C-reactive protein is essential for innate resistance to pneumococcal infection

Abstract

No deficiency of human C-reactive protein (CRP), or even structural polymorphism of the protein, has yet been reported so its physiological role is not known. Here we show for the first time that CRP-deficient mice are remarkably susceptible to Streptococcus pneumoniae infection and are protected by reconstitution with isolated pure human CRP, or by anti-pneumococcal antibodies. Autologous mouse CRP is evidently essential for innate resistance to pneumococcal infection before antibodies are produced. Our findings are consistent with the significant association between clinical pneumococcal infection and non-coding human CRP gene polymorphisms which affect CRP expression. Deficiency or loss of function variation in CRP may therefore be lethal at the first early-life encounter with this ubiquitous virulent pathogen, explaining the invariant presence and structure of CRP in human adults.

Keywords: C-reactive protein; anti-nuclear antibodies; host resistance; mouse knockout; pneumococcal infection.

Figure 1

Baseline concentrations of acute-phase proteins in sex and age matched Crp knockout and control wild-type C57BL/6 mice. Mean (SD), n = 7 per group, for (a) serum amyloid P component (SAP) and (b) serum amyloid A protein (SAA).

Figure 2

Spontaneous anti-nuclear antibody (ANA) production in sex-matched and age-matched Crp knockout and control wild-type C57BL/6 mice. Open columns, ANA-negative mice with titre < 1/80; solid columns, ANA-positive mice with titre > 1/80. (a) 3 months of age; (b) 6 months; (c) 9 months. Female Crp knockout mice had significantly more ANA by Fisher's exact test at 9 months (P = 0.03) and at 12 months (P = 0.002).

Figure 3

Lipopolysaccharide (LPS) lethality in sex-matched and age-matched Crp knockout and control wild-type C57BL/6 mice. There was no significant difference between survival of mice in the two groups after administration of LPS at 10 mg/kg (a), 20 mg/kg (b) or 40 mg/kg (c). The apparent trend to greater susceptibility in the Crp knockout mice receiving 40 mg/kg, which did not achieve statistical significance, is well within the typical variation in outcome after administration of LPS to mice. Biological significance is not supported by the fact that the knockouts were not more susceptible than wild-type mice to smaller doses of LPS.

Figure 4

Susceptibility to infection with Streptococcus pneumoniae. Kaplan–Meier survival analysis up to 72 hr after intraperitoneal inoculation of Crp knockout mice and wild-type littermate controls with 10⁵ colony-forming units (c.f.u.) per mouse of virulent S. pneumoniae type 6B, isolated from two different clinical cases of pneumococcal pneumonia, (a), isolate Tz7712-2 and (b) isolate RF200. The results are representative of three independent experiments. (c) Lethality at 72 hr of infection by intraperitoneal inoculation of different doses of virulent S. pneumoniae type 6B isolate Tz7712-2 in Crp knockout mice (open bars) and wild-type littermate controls (closed bars) (three to five mice per group). Wild-type mice were not tested with the lowest doses because they survived larger doses. For clarity, 0% survival is indicated by bars of 2%. The results are representative of four independent experiments with doses of 10³, 10⁴ and 10⁵ c.f.u.; for ethical reasons of animal welfare, doses of 10¹, 10², 10⁶ and 10⁷ c.f.u. were tested only in the experiment shown, as the results of further experiments clearly would not provide further information. (d) Kaplan–Meier survival analysis up to 72 hr after intraperitoneal inoculation of Crp knockout mice and wild-type littermate controls with 5 × 10⁴ c.f.u. per mouse of S. pneumoniae type 27 (strain HO 8452 0293) (single experiment). All P values are from log-rank tests.

Figure 5

Protection, by human C-reactive protein (hCRP) or previous immunization, of Crp knockout mice against lethal infection with Streptococcus pneumoniae. (a) Crp knockout mice received either 3.5 μg of isolated highly purified human CRP per mouse, or vehicle alone, immediately before intraperitoneal inoculation of 10³ colony-forming units (c.f.u.) per mouse of virulent S. pneumoniae type 6B (isolate Tz7712-2). The results are representative of two independent experiments. (b) Crp knockout mice received 1 mg of isolated highly purified human serum amyloid P component (hSAP) per mouse immediately before intraperitoneal inoculation with 10³ c.f.u. per mouse of virulent S. pneumoniae type 6B (isolate Tz7712-2), while another group of Crp knockouts, which received the same inoculum at the same time, had been immunized 4 weeks previously by intramuscular injection of 9 × 10⁷ heat-killed S. pneumoniae type 6B (isolate Tz7712-2) (single experiment). Kaplan–Meier survival curves; P-values are from log-rank tests.