Neonatal Exposure to Amoxicillin Alters Long-Term Immune Response Despite Transient Effects on Gut-Microbiota in Piglets

Abstract

Antibiotic exposure during neonatal development may result in transient or persistent alterations of key microbes that are vital for normal development of local and systemic immunity, potentially impairing immune competence later in life. To further elucidate the relationship between antibiotic exposure and immune development, newborn pigs were exposed to a therapeutic pediatric dose (30 mg/kg/day) of amoxicillin (AB) or placebo (PL) from post-natal day (PND) 0-14. Subsequently, immune cell phenotype, microbial composition, and immune response to an intraperitoneal (IP) challenge with Salmonella enterica serovar Typhimurium were evaluated. AB exposure caused significant changes in fecal microbial composition on PND 3 (P = 0.025). This stemmed from a 2-fold increase in Enterobacteriaceae with live cecal coliforms on PND 7 indicating at 10-fold increase (P = 0.036). Alterations in microbial composition were transient, and successional patterns were normalizing by PND 14 (P = 0.693). Differences in PBMC (peripheral blood mononuclear cell) immune cell subtypes were detected, with the percentage of CD3⁺CD4⁺ T cells among the broader T cell population (CD3⁺CD4⁺/CD3⁺) being significantly higher (P = 0.031) in AB pigs and the numbers of CD4+CD45RA+ (naïve) T cells per liter of blood were lower on PND 21 in AB pigs (P = 0.036). Meanwhile, PBMCs from AB pigs produced significantly more IFNγ upon stimulation with a T-cell mitogen on PND 21 and 49 (P = 0.021). When AB pigs were challenged with heat-killed Salmonella (IP) on PND 49, IFNγ gene expression in peripheral blood was upregulated compared to those treated with PL (P = 0.043). Additionally, AB pigs showed stronger activation among neutrophils infiltrating the peritoneal cavity after in vivo immune challenge, based on higher levels of NF-κB nuclear translocation (P = 0.001). Overall, our results indicate that early life treatment with a therapeutically relevant dose of a commonly prescribed antibiotic has a programming effect on the immune system. Despite antibiotics only causing a transient disruption in gut-associated microbial communities, implications were long-term, with antibiotic treated pigs mounting an upregulated response to an immune challenge. This research adds to the growing body of evidence indicating adverse immune outcomes of early life antibiotic exposures.

Keywords: Salmonella; antibiotic; immune development; microbiota; pig model.

Figure 1

In vitro cytokine secretion and cell sub-populations of PBMC were affected by antibiotic (AB) treatment. PBMCs from AB and placebo (PL) treated pigs were stimulated in vitro with phytohaemagglutinin (PHA), a T cell mitogen. (A) Secreted IFNγ was higher in AB vs. PL treated pigs (Two-way ANOVA, Treatment P = 0.006, Age P = 0.022, Interaction P = 0.472). (B) The ratio of IFNγ/IL-2 tended to be higher in AB vs. PL treated pigs and tended to increase with age (Two-way ANOVA, Treatment P = 0.059, Age P = 0.054, Interaction P = 0.780). (C) The ratio of IL-10/IL-2 was higher in AB vs. PL treated pigs and increased with age (Two-way ANOVA, Treatment P = 0.013, Age P = 0.003, Interaction P = 0.248). (D) In addition, immune cell subpopulation CD4+CD25-FoxP3+, identified by flow cytometry, tended to be higher in AB treated pigs (Two-way ANOVA, Treatment P = 0.100, Age P = 0.220, Interaction P = 0.106. (E) However, the CD4+CD25+FoxP3+ immune cell subpopulation was not affected by treatment or age (Two-way ANOVA, Treatment P = 0.752, Age P = 0.684, Interaction P = 0.500). PND 21: AB n = 7, PL n = 7; PND 49: AB n = 7, PL n = 8; PND 84: AB n = 8, PL n = 7.

Figure 2

Pigs treated with antibiotics (AB) from day 0–14 had transient changes in microbial community composition compared to those treated with placebo (PL), as determined with Bray-Curtis dissimilarity. (A) On PND 3 there is distinct clustering of pigs treated with AB vs. PL (Adonis, P = 0.025); AB n = 20, PL n = 21; (B) On PND 7 no distinct clustering was observed between pigs treated with AB and PL (Adonis, P = 0.693); AB n = 17, PL n = 20. (C) On PND 49 pigs treated with AB and PL were not distinctly clustered (Adonis, P = 1.0) (D) Alpha diversity was reduced in AB treated pigs and significantly increased overtime (Two-way ANOVA, Treatment P = 0.017; Time P < 0.0001; Interaction P = 0.444). PND 14: AB n = 18, PL n = 21; PND 21: AB n = 18, PL n = 21; PND 35: AB n = 20, PL n = 21; PND 49: AB n = 18, PL n = 21. (E) There was a significant interaction between Treatment and Day on cecal coliform counts. Pigs treated with AB on PND 7 had higher coliform counts vs. PL on PND 7, 14, and 21 and AB on PND 14 and 21 as indicated by an asterisk (Two-way ANOVA; Treatment P = 0.0257; Day P = 0.0103; Interaction P = 0.0358, post-hoc pair-wise comparisons made with Bonferroni correction), PND 7: AB n = 6, PL n = 6; PND 14: AB n = 8, PL n = 6; PND 21: AB n = 5, PL n = 5. The main effect of time is displayed below each figure and those PND not sharing a common letter differ significantly.

Figure 3

Gene expression of effector cytokines in whole blood was altered 0, 4, and 12 h post intraperitoneal Salmonella challenge in pigs treated with antibiotics (AB) vs. placebo (PL) as analyzed by a Two-way ANOVA (A) IFNγ expression was higher in AB vs. PL treated pigs (Treatment P = 0.043, Time P = 0.715, Interaction P = 0.647). (B) TNFα expression tended to be higher with AB treatment (Treatment P = 0.105, Time P = 0.192, Interaction P = 0.338). (C) IL-6 expression was also higher in AB vs. PL treated pigs (Treatment P = 0.007, Time P = 0.312, Interaction P = 0.125). (D) There was an interaction between time and treatment on the expression of IL-2 and was increased in AB treated pigs 12 h post-challenge compared to AB and PL treated pigs at all time points as indicated by an asterisk (Treatment P = 0.186; Time P = 0.040, Interaction P = 0.034) n = 7/treatment. The main effect of time is displayed below the figure and those time points not sharing a common letter differ significantly. Post-hoc pair-wise comparisons made with Bonferroni correction.

Figure 4

Antibiotic (AB) exposure induced a stronger leukocyte activation against an intraperitoneal Salmonella challenge in comparison to pigs treated with placebo (PL). (A) Total leukocyte infiltration (Two-way ANOVA Treatment P = 0.870, Time P < 0.001, Interaction P = 0.306) and (B) and total reactive oxygen species producing cells (Two-way ANOVA Treatment P = 0.963, Time P = 0.009, Interaction P = 0.845) increased progressively 4 and 12 h post IP Salmonella challenge. (C) Leukocyte infiltration tended to reduce over time (Two-way ANOVA Treatment P = 0.823, Time P = 0.055, Interaction P = 0.072). (D) Leukocyte activation detected through NF-κB nuclear translocation was higher in the AB treated pigs compared to PL post Salmonella challenge, and increased over time (Two-way ANOVA Treatment P = 0.001, Time P = 0.006, Interaction P = 0.410). (E) Exposure of peritoneal leukocytes in vitro to heat-killed Salmonella resulted in increased ROS production in both the AB and PL treated pigs between 0 and 4 h and 0 and 12 h with a tendency of AB treated piglets to have higher ROS production (Two-way ANOVA Treatment P = 0.073, Time P < 0.01, Interaction P = 0.064). n = 4–5/treatment/time. (F) Representative images of neutrophil NF-κB nuclear translocations (G) Representative images of ROS production in response to in vitro stimulation. The main effect of time is displayed below each figure and those PND not sharing a common subscript differ significantly.