TLR-mediated inflammatory responses to Streptococcus pneumoniae are highly dependent on surface expression of bacterial lipoproteins - PubMed (original) (raw)

Clinical Trial

. 2014 Oct 1;193(7):3736-45.

doi: 10.4049/jimmunol.1401413. Epub 2014 Aug 29.

Suneeta Chimalapati 2, Tracey Pollard 2, Thabo Lapp 1, Jonathan Cohen 3, Emilie Camberlein 2, Sian Stafford 2, Jimstan Periselneris 2, Christine Aldridge 4, Waldemar Vollmer 4, Capucine Picard 5, Jean-Laurent Casanova 6, Mahdad Noursadeghi 1, Jeremy Brown 7

Affiliations

Clinical Trial

TLR-mediated inflammatory responses to Streptococcus pneumoniae are highly dependent on surface expression of bacterial lipoproteins

Gillian Tomlinson et al. J Immunol. 2014.

Abstract

Streptococcus pneumoniae infections induce inflammatory responses that contribute toward both disease pathogenesis and immunity, but the host-pathogen interactions that mediate these effects are poorly defined. We used the surface lipoprotein-deficient ∆lgt pneumococcal mutant strain to test the hypothesis that lipoproteins are key determinants of TLR-mediated immune responses to S. pneumoniae. We show using reporter assays that TLR2 signaling is dependent on pneumococcal lipoproteins, and that macrophage NF-κB activation and TNF-α release were reduced in response to the ∆lgt strain. Differences in TNF-α responses between Δlgt and wild-type bacteria were abrogated for macrophages from TLR2- but not TLR4-deficient mice. Transcriptional profiling of human macrophages revealed attenuated TLR2-associated responses to ∆lgt S. pneumoniae, comprising many NF-κB-regulated proinflammatory cytokine and chemokine genes. Importantly, non-TLR2-associated responses were preserved. Experiments using leukocytes from IL-1R-associated kinase-4-deficient patients and a mouse pneumonia model confirmed that proinflammatory responses were lipoprotein dependent. Our data suggest that leukocyte responses to bacterial lipoproteins are required for TLR2- and IL-1R-associated kinase-4-mediated inflammatory responses to S. pneumoniae.

Copyright © 2014 The Authors.

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Figures

FIGURE 1.

FIGURE 1.

TLR2 activation by S. pneumoniae is dependent on surface lipoproteins. (A) Coomassie blue staining of Triton-X–extracted membrane proteins from the WT (TIGR4) and TIGR4_Δlgt_ strains separated by SDS-PAGE, confirming markedly reduced lipoprotein content for the Δlgt strain. A molecular mass marker (15–80 kDa) is also shown. (B and C) Mean relative absorbance (OD650) of supernatants from a TLR2/HEK reporter cell line incubated for 16 h with different MOI of live (empty columns) or lysed (gray columns, using deoxycholate) (B) TIGR4_Δlgt_ or TIGR4 and (C) D39_Δlgt_ or D39 S. pneumoniae. Error bars represent SEMs, and n = 3 with data representative of repeated experiments.

FIGURE 2.

FIGURE 2.

Macrophage TNF-α responses are dependent on S. pneumoniae lipoproteins. (A and B) Time course of TNF-α concentrations (measured by ELISA) in RAW cell culture supernatants after incubation with TIGR4 or TIGR4_Δlgt S. pneumoniae_ (MOI 5). (C) TNF-α concentrations in RAW cell culture supernatants after 4-h incubation with Pam2CSK4, WT, TIGR4_Δlgt_, TIGR4_ΔpabB_, or TIGR4_ΔlgtΔpabB S. pneumoniae_. Bacterial CFU after 4-h incubation are stated above each column. (D) TNF-α concentrations in RAW cell culture supernatants after incubation for 4 and 24 h with the TLR agonist Pam2CSK4, or sonicated TIGR4 or TIGR4_Δlgt S. pneumoniae_. (E) TNF-α concentrations in RAW cell culture supernatants after 4-h incubation with TIGR4 or TIGR4_Δlgt S. pneumoniae_ or their corresponding 3.3% Triton X-114 lipoprotein. For all panels, n = 3–4 and is representative of repeated experiments, data are presented as means, error bars represent SEMs, and p values were obtained using unpaired t tests.

FIGURE 3.

FIGURE 3.

Normal muropeptide profile for PGN from the TIGR4Δ_lgt_ strain. HPLC analysis of muropeptides isolated from cell wall preparations of the TIGR4 and TIGR4Δ_lgt_ strains. Major muropeptides are indicated by numbers, as follows: 1, Glc_N_-Mur_N_Ac-L-Ala-D-iGln-L-Lys; 2, Glc_N_Ac-Mur_N_Ac-L-Ala-D-iGln-L-Lys; 3, Glc_N_Ac-Mur_N_Ac-L-Ala-D-iGln-L-Lys-L-Ser-L-Ala; 4, Glc_N_Ac-Mur_N_Ac-L-Ala-D-iGln-L-Lys-D-Ala-L-Lys-D-iGln-L-Ala-Mur_N_Ac-Glc_N_Ac; 5, Glc_N_Ac-Mur_N_Ac-L-Ala-D-iGln-L-Lys-L-Ser-L-Ala-D-Ala-L-Lys-D-iGln-L-Ala-Mur_N_Ac-Glc_N_Ac. The structure of peak 1 (arrow) was confirmed by mass spectrometry. Glc_N_, glucosamine; Glc_N_Ac, _N_-acetylglucosamine; Mur_N_Ac, _N_-acetylmuramic acid.

FIGURE 4.

FIGURE 4.

S. pneumoniae lipoproteins induce proinflammatory cytokines via TLR2 pathway activation. Mean TNF-α concentrations in cell culture supernatants from BMDMs obtained from C57BL/6 mice incubated for 4 h with the TLR agonists (LPS and/or Pam2CSK4), or TIGR4 or TIGR4_Δlgt S. pneumoniae_. (A) Results for BMDMs obtained from WT or Myd88/trif−/− mice. (B) Results for BMDMs obtained from WT or TLR2−/− mice. (C) Results for BMDMs obtained from WT or TLR4−/− mice. For all panels, n = 3–4 and is representative of repeated experiments, error bars represent SEMs, and p values were obtained using unpaired t tests.

FIGURE 5.

FIGURE 5.

Pneumococcal lipoproteins are important determinants of TLR-induced NF-κB activation. (A) Quantification of nuclear NF-κB p65 using MSD (arbitrary units) for BMDMs from C57BL/6 WT and Myd88/trif−/− mice incubated for 1 h with Δlgt and TIGR4 S. pneumoniae (MOI of 5). Each symbol represents results for one well, the bar represents median values, and the p value for comparison of TIGR4 and Δlgt strains incubated with WT BMDMs was obtained using a Mann–Whitney U test. (B) Confocal immunofluorescence images of NF-κB RelA in human MDM from five different donors stimulated for 2 h with WT (TIGR4) or lipoprotein-deficient (Δlgt) S. pneumoniae (MOI 5 or 50) showing diminished nuclear translocation of RelA (pink nuclei) for cells incubated with the Δlgt strain. (C) Quantitative image analysis of the median (IQR) ratio of nuclear to cytoplasmic RelA staining (*p = 0.0079, Mann–Whitney U test).

FIGURE 6.

FIGURE 6.

Attenuated TLR2-associated macrophage transcriptional responses in response to lipoprotein-deficient S. pneumoniae. Comparison of MDM genome-wide transcriptional responses from at least three donors after 4-h stimulation with Pam2CSK4, WT (TIGR4), or Δlgt S. pneumoniae. (A) Total number of genes upregulated at least >2-fold. (B) Venn diagram of the overlap between the genes upregulated >2-fold by each stimulus. (C) PC analysis showing quantitative differences in expression levels of cocorrelated gene signatures in the global gene expression profile. The graph plots PC1 and PC2, responsible for the greatest differences, for each condition (data points represent mean ± SEM of ≥3 separate experiments). (D and E) The gene expression heat map shows the transcriptional response (compared with unstimulated MDM) for the top 50 genes that contribute to PC1 (D) and PC2 (E).

FIGURE 7.

FIGURE 7.

Effects of lipoproteins during early lung infection. CD1 mice (n = 5–6) were inoculated intranasally with 1 × 107 CFU TIGR4_ΔpabB_ or Δlgt/pabB and BALF obtained at 4 h. BALF TNF-α (A), IL-1β (B), and IL-6 (C) (PBS control data obtained during a separate infection experiment) levels measured by ELISA. (D) BALF total cell count. Each symbol represents results from a single mouse, and the bars medians. The p values were obtained using Mann–Whitney U tests.

FIGURE 8.

FIGURE 8.

IRAK4-dependent production of proinflammatory cytokines is largely lipoprotein dependent. PBMCs from three healthy volunteers and two IRAK4-deficient patients were stimulated for 4 h with TIGR4 or Δlgt S. pneumoniae (MOI 10). (A–C) Mean TNF-α (A), IL-1β (B), and IL-6 (C) concentrations measured by ELISA in cell culture supernatants. (D–F) qPCR of TNF-α (D), IL-1β (E), and IL-6 (F) gene expression (relative to GAPDH). Each symbol represents results from a single donor.

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