Iga Quantification in the Intestinal Wash
Methods
IgA quantification in the intestinal wash
IgA levels were quantified in the intestinal wash from small intestine, caecum and colon. Briefly, the organs were flushed with PBS (Gibco) containing Soybean trypsin inhibitor (Sigma-Aldrich), EDTA and Phenylmethanesulfonyl fluoride (Sigma-Aldrich). To measure IgA concentration, goat anti-mouse antibody (clone G18) was used for capture and alkaline-phosphatase-conjugated antibody (clone G41) for detection. Mouse IgA was used as standard. All antibodies were purchased from Southern Biotech (Birmingham, AL, USA).
Quantitative analysis of commensal bacteria
Pregnant C57BL/6 and TSLPR-/- females were housed in the same cage from the second week of pregnancy. After weaning, C57BL/6 and TSLPR-/- pups were housed in the same cage until sacrifice.
Quantitative PCR for 16S rDNA was performed on DNA purified from the ileal, caecal and colonic biofilm, and normalized to the total quantity of DNA recovered. DNA extraction was performed using a CTAB-based protocol1. Primer sequences were published by Bouskra et al.2.
FIGURE LEGENDS
Supplementary Figure 1. MP/DC phenotype is not affected by the absence of TSLP-TSLPR signaling. (A) Expression of the activation markers CD40, CD80 and CD86 was determined by flow cytometry in cells isolated from cLP of C57BL/6 and TSLPR-/- mice. MP/DC subsets were identified as shown in Figure 2. Results are shown from 1 representative experiment of 2 (n=4-5 per group).
Supplementary Figure 2. In the absence of intestinal flora, TSLPR-/- mice do not show lymphoid follicle hyperplasia. Quantification of the (A) number of immature and mature LFs and (B) size of mature LFs in C57BL/6 or TSLPR-/- germ-free mice. Data are shown from one representative experiment of two (n=5 per group).
Supplementary figure 3. The absence of TSLP signaling does not impact on IgA production or the composition of intestinal bacterial communities. Total IgA quantification by ELISA (left panel) and determination of the bacterial communities by 16S rDNA qPCR (right panel) from (A) small intestine and (B) colon of SPF C57BL/6 and TSLPR-/- mice. Results are shown from 1 representative experiment of 2 (n=4-6 per group).
Supplementary Figure 4. In the presence of SPF flora, TSLPR deficiency does not result in defective expansion of the Helios- Treg population. Quantification of Helios-/Helios+ ratios and Helios-Treg percentages in CD25+FoxP3+ cells from (A) cLP and (B) MLN of SPF mice. Results are shown from from one representative experiment of two (n=6 per group).
Supplementary Figure 5. TSLP neutralization using mAb treatment replicates observations from TSLPR deficient mice. (A) Representative plots of IL-17A and IFN production by CD4+ cells isolated from cLP of day 28 ASF colonized mice treated with isotype control or anti-TSLP antibody after PMA/Ionomycin restimulation. (B) Representative histograms of Helios staining in CD25+ FoxP3+ cells from cLP of day 28 ASF colonized mice treated with anti-TSLP or isotype control antibody. Results are shown from 1 experiment (n=3-4 per group).
Supplementary Figure 6. TSLP acts on CD4+ T cells to downregulate cytokine production in the MLN. Representative plots and quantification of TSLPR-/- (CD45.2+) and wild type CD45.1 (CD45.2-) FoxP3+ cells isolated from MLN of day 28 ASF RAG-/- mice reconstituted with CD4+ splenocyte from SPF TSLPR-/- or CD45.1 mice. Results from one experiment representative of two (n=5, per group per experiment).
REFERENCES
1.Minas, K., McEwan, N.R., Newbold, C.J. & Scott, K.P. Optimization of a high-throughput CTAB-based protocol for the extraction of qPCR-grade DNA from rumen fluid, plant and bacterial pure cultures. FEMS Microbiol Lett 325, 162-169 (2011).
2.Bouskra, D. et al. Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis. Nature 456, 507-510 (2008).
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