Studies on pneumococcal polysaccharides and their effect on immune cells

Abstract

Streptococcus pneumoniae is a major cause of morbidity and mortality in children and adults worldwide. The spectrum of infections caused by Streptococcus pneumoniae is well-characterized. The bacteria is transmitted via droplets/aerosols to the nasal cavity, from which it is spread locally to ears, sinuses, bronchi, lungs, blood (septicemia) and CNS (meningitis). In the perspective of increasing global antibiotic resistance and the reported shift of pneumococcal types post vaccination, alternative preventive actions might be warranted in the future. In order to prevent or cure pneumococcal infection, it is therefore important to investigate the adhesion capacity of the bacterium to cells/receptors and also to investigate the response of the immune system to Streptococcus pneumoniae. To investigate Streptococcus pneumoniae binding to human cell receptors, an ELISA was developed where the binding to two previously proposed receptors could be compared in the same test system. The results showed that Streptococcus pneumoniae adhered to the human cell receptor glycolipid asilao-GM1, in which the specific binding site was characterized as the disaccharide GalNAcβ1-4Gal. In response to the results showing asialo-GM1 as a human cell receptor for Streptococcus pneumoniae, further investigation aimed to determine the pneumococcal structure that would adhere to this receptor. One of the pneumococcal surface structures is the cell wall polysaccharide, CWPS, which contains phosphoryl choline.

To investigate whether CWPS was the pneumococcal ligand binding to asialo-GM1, carbohydrate material was extracted from an uncapsulated strain of pneumococci. The carbohydrate material in the extract was separated and fractions binding to the pneumococcal glycolipid receptor asialo-GM1 were detected. In nuclear magnetic resonance spectroscopy (NMR) analysis, these fractions exhibited a pattern consistent with a reference spectrum of pure CWPS. The purified CWPS adhered to asialo-GM1 without protein involvement as it was unaffected by CWPS exposure to proteinase K. Streptococcus pneumoniae grown under conditions where choline was replaced with ethanolamine did not bind to the host cell receptor asialo-GM1. This indicated that CWPS, with intact phosphoryl choline residues, is the ligand responsible for binding pneumococci to the glycolipid receptor asialo-GM1.

Having determined CWPS as the responsible ligand to host cell receptor asialo-GM1, the immune response toward this structure deserved more detailed analyses. This was investigated in regard to CWPS activation of a subset of immune cells. Apart from CWPS, Streptococcus pneumoniae also expose other important polysaccharides surrounding the bacterium, i.e. the capsule. The composition of the capsule differs between different Streptococcus pneumoniae bacteria and hence classifies these into different types. Different sets of capsules are used in commercial available pneumococcal vaccines. The present study examined CWPS as well as some capsular polysaccharides included in the pneumococcal vaccines, for their individual capacity to activate immune cells. CWPS, three different capsular polysaccharides (types 3, 9 and 23) and LPS (positive control) were used for in vitro stimulation of whole blood. CWPS and the three capsules activated the immune cells differently (measured as CD69 expression). Generally, NK cells and NK-like T cells exhibited the strongest activation followed by monocytes and T cells.

Among the three capsules, capsule type 23 induced the strongest activation and cytokine release, followed by type 9 and type 3. In these experiments, CWPS induction was well in the range of what was seen from type 23. CWPS is also a Toll Like Receptor (TLR) 2 ligand, and was investigated together with other TLR2 and TLR4 ligands for their capacity to induce gene expression and cytokine release from isolated monocytes and NK cells. Incubation of isolated peripheral blood monocytes with CWPS induced transcriptional upregulation and subsequent secretion of several major proinflammatory cytokines and chemokines, similar to the other TLR ligands investigated. CWPS as well as the other TLR ligands exhibited significant upregulation of CXCL8 expression in isolated NK cells.

With the results showing that CWPS is the ligand responsible for binding pneumococci to the host cell glycolipid receptor asialo-GM1 and also having confirmed that CWPS is an effective activator of immune cells, its impact in clinical settings (smokers) was investigated. Cigarette smoking is a well-known and high risk factor for infections of Streptococcus pneumoniae. The study investigated whether the TLR ligands (including CWPS) would induce a different immune response in smokers compared to non-smokers. In these experiments no difference in TLR gene expression could be detected between smokers and non-smokers in unstimulated cells. Following CWPS incubation with isolated monocytes, cells from smokers showed an increased upregulation of pro-inflammatory mediators as compared to non-smokers. Monocytes from non-smokers downregulated the immune regulatory molecules IL-10 and SOCS-1 after CWPS stimulation, while this was not found in smokers. The results suggest that the transcriptional activation of pro-inflammatory genes after TLR activation is dysregulated in smokers.