Recruitment of inflammatory cells in the MLN after CpG-ODN treatment.

<p>Eight-day-old neonatal mice received 20 µg/g of CpG-ODN by the oral route. Twenty-four hours later, MLN from 10 neonates were removed and pooled for FACS analysis. Four pools in each group were analyzed. These data were from two representative experiments. Mann-Whitney non parametric test ** p<0.005, * p<0.05.</p

Role of IL10 in the chemokine response induced by CpG-ODN stimulation.

<p>(A) Ilea of 8-day old neonatal (n = 8) and of adult (n = 6) mice were removed for RNA extraction. IL10 and FoxP3 mRNA levels were measured by real-time RT-PCR. Data were analyzed by the non-parametric Mann-Whitney test. (B) Neonates (n = 3) and adults (n = 3) IL10-deficient mice received PBS or 20 µg/g CpG-ODN orally and 10 µg/g intraperitoneally. They were killed 6 hours later and the ilea were removed. Total RNAs were extracted and RT-PCR analyses were performed for CXCL1, CXCL10, CCL2 and CCL7.</p

Chemokine responses of intestinal epithelial cells after CpG-ODN stimulation.

<p>(A) Intestinal epithelial cells (IEC) were purified from neonatal (n = 5) or adult mice (n = 5). RNA was extracted from the purified cells for TLR9 mRNA RT-PCR analysis. (B) Two murine intestinal epithelial cell lines (ICcl2 and CMT93) and purified IECs from adults and neonates were stimulated in vitro with 5 µg/ml of CpG-ODN for 2 hours, and RNAs were extracted for CXCL1 and CXCL10 measurement. (C) Neonatal (n = 30 for each group) and adult mice (n = 3 for each group) received CpG-ODN by the oral and IP route. They were killed 6 hours later, and the IECs were purified. RNAs were extracted and RT-PCR analyses were performed for CXCL1 and CXCL10 expression. Data were submitted to the non-parametric Mann-Whitney test. (*, p<0.05). ND, Not Done.</p

Recruitment of inflammatory cells in the intestine of neonates after CpG-ODN treatment.

<p>Eight-day-old neonatal mice received 20 µg/g of CpG-ODN orally. Twenty-four hours after treatment, ilea were removed to analyze the recruitment of inflammatory cells by immunohistology. Six to ten neonates from different litters (5 sections for each animal) per group were analyzed. Data were analyzed by non-parametric Mann-Whitney test.</p

IL12p40 and IFNγ responses after CpG-ODN treatment in the intestine.

<p>CpG-ODN was administered orally in 8-day-old WT and TLR9 KO neonate mice at a dose of 20 µg/g (black boxes). Control mice received PBS orally (white boxes). N = 5 neonates from two different litter for each group. Six hours later, ilea were removed for RNA extraction. RT-PCRs were performed for amplification of IL12p40 (A) and IFNγ (B) mRNA. These data are representative of two experiments. Data were analyzed by the non-parametric Mann-Whitney test.</p

Antibody production.

<p>(a) Schematic representation of recombinant IgA2 constructs bearing GFP or Luciferase (Luc) at the N terminus. Numbers in brackets correspond to the lanes of gels depicted in (b) and (c). Glycosylation sites on asparagine residues (N) are indicated in rectangles connected to alpha chain domains. (b) Recombinant Igs separated by SDS-PAGE under reducing conditions and stained with Coomassie blue. (c) Analysis of a selection of recombinant Igs by SDS-PAGE under nonreducing conditions and stained with Coomassie blue. The identification of the various assembled forms was determined based on immunodetection with Abs specific to the alpha and kappa chains, respectively (not shown). 1, human m-IgA2 (monomer); 2, human d-IgA2 (dimer carrying the J chain); 3, murine m-IgA (monomer); 4, human IgE; 5, human IgG; 6, human m-IgA1 (monomer); 7, human mI-gA2 (monomer); 8, human m-IgA2 G0 (no glycosylation – monomer); 9, human m-IgA2 G1 (1 glycosylation site (Asn²⁶³) – monomer); 10, human m-IgA2 G2 (2 glycosylations sites (Asn²⁶³ and Asn⁴⁶⁹) – monomer); 11, human m-IgA2 (monomer); 12, human m-IgA2 dPB (monomer lacking the basal part); 13, human m-IgA2 dCα3 (monomer lacking the basal part and Cα3); 14, human m-IgA2 dCα2/3 (monomer lacking the basal part, and domains Cα3 and Cα2); 15, human m-IgA2 Cα1 G0 (Cα1 domain only, no glycosylation); 16, human m-IgA2 Cα1 (Cα1domain only).</p

Validation and characterization of the inverted <i>in vitro</i> model of human FAE.

<p>(a) Schematic representation of the successive steps leading to the establishment of the M-like cell-containing Caco-2 cell monolayer. Molecular and cellular partners added as a function of the experimental setting are depicted. D, day. (b) Slight decrease of transepithelial electrical resistance (TEER) was observed during M cell conversion of polarized Caco-2 cell monolayer. (c) Localization by immunofluorescence of ZO-1 and CA19.9 in the mono- and co-culture models shows tight intercellular junctions around CA19.9⁺ M cells (top view). (d1) Identification of M-like cells by transmission electron microscopy (TEM). In co-culture, M-like cells were identified by the effacement of microvilli at the apical surface and the presence of enfolded lymphocytes (side view). Mono-cultures present a columnar shape as well as a brush border. (d2) Scanning electron microscopy (SEM) confirmed the presence of M-like cells and the lack of microvilli at their apical surface (top view). (e1) Identification and localization of M-like cells and enterocyte cells by immunofluorescence staining with anti-CA19.9-PE mAb and lectin UEA-1-FITC, respectively (top view). (e2) Co-localization evaluated by immunofluorescence microscopy of CA19.9-PE and IgA2-GFP indicates binding of the Ab to M-like cells (top view). (f) Increased apical-to-basolateral transport of 0.2 µm yellow-green-conjugated NPs across the co-cultures as compared to mono-cultures. Absence of transport at 4°C is indicative of an active membrane trafficking process. Each set of experiments was repeated at least twice.</p

Influence of glycosylation and sialylation on the uptake of m-IgA2 by M-like cells.

<p>(a) Mono- and co-cultures were incubated for 90 min at 37°C with various hypoglycosylated, or enzymatically deglycosylated, IgA2 constructs, and transported recombinant IgA (m-IgA2) was evaluated by associated luminescence (<i>n</i> = 4). PNGase, Peptide N:glycosidase; Neura, neuraminidase. (b) Same set of experiments performed with recombinant anti-CD20 m-IgA2, human colostrum SIgA, and plasma-purified IgA; transported IgA was measured by ELISA. (c) IgA deglycosylation and desialylation was monitored by Western blot analysis using detection with HRP-conjugated lectins Ulex europaeus-1 and wheat germ agglutinin, respectively. Lane content: 1, m-IgA2 G0; 2, m-IgA2 G2; 3, m-IgA2; 4, colostrum SIgA+PNGase; 5, colostrum SIgA.</p

IgA2 interacts with Dectin-1 and Siglec-5–expressing cells.

<p>(a) Recognition by m-IgA2 of recombinant Dectin-1 and Siglec-5 used as coating molecules (<i>n</i> = 6), as measured by ELISA. (b) Determination by immunofluorescence of the binding of m-IgA2 to Dectin-1 and Siglec-5 receptors expressed by double-transfected HEK cells. Co-localization resulted in the appearance of yellow areas in the cell periphery. In control experiments, no binding of IgA1 was detected, and nontransfected HEK cells did not stain (<i>n</i> = 2). (c) In flow cytometry analysis, cells were plotted according to the FSC and SSC profiles and gated to include only HEK cells. A second selection was performed to include only those cells positive for Dectin-1 and Siglec-5.</p

Dectin-1 on M cells is essential for SIgA uptake and elicitation of Ab responses against an associated Ag.

<p>(a) Incubation of p24-SIgA (red, indicated by arrowheads) for 90 min in a ligated intestinal loop containing a PP resulted in the targeting of the complex to Dectin-1⁺ M cells in the FAE, as indicated by the appearance of numerous yellow spots. V, villi. (b) Oral immunization of wt mice and chimeric-wt:KO (wt mice reconstituted with bone marrow cells from Dectin-1 KO mice) with HIVp24-SIgA resulted in the production of antigen-specific seric Abs, whereas Dectin-1 KO mice and chimeric-KO:wt mice (Dectin-1 KO mice reconstituted with bone marrow cells from wt mice) did not respond. (c) An identical pattern of Ag-specific Ab production was observed in the feces of immunized mice. Samples were collected 1 wk after the last immunization and Ab production was measured by ELISA. Horizontal bars show the mean value ± SEM (*<i>p</i><0.05).</p