Streptococcus pneumoniae is a natural colonizer of the human respiratory tract and an opportunistic pathogen. Although epithelial cells are among the first to encounter pneumococci, the cellular processes and contribution of epithelial cells to the host response are poorly understood. Here, we show that a S. pneumoniae serotype 6B ST90 strain, which does not cause disease in a murine infection model, induces a unique NF-κB signature response distinct from an invasive-disease-causing isolate of serotype 4 (TIGR4). This signature is characterized by activation of p65 and requires a histone demethylase KDM6B. We show, molecularly, that the interaction of the 6B strain with epithelial cells leads to chromatin remodelling within the IL-11 promoter in a KDM6B-dependent manner, where KDM6B specifically demethylates histone H3 lysine 27 dimethyl. Remodelling of the IL-11 locus facilitates p65 access to three NF-κB sites that are otherwise inaccessible when stimulated by IL-1β or TIGR4. Finally, we demonstrate through chemical inhibition of KDM6B with GSK-J4 inhibitor and through exogenous addition of IL-11 that the host responses to the 6B ST90 and TIGR4 strains can be interchanged both in vitro and in a murine model of infection in vivo. Our studies therefore reveal how a chromatin modifier governs cellular responses during infection.
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All data in the present study is available upon request from the corresponding author. The complete microarray dataset (ArrayExpress accession number E-MTAB-9603), PCA dataset and RT–qPCR ΔCT values are contained within Supplementary Table 1 and respective source data files. Full immunoblots are in Supplementary Figs. 1–3. Source data are provided with this paper.
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We thank E. Varon, B. H. Normark, M. M. Si-Tahar and T. Kohler for their generous gifts of S. pneumoniae strains. We are thankful to G. Dore (Institut Pasteur) for processing the microarray Affymetrix GeneChips. Biostatistics and R discussions with J. Nabuco (Institut Pasteur) and J. Camp (University of Veterinary Medicine Vienna) were greatly appreciated. M.G.C. is supported by the Pasteur Foundation Fellowship. T.M.N.C. is supported by Foundation pour la Recherche Médicale, grant no. PMJ201810007628 (Prix MARIANE JOSSO), and a donation from Credit Agricole. M.A.H. is supported by the Institut Pasteur, and the Agence National de la Recherche (ANREpiBActIn), and is a member of the IBEID LabEx. C.M.W. was supported by the Medical Research Council (MR/T016329/1). We thank R. S. Heyderman (UCL) for in-depth discussion on the manuscript, and he is supported by the MRC (MR/T016329/1). J.E. and L.B., members of the Dynamics of Host–Pathogen interactions Unit (Institut Pasteur), are supported by the European Commission (ERC-CoG-Endosubvert) and the ANR-HBPsensing, and are members of the IBEID and Milieu Interieur LabExes. R.J.L. is funded by NIH/NIDCR nos. DE01111, DE012505, DE017921 and DE023193.
The authors declare no competing interests.
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a, TIGR4 and 6B inocula for animal intranasal challenge model (n = 4 biological replicates; Geometric mean of 2 technical counts). Graphed as mean ± s.d. Analyzed by One way-ANOVA with Tukey’s multiple comparison post-hoc test, ***= pV≤0.001, ns=not significant. Exact pV are reported in in vivo source data. b, Human microarray of epithelial A549 cells 2 hrs post-challenge (MOI 20) with either 6B (blue) or TIGR4 (gray). All genes differentially regulated by ± 1.5 fold-change to uninfected condition. Circled 6B genes are NF-κB associated. c, Comparison of genes identified by microarray containing NF-κB sites between 6B and TIGR4. d, Clustering heat map, using ClustVis75, (Euclidean for genes, Correlation for cell line) of the median log2 relative expression RT-qPCR data for indicated genes from A549 (n = 5 biological replicates; 2–3 technicals per biological replicate), Detroit 562 (n = 1 biological replicate with 2 technicals per replicate) and Beas2b (n = 1 biological replicate with 2 technicals per replicate) cell lines. KDM6B and IL-11 cluster highlighted in red. e, Representative images of KDM6B (green) merged with nucleus stained with DAPI (false colored red for visual display). Scale = 10 µm. f, Quantified IL-11 ELISA (n = 5 biological replicates; 4–6 technicals per biological) 6 hrs post-infection of Detroit 562 cells. Median centered (solid red line) dot plot of all values. Dashed red line is the assay limit of detection. g, Total RNA 2 hrs post-challenge with 6B or TIGR4 from A549 cells. Histone demethylase panel RT-qPCR shown as ΔCt (n = 4 biological replicates; 2–3 technicals per biological replicate per group). Graphed as mean ± s.d. Two-tailed multiple T-test using Holm-Sidak correction. ***= pV≤0.001, ns=not significant. Exact pV are reported in Extended Data Fig. 1 source data.
a, RT-qPCR of NF-κB transcript profiles for IL-1β, 6B, or TIGR4 challenged A549 cells over a 2 hr time course (n = 3 biological replicates; 2 technicals per replicate). Displayed as a truncated violin plot with each data point. Graphed as the relative expression of each indicated transcript to matched uninfected/unstimulated control per time point. Gray line, when applicable, denotes uninfected/unstimulated level. b, A549 cells untreated or treated with 10 µM BAY 11-7082, 10 µM GSK-J4, or DMSO vehicle control (n = 4 biological replicates; 2-3 technicals per replicate). Cell viability determined by AlamarBlue. Expressed as % of untreated cells and graphed as mean ± s.d. One way-ANOVA with Dunnett’s multiple comparison post-hoc test to untreated, no significant difference observed (ns). Exact pV are reported in Fig. 2 and Extended Data Fig. 2 source data. c, Transcript levels for IL-11, KDM6B and PTGS2 determined by RT-qPCR. Displayed as ΔCt for comparison to untreated (n = 4 biological replicates; average of 2 ΔCt technical values per replicate). Graphed as mean ± s.d. All data analyzed by One way-ANOVA with Tukey’s multiple comparison post-hoc test, no significant difference observed (ns). Exact pV are reported in Extended Data Fig. 2 source data.
Chromatin obtained from untreated and 10 µM GSK-J4 treated A549 cells 2 hrs post-challenge with 6B (MOI 20), TIGR4 (MOI 20) or IL-1β. 10 µg chromatin input used for ChIP of p65, KDM6B, H3K27me3 and histone H3 (H3), followed by ChIP-qPCR at primer locations (P6, P3 & P2) spanning the NF-κB sites upstream of the transcriptional start site (TSS). a, Schematic of KDM6B promoter with ChIP-qPCR primer locations (P4 & P3) and the NF-κB sites. b, % recovery of input for p65 2 hrs post-infection with 6B (n = 3 untreated; n = 3 GSK-J4 treated). Two-tailed unpaired Student’s T-Test comparisons for untreated to GSK-J4 treated or 6B infected to uninfected, *= pV≤0.05, ***= pV≤0.001, ns=not significant. c, Schematic of IL-11 promoter with ChIP-qPCR primer locations (P6, P3 & P2) and the NF-κB sites. d–g, % recovery of input for p65, KDM6B, H3K27me3 normalized to H3, or H3 bound at P6, P3 & P2 in untreated and GSK-J4 treated samples (n = 3 untreated; n = 3 GSK-J4 treated). Graphed as Tukey box and whisker plot. Box boundaries are the upper and lower interquartile range (IQR), “whiskers” are ± 1.5 times IQR and the median (solid line). Dots represent outliers. One way-ANOVA with Tukey’s multiple comparison post-hoc test, no significant difference observed in comparison to uninfected/untreated or 6B samples from Fig. 3. Cell fractions from A549 cells 2 hrs post-challenge ±10 µM GSK-J4. h, Representative example of fraction purity. Full blots provided in Supplementary Information 2. i, Representative images of nuclear fractions and coomassie stained PVDF membranes. Full blots provided in Supplementary Information 2. j, Signal intensity for H3K27me2 or H3K27me3 for IL-1β, TIGR4 or 6B normalized to total protein as a ratio to uninfected/unstimulated control (n = 4; biological replicates). Bar graph with s.d. and each individual value displayed. Significance calculated between ±GSK-J4 groups by two-tailed Student’s T-test, p-values displayed. *= pV≤0.05.
a, Heat map represents fold change to either untreated or treatment matched controls per condition (n = 5 biologicals for untreated, and n = 3 biologicals per treatment; average 2 technicals per biological per group). Heat mapped difference between relative expression of treatments to untreated. Two-tailed uncorrected multiple T-Test of treatment groups to untreated with significant genes highlighted with “*”. IL-1β vs. IL-1β + GSK-J4: ICAM1 pV= 0.013, RELB pV= 0.150, TNF pV= 0.032, IL-6 pV=0.035, CXCL2 pV= 0.047. IL-1β vs. IL-1β + IL-11: CXCL2 pV=0.007, TNF pV=0.030. b & c, PCA variable correlation plots for GSJ-J4 PCA and IL-11 PCA. KDM6B and IL-11 highlighted in red. Exact pV are reported in Extended Data Fig. 4 source data.
a, % Cytotoxicity (LDH release) from A549 supernatants 2 hrs post-challenge with TIGR4 (MOI 20), 6B (MOI 20) or uninfected ± indicated treatments (n = 4 biological replicates; 2-3 technicals per replicate). UT = untreated. Tukey box and whisker plot with defined box boundaries being the upper and lower interquartile range (IQR), “whiskers” (fences) being ± 1.5 times IQR and the median depicted in the middle (solid line). All data was analyzed by One way-ANOVA with Tukey’s multiple comparison post-hoc test, **= pV≤0.01, ***= pV≤0.001, ns=not significant. Exact pV are reported in Extended Data Fig. 5 source data. b, Hemolytic activity of A549 cell culture supernatants ± 10 µM GSK-J4 2 hrs post-infection with either 6B (MOI 20) or TIGR4 (MOI 20) with uninfected control. All samples n = 4 biologicals with 2-3 technicals per biological. Graphed as mean ± s.d. Analyzed by One way-ANOVA with Tukey’s multiple comparison post-hoc test, ns=not significant. Exact pV are reported in Extended Data Fig. 5 source data. c, A549 cell protein lysates 2 hrs post-infection with TIGR4 (MOI 20; n = 7 biological replicates) or 6B (MOI 20; n = 7 biological replicates) probed for pneumolysin. Relative band intensity quantified with representative blot images. ΔPlyTIGR4 is a control for immunoblot. Scatter plot of all replicates with mean denoted in red. Data analyzed by two-tailed unpaired Student’s T-Test, ns=not significant. Full blots provided in Supplementary Information 3. d, Total RNA harvested from immortalized gingival keratinocytes 21 hrs post-challenge with Streptococcus gordonii, Streptococcus sanguinis, Streptococcus oralis, Eikenella corrodens and Fusobacterium nucleatum, at an MOI of 100. Transcript levels for IL-11 determined by RT-qPCR and represented as relative expression to uninfected (n = 1; average of two technicals).
a, 6B and TIGR4 ± GSK-J4 with matched DMSO controls. 24 hrs post-inoculation bacterial burden from the nasal lavage (NL), bronchoalveolar lavage fluid (BALF), lungs, and spleen of infected animals by conventional CFU counts on 5 µg/mL Gentamicin Columbia Blood agar selection plates (n = 9 animals; Geometric mean of 2 technical counts per lavage or organ). DMSO TIGR4 white, GSK-J4 TIGR4 gray, DMSO 6B light blue, GSK-J4 6B dark blue. All graphed as Tukey box and whisker plot with defined box boundaries being the upper and lower interquartile range (IQR), “whiskers” (fences) being ± 1.5 times IQR and the median depicted in the middle (solid line). Dots represent outliers. One way-ANOVA non-parametric Kruskal-Wallis with Dunn’s multiple comparison post-hoc test, *= pV≤0.05, **= pV≤0.01, ***= pV≤0.001, ns=not significant. Exact pV are reported in Extended Data Fig. 6 source data. CFU = colony forming unit. Dotted lines =Limit of detection (LD). LD for each organ: NL (50 CFU); BALF, Lung and Spleen (1000 CFU). b, TIGR4 and 6B inocula for animal intranasal challenge model in DMSO (vehicle control) or 5 mM GSK-J4 (n = 3 biological replicates; Geometric mean of 2 technical counts). Conventional CFU enumeration shows no significant difference. Graphed as mean ± s.d. Analyzed by One way-ANOVA with Tukey’s multiple comparison post-hoc test, ns=not significant. c, TIGR4 ± IL-11 inocula for animal intranasal challenge model (n = 3 biological replicates; 2 technical counts). Conventional CFU enumeration shows no significant difference. Graphed as mean ± s.d. Analyzed by two-tailed Student’s T-Test, ns=not significant. d, Absorbance (OD600) growth curve for mid-log TIGR4 ± IL-11 mouse inocula over 2 hrs (n = 4 biological replicates). No significant difference in growth observed. Mean (dot) ± s.d. Analyzed by two-tailed multiple T-Test with Holm-Sidak correction at each 20 min interval, ns=not significant. Exact F are reported in Extended Data Fig. 6 source data.
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Connor, M.G., Camarasa, T.M.N., Patey, E. et al. The histone demethylase KDM6B fine-tunes the host response to Streptococcus pneumoniae. Nat Microbiol 6, 257–269 (2021). https://doi.org/10.1038/s41564-020-00805-8
Nature Microbiology (2021)