Role of macrophages in the altered epithelial function during a type 2 immune response induced by enteric nematode infection

Abstract

Parasitic enteric nematodes induce a type 2 immune response characterized by increased production of Th2 cytokines, IL-4 and IL-13, and recruitment of alternatively activated macrophages (M2) to the site of infection. Nematode infection is associated with changes in epithelial permeability and inhibition of sodium-linked glucose absorption, but the role of M2 in these effects is unknown. Clodronate-containing liposomes were administered prior to and during nematode infection to deplete macrophages and prevent the development of M2 in response to infection with Nippostrongylus brasiliensis. The inhibition of epithelial glucose absorption that is associated with nematode infection involved a macrophage-dependent reduction in SGLT1 activity, with no change in receptor expression, and a macrophage-independent down-regulation of GLUT2 expression. The reduced transport of glucose into the enterocyte is compensated partially by an up-regulation of the constitutive GLUT1 transporter consistent with stress-induced activation of HIF-1α. Thus, nematode infection results in a "lean" epithelial phenotype that features decreased SGLT1 activity, decreased expression of GLUT2 and an emergent dependence on GLUT1 for glucose uptake into the enterocyte. Macrophages do not play a role in enteric nematode infection-induced changes in epithelial barrier function. There is a greater contribution, however, of paracellular absorption of glucose to supply the energy demands of host resistance. These data provide further evidence of the ability of macrophages to alter glucose metabolism of neighboring cells.

Figure 1. Macrophage depletion improves the N. brasiliensis-induced decrease in glucose absorption, but not in epithelial resistance.

WT mice were given PBS or clodronate (Cl₂MDP) containing liposomes to deplete macrophages and one day later treated with vehicle (VEH) or infected with N. brasiliensis (Nb). At day nine post inoculation, muscle-free mucosae were mounted in Ussing chambers to determine (A) mucosal resistance and (B) concentration dependent changes in epithelial sodium-linked glucose absorption. Values shown are means ± SE; *p<0.05 vs VEH Control.

Figure 2. Nippostrongylus. brasiliensis infection alters the expression of glucose transporters.

WT mice were treated with vehicle (VEH) or were infected with N. brasiliensis (Nb). At day nine post inoculation, full thickness sections of small intestine were prepared for (A) real-time qPCR to measure the mRNA expression of glucose transporters (B) Western blot analyses for glucose transporters, using actin as a loading control and (C) immunofluorescence staining for glucose transporters on the surface of villi of small intestine. The photomicrographs are representative of five different replicate slides. Values shown are means ± SE; p<0.05 vs VEH Control.

Figure 3. The effect of macrophage depletion on N. brasiliensis infection-induced changes in the expression of glucose transporters.

WT mice were given PBS or clodronate (Cl₂MDP) containing liposomes to deplete macrophages and one day later treated with vehicle (VEH) or infected with N. brasiliensis (Nb). At day nine post inoculation, full thickness sections of small intestine were prepared for real-time qPCR to measure the mRNA expression of glucose transporters. Values shown are means ± SE; *p<0.05 vs VEH Control; φp<0.05 vs N. brasiliensis infected mice treated with PBS-containing liposomes.

Figure 4. Immune regulation of glucose transporter expression and localization of GLUT1 and GLUT2 transporters.

WT or STAT6^−/− mice were treated with vehicle (VEH) or were infected with N. brasiliensis (Nb). At day nine post infection, full thickness sections of small intestine were prepared for real-time qPCR to determine the mRNA expression of (A) GLUT1 or (B) GLUT2. WT mice were treated with vehicle (VEH) or were infected with N. brasiliensis (Nb). At day nine post inoculation, sections of small intestine were prepared for laser capture micro-dissection (LCM) to determine (C) GLUT1 or (D) GLUT2 mRNA expression in epithelial cells or in the lamina propria. Values shown are means ± SE; **p<0.05 vs VEH Control; φp<0.05 vs WT N. brasiliensis.

Figure 5. Macrophage depletion alters expression of factors involved in small intestinal glucose homeostasis.

WT mice were given PBS or clodronate (Cl₂MDP) containing liposomes to deplete macrophages and one day later treated with vehicle (VEH) or infected with N. brasiliensis (Nb). At day nine post-inoculation, full thickness sections of small intestine were prepared for real-time qPCR to determine the mRNA expression of RELM-β leptin, or HIF-1α. Values shown are means ± SE, *p<0.01 vs. VEH Control; φp<0.05 vs N. brasiliensis-infected mice treated with PBS-containing liposomes.

Figure 6. Model for changes in epithelial glucose handling in response to N. brasiliensis infection.

(1) Infection induces a STAT6-dependent increase in mucosal permeability and (2) recruitment of alternatively activated macrophages (M2). (3) There is a M2-dependent inhibition of epithelial SGLT1 activity and M2-independent down-regulation of GLUT2. (4) This stresses the cell leading to activation of HIF-1α and up-regulation of the constitutive insulin-dependent GLUT1. This transporter serves as the major mechanism for glucose entry into the cell. Thus, parasitic nematode infection results in a “lean” epithelial phenotype as a result of a shift from insulin-dependent to insulin-independent glucose transporters. The increased permeability (1) enhances the contribution of paracellular absorption of glucose to maintain body weight during infection.