Fig 2: Hornigold K et al. Old mice have impaired immunity against E. coli infection and altered neutrophil recruitment, and the ability of their neutrophils to kill E. coli is reduced. (A) E. coli-induced septic peritonitis. Young (8-10 weeks, black symbols) and old (24 months, purple symbols) mice were infected i.p. with 1x104 pathogenic E. coli O18:K1 (filled symbols), or were mock-infected (open symbols), and culled humanely 3 h later. Peritoneal lavages were performed and assessed for bacterial burden by bacterial culture and quantification of CFU. Lavage neutrophils and total leukocytes were quantified by microscopy. Data are mean ± SEM of mice pooled from 3 independent experiments, with 1-2 mock-infected and 1-3 infected mice/age/experiment. (B) Aseptic peritonitis. Young and old mice were challenged i.p. with 0.25 ml sterile 3% thioglycollate (TGC, filled symbols), or were mock-treated (open symbols), and culled 3 h later. Peritoneal lavages were assessed for numbers of peritoneal neutrophils, macrophages and total leukocytes by hemocytometer and microscopy of cytospins. Data are mean ± SEM, pooled from 5 independent experiments, with 1-3 mock-treated and 1-4 TGC-treated mice/age/experiment. (C) LPS-induced septic peritonitis. Young and old mice were challenged i.p. with 250 ng LPS in 0.25 ml saline (filled symbols), or were mock-treated with saline (open symbols), and culled 3 h later. Peritoneal lavages were assessed as in (B). Data are mean ± SEM, pooled from 3 independent experiments, with 1-2 mock-treated and 2-3 LPS-treated mice/age/experiment; dots represent individual mice. Statistics in (A-C) are two-way ANOVA with Sidak’s multiple comparisons test on log-transformed raw data; black p-values are significant, grey p-values non-significant. (D) In vitro killing of E. coli. Neutrophils purified from young (8-10 weeks, black symbols) and old (24 months, purple symbols) mice were primed with 1 µg/ml LPS or with 20 ng/ml TNFa and 50 ng/ml GM-CSF, as indicated, or were heat-killed as a negative control, and were incubated with serum-opsonised E. coli DH5a (ratio of 2.5 bacteria per neutrophil) at 37°C for 30 or 120 min. Bacterial killing by neutrophils was quantified by comparing CFU between samples containing live and heat-killed neutrophils. Data are mean ± SEM of 3 independent experiments; each dot represents the mean of one experiment. Statistics are three-way ANOVA with Sidak’s multiple comparisons test; black p-values are significant, grey p-values non-significant.

Fig 3: TLR4 is a direct target of miR-93. (A) Putative binding site of miR-93 and TLR4. (B) Mutation was generated on the TLR4 3′UTR sequence in the complementary site for the seed region of miR-93. TLR4 3′UTR fragments containing the wild-type or mutant miR-93-binding sequence were cloned downstream of the luciferase reporter gene in pGL3-luc vector. (C) Luciferase assay of 293 cells co-transfected with firefly luciferase constructs containing TLR4 wt or mut 3′-UTRs and miR-93 mimics, mimics NC, miR-93 inhibitor or inhibitor NC, as indicated (n=3). Data are presented as the mean ± standard deviation of three independent experiments. **P<0.01, vs mimics NC, ##P<0.01, vs inhibitor NC. (D) mRNA and (E) protein expression of TLR4 following transfection with miR-93 mimics or miR-93 inhibitor was measured by RT-qPCR and western blot analyses. Data are presented as the mean ± standard deviation of three independent experiments. **P<0.01, vs, inhibitor NC, ##P<0.01, vs mimics NC. (F) Chondrocytes were transfected with miR-93 mimics or miR-93 inhibitor for 24 h and then treated with LPS (5 µg/ml) for 24 h. Expression of TLR4 was assessed by RT-qPCR analysis. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01, vs. control group; ##P<0.01, vs. LPS + mimics NC group; &&P<0.01, vs. LPS + inhibitor NC group. TLR4, Toll-like receptor 4; miR, microRNA; UTR, untranslated region; wt, wild-type; mut, mutant; NC, negative control; LPS, lipopolysaccharide; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Fig 4: Urolithin A reduced the pro-inflammatory cytokine production and mRNA expression in LPS-stimulated murine BMDMs. Murine BMDMs were stimulated by 1 µg/ml of LPS in the presence or absence of UA (25 µM or 50 µM). Subsequently, expression of the pro-inflammatory cytokines IL-1β (a), IL-6 (b), IL-12 (c), TNF-α (d), and NOS2 (e) were measured in BMDMs by qRT-PCR (over GAPDH) and ELISA at depicted time points. The unstimulated and untreated BMDMs were used as control. BMDMs treated with DMSO were used as negative control. Arithmetic means ± SEM from seven independent experiments are depicted. Two way ANOVA was used and *(p < 0.05), **(P 0.01), ***(p < 0.001), and ****(p < 0.0001) indicate statistically significant differences compared to respective control. Abbreviations: DMSO, dimethyl sulfoxide; UA, urolithin A; LPS, lipopolysaccharides; IL, Interleukin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; and TNF, tumor necrosis factor; NOS, nitric oxide synthase.

Fig 5: The TRIF-dependent phosphorylation of IRF-3 is augmented in Dab2-knockdown cells.(a–c) The shLuc and shDab2-B cells were treated with LPS (100 ng/ml) for the indicated time. The cell lysates were collected for Western blotting using the antibodies against the indicated proteins to determine the effect of Dab2 knockdown on LPS-stimulated phosphorylation of IRF-3 (panel A), p65-NF-κB (panel B), and MAPKs (p38, ERK1/2, and JNK1/2; panel C), and degradation of IRAK1 and IκBα (panel B). The expression of β-actin was used as a control for equal protein loading. Quantification for the changes of protein phosphorylation and degradation was performed by using the Image J analysis software. The full Western blot and the corresponding positions of the molecular weight protein markers are presented in Supplementary Fig. S3. Data represent the mean ± SEM of three independent experiments. ns, no significance. *P < 0.05; **P < 0.01; and ***P < 0.001.