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. 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). 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 the lungs 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 infected: n=1). Infected Tercko/ko mice were grouped into with systemic and without systemic infection. Data is displayed on a logarithmic scale.

B) Heatmap of overall gene expression in the different mice cohorts analyzed. Gene expression is displayed as the log2 of the fragments per kilobase of transcript per million fragments mapped (fpkm) of each individual gene. 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) Overview of the most differentially enriched KEGG pathways between infected young and Tercko/ko mice. The dot plot was constructed using R Studio. ns ≥ 0.05, *p < 0.05, **p < 0.01, ***p<0.001, ****p < 0.0001. P calculated by Kruskal-Wallis-test (A). 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 from young WT mice during phagocytosis of 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.

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. 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 young WT (n=3), old WT (n=3), and Tercko/ko mice (n=3). ns ≥ 0.05, *p < 0.05, **p < 0.01. P calculated by Kruskal-Wallis-test (B; D-F). 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. ns ≥ 0.05, *p < 0.05. P calculated by Kruskal-Wallis-test (D-E). Each replicate is displayed as a data point.

Tercko/ko mice with systemic infection display more severe pneumonia with bacteremia and increased mortality.

(A) Clinical score of infected Tercko/ko mice at the time of infection (0 dpi) and 24 hpi (1 dpi). The mice were divided into three groups according to their clinical score values: mice with mild infection (dark blue dots; n=1), mice with systemic infection (light blue dots; n=2), and mice where infection resulted in a fatal outcome (black dots; n=2). Mice with systemic infection were defined as mice with an increased clinical score and the presence of bacteria in organs other the lung.

(B) Bacterial load of kidney and liver tissue of the infected Tercko/ko mice (each n=3). Light blue data points represent mice with systemic infection, while dark blue dots represent the mouse with mild infection.

(C) Bacterial load of the lung of infected mice at 24 hpi. Young WT and old WT mice: n=5; Tercko/ko mice with systemic infection: n=2, Tercko/ko mice without systemic infection: n=1. Data is displayed as logarithmic.

(D) Relative body weight of young WT (each n=5), old WT (each n=5) and Tercko/ko mice (non-infected: n = 3; with systemic infection: n=2; without systemic infection: n =1). Relative body weight displays body weight at 24 hpi as a percentage of the body weight at the time of infection.

(E) Relative lung weight of y young WT (each n=5), old WT (each n=5) and Tercko/ko mice (non-infected: n = 3; with systemic infection: n=2; without systemic infection: n =1). Relative lung weight displays lung weight after 24 hpi as a percentage of the current body weight.

(F) Measurement of average telomere length per chromosome end in lungs of young WT (n=4), old WT (n=4) and Tercko/ko mice.

(G) PCA of sequenced lungs from young WT (each n=3), old WT (each n=3) and Tercko/ko mice (each n=3). The mice with systemic infection clustered separately from the other samples.

(H) Number of DEGs per comparison including up- and downregulated DEGs as well as the total count. 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. For each mouse cohort three biological replicates were sequenced and used for analysis. Infected Tercko/ko mice were divided into systemic and non-systemic infected mice for several analyses (C, D, and E). *p < 0.05. P calculated by Kruskal-Wallis-test (C-E). Each replicate is displayed as a data point.

Several pathways relevant for inflammation and adaptive and innate immune response are differentially expressed in infected Tercko/ko mice.

Heatmaps of DEGs related to the NLRP3 inflammasome pathway (A), macrophage marker (C), Chemokine signaling pathway (D), T helper cell marker (E), or TCR signaling (F). 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 comparisons are shown. B) Immunofluorescence staining of CD68 (red), actin (purple), and DAPI (blue) of AMs isolated from Tercko/ko and young WT mice. Representative pictures are shown for each group.

(A) Gating strategy for the immunophenotyping of cells from murine spleen. First, lymphocytes were selected, then duplicates and dead cells were excluded (APC-Cy7 negative). To avoid autofluorescence of macrophages, the F4/80 negative population was selected. For further calculations the CD3+ CD19- cell population was used.

(B) Most differentially enriched KEGG pathways of infected Tercko/ko vs. infected old WT, infected young WT vs. non-infected young WT and infected Tercko/ko vs. non-infected Tercko/ko mice. The dot plots were constructed using R Studio.

(C) Gating strategy for T cells isolated from murine spleen to asses CD247 expression. First, lymphocytes were selected, duplicates and dead cells were excluded before selecting CD3+ CD247+ cells. This population was used for further calculations.

(D) CD44 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. CD44 expression is displayed as geometric mean fluorescence intensity (gMFI).

(E) Heatmap of differentially regulated MSC markers. 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.

(A) Overview of the methods used in this publication. Illustration was created with Biorender.

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(A) Exemplary results of genotyping of Tercko/ko mice used for breeding. Knockout mice show a single band at 180 bp, while WT mice have a single band at 200 bp. Heterozygeous mice (WT/ko) display both bands. Results were analyzed using an Agilent 2200 TapeStation.