Innate immune functions of avian intestinal epithelial cells: Response to bacterial stimuli and localization of responding cells in the developing avian digestive tract

<div><p>Intestinal epithelial cells are multi-tasked cells that participate in digestion and absorption as well as in protection of the digestive tract. While information on the physiology and immune functions of intestinal epithelial cells in mammals is abundant, little is known of their immune function in birds and other species. Our main objectives were to study the development of anti-bacterial innate immune functions in the rapidly developing gut of the pre- and post-hatch chick and to determine the functional diversity of epithelial cells. After establishing primary intestinal epithelial cell cultures, we demonstrated their capacity to uptake and process bacteria. The response to bacterial products, LPS and LTA, induced expression of pro-inflammatory cytokine genes (IL-6, IL-18) as well as the expression of the acute phase proteins avidin, lysozyme and the secretory component derived from the polymeric immunoglobulin receptor. These proteins were then localized in gut sections, and the goblet cell was shown to store avidin, lysozyme as well as secretory component. Lysozyme staining was also located in a novel rod-shaped intestinal cell, situated at different loci along the villus, thus deviating from the classical Paneth cell in the mammal, that is restricted to crypts. Thus, in the chicken, the intestinal epithelium, and particularly goblet cells, are committed to innate immune protection. The unique role of the goblet cell in chicken intestinal immunity, as well as the unique distribution of lysozyme-positive cells highlight alternative solutions of gut protection in the bird.</p></div

E17-IEC cultures respond to bacterial LPS and LTA.

<p>Pooled IEC cultures (n = 20 embryos) were cultured with increasing dosages of <i>E</i>. <i>coli</i> LPS or <i>B</i>. <i>subtilis</i> LTA (1 and 10 μg/ml; control = 0) and gene expression was determined 6 hours later (TLR2, TLR4, MyD88 and CD14). Transcriptional levels were determined by quantitative real-time PCR using GAPDH and 18S mRNA as normalizing genes. Results are averages of at least 4 similar experiments ± SEM (* indicates significant responses above control p≤0.05).</p

Avidin staining in gut of pre- and post-hatch chicks.

<p>Two—3μm thick slides were stained by polyclonal rabbit anti-chicken avidin and color was developed by HRP-labeled goat ant rabbit IgG. A. Duodenum of E19 embryos (x200), B. Cecum of 2-day old chick (x400), C. Colon of 3-day old chick (x400). Insert in A–negative control (only secondary antibody).</p

Lysozyme staining in gut of pre- and post-hatch chicks.

<p>Two—3μm thick slides were stained by mouse anti-lysozyme and HRP-conjugated goat anti mouse IgG (H+L). A-D duodenum day 0 (x400), E-F duodenum day 0 (x1000). Arrow indications are described in Results. Negative control (secondary antibody only)—Insert in panel F.</p

Intestinal epithelial cells internalize and process bacteria conjugated bioparticles.

<p>Chick cecal IEC were pulsed with pHrodo™ Green E. Coli BioParticles® or pHrodo™ Red S. aureus BioParticles®. Micrographs were taken after 6 (<i>S</i>. <i>aureus</i>) or 36 hours (<i>E</i>. <i>coli</i>).</p

Development of secretory component (GGpIgR) in chick gut.

Panel A: Secretory component specific staining in intestine of 30-day old chicks. Two– 3μm slides were stained by mouse monoclonal anti-human secretory component and then HRP-conjugated goat anti mouse antibody (left and central micrographs x200; right micrograph x1000). Panel B: Temporal development of GGpIgR in pre- and post- hatch chicks (Staining–as above) (x200). C. GGpIgR mRNA Expression in pre- and post-hatch IEC. Expression of GGpIgR was determined in RNA prepared from stripped IEC (n = 5 for each time point). Transcriptional levels were determined by quantitative real-time PCR using GAPDH and 18S mRNA as normalizing genes. Results are averages of at least 3 similar experiments ± SEM (Different letters indicate significant differences in GGpIgR expression (PE. coli LPS or B. subtilis LTA (1 and 10 μg/ml; control = 0) and gene expression was determined 6 hours later. Transcriptional levels were determined by quantitative real-time PCR using GAPDH and 18S mRNA as normalizing genes. Results are averages of at least 4 similar experiments ± SEM (Different letters indicate significant differences in GGpIgR expression (P<0.05).</p

Avidin and lysozyme gene expression following stimulation by bacterial LPS and LTA.

<p>Pooled IEC cultures (n = 20 embryos) from either duodenum or cecum were cultured with or without <i>E</i>. <i>coli</i> LPS (basal and 1 μg/ml) or <i>B</i>. <i>subtilis</i> LTA (basal and10 μg/ml) and gene expression was determined 6 hours later. Transcriptional levels were determined by quantitative real-time PCR using GAPDH and 18S mRNA as normalizing genes (basal expression was 0.8–1.0). Results are averages of at least 4 similar experiments ± SEM (* indicates significant responses above control p≤0.05).</p

Morphological characterization of chick IEC cultures.

<p>Micrographs A & B show a typical epithelial monolayer under phase contrast microscopy. In A, the arrow head shows the intestinal fragment from which the monolayer originated. The monolayer gradually extended and assumed a typical mosaic structure (B) typical to epithelium. These structures are further characterized by light microscopy (May-Grunwald staining) (C & D). The typical mosaic structure is seen in the central area of D. The epithelium was stained with two FITC labeled polyclonal antibodies: mouse anti-villin antibody (E) and mouse anti-E-cadherin antibody (F). Goblet cells were demonstrated by staining with either goat polyclonal antibody to Muc2 (G) followed by staining with Alexa Fluor® 488 donkey anti goat IgG (H+L) or PAS (H; left x 4, right x 40).</p

PCR primer pairs.

<p>PCR primer pairs.</p

E17-IEC cultures respond to bacterial LPS and LTA.

<p>Pooled IEC cultures (n = 20 embryos) were cultured with increasing dosages of <i>E</i>. <i>coli</i> LPS or <i>B</i>. <i>subtilis</i> LTA (1 and 10 μg/ml; control = 0) and gene expression was determined 6 hours later (IL-6, IL-18, AvBD1). Transcriptional levels were determined by quantitative real-time PCR using GAPDH and 18S mRNA as normalizing genes. Results are averages of at least 4 similar experiments ± SEM (* indicates significant responses above control p≤0.05).</p