Fig 2: AzA’s impact on the immune-inflammation status in DSS-induced colitis mice. The relative mRNA expression levels of the inflammatory cytokines IL-1β (A), IL-6 (B), TNF-α (C), IL-17A (D), and IL-22 (E) were assessed using RT-PCR. (F) Serum IL-6 levels in each group. (G) MPO activity in the colon. (H) The cells of the MLNs were stained with mAbs against mouse CD4, CD25, and Foxp3. Flow cytometry was used to analyze the stained cells. (I) The number of CD4+CD25+Foxp3+Treg cells was calculated. Data is presented as the mean ± SEM (n = 6). Statistical analysis was performed with one-way ANOVA followed by the LSD test. #P < 0.05, ##P < 0.01, and ###P < 0.001 vs the water + 0.9% NaCl group and *P < 0.05 and **P < 0.01 vs the DSS + 0.9% NaCl group.

Fig 3: The gut microbiota is associated with Treg cell differentiation in Emu infection.a After depletion of the gut microbiota with antibiotic cocktail treatment (ABX), the proportion of colonic CD25+Foxp3+ Treg cells in the CD3+CD4+ cell population in Emu-infected (IN) mice was significantly decreased in comparison with that in the infected mice treated with Mock. Naive mice (N) with or without ABX treatment were included as controls. b Schematic showing the experimental design for fecal transplantation transfer (FMT). Recipient mice were pretreated with ABX cocktails for 2 weeks, followed by an FMT regime of 2 weeks from naive donors (ND) or helminth-infected donors (HID). c The proportion of colonic CD25+Foxp3+ Treg cells in the CD3+CD4+ cell population by flow cytometry analysis for the recipient mice. d The proportion of colonic Helios+ Treg cells in Foxp3+ Treg cells by flow cytometry analysis for the recipient mice. The flow cytometry analysis was performed at 4 weeks post-FMT engraftment. Experiments were repeated at least 2 times with similar results. The data shown are from a representative experiment (mean ± SD shown). ns, not significant; *P < 0.05, ***P < 0.001 using the two-sided Student t-test (n = 4–6 mice for panel a; n = 7 for panels (b–d)).

Fig 4: The L. reuteri isolate enhances Treg frequency and is promoted by Emu infection.a The schematic showing the experimental design for L. reuteri and other control microbe administration experiments. The mice were orally gavaged with each isolate daily for 2 weeks (on days 0–14) after antibiotics (ABX) treatment. b The level of each bacterium was measured by qPCR on day 28. c The frequency of colonic Foxp3+ Treg cells was quantitatively measured by Flow cytometry on day 42. The L. reuteri isolate (2 × 109 CFU per mouse) was administrated to the mice on day 60 post-Emu infection for panels d-e and the levels of the bacterium in feces were detected. d The level of L. reuteri in naive or Emu-infected Balb/c mice. e The level of L. reuteri in naive or Emu-infected Nude Balb/c mice. Data for each sample are presented as the mean expression level in arbitrary units. Experiments were repeated at least 2 times with similar results. The data shown are from a representative experiment (mean ± SD shown). ns, not significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 using the two-sided Student t-test for panels (n = 6 mice per group for panels b, c; n = 8 for panel d; n = 5 for panel e).