Induction of CXCL10 by IL-15 and poly I:C in the small intestine.

<p>Biopsies from celiac patients and non-CD controls were incubated for 3 h in the presence of IL-15 (<b>a</b>) or Poly I:C (<b>b</b>). A second biopsy from each patient was cultured with medium (NS). In non-CD controls, both IL-15 and poly I:C induced CXCL10 mRNA expression (p = 0.0100 and p = 0.0058, respectively; paired t-test). No significant changes were observed in the untreated CD group.</p

Role of CXCR3/CXCL10 Axis in Immune Cell Recruitment into the Small Intestine in Celiac Disease

<div><p>Lymphocytic infiltration in the <i>lamina propria</i> (LP), which is primarily composed of CD4⁺ Th1 cells and plasma cells, and increased numbers of intraepithelial lymphocytes (IELs), is a characteristic finding in active celiac disease (CD). Signals for this selective cell recruitment have not been fully established. CXCR3 and its ligands, particularly CXCL10, have been suggested to be one of the most relevant pathways in the attraction of cells into inflamed tissues. In addition, CXCR3 is characteristically expressed by Th1 cells. The aim of this work was to investigate the participation of the chemokine CXCL10/CXCR3 axis in CD pathogenesis. A higher concentration of CXCL10 was found in the serum of untreated CD patients. The mRNA levels of CXCL10 and CXCL11 but not CXCL9 were significantly higher in duodenal biopsies from untreated CD patients compared with non-CD controls or treated patients. The results demonstrate that CXCL10 is abundantly produced in untreated CD and reduced in treated patients, and the expression of CXCL10 was found to be correlated with the IFNγ levels in the tissue. Plasma cells and enterocytes were identified as CXCL10-producing cells. Moreover, the CXCL10 expression in intestinal tissues was upregulated by poly I:C and IL-15. IELs, LP T lymphocytes, and plasma cells, which infiltrate the intestinal mucosa in untreated CD, express CXCR3. The CXCR3/CXCL10 signalling axis is overactivated in the small intestinal mucosa in untreated patients, and this finding explains the specific recruitment of the major cell populations that infiltrate the epithelium and the LP in CD.</p></div

Infiltration of CXCR3⁺ cells in the <i>lamina propria</i> of small intestine mucosa.

<p><b>a.</b> Confocal immunofluorescence for CXCR3 was performed in sections of duodenal biopsies from controls (i), untreated celiac patients (ii), and treated celiac patients (iii). CXCR3 is shown in green, and nuclei are shown in red. Untreated celiac patients showed a higher number of positive cells infiltrating the <i>LP</i>. (Magnification, 630×). <b>b.</b> The number of CXCR3⁺ cells in the LP was higher in the duodenal mucosa of untreated celiac patients (n = 9) compared with control individuals (n = 6) and treated patients (n = 5) (unpaired t-test; p = 0.0089 and p = 0.0055, respectively). The positive cells in LP regions from sections of duodenal biopsies were counted using immunofluorescence microscopy. <b>c.</b> Representative flow cytometric analysis from the LP compartment of a duodenal sample of an untreated CD patient showing plasma cells (CD138⁺) that express CXCR3. <b>d.</b> Representative flow cytometric analysis from the LP compartment of a duodenal sample of an untreated CD patient showing LP lymphocytes (CD3⁺ or CD4⁺) that express CXCR3.</p

CXCL9, CXCL10, and CXCL11 mRNA levels in the duodenum.

<p><b>a.</b> The mRNA expression levels of CXCR3 ligands were determined by real-time PCR. Duodenal biopsies from celiac individuals at the time of diagnosis (n = 26), celiac individuals on a GFD (n = 6), and non-CD controls (n = 25) were included. The untreated celiac patients expressed significantly higher levels of CXCL10 and CXCL11 than the treated patients (p = 0.0436 and p = 0.0160, respectively) and controls (p = 0.0002 and p<0.0001, respectively). There was no difference in the CXCL9 mRNA levels between the groups. The results are shown as relative units in reference with the levels of the housekeeping gene β-actin. An unpaired t-test was used to assess the significance of the differences. <b>b.</b> The correlation between the mRNA levels of CXCL10 and the mRNA levels of CXCL11 in duodenal samples from CD patients (black circles) and non-CD controls (white circles) was analysed. The CXCL10 and CXCL11 expression levels were correlated significantly in untreated CD patients (r = 0.7463, p<0.0001) and in non-CD controls (r = 0.6690, p = 0.0003).</p

Cellular sources of CXCL10 in the small intestinal <i>lamina propria</i>.

<p>The cellular sources of CXCL10 in the duodenum were identified using immunofluorescence confocal microscopy. CD3⁺ cells were found to be negative for CXCL10 in both CD patients (i) and control subjects (ii). Numerous CD138⁺ cells that express CXCL10 were found in the celiac mucosa (iii). Plasma cells expressing CXCL10 were not found in the duodenum from non-CD controls (iv). HAM56⁺ cells did not produce CXCL10 in the celiac (v) and in the non-CD control intestinal mucosa (vi). CD3, CD138, and HAM56 are shown in green, CXCL10 is shown in red, and nuclei are shown in blue. (Magnification, 1071×).</p

Infiltration of CXCR3⁺ cells in the intraepithelial compartment.

<p><b>a.</b> Confocal immunofluorescence for CXCR3 was performed in sections of duodenal biopsies. (i) Intraepithelial lymphocyte CXCR3⁺ cells in untreated CD patients are indicated by arrows. (ii). CXCR3⁺ cells were rarely observed in the intraepithelial compartment in non-CD controls. The epithelium is delimited by a thin line. CXCR3 is shown in green, and nuclei are shown in red. (Magnifications, 630× (i) and 1071× (ii)). <b>b.</b> Representative flow cytometric analysis from the epithelial compartment of a duodenal sample of an untreated CD patient showing IELs (CD3⁺ CD103⁺) that express CXCR3.</p

Serum levels of CXCL10.

<p>The CXCL10 concentrations in serum samples from 21 non-celiac individuals, 26 untreated celiac patients, and nine CD patients on a GFD were assessed by quantitative ELISA. The untreated CD patients presented higher levels of CXCL10 than the controls (unpaired t test; p = 0.0007). The treated celiac patients presented lower levels of CXCL10 than the untreated patients, although the difference was not statistically significant.</p

Expression of IFNγ, IFNβ, and TNFα in the duodenum and their correlation with the levels of CXCR3 ligands.

<p><b>a.</b> The mRNA levels of IFNγ, IFNβ, and TNFα were determined by real-time PCR in the same set of samples that were previously tested: untreated celiac (n = 26), patients on a GFD (n = 6), and non-CD controls (n = 25). The IFNγ expression levels were significantly higher in the untreated celiac patients compared with the treated patients (p = 0.0123) and the controls (p<0.0001). The TNFα levels in the untreated celiac patients were lower than those found in the controls and the treated patients (unpaired t-test; p = 0.0054 and p = 0.0004, respectively). No significant difference was observed in the IFNβ expression levels between these groups. <b>b.</b> The correlation of the CXCL10 expression levels with the IFNγ levels in duodenal samples from untreated CD patients and non-CD controls was analysed. The IFNγ levels were positively correlated with the CXCL10 expression level in both untreated celiac patients (r = 04233, p = 0.0312) and non-CD controls (r = 0.5448, p = 0.0049). Linear regression analysis, Pearson’s coefficient, F-test.</p

Overexpression of CXCL10 in the duodenal mucosa of untreated CD patients.

<p>A representative immunofluorescence confocal microscopic analysis of CXCL10 expression in duodenal sections of untreated CD (i) and control subjects (ii) is shown. <b>a.</b> Active CD patients showed a massive expression of CXCL10 in the entire mucosa, whereas the controls only showed isolated CXCL10⁺ cells in the LP. CXCL10 is shown in green, and the nuclei are shown in red. (Magnification, 630×). <b>b.</b> The arrows indicate epithelial cells that produce CXCL10 in the duodenum from an untreated CD patient (i). In non-CD controls (ii), CXCL10 expression was not observed in the epithelium, and CXCL10⁺ cells were rarely found in the LP. (Magnification, 1071×).</p

<i>In vitro</i> stress treatments change the pattern of MICA/B expression. A. Induction of TIA-1⁺ granules in Caco-2 cells.

<p>Confocal microscopic analysis of Caco-2 cells treated during different periods of time with thapsigargin, sodium arsenite or fever-range temperature showing redistribution of TIA-1 (white) into stress granules. Nuclei (blue). (scan zoom 0,7, magnification 100×). <b>B.</b>- <b>Redistribution of MICA/B in treated Caco-2 cells</b>. Confocal microscopic analysis of Caco-2 cells treated during different periods of time with thapsigargin, sodium arsenite or fever-range temperature showing redistribution of MICA/B (red) in cytoplasmic aggregates. Nuclei (blue). (scan zoom 0,7, magnification 100×). <b>C.</b>- <b>Distribution of MICA/B and TIA-1 in Caco-2 treated cells.</b> Confocal microscopy of Caco-2 cells treated with thapsigargin (ER stress) or sodium arsenite (oxidative stress) for 1 hour, showing MICA/B (red) and TIA-1 (white) (magnification 100×). In both cases, MICA/B⁺ structures were not associated to stress TIA-1⁺ granules. (scan zoom 0,7, magnification 100×).</p