Innate secretory antibodies protect against natural Salmonella typhimurium infection

Abstract

The production of IgA is induced in an antigen-unspecific manner by commensal flora. These secretory antibodies (SAbs) may bind multiple antigens and are thought to eliminate commensal bacteria and self-antigens to avoid systemic recognition. In this study, we addressed the role of "innate" SAbs, i.e., those that are continuously produced in normal individuals, in protection against infection of the gastrointestinal tract. We used polymeric immunoglobulin receptor (pIgR-/-) knock-out mice, which are unable to bind and actively transport dimeric IgA and pentameric IgM to the mucosae, and examined the role of innate SAbs in protection against the invasive pathogen Salmonella typhimurium. In vitro experiments suggested that innate IgA in pIgR-/- serum bound S. typhimurium in a cross-reactive manner which inhibited epithelial cell invasion. Using a "natural" infection model, we demonstrated that pIgR-/- mice are profoundly sensitive to infection with S. typhimurium via the fecal-oral route and, moreover, shed more bacteria that readily infected other animals. These results imply an important evolutionary role for innate SAbs in protecting both the individual and the herd against infections, and suggest that the major role of SAbs may be to prevent the spread of microbial pathogens throughout the population, rather than protection of local mucosal surfaces.

Figure 1.

Innate IgA inhibits S. typhimurium invasion. (A) Naive mice were orally infected with 10⁷ CFUs S. typhimurium. Shown are the numbers of S. typhimurium detected in PP 6 or 12 h after oral infection, or in ileal mucosa 12 h after infection. Presented are the combined results of three independently performed experiments. *P = 0.011. (B) An in vitro invasion assay was used to determine the ability of serum from naive B6 (hatched columns) or pIgR^−/− mice (black columns) to inhibit S. typhimurium invasion. Presented is the mean invasion ± 1 SD of triplicate cultures of one representative out of three performed experiments. *P = 0.04; **, P < 0.001. (C) Goat anti–mouse IgG (hatched columns), goat anti–mouse IgA (black columns), or culture medium (white columns) was added to serum from pIgR^−/− mice before use in an in vitro invasion assay. As controls, B6 serum or goat antibodies added to the assay in the absence of serum was used. Presented is the mean invasion ± 1 SD of triplicate cultures. *, P = 0.4; **, P = 0.01. (D) Normal (white bars) and preabsorbed serum (hatched bars) from B6 or pIgR^−/− mice (pools of three) was assayed for its ability to block invasion of MDCK cells by S. typhimurium. Shown is the mean invasion ± 1 SD of quadruplicate cultures of one representative out of six independently performed experiments. *, P = 0.04; **, P = 0.01. (E) ELISA was used to determine the IgA titers specific for gut microflora in normal (white bars) and preabsorbed (hatched bars) serum from pIgR^−/− mice. Symbols represent serum pools formed from three or four mice (n = 10 in total). The dotted line indicates the detection limit of the assay.

Figure 2.

The S. typhimurium LD₅₀ is reduced in pIgR^−/− mice. Groups of five naive B6 (▪) or pIgR^−/− mice (◯) were orally infected with either 10⁶ (A), 10⁵ (B), or 10⁴ (C) CFUs S. typhimurium. For ethical reasons, mice were killed when they became moribund; these mice were deemed “nonsurvivors.”

Figure 3.

Naive pIgR^−/− mice are susceptible to “natural” infection with virulent S. typhimurium. One B6 mouse (□, A) or one pIgR^−/− mouse (◯, B) was orally inoculated with S. typhimurium and cohoused with naive B6 mi ce (▪) and naive pIgR^−/− mice (•). The course of the infection in all mice (n = 3, orally inoculated mice; n = 6, naive mice) was followed by measuring the number of bacteria shed in feces. Mice were killed when they became moribund, and the survival was recorded (C and D). The experiment was performed with three cages for each strain of mice, and the data represent the mean ± 1 SD.