The effects of antimicrobials and lipopolysaccharide on acute immune responsivity in pubertal male and female CD1 mice

Abstract

Exposure to stress during critical periods of development-such as puberty-is associated with long-term disruptions in brain function and neuro-immune responsivity. However, the mechanisms underlying the effect of stress on the pubertal neuro-immune response has yet to be elucidated. Therefore, the objective of the current study was to investigate the effect antimicrobial and lipopolysaccharide (LPS) treatments on acute immune responsivity in pubertal male and female mice. Moreover, the potential for probiotic supplementation to mitigate these effects was also examined. 240 male and female CD1 mice were treated with one week of antimicrobial treatment (mixed antimicrobials or water) and probiotic treatment (L. rhamnosis R0011 and L. helveticus R0052 or L. helveticus R0052 and B. longum R0175) or placebo at five weeks of age. At six weeks of age (pubertal stress-sensitive period), the mice received a single injection of LPS or saline. Sickness behaviours were assessed, and mice were euthanized 8 h post-injection. Brain, blood, and intestinal samples were collected. The results indicated that the antimicrobial treatment reduced sickness behaviours, and potentiated LPS-induced plasma cytokine concentrations and pro-inflammatory markers in the pre-frontal cortex (PFC) and hippocampus, in a sex-dependent manner. However, probiotics reduced LPS-induced plasma cytokine concentrations along with hippocampal and PFC pro-inflammatory markers in a sex-dependent manner. L. rhamnosis R0011 and L. helveticus R0052 treatment also mitigated antimicrobial-induced plasma cytokine concentrations and sickness behaviours. These findings suggest that the microbiome is an important modulator of the pro-inflammatory immune response during puberty.

Keywords: Inflammation; LPS; Microbiome; Probiotics; Puberty; Sex differences.

Fig. 1

Daily consumption volume of six-week-old (A) male water-treated mice, (B) male antimicrobial-treated mice, (C) female water-treated mice and (D) female antimicrobial-treated mice supplemented with either placebo, L. rhamnosis R0011 and L. helveticus R0052 (Lacidofil®), or L. helveticus R0052 and B. longum R0175 (Cerebiome®). Data represented as mean consumption (±SEM), n = 78–80/group. (a) denotes a significant difference between placebo and L. rhamnosis R0011 and L. helveticus R0052 (Lacidofil®) treatments (p < 0.05), (b) denotes a significant difference between placebo and L. helveticus R0052 and B. longum R0175 (Cerebiome®) treatments (p < 0.05) and (c) denotes a significant difference between L. rhamnosis R0011 and L. helveticus R0052 (Lacidofil®) and L. helveticus R0052 and B. longum R0175 (Cerebiome®) treatments (p < 0.05), (d) denotes a significant difference between antimicrobial and water-treated mice (p < 0.05).

Fig. 2

Percent body weight change in six-week-old (A) male and (B) female mice treated with water (CTL) or antimicrobials (AMNS) and supplemented with either placebo, L. rhamnosis R0011 and L. helveticus R0052 (Lacidofil®), or L. helveticus R0052 and B. longum R0175. Data represented as a mean percentage (±SEM), n = 18–20/group. (*) denotes a significant difference between antimicrobial-treated and water-treated mice (p < 0.05). Whole intestinal weights of (C) male and (D) female six-week-old mice treated with saline (SAL) or LPS, water (CTL-SAL, CTL-LPS) or antimicrobials (AMNS-SAL, AMNS-LPS), and supplemented with either placebo, L. rhamnosis R0011 and L. helveticus R0052 (Lacidofil®), or L. helveticus R0052 and B. longum R0175 (Cerebiome®). Data represented as mean (±SEM), n = 8–10/group. (*) denotes a significant difference between LPS and saline counterparts (p < 0.05), (a) denotes a significant difference between antimicrobial and water-treated counterparts (p < 0.05), (b) denotes a significant difference between male and female counterparts, and (c) denotes a significant difference between placebo and L. rhamnosis R0011 and L. helveticus R0052 (Lacidofil®) treatments.

Fig. 3

Acute sickness scores of six-week-old (A) male water-treated mice, (B) male antimicrobial-treated mice, (C) female water-treated mice, and (D) female antimicrobial-treated mice supplemented with either placebo, L. rhamnosis R0011 and L. helveticus R0052 (Lacidofil®), or L. helveticus R0052 and B. longum R0175 (Cerebiome®) and treated with saline (SAL-PLACEBO, SAL-LACTO, SAL-CEREBIOME) or LPS (LPS-PLACEBO, LPS-LACTO, LPS-CEREBIOME). Data represented as mean sickness scores (±SEM), n = 28–30/group. (*) denotes a significant difference between saline and LPS-treated mice (p < 0.05). (a) denotes a significant difference between antimicrobial and water-treated mice (p < 0.05). (b) denotes a significant difference between L. rhamnosis R0011 and L. helveticus R0052 (Lacidofil®) and L. helveticus R0052 and B. longum R0175 (Cerebiome®) treatments (p < 0.05).

Fig. 4

Acute hippocampal (A) IL1β mRNA expression of males, (B) IL1β mRNA expression of females, (C) TNFα mRNA expression of males, (D) TNFα mRNA expression of female mice, (E) IL6 mRNA expression of males, and (F) IL6 mRNA expression of female six-week-old mice treated with saline (SAL) or LPS, water (CTL-SAL, CTL-LPS) or antimicrobials (AMNS-SAL, AMNS-LPS), and supplemented with either placebo, L. rhamnosis R0011 and L. helveticus R0052 (Lacidofil®), or L. helveticus R0052 and B. longum R0175 (Cerebiome®). Data represented as mean fold change (±SEM), n = 18–20/group. The asterisks (*) denotes a significant difference between LPS and saline counterparts (p < 0.05), (a) denotes a significant difference between male and female counterparts (p < 0.05), (b) denotes a significant difference between water and antimicrobial treatments (c) denotes a significant difference from the L. rhamnosis R0011 and L. helveticus R0052 (Lacidofil®) experimental condition (p < 0.05), and (d) denotes a significant difference from the L. helveticus R0052 and B. longum R0175 (Cerebiome®) experimental condition (p < 0.05).

Fig. 5

Acute PFC (A) IL1β mRNA expression of males, (B) IL1β mRNA expression of females, (C) TNFα mRNA expression of males, (D) TNFα mRNA expression of female mice, (E) IL6 mRNA expression of males, and (F) IL6 mRNA expression of female six-week-old mice treated with saline (SAL) or LPS, water (CTL-SAL, CTL-LPS) or antimicrobials (AMNS-SAL, AMNS-LPS), and supplemented with placebo, L. rhamnosis R0011 and L. helveticus R0052 (Lacidofil®), or L. helveticus R0052 and B. longum R0175 (Cerebiome®). Data represented as mean fold change (±SEM), n = 18–20/group. The asterisks (*) denotes a significant difference between LPS and saline counterparts (p < 0.05), (a) denotes a significant difference between male and female counterparts (p < 0.05), (b) denotes a significant difference between water and antimicrobial treatments (c) denotes a significant difference from the L. rhamnosis R0011 and L. helveticus R0052 (Lacidofil®) experimental condition (p < 0.05), and (d) denotes a significant difference from the L. helveticus R0052 and B. longum R0175 (Cerebiome®) experimental condition (p < 0.05).

Fig. 6

Acute plasma IFNγ concentration of (A) males, and (B) females, acute plasma IL1β concentration of (C) males, and (D) females, acute IL6 plasma concentration of (E) males, and (F) females, acute plasma IL10 concentration of (G) males, and (H) females, acute plasma IL12 concentration of (I) males, and (J) females, and acute plasma TNFα concentration of (K) males, and (L) female six-week-old mice treated with saline (SAL) or lipopolysaccharide (LPS), water (CTL-SAL, CTL-LPS) or antimicrobials (AMNS-SAL, AMNS-LPS), and supplemented with either placebo, L. rhamnosis R0011 and L. helveticus R0052 (Lacidofil®), or L. helveticus R0052 and B. longum R0175 (Cerebiome®). Data represented as mean fold change (±SEM), n = 18–20/group. The asterisks (*) denotes a significant difference between LPS and saline counterparts (p < 0.05), (a) denotes a significant difference between male and female counterparts (p < 0.05), (b) denotes a significant difference between water and antimicrobial treatments, (c) denotes a significant difference from the L. rhamnosis R0011 and L. helveticus R0052 (Lacidofil®) experimental condition (p < 0.05), and (d) denotes a significant difference from the L. helveticus R0052 and B. longum R0175 (Cerebiome®) experimental condition (p < 0.0.