Development and Function of Immune Cells in an Adolescent Patient with a Deficiency in the Interleukin-10 Receptor

OBJECTIVE:: Monogenic defects in the interleukin-10 (IL-10) pathway are extremely rare and cause infantile-onset inflammatory bowel disease (IBD)-like pathology. Understanding how immune responses are dysregulated in monogenic IBD-like diseases can provide valuable insight in “classical” IBD pathogenesis. Here, we studied long-term immune cell development and function in an adolescent IL-10 receptor (IL10RA)-deficient patient who presented in infancy with severe colitis and fistulizing perianal disease and is currently treated with immune suppressants. METHODS:: Biomaterial was collected from the IL10RA-deficient patient, pediatric IBD patients and healthy controls. The frequency and phenotype of immune cells were determined in peripheral blood and intestinal biopsies by flow cytometry and immunohistochemistry. Functional changes in monocyte-derived dendritic cells and T cells were assessed by in vitro activation assays. RESULTS:: The IL10RA-deficient immune system developed normally with respect to numbers and phenotype of circulating immune cells. Despite normal co-stimulatory molecule expression, bacterial lipopolysaccharide-stimulated monocyte-derived dendritic cells from the IL10RA-deficient patient released increased amounts of TNFα compared to healthy controls. Upon T-cell receptor ligation, IL10RA-deficient peripheral blood mononuclear cells released increased amounts of T cell cytokines IFNγ and IL-17 agreeing with high numbers of T-bet and IL-17 cells in intestinal biopsies taken at disease onset. In vitro, the immunosuppressive drug thalidomide used to treat the patient decreased peripheral blood mononuclear cell-derived TNFα production. CONCLUSIONS:: With time and during immunosuppressive treatment the IL10RA- deficient immune system develops relatively normally. Upon activation, IL-10 is crucial for controlling excessive inflammatory cytokine release by dendritic cells and preventing IFNγ and IL-17-mediated T-cell responses

Electrical field stimulation causes oxidation of exogenous histamine in Krebs-Henseleit buffer:A potential source of error in studies of isolated airways

Electric field stimulation (EFS) relaxes human histamine-precontracted airways in vitro. This relaxation is only partly neurally mediated. Nonneural relaxation has been also shown in blood vessels and is due to the generation of oxygen radicals by EFS. In isolated airways the origin of the nonneural component of the relaxation is not clear. Because exogenous catecholamines are oxidized during EPS of carbogenated Krebs-Henseleit (K-H) buffer, we questioned whether this is also the case for exogenous histamine. Human airways precontracted with histamine or methacholine were exposed to either EFS-stimulated carbogenated K-H buffer that also contained histamine or methacholine or unstimulated buffer. Airways exposed to EFS-stimulated buffer that contained histamine relaxed, whereas airways exposed to buffer containing methacholine or exposed to unstimulated buffer did not. It appeared that the histamine concentrations in the organ baths decreased during 30 min of EFS. This decrease was significantly reduced in the presence of ascorbic acid. We conclude that EFS causes oxidation of histamine in carbogenated K-H buffer, and this may at least partly explain the nonneural component of EFS-induced relaxations of precontracted human isolated airways. Therefore, histamine should not be used to induce precontraction in EFS experiments.</p

Inhibition of cyclooxygenase activity reduces rotavirus infection at a postbinding step

Elevated levels of prostaglandins (PGs), products of cyclooxygenases (COXs), are found in the plasma and stool of rotavirus-infected children. We sought to determine the role of COXs, PGs, and the signal transduction pathways involved in rotavirus infection to elucidate possible new targets for antiviral therapy. Human intestinal Caco-2 cells were infected with human rotavirus Wa or simian rotavirus SA-11. COX-2 mRNA expression and secreted PGE(2) levels were determined at different time points postinfection, and the effect of COX inhibitors on rotavirus infection was studied by an immunofluorescence assay (IFA). To reveal the signal transduction pathways involved, the effect of MEK, protein kinase A (PKA), p38 mitogen-activated protein kinase (MAPK), and NF-κB inhibitors on rotavirus infection was analyzed. In infected Caco-2 cells, increased COX-2 mRNA expression and secreted PGE(2) levels were detected. Indomethacin (inhibiting both COX-1 and COX-2) and specific COX-1 and COX-2 inhibitors reduced rotavirus infection by 85 and 50%, respectively, as measured by an IFA. Indomethacin reduced virus infection at a postbinding step early in the infection cycle, inhibiting virus protein synthesis. Indomethacin did not seem to affect viral RNA synthesis. Inhibitors of MEK, PKA, p38 MAPK, and NF-κB decreased rotavirus infection by at least 40%. PGE(2) counteracted the effect of the COX and PKA inhibitors but not of the MEK, p38 MAPK, and NF-κB inhibitors. Conclusively, COXs and PGE(2) are important mediators of rotavirus infection at a postbinding step. The ERK1/2 pathway mediated by PKA is involved in COX induction by rotavirus infection. MAPK and NF-κB pathways are involved in rotavirus infection but in a PGE(2)-independent manner. This report offers new perspectives in the search for therapeutic agents in treatment of severe rotavirus-mediated diarrhea in children

Changes in Natural Foxp3⁺Treg but Not Mucosally-Imprinted CD62L^negCD38⁺Foxp3⁺Treg in the Circulation of Celiac Disease Patients

<div><p>Background</p><p>Celiac disease (CD) is an intestinal inflammation driven by gluten-reactive CD4⁺ T cells. Due to lack of selective markers it has not been determined whether defects in inducible regulatory T cell (Treg) differentiation are associated with CD. This is of importance as changes in numbers of induced Treg could be indicative of defects in mucosal tolerance development in CD. Recently, we have shown that, after encounter of retinoic acid during differentiation, circulating gut-imprinted T cells express CD62L^negCD38⁺. Using this new phenotype, we now determined whether alterations occur in the frequency of natural CD62L⁺Foxp3⁺ Treg or mucosally-imprinted CD62L^negCD38⁺Foxp3⁺ Treg in peripheral blood of CD patients. In particular, we compared pediatric CD, aiming to select for disease at onset, with adult CD.</p><p>Methods</p><p>Cell surface markers, intracellular Foxp3 and Helios were determined by flow cytometry. Foxp3 expression was also detected by immunohistochemistry in duodenal tissue of CD patients.</p><p>Results</p><p>In children, the percentages of peripheral blood CD4⁺Foxp3⁺ Treg were comparable between CD patients and healthy age-matched controls. Differentiation between natural and mucosally-imprinted Treg on the basis of CD62L and CD38 did not uncover differences in Foxp3. In adult patients on gluten-free diet and in refractory CD increased percentages of circulating natural CD62L⁺Foxp3⁺ Treg, but normal mucosally-imprinted CD62L^negCD38⁺Foxp3⁺ Treg frequencies were observed.</p><p>Conclusions</p><p>Our data exclude that significant numeric deficiency of mucosally-imprinted or natural Foxp3⁺ Treg explains exuberant effector responses in CD. Changes in natural Foxp3⁺ Treg occur in a subset of adult patients on a gluten-free diet and in refractory CD patients.</p></div

Demographic features of adult celiac disease (CD) patients and controls.

<p>CD, celiac disease; EmA, anti-endomysial antibodies; tTG anti-tissue transglutaminase antibodies; RCD, refractory celiac disease; EATL, enteropathy-associated T cell lymphoma; GFD, gluten free diet. All patients were tested for anti-tissue transglutaminase and anti-endomysial antibodies. * This patient was positive for anti-tissue transglutaminase antibodies.</p

Demographic features of pediatric celiac disease (CD) and controls.

<p>CD, celiac disease.</p

Distribution of CD62L/CD38 subsets within CD4⁺ peripheral blood lymphocytes; changes with age.

<p>Peripheral blood from pediatric CD patients was stained for flow cytometric analysis. (<b>a</b>) Representative gating strategy for analysis of CD62L/CD38 subsets within the CD4⁺ T cell population. Two representative CD62L/CD38 analyses of peripheral blood CD4⁺ T cells in CD patients aged 1 and 5 years old. (<b>b</b>) The percentages of cells in each of the four CD62L/CD38 T-cell subsets were calculated (Kruskal-Wallis test). CD, celiac disease.</p