The role of complement in innate and adaptive immunity to pneumococcal colonization and sepsis in a murine model
Introduction
Streptococcus pneumoniae is an important bacterial cause of sepsis, meningitis, pneumonia and otitis media. Risk groups for pneumococcal disease include infants, the elderly and immunocompromised persons, specifically people with B-cell defects, complement deficiencies, asplenia, interleukin-1 receptor defects and HIV infection [1]. Pneumococcal disease is generally preceded by mucosal colonization with the homologous strain; hence, resistance to colonization may be an important aspect of resistance to disease.
While a prerequisite for the development of invasive disease, colonization is usually asymptomatic. After a period of colonization, the host clears the pneumococcal strain from the nasopharynx [2]. Innate as well as adaptive immunity are believed to be involved in this process. An important role for complement has been implicated in both innate and adaptive immunity to many bacterial pathogens. Major functions of complement in host defense against infections are opsonization, leukocyte recruitment and activation, and lysis of bacteria and cells [3]. It is therefore not surprising that complement deficiencies in humans are correlated with an increased risk of pneumococcal invasive disease [1]. Animal studies using direct inoculation of pneumococci into normally sterile sites have confirmed this as well. Nakajima et al. showed the protective role of complement in the development of experimental pneumococcal pneumonia in mice [4]. Kerr et al. have shown that innate immune defenses against pneumococcal pneumonia require pulmonary complement component C3 [5]. Propst-Graham et al. have recently shown the role of complement in early (non-neutrophil-mediated) and late (neutrophil-mediated) protection against pneumococcal pneumonia [6]. In meningitis, complement components C1q and C3 seem to be critical for the innate immune response to pneumococci [7].
However, to our knowledge, there have been no studies that evaluate the role of complement in containment of pneumococcal colonization. Two colonization studies in mice have described the effect of complement depletion on pneumococcal colonization 48 h after challenge, but not on invasion [8], [9]. In the work described here, we wished to test the hypothesis that complement may serve to prevent colonizing bacteria in the nasopharynx from causing sepsis.
To this end, we studied the effect of complement depletion on colonization and disease following nasopharyngeal exposure in naïve mice. Our data show that although complement plays little or no role in controlling the initial density of pneumococcal colonization, it seems to be crucial for preventing the progression of colonization to sepsis. Furthermore, we evaluated whether acquired immunity to pneumococcal colonization was dependent on complement. To do so, we immunized mice with killed whole pneumococci and cholera toxin. This preparation is of interest both as a candidate whole cell vaccine (WCV) [10], [11], [12], and because the immunity induced appears to be very similar to that induced by mucosal exposure to live bacteria [10], [11], [12], and hence may mimic the acquired immune response to natural colonization. We found that mice immunized with WCV were protected against colonization even when complement-depleted at the time of challenge. This suggests that this form of acquired immunity can act in the absence of complement.
Section snippets
Bacteria for animal challenge
S. pneumoniae strain 0603 is a clinical isolate of capsular serotype 6B strain described previously [11]. This strain was grown to mid-log phase in Todd–Hewitt broth with 0.5% yeast extract, and stored at −80 °C, in THY with 10% glycerol. The bacteria were thawed just prior to challenge, centrifuged and resuspended in saline at a target concentration of 108 cfu/ml; the actual colony count was determined on blood agar.
Mouse model of colonization
Nasopharyngeal colonization (NP) was studied as described previously [11].
Colonization leads to sepsis in complement-depleted, but not neutrophil-depleted or control mice
To study the role of complement in containment of pneumococcal colonization in mice, we depleted mice of complement for a period of 7 days with repeated doses of CoVF (C3 depletion), and challenged the mice intranasally with a serotype 6B pneumococcal strain. Between 2 and 7 days post-challenge 60% of the complement-depleted mice developed invasive pneumococcal disease (blood cultures positive for pneumococci with for most mice bacterial titers of more than 104 cfu/ml) with rapid progression
Discussion
The natural route of pneumococcal infection in humans is nasopharyngeal colonization followed by invasion and disease. Humans with complement deficiency are known to be at increased risk of invasive disease [14]. We hypothesized that in mice exposed to pneumococci intranasally, complement depletion may result in invasive disease. To study this, we depleted mice of complement and challenged the mice intranasally with a serotype 6B pneumococcal strain that does not normally lead to bacteremia in
Acknowledgments
We thank Dan Weinberger and Gili Regev-Yochay for helpful discussions. This work was supported by grants from the Ter Meulen Fund, Royal Netherlands Academy of Arts and Sciences (D.B.) and by the National Institutes of Health grants R01 AI048935 (M.L.) and R01 AI066013 (R.M).
Presented in part at ISPPD-6, Reykjavik, Iceland, June 2008.
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Contributed equally.