Adaptive immune defense prevents Bartonella persistence upon trans-placental transmission

Abstract

Vertical transmission of Bartonella infection has been reported for several mammalian species including mice and humans. Accordingly, it is commonly held that acquired immunological tolerance contributes critically to the high prevalence of Bartonellae in wild-ranging rodent populations. Here we studied an experimental model of Bartonella infection in mice to assess the impact of maternal and newborn immune defense on vertical transmission and bacterial persistence in the offspring, respectively. Congenital infection was frequently observed in B cell-deficient mothers but not in immunocompetent dams, which correlated with a rapid onset of an antibacterial antibody response in infected WT animals. Intriguingly, B cell-deficient offspring with congenital infection exhibited long-term bacteremia whereas B cell-sufficient offspring cleared bacteremia within a few weeks after birth. Clearance of congenital Bartonella infection resulted in immunity against bacterial rechallenge, with the animals mounting Bartonella-neutralizing antibody responses of normal magnitude. These observations reveal a key role for humoral immune defense by the mother and offspring in preventing and eliminating vertical transmission. Moreover, congenital Bartonella infection does not induce humoral immune tolerance but results in anti-bacterial immunity, questioning the contribution of neonatal tolerance to Bartonella prevalence in wild-ranging rodents.

Fig 1. Efficient transplacental B. taylorii transmission in B cell-deficient but not wildtype mice.

(A) Schematic overview of the experimental setup for the analysis of extracted embryos (top) and living offspring (bottom). Female WT, Rag1-/- and μMT mice were infected with 10⁷ cfu of B. taylorii i.d. and were mated 10 days later. Embryos were extracted on day 18 of gestation and offspring were evaluated at 5 weeks of age. (B) Bacteremia of WT, Rag1-/- and μMT mice after inoculation with 10⁷ cfu B. taylorii i.d.. Symbols represent the mean ± SD of three mice per group. One representative of three experiments is shown. The time window for pregnancy of WT mice (upon mating on day 10 post infection) is indicated in grey. (C) The percentage of WT, Rag1-/- and μMT offspring harbouring B. taylorii was determined at embryonic day 18 (“extracted embryos”) and at four to five weeks after birth (“4–5 weeks of age”). Numbers above symbols in (C) indicate the percentage of litters containing at least one infected embryo or litter mate, respectively/number of litters assessed. Each symbol represents one litter, horizontal lines indicate the mean. Unpaired two-tailed Student’s t-tests were performed for statistical analysis. Related data are reported in S1 Fig.

Fig 2. Lack of correlation between bacterial burden in maternal blood, placentae and embryos.

Female WT mice were infected with 10⁷ cfu B. taylorii i.d. and mated with WT partners on day 10 as reported in Fig 1 (the same animals as in Fig 1C are shown). Embryos and placentae were extracted on day 18 of gestation. Female μMT and Rag1-/- mice were infected with B. taylorii 10⁷ cfu i.d. and mated continuously from day 10 onwards with partners of the same genotype. (A) Bacteremia of the mothers at the time point of mating (WT mice) or first mating (μMT and Rag1-/- mice) was determined. (B-C) The amount of cultivatable bacteria from placentae (B) and embryos (C) was also measured. Numbers above symbols in (A-C) indicate the number of bacteria-containing specimes/number of specimens tested. (D-F) The relationship between maternal bacterial blood titer, the bacterial burden in placentae and embryos is depicted for WT (D), μMT (E) and Rag1-/- (F) mothers. All symbols and lines represent individual mice, placentae and embryos, horizontal lines in (A-C) show the mean. Between-group differences were analyzed by two-way ANOVA with Tukey’s multiple comparisons test. Resulting P-values are indicated. Related data are reported in S1 Fig.

Fig 3. Immunocompetent offspring clear vertically transmitted Bartonella infection and develop protective immunity to re-infection.

(A) Schematic overview of homozygous (immunocompromized) and heterozygous (immunocompetent) crosses between infected Rag1-/- or μMT dams and uninfected males of the indicated genotypes. Female mice were infected i.d. with 10⁷ cfu of B. taylorii and were mated continuously with either WT, Rag1-/- or μMT-/- males. (B-C) Bacteremia of immunocompetent (μMT+/-, Rag1+/-) and immunocompromised (μMT-/-, Rag1-/-) pups born to Rag1-/- (B) and μMT (C) mothers. (D) The antibody response of immunocompetent μMT +/- offspring at 6 weeks of age was determined by means of an erythrocyte adhesion inhibition (EAI) assay [9], comparing animals with vertically transmitted B. taylorii infection and uninfected litter mates. (E) Schematic overview for the challenge experiment presented in (F-G): Offspring infected with B. taylorii by vertical transmission were re-challenged with with 10⁷ cfu of the same bacterial strain after they had cleared the congenital infection. Litter mates without detectable vertical B. taylorii infection served as controls. Bacteremia (F) and EAI antibody titers (G) were determined at the indicated time points. The data represents pooled results from: (B) 10 animals (4 Rag1-/- and 6 Rag+/-), (C) 13 animals (4 μMT -/- and 9 μMT +/-), (F-G) 26 animals (11 with congenital infection, 15 without congenital infection). Numbers above symbols in (D, G) indicate the number of animals with EAI antibody titers above technical cut-off/number of animals tested. Symbols in (B, C, D, G) show individual mice, data points in (F) represent the mean ± SD of 14 and 8 animals, respectively.