Elsevier

Immunobiology

Volume 217, Issue 4, April 2012, Pages 420-429
Immunobiology

Protection against Streptococcus pneumoniae serotype 1 acute infection shows a signature of Th17- and IFN-γ-mediated immunity

https://doi.org/10.1016/j.imbio.2011.10.012Get rights and content

Abstract

Acute pneumonia caused by Streptococcus pneumoniae is a major cause of child mortality. Antibodies are considered the main effectors of protection in this clinical presentation of pneumococcal invasive disease. To get new insights into the mechanisms involved in the protective immunity, we established a murine experimental model of protection against acute pneumococcal pneumonia and then evaluated the transcriptional, humoral and cellular responses in protected and non-protected animals. We found that intranasal inoculation of a sublethal dose of S. pneumoniae serotype 1 conferred complete protection against a subsequent challenge with a lethal dose of the same strain. Sublethal infection elicited a strong IgM and IgG antibody response against the capsular polysaccharide, as assessed one week later, and an exacerbated influx of neutrophils into the lungs immediately after the lethal challenge. Genome-wide microarray-based transcriptional analysis of whole lungs showed 149 differentially expressed genes among which we found upregulation of Il17a, Ifng and several IL-17A- and IFN-γ-related genes in protected versus non-protected mice. Kinetics analysis showed higher expression levels of Il17a in protected animals at all time points whereas Ifng was upregulated early in the protected mice and later in the non-protected animals. Intracelluar cytokine staining demonstrated that CD4+ T cells account for a great proportion of the IL-17A produced in the lungs of protected animals. Overall, these results showed that an upregulation of IL-17A- and a timely regulation of IFN-γ-related gene expression, together with development of a Th17 response, are relevant characteristics of the protective immunity against S. pneumoniae acute pneumonia.

Introduction

Streptococcus pneumoniae (pneumococcus) is the main etiological agent of human pneumonia and meningitis, otitis media and sinusitis; per year, about 1–2 millions of fatal pneumococcal diseases occur worldwide (van der Poll and Opal 2009). Pneumococcus colonizes asymptomatically the human nasopharynx and in some cases colonization may progress to pneumonia and invasive disease (Kayhty et al., 2006, Salyers, 2002). Based on differences in the capsular polysaccharide composition 91 pneumococcus serotypes have been described (van der Poll and Opal 2009). Some serotypes like serotype 1 are rarely associated with carriage and most frequently cause invasive infections (Hortal et al., 2008, Normark and Normark, 2002).

Innate immunity plays a critical role in the resistance to S. pneumoniae respiratory infection, both in human and mouse (Paterson and Mitchell 2006). Innate protection is characterized by an early influx of inflammatory cells, predominantly neutrophils (Kadioglu et al., 2000, Munoz et al., 2010, Rosseau et al., 2007), although lungs’ infiltrating CD4+ T cells are also involved in the early resistance to pneumococcal infection (Kadioglu et al., 2000, Kadioglu et al., 2004). Another key component of innate immunity is the deposition and activation of complement C3 component on the surface of pneumococcus and it has been shown to be impaired by capsular polysaccharides (Abeyta et al. 2003). Otherwise, activation of the classical complement pathway by natural IgM antibodies has been demonstrated to be important in preventing a rapid bacteremia during the infection with pneumococcus (Brown et al. 2002).

Activation of innate immunity is not only critical as the first line of defense against pneumococcus but also for the generation of appropriate adaptive immunity (Paterson and Mitchell 2006). It has been proposed that acquired immunity against pneumococcal nasopharyngeal colonization is mediated by CD4+ T-cells and independent of antibodies (Basset et al., 2007, Malley et al., 2005, Trzcinski et al., 2005, Trzcinski et al., 2008). Recently, it has been also demonstrated that IL-17A, produced by Th17 cells, is a key factor in protective immunity against pneumococcal colonization (Lu et al. 2008), being essential for the recruitment of macrophages and neutrophils to the nasopharynx (Zhang et al. 2009). However, other groups have shown evidences that antibodies and particularly IgA are necessary in protection against pneumococcal colonization (Fukuyama et al., 2010, Richards et al., 2010, Roche et al., 2007, Roche and Weiser, 2010). Overall, these data suggest that both antibodies and cellular responses may have an important role in protection against pneumococcal nasopharyngeal colonization (Richards et al. 2010).

Regarding pneumonia and invasive pneumococcal disease there is a consensus that anti-capsular antibodies play an essential role in protection (AlonsoDeVelasco et al., 1995, Lynch and Zhanel, 2009, Malley, 2010, WHO, 2008). In this sense, available pneumococcal vaccines are designed to elicit protective anti-capsular polysaccharide antibodies (Poolman 2004). The binding of specific antibodies to the bacterial surface facilitates its Fc- and complement-mediated opsonophagocytosis by neutrophils and macrophages (AlonsoDeVelasco et al. 1995).

To gain further insights into the mechanisms involved in protection against invasive pneumococcal disease, we established a murine experimental model of protection against acute pneumococcal pneumonia and then evaluated the transcriptional, humoral and cellular responses in protected and non-protected animals. We confirmed the elicitation of a specific humoral response together with an important influx of neutrophils into the lungs of protected mice. We also found upregulation of IL-17A- and IFN-γ-related genes and we confirmed the presence of Th17 cells in protected animals suggesting that these cells and IFN-γ mediated immunity are related to protection against acute pneumonia.

Section snippets

Ethics statement

All experiments involving animals complied with current national and institutional regulations and ethical guidelines from “Comisión Honoraria de Experimentación Animal” (CHEA) – Universidad de la República, Uruguay (approval ID: 08-05-10).

Animal studies

Female C57BL/6J mice, 6–8 weeks old, were obtained from the National Division of Veterinary Laboratories. Animals were maintained in individually ventilated cages and handled in a vertical laminar flow cabinet (ESCO, class II A2). Mice were anaesthetized by

A sublethal infection protects against acute S. pneumoniae serotype 1 pneumonia

We first set up an experimental model of acute pneumonia with a clinical isolate of S. pneumoniae serotype 1 (Pn1) based on intranasal (i.n.) administration of 2 × 107 CFU of Pn1 to C57BL/6J mice. The infection was lethal for animals within 4 days and was invasive since bacteria were found at 24 h in the blood. We then established a model of protection against the lethal challenge by prior intranasal administration of a sublethal dose of the same pneumococcus strain. Mice treated with 4 × 104 CFU of

Discussion

It is largely assumed that systemic antibodies to the capsular polysaccharide primarily determine the immunity against pneumococcal invasive disease (Malley 2010). To gain further insights into the molecular and cellular basis of this protection we established and characterized a murine model of protection against acute pneumonia using a clinical isolate of S. pneumoniae serotype 1. Then, we evaluated several aspects of transcriptional, humoral and cellular responses in protected and

Acknowledgments

This work was supported by a grant of the European Community (STREP grant SavinMucoPath INCO-CT-2006-032296) and ECOS-Sud (U08S02) as well as a grant from the Genopole Evry (to A.B.). F.X.P. was a recipient of a PhD fellowship from the Nausica combat sa Leucémie Association.

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    These authors contributed equally to this work.

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