AhR/IL-22 pathway as new target for the treatment of post-infectious irritable bowel syndrome symptoms

Figures & data

Table 1. Histological score of colon mice

Evaluation criteria	Score	Characteristic(s)
Infiltration of inflammatory cells	0	No inflammatory cells in the lamina propria
	1	Increased numbers of inflammatory cells, including neutrophils in the lamina propria
	2	Confluence of inflammatory cells extending into the submucosa
	3	Transmural extension of the inflammatory cell infiltrate
Goblet cells	0	Presence of goblet cells in sufficient number
	1	Loss of goblet cells number
	2	Absence of goblet cell
Oedema size	0	No oedema
	1	Presence of a little oedema
	2	Presence of a big oedema
Epithelial damage	0	Absence of mucosal damage
	1	Discrete focal lympho-epithelial lesions
	2	Mucosal erosion/ulceration
	3	Extensive mucosal damage and extension through deeper structures of the bowel wall
Hyperplasia	0	No hyperplasia
	1	Low hyperplasia
	2	Moderate hyperplasia
	3	Severe hyperplasia

Figure 1. Transient C. rodentium infection induces persistent perturbations in mice.

(a) Level of C. rodentium colonization followed by counting colony forming units (CFU) in the stools of infected mice (n = 8 per group) at different time points. (b) Low-grade inflammation assessed by measuring fecal lipocalin-2 at the peak of infection (7 DPI) and after clearance of the pathogen (23–24 DPI) (n = 12 per group). (c) Intestinal permeability assessed by measuring 4 kDa FITC-Dextran concentration in serum at the peak of infection (8 DPI) and after clearance of the pathogen (22 DPI) (n = 5–7 per group). (d-f) Colonic expression of (d) occludin, (e) claudin-2 and (f) ZO-1 mRNA in control (n = 10) and infected mice (n = 7–9) was quantified by RT-qPCR at 24 DPI. (g) Histological score and (h) histological analysis of colon by HPS staining in uninfected mice (left panel), C. rodentium-infected mice at 8 DPI (middle panel) and 23–24 DPI (right panel). Arrays represents crypts length. Scale bar = 50 µm. White circles represent control mice while black circles represent infected mice. Data are presented as mean ± SEM and are representative of two independent experiments. *p < 0.05 **p < 0.01 ****p < 0.0001.

Figure 2. Post-infectious visceral sensitivity and anxiety-like behavior induced by C. rodentium.

(a) Post-infectious colonic sensitivity assessed by measuring intracolonic pressure variations in response to CRD and (b) corresponding AUC at 24 DPI (n = 11–12 per group). (C-D) Post-infectious anxiety-like behavior assessed with the elevated plus maze test. (c) Entries frequency and (d) time spent in open arms of control and infected mice at 21 DPI (n = 8–9 per group). White circles represent control mice and black circles represent infected mice. Data are presented as mean ± SEM and are representative of at least two independent experiments. *p < 0.05; ***p < 0.001; ****p < 0.0001.

Figure 3. Colonic mucosa-associated microbiota dysbiosis after C. rodentium clearance in mice.

After clearance of C. rodentium (24 DPI), the composition of the colonic mucosa-associated microbiota was determined using 16S DNA sequencing (V4-V5 regions) (n = 10–11 per group). (a) Alpha diversity was determined by observed Operational Taxonomic Units (OTUs) measurement according to sequences per sample of control and infected mice. (b) Area under curve (AUC) of the observed OTUs for control and infected mice were calculated. (c) Principal coordinates analysis (PCoA) of the unweight UniFrac distance of control (red plots) and infected mice (blue plots). Significance was assessed with Adonis (p = 0,055). (d) Determination of Ratio Firmicutes/Bacteroidetes in control and infected mice. (e-f) Relative abundance (as a percentage) of phyla (e) and family (f) which are significantly modified in infected mice at 24 DPI. (g) Correlation between time spent in open arms (elevated plus maze test) and relative abundance of Lactobacillaceae (R²=0,3845; p = 0,003). (h) Fecal concentrations of SCFA were measured by gas chromatography for infected and control animals at the peak of infection (7 DPI) and after the clearance of C. rodentium (24 DPI) (n = 10–12 per group). White bars and circles represent control mice while black bars and circles represent infected mice. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4. AhR activity and tryptophan metabolic pathways are modified in post-infectious period.

Tryptophan and its catabolites levels were measured by liquid chromatography coupled with high resolution mass spectrometry in three different compartments of control and infected mice after the clearance of the pathogen (24 DPI) (n = 8 per group). (a) Fecal AhR activity, which reveal the amount of AhR ligands, was assessed in feces using HepG2-Lucia™ AhR reporter cells. Results were normalized on the basis of negative luciferase activity of the control, cytotoxicity measurement and feces weight. (b) Indole pathway metabolites were quantified in the serum, feces and caecum. (c) Tryptophol was quantified in feces and caecum. (d) Dose-effect of tryptophol on AhR activity was assessed. (e) Kynurenine pathway metabolites were quantified in serum, feces and caecum. (f) Kynurenine/tryptophan ratio in serum, feces and caecum was evaluated for each group. White circles represent control mice while black circles represent infected mice. Data are presented as mean ± SEM and each plot represent a mouse. * p < 0.05; ** p < 0.01.

Figure 5. IL-22 treatment reverses post-infectious anxiety-, cognition-like behaviors and ill-being induced by C. rodentium infection.

After a treatment with L. lactis^IL-22, anxiety-related behavior induced by a C. rodentium post-infection were determined by (a-b) EPM test at D21 (n = 7–8 per group). (a) Entries frequency and (b) time spent in open arms (monitoring for 5 minutes). (c) Number of head dips made on the hole board for 5 minutes were quantified (n = 10–12 per group). Cognition-related behaviors analysis were performed using (d) Y-maze test in which spontaneous alternation were measured for 10 minutes and (e-f) time spent exploring the novel object and the relocated object during novel object and relocation tests, respectively. (g-i) Mice specific behaviors assessed using PhenoTyper® at 23 DPI and recorded for 24 hours (n = 4–8 mice per group). (g) Time spent inside the hidden zone, (h) distance moved and (I) velocity were determined during the activity period (12 hours dark period). White squares represent uninfected mice treated with L. lactis^empty and light grey squares represent uninfected mice treated with L. lactis^IL-22 while black squares represent infected mice treated with L. lactis^empty and dark grey squares described infected mice treated with L. lactis^IL-22. Data are representative of two independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 ****p < 0.0001.

Figure 6. IL-22 treatment reverses post-infectious CHS and intestinal barrier alteration induced by C. rodentium infection independently of inflammation.

After a treatment with L. lactis^IL-22, (a) low-grade inflammation assessed by measuring fecal lipocalin-2 (24 DPI) (n = 9–12 per group). (b-c) Post-infectious colonic sensitivity using CRD response at D24 (n = 7–8 per group). (b) Intracolonic pressure variations in response to CRD and (c) corresponding AUC. Colonic expression of (d) occludin, (e) claudin-2 and (f) ZO-1 mRNA was quantified by RT-qPCR at 24 DPI (n = 7–8 per group). White squares represent uninfected mice treated with L. lactis^empty and light grey squares represent uninfected mice treated with L. lactis^IL-22 while black squares represent infected mice treated with L. lactis^empty and dark grey squares described infected mice treated with L. lactis^IL-22. Data are representative of two independent experiments. *p < 0.05; **p < 0.01.

Cryan JF, O’Riordan KJ, Cowan CSM, Sandhu KV, Bastiaanssen TFS, Boehme M, Codagnone MG, Cussotto S, Fulling C, Golubeva AV, et al. The microbiota-gut-brain axis. Physiol Rev. 2019;99(4):1877–2013. doi:10.1152/physrev.00018.2018.

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