Potentiation of polarized intestinal Caco-2 cell responsiveness to probiotics complexed with secretory IgA

Abstract

The precise mechanisms underlying the interaction between intestinal bacteria and the host epithelium lead to multiple consequences that remain poorly understood at the molecular level. Deciphering such events can provide valuable information as to the mode of action of commensal and probiotic microorganisms in the gastrointestinal environment. Potential roles of such microorganisms along the privileged target represented by the mucosal immune system include maturation prior, during and after weaning, and the reduction of inflammatory reactions in pathogenic conditions. Using human intestinal epithelial Caco-2 cell grown as polarized monolayers, we found that association of a Lactobacillus or a Bifidobacterium with nonspecific secretory IgA (SIgA) enhanced probiotic adhesion by a factor of 3.4-fold or more. Bacteria alone or in complex with SIgA reinforced transepithelial electrical resistance, a phenomenon coupled with increased phosphorylation of tight junction proteins zonula occludens-1 and occludin. In contrast, association with SIgA resulted in both enhanced level of nuclear translocation of NF-κB and production of epithelial polymeric Ig receptor as compared with bacteria alone. Moreover, thymic stromal lymphopoietin production was increased upon exposure to bacteria and further enhanced with SIgA-based complexes, whereas the level of pro-inflammatory epithelial cell mediators remained unaffected. Interestingly, SIgA-mediated potentiation of the Caco-2 cell responsiveness to the two probiotics tested involved Fab-independent interaction with the bacteria. These findings add to the multiple functions of SIgA and underscore a novel role of the antibody in interaction with intestinal bacteria.

FIGURE 1.

A, adhesion of L. rhamnosus LPR, B. lactis BL, and E. coli strains Nissle 1917 and TG1 to polarized intestinal Caco-2 cell monolayers. The number of bound bacteria per 100 Caco-2 cells is presented as a function of the concentration of freshly cultured bacteria added (CFU per ml). Bacterial counts were determined by plating of serial dilutions. Black bars, LPR; gray bars, BL; striped bars, Nissle 1917; white bars, E. coli TG 1. B, same as in A using 2 × 10⁷ bacteria alone (white bars) or associated with SIgA (black bars) prior to incubation with Caco-2 cell monolayers. Data were obtained from four independent experiments performed in triplicates. Significant statistical differences are indicated above the lanes.

FIGURE 2.

Laser-scanning confocal microscope imaging of the Fab-independent association of SIgAC5:Cy3 with LPR and BL. Bacteria are visualized by differential interference contrast (DIC, Nomarski), and bound Ab shows as co-localizing red fluorescent spots on the surface of bacteria. Formation of small strings is typical of the morphology of the microorganisms tested. One representative field obtained from 10 different observations after analysis of five different slides is shown. Bars: 5 μm.

FIGURE 3.

A, TER of epithelial Caco-2 cell monolayers exposed to 2 × 10⁷ LPR alone, 2 × 10⁷ BL alone, and in complexes with SIgA, determined at five time points. Description of symbols is given in the inset. SIgA used alone serve as control of the stability of the Caco-2 cell monolayer TER. Compilation of data from four independent experiments performed in triplicates is shown. Codes for symbols: light blue, LPR; dark blue, LPR + SIgA; pink, BL; purple, BL + SIgA; black, SIgA alone. B, overnight exposure of polarized Caco-2 cell monolayers to 2 × 10⁷ LPR or LPR-SIgA complexes increases phosphorylation of the tight junction proteins occludin and ZO-1. Cell lysates were immunoprecipitated (IP) with specific Ab, and detected by Western blot (Wb) with anti-phosphotyrosine, anti-phosphoserine, and Ab to occludin and ZO-1. C, quantification of phosphorylated occludin (P-occludin) and phosphorylated ZO-1 (P-ZO-1) after exposure of Caco-2 cells to 2 × 10⁷ LPR, 2 × 10⁷ BL, and in complexes with SIgA. Data were obtained from three independent experiments performed in triplicates. Comparative statistical analysis with the bar marked plain medium yielded p values < 0.002 for all experimental groups.

FIGURE 4.

Effect of LPR and BL818 along the NF-κB activation pathway. A, electrophoretic mobility shift assay performed with nuclear extracts from polarized Caco-2 cell monolayers incubated for 16 h with 2 × 10⁷ LPR alone, 2 × 10⁷ BL alone, or in association with SIgA, as indicated at the bottom of the lanes. Comp corresponds to a 20-fold molar excess of unlabeled NF-κB oligoNT probe to identify the specific retarded complex. NF-κB-probe complexes obtained when using nuclear extracts from cells stimulated with 2 × 10⁷ enteropathogic S. flexneri are shown for comparison. B, immunoblotting of IκBα in cytoplasmic extracts from Caco-2 cells incubated as in A with BL and Ab, or S. flexneri for comparison (upper panel). Nuclear translocation of NF-κB subunits p50 and p65/relA induced by the contact of Caco-2 cells with 2 × 10⁷ BL alone and in complex with SIgA, or 2 × 10⁷ S. flexneri for comparison (lower panels). Immunoblotting was carried out on Caco-2 cell nuclear extracts. Identical amounts of nuclear and cellular extracts based on protein concentration were used for each set of experiments. Panels are representative of one individual triplicate experiment performed three times.

FIGURE 5.

A, Western blot analysis of lysates recovered from polarized Caco-2 cell monolayers exposed to 2 × 10⁷ LPR alone, 2 × 10⁷ BL alone, and in the form of complexes with SIgA, as indicated on the top of the lanes. Detection was performed with rabbit anti-serum against SC recognizing both SC and the precursor pIgR. Signals caused by β-actin were obtained using a specific antiserum. B, densitometric analysis of two independent experiments was carried out with standardization based on the β-actin signal. The lane content is indicated below the plot. Significant statistical differences are indicated above the lanes. C, quantification of pIgR and converted SC was assessed by ELISA and is reported as a function of the amount measured expressed in ng per mg of protein in Caco-2 cell lysates. Data were gathered from three independent experiments performed in triplicates. Codes for symbols: light blue, LPR; dark blue, LPR + SIgA; pink, BL; purple, BL + SIgA; black, SIgA alone; circle, plain medium; diamond, S. flexneri.