Antibiotic administration early in life impairs specific humoral responses to an oral antigen and increases intestinal mast cell numbers and mediator concentrations

Abstract

In this study, we assessed the effect of administering the antibiotic amoxicillin to rat pups on the immune response to orally fed ovalbumin (OVA). We first established that amoxicillin administration durably altered the gut microbiota of these animals. In parallel, we observed that the induction of the specific humoral response to ovalbumin was impaired when it occurred during antibiotic administration to the rat pups. We also examined the consequences of those observations on further allergic reactions. Amoxicillin administration had no significant impact on subsequent sensitization to OVA, as nonexacerbated systemic allergic responses were induced in antibiotic-treated animals. However, increased rat mast cell protease II levels and higher mast cell numbers were detected in their small intestines, independently of the antigen administration. Globally, our data suggest that antibiotic administration early in life negatively affects the specific immune response to a luminal antigen when it is first introduced during antibiotic administration. The increased mast cell numbers and mediator concentrations in the intestinal mucosae of the antibiotic-treated animals may testify to the early stages of an altered immune system homeostasis.

FIG. 1.

Effect of amoxicillin (Clamoxyl) on the colonic microbiota during and after antibiotic treatment. (A) Concentrations of lactobacilli, enterobacteria, and enterococci in colonic contents of saline-treated rats (closed diamonds) and amoxicillin-treated (PND7 to PND18) rats (open squares) at PNDs 15, 21, 36, 42, and 56 (the results of one of five representative experiments are presented; n = 5 to 8 per group). (B) Concentrations of lactobacilli, enterobacteria, and enterococci at PND25 in the colonic contents of saline-treated rats (gray bars) and amoxicillin-treated rats (PND7 to PND25) (white bars) (n = 8 per group; the results of one of two representative experiments are represented). Data are expressed as log CFU/g of luminal content (mean ± standard error of the mean). *, significant differences between saline- and amoxicillin-treated groups (P < 0.05). The detection limit was 3 log CFU/g intestinal content.

FIG. 2.

Effect of amoxicillin (Clamoxyl) on specific humoral responses induced during antibiotic treatment. (A) Experimental design of OT induction in amoxicillin-treated rats. Amoxicillin was administered to the pups from PND7 to PND18. OT was induced by intragastric administration of one dose of 100 mg OVA at PND15. Challenge was performed by subcutaneous injection of 100 μg OVA and 100 μg of KLH in Al(OH)₃ at PND21. The animals were killed at PND36. (B) OVA-specific IgE, IgG, and IgG1 titers in sera of rats at PND36. (C) Total IgE levels (ng/ml) in sera of rats at PND36. (D) KLH-specific IgE and IgG titers in sera of rats at PND36. Rats were treated with saline (gray bars) or amoxicillin (white bars) and force-fed water (nonfed animals, nontolerized [NT]) or OVA (fed animals, tolerized [T]). Data are expressed as the mean log₅ titers ± standard errors of the means (n = 8 to 18 rats/group from two independent experiments). Differences were considered significant when P was <0.05 (*).

FIG. 3.

Effect of amoxicillin (Clamoxyl) on specific humoral immune responses induced after antibiotic treatment and on OT maintenance. (A and C) Experimental design of OT induction in amoxicillin-treated rats. Amoxicillin was administered to the pups from PND7 to PND18. OT was induced by the intragastric administration of one dose of 100 mg OVA at PND15 (A) or PND43 (C). One challenge (C) or two challenges (A) were performed by subcutaneous injection of 100 μg OVA and 100 μg of KLH in Al(OH)₃ at PND21 and PND41 (A) or PND49 (C). The animals were killed at PND56 (A) or PND64 (C). (B and D) OVA-specific IgE titers in the sera of rats at PND56 (B) or PND64 (D). Rats were treated with saline (gray bars) or amoxicillin (white bars) and force-fed water (nontolerized [NT]animals) or OVA (tolerized [T]animals). Data are expressed as the mean log₅ titers ± standard errors of the means (one experiment; n = 8 to 10 rats/group). Differences were considered significant when P was <0.05 (*).

FIG. 4.

Effect of amoxicillin (Clamoxyl) on the severity of the allergic reaction. (A) Experimental design of sensitization in amoxicillin-treated rats. Antibiotics were administered to the pups from PND7 to PND25. Sensitization was performed by subcutaneous injection of 50 μg OVA in Al(OH)₃ at PND11. Challenge was performed by intragastric gavage of 100 mg OVA at PND25. The animals were killed 2 h after the challenge. (B) IgE and IgG levels in amoxicillin- and saline-treated rats (log₅ titers). (C and D) Plasma (C) and small intestinal (D) RMCPII levels in rats treated with saline (gray bars) or amoxicillin (white bars), sensitized with OVA, and force-fed saline solution (nonchallenged) or OVA (OVA challenged). (E and F) Light micrographs of the small intestine sampled 2 h after challenge and stained either with chloroacetate esterase activity for mast cell visualization (E) or with hematoxylin and eosin (F) (magnification, ×100) in (a) saline-treated nonchallenged, (b) saline-treated challenged, (c) amoxicillin-treated nonchallenged, and (d) amoxicillin-treated challenged animals. Data are expressed as the means ± standard errors of the means (one experiment; n = 12). Differences were considered significant when P was <0.05 (*).