Tercko/ko mice exhibit more severe pneumonia with increased mortality and a higher bacterial load.

A) Clinical score of non-infected and infected young WT (n =10, 5 non-infected and 5 infected), old WT (n =10, 5 non-infected and 5 infected), and Tercko/ko (n =8, 3 non-infected and 5 infected) mice at 24 hpi. The age and genetic background of the different groups is depicted below.

B) Bacterial load of the lungs of infected mice at 24 hpi. Data is displayed as logarithmic.

C) Relative body weight of young WT, old WT and Tercko/ko mice. Relative body weight displays body weight at 24 hpi as a percentage of the body weight at the time of infection.

D) Relative lung weight of young WT, old WT, and Tercko/ko mice. Relative lung weight displays lung weight at 24 hpi as a percentage of the current body weight.

E) Immunofluorescence staining of lung tissue of infected young (WT 8 weeks), old (WT 2 years), and Tercko/ko mice. Lungs were stained for S. aureus (green), actin (red), and DAPI (blue). Representative pictures are shown for each group.

*p < 0.05, **p < 0.01, ***p<0.001. P calculated by Kruskal-Wallis-test (A-D). Data is displayed as violin plot showing the median as well as lower and upper percentile of each dataset. Each replicate is displayed as a data point.

Lungs of Tercko/ko mice display excessive inflammation and NLRP3 inflammasome activation.

A) Pro-inflammatory cytokines measured in lung homogenates of non-infected and infected young WT (each n=3), old WT (each n=4) and Tercko/ko mice (non-infected: n=3, with systemic infection: n=2 and without systemic infection: n=1). Infected Tercko/ko mice were grouped into mice with systemic and without systemic infection. Cytokines were measured using a flow cytometry based LEGENDPlexTM mouse inflammation panel. Data is displayed on a logarithmic scale.

B) Heatmap of overall gene expression in the lungs of the different mice cohorts at 24 hpi. Gene expression is displayed as the log2 of the fragments per kilobase of transcript per million fragments mapped (fpkm) of each individual gene. Gene expression is normalized to sequencing depth and transcript length. Red indicates upregulation, and green indicates downregulation of the gene expression. The respective mouse groups are visualized above the heatmap. For each mouse cohort three biological replicates were sequenced.

C) Heatmap of differentially expressed genes (DEG) belonging to the NLRP3 inflammasome pathway. Red indicates upregulation, and blue indicates downregulation of the gene. Differential gene expression analysis was conducted using the DESeq2 software as well as negative binomial distribution. For FDR calculation the Benjamini-Hochberg procedure was used. Heatmaps were constructed using R Studio. Only significant DEGs (p-value < 0.05) were displayed in the heatmap. Non-significant genes were set to zero and are shown as white. DEGs are displayed as log2fold-change. For each group three biological replicates were sequenced and used for analysis. For clarity, the heatmap has been split in half, and no column clustering has been performed. The respective groups used for the comparison are indicated below each column.

D) KEGG pathway enrichment analysis comparing infected young and Tercko/ko mice at 24 hpi. The gene ratio describes the ratio of differentially expressed genes to all genes of the respective pathway. The dot plot was constructed using R Studio.

All sequencing data displays mRNA sequencing data of non-infected and infected lungs of the three different mice cohorts at 24 hpi. ns ≥ 0.05, *p < 0.05, **p < 0.01,

***p<0.001, ****p < 0.0001. P calculated by Kruskal-Wallis-test (A). Data is presented as mean ±SD. Each replicate is displayed as a data point.

Elevated expression of pro-inflammatory M1 macrophages markers and chemokines are present in the lungs of infected Tercko/ko mice.

A) Colorized SEM picture of an AM isolated from young WT mice phagocytosing S. aureus.

Heatmap of differentially expressed macrophage markers (B) and DEGs belonging to the chemokine signaling pathway (C). Red indicates upregulation, and blue indicates downregulation of the gene. Differential gene expression analysis was conducted using the DESeq2 software as well as negative binomial distribution. For FDR calculation the Benjamini-Hochberg procedure was used. Heatmaps were constructed using R Studio. Only significant DEGs (p-value < 0.05) were displayed in the heatmap. Non-significant genes were set to zero and are shown as white. DEGs are displayed as log2fold-change. For each group three biological replicates were sequenced and used for analysis. The respective groups used for the comparison are indicated below each column. All heatmaps display mRNA sequencing data of non-infected and infected lungs of the three different mice cohorts at 24 hpi.

TCR in infected Tercko/ko mice is partially downregulated.

Heatmap of differentially expressed T helper cell markers (A) and DEGs belonging to the TCR signaling pathway (C). Red indicates upregulation, and blue indicates downregulation of the gene. Differential gene expression analysis was conducted using the DESeq2 software as well as negative binomial distribution. For FDR calculation the Benjamini-Hochberg procedure was used. Heatmaps were constructed using R Studio. Only significant DEGs (p-value < 0.05) were displayed in the heatmap. Non-significant genes were set to zero and are shown as white. DEGs are displayed as log2fold-change. For each mouse cohort three biological replicates were sequenced and used for analysis. The respective groups used for the comparison are indicated below each column. All heatmaps display mRNA sequencing data of non-infected and infected lungs of the three different mice cohorts at 24 hpi.

B) Percentage of CD3+ -living cells in non-infected young WT (n=3), old WT (n=3), and Tercko/ko mice (n=5) spleen. Cells were analyzed with flow cytometry.

Gene expression levels in fpkm of CD247 (D), PDCD1 (E), and CD4 (F) in the lungs of non-infected and infected young WT (n=3), old WT (n=3), and Tercko/ko mice (n=3) at 24 hpi.

ns ≥ 0.05, *p < 0.05, **p < 0.01. P calculated by Kruskal-Wallis-test (B; D-F). Data is displayed as violin or boxplot showing the median as well as lower and upper percentile of each dataset. Each replicate is displayed as a data point.

T cells of Tercko/ko mice do not adequately react to bacterial challenge.

(A) Schematic representation of experiment. Murine T cells and AMs were isolated, co-cultured, and stimulated with heat-killed S. aureus with an MOI5 for 24 hours.

(B) Colorized SEM picture of the experimental setup. An AM close to a T cell, is shown.

(C) Immunofluorescence staining of infected and non-infected AMs from young WT and Tercko/ko mice. AMs were stained with antibodies for S. aureus (red), actin (purple), and DAPI (blue).

(D) CD247 expression measured with flow cytometry of non-infected and infected T cells (left) and co-cultured T cells and AMs (right) of young WT (each n=3) and Tercko/ko mice (each n=3) after 24 hpi. CD247 expression is displayed as geometric mean fluorescence intensity (gMFI).

(E) IL-1α from supernatants of T cells (left) and co-cultured T cells and AMs (right) of young WT (each n=3) and Tercko/ko mice (each n=3) at 2 and 24 hpi. IL-1α levels were measured using a flow cytometry based LEGENDPlexTM mouse T helper cytokine panel.

ns ≥ 0.05, *p < 0.05. P calculated by Kruskal-Wallis-test (D-E). Data is displayed as violin plot showing the median as well as lower and upper percentile of each dataset. Each replicate is displayed as a data point.