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. 2021 Oct 19;10:e66793. doi: 10.7554/eLife.66793

Figure 3. Antimicrobial S. felis C4 extract and PSMβ suppress TLR-mediated inflammation.

mRNA transcript abundance of hBD2 (A), IL-6 (B) and CXCL10 (C) as measured by qPCR in NHEKs stimulated with or without TLR2/6 agonist MALP-2 (M-2) (200 ng/ml) or TLR3 agonist Poly I:C (PI:C) (0.4 μg/ml) in the presence or absence of S. felis C4 extract, PSMβ2 or PSMβ3 (10 μg/ml) or DMSO control (0.1%) at 4 hr post-treatment. (D) Quantification of CXCL10 protein by ELISA from the supernatant of NHEKs stimulated with MALP-2 or Poly I:C in the presence or absence of S. felis extract, PSMβ2, PSMβ3 or DMSO control 24 hr post-treatment. (A–D) Error bars indicate SEM. One-way ANOVA with multiple corrections (Tukey’s correction) was performed. p values: * p < 0.05; ** p < 0.01; *** p < 0.001. (E) Time-course of the TLR3 signaling cascade by immunoblot of phosphorylated TBK1 (P-TBK1) and IRF-3 (P-IRF-3) proteins after stimulation of NHEKs with Poly I:C (PI:C), PSMβ2 (β2), or co-treatment with Poly I:C and PSMβ2 (PI:C+β2). (F) Gene ontology (GO) pathway analysis of genes downregulated in NHEKs after 4 hr co-treatment with Poly I:C and PSMβ2 versus treatment with Poly I:C alone. (G) Hierarchical clustering and Heatmap visualization of selected genes from GO enriched ‘immune response’ pathway (1.5-fold change) 4 hr post-treatment with DMSO, PSMβ2 or Poly I:C alone or with Poly I:C and PSMβ2 cotreatment. (H) Growth of S. pseudintermedius ST71 (OD600 nm) after 18 hr incubation with increasing concentrations of S. felis C4 extract, formylated peptides PSMβ1, PSMβ2, PSMβ3, EFHAND domain-containing peptide or non-formylated PSMβ2. Error bars indicate SEM. Representative of two independent experiments.

Figure 3—source data 1. Source data for Figure 3A–C (gene expression values measured by qPCR), Figure 3D (secreted CXCL10 values measured by ELISA), Figure 3F (gene list for GO terms) and Figure 3H (growth of S.p ST71 in the presence of peptides and extract).
Figure 3—source data 2. Source data for Figure 3E (labeled and unlabeled western blots of (P) TBK1, (P) IRF3 and COXIV).

Figure 3.

Figure 3—figure supplement 1. S. felis C4 PSMβ2 reduces TLR2- and TLR4-stimulated transcripts in THP-1 macrophages.

Figure 3—figure supplement 1.

Transcript abundance of inflammatory cytokines IL-6 and TNFα in THP-1 cells stimulated with or without TLR2/6 agonist MALP-2 (200 ng/ml) or TLR4 agonist LPS (1 μg/ml) in the presence or absence of S. felis C4 extract, PSMβ2 or PSMβ3 (10 μg/ml) or DMSO control (0.1%) 4 hr post-treatment. Error bars indicate SEM. One-way ANOVA with multiple corrections (Tukey’s correction) was performed. p values: * p < 0.05; **, p < 0.01; *** p < 0.001.
Figure 3—figure supplement 2. S. felis C4 PSMβ2 downregulates transcripts associated with cytokine signaling.

Figure 3—figure supplement 2.

GO pathway analysis of genes down-regulated in NHEKs at 24 hr post treatment with PSMβ2 versus DMSO control.
Figure 3—figure supplement 3. PSMβ do not exhibit synergistic antimicrobial activity.

Figure 3—figure supplement 3.

Growth of S. pseudintermedius ST71 (OD600), normalized to TSB alone at 100% at 18 hr post-treatment with N-formylated and non-N-formylated (no N-f) PSMβ peptides alone or in combination. Error bars indicated SEM.