Toll-like receptor signaling in small intestinal epithelium promotes B-cell recruitment and IgA production in lamina propria

Abstract

Background & aims: Several lines of evidence support a role for Toll-like receptor (TLR) signaling to protect the intestine from pathogenic infection. We hypothesized that TLR signaling at the level of the intestinal epithelium is critical for mucosal immune responses.

Methods: We generated transgenic mice that express a constitutively active form of TLR4 in the intestinal epithelium (V-TLR4 mice). Lamina propria cellularity was evaluated by immunostaining and flow cytometry. Immunoglobulin (Ig) A levels in the stool and serum were measured by enzyme-linked immunosorbent assay. Chemokine and cytokine expression were analyzed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay.

Results: V-TLR4 transgenic mice reproduced normally and had a normal life span. Constitutive activity of TLR4 in the intestinal epithelium promoted recruitment of B cells and an increase in fecal IgA levels. Intestinal epithelial cells of V-TLR4 mice expressed higher levels of CCL20 and CCL28, chemokines known to be involved in B-cell recruitment, and of a proliferation-inducing ligand (APRIL), a cytokine that promotes T-cell-independent class switching of B cells to IgA. The changes in B-cell numbers and IgA levels were blocked by simultaneous expression in intestinal epithelial cells of M3, a herpes virus protein that binds and inhibits multiple chemokines.

Conclusions: TLR signaling in the intestinal epithelial cells significantly elevated the production of IgA in the intestine. This effect was mediated by TLR-induced expression of a specific set of chemokines and cytokines that promoted both recruitment of B cells into the lamina propria and IgA class switching of B cells.

Figure 1. Expression of the CD4-TLR4 transgene in V-TLR4 mice

A) Diagram of the V-TLR4 transgene. The transgene encodes a fusion protein containing the mouse CD4 (mCD4) extracellular domain and human TLR4 signaling domain and is driven by the mouse villin promoter (mVillin). p(A) represents SV40 poly A sequences. B) CD4-TLR4 mRNA expression in different tissues of V-TLR4 mice. The values were standardized to ubiquitin levels in each sample. H: Heart; Lu: Lung; K: kidney; Li: liver; P: Pancreas; M: Skeletal Muscle; Sk: Skin; T: Testis; Br: Brain; LI: Large intestine; SI: Small intestine. C–D). Representative immunostaining for CD4 (red) and DAPI (blue) in small intestine of WT (C) and V-TLR4 (D) mice. E) Representative immunostaining for CD3 (red) and CD4 (green) in small intestine of V-TLR4 mice. Note that the transgene expression is restricted to epithelial cells. F) FACS analysis of CD4-TLR4 expression in epithelial cells isolated from small intestine. Cells were gated on the CD45⁻/PI⁻.

Figure 2. Activation of the TLR signaling pathway in IECs of V-TLR4 mice

A) DNA binding ELISA analysis of p65 in IECs isolated from the small intestine of WT and V-TLR4 mice (n=10/group, *p<0.05). B–D). Relative mRNA expression of TNFα (B), iNOS (C), and A20 (D) in IECs isolated from small intestine of WT and V-TLR4 mice (n=3/group; * p<0.05, ** p<0.01).

Figure 3. Transgene expression in IECs increases the number of B cells in the lamina propria

A–B) FACS analysis of CD19⁺ B cells (A) and CD3⁺ T cells (B) in lamina propria of WT and V-TLR4 mice. Values indicate the absolute number of each subset (n=4/group). Note the increased number of B cells in the proximal small bowel of V-TLR4 mice (** p<0.01).

Figure 4. Increased expression of chemokines in the proximal small intestine of V-TLR4 mice

A–B) Q-PCR analysis of CCL20, CCL28 (A) and CXCL16 (B) expression in the proximal small intestine of WT and V-TLR4 mice (n=7/group; ** p<0.01, *** p<0.001). C–D) CCL20 (C) and CCL28 (D) protein levels in small intestine of V-TLR4 mice and WT mice (WT n=12, V-TLR4 n=15; *** p< 0.001).

Figure 5. Increased IgA production in the lamina propria of V-TLR4 mice

A–B). FACS analysis of B cell (A) and plasma cell subsets (B) in lamina propria of proximal small intestine of WT and V-TLR4 mice (WT n=3, V-TLR4 n=4; * p< 0.05, ** p< 0.01). C & D). Immunostaining of IgA⁺ cells in duodenal lamina propria of WT (C) and V-TLR4 (D) mice. E). IgA levels in feces of WT (n=24) and V-TLR4 (n=50) mice (** p<0.01). F). Q-PCR analysis of pIgR expression in IECs of WT and V-TLR4 mice (n=4/group).

Figure 6. Increase in APRIL expression in IECs and B cell class switching to IgA in V-TLR4 mice

A). Q-PCR analysis of APRIL, BAFF and TSLP expression in proximal small intestinal epithelial cells of WT and V-TLR4 mice (n=3/group; **, p<0.01). B). Western blot of APRIL protein in proximal small intestinal epithelial cells of WT and V-TLR4 mice. C & D). Immunostaining of APRIL in duodenum of WT (C) and V-TLR4 (D) mice (red, APRIL; blue, DAPI). E). FACS analysis of IgM⁺/IgA⁺ cells in lamina propria of proximal small intestine of WT and V-TLR4 mice (WT n=3, V-TLR4 n=4; * p< 0.05). F). RT-PCR analysis of post switch circular transcript (α-CT) expression in lamina propria B cells from WT and V-TLR4 mice. Data represented pooled B cells from 20 WT or 10 V-TLR4 mice. Different bands represent different splice variants of α-CT as previously described .

Figure 7. Blockade of B cell recruitment and IgA production in the lamina propria of V-TLR4 mice by co-expression of chemokine binding protein M3

A). Absolute number of CD19⁺ B cells in the lamina propria of WT, V-TLR4, V-M3 and double transgenic mice (n=4/group; ** p<0.01, * p<0.05). B). Absolute number of IgA⁺ cells in the lamina propria of the proximal small intestine of WT, V-TLR4, V-M3 and double transgenic mice (n=4/group; ** p<0.01, * p<0.05). C). IgA levels in feces of WT, V-TLR4, V-M3 and double transgenic mice (WT n=36, V-TLR4 n=51, V-M3 n=18, V-M3/V-TLR4 double TG n=11; ** p<0.01, * p<0.05). D). Q-PCR analysis of CCL20, CCL28 and CXCL16 expression in the proximal small intestine of WT, V-TLR4, V-M3 and double transgenic mice (n=4/group).