Intraluminal poly I:C generates a proinflammatory response and the induction of MDA5, RIG-I and TLR3 mRNA expression in small intestine.

<p>Real Time-PCR analysis of small intestinal samples from C57BL/6 mice after intraluminal administration of poly I:C or PBS was performed. Plots shows mRNA expression after 2 to 12h of poly I:C (black dots) or PBS (empty dots) treatment. IFNβ, CXCL10, and TNFα mRNA expression (A). MDA5, RIG-I, and TLR3 mRNA expression (B). All values were normalized with the housekeeping mRNA expression (HPRT). Results were expressed as fold increase of PBS and poly I:C treatment versus the mean of PBS treatment in every time point (2^-ΔΔCt method). Stats: N =  4 mice per group, Unpaired t test, *P<0.05, **P<0.01, ***P<0.001, poly I:C treated mice versus PBS control in the same time point.</p

Intraluminal poly I:C induces enteropathy.

<p>Representative H&E stained sections of proximal small intestine of C57BL/6 mice after 12hs and 72hs of intraluminal administration of poly I:C or PBS (<b>A</b>). Morphological analysis of small intestine from C57BL/6 mice: villus-to-crypt (V/C) ratio, number of IELs and histological score after 12h (<b>B</b>) and 72h (<b>C</b>) (Stats: N =  4 mice per group, Unpaired t test, *P<0.05, **P<0.01, ***P<0.001).</p

Increased serum levels of TNFα, MCP-1, and IL-6 after intraluminal poly I:C administration.

<p>TNFα, MCP-1, and IL-6 concentration was assessed in serum after 3h of poly I:C or PBS treatment, using a cytometric bead array inflammation kit. The dotted line represents the limit of detection. Stats: N =  4 mice per group, Unpaired t test, *P<0.05, **P<0.01.</p

Intraluminal administration of poly I:C induces enteropathy in NOD-DQ8 mice.

<p>Representative H&E-stained sections of the proximal small intestine in PBS and poly I:C-treated NOD-DQ8 mice at 12h post-treatment (<b>A</b>). Morphological analysis of small intestine from NOD-DQ8 mice: villus-to-crypt (V/C) ratio, number of IELs and histological score after 12h. Stats: N =  4 mice per group, Unpaired t test, *P<0.05, **P<0.01 (<b>B</b>).</p

Intraluminal Administration of Poly I:C Causes an Enteropathy That Is Exacerbated by Administration of Oral Dietary Antigen

<div><p>Systemic administration of polyinosinic:polycytidylic acid (poly I:C), mimics virally-induced activation of TLR3 signalling causing acute small intestine damage, but whether and how mucosal administration of poly I:C causes enteropathy is less clear. Our aim was to investigate the inflammatory pathways elicited after intraluminal administration of poly I:C and determine acute and delayed consequences of this locally induced immune activation. Intraluminal poly I:C induced rapid mucosal immune activation in C57BL/6 mice involving IFNβ and the CXCL10/CXCR3 axis, that may drive inflammation towards a Th1 profile. Intraluminal poly I:C also caused enteropathy and gut dysfunction in gliadin-sensitive NOD-DQ8 mice, and this was prolonged by concomitant oral administration of gliadin. Our results indicate that small intestine pathology can be induced in mice by intraluminal administration of poly I:C and that this is exacerbated by subsequent oral delivery of a relevant dietary antigen.</p></div

Primers used for quantitative PCR.

<p>Primers used for quantitative PCR.</p

Gliadin induces barrier dysfunction and proinflammatory cytokines in poly I:C-treated NOD-DQ8 mice.

<p>After 24h of the final gavage, sections of small intestine were mounted in Ussing chambers and tissue conductance (mS/cm2) and isotopic flux (% Hot/h/cm²) were measured. Each dot represents an individual mouse and is a mean of 2 independent tissues. Stats: N =  3-4 mice per group, Two way ANOVA and Bonferroni post-test, *P<0.05 (<b>A</b>). Analysis of IL-12p70, TNFα and IL-10 in serum samples from poly I:C or PBS-treated mice on a gluten-free diet (GFD) or gavaged with gliadin for 2 weeks. IL-12p70/IL-10 ratio was determined. Serum concentration of cytokines was determined by a cytometric bead array inflammation kit. The dotted line represents the limit of detection. Stats: N =  4-5 mice per group, Two way ANOVA and Bonferroni post-test, *P<0.05, **P<0.01 gliadin versus GFD mice; #P<0.05 poly I:C versus PBS in Gliadin-gavaged mice (<b>B</b>).</p

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

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