may lead to abnormalities and disease. Blood vessels are particularly regulated via mechanical stimulation. Endothelial cells that line arterial, venular and capillary inner walls are under continuous strain by the hemodynamic forces as a result of blood flow. Alterations in the mechanical signals received by endothelial cells are frequent. The endothelium provides a bridge and a barrier between the bloodstream and the underlying vascular tissue. Consequently, mechanical signaling at the endothelium has an influence on overall vascular maintenance; mostly regulating the level of vessel dilation via the production of nitric oxide. The most studied mechanical force on endothelial cells is shear stress, the parallel frictional force caused by the flow of a fluid. Studies on shear stress have revealed that it has a major role in maintaining endothelial barrier and in preventing vascular diseases. Hemodynamic shear stress have a regular pattern that can locally variate in case of a damaged vessel, or in sites of branching geometries such as bifurcations and curvatures. In the branching geometries, laminar blood flow is disrupted and separated into secondary flows of oscillatory or reversing behavior, creating low shear stress in the local area. Mechanobiology is the discipline that studies how mechanical forces can affect cellular functions. Studying vascular mechanobiology is particularly challenging in that it requires establishment of the three-dimensional vascular microenvironment and generation of fluid flow. Recently, microfluidic channels have come into use to enable creating such research environments. Culturing of endothelial cells in microdevices of unique channel geometries allows to study cell behavior under controlled shear environments. Atherosclerosis, formation of fatty plaques in arteries, draws much attention because it is a common disease that has been linked to local alterations in shear stress. The cytoskeleton is at the core of mechanotransduction. Elements of the cytoskeleton are connected to each other and to membrane proteins, providing the balance and transmission of tensional forces. The shearing force initially deforms the cell surface and activates membrane ion channels, followed by transmission of the signal by secondary messengers assisted by the cytoskeleton. Then the message is converted into biochemical activity and is carried to distant regions of the cell. Transduction of the signal activates intracellular signaling pathways to promote gene transcription or regulates cell adhesion interactions. Upon exposure to laminar shear stress, endothelial cells elongate and align their cytoskeleton in the direction of flow. Low shear stress has been shown to increase the expression of endothelial adhesion molecule ICAM-1 (intercellular adhesion molecule 1), promoting endothelial permeability to leukocytes. Studies on human aortas suggest that monocytes accumulate greatly at the endothelium in the outer walls of bifurcations that are susceptible to disturbed shear stress patterns. Monocytes are phagocytic innate immune cells that remove bacteria, viruses and toxic metabolites. Monocytes circulate in the bloodstream, but they enter tissues via diapedesis and differentiate into macrophages in order to participate in homeostasis or inflammation via phagocytosis and antigen presentation. Monocytes play a central role in long-term, tissue-damaging inflammatory conditions such as atherosclerosis. Atherosclerosis is a chronic inflammatory response to endothelial injury characterized by formation of fat-laden plaques in artery walls. Progression and perchance rupture of atherosclerotic plaques can lead to cardiovascular disease. The occurrence of atherosclerosis depends on the nature of local blood flow as well as the presence of biochemical risk factors. While high laminar shear stress in straight blood vessels is atheroprotective, low shear stress and oscillatory/reversing blood flow in vessel curvatures bring about atheroprone sites. In addition, the blood vessel becomes narrower as the atherosclerotic plaque grows, changing the local shear stress at the plaque site. The significance of physiological shear stress and the response of endothelial cells to alterations in shear stress have been well studied to date. However, no studies yet have analyzed the biological effects of disturbed flow on endothelial cells applying a model that can mimic the atherosclerotic plaque microenvironment with high-to-low alteration of shear stress. In this study, a unique design of cavitated microfluidic channel mimicking the atherosclerotic plaque geometry was used for the first time to study the effects of disturbed flow on HUVECs (Human Umbilical Vein Endothelial Cells). The first part of the study describes methods to obtain a confluent endothelial monolayer within straight and cavitated microfluidic channels and a method to apply shear stress on cells. In the preliminary studies, disturbed shear effects on endothelial F-actin network and ICAM-1 expression were investigated. Low-level disturbed shear stress was shown to cause F-actin accumulation at the cell cortex and F-actin reorganization in the direction of flow. This effect was higher in the narrower channel regions where shear stress was relatively high. The more random alignment of HUVECs in the low shear-harboring cavities may be related to the local disturbance of flow within the region. In addition, low-level disturbed shear stress was found to upregulate ICAM-1 mRNA expression, although a less increase in the expression was obtained when compared to TNFα activation. Taken together, disturbed shear stress at atheroprone regions may contribute to monocyte recruitment at the endothelium via upregulation of the endothelial adhesion molecule ICAM-1, driving the effect further with local presence of TNFα. Since this study involved only static cells as a reference, further studies may include application of laminar shear stress on endothelial cells in straight channels as an additional control. The individual effect of shear stress on monocyte behavior has not been studied to date. In this study, THP-1 monocytic cells were subjected to sequential high and low shear stress in cavitated channels at an average of 4 Pa as normal, 15 Pa as high or 45 Pa as very high shear stress and changes in F-actin levels were analyzed by fluorescence microscopy and flow cytometry. High level (15 Pa) disturbed shear stress was determined to increase F-actin content, whereas very high (45 Pa) level of disturbed shear stress was concluded to decrease the F-actin content in the long term in THP-1 monocytes. Furthermore, the effect of disturbed shear on F-actin in shorter time intervals may be assessed to fully characterize time-dependent changes in the F-actin.
Sistemik lupus eritematozus ve skleroderma hastalarında anti-insan lökosit antijen antikorlarının incelenmesi
Human leukocyte antigen (HLA) is the human form of Major Histocompatibility Complex (MHC). Generally, HLA is a system of gene locus that encode for proteins on the surface of cells that are responsible for the regulation of the immune system. It is located on chromosome 6, encoded cell surface antigen presenting proteins. Anti-HLA antibodies are normally not occurring produced against self or non-self HLA antigens to form a immunologic response in blood transfusion, pregnancy and organ or tissue transplantation. Systemic lupus erythematosus (SLE) is an autoimmunity disorder which inflammation along with tissue damage affecting multi organs such as skin, kidney, joint are the main cause of it. SLE is a multisystem autoimmune disorder that has variable clinic manifestation. Prior physiologic mechanism of SLE is polyclonal B cell activation and production of autoantibodies against nucleus, cytoplasmic and cell surface antigens. Although the main cause of SLE pathogenesis remains unclear, there have been several clinical and serological factors that define predisposition and risk for SLE. Systemic Sclerosis (SSc) that is characterized with vascular damage caused by immune system dysfunctions and collagen accumulation in several tissues and organs can cause smooth muscle atrophy result of neurological function loss and then motor function loss. Generally anti-HLA antibodies can cause acute and chronic rejection in transplantation. Lately, there have some studies that associated between autoimmunity and anti-HLA antibodies. According to new theory, it has been indicated that there may be a similarity between alloimmune responses occur during transplantation and autoimmunity for self-antigen. Microchimerism is a well-known phenomenon effecting after solid transplantation, pregnancy and blood transfusion. The analogy between autoimmune diseases and alloimmune response in transplantation may explain role of microchimerism related with fetal or maternal origin. Therefore, in our project, SLE and SSc were selected as a model autoimmune disease to investigate the relationship between autoimmunity and anti-HLA antibodies. First of all, three different group including SLE, SSc patients and healthy controls were formed. The clinical and demographic data were collected and analyzed. Then, anti-HLA antibodies screening were done to all groups with flourometric methods. After analysis of anti-HLA antibodies screening, statistical analysis were done. Also, anti-HLA antibody identification analysis has been applied to anti-HLA antibody positive SLE, SSc patients and healthy controls. PRA ratios and CREG groups were determined statistically. According to our findings, the frequencies of both class I and II anti-HLA antibodies were increased in both SLE and SSc patients when compared to controls. Also, anti-HLA antibodies frequencies were similar in both SLE and SSc patients. We also found that typical parameters for SLE patients such as photosensitivity, thrombocytopenia, anti-RNP positivity, anti-Ro positivity, anti-nuclear antibody (ANA) had slightly higher frequency than SSc patients. On the other hand, SSc parameters frequency such as digital ulcers, anti-centromere antibody, pulmonary hypertension, and pulmonary fibrosis were higher in SSc patients. Also, in comparison of degree of positivity of anti-HLA antibodies we found that patients with high positivity for anti-HLA antibodies in both SLE and SSc group was found higher than low positivity individuals in same groups. In conclusion, anti-HLA class I and II antibodies were found as a significantly increased in patients of SLE and SSc that were chosen as model autoimmune diseases than healthy controls in which microchimerism may play a role. In order to focus the role of microchimerism in this relationship, more targeted studies should be done.
Helicobacter-aktive B(HAKT-B) hücrelerinin CD4+ T hücreleri ile fonksiyonel etkileşimleri
Helicobacter pylori is a spiral-shaped, gram-negative bacterium that infects the gastric mucosa of more than half of the world's population. The infection initially occurs in childhood, becomes persistent and the chronic infection leads to gastric inflammation. A major virulence factor of H. pylori is the cytotoxin-associated gene A (CagA) protein and this CagA protein interacts with several intracellular components of signal transduction and activates some crucial signaling pathways. Helicobacter has developed a variety of mechanisms to persist in the gastric mucosa. Gastric epithelial cells (GECs) are primary target for H. pylori infection, therefore they are the first point of contact for H. pylori and activate an innate immune response through TLRs. Rather than being a strong TLR4 ligand, H. pylori LPS is thought to activate TLR2 on gastric epithelial cells. Animal and cell culture experiments suggested that ligands in Helicobacter species can bind to TLR2 and activate NF-κB in epithelial cells. In mouse studies, Helicobacter felis (H. felis) is mainly used because it is more immunogenic than H.pylori in mice. B cells and their cytokines have important roles in Helicobacter infections by balancing between the infection and T cell driven gastric immunopathology. The ability of B cells to interact with pathogenic T cells and to produce anti-inflammatory cytokines such as IL-10 is crucial to dampen harmful immune responses. It has been found that B-cells exposed to Helicobacter sonicate produced large amounts of the regulatory cytokine IL-10. Using mouse models of Helicobacter-induced gastric premalignant pathology, it is shown that IL-10 secretion by purified B cells absolutely required MyD88 signaling and TLR-2. The TLR-2- dependent Helicobacter activation of B cells differentiates them into IL-10 and TGF-β producing regulatory B cells. Both IL-10 and TGF-β have crucial effects on T cell differentiation. When co-cultured with Helicobacter-activated B cells, naive CD4+ T-cells are shown to produce IL-10 and differentiate into T regulatory 1 (Tr1)-like cells. In addition to that, it is suggested that Breg cells contribute to regulatory T-cell induction by producing TGF-β. At the same time, studies have shown convincingly that TGF-β is required for Th17 differentiation in vitro and in vivo. In previous studies in our laboratory, Helicobacter-activated total B cells were separated into two subgroups: IL-10+ B cells and IL-10- B cells. The experimental results showed that Helicobacter-activated IL-10+ B cells are the source of the IL-10 production while Helicobacter-activated IL-10-B cells are mostly TGF-β positive. However, it was not clear if the Helicobacter-acitvated-IL-10+ B cells or the Helicobacter-activated-IL-10- B were specifically causing the Tr1 differentiation. Taking account that Bregs are known for producing IL-10 and TGF-β which are key cytokines in T cell differentiation, the interaction between Helicobacter-activated B cell subgroups and CD4+ T cell differentiation was investigated. By magnetic isolation techniques, CD19+B cells and CD4+T cells were isolated from the spleens of C57BL/6 mice with high purities (with an average of 90% and 93%, respectively). Following the B cell isolation, cells were treated with Helicobacter felis sonicate (10 µg/ml) for 24 hours. For the last 5 hours of incubation, to induce an optimal IL-10 production and secretion, PMA (50 ng/ml) and ionomycin (500 ng/ml) were added. After the in vitro stimulation of B cells, IL-10 producing B cells were labeled and the IL-10+B and IL-10-B cells were separated. To observe the interaction between the Helicobacter-activated-B cell subgroups and CD4+ T cells, isolated CD4+ T cells were put on co-culture in 1:1 ratio with the Helicobacter-activated-IL-10+ B cells and Helicobacter-activated-IL-10- B cells, for 24 hours. The cell surface markers and intracellular cytokine productions were examined by flow cytometry. While the supernatants of the co-culture groups were subjected to ELISA tests, the cell pellets were used for gene expression analyses. The intracellular staining of IL-10 of T cells co-cultured with Helicobacter-activated B cell subgroups showed that about 15% of T cells co-cultured with HACT-IL-10+ B cells produced IL-10 while almost 20% of the T cell population was IL-10 positive when T cells were co-cultured with HACT-IL-10- B cells. In addition, according to IL-10 ELISA results, CD4+T cells co-cultured with IL-10+B cells and IL-10-B cells secreted twice IL-10 when compared to only T cells. For IL-10+B cell and T cell co-culture, a part of secreted IL-10 came from B cells while most of the IL-10 secreted from IL-10-B cell and T cell co-culture originated is suggested to be from T cells. CD25 has been used as a marker to identify activated T cells as well as some regulatory T cell subsets in mice; while CD62L has been known to rapidly shed from lymphocytes upon cellular activation. Both CD25 and CD62L levels in co-culture groups showed significant differences compared to T only groups. Furthermore, the differences in T cell CD25 and CD62L levels together might indicate that T cells co-cultured with Helicobacter-activated IL-10-B cells are more activated/differentiated compared to the T cells interacting with IL-10+B cells. In literature, it has been shown that B cells which were activated by Helicobacter induce IL-10–producing CD4+CD25+ Tr1–like cells in vitro. In addition, it has been identified that the co-expression of CD49b and LAG-3 distinguishes Tr1 cells from Th1, Th2 and Th17 cells. Flow cytometry results of CD4-CD49b-LAG3 stainings showed that almost 20% of T cells co-cultured with IL-10+B cells express CD4-CD49b-LAG3 surface markers while only around 10% of CD4+ T cells co-cultured with IL-10-B cells express CD49b and LAG3 together. CD4+ T cells obtain distinct functional properties in response to signals sent by commensal and pathogenic microbe-activated cells of the innate immune system. Th17 cells secrete interleukin-17 (IL-17), IL-17F, and IL-22 and have significant roles in protecting the host from bacterial and fungal infections, particularly at mucosal surfaces. Data of four independent experiments showed that the approximately 15% of CD4+ T cells co-cultured with IL10-B cells turned into IL17 producing CD4+ T cells while surprisingly about 8% of T cells also produced IL-17 when co-cultured with IL10+B cells. IL-17 ELISA results were parallel with the IL-17 intracellular cytokine staining analyses. Ror gamma T and IL-17 relative gene expression levels in co-culture groups showed similar results with flow cytometry and ELISA results. Main research focus of this study was to investigate the interaction between HACT-B cell subgroups and CD4+ T cells and understand the effects of these B cell subgroups on T cell differentiation ex vivo. The results revealed that both Helicobacter-activated IL-10+B and IL-10-B cells induce IL-10 production from CD4+T cells and it might suggest that T cells differentiate into Tr1-like cells in both co-culture conditions. Although the CD25 and CD62L expression on T cell surfaces show significantly higher activation/differentiation in Helicobacter-activated IL-10-B and T cell co-cultures, regulatory type indicator CD4-CD49b-LAG3 surface marker co-expressions were higher on T cells in the Helicobacter-activated IL-10+B and T cell co-cultures. Furthermore, intracellular IL-17A levels and relative gene expression experiments of IL-17A and RorgammaT showed significantly higher results in IL-10- B and T cell co-culture groups, as expected. On the other hand, interestingly IL-17 production was also observed in T cells co-cultured with IL-10- B cells. The reason behind the IL-17 and RorgammaT expressions in IL-10+ B–T cell co-culture groups was suggested to be the IL-6 and TGF- β produced by IL-10 negative B cell population in the IL-10 positive co-culture group. This study has contributed to the literature through providing a first step to show the ex vivo interactions of Helicobacter-activated B cells subgroups, IL10+ HACT B cells and IL-10- HACT B cells, with CD4+ T cells.
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.