Impaired antibacterial host defense in mice lacking the N-formylpeptide receptor

Abstract

N-formylpeptides derive from bacterial and mitochondrial proteins, and bind to specific receptors on mammalian phagocytes. Since binding induces chemotaxis and activation of phagocytes in vitro, it has been postulated that N-formylpeptide receptor signaling in vivo may be important in antimicrobial host defense, although direct proof has been lacking. Here we test this hypothesis in mice lacking the high affinity N-formylpeptide receptor (FPR), created by targeted gene disruption. FPR-/- mice developed normally, but had increased susceptibility to challenge with Listeria monocytogenes, as measured by increased mortality compared with wild-type littermates. FPR-/- mice also had increased bacterial load in spleen and liver 2 d after infection, which is before development of a specific cellular immune response, suggesting a defect in innate immunity. Consistent with this, neutrophil chemotaxis in vitro and neutrophil mobilization into peripheral blood in vivo in response to the prototype N-formylpeptide fMLF (formyl-methionyl-leucyl-phenylalanine) were both absent in FPR-/- mice. These results indicate that FPR functions in antibacterial host defense in vivo.

Figure 1

Genetic inactivation of mouse FPR. (a) Mutagenesis strategy. Maps of the wild-type and mutant alleles are shown above and below the map of the targeting construct, respectively, and corresponding sites are connected by broken lines. The neomycin resistance gene (neo^r) replaced 150 bp of the FPR ORF and was used to select for targeted events. The Herpes simplex virus thymidine kinase gene (HSV-tk) was used for counterselection of nontargeted events. Horizontal bars, ORFs of the indicated genes; arrows, sense orientation of the indicated ORF; X, XhoI; B, BamHI; K, KpnI. (b) Genotypic analysis of tail DNA derived from progeny of heterozygous crosses. The mutated allele (8.5-kb XbaI fragment) is distinguished from the wild-type allele (7.8-kb XbaI fragment) using a 1-kb probe, whose location is given in panel a. (c) Reverse transcription PCR analysis of total RNA from TP neutrophils from mice with the FPR genotype indicated at the top of each lane. Mutated mRNA (430-bp band) is distinguished from the wild-type mRNA (350-bp band) by reverse transcription PCR with the primers indicated in the text.

Figure 2

Loss of FPR function in neutrophils from FPR^−/− mice. (a) Calcium flux response. [Ca²⁺]_i was monitored in real time in FURA-2–loaded peritoneal neutrophils stimulated with 1 μM fMLF and 100 nM MIP-1α. Each tracing represents analysis of 3 × 10⁶ cells from a single mouse with the FPR genotype indicated at the top (+/+, wild-type mice; −/−, knockout mice). Agonists were added at the times indicated by the arrows. Results are from a single experiment representative of at least four separate experiments. (b) Neutrophil chemotaxis. Results are the mean ± SEM of triplicate determinations in a single experiment using peritoneal neutrophils and fMLF in vitro and are representative of three separate experiments. There was no difference in MIP-1α–induced chemotaxis between neutrophils from FPR^−/− and from FPR^+/+ mice (data not shown). (c) Neutrophil mobilization to peripheral blood. Each pair of bars represents the peripheral blood neutrophil concentration for a single mouse before and 90 min after subcutaneous injection with 200 μl of 2 μM fMLF. Results are from a single experiment representative of three separate experiments.

Figure 3

Anti-listerial host defense in mice lacking FPR. (a) Accelerated lethality in FPR^−/− mice challenged with L. monocytogenes. Mice were injected with 2 × 10⁴ CFU in the tail vein. Results are from a single experiment with −/− and +/+ sex-matched littermates (n = 15 in each group), and are representative of three separate experiments with a consistent pattern. P = 0.003 for +/+ versus −/−; P = 0.038 for +/− versus −/− (log-rank test). (b) Listeria clearance. Under the same conditions as in panel a, the bacterial burden from whole spleen and liver was determined 2 d after infection. The mean values are indicated as solid bars. +/+, wild-type mice; −/−, knockout mice.