Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: Killing efficiency of different physicochemical treatments on recombinant L. lactis strains displaying different binders of proinflammatory cytokines on their surface. The killing efficiency was determined by culturing and counting colonies on agar plates. Complete killing of bacteria i.e., 0 CFU/mL (no bacterial colonies on agar plates) was considered as 100% efficiency. pSD-ZIL6, L. lactis displaying anti-IL6 affibody; pSD-ZTNF, L. lactis displaying anti-TNF affibody; pSD-Fyn17, L. lactis displaying anti-IL17 fynomer; pSD-EVA, L. lactis displaying anti-IL8 evasin; pNZ8148, L. lactis harboring empty plasmid. The results presented are means ± standard deviation (SD) of three technical replicates from a representative experiment.

Article Snippet: Briefly, serial dilutions of IL6 (0.32, 0.65, 1.31, 2.62, 5.25, 10.5, 21, 42 nM; HZ-1019, Proteintech) were incubated with a fixed number of recombinant bacteria (8 × 10 6 CFU) for 2 h in a final volume of 200 μl.

Techniques: Recombinant, Bacteria, Plasmid Preparation, Standard Deviation

Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: Cytokine binding ability of recombinant L. lactis bacteria displaying different binders of proinflammatory cytokines on their surface upon exposure to bacteria-killing treatments. ELISA-determined concentrations of recombinant IL6 (A) , TNF (B) , IL17 (C) , and IL8 (D) that remained in the solution following incubation with the corresponding strain of bacteria before and after treatment with heat (70 °C, 40 min or 100 °C, 30 min), sonication, ethanol, UV, and gamma irradiation. pSD-ZIL6, L. lactis displaying ZIL6 affibody; pSD-ZTNF, L. lactis displaying anti-TNF affibody; pSD-Fyn17, L. lactis displaying anti-IL17 fynomer; pSD-EVA, L. lactis displaying anti-IL8 evasin; Ctrl: L. lactis control cells containing empty plasmid pNZ8148. The results are presented as means ± SD of three technical replicates of a representative experiment. ns, p = 0.09; *, p ≤ 0.05; **, p = 0.002; ***, p < 0.001 (unpaired two-tailed t -test).

Article Snippet: Briefly, serial dilutions of IL6 (0.32, 0.65, 1.31, 2.62, 5.25, 10.5, 21, 42 nM; HZ-1019, Proteintech) were incubated with a fixed number of recombinant bacteria (8 × 10 6 CFU) for 2 h in a final volume of 200 μl.

Techniques: Binding Assay, Recombinant, Bacteria, Enzyme-linked Immunosorbent Assay, Incubation, Sonication, Irradiation, Control, Plasmid Preparation, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: Determination of binding affinity of ZIL6-displaying L. lactis for human IL6. A constant number of 8 × 10 6 CFU bacteria was incubated with increasing concentrations of human IL6 for 2 h. MFI values were measured by flow cytometry and binding curves were fitted to the data using the Hill equation in the GraphPad Prism v.10.3.1. The dissociation constants (Kd) values were calculated from the binding curves for non-treated, live ZIL6-displaying L. lactis bacteria and bacteria treated by heat (70 °C, 40 min or 100 °C, 30 min), sonication, ethanol, and UV irradiation. Data are means ± SD of three technical replicates from a representative experiment.

Article Snippet: Briefly, serial dilutions of IL6 (0.32, 0.65, 1.31, 2.62, 5.25, 10.5, 21, 42 nM; HZ-1019, Proteintech) were incubated with a fixed number of recombinant bacteria (8 × 10 6 CFU) for 2 h in a final volume of 200 μl.

Techniques: Binding Assay, Bacteria, Incubation, Flow Cytometry, Sonication, Irradiation

Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: Quantification of the maximum binding capacity of ZIL6-displaying L. lactis . Increasing concentrations of recombinant human IL6 (0.45, 4.5, 45 and 450 ng) were incubated with a constant number of bacteria (4.5 × 10 7 CFU equivalent to 0.1 mg dry cell weight) in 450 μl. Residual IL6 in the supernatants was quantified by ELISA for live non-treated bacteria and the cells treated by heat (70 °C, 40 min or 100 °C, 30 min), sonication, ethanol, and UV irradiation. The percentage of bound IL6 was calculated from the difference measured in the presence of ZIL6-displaying L. lactis and control bacteria (carrying empty plasmid pNZ8148). The data are means ± SD of two biological replicates. The asterisks denote statistically significant differences between treated and non-treated bacteria for each concentration. ***, p < 0.001 (one-way ANOVA with Dunnett multiple comparison test).

Article Snippet: Briefly, serial dilutions of IL6 (0.32, 0.65, 1.31, 2.62, 5.25, 10.5, 21, 42 nM; HZ-1019, Proteintech) were incubated with a fixed number of recombinant bacteria (8 × 10 6 CFU) for 2 h in a final volume of 200 μl.

Techniques: Binding Assay, Recombinant, Incubation, Bacteria, Enzyme-linked Immunosorbent Assay, Sonication, Irradiation, Control, Plasmid Preparation, Concentration Assay, Comparison

Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: (A) Inhibition of STAT3 signaling in HEKblue-IL6R cells by non-viable ZIL6-displaying L. lactis bacteria in comparison to live non-treated strain. ZIL6-displaying L. lactis cells (1 × 10 8 CFU/mL or 1 × 10 9 CFU/mL) were preincubated with IL6 (1 ng/mL) for 2 h. After removal of bacterial cells, the cell-free supernatant containing remainder of IL6 was added to HEKblue-IL6R cells to induce the reporter system. The percentage of STAT3 inhibition was calculated relative to the STAT3 signaling induced by IL6 in the absence of bacteria. The inhibition of STAT3 signaling was determined for live non-treated bacteria and the cells treated by heat (70 °C, 40 min or 100 °C, 30 min), sonication, ethanol, UV, and gamma irradiation. pSD-ZIL6, L. lactis displaying ZIL6 affibody; Ctrl: L. lactis control cells containing empty plasmid pNZ8148. An anti-IL6 monoclonal antibody (Ab) was used as a positive control. The data are means ± SD of four biological replicates. The asterisks denote statistically significant differences between treated and non-treated bacteria for each concentration. ***, p < 0.001 (one-way ANOVA with Dunnett multiple comparison test). (B) The effect of lactic acid production by L. lactis on viability of HEKblue-IL6R cells. HEKblue-IL6R cells (100 000 cells/well) were incubated with ZIL6-displaying L. lactis (2 × 10 7 bacteria/well) for 6, 12, and 24 h, and the viability of HEKblue-IL6R cells was determined with trypan blue. The data are means ± SD of three technical replicates of a representative experiment.

Article Snippet: Briefly, serial dilutions of IL6 (0.32, 0.65, 1.31, 2.62, 5.25, 10.5, 21, 42 nM; HZ-1019, Proteintech) were incubated with a fixed number of recombinant bacteria (8 × 10 6 CFU) for 2 h in a final volume of 200 μl.

Techniques: Inhibition, Bacteria, Comparison, Sonication, Irradiation, Control, Plasmid Preparation, Positive Control, Concentration Assay, Incubation

Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: Killing efficiency of different physicochemical treatments on recombinant L. lactis strains displaying different binders of proinflammatory cytokines on their surface. The killing efficiency was determined by culturing and counting colonies on agar plates. Complete killing of bacteria i.e., 0 CFU/mL (no bacterial colonies on agar plates) was considered as 100% efficiency. pSD-ZIL6, L. lactis displaying anti-IL6 affibody; pSD-ZTNF, L. lactis displaying anti-TNF affibody; pSD-Fyn17, L. lactis displaying anti-IL17 fynomer; pSD-EVA, L. lactis displaying anti-IL8 evasin; pNZ8148, L. lactis harboring empty plasmid. The results presented are means ± standard deviation (SD) of three technical replicates from a representative experiment.

Article Snippet: Bacteria (1 × 10 8 CFU/mL) were incubated with recombinant human IL6 (1,000 pg/mL; HZ-1019; Proteintech) for various time intervals (10, 20, 30, 40, 60, 90, 120, 150, 160, 180 min).

Techniques: Recombinant, Bacteria, Plasmid Preparation, Standard Deviation

Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: Cytokine binding ability of recombinant L. lactis bacteria displaying different binders of proinflammatory cytokines on their surface upon exposure to bacteria-killing treatments. ELISA-determined concentrations of recombinant IL6 (A) , TNF (B) , IL17 (C) , and IL8 (D) that remained in the solution following incubation with the corresponding strain of bacteria before and after treatment with heat (70 °C, 40 min or 100 °C, 30 min), sonication, ethanol, UV, and gamma irradiation. pSD-ZIL6, L. lactis displaying ZIL6 affibody; pSD-ZTNF, L. lactis displaying anti-TNF affibody; pSD-Fyn17, L. lactis displaying anti-IL17 fynomer; pSD-EVA, L. lactis displaying anti-IL8 evasin; Ctrl: L. lactis control cells containing empty plasmid pNZ8148. The results are presented as means ± SD of three technical replicates of a representative experiment. ns, p = 0.09; *, p ≤ 0.05; **, p = 0.002; ***, p < 0.001 (unpaired two-tailed t -test).

Article Snippet: Bacteria (1 × 10 8 CFU/mL) were incubated with recombinant human IL6 (1,000 pg/mL; HZ-1019; Proteintech) for various time intervals (10, 20, 30, 40, 60, 90, 120, 150, 160, 180 min).

Techniques: Binding Assay, Recombinant, Bacteria, Enzyme-linked Immunosorbent Assay, Incubation, Sonication, Irradiation, Control, Plasmid Preparation, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: Determination of binding affinity of ZIL6-displaying L. lactis for human IL6. A constant number of 8 × 10 6 CFU bacteria was incubated with increasing concentrations of human IL6 for 2 h. MFI values were measured by flow cytometry and binding curves were fitted to the data using the Hill equation in the GraphPad Prism v.10.3.1. The dissociation constants (Kd) values were calculated from the binding curves for non-treated, live ZIL6-displaying L. lactis bacteria and bacteria treated by heat (70 °C, 40 min or 100 °C, 30 min), sonication, ethanol, and UV irradiation. Data are means ± SD of three technical replicates from a representative experiment.

Article Snippet: Bacteria (1 × 10 8 CFU/mL) were incubated with recombinant human IL6 (1,000 pg/mL; HZ-1019; Proteintech) for various time intervals (10, 20, 30, 40, 60, 90, 120, 150, 160, 180 min).

Techniques: Binding Assay, Bacteria, Incubation, Flow Cytometry, Sonication, Irradiation

Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: Quantification of the maximum binding capacity of ZIL6-displaying L. lactis . Increasing concentrations of recombinant human IL6 (0.45, 4.5, 45 and 450 ng) were incubated with a constant number of bacteria (4.5 × 10 7 CFU equivalent to 0.1 mg dry cell weight) in 450 μl. Residual IL6 in the supernatants was quantified by ELISA for live non-treated bacteria and the cells treated by heat (70 °C, 40 min or 100 °C, 30 min), sonication, ethanol, and UV irradiation. The percentage of bound IL6 was calculated from the difference measured in the presence of ZIL6-displaying L. lactis and control bacteria (carrying empty plasmid pNZ8148). The data are means ± SD of two biological replicates. The asterisks denote statistically significant differences between treated and non-treated bacteria for each concentration. ***, p < 0.001 (one-way ANOVA with Dunnett multiple comparison test).

Article Snippet: Bacteria (1 × 10 8 CFU/mL) were incubated with recombinant human IL6 (1,000 pg/mL; HZ-1019; Proteintech) for various time intervals (10, 20, 30, 40, 60, 90, 120, 150, 160, 180 min).

Techniques: Binding Assay, Recombinant, Incubation, Bacteria, Enzyme-linked Immunosorbent Assay, Sonication, Irradiation, Control, Plasmid Preparation, Concentration Assay, Comparison

Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: (A) Inhibition of STAT3 signaling in HEKblue-IL6R cells by non-viable ZIL6-displaying L. lactis bacteria in comparison to live non-treated strain. ZIL6-displaying L. lactis cells (1 × 10 8 CFU/mL or 1 × 10 9 CFU/mL) were preincubated with IL6 (1 ng/mL) for 2 h. After removal of bacterial cells, the cell-free supernatant containing remainder of IL6 was added to HEKblue-IL6R cells to induce the reporter system. The percentage of STAT3 inhibition was calculated relative to the STAT3 signaling induced by IL6 in the absence of bacteria. The inhibition of STAT3 signaling was determined for live non-treated bacteria and the cells treated by heat (70 °C, 40 min or 100 °C, 30 min), sonication, ethanol, UV, and gamma irradiation. pSD-ZIL6, L. lactis displaying ZIL6 affibody; Ctrl: L. lactis control cells containing empty plasmid pNZ8148. An anti-IL6 monoclonal antibody (Ab) was used as a positive control. The data are means ± SD of four biological replicates. The asterisks denote statistically significant differences between treated and non-treated bacteria for each concentration. ***, p < 0.001 (one-way ANOVA with Dunnett multiple comparison test). (B) The effect of lactic acid production by L. lactis on viability of HEKblue-IL6R cells. HEKblue-IL6R cells (100 000 cells/well) were incubated with ZIL6-displaying L. lactis (2 × 10 7 bacteria/well) for 6, 12, and 24 h, and the viability of HEKblue-IL6R cells was determined with trypan blue. The data are means ± SD of three technical replicates of a representative experiment.

Article Snippet: Bacteria (1 × 10 8 CFU/mL) were incubated with recombinant human IL6 (1,000 pg/mL; HZ-1019; Proteintech) for various time intervals (10, 20, 30, 40, 60, 90, 120, 150, 160, 180 min).

Techniques: Inhibition, Bacteria, Comparison, Sonication, Irradiation, Control, Plasmid Preparation, Positive Control, Concentration Assay, Incubation

Journal: The Journal of Clinical Investigation

Article Title: A20’s linear ubiquitin–binding motif restrains pathogenic activation of Th17 cells and IL-22–driven enteritis

doi: 10.1172/JCI187499

Figure Lengend Snippet: ( A ) Gene ontology enrichment of genes that are significantly upregulated in A20 ZF7 versus WT intestines by bulk RNA-Seq. Red bars highlight categories related to Th17 differentiation. Blue bars highlight categories related to cellular proliferation. ( B ) qPCR analyses of mRNA expression from intact small intestine. ( C ) Volcano plot of all annotated UCSC RefSeq genes from bulk RNA-Seq analyses of A20 ZF7 versus WT small intestines. Horizontal dotted line indicates adjusted P value (FDR) of 0.01. ( D ) Representative immunohistochemical analyses of CD4 expression in WT and A20 ZF7 small intestines. Data are representative of 3 mice from each genotype. Scale bars: 100 μm. ( E ) Flow cytometry of small intestinal lamina propria (SILP) cells from WT and A20 ZF7 mice. SILP yields from the proximal 10 cm of small intestine typically range from 1 million to 2 million cells from WT mice and 3 million to 7 million cells from A20 ZF7 mice. Data are shown as mean ± SEM. Statistics were calculated using unpaired 2-tailed Student’s t test with Welch’s correction. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cells were subsequently differentiated into Th17 cells in plates precoated with 5 μg/mL anti-CD3 (Bio X Cell BE0001-2, clone OKT3) in Th17 medium (Immunocult XF serum-free medium with 50 μM β-mercaptoethanol, 30 ng/mL recombinant human IL-6 [Proteintech HZ-1019], 2.5 ng/mL recombinant human TGF-β1 [PeproTech 100-21], 10 ng/mL recombinant human IL-1β [Proteintech HZ-1164], 10 ng/mL recombinant human IL-23 [Proteintech HZ-1254], 10 μg/mL anti–IL-4 [Bio X Cell BE0240, clone MP4-25D2], and 10 μg/mL anti–IFN-γ [Bio X Cell BE0235, clone B133.5]) at a density of 1 million cells/mL.

Techniques: RNA Sequencing, Expressing, Immunohistochemical staining, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: A20’s linear ubiquitin–binding motif restrains pathogenic activation of Th17 cells and IL-22–driven enteritis

doi: 10.1172/JCI187499

Figure Lengend Snippet: ( A and B ) UMAP clusters of scRNA-Seq analyses of SILP from WT and A20 ZF7 mice. Data represent pooled mixtures of 2 WT and 2 A20 ZF7 mice. ( C ) Relative proportions of lymphoid subsets in WT and A20 ZF7 SILP. Th17 subset includes both proliferative (mustard yellow) and non-proliferative (sky blue) compartments. ( D ) Projection of Il17a and Il22 expression onto UMAP clusters shown in B . ( E ) Violin plots of Il17a and Il22 expression in Th17 cells from indicated genotypes of mice. Statistics were calculated using unpaired 2-tailed Wilcoxon’s rank sum test. ** P < 0.01, *** P < 0.001.

Article Snippet: Cells were subsequently differentiated into Th17 cells in plates precoated with 5 μg/mL anti-CD3 (Bio X Cell BE0001-2, clone OKT3) in Th17 medium (Immunocult XF serum-free medium with 50 μM β-mercaptoethanol, 30 ng/mL recombinant human IL-6 [Proteintech HZ-1019], 2.5 ng/mL recombinant human TGF-β1 [PeproTech 100-21], 10 ng/mL recombinant human IL-1β [Proteintech HZ-1164], 10 ng/mL recombinant human IL-23 [Proteintech HZ-1254], 10 μg/mL anti–IL-4 [Bio X Cell BE0240, clone MP4-25D2], and 10 μg/mL anti–IFN-γ [Bio X Cell BE0235, clone B133.5]) at a density of 1 million cells/mL.

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: A20’s linear ubiquitin–binding motif restrains pathogenic activation of Th17 cells and IL-22–driven enteritis

doi: 10.1172/JCI187499

Figure Lengend Snippet: ( A and B ) Flow nucleometry of RORγt ( A ) or phosphorylated Stat3 (Tyr705) ( B ) expression in nuclei isolated from in vitro–differentiated Th17 cells from WT and A20 ZF7 mice. Histograms are representative of n = 2–3 biologically independent experiments. ( C ) qPCR analyses of Il17a and Il22 expression in cells generated as in A . “Th17” indicates Th17 differentiation conditions. “FICZ” indicates treatment with Ahr agonist FICZ. ( D ) ELISA of IL-22 secretion from cells generated as in A . ( E ) ATAC-seq of genomic loci at or near the Il22 locus in cells generated as in A . ( F ) ChIP of acetylated H3K27 at indicated Il22 loci (loci a–c in E ) in cells generated as in A . ( G ) ChIP of histone H3 at the Il22 promoter and enhancer loci (loci a–c in E ). Cells were generated as in A and treated with the RORγt inhibitor GSK805. H3 ChIP directly assesses nucleosome occupancy and, thus, chromatin accessibility. ( H ) ChIP of acetylated H3K27 at Il22 enhancer loci (loci a–c in E ). Cells were generated as in A and treated with the RORγt inhibitor GSK805. ( I ) Flow cytometric analyses of RORγt expression in CRISPR/Cas9–edited primary human T cells differentiated in vitro using Th17 conditions. ( J ) qPCR analyses of expression of indicated genes in paired isogenic human Th17 cells engineered with CRISPR/Cas9 and either A20 ZF7 -targeted or nontargeting guide RNAs. Three pairs of isogenic samples from 2 healthy donors are shown. Data are shown as mean ± SEM. Statistics were calculated using unpaired 2-tailed Student’s t test with Welch’s correction ( C , D , and F ), 2-way ANOVA with post hoc Tukey’s multiple-comparison correction with simple effects ( G and H ), or paired-ratio t test ( J ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cells were subsequently differentiated into Th17 cells in plates precoated with 5 μg/mL anti-CD3 (Bio X Cell BE0001-2, clone OKT3) in Th17 medium (Immunocult XF serum-free medium with 50 μM β-mercaptoethanol, 30 ng/mL recombinant human IL-6 [Proteintech HZ-1019], 2.5 ng/mL recombinant human TGF-β1 [PeproTech 100-21], 10 ng/mL recombinant human IL-1β [Proteintech HZ-1164], 10 ng/mL recombinant human IL-23 [Proteintech HZ-1254], 10 μg/mL anti–IL-4 [Bio X Cell BE0240, clone MP4-25D2], and 10 μg/mL anti–IFN-γ [Bio X Cell BE0235, clone B133.5]) at a density of 1 million cells/mL.

Techniques: Expressing, Isolation, In Vitro, Generated, Enzyme-linked Immunosorbent Assay, CRISPR, Comparison