Journal: Nature Communications

Article Title: Angptl4 integrates dietary and microbial signals to disrupt gut barrier function in MASH

doi: 10.1038/s41467-026-72575-6

Figure Lengend Snippet: A Transepithelial electrical resistance (TEER) measurement of human intestinal epithelial cells (hIECs) treated with recombinant cAngptl4 (rcAngptl4) at varying concentrations and in combination with an anti-cAngptl4 monoclonal antibody (mAb), mAb11F6C4. The graph shows the impact of cAngptl4 on cell barrier function over time. n = 3 independent experiments. B Relative expression of Angptl4 mRNA (top panel) and protein (bottom panel) in hIECs treated with different fatty acids or vehicle control. Representative immunoblots of cAngptl4 (35 kDa) and β-actin (42 kDa) are shown. n = 3 independent experiments. C Chromatin immunoprecipitation (ChIP) assays to assess PPARα binding to the PPRE element in hIECs treated with C18:2 fatty acid or vehicle. Data are normalized to IgG control and presented as fold change. D Relative Angptl4 mRNA expression in hIECs treated with C18:2 and 10 μM fenofibrate as PPARα agonizts. These are co-treated with either si-scrambled or si PPARα cells. n = 4 independent experiments. E Relative mRNA levels of TLR2, 4, 5, 6 across different regions of the small intestine of mice fed with LIDPAD or control diet for two weeks. n = 5 mice per group. For duodenum, p -values = 0.93 (TLR2), 0.047 (TLR4), 0.0384 (TLR5), 0.33 (TLR6); for jejunum, p -values = 0.0154 (TLR2), 0.0019 (TLR4), 0.0015 (TLR5), 0.477 (TLR6); for ileum, p -values = 0.0430 (TLR2), 0.044 (TLR4), 0.0005 (TLR5), 0.476(TLR6); F , G Relative expression of Angptl4 in ex vivo intestinal explants ( F ) and in vitro hIECs ( G ) treated with various TLR agonizts and vehicle control. For ( F , G ), n = 5 independent experiments. For ( F ) p -values (compared with vehicle group) = 0.0038 (LPS), 0.0007 (Pam3CSK4), 0.069 (HKLM), 0.8046 (FSL-1), 0.026 (FLA). For G, p -values (compared with vehicle group) = 0.476 (LPS), 0.0159 (Pam3CSK4), 0.78576 (HKLM), 0.2738 (FSL-1), 0.000099 (FLA). H Relative Angptl4 mRNA expression in cells transfected with control siRNA (si-scrambled, white bars) or TLR4-targeting siRNA (siTLR4, black bars) and stimulated with LPS (+). n = 3 independent experiments. I Relative expression of Angptl4 in in vitro human intestinal epithelial cells treated with Salmonella typhi flagellin (FLA) and various inhibitors against downstream kinases of TLR5 pathways. n = 4 independent experiments. p -values = 0.476 (FLA vs vehicle group), 0.0011 (FLA + GSKi vs FLA), 0.000099 (FLA + JNKi vs FLA). J ChIP assays to assess EGR1 binding to its cognate binding sites in human intestinal epithelial cells treated with flagellin or vehicle. Data are normalized to IgG control and presented as fold enrichment. n = 3 independent experiments. K Relative Angptl4 mRNA expression (normalized to 18S) in hIECs transfected with scrambled siRNA (si-scrambled, white bars), siEGR1 (black bars), and stimulated with Salmonella typhi flagellin (FLA). n = 3 independent experiments. L , M Box plot of the ratio of Gram-negative to Gram-positive bacteria ( L ) and stimulator-to-silent flagellin ( M ) on control and LIDPAD diet for 2 weeks. All box plots need to be defined in the legends in terms of minima, maxima, center, bounds of box and whiskers and percentile. n = 3 biological replicates. ( N ) Relative expression of Angptl4 in patient-derived intestinal organoids incubated with recombinant flagellins from Roseburia hominis, Romboutsia hominis and Salmonella typhi . n = 3 independent experiments. p -values (compared with vehicle control) = 0.459 ( Roseburia hominis ), 0.0346 ( Romboutsia hominis ), 0.0379 ( Salmonella typhi ). O TEER measurements of patient-derived intestinal organoids incubated with recombinant flagellins from Salmonella typhi, Roseburia hominis, Romboutsia hominis, Salmonella typhi with anti-cAngptl4 mAb, Romboutsia hominis with anti-cAngptl4 mAb (mAb11F6C4). The graph shows the impact of stimulator and silent flagellins as well as stimulator flagellins with cAngptl4 mAb (mAb11F6C4) treatment on cell barrier function over time. n = 3 independent experiments. P FITC-Dextran levels (Δ ng/μL) represent the change in gut permeability before and after bacterial transplantation in germ-free mice colonized with Roseburia hominis or Romboutsia hominis . Mice colonized with Romboutsia exhibited a significantly greater increase in FITC-Dextran levels, indicating heightened gut permeability compared to Roseburia -colonized mice. Data are presented as box-and-whisker plots, with individual data points shown, n = 8 independent biological replicates. Statistical significance was determined using an unpaired two-sided t test ( p < 0.001). Q Quantification of 4HNE positive area (%) in livers of mice colonized with Roseburia hominis and Romboutsia hominis after 2 weeks of control diet feeding. n = 3 biological replicates, p -value = 0.027. R Schematic illustrates how dietary fats and MAMPs influence gut permeability and hepatic oxidative stress in MASLD. Dietary fats stimulate PPARα, which directly enhances Angptl4 expression. Meanwhile, gut dysbiosis alters microbial composition in two key ways: (1) a decrease in the Gram-negative:Gram-positive ratio, which suggests a reduction in overall available LPS levels, and (2) an increase in the stimulator-to-silent flagellin ratio, indicating a shift toward more stimulator flagellins. As a result, dysbiotic bacteria have higher stimulator flagellin, which activates TLR5, leading to downstream signaling through GSK3 and JNK, ultimately inducing EGR1, which transcriptionally upregulates Angptl4 expression. In parallel, the reduced LPS exposure from Gram-negative bacteria leads to a diminished TLR4-mediated repression of Angptl4. The net effect is an increase in gut permeability and hepatic oxidative stress. The gray line denotes diminished regulatory effects. Figures referenced in parentheses correspond to experimental data supporting each molecular interaction. Created in BioRender. Li, L. (2026) https://BioRender.com/jo5i54i . For ( A – K , N , O , Q ), data are expressed as mean ± SD. For ( L , M , P ), boxplots show the median (center line), the 25 th and 75 th percentiles (bounds of the box), and whiskers extending to maxima and minima. *** p < 0.001, ** p < 0.01, * p < 0.05 (two-sided unpaired t test). n.s. denotes not significant.

Article Snippet: TLR agonist treatments were performed using a human TLR1-9 agonist kit per their recommended protocol (Invivogen, France, #tlrl-kit1hw).

Techniques: Recombinant, Expressing, Control, Western Blot, Chromatin Immunoprecipitation, Binding Assay, Ex Vivo, In Vitro, Transfection, Bacteria, Derivative Assay, Incubation, Permeability, Transplantation Assay, Whisker Assay

Journal: Infection and Immunity

Article Title: Lipidated Protein D vaccination elicits humoral and cellular responses and protects mice against challenge with non-typeable Haemophilus influenzae

doi: 10.1128/iai.00647-25

Figure Lengend Snippet: Lipidation of PD enhances immunogenicity. Mice were vaccinated with three doses via subcutaneous injection of alum alone, lipidated (L)-PD + Alum, L-PD, nonlipidated (NL)-PD + Alum, or NL-PD. Mice were challenged with influenza X31, then with NTHi 86-028 (5 × 10 6 CFU) 3 days later. ( A ) Total IgG to PD was measured by ELISA using sera collected from vaccinated mice 4 days after challenge with NTHi 86-028. Mean ± SEM of n = 5 per group from two independent experiments. Welch’s ANOVA comparison to the control alum group was performed with P < 0.05 considered significant. Lymphocytes from (B and C) cervical lymph nodes (CLN) and (D and E) splenocytes of vaccinated and NTHi 86-028-challenged mice were stimulated in vitro with 1 × 10 5 CFU/mL heat-killed (HK)- NTHi 86-028 for 5 days. Live CD4+ T cells from CLN were analyzed for the expression of ( B ) CD69 and IL-17A to identify activated Th17 cells. ( C ) The percentage of T-helper (Th) effector memory cells (Tem) defined as CD44hiCD62L-CCR7- was compared after stimulation. Live CD4+ T splenocytes were analyzed for the expression of ( D ) CD69 and IL17A to identify activated Th17 cells and ( E ) CD44hiCD62L-CCR7- to identify Tem cells. Mean ± SEM of n = 3 is shown. An ordinary one-way ANOVA comparison to the control alum group was performed, with P < 0.05 considered significant.

Article Snippet: HEK-Blue hTLR2-TLR1 cells (Invivogen) were used to determine triacyl lipidation status, and hTLR2-TLR6 reporter cells (Invivogen) were used to determine diacyl lipidation status as previously described ( ).

Techniques: Immunopeptidomics, Injection, Enzyme-linked Immunosorbent Assay, Comparison, Control, In Vitro, Expressing, FACS