Figure 1.
Tnf, Il6, and Il10 mRNA expression.
Hepatic mRNA levels of Tnf (A), Il6 (B), and Il10 (C) were measured in WT, TLR2−/−, and TLR4−/− mice at 0 h (healthy control; HC), 6 h and 24 h PI in S. aureus sepsis. For each strain, n≥3 mice at each time point were compared with HC of the same strain. * P<0.05, ** P<0.01; # indicates higher and ## lower values than WT. Vertical bars are SD.
Figure 2.
Nuclear p65, p50, and c-rel, and whole-cell phospho-p65.
Immunoblots are shown for NF-κB family members in nuclear extracts and in whole-liver extracts from WT, TLR2−/−, and TLR4−/− mice in HC and at 6 h PI (A). Ppargc1a and Tnf mRNA levels in S. aureus sepsis (B). Ppargc1a and Tnf mRNA levels at 6 h PI (compared to HC) were measured in WT, p50−/−, and BAY-11-7082-treated mice (n = 3 mice of each strain); * P<0.01 compared with WT Tnf levels at 6 h PI. Vertical bars are SD.
Figure 3.
Ppargc1a, Ppargc1b, and Tnf mRNA levels in S. aureus sepsis.
Ppargc1a (A) and Ppargc1b (B) mRNA levels were measured in WT, MyD88−/−, and MAL−/− mice in healthy controls (HC) and at 6 h and 24 h PI by Q-PCR, together with Tnf mRNA levels (C) at 6 h PI (fold-induction compared to HCs; n≥3 mice at each point for each strain); * P<0.05, ** P<0.01 compared to HC of the same strain. Vertical bars are SD.
Figure 4.
Ppargc1a, Ppargc1b, and Tnf mRNA levels in S. aureus sepsis.
Hepatic mRNA levels of (A) Ppargc1a and (B) Ppargc1b were measured in WT, TRAM−/−, and TRIF−/− mice in healthy controls (HC) and at 6 h and 24 h PI by Q-PCR, compared with mRNA levels of (C) Tnf at 6 h PI (fold-induction compared to HC; n≥3 mice at each point for each strain); * P<0.05 compared to HC of the same strain; #, P<0.05, ##, P<0.01 compared to WT data at 6 h. Bars are SD.
Figure 5.
Nuclear immunoblots for IRF-3 (A) and IRF-7 (B).
Immunoblots are shown for IRF-3 and IRF-7 in nuclear extracts from WT, TLR2−/−, and TLR4−/− mice in HC and at 6 h PI (One of duplicate experiments with two mice per strain). (C) Immunoblots for PGC-1α protein in WT, TLR2−/− and TLR4−/− mice at 0, 6, and 24 h after S aureus inoculation. Equal protein loading was confirmed by Coomassie blue staining. (D) Immunoblots for the mitochondrial VLCAD fatty acid oxidation enzyme in HC and at 6 h PI in WT, TLR2−/−, and TLR4−/− mice. Porin is a mitochondrial reference protein. (E) Chromatin Immunoprecipitation. ChIP for IRF7 binding on the Ppargc1a promoter at −289 bp from TSS. WT and TLR2−/− mice (HC, 6 h PI, and 24 h PI) were tested. Arrow shows the position of the binding. Pol II pull-downs on EF1a are shown as loading controls.
Figure 6.
Ppargc1a and Ppargc1b mRNA levels in S. aureus sepsis.
Hepatic levels of (A) Ppargc1a and (B) Ppargc1b mRNA were measured in WT and IRF3/7−/− mice in healthy controls (HC) and at 6 h and 24 h PI (n≥3 at each point for each strain). (C) Ppargc1a mRNA levels after PolyI:C treatment with or without S. aureus sepsis. Ppargc1a mRNA levels were measured in WT and TLR2−/− mice in healthy controls (HC), in mice dosed with 400 ug PolyI:C, and in mice given PolyI:C plus S. aureus sepsis at 6 h and 24 h PI (n = 3 mice at each point for each strain *, P<0.05 compared to HC of the same strain; ##, P<0.05, #, P = 0.08 compared to WT at 6 h). Vertical bars are SD.
Figure 7.
A. TLR2 and TLR4 localization in WT mouse liver by immunofluorescence microscopy.
Representative paraffin sections were stained for TLR2 in HC (top left) and 6 h PI (bottom left) and for TLR4 in HC (top right) and 6 h PI (bottom right). TLR staining is red; nuclear staining with DAPI is blue. B. Blue native PAGE on whole liver extracts from WT mice at 6 h after inoculation with S. aureus. Each blot shows three lanes: Lane 1, NativeMark molecular weight standard; Lane 2, sample in 0.5% DDM with no DTT or heating; lane 3, sample in 4% SDS with 100 mM DTT, boiled at 95°C for 5 min. At the left, Coomassie staining of entire blot showing molecular markers. Western blots were performed with anti-TLR2, TLR4, or TRAM. A complex near 300 kD was identified by all three primary antibodies (arrows) suggesting a possible interaction among the three proteins.
Figure 8.
Ppargc1a, Ppargc1b, Il10, and Tnf mRNA levels in Unc93b1−/− mice.
Hepatic mRNA levels of Ppargc1a, Ppargc1b, Il10, and Tnf were measured in healthy controls (HC) and in S. aureus sepsis at 6 h PI in WT and Unc93b1−/− mice. There was no significant difference between induction levels in WT and Unc93b1−/− mice for the four genes (n≥3 mice at each point for each strain). Vertical bars are SD.
Figure 9.
Potential TLR signaling pathways for Ppargc1 metabolic co-activator gene activation after S. aureus infection.
Pathway 1 shows the canonical TLR2 MyD88-dependent signaling pathway that activates NF-kB after S. aureus. Pathway 2 shows TLR4 MyD88-dependent signaling to NF-kB and MyD88–independent signaling to TRIF/TRAM. Both MyD88 pathways have been excluded as causes of the Ppargc1a gene expression. Pathway 3 shows a putative TLR2-TLR4 heterodimer interacting with TRIF/TRAM. Pathway 4 indicates TLR2 in the TLR4 null state, as a homodimer or a heterodimer involving a non-TLR4 partner such as TLR1 or 6, interacting with TRIF/TRAM and unmasking the innate immune regulation of Ppargc1a expression. Pathway 5 shows canonical TLR3 endosome signaling also excluded in Ppargc1 gene regulation after S. aureus; however, independent TLR3 activation partially rescues the Ppargc1 phenotype in mice. TIRAP is Toll/interleukin-1 receptor domain-containing adapter protein (MAL); IRAK4 is Interleukin-1 receptor-associated kinase 4; TRAF3 and TRAF6 are TNF receptor-associated factor 3 and 6; TAK1 is TGF-beta-activated kinase 1 and TBK1 is NF-kappa-B-activating kinase.
Table 1.
Antibodies and Primers.