Fig 2: A-SAA and TLR2 expression is dependent on STING activation.(A and B) IMR90 ER:RAS cells were treated with 4OHT and repeatedly transfected with indicated pooled siRNA and nontarget siRNA as control for 8 days. TLR2, SAA1, and SAA2 transcripts were measured by qRT-PCR. Results are expressed as means ± SEM of three independent experiments. (C) IMR90 cells were transfected with siRNA targeting RELA, IRF3, TLR2, and STING for 2 days, followed by transfection with 2.5 µg of herrings-testes DNA (HT-DNA) for 24 hours. TLR2 transcripts were measured by qRT-PCR. Results are expressed as means ± SEM of three independent experiments. (D) Western blot for STING dimerization. HT-DNA transfection of IMR90 cells were used as positive control for STING dimerization. IMR90 ER:RAS cells were transfected with siRNA targeting TLR2, TLR10, and STING for 8 days with 4OHT. All statistical significance was calculated using a one-way ANOVA. ***P < 0.001, **P < 0.01, and *P < 0.05.

Fig 3: Co-activation of CLEC5A and TLR2 by L. monocytogenes enhances host immunity. Listeria activates CLEC5A and TLR2 simultaneously to activate both MyD88-p38 and Syk-AKT signaling pathways. Co-activation of CLEC5A and TLR2 in neutrophils enhances CXCL8 and ROS production, and NET formation, whereas co-activation of CLEC5A and TLR2 in macrophages induces inflammasome activation and production of IL-1ß to stimulate the development of IL-17A proudcing TCR ?d T-cells

Fig 4: TLR2 expression is induced during OIS.(A) Schematic showing IMR90 ER:RAS cells treated with 4OHT undergo OIS. IMR90 ER:STOP cells serve as a control and retain proliferative capacity with 4OHT. (B) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of TLR family member expression in IMR90 ER:RAS and ER:STOP cells treated with 4OHT for 5 and 8 days. (C) Western blot of TLR2 expression in IMR90 ER:RAS and ER:STOP (ER:S) cells with up to 10 days of 4OHT treatment (top). The 5-bromo-2'-deoxyuridine (BrdU) incorporation in IMR90 ER:RAS cells treated with 4OHT for up to 8 days, as indicated. (D) RNA was extracted from snap-frozen liver samples from wild-type (WT) mice 6 days following hydrodynamic delivery of NrasG12V/D38A (n = 5) and NrasG12V (n = 4) transposons. tlr2 mRNA expression was measured using qRT-PCR. Scatter plots represents value per animal, with the horizontal line representing group means ± SEM. Statistical significance was calculated using Students two-tailed t test. **P < 0.01. (E) Immunohistochemical staining for Nras and Tlr2 in consecutive liver sections from corresponding mice in (D) showing that oncogenic NrasG12V expressing, but not NrasG12V/D38A expressing, hepatocytes express Tlr2. Scale bars, 50 µm.

Fig 5: Attenuation of SARS-CoV-2-induced thromboinflammation in CLEC5A and TLR2 deficient mice. C57BL/6 mice (WT) and clec5a-/-tlr2-/- mice were inoculated with AAV-hACE2 for 14 days, followed by intranasal inoculation of SARS-CoV-2 (8 × 104 PFU/per mice). Tissues were collected at 3 days and 5 days post-infection. N = 3 of each group a The level of proinflammatory cytokines and chemokines were measured by real-time PCR and presented as fold change (compared to AAV-hACE2 uninfected mice/mock). b–d NET structure and thrombus were detected by Hoechst. 33342 (blue), anti-MPO antibody (green), anti-citrullinated histone H3 (red), anti-CD42b antibody (yellow) (b), and images were captured by a confocal microscope and subjected to determine the area of MPO (c) and CD42b (d) using MetaMorph™ software. e Cell infiltrated to lung at 3 d.p.i.. Interstitial macrophage (interstitial M?) was defined as CD11b+CD64+F4/80+ cells; monocyte-derived dendritic cell (DC)/macrophage (M?) was defined as CD11b+CD64+Ly6C+; Ly6C+ monocyte was defined as Ly6C+. The cell number of each cell population was calculated using the multiple fluorescent staining image and analyzed by software MetaMorph™, and the data was presented as cell number/ per 664225 (815 × 815). Scale bar is 200 µm. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001 (Student’s t-test)