Journal: Neural Regeneration Research

Article Title: Porcine decellularized nerve matrix hydrogel attenuates neuroinflammation after peripheral nerve injury by inhibiting the TLR4/MyD88/NF-κB axis

doi: 10.4103/NRR.NRR-D-24-00302

Figure Lengend Snippet: TLR4/MyD88/NF-κB pathway plays an indispensable role in regulating pDNM-gel-mediated macrophage polarization and anti-inflammatory reactions in vitro . (A) WB detection of TLR4, MyD88, p-NF-κB, NF-κB, p-IκBα, and IκBα in RAW264.7 cells treated with LPS (100 ng/mL) + pDNM-gel (0.5%) for 24 hours, followed by the addition of CRX-527 (500 µM), TAK242 (100 nM), CL075 (2 µM), or T6167923 (100 µM) for another 24 hours. (B–E) Quantitative analysis of the expression levels of these four proteins from A. (F, G) Co-staining of CD68 (red) with iNOS (green) or Arg1 (green) in the LPS + pDNM-gel, LPS + pDNM-gel + CRX-527, LPS + pDNM-gel + TAK242, LPS + pDNM-gel + CL075, and LPS + pDNM-gel + T6167923 treatment groups. Nuclei were stained with DAPI (blue). Scale bars: 10 μm. (H, I) Quantification of the fluorescence intensity of iNOS and Arg1 from F and G. (J, K) Flow cytometry detection of M1-type and M2-type macrophages in each group. (L, M) Quantitative analysis of the percentages of double-positive CD68 + CD86 + and CD68 + CD206 + cells from J and K. (N, O) qRT-PCR analysis of the mRNA expression of pro-inflammatory signature genes TNF-α , IL-1 β, and IL-6 , and anti-inflammatory signature genes IL-4 , IL-10 , and TGF- β, in each group. (P, Q) ELISAs of inflammatory cytokine levels in RAW264.7 cell culture supernatants in each group. Data are expressed as mean ± SEM from three independent assays in vitro ; * P < 0.05, ** P < 0.01, *** P < 0.001. Arg1: Arginase 1; CD206: cluster of differentiation protein 206; CD68: cluster of differentiation protein 68; CD86: cluster of differentiation protein 86; DAPI: 4′,6-Diamidino-2-phenylindole; ELISA: Enzyme-linked immunosorbent assay; IL-10: interleukin-10; IL-1β: interleukin-1β; IL-4: interleukin-4; IL-6: interleukin-6; iNOS: inducible nitric oxide synthase; IκBα: nuclear factor kappa B inhibitory protein alpha; LPS: lipopolysaccharides; MyD88: myeloid differentiation factor 88; NF-κB: nuclear factor-κB; n.s,: not significant; pDNM-gel: porcine decellularized nerve matrix hydrogel; p-IκBα: phosphorylated nuclear factor kappa B inhibitory protein alpha; p-NF-κB: phosphorylated nuclear factor-κB; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; TGF-β: transforming growth factor-beta; TLR4: Toll-like receptor 4; TNF-α: tumor necrosis factor-alpha.

Article Snippet: Then, the medium was added with one of the following agonists/inhibitors and treated the cells for a further 24 hours: 500 μM CRX-527 (InvivoGen, Toulouse, France, Cat# tlrl-crx527), 100 nM TAK242 (MedChemExpress, Monmouth Junction, NJ, USA, Cat# HY-11109), 2 μM CL075 (MedChemExpress, Cat# HY-117066), or 100 μM T6167923 (MedChemExpress, Cat# HY-19744; Additional Figure 3 ).

Techniques: In Vitro, Expressing, Staining, Fluorescence, Flow Cytometry, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: Porcine decellularized nerve matrix hydrogel attenuates neuroinflammation after peripheral nerve injury by inhibiting the TLR4/MyD88/NF-κB axis

doi: 10.4103/NRR.NRR-D-24-00302

Figure Lengend Snippet: Promotional effect of pDNM-gel on nerve regeneration is abolished using TLR4/MyD88/NF-κB agonists in vivo . (A) Immunofluorescence staining for neurofilament-H (green) and MBP (red) in sciatic nerves from rats treated with pDNM-gel, pDNM-gel + CRX-527, or pDNM-gel + CL075 at 21 dpi ( n = 5/group). Nuclei were stained with DAPI (blue). Scale bars: 20 μm. (B) Representative TEM images of regenerated sciatic nerve cross-sections from the three groups at 21 dpi. Scale bars: 10 μm. (C, D) Calculated area (%) of positive staining of neurofilament-H and MBP per 100 μm 2 in the immunofluorescence images for the three groups ( n = 5/group). (E, F) Statistical analysis of the number and thickness of myelin sheaths in the three groups using ImageJ software ( n = 3/group). Data are expressed as mean ± SEM; ** P < 0.01, *** P < 0.001. DAPI: 4′,6-Diamidino-2-phenylindole; dpi: days post-injury; MBP: myelin basic protein; MyD88: myeloid differentiation factor 88; NF-κB: nuclear factor-κB; pDNM-gel: porcine decellularized nerve matrix hydrogel; PNI: peripheral nerve injury; SEM: standard error of the mean; TEM: transmission electron microscopy; TLR4: Toll-like receptor 4.

Article Snippet: Then, the medium was added with one of the following agonists/inhibitors and treated the cells for a further 24 hours: 500 μM CRX-527 (InvivoGen, Toulouse, France, Cat# tlrl-crx527), 100 nM TAK242 (MedChemExpress, Monmouth Junction, NJ, USA, Cat# HY-11109), 2 μM CL075 (MedChemExpress, Cat# HY-117066), or 100 μM T6167923 (MedChemExpress, Cat# HY-19744; Additional Figure 3 ).

Techniques: In Vivo, Immunofluorescence, Staining, Software, Transmission Assay, Electron Microscopy

Journal: Frontiers in Immunology

Article Title: B-type natriuretic peptide attenuates TLR-induced cytokine and chemokine secretion in monocyte-derived Langerhans cells

doi: 10.3389/fimmu.2026.1791284

Figure Lengend Snippet: The effects of BNP on activated moLCs differentiation and activation markers. (A) Representative density plot of the main markers of moLCs, CD1a and CD207 expression measured by flow cytometry. Monocytes were cultured in the presence of GM-CSF, TNF-α, and TGF-β for 5 days supplemented with IL-4 for the first 48 hrs to differentiate moLCs, and treated with 10 nM BNP throughout the entire differentiation process. On day 4 moLCs were activated with CL075 and poly(I:C) and a combination of both for 24 hrs. (B) A percentage of positive CD207 and CD1a moLCs. N = 6 (C) Histograms of moLCs activation markers, CD83, CD86, and HLA-DQ. (D) A percentage of positive CD83, CD86 and HLA-DQ moLCs. Symbols with different colors represent individual donors. Data are presented as individual values with mean ± SEM. Statistical significance was determined by One-way ANOVA followed by Tukey’s post-hoc test. Homogeneity of variances was confirmed by the Brown-Forsythe test. n=6 biological replicates per group. *P<0.05, **P<0.01, *** P<0.001, **** P< 0.0001. BNP, B-type natriuretic peptide; CL075, TLR7/8 agonist, thiazoquinoline compound; CTRL, Control; Poly(I:C), TLR3 agonist, polyinosinic:polycytidylic acid.

Article Snippet: MoLCs were activated with 0.5 μg/ml CL075 (TLR7/8 agonist) and 20 μg/ml poly(I:C) (TLR3 agonist) and with their combination at day 4 for 24 hours (both were obtained from Invivogen, San Diego, CA, USA).

Techniques: Activation Assay, Expressing, Flow Cytometry, Cell Culture, Control

Journal: Frontiers in Immunology

Article Title: B-type natriuretic peptide attenuates TLR-induced cytokine and chemokine secretion in monocyte-derived Langerhans cells

doi: 10.3389/fimmu.2026.1791284

Figure Lengend Snippet: BNP inhibited the TLR activated moLCs IL-6, TNF-α, IL-12, IL-10 and IFN-β cytokine production. Monocytes were cultured in the presence of GM-CSF, TNF-α, and TGF-β for 5 days supplemented with IL-4 for the first 48 hrs to differentiate moLCs, and treated with 10 nM BNP throughout the entire differentiation process. On day 4 moLCs were activated with CL075 and poly(I:C) and a combination of both for 24 hrs. Supernatants were collected on day 5 from the cells. (A–F) Levels of different pro-inflammatory cytokines IL-6 and IL-8, TNF-α, cytokines during viral response IL-12 and IFN-β, and an anti-inflammatory IL-10, were measured by ELISA. Symbols with different colors represent individual donors, lines marks mean expression levels. Data is presented as Mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparison test. Normality of residuals was confirmed by QQ plot analysis. n=6–10 biological replicates per group *P<0.05, **P<0.01, *** P<0.001, **** P< 0.0001. BNP, B-type natriuretic peptide; CL075; TLR7/8 agonist, thiazoquinoline compound; CTRL, Control; moLC, monocyte-derived Langerhans cells; Poly(I:C), TLR3 agonist, polyinosinic:polycytidylic acid.

Article Snippet: MoLCs were activated with 0.5 μg/ml CL075 (TLR7/8 agonist) and 20 μg/ml poly(I:C) (TLR3 agonist) and with their combination at day 4 for 24 hours (both were obtained from Invivogen, San Diego, CA, USA).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Comparison, Control, Derivative Assay

Journal: Frontiers in Immunology

Article Title: B-type natriuretic peptide attenuates TLR-induced cytokine and chemokine secretion in monocyte-derived Langerhans cells

doi: 10.3389/fimmu.2026.1791284

Figure Lengend Snippet: BNP reduced the T cell proliferation induction ability in Poly(I:C) and CL075 activated moLCs. (A) Proliferating T cells were measured with flow cytometry by the CFSE intensity at day 5 of coculture. Monocytes were cultured in the presence of GM-CSF, TNF-α, and TGF-β for 5 days, supplemented with IL-4 for the first 48 hrs to differentiate moLCs, and treated with 10 nM BNP throughout the differentiation process. The moLCs were activated for 24 hrs with CL075, and poly(I:C), and a combination of both. Naїve CD4 + T cells were isolated with CD4 + magnetic beads and made a coculture with fully differentiated moLCs for 5 days. n=6 (B) Representative histograms of CFSE intensity from a coculture of T cells and moLCs. (C) Representative Western blot of IDO1 expression of moLCs. Cells were treated with 10 nM BNP and activated for 24 hrs in the presence of CL075 and poly(I:C) at day 4. Symbols with different colors represent individual donors. Data are presented as individual values with mean ± SEM. Statistical significance was determined by One-way ANOVA followed by Tukey’s post-hoc test. Homogeneity of variances was confirmed by the Brown-Forsythe test. n=2 biological replicates per group *P<0.05, **P<0.01, *** P<0.001, **** P< 0.0001. BNP, B-type natriuretic peptide; CFSE, Carboxyfluorescein succinimidyl ester; CL075; TLR7/8 agonist, thiazoquinoline compound; CTRL, Control; IDO1, Indoleamine 2,3-dioxygenase; Poly(I:C), TLR3 agonist, polyinosinic:polycytidylic acid.

Article Snippet: MoLCs were activated with 0.5 μg/ml CL075 (TLR7/8 agonist) and 20 μg/ml poly(I:C) (TLR3 agonist) and with their combination at day 4 for 24 hours (both were obtained from Invivogen, San Diego, CA, USA).

Techniques: Flow Cytometry, Cell Culture, Isolation, Magnetic Beads, Western Blot, Expressing, Control

Journal: Frontiers in Immunology

Article Title: B-type natriuretic peptide attenuates TLR-induced cytokine and chemokine secretion in monocyte-derived Langerhans cells

doi: 10.3389/fimmu.2026.1791284

Figure Lengend Snippet: RNA-Seq analysis showed an upregulation of migratory genes and the downregulation of inflammatory cytokines and chemokines in activated moLCs. Monocytes were cultured in the presence of GM-CSF, TNF-α, and TGF-β for 5 days supplemented with IL-4 for the first 48 hrs to differentiate moLCs, and treated with 10 nM BNP throughout the differentiation process. The moLCs were activated for 24 hrs with CL075, and poly(I:C), and a combination of both. [( A–C) , left] Volcano plot analysis of poly(I:C) and CL075 and BNP treated moLCs compared to vehicle-treated controls or each other. The top 15 most significantly changed gene symbols are highlighted. [ (A–C , middle, right] GO annotation of the biological process of up- and down-regulated genes between the groups marked. The size of the circle corresponds to the number of induced genes, while the color of the line corresponds to the -log10 of the False Discovery Rate (FDR). (A) Poly(I:C) and CL075 vs. CTRL (B) BNP, Poly(I:C) and CL075 vs. CTRL (C) BNP, Poly(I:C) and CL075 vs. Poly(I:C) and CL075. BNP, B-type natriuretic peptide; CL075; TLR7/8 agonist, thiazoquinoline compound; CTRL, Control; GO, gene ontology; Poly(I:C), TLR3 agonist, polyinosinic:polycytidylic acid.

Article Snippet: MoLCs were activated with 0.5 μg/ml CL075 (TLR7/8 agonist) and 20 μg/ml poly(I:C) (TLR3 agonist) and with their combination at day 4 for 24 hours (both were obtained from Invivogen, San Diego, CA, USA).

Techniques: RNA Sequencing, Cell Culture, Control

Journal: Frontiers in Immunology

Article Title: B-type natriuretic peptide attenuates TLR-induced cytokine and chemokine secretion in monocyte-derived Langerhans cells

doi: 10.3389/fimmu.2026.1791284

Figure Lengend Snippet: BNP treatment increased moLCs migration toward CCL21 and reduced chemokine production in activated moLCs. Monocytes were cultured in the presence of GMCSF, TNF-α, and TGF-β for 5 days supplemented with IL-4 for the first 48 hrs to differentiate moLCs, and treated with 10 nM BNP throughout the entire differentiation process. On day 4, moLCs were activated with CL075 and poly(I:C) and a combination of both for 24 hrs. (A) In Transwell migration assay, 1*10 6 cells were applied in the upper well of the Transwell plate, and in the bottom of the plate, it contains RPMI supplemented with CCL19 and CCL21. Migration of the cells was measured with flow cytometry. Data are presented as individual values with mean ± SEM. Statistical significance was determined by Two-way ANOVA followed by Tukey’s multiple comparison test. Normality of residuals was confirmed by QQ plot analysis. n=8 biological replicates per group (B) A chemokine array was performed using supernatants from BNP treated and TLR activated cells. Symbols with different colors represent individual donors. Data is presented as Mean ± SEM. One-way ANOVA with Tukey’s multiple comparison test was used for statistical analysis. n=2 biological replicates per group *P<0.05, **P<0.01, *** P<0.001, **** P< 0.0001. BNP, B-type natriuretic peptide; CL075; TLR7/8 agonist, thiazoquinoline compound; CTRL, Control; moLC, monocyte-derived Langerhans cells; Poly(I:C), TLR3 agonist, polyinosinic:polycytidylic acid.

Article Snippet: MoLCs were activated with 0.5 μg/ml CL075 (TLR7/8 agonist) and 20 μg/ml poly(I:C) (TLR3 agonist) and with their combination at day 4 for 24 hours (both were obtained from Invivogen, San Diego, CA, USA).

Techniques: Migration, Cell Culture, Transwell Migration Assay, Flow Cytometry, Comparison, Control, Derivative Assay

Journal: Frontiers in Immunology

Article Title: B-type natriuretic peptide attenuates TLR-induced cytokine and chemokine secretion in monocyte-derived Langerhans cells

doi: 10.3389/fimmu.2026.1791284

Figure Lengend Snippet: BNP treated moLCs supernatant inhibited NK cell migration. Monocytes were cultured in the presence of GM-CSF, TNF-α, and TGF-β for 5 days supplemented with IL-4 for the first 48 hrs to differentiate moLCs, and treated with 10 nM BNP throughout the entire differentiation process. On day 4 moLCs were activated with CL075 and poly(I:C) and a combination of both for 24 hrs. Transwell plates were used to perform the migration experiments, and cell numbers were determined by flow cytometry. (A) A flow cytometry gating strategy was used to distinguish between the PBL and monocyte populations based on the FSC-H and SSC-H. In the histograms, CD3 + cells represent the T cell population. CD3 - cells selected to determine the CD56 + population, which represented the NK cells. (B–F) Flow cytometry was used to measure the number of migrating cells in the lower wells of the Transwell plate. (B) PBL, (C) CD3 + , (D) CD3 - , CD56 + , (E) CD3 - , CD56 - , (F) monocytes. Symbols represent individual donors. Data are presented as individual values with mean ± SEM. Statistical significance was determined by Two-way ANOVA followed by Tukey’s multiple comparison test. Normality of residuals was confirmed by QQ plot analysis. n=10–12 biological replicates per group *P<0.05, **P<0.01, **** P< 0.0001. BNP, B-type natriuretic peptide; CL075; TLR7/8 agonist, thiazoquinoline compound; CTRL, Control; moLC, monocyte-derived Langerhans cells; NK cells, Natural Killer cells; Poly(I:C), TLR3 agonist, polyinosinic:polycytidylic acid.

Article Snippet: MoLCs were activated with 0.5 μg/ml CL075 (TLR7/8 agonist) and 20 μg/ml poly(I:C) (TLR3 agonist) and with their combination at day 4 for 24 hours (both were obtained from Invivogen, San Diego, CA, USA).

Techniques: Migration, Cell Culture, Flow Cytometry, Comparison, Control, Derivative Assay

Journal: bioRxiv

Article Title: Pregnancy loss due to early developmental defects in lupus mice expressing human TLR8

doi: 10.64898/2026.02.07.701591

Figure Lengend Snippet: HuTLR8 induces fetal resorption and spontaneous pregnancy loss in Sle1 mice. A) C57Bl/6.huTLR8tg mice were crossed with Yaa males and then with Sle1 females . Sle1.huTLR8tg female mice express one (Sle1.huTLR8 +/- ) or two (Sle1.huTLR8 +/+ ) copies of the single human TLR8 (huTLR8) transgenic locus. B. Outcomes of observed pregnancies. Adverse outcome was defined as maternal distress or death at delivery with evidence of an impacted pup or birth of dead or runted pups. p values calculated using Chi square analysis. C. I. An impacted pup in a Sle1.huTLR8 +/+ female II. An impacted pup together with an unaffected littermate; III. Representative uteri from Sle1 (top) and Sle1.huTLR8 +/+ (bottom) mice; IV. Growth retarded pup with an associated small placenta (*); V. A normal sized fetus and a resorbed fetus from the same pregnancy. D. Fetal weights (in gram) are depicted for full term Sle1 (n=5) and C57BL/6.huTLR8tg control live pregnancies and Sle1.huTLR8 +/- (n=16) and Sle1.huTLR8 +/+ (n=18) pregnancies with dystocia. Impacted/resorbed/dead fetuses are depicted in red. Maternal genotype and number of pregnancies are shown below the x axis. E, F. Total number of pups (E) and number of male (blue bars) and female (pink bars) pups (F) per live litter. Maternal genotype and number of pregnancies are shown below the x axis. Each symbol represents an individual pregnancy; bars represent the mean + SD. D-F: p values calculated using Kruskal Wallis ANOVA with Dunn’s correction for multiple analyses. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Article Snippet: Bone marrow neutrophils were isolated from 3– and 6-month-old homozygous female Sle1.huTLR8tg, Sle1 and C57BL/6 controls using EasySep mouse neutrophil enrichment kit (STEMCELL) and were treated with PMA 50nM for 4 hours at 37°C with or without pre-treatment with PMA or TLR8 agonist CL075 (Invivogen) 10mg/ml for 20 minutes.

Techniques: Transgenic Assay, Control

Journal: bioRxiv

Article Title: SLAMF1-peptide mediated epigenetic priming reprograms innate immune responses in sepsis

doi: 10.64898/2025.12.29.696918

Figure Lengend Snippet: Volcano plots comparing cytokine secretion by PBMCs primed with vehicle or P7-Pen and subsequently stimulated with LPS ( A ) or CL075 ( B ) for 2 h. Plots were generated by plotting log 2 fold change against −log 10 FDR-adjusted p values (q < 0.05). (C) Quantification of the most strongly affected LPS-induced cytokines in PBMCs primed with vehicle or P7-Pen. Statistical significance for panels (A–C) was assessed using multiple unpaired t -tests ( p < 0.05, * p < 0.01, ** p < 0.001).

Article Snippet: Ultrapure K12 LPS (#tlrl-peklps) from E. coli , ultrapure thiazoloquinoline compound CL075 (#tlrl-c75), benzazepine analog TL8-506 (#tlrl-tl8506) and FSL-1 TLR2/TLR6 ligand (#tlrl-fsl) were from InvivoGen (San Diego, CA, USA).

Techniques: Generated

Journal: bioRxiv

Article Title: SLAMF1-peptide mediated epigenetic priming reprograms innate immune responses in sepsis

doi: 10.64898/2025.12.29.696918

Figure Lengend Snippet: Western blot analysis shows histone H3 acetylation patterns together with quantitative densitometry for all analyzed samples. (A, B) Representative immunoblots of PBMC lysates from healthy donors ( A ) or sepsis patients ( B ) showing levels of H3K9ac, H3K27ac, and H3K56ac following priming with vehicle (water), 15 µM control peptide C3-Pen, or 15 µM P7-Pen for 20–22 h. (C) Representative immunoblots (2 of 10 donors per group) showing H3K27ac levels in PBMCs primed with water or P7-Pen and subsequently stimulated with LPS or CL075 for 2 h. Quantification shown in the graphs represents signal intensity normalized to the corresponding loading control (β-tubulin). Data are presented as mean relative fold change ± SEM. Statistical significance was assessed using the nonparametric Wilcoxon matched-pairs signed-rank test ( p < 0.05, * p < 0.01, ** p < 0.001).

Article Snippet: Ultrapure K12 LPS (#tlrl-peklps) from E. coli , ultrapure thiazoloquinoline compound CL075 (#tlrl-c75), benzazepine analog TL8-506 (#tlrl-tl8506) and FSL-1 TLR2/TLR6 ligand (#tlrl-fsl) were from InvivoGen (San Diego, CA, USA).

Techniques: Western Blot, Control