Presence of intestinal Mycobacterium avium subspecies paratuberculosis (MAP) DNA is not associated with altered MMP expression in ulcerative colitis

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium avium </it>subspecies <it>paratuberculosis </it>(MAP) is suspected to be a causative agent in human Crohn's disease (CD). Recent evidence suggests that pathogenic mycobacteria and MAP can induce the expression of Matrix Metalloproteinases (MMP), which are the main proteases in the pathogenesis of mucosal ulcerations in inflammatory bowel disease (IBD). Within this study we assessed the prevalence of intestinal MAP specific DNA in patients with Crohn's disease, ulcerative colitis (UC), and healthy controls. We further analysed regulation patterns of MMPs in mucosal tissues of UC patients with and without intestinal MAP DNA detection.</p> <p>Methods</p> <p>Colonic biopsy samples were obtained from 63 Norwegian and German IBD patients and 21 healthy controls. RNA was quantified by quantitative real-time polymerase chain reaction (PCR) to study MMP gene expression in both pathological and healthy mucosal specimens. The presence of MAP DNA in colonic mucosa was examined using MAP specific PCR.</p> <p>Results</p> <p>MAP DNA was detected in 20% of UC patients and 33% of healthy controls but only in 7% of patients with CD. UC patients treated with corticosteroids exhibited a significantly increased frequency of intestinal MAP DNA compared to those not receiving corticosteroids. Expression of MMP-1, -2, -7, -9, -13, -19, -28 and TNF-α did not differ between UC patients with presence of intestinal MAP DNA compared to those without. MMP-2, MMP-9 and MMP-13 were significantly decreased in UC patients receiving corticosteroids.</p> <p>Conclusions</p> <p>The presence of intestinal MAP specific DNA is not associated with altered MMP expression in UC <it>in vivo</it>. Corticosteroids are associated with increased detection of intestinal MAP DNA and decreased expression of certain MMPs. Frequent detection of MAP DNA in healthy controls might be attributable to the wide environmental distribution of MAP and its presence in the food-chain.</p

Orientia tsutsugamushi Stimulates an Original Gene Expression Program in Monocytes: Relationship with Gene Expression in Patients with Scrub Typhus

Orientia tsutsugamushi is the causal agent of scrub typhus, a public health problem in the Asia-Pacific region and a life-threatening disease. O. tsutsugamushi is an obligate intracellular bacterium that mainly infects endothelial cells. We demonstrated here that O. tsutsugamushi also replicated in monocytes isolated from healthy donors. In addition, O. tsutsugamushi altered the expression of more than 4,500 genes, as demonstrated by microarray analysis. The expression of type I interferon, interferon-stimulated genes and genes associated with the M1 polarization of macrophages was significantly upregulated. O. tsutsugamushi also induced the expression of apoptosis-related genes and promoted cell death in a small percentage of monocytes. Live organisms were indispensable to the type I interferon response and apoptosis and enhanced the expression of M1-associated cytokines. These data were related to the transcriptional changes detected in mononuclear cells isolated from patients with scrub typhus. Here, the microarray analyses revealed the upregulation of 613 genes, which included interferon-related genes, and some features of M1 polarization were observed in these patients, similar to what was observed in O. tsutsugamushi-stimulated monocytes in vitro. This is the first report demonstrating that monocytes are clearly polarized in vitro and ex vivo following exposure to O. tsutsugamushi. These results would improve our understanding of the pathogenesis of scrub typhus, during which interferon-mediated activation of monocytes and their subsequent polarization into an M1 phenotype appear critical. This study may give us a clue of new tools for the diagnosis of patients with scrub typhus

Homing commitment of lymphocytes activated in the human gastric and intestinal mucosa

BACKGROUND—Gastric infection with the human pathogen Helicobacter pylori results in a large accumulation of IgA and IgM secreting cells in the gastric mucosa. The molecular mechanisms resulting in B cell migration to the gastric mucosa in H pylori infection are however not known.
AIMS—To examine expression of the mucosal homing receptor integrin α4β7 and the homing receptor for secondary lymphoid tissues, L-selectin, on lymphocytes activated by gastric, intestinal, or systemic antigens. Furthermore, to examine gastric expression of the mucosal addressin cellular adhesion molecule 1 (MAdCAM-1), the endothelial counter-receptor to integrin α4β7.
SUBJECTS AND METHODS—H pylori infected individuals were immunised by either gastric (n=8) or intestinal (n=8) delivery of an inactivated cholera vaccine. The resulting circulating vaccine specific B cells were sorted according to α4β7 and L-selectin expression and assayed for production of IgA and IgG using an enzyme linked immunospot assay. In addition, circulating CD4+ T cells from seven H pylori infected individuals were fractionated according to α4β7 and L-selectin expression. The resulting T cell fractions were then assayed for specific proliferation against H pylori or the systemic antigen tetanus toxoid. Finally, gastric expression of MAdCAM-1 was determined by immunohistochemistry in H pylori infected (n=16) and uninfected (n=8) individuals.
RESULTS—Virtually all B cells induced by both gastric and intestinal antigen delivery expressed α4β7 whereas less then half coexpressed L-selectin. Furthermore, H pylori reactive T cells were mainly found in the α4β7+L-selectin+ T cell fraction whereas tetanus specific T cells were largely α4β7−L-selectin+. MAdCAM-1 was present in similar amounts in gastric mucosa from H pylori infected and uninfected individuals.
CONCLUSIONS—B cells and T cells activated by antigens delivered to the gastric mucosa express the mucosal homing receptor integrin α4β7, as do cells activated in the intestine. Together with the observation that gastric endothelial cells express MAdCAM-1, this may partly explain the homing of lymphocytes activated in the stomach or in the small intestine to the gastric mucosa.


Keywords: lymphocyte trafficking; integrin α4β7; L-selectin; stomach; Helicobacter pylor

Human gastric B cell responses can be induced by intestinal immunisation

BACKGROUND—In a previous study, we found that oral vaccination induces strong B cell responses in the stomach of Helicobacter pylori infected but not of uninfected individuals. In this study, we have evaluated the possibility of inducing gastric immune responses in H pylori infected volunteers by intestinal and gastric immunisation.
METHODS—H pylori infected subjects were given two doses of an inactivated cholera vaccine, either intestinally via an endoscope approximately 30 cm distal to the pylorus sphincter or intragastrically as small droplets applied directly onto the stomach mucosa. Uninfected individuals received the vaccine by standard oral procedure. Vaccine specific antibody secreting cells in antral and duodenal biopsies were detected by the enzyme linked immunospot assay technique before and seven days after the second immunisation.
RESULTS—Intestinal immunisations resulted in induction of vaccine specific gastric IgA secreting cells in five of eight volunteers. This immunisation schedule also gave rise to specific duodenal antibody secreting cells in seven of eight individuals. Local gastric immunisation resulted in the induction of specific B cells in the gastric mucosa of four of eight volunteers. Gastric antigen application also resulted in B cell responses in the duodenum in all volunteers. Uninfected volunteers receiving the vaccine perorally responded in the duodenum but not in the stomach.
CONCLUSIONS—H pylori infection increases the ability of the gastric mucosa to serve as an expression site for intestinally induced B cell responses. These findings are of importance when designing a therapeutic H pylori vaccine, and based on our results such a vaccine can be delivered along the whole upper gastrointestinal tract.


Keywords: Helicobacter pylori; vaccine; stomach; B cell; lymphocyte traffickin

Differential expression of chemokine receptors on human IgA+ and IgG+ B cells

Organ-specific lymphocyte homing is dependent on the expression of tissue-specific homing receptors and selected chemokine receptors. During the effector phase of an immune response, IgA and IgG antibody-secreting cells (ASC) are differently distributed in the body. Still, B cell expression of L-selectin and the mucosal homing receptor integrin α4β7 is not related to the isotype produced, but only to the site of antigen encounter. In this study, we examined if differences in chemokine responsiveness between IgA+ and IgG+ B cells could explain their different tissue localization. Circulating CD19+ B cells were isolated and their expression of IgA, IgG, and selected chemokine receptors was determined by flow cytometry. Few Ig+ cells expressed CCR2, CCR3, or CCR9, and there was no difference in the expression of these receptors between IgA+ and IgG+ cells. In contrast, CCR4, CCR5, and CXCR3 was expressed on significantly more IgG+ than IgA+ cells. The function of chemokine receptors on memory B cells and ASC was then tested in the transwell system. IgG+ memory cells migrated to a higher extent than IgA+ cells towards the CXCR3 ligand CXCL11/I-TAC, while there was only a small migration towards the CCR4 ligand CCL17/TARC and the CCR9 ligand CCL25/TECK. ASC migrated poorly to all chemokines tested. In conclusion, this study shows that IgG+ and IgA+ memory B cells have a differential expression of the Th1 associated chemokine receptor CXCR3, as well as of CCR4 and CCR5. In contrast, none of the studied chemokine receptors was preferentially expressed by IgA+ cells

Lipoxins modulate neutrophil oxidative burst, integrin expression and lymphatic transmigration differentially in human health and atherosclerosis

Dysregulated chronic inflammation plays a crucial role in the pathophysiology of atherosclerosis and may be a result of impaired resolution. Thus, restoring levels of specialized pro-resolving mediators (SPMs) to promote the resolution of inflammation has been proposed as a therapeutic strategy for patients with atherosclerosis, in addition to standard clinical care. Herein, we evaluated the effects of the SPM lipids, lipoxin A4 (LXA4) and lipoxin B4 (LXB4), on neutrophils isolated from patients with atherosclerosis compared with healthy controls. Patients displayed altered endogenous SPM production, and we demonstrated that lipoxin treatment in whole blood from atherosclerosis patients attenuates neutrophil oxidative burst, a key contributor to atherosclerotic development. We found the opposite effect in neutrophils from healthy controls, indicating a potential mechanism whereby lipoxins aid the endogenous neutrophil function in health but reduce its excessive activation in disease. We also demonstrated that lipoxins attenuated upregulation of the high-affinity conformation of the CD11b/CD18 integrin, which plays a central role in clot activation and atherosclerosis. Finally, LXB4 enhanced lymphatic transmigration of human neutrophils isolated from patients with atherosclerosis. This finding is noteworthy, as impaired lymphatic function is now recognized as an important contributor to atherosclerosis. Although both lipoxins modulated neutrophil function, LXB4 displayed more potent effects than LXA4 in humans. This study highlights the therapeutic potential of lipoxins in atherosclerotic disease and demonstrates that the effect of these SPMs may be specifically tailored to the need of the individual.Funding Agencies: Vetenskapsrådet (VR) Swedish Research Council [2016/82]; Svenska Sällskapet for Medicinsk Forskning (SSMF) [S150086]; EC \ H2020 \ H2020 Priority Excellent Science \ H2020 European Research Council (ERC) [804418]; Knut och Alice Wallenbergs Stiftelse (Knut and Alice Wallenberg Foundation)</p

Induced sputum MMP-1, -3 & -8 concentrations during treatment of tuberculosis.

INTRODUCTION: Tuberculosis (TB) destroys lung tissues and this immunopathology is mediated in part by Matrix Metalloproteinases (MMPs). There are no data on the relationship between local tissue MMPs concentrations, anti-tuberculosis therapy and sputum conversion. MATERIALS AND METHODS: Induced sputum was collected from 68 TB patients and 69 controls in a cross-sectional study. MMPs concentrations were measured by Luminex array, TIMP concentrations by ELISA and were correlated with a disease severity score (TBscore). 46 TB patients were then studied longitudinally at the 2nd, 8th week and end of treatment. RESULTS: Sputum MMP-1,-2,-3,-8,-9 and TIMP-1 and -2 concentrations are increased in TB. Elevated MMP-1 and -3 concentrations are independently associated with higher TB severity scores (p<0.05). MMP-1, -3 and -8 concentrations decreased rapidly during treatment (p<0.05) whilst there was a transient increase in TIMP-1/2 concentrations at week 2. MMP-2, -8 and -9 and TIMP-2 concentrations were higher at TB diagnosis in patients who remain sputum culture positive at 2 weeks and MMP-3, -8 and TIMP-1 concentrations were higher in these patients at 2nd week of TB treatment. CONCLUSIONS: MMPs are elevated in TB patients and associate with disease severity. This matrix-degrading phenotype resolves rapidly with treatment. The MMP profile at presentation correlates with a delayed treatment response

Human tonsil implants xenotransplanted in SCID mice display broad lymphocytic diversity and cellular activation profile similar to those in the original lymphoid organ.

BACKGROUND: Models consisting of human immune cells in suspension transferred to severe combined immune deficient (SCID) mice have been invaluable for studying immune response, autoimmunity, and lymphomagenesis. The dissemination of human cells within the mouse body hampers immune functionality with time and favorites the development of human graft vs. mouse host (GvH) disease. To circumvent these limitations we surgically implanted tonsil pieces subcutaneously in SCID animals (hu-ton-SCID mice). Recall humoral responses was elicited and animals did not suffer from signs of GvH disease. A detailed cell subset and cell activation analysis of implants has not yet been reported. METHODS: Implants from 86 hu-ton-SCID mice were evaluated by immunohistochemistry and flow cytometry analyses to assess human lymphoid cell subpopulation surviving with time after implantation, and to evaluate status of human cell activation. RESULTS: B cells persist over 3 months in implants. The proportion of class and type-specific Ig+ cells varied between implants, but as a whole IgG+ cells were more abundant than IgA+, and IgM+ cells, and kappa+ cells predominated over lambda+ cells. The mean proportions of these cells resemble those in the original tonsil. Fine analysis of CD19+ B cells demonstrated no expansion of activated (CD5+, CD23+, CD69+) B cells in implants compared with tonsils, and a decrease of CD19+CD77+ B cells corresponding to a centroblastic phenotype, which is consistent with the disappearance of follicular structure in implants. Double positive CD20+CD27+ memory B cells were detected in implants by immunohistochemistry. T cell CD4+CD8-/CD4-CD8+ ratios were about 4 in implants, that is similar to those in tonsils, and there was no expansion of CD3+CD4+CD8+ and of CD3+CD4-CD8- T-cell subpopulations. T cells activation markers (CD25, CD69) were similarly expressed in implants and tonsils, and implants contained cells with a memory T cell phenotype (CD45RO). Finally cells within implants depicted a low rate of proliferation when assessed by Ki-67 expression levels. CONCLUSIONS: Compared with original tonsils, tonsil implants in hu-ton-SCID mice lose the germinal center architecture, which is correlated with the decrease of CD77+ B cells, but conserve T and B cell subpopulation diversity, notably memory cells. In addition, implant T and B cells are not differently activated when compared with those in original tonsils and do not proliferate extensively. These observations indicate indirectly absence of GvH reaction at the cellular level in this model. Collectively, the detailed implant cellular characterization in the hu-ton-SCID model provides a strong rationale for the use of this model in the study of human recall antibody response