Sex differences in anatomic plasticity of gut neuronal-mast cell interactions

Abstract

The gut wall houses mast cells that are anatomically situated near enteric neuronal fibers. Roles of specific neuropeptides in modulating function of immune components like mast cells in response to challenge with bacterial components are relatively unknown. Investigating such interactions requires models that include diverse cellular elements in native anatomic arrangements. Using an organotypic slice model that maintains gut wall cellular diversity ex vivo, the present study compared responses between tissues derived from male and female mice to examine neural-immune signaling in the gut wall after selected treatments. Ileum slices were treated with pharmacological reagents that block neuronal function (e.g., tetrodotoxin) or vasoactive intestinal peptide (VIP) receptors prior to challenge with lipopolysaccharide (LPS) to assess their influence on anatomic plasticity of VIP fibers and activation of mast cells. Sex differences were observed in the number of mucosal mast cells (c-kit/ACK2 immunoreactive) at baseline, regardless of treatment, with female ileum tissue having 46% more ACK2-IR mast cells than males. After challenge with LPS, male mast cell counts rose to female levels. Furthermore, sex differences were observed in the percentage of ACK2-IR cells within 1 µm of a VIP+ neuronal fiber, and mast cell size, a metric previously tied to activation, with females having larger cells at baseline. Male mast cell sizes reached female levels after LPS challenge. This study suggests sex differences in neural-immune plasticity and in mast cell activation both basally and in response to challenge with LPS. These sex differences could potentially impact functional neuroimmune response to pathogens.

Keywords: VIP; gut; intestine; mast cell; mucosa.

FIGURE 1

Basal sex difference in mast cell count per crypt‐villus axis (CVA) and mean ACK2 cell size were observed. Representative images from male (a) and female (b) ileum samples with arrows denoting representative ACK2 immunoreactive (IR) cells (red) not near a VIP‐immunoreactive fiber (green), while arrow heads point to exemplary ACK2‐IR cells within 1 μm of a VIP‐immunoreactive fiber. Cropped images in (a’) and (b’) show zoomed views of ACK2 cell proximity with VIP neuronal fibers. (c) quantification of ACK2 cell counts per CVA. Quantification of ACK2 cell size (d) and the percentage of ACK2‐IR cells within 1 μm of a VIP‐IR fiber (e). n = 6 males, 6 females. ‘sm’ denotes submucosa, ‘c’ crypt, and ‘me’ muscularis externa. Scale bars in (a) and (b) are both 25 μm

FIGURE 2

Sex and LPS effects were observed in ACK2‐IR cell counts per CVA regardless of treatment, and in the percentage of ACK2‐IR cells within 1 μm of a fiber. (a‐d) representative images from a male ileum treated with vehicle (a) and vehicle +LPS (B) showing ACK2‐IR cells (red) and VIP‐IR fibers (green). Representative images from female ileum treated with vehicle (c) or vehicle +LPS (d). (e) is a 3‐way ANOVA quantitation showing effects of sex and LPS treatment on ACK2 cell counts. (f) is a 3‐way ANOVA quantitation showing effects of sex and LPS treatment on the percentage of ACK2‐IR cells within 1 μm of a VIP‐IR fiber. Vertical significance bars in (e) and (f) denote an observed effect of LPS while horizontal significance bars denote observed sex effects. n = 4 male, 4 female in ‘NO LPS’ groups, and n = 5 male, 5 female in ‘LPS’ groups Arrow heads in (a‐d) point towards exemplary ACK2‐IR cells within 1 μm of a VIP‐IR fiber. ‘v’ denotes a villus, ‘L’ lumen. Scale bars are 25 μm in (a‐d). ‘*’ denotes a p < 0.05, and ‘**’ a p < 0.01

FIGURE 3

Female ACK2‐IR cells were larger than males, an effect lost when slices were challenged with LPS. (a/e) and (b/f) are representative images of ACK2‐immunoreactive cells from male and female ileum slices treated with vehicle (a/b) or vehicle + LPS (E/F) respectively. (c) and (d) are quantification of mean ACK2‐ IR cell size across treatments. (g) and (h) are quantification of mean ACK2‐IR cell size in slices challenged with LPS ex vivo. n = 4 male, 4 female in ‘NO LPS’ groups, and n = 5 male, 5 female in ‘LPS’ groups Scale bars in (a/e) and (b/f) are all 5 μm

FIGURE 4

ACK2‐IR cells within 1 μm of a VIP‐IR neuronal fiber were larger than those not within 1 μm of a VIP‐IR fiber, regardless of treatment or challenge with LPS. (a/e) and (b/f) are representative images of ACK2‐immunoreactive cells (red) and VIP‐immunoreactive fibers (green) from a cell <1 μm from a VIP fiber or >1 μm from a fiber, respectively. Quantification in (c) and (d) of the mean ACK2 cell size in μm2 by treatment. (g) and (h) are quantification in the same fashion as (c/d) but in slices challenged with LPS. n = 8 animals Scale bars in (a/e) and (b/f) are all 5 μm

FIGURE 5

Slices from female ileums had more ACK2‐IR cells also immunoreactive to VIP in response to LPS challenge than males. (a/e) and (b/f) are representative images of ACK2 immunoreactivity (red) and VIP immunoreactivity (green) with arrows pointing towards exemplary cells immunoreactive to both ACK2 and VIP. (c) and (d) are quantifications of the percentage of ACK2‐IR cells that were greater than 20% of their area immunoreactive for VIP. (g) and (h) are quantifications the same as in (c/d) but in slices challenged with LPS. n = 4 male, 4 female in ‘NO LPS’ groups, and n = 5 male, 5 female in ‘LPS’ groups Scale bars in (a/e) and (b/f) are all 10 μm