Fig 2: Tenascin-C Posttranscriptionally Regulates the Synthesis of Specific Cytokine Subsets in BMDMs(A) ELISA of TNF-a, IL-6, CXCL1, IL-10, IL-1ß, IL-12, and TGF-ß1 secreted by tnc+/+ and tnc-/- BMDMs stimulated for 1.5, 4, 8, 24, and 48 hr with 100 ng/ml LPS. Data are from six to nine independent experiments (mean ± SEM). ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05.(B) Quantitative RT-PCR analysis of TNF-a, IL-6, CXCL1, and IL-10 mRNA in tnc+/+ and tnc-/- BMDMs stimulated for 0.5, 1, 1.5, 4, 8, 24, and 48 hr with 100 ng/ml LPS. Results are presented relative to those of untreated BMDMs. Data are from four to five independent experiments (mean ± SEM).See also Figure S3.

Fig 3: Tenascin-C Expression Is Transiently Induced by LPS(A) Quantitative RT-PCR analysis of tenascin-C mRNA in BMDMs stimulated for 1, 1.5, 4, 8, 24, and 48 hr with 100 ng/ml LPS. Results are presented relative to those of untreated BMDMs. Data are from seven independent experiments (mean ± SEM). ***p < 0.001 and **p < 0.01 compared to nonstimulated cells.(B) ELISA of cellular tenascin-C in BMDMs stimulated for 1.5, 4, 8, 24, 48, and 72 hr with 100 ng/ml LPS. Results are normalized to the cell number. Data are from six independent experiments (mean ± SEM). ***p < 0.001 compared to nonstimulated cells.(C) ELISA of secreted tenascin-C by BMDMs stimulated for 1.5, 4, 8, 24, 48, and 72 hr with 100 ng/ml LPS. Data are from six independent experiments (mean ± SEM). **p < 0.01 and *p < 0.05 compared to nonstimulated cells.See also Figure S2.

Fig 4: Tnc-/- Mice Are Less Susceptible to LPS-Induced Sepsis(A and B) Body weight (A) and mobility (B) loss in tnc+/+ and tnc-/- mice upon LPS injection, monitored over 8 hr. Data in (A) are presented as percent loss relative to noninjected mice (n = 17 (A) or 10 (B) mice per group; mean ± SEM). ****p < 0.0001 and ***p < 0.001 compared to noninjected mice (A). Data are from three (A) or two (B) independent experiments.(C) Time course analysis of TNF-a, IL-6, and HMGB1 in plasma from tnc+/+ and tnc-/- mice after LPS injection by ELISA (n = 3–5 mice per group; mean ± SEM). ****p < 0.0001, ***p < 0.001, and ** p < 0.01. Data are representative of three independent experiments.(D) Immunohistochemical analysis of neutrophil elastase+ cell infiltration in lungs from tnc+/+ and tnc-/- mice 0, 1.5, 4, and 8 hr after LPS injection (n = 4 LPS-injected and 3 noninjected mice per genotype). Scale bars, 100 µm. Data are representative of three independent experiments.(E and F) Immunoblot analysis of plasma tenascin-C (TN-C) in tnc+/+ (one to three) and tnc-/- (four to six) mice 8 hr (E) and 0, 1.5, 4, and 8 hr (F) after LPS injection. Results are representative of three independent experiments.(G) ELISA of plasma tenascin-C in tnc+/+ mice 0, 1.5, 4, and 8 hr after LPS injection (n = 7; mean ± SEM). Data are from two independent experiments.Related to Figure S1.

Fig 5: Tnc-/- Mice Develop Less-Severe Symptoms upon LPS Injection, Related to Figure 1(A) Representative images of the eyes of tnc+/+ and tnc-/- mice 0, 4 and 8 hr after LPS injection. Data are representative of three independent experiments.(B) Fur ruffling in tnc+/+ and tnc-/- mice upon LPS injection, monitored over 8 hr. (n = 10 per genotype; mean ± SEM); **** = p < 0.0001. Data are from two independent experiments.(C) Immunohistochemical analysis of neutrophil elastase+ cell infiltration in liver from tnc+/+ and tnc-/- mice 0, 1.5, 4 and 8 hr after LPS injection (n = 4 LPS-injected mice per genotype and 3 non-injected mice per genotype); scale bars, 100 µm. Results are representative of three independent experiments.(D) Neutrophil elastase+ cell count in lungs and liver from tnc+/+ and tnc-/- mice 0, 1.5, 4 and 8 hr after LPS injection. Values are expressed as mean ± SD (n). Data are representative of three independent experiments.