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recombinant s100a8  (MedChemExpress)


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    MedChemExpress recombinant s100a8
    ( A ) Fecal microbiota from Clec4n –/– mice were cultured on MRS medium supplemented with neomycin for 3 days. Single colonies were isolated and identified by PCR amplification and sequencing of bacterial 16S rDNA. ( B – F ) L.j . WXY strain was isolated from mouse feces. WT mice were treated with antibiotics (ABX), followed by oral transfer of the indicated bacterial strains twice over 7 days. Whole fecal microbiota from normal WT mice were then transferred back for 3 days, followed by DSS treatment for 7 days and sacrifice on day 10. Body weight loss ( B ), DAI ( C ), colon length measurement ( D ), distal colon histology ( E ), and cLP neutrophil and Treg frequencies ( F ) ( B – D , and F , PBS, n = 7; L . j . WXY, n = 7; Lactobacillus mixture, n = 8; E . coli , n = 7; E , n = 3 /group). ( G ) Correlation between fecal Lactobacillus abundance and colonic <t>S100a8</t> / S100a9 mRNA expression in C57BL/6J mice under steady-state conditions ( n = 18). ( H ) L.j . WXY was cultured anaerobically at 37°C with recombinant S100A8 + S100A9 (1:1 mixture, 5 μg/mL each). Bacterial growth was quantified spectrophotometrically at 6 and 24 hours ( n = 6 technical replicates/group). ( I ) Clec4n –/– mice received intrarectal administration of recombinant S100A8 + S100A9 for 5 hours, followed by quantification of fecal L.j . abundance by qPCR ( n = 10). ( J and K ) WT and Clec4n –/– recipient mice were lethally irradiated and reconstituted with BM cells from WT or Clec4n –/– donors. After 30 days, colonic expression of S100a8 and S100a9 was assessed by qPCR ( J ), and fecal Lactobacillus abundance was measured by qPCR on days 10, 20, and 30 after transfer ( K ) (WT→WT, n = 8; WT→ Clec4n –/– , n = 7; Clec4n –/– →WT, n = 8; Clec4n –/– → Clec4n –/– , n = 8.). ( L – N ) WT and Clec4n –/– mice were treated intraperitoneally with anti-Ly6G neutralizing Ab or isotype control IgG (100 μg/mouse) every other day for 5 doses. Two days after the final injection, colonic tissues and feces were collected. S100A8 and S100A9 protein levels in colon lysates were measured by ELISA ( L ), fecal L.j . abundance was determined by qPCR ( M ), and colonic Il6 and Tnf expression was assessed by qPCR ( N ) (control [con] IgG, n = 3–4; anti-Ly6G, n = 3–4). ( O – S ) WT mice were orally administered culture supernatant (sup.) from L.j . WXY or heat-killed (hk) bacteria daily for 3 days before and throughout 7 days of DSS treatment ( n = 10 total administrations). Body weight loss ( O ), DAI ( P ), colon length measurement at sacrifice on day 9 ( Q ), distal colon histology ( R ), and cLP Treg frequencies ( S ) (PBS n = 8; WXY-sup. n = 8; WXY-hk n = 9). ( T ) CD11b + and CD11c + cells isolated from WT cLP were stimulated in vitro with L.j . WXY, Lactobacillus mixture, or E . coli . After 12 hours, Il10 and Tgfb1 mRNA expression was quantified by qPCR ( n = 4 technical replicates/group). ( U ) CD62L + naive CD4 + T cells isolated from WT spleen and lymph nodes were polarized toward Treg differentiation in the presence of L.j . WXY culture supernatant. After 6 days, Il10 and Tgfb1 expression was measured by qPCR ( n = 9 technical replicates from 3 biological replicates/group). ( V and W ) ABX-treated WT mice received oral L.j . WXY or PBS, followed by fecal microbiota transplantation from normal WT mice and subsequent DSS treatment. Cecal contents were collected on day 10 for untargeted metabolomic analysis by liquid chromatography. Histogram ( V ) shows the top 20 enriched metabolites, and volcano plot ( W ) highlights significantly altered metabolites in WXY-treated mice. ( X ) Targeted metabolomic analysis of l -glutamic acid levels in cecal contents from 8-week-old WT and Clec4n –/– mice under physiological conditions ( n = 3/group). ( Y ) l -Glutamic acid concentrations in culture supernatants of L.j . WXY, Lactobacillus mixture, or E . coli measured by targeted metabolomics ( n = 3 replicates/group). Data in B – F , H – K , and O – S are pooled from 2 independent experiments. Data in B – F , J , L – U , X , and Y are presented as mean ± SD. Statistical analysis: 1-way ANOVA with Bonferroni’s multiple-comparison test ( B , C , K , O , and P ); 1-way ANOVA with Tukey’s multiple-comparison test ( D – F , J , L – N , Q – T , and Y ); Spearman’s correlation test ( G ); 2-way ANOVA test with repeated measures ( H ); paired Student’s t test ( I ); or 2-tailed unpaired Student’s t test ( U and X ).
    Recombinant S100a8, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/recombinant+s100a8/pmc13078882-243-12-21?v=MedChemExpress
    Average 94 stars, based on 3 article reviews
    recombinant s100a8 - by Bioz Stars, 2026-08
    94/100 stars

    Images

    1) Product Images from "Colonic Engyodontium fungus triggers neutrophil antimicrobial activity to suppress Lactobacillus johnsonii –derived glutamic acid–maintained Tregs"

    Article Title: Colonic Engyodontium fungus triggers neutrophil antimicrobial activity to suppress Lactobacillus johnsonii –derived glutamic acid–maintained Tregs

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI196788

    ( A ) Fecal microbiota from Clec4n –/– mice were cultured on MRS medium supplemented with neomycin for 3 days. Single colonies were isolated and identified by PCR amplification and sequencing of bacterial 16S rDNA. ( B – F ) L.j . WXY strain was isolated from mouse feces. WT mice were treated with antibiotics (ABX), followed by oral transfer of the indicated bacterial strains twice over 7 days. Whole fecal microbiota from normal WT mice were then transferred back for 3 days, followed by DSS treatment for 7 days and sacrifice on day 10. Body weight loss ( B ), DAI ( C ), colon length measurement ( D ), distal colon histology ( E ), and cLP neutrophil and Treg frequencies ( F ) ( B – D , and F , PBS, n = 7; L . j . WXY, n = 7; Lactobacillus mixture, n = 8; E . coli , n = 7; E , n = 3 /group). ( G ) Correlation between fecal Lactobacillus abundance and colonic S100a8 / S100a9 mRNA expression in C57BL/6J mice under steady-state conditions ( n = 18). ( H ) L.j . WXY was cultured anaerobically at 37°C with recombinant S100A8 + S100A9 (1:1 mixture, 5 μg/mL each). Bacterial growth was quantified spectrophotometrically at 6 and 24 hours ( n = 6 technical replicates/group). ( I ) Clec4n –/– mice received intrarectal administration of recombinant S100A8 + S100A9 for 5 hours, followed by quantification of fecal L.j . abundance by qPCR ( n = 10). ( J and K ) WT and Clec4n –/– recipient mice were lethally irradiated and reconstituted with BM cells from WT or Clec4n –/– donors. After 30 days, colonic expression of S100a8 and S100a9 was assessed by qPCR ( J ), and fecal Lactobacillus abundance was measured by qPCR on days 10, 20, and 30 after transfer ( K ) (WT→WT, n = 8; WT→ Clec4n –/– , n = 7; Clec4n –/– →WT, n = 8; Clec4n –/– → Clec4n –/– , n = 8.). ( L – N ) WT and Clec4n –/– mice were treated intraperitoneally with anti-Ly6G neutralizing Ab or isotype control IgG (100 μg/mouse) every other day for 5 doses. Two days after the final injection, colonic tissues and feces were collected. S100A8 and S100A9 protein levels in colon lysates were measured by ELISA ( L ), fecal L.j . abundance was determined by qPCR ( M ), and colonic Il6 and Tnf expression was assessed by qPCR ( N ) (control [con] IgG, n = 3–4; anti-Ly6G, n = 3–4). ( O – S ) WT mice were orally administered culture supernatant (sup.) from L.j . WXY or heat-killed (hk) bacteria daily for 3 days before and throughout 7 days of DSS treatment ( n = 10 total administrations). Body weight loss ( O ), DAI ( P ), colon length measurement at sacrifice on day 9 ( Q ), distal colon histology ( R ), and cLP Treg frequencies ( S ) (PBS n = 8; WXY-sup. n = 8; WXY-hk n = 9). ( T ) CD11b + and CD11c + cells isolated from WT cLP were stimulated in vitro with L.j . WXY, Lactobacillus mixture, or E . coli . After 12 hours, Il10 and Tgfb1 mRNA expression was quantified by qPCR ( n = 4 technical replicates/group). ( U ) CD62L + naive CD4 + T cells isolated from WT spleen and lymph nodes were polarized toward Treg differentiation in the presence of L.j . WXY culture supernatant. After 6 days, Il10 and Tgfb1 expression was measured by qPCR ( n = 9 technical replicates from 3 biological replicates/group). ( V and W ) ABX-treated WT mice received oral L.j . WXY or PBS, followed by fecal microbiota transplantation from normal WT mice and subsequent DSS treatment. Cecal contents were collected on day 10 for untargeted metabolomic analysis by liquid chromatography. Histogram ( V ) shows the top 20 enriched metabolites, and volcano plot ( W ) highlights significantly altered metabolites in WXY-treated mice. ( X ) Targeted metabolomic analysis of l -glutamic acid levels in cecal contents from 8-week-old WT and Clec4n –/– mice under physiological conditions ( n = 3/group). ( Y ) l -Glutamic acid concentrations in culture supernatants of L.j . WXY, Lactobacillus mixture, or E . coli measured by targeted metabolomics ( n = 3 replicates/group). Data in B – F , H – K , and O – S are pooled from 2 independent experiments. Data in B – F , J , L – U , X , and Y are presented as mean ± SD. Statistical analysis: 1-way ANOVA with Bonferroni’s multiple-comparison test ( B , C , K , O , and P ); 1-way ANOVA with Tukey’s multiple-comparison test ( D – F , J , L – N , Q – T , and Y ); Spearman’s correlation test ( G ); 2-way ANOVA test with repeated measures ( H ); paired Student’s t test ( I ); or 2-tailed unpaired Student’s t test ( U and X ).
    Figure Legend Snippet: ( A ) Fecal microbiota from Clec4n –/– mice were cultured on MRS medium supplemented with neomycin for 3 days. Single colonies were isolated and identified by PCR amplification and sequencing of bacterial 16S rDNA. ( B – F ) L.j . WXY strain was isolated from mouse feces. WT mice were treated with antibiotics (ABX), followed by oral transfer of the indicated bacterial strains twice over 7 days. Whole fecal microbiota from normal WT mice were then transferred back for 3 days, followed by DSS treatment for 7 days and sacrifice on day 10. Body weight loss ( B ), DAI ( C ), colon length measurement ( D ), distal colon histology ( E ), and cLP neutrophil and Treg frequencies ( F ) ( B – D , and F , PBS, n = 7; L . j . WXY, n = 7; Lactobacillus mixture, n = 8; E . coli , n = 7; E , n = 3 /group). ( G ) Correlation between fecal Lactobacillus abundance and colonic S100a8 / S100a9 mRNA expression in C57BL/6J mice under steady-state conditions ( n = 18). ( H ) L.j . WXY was cultured anaerobically at 37°C with recombinant S100A8 + S100A9 (1:1 mixture, 5 μg/mL each). Bacterial growth was quantified spectrophotometrically at 6 and 24 hours ( n = 6 technical replicates/group). ( I ) Clec4n –/– mice received intrarectal administration of recombinant S100A8 + S100A9 for 5 hours, followed by quantification of fecal L.j . abundance by qPCR ( n = 10). ( J and K ) WT and Clec4n –/– recipient mice were lethally irradiated and reconstituted with BM cells from WT or Clec4n –/– donors. After 30 days, colonic expression of S100a8 and S100a9 was assessed by qPCR ( J ), and fecal Lactobacillus abundance was measured by qPCR on days 10, 20, and 30 after transfer ( K ) (WT→WT, n = 8; WT→ Clec4n –/– , n = 7; Clec4n –/– →WT, n = 8; Clec4n –/– → Clec4n –/– , n = 8.). ( L – N ) WT and Clec4n –/– mice were treated intraperitoneally with anti-Ly6G neutralizing Ab or isotype control IgG (100 μg/mouse) every other day for 5 doses. Two days after the final injection, colonic tissues and feces were collected. S100A8 and S100A9 protein levels in colon lysates were measured by ELISA ( L ), fecal L.j . abundance was determined by qPCR ( M ), and colonic Il6 and Tnf expression was assessed by qPCR ( N ) (control [con] IgG, n = 3–4; anti-Ly6G, n = 3–4). ( O – S ) WT mice were orally administered culture supernatant (sup.) from L.j . WXY or heat-killed (hk) bacteria daily for 3 days before and throughout 7 days of DSS treatment ( n = 10 total administrations). Body weight loss ( O ), DAI ( P ), colon length measurement at sacrifice on day 9 ( Q ), distal colon histology ( R ), and cLP Treg frequencies ( S ) (PBS n = 8; WXY-sup. n = 8; WXY-hk n = 9). ( T ) CD11b + and CD11c + cells isolated from WT cLP were stimulated in vitro with L.j . WXY, Lactobacillus mixture, or E . coli . After 12 hours, Il10 and Tgfb1 mRNA expression was quantified by qPCR ( n = 4 technical replicates/group). ( U ) CD62L + naive CD4 + T cells isolated from WT spleen and lymph nodes were polarized toward Treg differentiation in the presence of L.j . WXY culture supernatant. After 6 days, Il10 and Tgfb1 expression was measured by qPCR ( n = 9 technical replicates from 3 biological replicates/group). ( V and W ) ABX-treated WT mice received oral L.j . WXY or PBS, followed by fecal microbiota transplantation from normal WT mice and subsequent DSS treatment. Cecal contents were collected on day 10 for untargeted metabolomic analysis by liquid chromatography. Histogram ( V ) shows the top 20 enriched metabolites, and volcano plot ( W ) highlights significantly altered metabolites in WXY-treated mice. ( X ) Targeted metabolomic analysis of l -glutamic acid levels in cecal contents from 8-week-old WT and Clec4n –/– mice under physiological conditions ( n = 3/group). ( Y ) l -Glutamic acid concentrations in culture supernatants of L.j . WXY, Lactobacillus mixture, or E . coli measured by targeted metabolomics ( n = 3 replicates/group). Data in B – F , H – K , and O – S are pooled from 2 independent experiments. Data in B – F , J , L – U , X , and Y are presented as mean ± SD. Statistical analysis: 1-way ANOVA with Bonferroni’s multiple-comparison test ( B , C , K , O , and P ); 1-way ANOVA with Tukey’s multiple-comparison test ( D – F , J , L – N , Q – T , and Y ); Spearman’s correlation test ( G ); 2-way ANOVA test with repeated measures ( H ); paired Student’s t test ( I ); or 2-tailed unpaired Student’s t test ( U and X ).

    Techniques Used: Cell Culture, Isolation, Amplification, Sequencing, Expressing, Recombinant, Irradiation, Control, Injection, Enzyme-linked Immunosorbent Assay, Bacteria, In Vitro, Transplantation Assay, Metabolomic, Liquid Chromatography, Comparison

    ( A ) Phylum- and genus-level composition of human fecal commensal fungi isolated using Dectin-2–Fc and identified by qPCR with species-specific primers. ( B ) Relative contents of Engyodontium sp. in fecal fungi and L.j . in fecal bacteria from patients with CD and healthy individuals (HC), determined by qPCR ( Engyodontium : HC, n = 59; CD, n = 62. Lacutobacillus : HC, n = 62; CD, n = 63). ( C ) Relative contents of Engyodontium sp. in fecal fungi and L.j . in fecal bacteria from patients with UC and healthy individuals, determined by qPCR (HC, n = 36; UC, n = 25). ( D ) Correlation between Ct value of L.j . and Engyodontium sp. from human individuals as described in B ( n = 97 [including HC, n = 48 and CD, n = 49]). ( E ) Correlation between Ct value of L.j . and Engyodontium sp. from human individuals as described in C (n=61 [including HC, n=36 and UC, n=25]). ( F ) C57BL/6J SPF mice were orally colonized with Engyodontium sp. ( Engyod. ) every other day for 3 administrations. Thirteen days later, fecal samples were collected, and the relative abundance of fecal L.j . was quantified by qPCR ( n = 10 /group). ( G ) Relative expression of CLEC6A , S100A8 , and S100A9 in colon tissues from patients with CD and healthy control (HC) individuals, determined by bulk RNA-Seq analysis (HC, n = 47; CD, n = 57). ( H ) Correlation between CLEC6A and S100A8 or S100A9 expression based on bulk RNA-Seq data described in G . ( I ) Correlation between CLEC6A and S100A8 or S100A9 relative expression in colon tissues of patients with UC, determined by qPCR ( n = 25). ( J ) IHC staining for DECTIN-2 and S100A8/S100A9 proteins in serial inflammatory colon sections obtained from a patient with CD after surgical resection. ( K ) IHC staining for human DECTIN-2 in paired normal and inflamed colon regions from the patient with CD shown in J . ( L ) Transcriptional levels of CLEC6A in colon tissues from non-IBD control individuals and patients with colonic CD (cCD), ileal CD (iCD), or UC, analyzed by reprocessing a public bulk RNA-Seq dataset ( GSE117993 ) (not IBD, n = 55; cCD, n = 31; iCD, n = 60; UC, n = 43). ( M ) Colon tissues from 3 patients with IBD were obtained after resection. cLP CD11b + cells were isolated and stimulated with α-mannan in vitro for 6 hours. S100A8 and S100A9 relative expression was quantified by qPCR ( n = 7). ( N ) Neutrophils isolated from peripheral blood of 2 patients with IBD were stimulated with α-mannan in vitro for 6 hours, and S100A8 and S100A9 relative expression was measured by qPCR ( n = 7 technical replicates from 2 biological replicates/group). ( O ) Correlation between fecal Engyodontium sp. abundance and colonic expression of CLEC6A , S100A8 , or S100A9 in patients with UC ( n = 25). ( P ) Correlation between fecal L.j . abundance and colonic expression of CLEC6A , S100A8 , or S100A9 in patients with UC ( n = 25). ( Q ) CD11b + cells isolated from cLP of resected colon tissues from 2 patients with IBD were stimulated with Engyodontium sp. for 24 hours in vitro, followed by qPCR analysis of S100A8 and S100A9 expression ( n = 6 technical replicates from 2 biological replicates/group). non-stimu., nonstimulated. ( R ) Engyodontium sp. was cultured at room temperature with recombinant S100A8 + S100A9 (1:1 mixture, 5 μg/mL each) for 12, 24, and 48 hours. Fungus growth was quantified by CFU enumeration on PDA plates ( n = 3 replicates/group). ( S ) Relative amount of glutamic acid in fecal samples from patients with CD and healthy control individuals, assessed by liquid chromatography for untargeted metabolomics (HC, n = 80; CD, n = 65). ( T ) Correlation between 1/Ct value of L.j . and fecal glutamic acid levels in patients with CD ( n = 8). ( U ) Correlation between 1/Ct value of L.j . and glutamic acid concentration in colon tissues from patients with UC ( n = 25). ( V ) Peripheral blood leukocytes from 2 healthy donors were isolated and induced toward Treg differentiation in the presence of l -glutamic acid. After 5 days, the proportion of Foxp3 + CD4 + among CD45 + leukocytes was analyzed by flow cytometry ( n = 6 technical replicates from 2 biological replicates/group). Data in F , M , N , Q , and V are pooled from 2 independent experiments. Data in B , C , G , and L are presented as mean ± SEM, and in F , N , Q , R , S , and V as mean ± SD. Statistical analysis: 2-tailed Mann-Whitney test ( B , C , and G ), 2-tailed unpaired Student’s t test ( F , N , Q – S , and V ), 1-way ANOVA with Kruskal-Wallis and Dunn’s test ( L ), Spearman’s correlation test ( D , E , H , I , O , P , T , and U ), or paired Student’s t test ( M ).
    Figure Legend Snippet: ( A ) Phylum- and genus-level composition of human fecal commensal fungi isolated using Dectin-2–Fc and identified by qPCR with species-specific primers. ( B ) Relative contents of Engyodontium sp. in fecal fungi and L.j . in fecal bacteria from patients with CD and healthy individuals (HC), determined by qPCR ( Engyodontium : HC, n = 59; CD, n = 62. Lacutobacillus : HC, n = 62; CD, n = 63). ( C ) Relative contents of Engyodontium sp. in fecal fungi and L.j . in fecal bacteria from patients with UC and healthy individuals, determined by qPCR (HC, n = 36; UC, n = 25). ( D ) Correlation between Ct value of L.j . and Engyodontium sp. from human individuals as described in B ( n = 97 [including HC, n = 48 and CD, n = 49]). ( E ) Correlation between Ct value of L.j . and Engyodontium sp. from human individuals as described in C (n=61 [including HC, n=36 and UC, n=25]). ( F ) C57BL/6J SPF mice were orally colonized with Engyodontium sp. ( Engyod. ) every other day for 3 administrations. Thirteen days later, fecal samples were collected, and the relative abundance of fecal L.j . was quantified by qPCR ( n = 10 /group). ( G ) Relative expression of CLEC6A , S100A8 , and S100A9 in colon tissues from patients with CD and healthy control (HC) individuals, determined by bulk RNA-Seq analysis (HC, n = 47; CD, n = 57). ( H ) Correlation between CLEC6A and S100A8 or S100A9 expression based on bulk RNA-Seq data described in G . ( I ) Correlation between CLEC6A and S100A8 or S100A9 relative expression in colon tissues of patients with UC, determined by qPCR ( n = 25). ( J ) IHC staining for DECTIN-2 and S100A8/S100A9 proteins in serial inflammatory colon sections obtained from a patient with CD after surgical resection. ( K ) IHC staining for human DECTIN-2 in paired normal and inflamed colon regions from the patient with CD shown in J . ( L ) Transcriptional levels of CLEC6A in colon tissues from non-IBD control individuals and patients with colonic CD (cCD), ileal CD (iCD), or UC, analyzed by reprocessing a public bulk RNA-Seq dataset ( GSE117993 ) (not IBD, n = 55; cCD, n = 31; iCD, n = 60; UC, n = 43). ( M ) Colon tissues from 3 patients with IBD were obtained after resection. cLP CD11b + cells were isolated and stimulated with α-mannan in vitro for 6 hours. S100A8 and S100A9 relative expression was quantified by qPCR ( n = 7). ( N ) Neutrophils isolated from peripheral blood of 2 patients with IBD were stimulated with α-mannan in vitro for 6 hours, and S100A8 and S100A9 relative expression was measured by qPCR ( n = 7 technical replicates from 2 biological replicates/group). ( O ) Correlation between fecal Engyodontium sp. abundance and colonic expression of CLEC6A , S100A8 , or S100A9 in patients with UC ( n = 25). ( P ) Correlation between fecal L.j . abundance and colonic expression of CLEC6A , S100A8 , or S100A9 in patients with UC ( n = 25). ( Q ) CD11b + cells isolated from cLP of resected colon tissues from 2 patients with IBD were stimulated with Engyodontium sp. for 24 hours in vitro, followed by qPCR analysis of S100A8 and S100A9 expression ( n = 6 technical replicates from 2 biological replicates/group). non-stimu., nonstimulated. ( R ) Engyodontium sp. was cultured at room temperature with recombinant S100A8 + S100A9 (1:1 mixture, 5 μg/mL each) for 12, 24, and 48 hours. Fungus growth was quantified by CFU enumeration on PDA plates ( n = 3 replicates/group). ( S ) Relative amount of glutamic acid in fecal samples from patients with CD and healthy control individuals, assessed by liquid chromatography for untargeted metabolomics (HC, n = 80; CD, n = 65). ( T ) Correlation between 1/Ct value of L.j . and fecal glutamic acid levels in patients with CD ( n = 8). ( U ) Correlation between 1/Ct value of L.j . and glutamic acid concentration in colon tissues from patients with UC ( n = 25). ( V ) Peripheral blood leukocytes from 2 healthy donors were isolated and induced toward Treg differentiation in the presence of l -glutamic acid. After 5 days, the proportion of Foxp3 + CD4 + among CD45 + leukocytes was analyzed by flow cytometry ( n = 6 technical replicates from 2 biological replicates/group). Data in F , M , N , Q , and V are pooled from 2 independent experiments. Data in B , C , G , and L are presented as mean ± SEM, and in F , N , Q , R , S , and V as mean ± SD. Statistical analysis: 2-tailed Mann-Whitney test ( B , C , and G ), 2-tailed unpaired Student’s t test ( F , N , Q – S , and V ), 1-way ANOVA with Kruskal-Wallis and Dunn’s test ( L ), Spearman’s correlation test ( D , E , H , I , O , P , T , and U ), or paired Student’s t test ( M ).

    Techniques Used: Isolation, Bacteria, Expressing, Control, RNA Sequencing, Immunohistochemistry, In Vitro, Cell Culture, Recombinant, Liquid Chromatography, Concentration Assay, Flow Cytometry, MANN-WHITNEY



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    ( A ) Fecal microbiota from Clec4n –/– mice were cultured on MRS medium supplemented with neomycin for 3 days. Single colonies were isolated and identified by PCR amplification and sequencing of bacterial 16S rDNA. ( B – F ) L.j . WXY strain was isolated from mouse feces. WT mice were treated with antibiotics (ABX), followed by oral transfer of the indicated bacterial strains twice over 7 days. Whole fecal microbiota from normal WT mice were then transferred back for 3 days, followed by DSS treatment for 7 days and sacrifice on day 10. Body weight loss ( B ), DAI ( C ), colon length measurement ( D ), distal colon histology ( E ), and cLP neutrophil and Treg frequencies ( F ) ( B – D , and F , PBS, n = 7; L . j . WXY, n = 7; Lactobacillus mixture, n = 8; E . coli , n = 7; E , n = 3 /group). ( G ) Correlation between fecal Lactobacillus abundance and colonic <t>S100a8</t> / S100a9 mRNA expression in C57BL/6J mice under steady-state conditions ( n = 18). ( H ) L.j . WXY was cultured anaerobically at 37°C with recombinant S100A8 + S100A9 (1:1 mixture, 5 μg/mL each). Bacterial growth was quantified spectrophotometrically at 6 and 24 hours ( n = 6 technical replicates/group). ( I ) Clec4n –/– mice received intrarectal administration of recombinant S100A8 + S100A9 for 5 hours, followed by quantification of fecal L.j . abundance by qPCR ( n = 10). ( J and K ) WT and Clec4n –/– recipient mice were lethally irradiated and reconstituted with BM cells from WT or Clec4n –/– donors. After 30 days, colonic expression of S100a8 and S100a9 was assessed by qPCR ( J ), and fecal Lactobacillus abundance was measured by qPCR on days 10, 20, and 30 after transfer ( K ) (WT→WT, n = 8; WT→ Clec4n –/– , n = 7; Clec4n –/– →WT, n = 8; Clec4n –/– → Clec4n –/– , n = 8.). ( L – N ) WT and Clec4n –/– mice were treated intraperitoneally with anti-Ly6G neutralizing Ab or isotype control IgG (100 μg/mouse) every other day for 5 doses. Two days after the final injection, colonic tissues and feces were collected. S100A8 and S100A9 protein levels in colon lysates were measured by ELISA ( L ), fecal L.j . abundance was determined by qPCR ( M ), and colonic Il6 and Tnf expression was assessed by qPCR ( N ) (control [con] IgG, n = 3–4; anti-Ly6G, n = 3–4). ( O – S ) WT mice were orally administered culture supernatant (sup.) from L.j . WXY or heat-killed (hk) bacteria daily for 3 days before and throughout 7 days of DSS treatment ( n = 10 total administrations). Body weight loss ( O ), DAI ( P ), colon length measurement at sacrifice on day 9 ( Q ), distal colon histology ( R ), and cLP Treg frequencies ( S ) (PBS n = 8; WXY-sup. n = 8; WXY-hk n = 9). ( T ) CD11b + and CD11c + cells isolated from WT cLP were stimulated in vitro with L.j . WXY, Lactobacillus mixture, or E . coli . After 12 hours, Il10 and Tgfb1 mRNA expression was quantified by qPCR ( n = 4 technical replicates/group). ( U ) CD62L + naive CD4 + T cells isolated from WT spleen and lymph nodes were polarized toward Treg differentiation in the presence of L.j . WXY culture supernatant. After 6 days, Il10 and Tgfb1 expression was measured by qPCR ( n = 9 technical replicates from 3 biological replicates/group). ( V and W ) ABX-treated WT mice received oral L.j . WXY or PBS, followed by fecal microbiota transplantation from normal WT mice and subsequent DSS treatment. Cecal contents were collected on day 10 for untargeted metabolomic analysis by liquid chromatography. Histogram ( V ) shows the top 20 enriched metabolites, and volcano plot ( W ) highlights significantly altered metabolites in WXY-treated mice. ( X ) Targeted metabolomic analysis of l -glutamic acid levels in cecal contents from 8-week-old WT and Clec4n –/– mice under physiological conditions ( n = 3/group). ( Y ) l -Glutamic acid concentrations in culture supernatants of L.j . WXY, Lactobacillus mixture, or E . coli measured by targeted metabolomics ( n = 3 replicates/group). Data in B – F , H – K , and O – S are pooled from 2 independent experiments. Data in B – F , J , L – U , X , and Y are presented as mean ± SD. Statistical analysis: 1-way ANOVA with Bonferroni’s multiple-comparison test ( B , C , K , O , and P ); 1-way ANOVA with Tukey’s multiple-comparison test ( D – F , J , L – N , Q – T , and Y ); Spearman’s correlation test ( G ); 2-way ANOVA test with repeated measures ( H ); paired Student’s t test ( I ); or 2-tailed unpaired Student’s t test ( U and X ).
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    Sino Biological recombinant s100a8 a9 heterodimer
    ( A ) Fecal microbiota from Clec4n –/– mice were cultured on MRS medium supplemented with neomycin for 3 days. Single colonies were isolated and identified by PCR amplification and sequencing of bacterial 16S rDNA. ( B – F ) L.j . WXY strain was isolated from mouse feces. WT mice were treated with antibiotics (ABX), followed by oral transfer of the indicated bacterial strains twice over 7 days. Whole fecal microbiota from normal WT mice were then transferred back for 3 days, followed by DSS treatment for 7 days and sacrifice on day 10. Body weight loss ( B ), DAI ( C ), colon length measurement ( D ), distal colon histology ( E ), and cLP neutrophil and Treg frequencies ( F ) ( B – D , and F , PBS, n = 7; L . j . WXY, n = 7; Lactobacillus mixture, n = 8; E . coli , n = 7; E , n = 3 /group). ( G ) Correlation between fecal Lactobacillus abundance and colonic <t>S100a8</t> / S100a9 mRNA expression in C57BL/6J mice under steady-state conditions ( n = 18). ( H ) L.j . WXY was cultured anaerobically at 37°C with recombinant S100A8 + S100A9 (1:1 mixture, 5 μg/mL each). Bacterial growth was quantified spectrophotometrically at 6 and 24 hours ( n = 6 technical replicates/group). ( I ) Clec4n –/– mice received intrarectal administration of recombinant S100A8 + S100A9 for 5 hours, followed by quantification of fecal L.j . abundance by qPCR ( n = 10). ( J and K ) WT and Clec4n –/– recipient mice were lethally irradiated and reconstituted with BM cells from WT or Clec4n –/– donors. After 30 days, colonic expression of S100a8 and S100a9 was assessed by qPCR ( J ), and fecal Lactobacillus abundance was measured by qPCR on days 10, 20, and 30 after transfer ( K ) (WT→WT, n = 8; WT→ Clec4n –/– , n = 7; Clec4n –/– →WT, n = 8; Clec4n –/– → Clec4n –/– , n = 8.). ( L – N ) WT and Clec4n –/– mice were treated intraperitoneally with anti-Ly6G neutralizing Ab or isotype control IgG (100 μg/mouse) every other day for 5 doses. Two days after the final injection, colonic tissues and feces were collected. S100A8 and S100A9 protein levels in colon lysates were measured by ELISA ( L ), fecal L.j . abundance was determined by qPCR ( M ), and colonic Il6 and Tnf expression was assessed by qPCR ( N ) (control [con] IgG, n = 3–4; anti-Ly6G, n = 3–4). ( O – S ) WT mice were orally administered culture supernatant (sup.) from L.j . WXY or heat-killed (hk) bacteria daily for 3 days before and throughout 7 days of DSS treatment ( n = 10 total administrations). Body weight loss ( O ), DAI ( P ), colon length measurement at sacrifice on day 9 ( Q ), distal colon histology ( R ), and cLP Treg frequencies ( S ) (PBS n = 8; WXY-sup. n = 8; WXY-hk n = 9). ( T ) CD11b + and CD11c + cells isolated from WT cLP were stimulated in vitro with L.j . WXY, Lactobacillus mixture, or E . coli . After 12 hours, Il10 and Tgfb1 mRNA expression was quantified by qPCR ( n = 4 technical replicates/group). ( U ) CD62L + naive CD4 + T cells isolated from WT spleen and lymph nodes were polarized toward Treg differentiation in the presence of L.j . WXY culture supernatant. After 6 days, Il10 and Tgfb1 expression was measured by qPCR ( n = 9 technical replicates from 3 biological replicates/group). ( V and W ) ABX-treated WT mice received oral L.j . WXY or PBS, followed by fecal microbiota transplantation from normal WT mice and subsequent DSS treatment. Cecal contents were collected on day 10 for untargeted metabolomic analysis by liquid chromatography. Histogram ( V ) shows the top 20 enriched metabolites, and volcano plot ( W ) highlights significantly altered metabolites in WXY-treated mice. ( X ) Targeted metabolomic analysis of l -glutamic acid levels in cecal contents from 8-week-old WT and Clec4n –/– mice under physiological conditions ( n = 3/group). ( Y ) l -Glutamic acid concentrations in culture supernatants of L.j . WXY, Lactobacillus mixture, or E . coli measured by targeted metabolomics ( n = 3 replicates/group). Data in B – F , H – K , and O – S are pooled from 2 independent experiments. Data in B – F , J , L – U , X , and Y are presented as mean ± SD. Statistical analysis: 1-way ANOVA with Bonferroni’s multiple-comparison test ( B , C , K , O , and P ); 1-way ANOVA with Tukey’s multiple-comparison test ( D – F , J , L – N , Q – T , and Y ); Spearman’s correlation test ( G ); 2-way ANOVA test with repeated measures ( H ); paired Student’s t test ( I ); or 2-tailed unpaired Student’s t test ( U and X ).
    Recombinant S100a8 A9 Heterodimer, supplied by Sino Biological, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    s100a8 9  (R&D Systems)
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    <t>Increased</t> <t>S100A8/9</t> expression in the skin and serum of patients with BP and AD. ( a ) Immunohistochemical analysis with anti-S100A8/9 antibody showing the expression of S100A8/9 in keratinocytes and infiltrating inflammatory cells of lesional skin of patients with BP and AD. S100A8/9 expression was not observed in the healthy skin. Scale bar: 100 μm. (b) Quantification of the expression level of S100A8/9. (control, n = 2; BP, n = 5; AD n = 2). Inter-patient heterogeneity of the expression level of S100A8/9 was observed in the BP skin samples, and representative images of both low and high S100A8/9 expression were shown in A. (c) ELISA analysis showing increased S100A8/9 concentrations in the serum of patients with BP. (control, n = 8; BP, n = 16; P = .0087, Mann-Whitney test). Data are reported as mean ± SD. AD, atopic dermatitis; BP, bullous pemphigoid.
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    recombinant human s100a8 a9 protein  (R&D Systems)
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    Klebsiella pneumoniae infection directly <t>induces</t> <t>S100A8/A9</t> expression and secretion in HBE cells. Primary HBE cells were infected with WT K. pneumoniae or an isogenic acapsular mutant (Δ cps ). (A,B) Expression of S100A8 and S100A9 was determined by qRT-PCR at 8 h post-infection with various MOIs of K. pneumoniae , or 100 MOI K. pneumoniae for 4–12 h (C,D) . Data are normalized to the housekeeping gene GAPDH and expressed as fold change relative to uninfected control cells. (E) Secretion of S100A8/A9 heterodimer induced by WT K. pneumoniae and the isogenic Δ cps mutant at indicated MOIs after 24 h. All data are presented as mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01 vs. uninfected cells (A–D) . Statistical differences between the WT and Δ cps groups were determined by Two-way ANOVA followed by Tukey’s post-hoc test. * p < 0.05 compared between strains at the same MOI (E) .
    Recombinant Human S100a8 A9 Protein, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    s100a8 a9 recombinant protein  (R&D Systems)
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    Klebsiella pneumoniae infection directly <t>induces</t> <t>S100A8/A9</t> expression and secretion in HBE cells. Primary HBE cells were infected with WT K. pneumoniae or an isogenic acapsular mutant (Δ cps ). (A,B) Expression of S100A8 and S100A9 was determined by qRT-PCR at 8 h post-infection with various MOIs of K. pneumoniae , or 100 MOI K. pneumoniae for 4–12 h (C,D) . Data are normalized to the housekeeping gene GAPDH and expressed as fold change relative to uninfected control cells. (E) Secretion of S100A8/A9 heterodimer induced by WT K. pneumoniae and the isogenic Δ cps mutant at indicated MOIs after 24 h. All data are presented as mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01 vs. uninfected cells (A–D) . Statistical differences between the WT and Δ cps groups were determined by Two-way ANOVA followed by Tukey’s post-hoc test. * p < 0.05 compared between strains at the same MOI (E) .
    S100a8 A9 Recombinant Protein, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    recombinant mouse anti s100a8 monoclonal antibody  (Santa Cruz Biotechnology)
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    Klebsiella pneumoniae infection directly <t>induces</t> <t>S100A8/A9</t> expression and secretion in HBE cells. Primary HBE cells were infected with WT K. pneumoniae or an isogenic acapsular mutant (Δ cps ). (A,B) Expression of S100A8 and S100A9 was determined by qRT-PCR at 8 h post-infection with various MOIs of K. pneumoniae , or 100 MOI K. pneumoniae for 4–12 h (C,D) . Data are normalized to the housekeeping gene GAPDH and expressed as fold change relative to uninfected control cells. (E) Secretion of S100A8/A9 heterodimer induced by WT K. pneumoniae and the isogenic Δ cps mutant at indicated MOIs after 24 h. All data are presented as mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01 vs. uninfected cells (A–D) . Statistical differences between the WT and Δ cps groups were determined by Two-way ANOVA followed by Tukey’s post-hoc test. * p < 0.05 compared between strains at the same MOI (E) .
    Recombinant Mouse Anti S100a8 Monoclonal Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ( A ) Fecal microbiota from Clec4n –/– mice were cultured on MRS medium supplemented with neomycin for 3 days. Single colonies were isolated and identified by PCR amplification and sequencing of bacterial 16S rDNA. ( B – F ) L.j . WXY strain was isolated from mouse feces. WT mice were treated with antibiotics (ABX), followed by oral transfer of the indicated bacterial strains twice over 7 days. Whole fecal microbiota from normal WT mice were then transferred back for 3 days, followed by DSS treatment for 7 days and sacrifice on day 10. Body weight loss ( B ), DAI ( C ), colon length measurement ( D ), distal colon histology ( E ), and cLP neutrophil and Treg frequencies ( F ) ( B – D , and F , PBS, n = 7; L . j . WXY, n = 7; Lactobacillus mixture, n = 8; E . coli , n = 7; E , n = 3 /group). ( G ) Correlation between fecal Lactobacillus abundance and colonic S100a8 / S100a9 mRNA expression in C57BL/6J mice under steady-state conditions ( n = 18). ( H ) L.j . WXY was cultured anaerobically at 37°C with recombinant S100A8 + S100A9 (1:1 mixture, 5 μg/mL each). Bacterial growth was quantified spectrophotometrically at 6 and 24 hours ( n = 6 technical replicates/group). ( I ) Clec4n –/– mice received intrarectal administration of recombinant S100A8 + S100A9 for 5 hours, followed by quantification of fecal L.j . abundance by qPCR ( n = 10). ( J and K ) WT and Clec4n –/– recipient mice were lethally irradiated and reconstituted with BM cells from WT or Clec4n –/– donors. After 30 days, colonic expression of S100a8 and S100a9 was assessed by qPCR ( J ), and fecal Lactobacillus abundance was measured by qPCR on days 10, 20, and 30 after transfer ( K ) (WT→WT, n = 8; WT→ Clec4n –/– , n = 7; Clec4n –/– →WT, n = 8; Clec4n –/– → Clec4n –/– , n = 8.). ( L – N ) WT and Clec4n –/– mice were treated intraperitoneally with anti-Ly6G neutralizing Ab or isotype control IgG (100 μg/mouse) every other day for 5 doses. Two days after the final injection, colonic tissues and feces were collected. S100A8 and S100A9 protein levels in colon lysates were measured by ELISA ( L ), fecal L.j . abundance was determined by qPCR ( M ), and colonic Il6 and Tnf expression was assessed by qPCR ( N ) (control [con] IgG, n = 3–4; anti-Ly6G, n = 3–4). ( O – S ) WT mice were orally administered culture supernatant (sup.) from L.j . WXY or heat-killed (hk) bacteria daily for 3 days before and throughout 7 days of DSS treatment ( n = 10 total administrations). Body weight loss ( O ), DAI ( P ), colon length measurement at sacrifice on day 9 ( Q ), distal colon histology ( R ), and cLP Treg frequencies ( S ) (PBS n = 8; WXY-sup. n = 8; WXY-hk n = 9). ( T ) CD11b + and CD11c + cells isolated from WT cLP were stimulated in vitro with L.j . WXY, Lactobacillus mixture, or E . coli . After 12 hours, Il10 and Tgfb1 mRNA expression was quantified by qPCR ( n = 4 technical replicates/group). ( U ) CD62L + naive CD4 + T cells isolated from WT spleen and lymph nodes were polarized toward Treg differentiation in the presence of L.j . WXY culture supernatant. After 6 days, Il10 and Tgfb1 expression was measured by qPCR ( n = 9 technical replicates from 3 biological replicates/group). ( V and W ) ABX-treated WT mice received oral L.j . WXY or PBS, followed by fecal microbiota transplantation from normal WT mice and subsequent DSS treatment. Cecal contents were collected on day 10 for untargeted metabolomic analysis by liquid chromatography. Histogram ( V ) shows the top 20 enriched metabolites, and volcano plot ( W ) highlights significantly altered metabolites in WXY-treated mice. ( X ) Targeted metabolomic analysis of l -glutamic acid levels in cecal contents from 8-week-old WT and Clec4n –/– mice under physiological conditions ( n = 3/group). ( Y ) l -Glutamic acid concentrations in culture supernatants of L.j . WXY, Lactobacillus mixture, or E . coli measured by targeted metabolomics ( n = 3 replicates/group). Data in B – F , H – K , and O – S are pooled from 2 independent experiments. Data in B – F , J , L – U , X , and Y are presented as mean ± SD. Statistical analysis: 1-way ANOVA with Bonferroni’s multiple-comparison test ( B , C , K , O , and P ); 1-way ANOVA with Tukey’s multiple-comparison test ( D – F , J , L – N , Q – T , and Y ); Spearman’s correlation test ( G ); 2-way ANOVA test with repeated measures ( H ); paired Student’s t test ( I ); or 2-tailed unpaired Student’s t test ( U and X ).

    Journal: The Journal of Clinical Investigation

    Article Title: Colonic Engyodontium fungus triggers neutrophil antimicrobial activity to suppress Lactobacillus johnsonii –derived glutamic acid–maintained Tregs

    doi: 10.1172/JCI196788

    Figure Lengend Snippet: ( A ) Fecal microbiota from Clec4n –/– mice were cultured on MRS medium supplemented with neomycin for 3 days. Single colonies were isolated and identified by PCR amplification and sequencing of bacterial 16S rDNA. ( B – F ) L.j . WXY strain was isolated from mouse feces. WT mice were treated with antibiotics (ABX), followed by oral transfer of the indicated bacterial strains twice over 7 days. Whole fecal microbiota from normal WT mice were then transferred back for 3 days, followed by DSS treatment for 7 days and sacrifice on day 10. Body weight loss ( B ), DAI ( C ), colon length measurement ( D ), distal colon histology ( E ), and cLP neutrophil and Treg frequencies ( F ) ( B – D , and F , PBS, n = 7; L . j . WXY, n = 7; Lactobacillus mixture, n = 8; E . coli , n = 7; E , n = 3 /group). ( G ) Correlation between fecal Lactobacillus abundance and colonic S100a8 / S100a9 mRNA expression in C57BL/6J mice under steady-state conditions ( n = 18). ( H ) L.j . WXY was cultured anaerobically at 37°C with recombinant S100A8 + S100A9 (1:1 mixture, 5 μg/mL each). Bacterial growth was quantified spectrophotometrically at 6 and 24 hours ( n = 6 technical replicates/group). ( I ) Clec4n –/– mice received intrarectal administration of recombinant S100A8 + S100A9 for 5 hours, followed by quantification of fecal L.j . abundance by qPCR ( n = 10). ( J and K ) WT and Clec4n –/– recipient mice were lethally irradiated and reconstituted with BM cells from WT or Clec4n –/– donors. After 30 days, colonic expression of S100a8 and S100a9 was assessed by qPCR ( J ), and fecal Lactobacillus abundance was measured by qPCR on days 10, 20, and 30 after transfer ( K ) (WT→WT, n = 8; WT→ Clec4n –/– , n = 7; Clec4n –/– →WT, n = 8; Clec4n –/– → Clec4n –/– , n = 8.). ( L – N ) WT and Clec4n –/– mice were treated intraperitoneally with anti-Ly6G neutralizing Ab or isotype control IgG (100 μg/mouse) every other day for 5 doses. Two days after the final injection, colonic tissues and feces were collected. S100A8 and S100A9 protein levels in colon lysates were measured by ELISA ( L ), fecal L.j . abundance was determined by qPCR ( M ), and colonic Il6 and Tnf expression was assessed by qPCR ( N ) (control [con] IgG, n = 3–4; anti-Ly6G, n = 3–4). ( O – S ) WT mice were orally administered culture supernatant (sup.) from L.j . WXY or heat-killed (hk) bacteria daily for 3 days before and throughout 7 days of DSS treatment ( n = 10 total administrations). Body weight loss ( O ), DAI ( P ), colon length measurement at sacrifice on day 9 ( Q ), distal colon histology ( R ), and cLP Treg frequencies ( S ) (PBS n = 8; WXY-sup. n = 8; WXY-hk n = 9). ( T ) CD11b + and CD11c + cells isolated from WT cLP were stimulated in vitro with L.j . WXY, Lactobacillus mixture, or E . coli . After 12 hours, Il10 and Tgfb1 mRNA expression was quantified by qPCR ( n = 4 technical replicates/group). ( U ) CD62L + naive CD4 + T cells isolated from WT spleen and lymph nodes were polarized toward Treg differentiation in the presence of L.j . WXY culture supernatant. After 6 days, Il10 and Tgfb1 expression was measured by qPCR ( n = 9 technical replicates from 3 biological replicates/group). ( V and W ) ABX-treated WT mice received oral L.j . WXY or PBS, followed by fecal microbiota transplantation from normal WT mice and subsequent DSS treatment. Cecal contents were collected on day 10 for untargeted metabolomic analysis by liquid chromatography. Histogram ( V ) shows the top 20 enriched metabolites, and volcano plot ( W ) highlights significantly altered metabolites in WXY-treated mice. ( X ) Targeted metabolomic analysis of l -glutamic acid levels in cecal contents from 8-week-old WT and Clec4n –/– mice under physiological conditions ( n = 3/group). ( Y ) l -Glutamic acid concentrations in culture supernatants of L.j . WXY, Lactobacillus mixture, or E . coli measured by targeted metabolomics ( n = 3 replicates/group). Data in B – F , H – K , and O – S are pooled from 2 independent experiments. Data in B – F , J , L – U , X , and Y are presented as mean ± SD. Statistical analysis: 1-way ANOVA with Bonferroni’s multiple-comparison test ( B , C , K , O , and P ); 1-way ANOVA with Tukey’s multiple-comparison test ( D – F , J , L – N , Q – T , and Y ); Spearman’s correlation test ( G ); 2-way ANOVA test with repeated measures ( H ); paired Student’s t test ( I ); or 2-tailed unpaired Student’s t test ( U and X ).

    Article Snippet: Lactobacillus cultures were incubated in MRS medium containing a 1:1 mixture of recombinant S100A8 and S100A9 peptides (5 μg/mL each; HY-P71076, MCE) for 6 or 24 hours under anaerobic conditions.

    Techniques: Cell Culture, Isolation, Amplification, Sequencing, Expressing, Recombinant, Irradiation, Control, Injection, Enzyme-linked Immunosorbent Assay, Bacteria, In Vitro, Transplantation Assay, Metabolomic, Liquid Chromatography, Comparison

    ( A ) Phylum- and genus-level composition of human fecal commensal fungi isolated using Dectin-2–Fc and identified by qPCR with species-specific primers. ( B ) Relative contents of Engyodontium sp. in fecal fungi and L.j . in fecal bacteria from patients with CD and healthy individuals (HC), determined by qPCR ( Engyodontium : HC, n = 59; CD, n = 62. Lacutobacillus : HC, n = 62; CD, n = 63). ( C ) Relative contents of Engyodontium sp. in fecal fungi and L.j . in fecal bacteria from patients with UC and healthy individuals, determined by qPCR (HC, n = 36; UC, n = 25). ( D ) Correlation between Ct value of L.j . and Engyodontium sp. from human individuals as described in B ( n = 97 [including HC, n = 48 and CD, n = 49]). ( E ) Correlation between Ct value of L.j . and Engyodontium sp. from human individuals as described in C (n=61 [including HC, n=36 and UC, n=25]). ( F ) C57BL/6J SPF mice were orally colonized with Engyodontium sp. ( Engyod. ) every other day for 3 administrations. Thirteen days later, fecal samples were collected, and the relative abundance of fecal L.j . was quantified by qPCR ( n = 10 /group). ( G ) Relative expression of CLEC6A , S100A8 , and S100A9 in colon tissues from patients with CD and healthy control (HC) individuals, determined by bulk RNA-Seq analysis (HC, n = 47; CD, n = 57). ( H ) Correlation between CLEC6A and S100A8 or S100A9 expression based on bulk RNA-Seq data described in G . ( I ) Correlation between CLEC6A and S100A8 or S100A9 relative expression in colon tissues of patients with UC, determined by qPCR ( n = 25). ( J ) IHC staining for DECTIN-2 and S100A8/S100A9 proteins in serial inflammatory colon sections obtained from a patient with CD after surgical resection. ( K ) IHC staining for human DECTIN-2 in paired normal and inflamed colon regions from the patient with CD shown in J . ( L ) Transcriptional levels of CLEC6A in colon tissues from non-IBD control individuals and patients with colonic CD (cCD), ileal CD (iCD), or UC, analyzed by reprocessing a public bulk RNA-Seq dataset ( GSE117993 ) (not IBD, n = 55; cCD, n = 31; iCD, n = 60; UC, n = 43). ( M ) Colon tissues from 3 patients with IBD were obtained after resection. cLP CD11b + cells were isolated and stimulated with α-mannan in vitro for 6 hours. S100A8 and S100A9 relative expression was quantified by qPCR ( n = 7). ( N ) Neutrophils isolated from peripheral blood of 2 patients with IBD were stimulated with α-mannan in vitro for 6 hours, and S100A8 and S100A9 relative expression was measured by qPCR ( n = 7 technical replicates from 2 biological replicates/group). ( O ) Correlation between fecal Engyodontium sp. abundance and colonic expression of CLEC6A , S100A8 , or S100A9 in patients with UC ( n = 25). ( P ) Correlation between fecal L.j . abundance and colonic expression of CLEC6A , S100A8 , or S100A9 in patients with UC ( n = 25). ( Q ) CD11b + cells isolated from cLP of resected colon tissues from 2 patients with IBD were stimulated with Engyodontium sp. for 24 hours in vitro, followed by qPCR analysis of S100A8 and S100A9 expression ( n = 6 technical replicates from 2 biological replicates/group). non-stimu., nonstimulated. ( R ) Engyodontium sp. was cultured at room temperature with recombinant S100A8 + S100A9 (1:1 mixture, 5 μg/mL each) for 12, 24, and 48 hours. Fungus growth was quantified by CFU enumeration on PDA plates ( n = 3 replicates/group). ( S ) Relative amount of glutamic acid in fecal samples from patients with CD and healthy control individuals, assessed by liquid chromatography for untargeted metabolomics (HC, n = 80; CD, n = 65). ( T ) Correlation between 1/Ct value of L.j . and fecal glutamic acid levels in patients with CD ( n = 8). ( U ) Correlation between 1/Ct value of L.j . and glutamic acid concentration in colon tissues from patients with UC ( n = 25). ( V ) Peripheral blood leukocytes from 2 healthy donors were isolated and induced toward Treg differentiation in the presence of l -glutamic acid. After 5 days, the proportion of Foxp3 + CD4 + among CD45 + leukocytes was analyzed by flow cytometry ( n = 6 technical replicates from 2 biological replicates/group). Data in F , M , N , Q , and V are pooled from 2 independent experiments. Data in B , C , G , and L are presented as mean ± SEM, and in F , N , Q , R , S , and V as mean ± SD. Statistical analysis: 2-tailed Mann-Whitney test ( B , C , and G ), 2-tailed unpaired Student’s t test ( F , N , Q – S , and V ), 1-way ANOVA with Kruskal-Wallis and Dunn’s test ( L ), Spearman’s correlation test ( D , E , H , I , O , P , T , and U ), or paired Student’s t test ( M ).

    Journal: The Journal of Clinical Investigation

    Article Title: Colonic Engyodontium fungus triggers neutrophil antimicrobial activity to suppress Lactobacillus johnsonii –derived glutamic acid–maintained Tregs

    doi: 10.1172/JCI196788

    Figure Lengend Snippet: ( A ) Phylum- and genus-level composition of human fecal commensal fungi isolated using Dectin-2–Fc and identified by qPCR with species-specific primers. ( B ) Relative contents of Engyodontium sp. in fecal fungi and L.j . in fecal bacteria from patients with CD and healthy individuals (HC), determined by qPCR ( Engyodontium : HC, n = 59; CD, n = 62. Lacutobacillus : HC, n = 62; CD, n = 63). ( C ) Relative contents of Engyodontium sp. in fecal fungi and L.j . in fecal bacteria from patients with UC and healthy individuals, determined by qPCR (HC, n = 36; UC, n = 25). ( D ) Correlation between Ct value of L.j . and Engyodontium sp. from human individuals as described in B ( n = 97 [including HC, n = 48 and CD, n = 49]). ( E ) Correlation between Ct value of L.j . and Engyodontium sp. from human individuals as described in C (n=61 [including HC, n=36 and UC, n=25]). ( F ) C57BL/6J SPF mice were orally colonized with Engyodontium sp. ( Engyod. ) every other day for 3 administrations. Thirteen days later, fecal samples were collected, and the relative abundance of fecal L.j . was quantified by qPCR ( n = 10 /group). ( G ) Relative expression of CLEC6A , S100A8 , and S100A9 in colon tissues from patients with CD and healthy control (HC) individuals, determined by bulk RNA-Seq analysis (HC, n = 47; CD, n = 57). ( H ) Correlation between CLEC6A and S100A8 or S100A9 expression based on bulk RNA-Seq data described in G . ( I ) Correlation between CLEC6A and S100A8 or S100A9 relative expression in colon tissues of patients with UC, determined by qPCR ( n = 25). ( J ) IHC staining for DECTIN-2 and S100A8/S100A9 proteins in serial inflammatory colon sections obtained from a patient with CD after surgical resection. ( K ) IHC staining for human DECTIN-2 in paired normal and inflamed colon regions from the patient with CD shown in J . ( L ) Transcriptional levels of CLEC6A in colon tissues from non-IBD control individuals and patients with colonic CD (cCD), ileal CD (iCD), or UC, analyzed by reprocessing a public bulk RNA-Seq dataset ( GSE117993 ) (not IBD, n = 55; cCD, n = 31; iCD, n = 60; UC, n = 43). ( M ) Colon tissues from 3 patients with IBD were obtained after resection. cLP CD11b + cells were isolated and stimulated with α-mannan in vitro for 6 hours. S100A8 and S100A9 relative expression was quantified by qPCR ( n = 7). ( N ) Neutrophils isolated from peripheral blood of 2 patients with IBD were stimulated with α-mannan in vitro for 6 hours, and S100A8 and S100A9 relative expression was measured by qPCR ( n = 7 technical replicates from 2 biological replicates/group). ( O ) Correlation between fecal Engyodontium sp. abundance and colonic expression of CLEC6A , S100A8 , or S100A9 in patients with UC ( n = 25). ( P ) Correlation between fecal L.j . abundance and colonic expression of CLEC6A , S100A8 , or S100A9 in patients with UC ( n = 25). ( Q ) CD11b + cells isolated from cLP of resected colon tissues from 2 patients with IBD were stimulated with Engyodontium sp. for 24 hours in vitro, followed by qPCR analysis of S100A8 and S100A9 expression ( n = 6 technical replicates from 2 biological replicates/group). non-stimu., nonstimulated. ( R ) Engyodontium sp. was cultured at room temperature with recombinant S100A8 + S100A9 (1:1 mixture, 5 μg/mL each) for 12, 24, and 48 hours. Fungus growth was quantified by CFU enumeration on PDA plates ( n = 3 replicates/group). ( S ) Relative amount of glutamic acid in fecal samples from patients with CD and healthy control individuals, assessed by liquid chromatography for untargeted metabolomics (HC, n = 80; CD, n = 65). ( T ) Correlation between 1/Ct value of L.j . and fecal glutamic acid levels in patients with CD ( n = 8). ( U ) Correlation between 1/Ct value of L.j . and glutamic acid concentration in colon tissues from patients with UC ( n = 25). ( V ) Peripheral blood leukocytes from 2 healthy donors were isolated and induced toward Treg differentiation in the presence of l -glutamic acid. After 5 days, the proportion of Foxp3 + CD4 + among CD45 + leukocytes was analyzed by flow cytometry ( n = 6 technical replicates from 2 biological replicates/group). Data in F , M , N , Q , and V are pooled from 2 independent experiments. Data in B , C , G , and L are presented as mean ± SEM, and in F , N , Q , R , S , and V as mean ± SD. Statistical analysis: 2-tailed Mann-Whitney test ( B , C , and G ), 2-tailed unpaired Student’s t test ( F , N , Q – S , and V ), 1-way ANOVA with Kruskal-Wallis and Dunn’s test ( L ), Spearman’s correlation test ( D , E , H , I , O , P , T , and U ), or paired Student’s t test ( M ).

    Article Snippet: Lactobacillus cultures were incubated in MRS medium containing a 1:1 mixture of recombinant S100A8 and S100A9 peptides (5 μg/mL each; HY-P71076, MCE) for 6 or 24 hours under anaerobic conditions.

    Techniques: Isolation, Bacteria, Expressing, Control, RNA Sequencing, Immunohistochemistry, In Vitro, Cell Culture, Recombinant, Liquid Chromatography, Concentration Assay, Flow Cytometry, MANN-WHITNEY

    Increased S100A8/9 expression in the skin and serum of patients with BP and AD. ( a ) Immunohistochemical analysis with anti-S100A8/9 antibody showing the expression of S100A8/9 in keratinocytes and infiltrating inflammatory cells of lesional skin of patients with BP and AD. S100A8/9 expression was not observed in the healthy skin. Scale bar: 100 μm. (b) Quantification of the expression level of S100A8/9. (control, n = 2; BP, n = 5; AD n = 2). Inter-patient heterogeneity of the expression level of S100A8/9 was observed in the BP skin samples, and representative images of both low and high S100A8/9 expression were shown in A. (c) ELISA analysis showing increased S100A8/9 concentrations in the serum of patients with BP. (control, n = 8; BP, n = 16; P = .0087, Mann-Whitney test). Data are reported as mean ± SD. AD, atopic dermatitis; BP, bullous pemphigoid.

    Journal: JID Innovations

    Article Title: A neuroimmune axis linking S100A8/9 to itch sensitization in both bullous pemphigoid and atopic dermatitis

    doi: 10.1016/j.xjidi.2026.100470

    Figure Lengend Snippet: Increased S100A8/9 expression in the skin and serum of patients with BP and AD. ( a ) Immunohistochemical analysis with anti-S100A8/9 antibody showing the expression of S100A8/9 in keratinocytes and infiltrating inflammatory cells of lesional skin of patients with BP and AD. S100A8/9 expression was not observed in the healthy skin. Scale bar: 100 μm. (b) Quantification of the expression level of S100A8/9. (control, n = 2; BP, n = 5; AD n = 2). Inter-patient heterogeneity of the expression level of S100A8/9 was observed in the BP skin samples, and representative images of both low and high S100A8/9 expression were shown in A. (c) ELISA analysis showing increased S100A8/9 concentrations in the serum of patients with BP. (control, n = 8; BP, n = 16; P = .0087, Mann-Whitney test). Data are reported as mean ± SD. AD, atopic dermatitis; BP, bullous pemphigoid.

    Article Snippet: Chemicals used are listed as follows: S100A8, S100A9, and S100A8/9 (100 ng/ml, R&D Systems, 9877-S8, 2065-S9, 8916-S8), Bam8-22 (1 μM, custom synthesized by Genscript), and capsaicin (Sigma M2028, 1 μM).

    Techniques: Expressing, Immunohistochemical staining, Control, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    S100A8/9 directly activates small-diameter, nociceptive DRG sensory neurons. ( a ) Representative fluorescent images of cultured DRG sensory neurons isolated from Pirt GCaMP3/+ mice before and after S100A8/9 (100 ng/ml) application. Arrows point to sensory neurons showing increased GCaMP3 fluorescence levels after S100A8/9 application. (b) Histogram showing size distribution of S100A8/9-responsive neurons. (c–e) Representative traces of DRG neurons evoked by indicated chemicals, including S100A8/9 (100 ng/ml), S100A8 (100 ng/ml), S100A9 (100 ng/ml), capsaicin (1 μM), and KCl (75 mM) in a calcium imaging assay. All S100A8/9-sensitive neurons responded to capsaicin and KCl. The majority of S100A8- and S100A9-responsive neurons also responded to S100A8/9. The three different colors (Red, Green, and Blue) represent individual cells in C-E. (f) TAK-242 inhibited S100A8/9-induced calcium responses in sensory neurons. (n = 3 for both groups P = .018, Welch’s t -test). Data are reported as mean ± SD. AD, atopic dermatitis; BP, bullous pemphigoid; DRG, dorsal root ganglia.

    Journal: JID Innovations

    Article Title: A neuroimmune axis linking S100A8/9 to itch sensitization in both bullous pemphigoid and atopic dermatitis

    doi: 10.1016/j.xjidi.2026.100470

    Figure Lengend Snippet: S100A8/9 directly activates small-diameter, nociceptive DRG sensory neurons. ( a ) Representative fluorescent images of cultured DRG sensory neurons isolated from Pirt GCaMP3/+ mice before and after S100A8/9 (100 ng/ml) application. Arrows point to sensory neurons showing increased GCaMP3 fluorescence levels after S100A8/9 application. (b) Histogram showing size distribution of S100A8/9-responsive neurons. (c–e) Representative traces of DRG neurons evoked by indicated chemicals, including S100A8/9 (100 ng/ml), S100A8 (100 ng/ml), S100A9 (100 ng/ml), capsaicin (1 μM), and KCl (75 mM) in a calcium imaging assay. All S100A8/9-sensitive neurons responded to capsaicin and KCl. The majority of S100A8- and S100A9-responsive neurons also responded to S100A8/9. The three different colors (Red, Green, and Blue) represent individual cells in C-E. (f) TAK-242 inhibited S100A8/9-induced calcium responses in sensory neurons. (n = 3 for both groups P = .018, Welch’s t -test). Data are reported as mean ± SD. AD, atopic dermatitis; BP, bullous pemphigoid; DRG, dorsal root ganglia.

    Article Snippet: Chemicals used are listed as follows: S100A8, S100A9, and S100A8/9 (100 ng/ml, R&D Systems, 9877-S8, 2065-S9, 8916-S8), Bam8-22 (1 μM, custom synthesized by Genscript), and capsaicin (Sigma M2028, 1 μM).

    Techniques: Cell Culture, Isolation, Fluorescence, Imaging

    S100A8/9 potentiate histamine-induced itch. ( a ) Subcutaneous injection of S100A8/9 (20 μg/ml) into the nape of the neck of wild-type mice does not induce scratching behavior. (n = 7 for both groups, P = .97, Welch’s t test). (b) Co-injection of S100A8/9 increased histamine (20 mM)-induced scratching behavior. (n = 11 for both groups, P = .038, Welch’s t test). Data are reported as mean ± SD.

    Journal: JID Innovations

    Article Title: A neuroimmune axis linking S100A8/9 to itch sensitization in both bullous pemphigoid and atopic dermatitis

    doi: 10.1016/j.xjidi.2026.100470

    Figure Lengend Snippet: S100A8/9 potentiate histamine-induced itch. ( a ) Subcutaneous injection of S100A8/9 (20 μg/ml) into the nape of the neck of wild-type mice does not induce scratching behavior. (n = 7 for both groups, P = .97, Welch’s t test). (b) Co-injection of S100A8/9 increased histamine (20 mM)-induced scratching behavior. (n = 11 for both groups, P = .038, Welch’s t test). Data are reported as mean ± SD.

    Article Snippet: Chemicals used are listed as follows: S100A8, S100A9, and S100A8/9 (100 ng/ml, R&D Systems, 9877-S8, 2065-S9, 8916-S8), Bam8-22 (1 μM, custom synthesized by Genscript), and capsaicin (Sigma M2028, 1 μM).

    Techniques: Injection

    Klebsiella pneumoniae infection directly induces S100A8/A9 expression and secretion in HBE cells. Primary HBE cells were infected with WT K. pneumoniae or an isogenic acapsular mutant (Δ cps ). (A,B) Expression of S100A8 and S100A9 was determined by qRT-PCR at 8 h post-infection with various MOIs of K. pneumoniae , or 100 MOI K. pneumoniae for 4–12 h (C,D) . Data are normalized to the housekeeping gene GAPDH and expressed as fold change relative to uninfected control cells. (E) Secretion of S100A8/A9 heterodimer induced by WT K. pneumoniae and the isogenic Δ cps mutant at indicated MOIs after 24 h. All data are presented as mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01 vs. uninfected cells (A–D) . Statistical differences between the WT and Δ cps groups were determined by Two-way ANOVA followed by Tukey’s post-hoc test. * p < 0.05 compared between strains at the same MOI (E) .

    Journal: Frontiers in Microbiology

    Article Title: Klebsiella pneumoniae infection induces an S100A8/A9-mediated autocrine loop in human airway epithelium to amplify inflammation

    doi: 10.3389/fmicb.2026.1768140

    Figure Lengend Snippet: Klebsiella pneumoniae infection directly induces S100A8/A9 expression and secretion in HBE cells. Primary HBE cells were infected with WT K. pneumoniae or an isogenic acapsular mutant (Δ cps ). (A,B) Expression of S100A8 and S100A9 was determined by qRT-PCR at 8 h post-infection with various MOIs of K. pneumoniae , or 100 MOI K. pneumoniae for 4–12 h (C,D) . Data are normalized to the housekeeping gene GAPDH and expressed as fold change relative to uninfected control cells. (E) Secretion of S100A8/A9 heterodimer induced by WT K. pneumoniae and the isogenic Δ cps mutant at indicated MOIs after 24 h. All data are presented as mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01 vs. uninfected cells (A–D) . Statistical differences between the WT and Δ cps groups were determined by Two-way ANOVA followed by Tukey’s post-hoc test. * p < 0.05 compared between strains at the same MOI (E) .

    Article Snippet: For S100A8/A9 stimulating experiment, cells were incubated with endotoxin-free recombinant human S100A8/A9 protein (R&D Systems) at the indicated concentrations for specified durations.

    Techniques: Infection, Expressing, Mutagenesis, Quantitative RT-PCR, Control

    Extracellular S100A8/A9 functions as a potent pro-inflammatory stimulus for primary HBE cells. (A) Primary HBE cells were infected with WT K. pneumoniae (MOI 100) for the indicated time points. Relative mRNA expression of TLR4 was determined by qRT-PCR, with data calibrated to GAPDH and shown as fold induction over the baseline (0 h). (B) Representative western blot showing total TLR4 protein levels. β -Actin served as the housekeeping protein. (C) Quantification of TLR4 protein expression from three independent replicates, with values standardized to β-Actin. (D,E) HBE cells were treated for 24 h with the specified doses of endotoxin-free recombinant human S100A8/A9 (rS100A8/A9). The release of (D) IL-8 and (E) IL-6 was detected by ELISA. Data are presented as mean ± SEM of three independent experiments. * p < 0.05 versus the untreated control (A,C,D,E) .

    Journal: Frontiers in Microbiology

    Article Title: Klebsiella pneumoniae infection induces an S100A8/A9-mediated autocrine loop in human airway epithelium to amplify inflammation

    doi: 10.3389/fmicb.2026.1768140

    Figure Lengend Snippet: Extracellular S100A8/A9 functions as a potent pro-inflammatory stimulus for primary HBE cells. (A) Primary HBE cells were infected with WT K. pneumoniae (MOI 100) for the indicated time points. Relative mRNA expression of TLR4 was determined by qRT-PCR, with data calibrated to GAPDH and shown as fold induction over the baseline (0 h). (B) Representative western blot showing total TLR4 protein levels. β -Actin served as the housekeeping protein. (C) Quantification of TLR4 protein expression from three independent replicates, with values standardized to β-Actin. (D,E) HBE cells were treated for 24 h with the specified doses of endotoxin-free recombinant human S100A8/A9 (rS100A8/A9). The release of (D) IL-8 and (E) IL-6 was detected by ELISA. Data are presented as mean ± SEM of three independent experiments. * p < 0.05 versus the untreated control (A,C,D,E) .

    Article Snippet: For S100A8/A9 stimulating experiment, cells were incubated with endotoxin-free recombinant human S100A8/A9 protein (R&D Systems) at the indicated concentrations for specified durations.

    Techniques: Infection, Expressing, Quantitative RT-PCR, Western Blot, Recombinant, Enzyme-linked Immunosorbent Assay, Control

    Epithelial-derived S100A8/A9 is functionally required for the full inflammatory response to K. pneumoniae infection. Primary HBE cells were transfected with control siRNA (siCtrl) or S100A9 siRNA (siA9). (A) Knockdown efficiency was confirmed 48 h post-transfection by qRT-PCR analysis of S100A9 mRNA levels following a 12-h infection with WT K. pneumoniae (MOI 100), negative values represent fold reduction calculated as the negative inverse of 2 −ΔΔCt following the Schmittgen and Livak protocol. (B) Functional knockdown was confirmed by measuring S100A8/A9 protein secretion by ELISA in supernatants from infected cells at 24 h. (C,D) Control and S100A9 -deficient cells were infected with WT K. pneumoniae (MOI 100) for 24 h, and the secretion of cytokines were quantified by ELISA. Data are presented as mean ± SEM of three independent experiments. * p < 0.05 vs. Kp + siCtrl (A,B) or uninfected cells (C,D) , # p < 0.05 vs. Kp + siCtrl (C,D) .

    Journal: Frontiers in Microbiology

    Article Title: Klebsiella pneumoniae infection induces an S100A8/A9-mediated autocrine loop in human airway epithelium to amplify inflammation

    doi: 10.3389/fmicb.2026.1768140

    Figure Lengend Snippet: Epithelial-derived S100A8/A9 is functionally required for the full inflammatory response to K. pneumoniae infection. Primary HBE cells were transfected with control siRNA (siCtrl) or S100A9 siRNA (siA9). (A) Knockdown efficiency was confirmed 48 h post-transfection by qRT-PCR analysis of S100A9 mRNA levels following a 12-h infection with WT K. pneumoniae (MOI 100), negative values represent fold reduction calculated as the negative inverse of 2 −ΔΔCt following the Schmittgen and Livak protocol. (B) Functional knockdown was confirmed by measuring S100A8/A9 protein secretion by ELISA in supernatants from infected cells at 24 h. (C,D) Control and S100A9 -deficient cells were infected with WT K. pneumoniae (MOI 100) for 24 h, and the secretion of cytokines were quantified by ELISA. Data are presented as mean ± SEM of three independent experiments. * p < 0.05 vs. Kp + siCtrl (A,B) or uninfected cells (C,D) , # p < 0.05 vs. Kp + siCtrl (C,D) .

    Article Snippet: For S100A8/A9 stimulating experiment, cells were incubated with endotoxin-free recombinant human S100A8/A9 protein (R&D Systems) at the indicated concentrations for specified durations.

    Techniques: Derivative Assay, Infection, Transfection, Control, Knockdown, Quantitative RT-PCR, Functional Assay, Enzyme-linked Immunosorbent Assay

    TLR4 is the essential receptor mediating S100A8/A9-induced inflammation in HBE cells during K. pneumoniae infection. (A,B) HBE cells were pre-incubated with neutralizing antibodies against TLR4 (10 μg/mL), RAGE (100 μg/mL), or an isotype control (Iso Ctrl) for 1 h, followed by stimulation with recombinant human S100A8/A9 (20 μg/mL) for 24 h. Levels of (A) IL-8 and (B) IL-6 in supernatants were determined by ELISA. (C,D) HBE cells were pre-treated with the indicated antibodies for 1 h and subsequently infected with WT K. pneumoniae (MOI 100) for 24 h. Secretion of (C) IL-8 and (D) IL-6 was quantified by ELISA. All results are expressed as mean ± SEM of three independent experiments. * p < 0.05 compared with the indicated groups.

    Journal: Frontiers in Microbiology

    Article Title: Klebsiella pneumoniae infection induces an S100A8/A9-mediated autocrine loop in human airway epithelium to amplify inflammation

    doi: 10.3389/fmicb.2026.1768140

    Figure Lengend Snippet: TLR4 is the essential receptor mediating S100A8/A9-induced inflammation in HBE cells during K. pneumoniae infection. (A,B) HBE cells were pre-incubated with neutralizing antibodies against TLR4 (10 μg/mL), RAGE (100 μg/mL), or an isotype control (Iso Ctrl) for 1 h, followed by stimulation with recombinant human S100A8/A9 (20 μg/mL) for 24 h. Levels of (A) IL-8 and (B) IL-6 in supernatants were determined by ELISA. (C,D) HBE cells were pre-treated with the indicated antibodies for 1 h and subsequently infected with WT K. pneumoniae (MOI 100) for 24 h. Secretion of (C) IL-8 and (D) IL-6 was quantified by ELISA. All results are expressed as mean ± SEM of three independent experiments. * p < 0.05 compared with the indicated groups.

    Article Snippet: For S100A8/A9 stimulating experiment, cells were incubated with endotoxin-free recombinant human S100A8/A9 protein (R&D Systems) at the indicated concentrations for specified durations.

    Techniques: Infection, Incubation, Control, Recombinant, Enzyme-linked Immunosorbent Assay

    The S100A8/A9 autocrine loop drives NF-κB activation and subsequent cytokine release in HBE cells. (A) Representative immunoblots showing degradation of IκBα in primary HBE cells stimulated with rS100A8/A9 (20 μg/mL) for 60 min. β-Actin served as a loading control. (B) Densitometric quantification of IκBα protein levels from (A) . (C) Representative immunofluorescence images showing subcellular localization of the p65 subunit (green) at 60 min post-stimulation with rS100A8/A9. Nuclei were counterstained with DAPI (blue). (D) HBE cells were transfected with control siRNA (siCtrl) or siRNA targeting S100A9 (siS100A9) and subsequently infected with wild-type K. pneumoniae (MOI 100). Representative immunoblots of IκBα levels are shown at 60 min post-infection. (E) Densitometric quantification of IκBα degradation from (D) . (F) Representative immunofluorescence images showing p65 localization (green) in siCtrl- or siS100A9-transfected HBE cells at 60 min post- K. pneumoniae infection. Nuclei were stained with DAPI (blue). (G) Secretion of IL-8 and IL-6 by HBE cells. Cells were pre-treated with or without the NF-κB inhibitor BAY 11–7,082 (10 μM) for 60 min, followed by stimulation with rS100A8/A9 (20 μg/mL) for 24 h. Cytokine levels in supernatants were measured by ELISA. All graphs display mean ± SEM, * p < 0.05 compared with the untreated control (B,E,G) ; # p < 0.05 compared with siCtrl + Kp or the S100A8/A9-stimulated group without inhibitor (E,G) .

    Journal: Frontiers in Microbiology

    Article Title: Klebsiella pneumoniae infection induces an S100A8/A9-mediated autocrine loop in human airway epithelium to amplify inflammation

    doi: 10.3389/fmicb.2026.1768140

    Figure Lengend Snippet: The S100A8/A9 autocrine loop drives NF-κB activation and subsequent cytokine release in HBE cells. (A) Representative immunoblots showing degradation of IκBα in primary HBE cells stimulated with rS100A8/A9 (20 μg/mL) for 60 min. β-Actin served as a loading control. (B) Densitometric quantification of IκBα protein levels from (A) . (C) Representative immunofluorescence images showing subcellular localization of the p65 subunit (green) at 60 min post-stimulation with rS100A8/A9. Nuclei were counterstained with DAPI (blue). (D) HBE cells were transfected with control siRNA (siCtrl) or siRNA targeting S100A9 (siS100A9) and subsequently infected with wild-type K. pneumoniae (MOI 100). Representative immunoblots of IκBα levels are shown at 60 min post-infection. (E) Densitometric quantification of IκBα degradation from (D) . (F) Representative immunofluorescence images showing p65 localization (green) in siCtrl- or siS100A9-transfected HBE cells at 60 min post- K. pneumoniae infection. Nuclei were stained with DAPI (blue). (G) Secretion of IL-8 and IL-6 by HBE cells. Cells were pre-treated with or without the NF-κB inhibitor BAY 11–7,082 (10 μM) for 60 min, followed by stimulation with rS100A8/A9 (20 μg/mL) for 24 h. Cytokine levels in supernatants were measured by ELISA. All graphs display mean ± SEM, * p < 0.05 compared with the untreated control (B,E,G) ; # p < 0.05 compared with siCtrl + Kp or the S100A8/A9-stimulated group without inhibitor (E,G) .

    Article Snippet: For S100A8/A9 stimulating experiment, cells were incubated with endotoxin-free recombinant human S100A8/A9 protein (R&D Systems) at the indicated concentrations for specified durations.

    Techniques: Activation Assay, Western Blot, Control, Immunofluorescence, Transfection, Infection, Staining, Enzyme-linked Immunosorbent Assay

    The epithelial S100A8/A9 autocrine loop is a critical driver of neutrophil chemotaxis in response to K. pneumoniae infection. Neutrophil chemotaxis was assessed using a Transwell assay. (A) Comparison of the chemotactic activity of supernatants from uninfected HBE cells (conditioned medium, CM), HBE cells infected with WT K. pneumoniae (MOI 100), and fMLP (positive control) on primary human neutrophils. (B) Comparison of the chemotactic activity of supernatants from infected control (siCtrl) HBE cells versus infected S100A9-deficient (siS100A9) HBE cells. Flow cytometry was used to determine the absolute number of migrated neutrophils. Data are shown as mean ± SEM from three separate experiments. * p < 0.05 vs. uninfected CM, # p < 0.05 vs. Kp + siCtrl.

    Journal: Frontiers in Microbiology

    Article Title: Klebsiella pneumoniae infection induces an S100A8/A9-mediated autocrine loop in human airway epithelium to amplify inflammation

    doi: 10.3389/fmicb.2026.1768140

    Figure Lengend Snippet: The epithelial S100A8/A9 autocrine loop is a critical driver of neutrophil chemotaxis in response to K. pneumoniae infection. Neutrophil chemotaxis was assessed using a Transwell assay. (A) Comparison of the chemotactic activity of supernatants from uninfected HBE cells (conditioned medium, CM), HBE cells infected with WT K. pneumoniae (MOI 100), and fMLP (positive control) on primary human neutrophils. (B) Comparison of the chemotactic activity of supernatants from infected control (siCtrl) HBE cells versus infected S100A9-deficient (siS100A9) HBE cells. Flow cytometry was used to determine the absolute number of migrated neutrophils. Data are shown as mean ± SEM from three separate experiments. * p < 0.05 vs. uninfected CM, # p < 0.05 vs. Kp + siCtrl.

    Article Snippet: For S100A8/A9 stimulating experiment, cells were incubated with endotoxin-free recombinant human S100A8/A9 protein (R&D Systems) at the indicated concentrations for specified durations.

    Techniques: Chemotaxis Assay, Infection, Transwell Assay, Comparison, Activity Assay, Positive Control, Control, Flow Cytometry

    Proposed model of the S100A8/A9-mediated autocrine amplification loop in human airway epithelial cells during K. pneumoniae infection. Initial recognition of encapsulated K. pneumoniae by the airway epithelium triggers a primary transcriptional response, leading to the synthesis and secretion of the alarmin S100A8/A9 and a simultaneous upregulation of its cognate receptor, TLR4. This dual mechanism creates a primed state within the epithelium. The endogenously produced S100A8/A9 then acts back on the enriched TLR4 receptors in an autocrine or paracrine manner, activating the canonical NF-κB signaling pathway (characterized by IκB degradation and p65 nuclear translocation). This positive feedback loop significantly magnifies the production of pro-inflammatory cytokines such as IL-6 and IL-8, ultimately orchestrating massive neutrophil recruitment and driving the hyper-inflammation observed in severe pneumonia. Created in BioRender [You (2026) https://BioRender.com/qo9szis ].

    Journal: Frontiers in Microbiology

    Article Title: Klebsiella pneumoniae infection induces an S100A8/A9-mediated autocrine loop in human airway epithelium to amplify inflammation

    doi: 10.3389/fmicb.2026.1768140

    Figure Lengend Snippet: Proposed model of the S100A8/A9-mediated autocrine amplification loop in human airway epithelial cells during K. pneumoniae infection. Initial recognition of encapsulated K. pneumoniae by the airway epithelium triggers a primary transcriptional response, leading to the synthesis and secretion of the alarmin S100A8/A9 and a simultaneous upregulation of its cognate receptor, TLR4. This dual mechanism creates a primed state within the epithelium. The endogenously produced S100A8/A9 then acts back on the enriched TLR4 receptors in an autocrine or paracrine manner, activating the canonical NF-κB signaling pathway (characterized by IκB degradation and p65 nuclear translocation). This positive feedback loop significantly magnifies the production of pro-inflammatory cytokines such as IL-6 and IL-8, ultimately orchestrating massive neutrophil recruitment and driving the hyper-inflammation observed in severe pneumonia. Created in BioRender [You (2026) https://BioRender.com/qo9szis ].

    Article Snippet: For S100A8/A9 stimulating experiment, cells were incubated with endotoxin-free recombinant human S100A8/A9 protein (R&D Systems) at the indicated concentrations for specified durations.

    Techniques: Amplification, Infection, Produced, Translocation Assay