Journal: Molecular Therapy. Nucleic Acids

Article Title: Hemoglobin inhibits fibroblast-to-cardiomyocyte reprogramming via TLR2/TLR4-dependent chromatin compaction

doi: 10.1016/j.omtn.2026.102900

Figure Lengend Snippet: Hemoglobin mediates gene repression through TLR2 and TLR4 (A and B) A study was conducted to determine the effect of hemoglobin (Hb) on (A) fibroblast-to-cardiomyocyte reprogramming and (B) fibroblast gene expression. With respect to fibroblast-to-cardiomyocyte reprogramming, cardiac fibroblasts were transfected with either miR combo or a non-targeting control miR. 24 h later, the cells were incubated with vehicle, a TLR2 pharmacological inhibitor, a TLR4 pharmacological inhibitor, or a combination of both pharmacological inhibitors for 3 h. After incubation with the indicated pharmacological inhibitors, Hb was added (5 mg/mL) to the media. All media was replaced the next day. Fourteen days after miR transfection, cells were analyzed for expression of the indicated cardiomyocyte specific genes by qPCR. Expression values were normalized to the housekeeping gene Gapdh. N = 6–10. One-sample t tests were used to compare groups to the control group (∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05; ns, not significant). t test was used to determine the significance between the miR combo groups (## p < 0.01, # p < 0.05; ns, not significant). With respect to fibroblast gene expression, cardiac fibroblasts were incubated with vehicle, a TLR2 pharmacological inhibitor, a TLR4 pharmacological inhibitor, or a combination of both pharmacological inhibitors for 3 h. After incubation with the indicated pharmacological inhibitors, Hb was added (5 mg/mL) to the media. All media was replaced the next day. Expression of the indicated fibroblast-specific genes was determined by qPCR and normalized to the housekeeping gene Gapdh. N = 10. One-sample t tests were used to compare groups to the control group (∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05; ns, not significant).

Article Snippet: Hb (Millipore Sigma, H2625), TLR2 signaling inhibitor-TL2-C29 (InvivoGen, catalog no. inh-c29), and TLR4 inhibitor-CLI-095 (InvivoGen, catalog no. tlrl-cli95-4) were used.

Techniques: Gene Expression, Transfection, Control, Incubation, Expressing

Journal: bioRxiv

Article Title: Liver sinusoidal endothelial cells integrate metabolic and immune signals for MAPK-dependent BMP6 regulation and hepcidin induction

doi: 10.64898/2026.05.07.723498

Figure Lengend Snippet: (A) Bmp6 mRNA expression in primary LSECs treated with 5 ng/mL LPS or 2.5 μM heme for 6 h in the presence or absence of hepatocyte-conditioned medium. (B) Bmp6 mRNA expression in primary LSECs treated with various TLR ligands: Pam3CSK4 (TLR1/2), PGN (TLR2), Poly I:C (TLR3), LPS (TLR4), FLA-ST (TLR5), FSL1 (TLR2/6), R848 (TLR7/8), and ODN (TLR9) or vehicle control (NT, non-treated) for 6 h. (C-D) Bmp6 mRNA expression in primary LSECs treated with increasing concentrations of LPS for 6 h or with 5 ng/mL LPS for 2, 4, and 6 h. (E) Bmp6 mRNA expression in LSECs treated with 2.5 μM heme or protoporphyrin IX (PPIX) for 6 h. (F-G) Bmp6 mRNA expression in primary LSECs treated with increasing concentrations of heme for 6 h or treated with 2.5 μM heme for 2, 4, or 6 h. (H) Bmp6 mRNA expression in primary LSECs pre-treated with TAK242 (5 μM) or DMSO for 1 h, followed by 5 ng/mL LPS or 2.5 μM heme treatment for 6 h. (I) Transcription factor activity analysis by RNA-seq of LSECs treated with heme with respect to untreated controls (contrast-wise), in presence of hepatocyte-conditioned medium. (J) Bmp6 mRNA expression in primary LSECs pre-treated with CHX (5 μM) or DMSO for 1 h, followed by 5 ng/mL LPS treatment for 6 h. Cell culture experiments, except those in panel A, were always conducted in the presence of hepatocyte-conditioned medium. Gene expression levels were assessed by RT-qPCR, normalized to the housekeeping gene Rpl19 , and expressed as fold change relative to vehicle-treated controls. The dashed line (ut) represents the mRNA expression of LSECs treated with the conditions shown, in the absence of LPS or heme. Data are obtained from three or four independent experiments and displayed as mean ± SD. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA or Student’s t-test. CM, conditioned medium; PPIX, protoporphyrin IX; CHX, cycloheximide.

Article Snippet: The TLR ligands Pam3CSK4 (TLR2:1) (#tlrl-pms), PGN-SA (TLR2) (#tlrl-pgns2), Poly I:C (TLR3) (#tlrl-picw), FLA-ST (TLR5) (#tlrl-stfla), FSL1 (TLR2:TLR6) (tlrl-fsl), R848 (TLR7:8) (#tlrl-r848-1), ODN (TLR9) (#tlrl-1826) and the MAPK inhibitor SP600125 (#tlrl-sp60) were purchased form Invivogen and diluted in PBS (TLR ligands) or DMSO (SP600125).

Techniques: Expressing, Control, Activity Assay, RNA Sequencing, Cell Culture, Gene Expression, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Liver sinusoidal endothelial cells integrate metabolic and immune signals for MAPK-dependent BMP6 regulation and hepcidin induction

doi: 10.64898/2026.05.07.723498

Figure Lengend Snippet: (A) Bmp6 mRNA expression in primary LSECs treated with 5 ng/mL LPS or 2.5 μM heme for 6 h in the presence or absence of hepatocyte-conditioned medium. (B) Bmp6 mRNA expression in primary LSECs treated with various TLR ligands: Pam3CSK4 (TLR1/2), PGN (TLR2), Poly I:C (TLR3), LPS (TLR4), FLA-ST (TLR5), FSL1 (TLR2/6), R848 (TLR7/8), and ODN (TLR9) or vehicle control (NT, non-treated) for 6 h. (C-D) Bmp6 mRNA expression in primary LSECs treated with increasing concentrations of LPS for 6 h or with 5 ng/mL LPS for 2, 4, and 6 h. (E) Bmp6 mRNA expression in LSECs treated with 2.5 μM heme or protoporphyrin IX (PPIX) for 6 h. (F-G) Bmp6 mRNA expression in primary LSECs treated with increasing concentrations of heme for 6 h or treated with 2.5 μM heme for 2, 4, or 6 h. (H) Bmp6 mRNA expression in primary LSECs pre-treated with TAK242 (5 μM) or DMSO for 1 h, followed by 5 ng/mL LPS or 2.5 μM heme treatment for 6 h. (I) Transcription factor activity analysis by RNA-seq of LSECs treated with heme with respect to untreated controls (contrast-wise), in presence of hepatocyte-conditioned medium. (J) Bmp6 mRNA expression in primary LSECs pre-treated with CHX (5 μM) or DMSO for 1 h, followed by 5 ng/mL LPS treatment for 6 h. Cell culture experiments, except those in panel A, were always conducted in the presence of hepatocyte-conditioned medium. Gene expression levels were assessed by RT-qPCR, normalized to the housekeeping gene Rpl19 , and expressed as fold change relative to vehicle-treated controls. The dashed line (ut) represents the mRNA expression of LSECs treated with the conditions shown, in the absence of LPS or heme. Data are obtained from three or four independent experiments and displayed as mean ± SD. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA or Student’s t-test. CM, conditioned medium; PPIX, protoporphyrin IX; CHX, cycloheximide.

Article Snippet: The TLR ligands Pam3CSK4 (TLR2:1) (#tlrl-pms), PGN-SA (TLR2) (#tlrl-pgns2), Poly I:C (TLR3) (#tlrl-picw), FLA-ST (TLR5) (#tlrl-stfla), FSL1 (TLR2:TLR6) (tlrl-fsl), R848 (TLR7:8) (#tlrl-r848-1), ODN (TLR9) (#tlrl-1826) and the MAPK inhibitor SP600125 (#tlrl-sp60) were purchased form Invivogen and diluted in PBS (TLR ligands) or DMSO (SP600125).

Techniques: Expressing, Control, Activity Assay, RNA Sequencing, Cell Culture, Gene Expression, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Liver sinusoidal endothelial cells integrate metabolic and immune signals for MAPK-dependent BMP6 regulation and hepcidin induction

doi: 10.64898/2026.05.07.723498

Figure Lengend Snippet: (A) Bmp6 mRNA expression in primary LSECs treated with 5 ng/mL LPS or 2.5 μM heme for 6 h in the presence or absence of hepatocyte-conditioned medium. (B) Bmp6 mRNA expression in primary LSECs treated with various TLR ligands: Pam3CSK4 (TLR1/2), PGN (TLR2), Poly I:C (TLR3), LPS (TLR4), FLA-ST (TLR5), FSL1 (TLR2/6), R848 (TLR7/8), and ODN (TLR9) or vehicle control (NT, non-treated) for 6 h. (C-D) Bmp6 mRNA expression in primary LSECs treated with increasing concentrations of LPS for 6 h or with 5 ng/mL LPS for 2, 4, and 6 h. (E) Bmp6 mRNA expression in LSECs treated with 2.5 μM heme or protoporphyrin IX (PPIX) for 6 h. (F-G) Bmp6 mRNA expression in primary LSECs treated with increasing concentrations of heme for 6 h or treated with 2.5 μM heme for 2, 4, or 6 h. (H) Bmp6 mRNA expression in primary LSECs pre-treated with TAK242 (5 μM) or DMSO for 1 h, followed by 5 ng/mL LPS or 2.5 μM heme treatment for 6 h. (I) Transcription factor activity analysis by RNA-seq of LSECs treated with heme with respect to untreated controls (contrast-wise), in presence of hepatocyte-conditioned medium. (J) Bmp6 mRNA expression in primary LSECs pre-treated with CHX (5 μM) or DMSO for 1 h, followed by 5 ng/mL LPS treatment for 6 h. Cell culture experiments, except those in panel A, were always conducted in the presence of hepatocyte-conditioned medium. Gene expression levels were assessed by RT-qPCR, normalized to the housekeeping gene Rpl19 , and expressed as fold change relative to vehicle-treated controls. The dashed line (ut) represents the mRNA expression of LSECs treated with the conditions shown, in the absence of LPS or heme. Data are obtained from three or four independent experiments and displayed as mean ± SD. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA or Student’s t-test. CM, conditioned medium; PPIX, protoporphyrin IX; CHX, cycloheximide.

Article Snippet: The TLR ligands Pam3CSK4 (TLR2:1) (#tlrl-pms), PGN-SA (TLR2) (#tlrl-pgns2), Poly I:C (TLR3) (#tlrl-picw), FLA-ST (TLR5) (#tlrl-stfla), FSL1 (TLR2:TLR6) (tlrl-fsl), R848 (TLR7:8) (#tlrl-r848-1), ODN (TLR9) (#tlrl-1826) and the MAPK inhibitor SP600125 (#tlrl-sp60) were purchased form Invivogen and diluted in PBS (TLR ligands) or DMSO (SP600125).

Techniques: Expressing, Control, Activity Assay, RNA Sequencing, Cell Culture, Gene Expression, Quantitative RT-PCR

Journal: Nature Communications

Article Title: A tissue-intrinsic mechanism sensitizes HIV-1 particles for TLR-triggered innate immune responses

doi: 10.1038/s41467-026-72586-3

Figure Lengend Snippet: a Schematic representation of PRRs and their cognate ligands. PRRs: green, adapter proteins: pink, PAMPs: red, transcription factors: blue. b PRR inhibitor screening. MDMs were challenged with S or DC HIV-1 NL4.3 R5 virions in presence or absence of PRR inhibitors. IL-6 release was measured by ELISA from MDM supernatants. Data is normalized to mock infected condition (gray dotted line). c Measurement of IL-6 release from the supernatants of MDMs challenged with S or DC HIV-1 virions in presence or absence of the indicated inhibitors. d Volcano plot illustrating the differential gene expression between MDMs challenged with DC virus and mock challenged cells for 6 h. The number of down- and upregulated genes are indicated. e Volcano plot as in d. with highlighted gene signatures. Genes induced by TLR2 alone (blue), TLR8 alone (brown), co-regulated (light green) or associated with “Innate Immune Response” genes (red). f Volcano plot illustrating the differential gene expression between MDMs challenged with suspension or collagen primed HIV-1 virus for 6 h. The number of down- and upregulated genes are indicated. g Volcano plot as in f. with highlighted gene signatures. Genes induced by TLR2 alone (blue), TLR8 alone (brown), co-regulated (light green) or associated with Innate Immune Response genes (red). Top deregulated genes belonging to different gene ontology terms are highlighted. h Gene ontology analysis of biological processes repressed or induced in MDMs challenged with collagen primed virions as compared to cells challenged with suspension virus for 6 h. FDR is indicated (only upregulated pathways have a q -value < 0.05). Top deregulated genes for each pathway are highlighted. Results represent the mean ± SD of 3 independent donors. Symbols indicate individual donors ( b , c ) or individual genes ( d–g ). Significance is indicated by p-values, and was calculated by two-tailed paired t-tests or Wilcoxon matched-pairs signed rank test ( b , c ), differential gene expression analysis was performed using DESeq2: genes with adjusted p-value < 0.05 were considered significant ( d–g ), for GSEA analysis, p -values were corrected using Benjamini-Hochberg FDR ( h ). Source data are provided as a file.

Article Snippet: 5 μM of cGAS inhibitor (G140, Invivogen), 8 μM of TLR 1/2 inhibitor (Cu-CPT22, Selleckchem), 49 μM of TLRs 4& 2/6 inhibitor (GIT27, Tocris), 2 μM of TLR4 inhibitor (CLI-095, Invivogen), 100 μM of TLR2 inhibitor (TL2-C29, Invivogen) or 10 μM of TLR8 inhibitor (Cu-CPT9a, Invivogen) were incubated with MDMs 3 h prior to infection.

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Gene Expression, Virus, Suspension, Two Tailed Test

Journal: Nature Communications

Article Title: A tissue-intrinsic mechanism sensitizes HIV-1 particles for TLR-triggered innate immune responses

doi: 10.1038/s41467-026-72586-3

Figure Lengend Snippet: a Representative structure of an HIV-1 Env trimer. Epitopes targeted by different bNAbs or nNAbs as well as sCD4 are highlighted (based upon a prediction of NL4.3 Env trimer structure using AlphaFold 3). b Experimental workflow. HIV-1 NL4.3 R5 Vpr.Int.GFP virions harvested from suspension or collagen cultures were sequentially incubated with primary, epitope-specific anti-gp120 antibodies, then with AF568 coupled secondary antibodies prior to processing for microscopy analysis. c . Representative micrographs of stained virions. Vpr.Int.GFP positive spots (green) can be seen bound by different antibodies or sCD4-PE (red). Scale bar: 0.5 µm. d Spider plot representing the binding frequency of antibody binding to HIV-1 NL4.3 R5 as in (c). e . sTLR2 is retained at the surface of NL4.3 R5 but not NL4.3 ΔEnv virions after collagen priming. One representative Western Blot analysis showing the ratio from HIV-1 NL4.3 virions cultured in S, DC or LC. The TLR2/p24 ratios are indicated below the blots after correction, after subtraction of the TLR2 background signals from the last two lanes. f Quantification of western blot analyses from three independent experiments as in e . g . Representative micrographs. MDMs challenged with HIV1 NL4.3 R5 Vpr.Int.GFP virions after culture in suspension or in DC. Yellow arrows indicate Vpr.Int.GFP/TLR8 colocalization. Scale bar: 5 µm. h Quantification of Virus/TLR8 colocalization from micrographs as in d. Results represent the mean ± SD of three independent experiments. Significance is indicated by p -values, and was calculated by two-tailed paired t-tests ( d , f , h ). Source data are provided as a file.

Article Snippet: 5 μM of cGAS inhibitor (G140, Invivogen), 8 μM of TLR 1/2 inhibitor (Cu-CPT22, Selleckchem), 49 μM of TLRs 4& 2/6 inhibitor (GIT27, Tocris), 2 μM of TLR4 inhibitor (CLI-095, Invivogen), 100 μM of TLR2 inhibitor (TL2-C29, Invivogen) or 10 μM of TLR8 inhibitor (Cu-CPT9a, Invivogen) were incubated with MDMs 3 h prior to infection.

Techniques: Suspension, Incubation, Microscopy, Staining, Binding Assay, Western Blot, Cell Culture, Virus, Two Tailed Test

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a ) Plate-based epithelial single cell re-analysis of Toll-like receptors (TLRs). Dot plot of 11 murine TLRs expressed in different IECs under homeostasis, taken from a publicly available dataset . The dot size indicates the proportion of cells expressing a gene, and the color indicates the mean expression levels. ( b ) Representative flow-cytometry plot of Lgr5+-ISC population in Lgr5-GFP mouse model (left) and Relative quantification (RQ) of Tlr2 expression in murine Lgr5 + cells under homeostasis using quantitative PCR (qPCR) (Right). Data are presented as mean ± SEM. n =6 mice per condition, * p <0.05, two-tailed Student’s t-test . ( c-d ) Tlr2 expression on ileal Lgr5 + -ISC under homeostasis. (c) Co-localization of Lgr5 and Tlr2 molecules in ISCs. Single-molecule fluorescence in situ hybridization (smFISH) of Tlr2 (green) and Lgr5 (red), co-stained with E-Cadherin and DAPI (blue). E-Cadherin stains IEC boundaries, and DAPI stains nuclei. Scale bar, 20μm. Single channel of each color (red or green) or merged images (lower panels); WT crypt (left panels) and TLR2 KO crypt (right panels). Insets x2 magnification. (d) Experimental scheme of the different segments of the small intestine for villi and crypts isolation (left) and RQ of Tlr2 expression levels using qPCR (right). Data are presented as mean ± SEM. n =3 mice per region; ns (non-significant), * p <0.05, ** p <0.01, one-way ANOVA . ( e ) Primary mouse SI spheroids and organoids from ileal crypts. Representative pictures of stem-enriched spheroids (upper panel) and differentiated organoids (lower panel) (left). Scale bar, 500μm. RQ of Tlr2 and Lgr5 expression levels in spheroids and organoids using qPCR (right). Data are presented as mean ± SEM. n =3 biological repetitions for spheroids or 2 for organoids, * p <0.05, two-tailed Student’s t-test . ( f ) RQ of Cxcl2 and Tnfα expression levels in spheroids generated from WT vs. TLR2 cKO mice after 4h or 8h of ex vivo activation with TLR2/TLR1 agonist (Pam3Csk4, 1μg/mL) or without (control) using qPCR. Data are presented as mean ± SEM. n =3 biological repetitions, * p <0.05, *** p <0.001, two-way ANOVA. ( g ) Heatmap of top TLR2 target genes of bulk RNA-seq from WT vs TLR2 cKO stem-enriched spheroids after 4h of activation with or without Pam3Csk4 (1μg/mL). Mean Z score of log 2 (TPM+1), color bar. n =3 biological repetitions for WT and 2 for TLR2 cKO. ( h ) NFĸB pathway activation. Representative images of immunofluorescence assay (IFA) of NFĸB subunit (p65, red) and DAPI of stem-enriched spheroids in WT (top panels) or TLR2 cKO (lower panels) at time 0 or 1 hour after Pam3Csk4 induction (1μg/mL). Scale bar, 20μm. Insets, x2 magnification. Arrows show p65 localization to the nucleus ( i ). Tlr2 activation of Lgr5+-ISC ex vivo scheme (upper panel). Heatmap of TLR2 target genes (inflammatory) and canonical NFĸB targets from sorted WT Lgr5+-ISCs after 4h of ex vivo activation with or without Pam3Csk4 (1μg/mL) by bulk RNA-seq. Average relative expression (mean Z score of log 2 (TPM+1), color bar). n =3 biological repetitions for control and 2 for Pam3Csk4 activation.

Article Snippet: For TLR2 stimulation, the TLR2 agonist Pam3Csk4 (1μg/mL, InvivoGen) was added to the growth medium 4 and 8 hours prior to RNA harvesting.

Techniques: Single Cell, Expressing, Flow Cytometry, Quantitative Proteomics, Real-time Polymerase Chain Reaction, Two Tailed Test, Fluorescence, In Situ Hybridization, Staining, Isolation, Generated, Ex Vivo, Activation Assay, Control, RNA Sequencing, Immunofluorescence

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a ) A representative flow cytometry gating strategy for epithelial cells. ( b ) Relative quantification (RQ) of Cxcl2 and Tnfα expression levels in spheroids vs. organoids generated from WT mice after 4h or 8h of ex vivo activation with TLR2/TLR1 agonist (Pam3Csk4, 1μg/mL) or without (control) using qPCR. Data are presented as mean ± SEM. n =3 biological repetitions per group, * p <0.05, *** p <0.001, **** p < 0.0001, two-way ANOVA. ( c ) Relative quantification (RQ) of Tlr2 expression in spheroids generated from WT or TLR2 cKO mice after 4h or 8h of ex vivo activation with TLR2/TLR1 agonist (Pam3Csk4, 1μg/mL) or without (control) using qPCR. Data are presented as mean ± SEM. n =3 biological repetitions per group, except for the control time 0 with 2 biological repetitions, * p <0.05, ** p <0.01, *** p <0.001, two-way ANOVA. ( d ) NFĸB pathway activation. Representative images of IFA of NFĸB subunit (p65, red) and DAPI in WT (upper panel) and TLR2 cKO (lower panel) stem-enriched spheroids after 30 minutes of Pam3Csk4 (1μg/mL) induction. Scale bar, 20μm. Insets, x2 magnification. ( e ) Relative quantification (RQ) of Cxcl2 and Tnfα expression levels in isolated Lgr5 + ileal stem cells from WT mice after 4h of ex vivo activation with TLR2/TLR1 agonist (Pam3Csk4, 1μg/mL) or without (control) using qPCR. Data are presented as mean ± SEM. n =3 biological repetitions, * p <0.05, two-tailed Student’s t-test .

Article Snippet: For TLR2 stimulation, the TLR2 agonist Pam3Csk4 (1μg/mL, InvivoGen) was added to the growth medium 4 and 8 hours prior to RNA harvesting.

Techniques: Flow Cytometry, Quantitative Proteomics, Expressing, Generated, Ex Vivo, Activation Assay, Control, Isolation, Two Tailed Test

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a ) Polarized monolayer generation from primary mouse SI spheroids scheme. ( b ) Representative images of primary mouse SI polarized monolayers using H&E staining (upper panel); scale bar, 75μm. IFA of primary monolayer (mid) or top of the villus (lower) stained with Villin (red), CD138 (green), and DAPI (blue); scale bar, 20μm. ( c ) Schematic representation of monolayer integrity assay, with FITC-dextran permeability quantification (left) or transepithelial electrical resistance (TEER) measurements (right) over 30 days of primary monolayer cultures. ( d-e ) Relative quantification (RQ) of TLR2 target genes, Cxcl2 and Tnfα expression levels in stem-enriched monolayers after 4h or 24h of ex vivo apical (d) and basolateral (e) activation with TLR2/TLR1 agonist (Pam3Csk4, 1μg/mL) or without (control) using qPCR. Data are presented as mean ± SEM. n =5 biological repetitions per condition, ns (non-significant), * p <0.05, one-way ANOVA. ( f ) Heatmap of top TLR2 target genes from WT vs TLR2 cKO stem-enriched monolayers after 4h of ex vivo apical activation with or without Pam3Csk4 induction (1μg/mL), tested by bulk RNA-seq. Average relative expression (mean Z score of log 2 (TPM+1), color bar). n =3 vs 2 for WT or 3 vs 3 for TLR2 cKO biological repetitions for monolayers with or without Pam3Csk4 induction for 4h, respectively.

Article Snippet: For TLR2 stimulation, the TLR2 agonist Pam3Csk4 (1μg/mL, InvivoGen) was added to the growth medium 4 and 8 hours prior to RNA harvesting.

Techniques: Staining, Integrity Assay, Permeability, Quantitative Proteomics, Expressing, Ex Vivo, Activation Assay, Control, RNA Sequencing

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a ) Relative quantification (RQ) of Tlr2 expression in stem-enriched monolayers derived from WT vs TLR2 cKO using qPCR. Data are presented as mean ± SEM. n =3 biological repetitions, ** p <0.01, two-tailed Student’s t-test . ( b ) Monolayer barrier integrity assay using TEER measurements of WT or TLR2 cKO. Data are presented as mean ± SEM. n =3 biological repetitions; *** p <0.001, **** p < 0.0001, two-tailed Student’s t-test . ( c ) Relative quantification (RQ) of Cxcl2 expression levels in stem-enriched monolayers from WT or TLR2 cKO mice after 4h of activation with TLR2/TLR1 agonist (Pam3Csk4, 1μg/mL) or without (control) using qPCR. Data are presented as mean ± SEM; n =3 biological repetitions; ns , ** p <0.01, two-way ANOVA.

Article Snippet: For TLR2 stimulation, the TLR2 agonist Pam3Csk4 (1μg/mL, InvivoGen) was added to the growth medium 4 and 8 hours prior to RNA harvesting.

Techniques: Quantitative Proteomics, Expressing, Derivative Assay, Two Tailed Test, Integrity Assay, Activation Assay, Control

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a ) Representative H&E images of SI ileum sections from WT, TLR2 cKO, or TLR4 cKO mice; scale bars, 20μm; yellow arrows show the changes in granular cells at the bottom of the crypt in TLR2 cKO mice. ( b-d ) Comparison of Lgr5 + -ISCs from WT or TLR2 cKO mice using bulk-RNA sequencing. (b) Experimental scheme for GFP-labeled Lgr5 + -ISC sort (top) and volcano plot showing log2fc estimates and -log(FDR q-value) of top ten upregulated (right) and downregulated (left) differentially expressed genes (DEG) in Lgr5 + -ISCs of TLR2 cKO ( n =2) compared to WT ( n =3) mice. Colored points correspond to padj ≤ 0.05 and |log2FoldChange| ≥ 1. Color coding is shown in the bottom-right corner. (c) Gene ontogeny (GO) biological processes showing hypergeometric enrichment of significantly upregulated and downregulated DEGs in Lgr5+-ISCs of TLR2 cKO ( n =2) vs. WT ( n =3) mice. Plotted is the negative log10 of enrichment padj. The direction of enrichment is shown as positive for biological processes enriched in TLR2 cKO, and negative for those enriched in WT. (d) Heatmap of the top DE antimicrobial peptides from WT ( n =3) vs. TLR2 cKO ( n =2) mice. Average relative expression (mean Z score of log 2 (TPM+1), color bar). ( e-g ) Goblet-Paneth intermediate cells accumulation is observed in TLR2 cKO. (e) Representative images of Periodic-Acid Schiff (PAS) and Alcian Blue (AB) staining of ileal sections from WT (top), TLR2 cKO (mid), and TLR2 cKO chimeric (CH, bottom) mice. The number of Paneth cells and goblet-Paneth intermediate cells per crypt was quantified (right); n = 10 fields per mouse, 2 mice per group; data are mean ± SEM; *** p < 0.001, one-way ANOVA. scale bar, 50μm. Inset, x4 magnification. (f) RQ of Lyz1 and Mptx2 expression levels in WT vs. TLR2 cKO using qPCR. Data are presented as mean ± SEM. n =5 WT vs 4 of TLR4 cKO mice, * p <0.05, two-tailed Student’s t-test . (g) Transmission electron microscopy (TEM) of Paneth cell eosinophilic granules in SI ileal segments of WT (left) and TLR2 cKO (right) mice; Scale bar, 5μm. (h) Mature Paneth cells staining in SI ileal crypts of WT (left), TLR2 cKO (mid), or TLR2 cKO chimera (CH, right). Single-molecule fluorescence in situ hybridization (smFISH) of Mptx2 (green) co-stained with Lyz1 antibody (red). E-Cadherin stains IEC boundaries, while DAPI stains the nucleus (blue). Scale bar, 20μm. Insets, x1.4 magnification. Lyz1+ Mptx2 + Paneth cells per crypt were quantified (right); n =10 fields per mouse, 2 mice per group. Data are presented as mean ± SEM. ns , non-significant, ** p <0.01, one-way ANOVA.

Article Snippet: For TLR2 stimulation, the TLR2 agonist Pam3Csk4 (1μg/mL, InvivoGen) was added to the growth medium 4 and 8 hours prior to RNA harvesting.

Techniques: Comparison, RNA Sequencing, Labeling, Expressing, Staining, Two Tailed Test, Transmission Assay, Electron Microscopy, Fluorescence, In Situ Hybridization

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a-b ) Representative flow cytometry plot (a), and quantification of the percentage of Lgr5 + ISC GFP high (left) and GFP low (right) EpCAM + cells from ileal crypts in WT or TLR2 cKO (b). Data are presented as mean ± SEM. n =3 mice per group; * p < 0.05, ** p < 0.01, two-tailed Student’s t-test. ( c ) schematic representation of the TLR2 KO chimeric mice model. TLR2 cKO mice were irradiated with a 500 Rad dose twice, followed by bone marrow transplantation (BMT) from GFP/CD45.1 mice 24h later. ( d-e ) Confirmation of BMT in the TLR2 cKO chimeric mouse model. (d) Representative flow cytometry plot of GFP-labeled immune cells in the LP. (e) immunofluorescence assay (IFA) of GFP (green), E-cadherin (blue), and DAPI from TLR2 cKO or chimeric TLR2 cKO (TLR2 cKO CH) mice. E-Cadherin (blue) stains IEC boundaries. Scale bar, 20μm. ( f ) Mptx2 staining in Paneth cells from SI ileal crypts of WT (left), TLR2 cKO (mid), and chimeric TLR2 cKO (TLR2 cKO CH). IFA of Mptx2 (green), E-Cadherin (blue), and DAPI. E-Cadherin (blue) stains IEC boundaries. Scale bar, 20μm. Cells with Mptx2+ granules were quantified per crypt (right); 10 crypts per mouse. Data are presented as mean ± SEM. **** p < 0.0001, one-way ANOVA.

Article Snippet: For TLR2 stimulation, the TLR2 agonist Pam3Csk4 (1μg/mL, InvivoGen) was added to the growth medium 4 and 8 hours prior to RNA harvesting.

Techniques: Flow Cytometry, Two Tailed Test, Irradiation, Transplantation Assay, Labeling, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a ) Two-dimensional Uniform Manifold Approximation and Projection (UMAP) graphical representation of EpCAM + single cell RNA-sequencing (scRNA-seq), colored by graph clustering per different clusters from chimeric (CH) WT and TLR2-KO mice (left) and stacked bar of relative cell subset abundance in chimeric (CH) WT and TLR2 cKO (right). Data are presented as mean ± SEM. n =2 mice per condition. TA, transient amplifying cells; EEC, enteroendocrine. ( b ) Box-and-whisker plot of cell cycle signature score in chimeric (CH) WT vs TLR2 cKO. n =2 mice per condition. Boxes represent median and Interquartile Range (IQR); whiskers extend to 1.5× IQR. Mann-Whitney U test; **** p <0.0001. ( c ) Diffusion pseudotime (dpt) score comparing the differentiation state of chimeric (CH) WT (blue) and TLR2 cKO (orange). Dpt scores showing a right-shift peak in TLR2 cKO cells, indicating accelerated differentiation compared to WT. ( d ) Diffusion map of enterocytes differentiation trajectory colored by cell subset in chimeric (CH) WT and TLR2 cKO (left) and diffusion pseudotime (dpt) score comparing the enterocytes differentiation state of chimeric (CH) WT (blue) and TLR2 cKO (orange) (right). Dpt scores showing a right-shift peak in TLR2 cKO cells, indicating accelerated differentiation compared to WT. ( e ) Dot plot of immature and mature enterocyte cell markers comparing enterocyte subsets of chimeric (CH) WT (blue) vs. TLR2 cKO (orange) mice. The size of the dot indicates the proportion of cells expressing a gene, while the color indicates the mean expression level. ( f ) Volcano plot showing log2fc estimates and the negative log10 of enrichment padj. of top upregulated (right) and downregulated (left) DEG in mature enterocytes of chimeric (CH) WT ( n =2) compared to TLR2 cKO ( n =2) chimeras. Colored points correspond to padj ≤ 0.05 and |log2FoldChange| ≥ 1. Color coding is shown on the top left. ( g ) Distribution of proximal (left) and distal (right) enterocyte signatures in mature enterocytes of chimeric (CH) WT (blue) vs. TLR2 cKO (orange) mice; Significance was determined by Pearson’s r test, **** p <0.0001. ( h ) Scatter plot comparing Pearson correlation coefficients (r) of proximal and distal enterocyte gene signatures in chimeric (CH) WT and TLR2 cKO mature enterocyte cells. The x-axis represents the Pearson r values for proximal enterocyte signature genes, and the y-axis shows Pearson r values for distal enterocyte signature genes. Each point corresponds to a cell within the respective signature. The colors represent different conditions: blue for WT and orange for TLR2 cKO. ( i-j ) Antimicrobial peptides expression in Paneth cells. (i) Dot plot of top DEG in Paneth cells from chimeric (CH) WT vs. TLR2 cKO mice. The size of the dot indicates the proportion of cells expressing a gene, while the color indicates the average relative expression (mean Z score of log2(TPM+1)). (j) Reduction of AMP expressed by Paneth cells in TLR2 cKO. IFA of Lyz1 (green), Mptx2 (red), E-Cadherin, and DAPI co-staining in chimeric (CH) WT (left) and TLR2 cKO (right) mice. (k) Uncharacterized top AMP-encrypted candidates in mature enterocytes derived from DEG upregulated in chimeric WT compared to TLR2 cKO. X-axis: gene list, Y-axis: absolute Torres score.

Article Snippet: For TLR2 stimulation, the TLR2 agonist Pam3Csk4 (1μg/mL, InvivoGen) was added to the growth medium 4 and 8 hours prior to RNA harvesting.

Techniques: Single Cell, RNA Sequencing, Whisker Assay, MANN-WHITNEY, Diffusion-based Assay, Expressing, Staining, Derivative Assay

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a ) Epithelial cell subset markers used for cluster annotation. Dot plot of five known markers per epithelial cell subset derived from publicly available data . The size of the dot indicates the proportion of cells expressing a gene, while the color indicates the mean expression level. TA, transit amplifying; E. Immature, immature enterocyte; E. Mature, mature enterocyte; E. Mature T, mature top-villus enterocytes; G/P Progenitors, goblet-Paneth progenitors; EEC, enteroendocrine. ( b ) Box-and-whisker plot of cell cycle signature score in different epithelial clusters from chimeric (CH) WT (blue) vs TLR2 cKO (orange) mice. n =2 per condition. EEC, enteroendocrine. Boxes represent median and Interquartile Range (IQR); whiskers extend to 1.5× IQR; ns (non-significant), * p < 0.05, ** p <0.01, *** p <0.001, Wilcox-test . ( c ) Volcano plot showing log2fc estimates and the negative log10 of enrichment padj. of top upregulated in WT (blue, right) and downregulated (orange, left) DEG in stem cells compared to TLR2 cKO ( n =2 mice per condition). Colored points correspond to padj ≤ 0.05 and |log2FoldChange| ≥ 1. Color coding is shown in the top-right corner. ( d ) Proliferation analysis in SI ileal crypts of WT (left panel), TLR2 cKO (middle panel), and TLR2 cKO chimera (right panel). IFA of Ki67 (white), Olfm4 (green), E-Cadherin (blue), and DAPI. E-cadherin (blue) stains IEC boundaries. Scale bar, 20μm. Ki67 + Cells were quantified per crypt (right); 10 crypts per mouse. Data are presented as mean ± SEM. * p <0.05, one-way ANOVA. ( e ) Apoptosis analysis in SI ileal crypts of positive control (24h post-irradiation, left), chimeric (CH) WT (middle), or TLR2 cKO (right) mice. IFA of Cleaved caspase 3 (CC3) (red), Ki67 (green), E-cadherin (white) and DAPI. E-cadherin (white) stains IEC boundaries. Yellow arrows show CC3+ cells. Scale bar, 20μm. ( f ) Diffusion map of differentiation trajectory to the main epithelial lineages colored by cell subset (left) or by cell cycle state (right) in chimeric (CH) WT and TLR2 cKO. Color coding is shown on the right. TA, transit amplifying; EEC, Enteroendocrine. ( g ) Diffusion pseudotime (dpt) score comparing the enterocyte subsets differentiation state of chimeric (CH) WT (blue) and TLR2 cKO (orange). Dpt scores showing an accelerated differentiation in TLR2 cKO CH. ( h ) Distribution of Antimicrobial humoral immune response mediated by antimicrobial peptide-signature score in Paneth cells from chimeric WT vs. TLR2 cKO; significance was determined by Wilcox-test, **** p <0.0001. ( i ) Top AMP-encrypted candidates in Paneth cells derived from DEG upregulated in WT CH compared to TLR2 cKO CH. X-axis: gene list, Y-axis: absolute Torres score. ( j ) Distribution of proximal (left) and distal (right) Paneth signatures in Paneth cells of chimeric (CH) WT (blue) vs. TLR2 cKO (orange) mice; Significance was determined by Pearson’s r test, ** p <0.01, **** p <0.0001.

Article Snippet: For TLR2 stimulation, the TLR2 agonist Pam3Csk4 (1μg/mL, InvivoGen) was added to the growth medium 4 and 8 hours prior to RNA harvesting.

Techniques: Derivative Assay, Expressing, Whisker Assay, Positive Control, Irradiation, Diffusion-based Assay

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a-c ) Epithelial-specific TLR2 deletion (TLR2 ΔIEC ) mouse model. (a) Experimental scheme for the generation of epithelial-specific TLR2 deletion by crossing Vil-Cre to TLR2 fl/fl mouse model (TLR2 ΔIEC ). (b) Representative images of PAS and AB staining of ileum sections from TLR2 fl/fl (upper panel) and TLR2 ΔIEC (lower panel). scale bar: 20 μm. Insets, x5 magnification. (c) AMP (Lyz1 and Mptx2) expressed by Paneth cells staining in TLR2 fl/fl (left) and TLR2 ΔIEC (right) ileal sections. IFA of Lyz1 (green), Mptx2 (red), E-cadherin (white) and DAPI. E-cadherin (white) stains IEC boundaries; scale bar: 20 μm. ( d-g ) Lgr5+-ISC-specific TLR2 deletion (TLR2 ΔISC ) mouse model. (d) Schematic representation of Lgr5+-ISC-specific TLR2 deletion lineage tracing (TLR2 DISC+tdTom ) generation by crossing Lgr5+-ISC-CreER T -TLR2 fl/fl mice to Rosa26-tdTomato mice. TdTomato cells are progeny of TLR2-deleted Lgr5+-ISC after one injection of Tamoxifen (2mg) at 48h (d-f) or 5 days (g). (e) Representative flow cytometry plot of the percentage of TdTomato + EpCAM + cells among EpCAM + cells in TLR2 fl/fl vs. TLR2 ΔISC mice (left panel); quantification of TdTomato + EpCAM + percentages in both groups is depicted on a bar graph (right panel); n =3 mice per TLR2 fl/fl and 2 mice per TLR2 ΔISC . Data are presented as mean ± SEM. * p <0.05, two-tailed Student’s t-test . (f) IFA of tdTomato (red), Ki67 (green), E-cadherin (white), and DAPI of distal SI, 48h after Tamoxifen induction. E-cadherin (white) stains IEC boundaries; scale bar, 20 μm. (left panel). Quantification of tdTom+ cells (mid) and of Ki67+ tdTom+ cells (Right) per crypt. Data are presented as mean ± SEM. 10 fields (tdTom+) or 20 fields (tdTom+ Ki67+) per mouse. **** p < 0.001, two-tailed Student’s t-test . (g) IFA of tdTomato (red), Lyz1 (green), E-cadherin+DAPI (blue) of distal SI, 5 days after Tamoxifen induction; scale bar, 20 μm. (left panel). Quantification of tdTom+ Lyz1+ cells (right panel) per crypt. Data are presented as mean ± SEM. 20 fields per mouse and n =2 mice. * p < 0.05, two-tailed Student’s t-test .

Article Snippet: For TLR2 stimulation, the TLR2 agonist Pam3Csk4 (1μg/mL, InvivoGen) was added to the growth medium 4 and 8 hours prior to RNA harvesting.

Techniques: Staining, Injection, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a ) Representative H&E images of SI ileum sections from TLR2 fl/fl and TLR2 ΔIEC ; scale bars, 75μm; yellow arrows show the changes in granular cells at the bottom of the crypt in TLR2 ΔIEC mice. ( b ) Relative quantification (RQ) of Tlr2 expression in TLR2 fl/fl and TLR2 ΔIEC mice using qPCR. Data are presented as mean ± SEM. n =4 mice per group, ** p <0.01, two-tailed Student’s t-test. ( c ) Goblet-Paneth intermediate cells accumulation is observed in TLR2 ΔIEC . The number of Paneth (left) and goblet-Paneth intermediate (right) cells per crypt was quantified (right); 25 crypts per mouse, 2 mice per group; data are mean ± SEM; ** p < 0.01, two-tailed Student’s t-test . ( d ) TUNEL assay analysis of TLR2 ΔISC SI ileal sections shows no signs of cell death after 48h of TLR2 deletion induction. Crypts of positive control (24h post-irradiation (1000 rad), left), TLR2 fl/fl (middle), or TLR2 ΔISC (right) mice. IFA of tdTomato cells (red), BrdU (TUNEL, white), and DAPI (blue). Scale bar, 20μm. ( e ) Relative quantification (RQ) of Tlr2 expression levels in TLR2fl/fl ( n =4 mice) or TLR2 ΔISC ( n =3 mice) using qPCR (right). Data are presented as mean ± SEM; * p <0.05, two-tailed Student’s t-test . ( f ) IFA of tdTomato (red), Ki67 (white), E-cadherin and DAPI (blue) of distal SI TLR2 fl/fl or TLR2 ΔISC , 5 days after Tamoxifen induction; scale bar, 20μm. (left panel). Quantification of Ki67+ tdTomato+ cells (Right) per crypt. Data are presented as mean ± SEM. 20 crypts per mouse, 2 mice per group. * p < 0.05, two-tailed Student’s t-test .

Article Snippet: For TLR2 stimulation, the TLR2 agonist Pam3Csk4 (1μg/mL, InvivoGen) was added to the growth medium 4 and 8 hours prior to RNA harvesting.

Techniques: Quantitative Proteomics, Expressing, Two Tailed Test, TUNEL Assay, Positive Control, Irradiation

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a ) Representative flow cytometry plot (left) and quantification (right) of the percentage of CD24 + side-scattered low (SSC low ) cells among EpCAM + cells in WT vs. TLR2 cKO mice (left panel); quantification of CD24 + SSC low percentages in both groups are depicted on a bar graph (right panel); n =3 mice per group. Data are presented as mean ± SEM. ** p <0.01, two-tailed Student’s t-test . ( b ) Relative quantification (RQ) of Dclk1 expression levels in EpCAM + cells from WT or TLR2 cKO using qPCR. Data are presented as mean ± SEM. n =5 mice per group, * p <0.05, two-tailed Student’s t-test . ( c ) Heatmap of the top tuft cell markers (Tuft 1 or 2 subsets) in EpCAM + cells from WT ( n =3) vs. TLR2 cKO ( n =2) mice out of the DEG using bulk RNA-seq. Average relative expression (mean Z score of log 2 (TPM+1), color bar). ( d-e ) elevation in Tuft 1 and 2 in TLR2 deletion. (d) IFA of DCLK1 (red), CD45 (white), E-cadherin (green), and DAPI. E-Cadherin (green) stains IEC boundaries; scale bar, 20 μm. Inset, x2 magnification. (e) The number of Tuft 1 (DCLK1 + CD45 - ) and Tuft 2 (DCLK + CD45 + ) cells per villus-crypt axis was quantified. Data are presented as mean ± SEM. 10 fields per mouse, **** p < 0.0001, two-tailed Student’s t-test . ( f-g ) Pseudotime analysis for tuft cell differentiation trajectory in chimeric (CH) WT or TLR2 cKO at single cell resolution. (f) Diffusion pseudotime ( dpt ) score comparing the tuft cell differentiation state of chimeric (CH) WT (blue) and TLR2 cKO (orange). Dpt scores showing a left-shift peak in TLR2 cKO cells, indicating accelerated differentiation compared to WT. (g) Scatter plot comparing log2 fold-change between chimeric (CH) WT or TLR2 cKO (y-axis) in tuft cells and log2 fold-change between Tuft1 or Tuft2 (x-axis) clusters. Each point represents one gene. The colors represent different conditions. Highlighted genes (blue) represent Tuft-2–specific bacterial-sensing receptors.

Article Snippet: For TLR2 stimulation, the TLR2 agonist Pam3Csk4 (1μg/mL, InvivoGen) was added to the growth medium 4 and 8 hours prior to RNA harvesting.

Techniques: Flow Cytometry, Two Tailed Test, Quantitative Proteomics, Expressing, RNA Sequencing, Cell Differentiation, Single Cell, Diffusion-based Assay

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a-d ) The involvement of microbiota in TLR2 activation and Paneth differentiation. (a) Representative flow cytometry plot (left) and quantification (right) of the percentage of Lyz1 + cells among EpCAM + cells in ileal crypts of SPF vs. GF mice. n =6 mice per group; data are presented as mean ± SEM. * p < 0.05, two-tailed Student’s t-test . (b) Representative images of PAS and AB staining of ileum sections from SPF (upper panel) and GF (lower panel) mice; Scale bar, 20μm. Inset, x5 magnification. (c) Relative quantification (RQ) of Mptx2 and Reg3γ expression levels, key Paneth and enterocyte AMPs, in EpCAM + cells of GF ( n =5) vs. SPF ( n =4) mice using qPCR. Data are presented as mean ± SEM; ns (non-significant), * p <0.05, ** p <0.01, two-tailed Student’s t-test . (d) GF colonization experiment. Representative images of PAS and AB staining of ileum sections (Left) from GF (upper panel) or GF colonized with WT bacteria (lower panel); n =4 mice per group, scale bar, 20μm. Inset, x5 magnification. Quantification of Paneth cells per crypt of GF vs GF colonized with WT bacteria (right). 6 fields of view per condition; data are presented as mean ± SEM. **** p < 0.0001, two-tailed Student’s t-test . ( e-g ) Human ileal spheroids in vitro activation assay. (e) Experimental scheme of generating spheroids from human ileum biopsies and activation with TLR2/TLR1 agonist (Pam3Csk4). (f) Relative quantification (RQ) of TLR2 and IL8 expression levels in spheroids generated from ileal biopsies after 4h or 8h of ex vivo activation with TLR2/TLR1 agonist (Pam3Csk4, 1μg/mL) or without (control) using qPCR; n =4 biological repetitions per group. Data are presented as mean ± SEM. ns (non-significant), * p <0.05, ** p <0.01, *** p <0.001, one-way ANOVA. (g) Human Paneth cell expansion following Pam3Csk4 induction. IFA of LYZ1 (red), E-cadherin (blue), and DAPI in fixed differentiated spheroids after 4h, 24h, and 72h of ex vivo activation with TLR2/TLR1 agonist (Pam3Csk4, 1μg/mL) or without (control); scale bar, 50μm. Yellow arrows, LYZ1 + crypts. ( h ) Suggested model for the symbiotic relationship of the Lgr5+-ISC with microbiota mediated by TLR2 signaling.

Article Snippet: For TLR2 stimulation, the TLR2 agonist Pam3Csk4 (1μg/mL, InvivoGen) was added to the growth medium 4 and 8 hours prior to RNA harvesting.

Techniques: Activation Assay, Flow Cytometry, Two Tailed Test, Staining, Quantitative Proteomics, Expressing, Bacteria, In Vitro, Generated, Ex Vivo, Control

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a ) Relative quantification (RQ) of Lgr5 and TLR2-related genes ( Tlr2, Tlr1 and Tnfα ) expression levels in SPF ( n =4 mice) or GF ( n =5 mice) using qPCR (right). Data are presented as mean ± SEM; * p <0.05, **p<0.01, two-tailed Student’s t-test . ( b-c ) GF colonization experiment. (b) Experimental scheme of colonization experiment in GF mice. (c) Representative flow cytometry plots (left) and quantification (right) of the percentage of Lyz1 + cells in ileal crypts of GF (left) or GF colonized with WT microbiota (right). n =4 biological repetitions. Data are presented as mean ± SEM; ns (non-significant), two-tailed Student’s t-test . ( d ) droplet-based epithelial single cell re-analysis of Toll-like receptors (TLRs). Dot plot of 7 human TLRs expressed in different IECs under homeostasis, taken from a publicly available dataset . The dot size indicates the proportion of cells expressing a gene, and the color indicates the mean expression levels. TA, transient amplifying cells.

Article Snippet: For TLR2 stimulation, the TLR2 agonist Pam3Csk4 (1μg/mL, InvivoGen) was added to the growth medium 4 and 8 hours prior to RNA harvesting.

Techniques: Quantitative Proteomics, Expressing, Two Tailed Test, Flow Cytometry, Single Cell

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a ) Plate-based epithelial single cell re-analysis of Toll-like receptors (TLRs). Dot plot of 11 murine TLRs expressed in different IECs under homeostasis, taken from a publicly available dataset . The dot size indicates the proportion of cells expressing a gene, and the color indicates the mean expression levels. ( b ) Representative flow-cytometry plot of Lgr5+-ISC population in Lgr5-GFP mouse model (left) and Relative quantification (RQ) of Tlr2 expression in murine Lgr5 + cells under homeostasis using quantitative PCR (qPCR) (Right). Data are presented as mean ± SEM. n =6 mice per condition, * p <0.05, two-tailed Student’s t-test . ( c-d ) Tlr2 expression on ileal Lgr5 + -ISC under homeostasis. (c) Co-localization of Lgr5 and Tlr2 molecules in ISCs. Single-molecule fluorescence in situ hybridization (smFISH) of Tlr2 (green) and Lgr5 (red), co-stained with E-Cadherin and DAPI (blue). E-Cadherin stains IEC boundaries, and DAPI stains nuclei. Scale bar, 20μm. Single channel of each color (red or green) or merged images (lower panels); WT crypt (left panels) and TLR2 KO crypt (right panels). Insets x2 magnification. (d) Experimental scheme of the different segments of the small intestine for villi and crypts isolation (left) and RQ of Tlr2 expression levels using qPCR (right). Data are presented as mean ± SEM. n =3 mice per region; ns (non-significant), * p <0.05, ** p <0.01, one-way ANOVA . ( e ) Primary mouse SI spheroids and organoids from ileal crypts. Representative pictures of stem-enriched spheroids (upper panel) and differentiated organoids (lower panel) (left). Scale bar, 500μm. RQ of Tlr2 and Lgr5 expression levels in spheroids and organoids using qPCR (right). Data are presented as mean ± SEM. n =3 biological repetitions for spheroids or 2 for organoids, * p <0.05, two-tailed Student’s t-test . ( f ) RQ of Cxcl2 and Tnfα expression levels in spheroids generated from WT vs. TLR2 cKO mice after 4h or 8h of ex vivo activation with TLR2/TLR1 agonist (Pam3Csk4, 1μg/mL) or without (control) using qPCR. Data are presented as mean ± SEM. n =3 biological repetitions, * p <0.05, *** p <0.001, two-way ANOVA. ( g ) Heatmap of top TLR2 target genes of bulk RNA-seq from WT vs TLR2 cKO stem-enriched spheroids after 4h of activation with or without Pam3Csk4 (1μg/mL). Mean Z score of log 2 (TPM+1), color bar. n =3 biological repetitions for WT and 2 for TLR2 cKO. ( h ) NFĸB pathway activation. Representative images of immunofluorescence assay (IFA) of NFĸB subunit (p65, red) and DAPI of stem-enriched spheroids in WT (top panels) or TLR2 cKO (lower panels) at time 0 or 1 hour after Pam3Csk4 induction (1μg/mL). Scale bar, 20μm. Insets, x2 magnification. Arrows show p65 localization to the nucleus ( i ). Tlr2 activation of Lgr5+-ISC ex vivo scheme (upper panel). Heatmap of TLR2 target genes (inflammatory) and canonical NFĸB targets from sorted WT Lgr5+-ISCs after 4h of ex vivo activation with or without Pam3Csk4 (1μg/mL) by bulk RNA-seq. Average relative expression (mean Z score of log 2 (TPM+1), color bar). n =3 biological repetitions for control and 2 for Pam3Csk4 activation.

Article Snippet: C57BL/6J wild type (WT), TLR2 cKO (strain name: B6.129-Tlr2tm1Kir/J; stock number 004650), TLR4 cKO (strain name: B6(Cg)-Tlr4tm1.2Karp/J; stock number: 029015), CD45.1 (strain name: B6.SJL-Ptprca Pepcb/BoyJ; stock number: 002014), GFP (strain name: C57BL/6-Tg(UBC-GFP)30Scha/J; stock number: 004353) and Villin-Cre (strain name: B6.Cg-Tg(Vil1-cre)997Gum/J; stock number: 004586) mice were purchased from the Jackson Laboratory.

Techniques: Single Cell, Expressing, Flow Cytometry, Quantitative Proteomics, Real-time Polymerase Chain Reaction, Two Tailed Test, Fluorescence, In Situ Hybridization, Staining, Isolation, Generated, Ex Vivo, Activation Assay, Control, RNA Sequencing, Immunofluorescence

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a ) A representative flow cytometry gating strategy for epithelial cells. ( b ) Relative quantification (RQ) of Cxcl2 and Tnfα expression levels in spheroids vs. organoids generated from WT mice after 4h or 8h of ex vivo activation with TLR2/TLR1 agonist (Pam3Csk4, 1μg/mL) or without (control) using qPCR. Data are presented as mean ± SEM. n =3 biological repetitions per group, * p <0.05, *** p <0.001, **** p < 0.0001, two-way ANOVA. ( c ) Relative quantification (RQ) of Tlr2 expression in spheroids generated from WT or TLR2 cKO mice after 4h or 8h of ex vivo activation with TLR2/TLR1 agonist (Pam3Csk4, 1μg/mL) or without (control) using qPCR. Data are presented as mean ± SEM. n =3 biological repetitions per group, except for the control time 0 with 2 biological repetitions, * p <0.05, ** p <0.01, *** p <0.001, two-way ANOVA. ( d ) NFĸB pathway activation. Representative images of IFA of NFĸB subunit (p65, red) and DAPI in WT (upper panel) and TLR2 cKO (lower panel) stem-enriched spheroids after 30 minutes of Pam3Csk4 (1μg/mL) induction. Scale bar, 20μm. Insets, x2 magnification. ( e ) Relative quantification (RQ) of Cxcl2 and Tnfα expression levels in isolated Lgr5 + ileal stem cells from WT mice after 4h of ex vivo activation with TLR2/TLR1 agonist (Pam3Csk4, 1μg/mL) or without (control) using qPCR. Data are presented as mean ± SEM. n =3 biological repetitions, * p <0.05, two-tailed Student’s t-test .

Article Snippet: C57BL/6J wild type (WT), TLR2 cKO (strain name: B6.129-Tlr2tm1Kir/J; stock number 004650), TLR4 cKO (strain name: B6(Cg)-Tlr4tm1.2Karp/J; stock number: 029015), CD45.1 (strain name: B6.SJL-Ptprca Pepcb/BoyJ; stock number: 002014), GFP (strain name: C57BL/6-Tg(UBC-GFP)30Scha/J; stock number: 004353) and Villin-Cre (strain name: B6.Cg-Tg(Vil1-cre)997Gum/J; stock number: 004586) mice were purchased from the Jackson Laboratory.

Techniques: Flow Cytometry, Quantitative Proteomics, Expressing, Generated, Ex Vivo, Activation Assay, Control, Isolation, Two Tailed Test

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a ) Polarized monolayer generation from primary mouse SI spheroids scheme. ( b ) Representative images of primary mouse SI polarized monolayers using H&E staining (upper panel); scale bar, 75μm. IFA of primary monolayer (mid) or top of the villus (lower) stained with Villin (red), CD138 (green), and DAPI (blue); scale bar, 20μm. ( c ) Schematic representation of monolayer integrity assay, with FITC-dextran permeability quantification (left) or transepithelial electrical resistance (TEER) measurements (right) over 30 days of primary monolayer cultures. ( d-e ) Relative quantification (RQ) of TLR2 target genes, Cxcl2 and Tnfα expression levels in stem-enriched monolayers after 4h or 24h of ex vivo apical (d) and basolateral (e) activation with TLR2/TLR1 agonist (Pam3Csk4, 1μg/mL) or without (control) using qPCR. Data are presented as mean ± SEM. n =5 biological repetitions per condition, ns (non-significant), * p <0.05, one-way ANOVA. ( f ) Heatmap of top TLR2 target genes from WT vs TLR2 cKO stem-enriched monolayers after 4h of ex vivo apical activation with or without Pam3Csk4 induction (1μg/mL), tested by bulk RNA-seq. Average relative expression (mean Z score of log 2 (TPM+1), color bar). n =3 vs 2 for WT or 3 vs 3 for TLR2 cKO biological repetitions for monolayers with or without Pam3Csk4 induction for 4h, respectively.

Article Snippet: C57BL/6J wild type (WT), TLR2 cKO (strain name: B6.129-Tlr2tm1Kir/J; stock number 004650), TLR4 cKO (strain name: B6(Cg)-Tlr4tm1.2Karp/J; stock number: 029015), CD45.1 (strain name: B6.SJL-Ptprca Pepcb/BoyJ; stock number: 002014), GFP (strain name: C57BL/6-Tg(UBC-GFP)30Scha/J; stock number: 004353) and Villin-Cre (strain name: B6.Cg-Tg(Vil1-cre)997Gum/J; stock number: 004586) mice were purchased from the Jackson Laboratory.

Techniques: Staining, Integrity Assay, Permeability, Quantitative Proteomics, Expressing, Ex Vivo, Activation Assay, Control, RNA Sequencing

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a ) Relative quantification (RQ) of Tlr2 expression in stem-enriched monolayers derived from WT vs TLR2 cKO using qPCR. Data are presented as mean ± SEM. n =3 biological repetitions, ** p <0.01, two-tailed Student’s t-test . ( b ) Monolayer barrier integrity assay using TEER measurements of WT or TLR2 cKO. Data are presented as mean ± SEM. n =3 biological repetitions; *** p <0.001, **** p < 0.0001, two-tailed Student’s t-test . ( c ) Relative quantification (RQ) of Cxcl2 expression levels in stem-enriched monolayers from WT or TLR2 cKO mice after 4h of activation with TLR2/TLR1 agonist (Pam3Csk4, 1μg/mL) or without (control) using qPCR. Data are presented as mean ± SEM; n =3 biological repetitions; ns , ** p <0.01, two-way ANOVA.

Article Snippet: C57BL/6J wild type (WT), TLR2 cKO (strain name: B6.129-Tlr2tm1Kir/J; stock number 004650), TLR4 cKO (strain name: B6(Cg)-Tlr4tm1.2Karp/J; stock number: 029015), CD45.1 (strain name: B6.SJL-Ptprca Pepcb/BoyJ; stock number: 002014), GFP (strain name: C57BL/6-Tg(UBC-GFP)30Scha/J; stock number: 004353) and Villin-Cre (strain name: B6.Cg-Tg(Vil1-cre)997Gum/J; stock number: 004586) mice were purchased from the Jackson Laboratory.

Techniques: Quantitative Proteomics, Expressing, Derivative Assay, Two Tailed Test, Integrity Assay, Activation Assay, Control

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a ) Representative H&E images of SI ileum sections from WT, TLR2 cKO, or TLR4 cKO mice; scale bars, 20μm; yellow arrows show the changes in granular cells at the bottom of the crypt in TLR2 cKO mice. ( b-d ) Comparison of Lgr5 + -ISCs from WT or TLR2 cKO mice using bulk-RNA sequencing. (b) Experimental scheme for GFP-labeled Lgr5 + -ISC sort (top) and volcano plot showing log2fc estimates and -log(FDR q-value) of top ten upregulated (right) and downregulated (left) differentially expressed genes (DEG) in Lgr5 + -ISCs of TLR2 cKO ( n =2) compared to WT ( n =3) mice. Colored points correspond to padj ≤ 0.05 and |log2FoldChange| ≥ 1. Color coding is shown in the bottom-right corner. (c) Gene ontogeny (GO) biological processes showing hypergeometric enrichment of significantly upregulated and downregulated DEGs in Lgr5+-ISCs of TLR2 cKO ( n =2) vs. WT ( n =3) mice. Plotted is the negative log10 of enrichment padj. The direction of enrichment is shown as positive for biological processes enriched in TLR2 cKO, and negative for those enriched in WT. (d) Heatmap of the top DE antimicrobial peptides from WT ( n =3) vs. TLR2 cKO ( n =2) mice. Average relative expression (mean Z score of log 2 (TPM+1), color bar). ( e-g ) Goblet-Paneth intermediate cells accumulation is observed in TLR2 cKO. (e) Representative images of Periodic-Acid Schiff (PAS) and Alcian Blue (AB) staining of ileal sections from WT (top), TLR2 cKO (mid), and TLR2 cKO chimeric (CH, bottom) mice. The number of Paneth cells and goblet-Paneth intermediate cells per crypt was quantified (right); n = 10 fields per mouse, 2 mice per group; data are mean ± SEM; *** p < 0.001, one-way ANOVA. scale bar, 50μm. Inset, x4 magnification. (f) RQ of Lyz1 and Mptx2 expression levels in WT vs. TLR2 cKO using qPCR. Data are presented as mean ± SEM. n =5 WT vs 4 of TLR4 cKO mice, * p <0.05, two-tailed Student’s t-test . (g) Transmission electron microscopy (TEM) of Paneth cell eosinophilic granules in SI ileal segments of WT (left) and TLR2 cKO (right) mice; Scale bar, 5μm. (h) Mature Paneth cells staining in SI ileal crypts of WT (left), TLR2 cKO (mid), or TLR2 cKO chimera (CH, right). Single-molecule fluorescence in situ hybridization (smFISH) of Mptx2 (green) co-stained with Lyz1 antibody (red). E-Cadherin stains IEC boundaries, while DAPI stains the nucleus (blue). Scale bar, 20μm. Insets, x1.4 magnification. Lyz1+ Mptx2 + Paneth cells per crypt were quantified (right); n =10 fields per mouse, 2 mice per group. Data are presented as mean ± SEM. ns , non-significant, ** p <0.01, one-way ANOVA.

Article Snippet: C57BL/6J wild type (WT), TLR2 cKO (strain name: B6.129-Tlr2tm1Kir/J; stock number 004650), TLR4 cKO (strain name: B6(Cg)-Tlr4tm1.2Karp/J; stock number: 029015), CD45.1 (strain name: B6.SJL-Ptprca Pepcb/BoyJ; stock number: 002014), GFP (strain name: C57BL/6-Tg(UBC-GFP)30Scha/J; stock number: 004353) and Villin-Cre (strain name: B6.Cg-Tg(Vil1-cre)997Gum/J; stock number: 004586) mice were purchased from the Jackson Laboratory.

Techniques: Comparison, RNA Sequencing, Labeling, Expressing, Staining, Two Tailed Test, Transmission Assay, Electron Microscopy, Fluorescence, In Situ Hybridization

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a-b ) Representative flow cytometry plot (a), and quantification of the percentage of Lgr5 + ISC GFP high (left) and GFP low (right) EpCAM + cells from ileal crypts in WT or TLR2 cKO (b). Data are presented as mean ± SEM. n =3 mice per group; * p < 0.05, ** p < 0.01, two-tailed Student’s t-test. ( c ) schematic representation of the TLR2 KO chimeric mice model. TLR2 cKO mice were irradiated with a 500 Rad dose twice, followed by bone marrow transplantation (BMT) from GFP/CD45.1 mice 24h later. ( d-e ) Confirmation of BMT in the TLR2 cKO chimeric mouse model. (d) Representative flow cytometry plot of GFP-labeled immune cells in the LP. (e) immunofluorescence assay (IFA) of GFP (green), E-cadherin (blue), and DAPI from TLR2 cKO or chimeric TLR2 cKO (TLR2 cKO CH) mice. E-Cadherin (blue) stains IEC boundaries. Scale bar, 20μm. ( f ) Mptx2 staining in Paneth cells from SI ileal crypts of WT (left), TLR2 cKO (mid), and chimeric TLR2 cKO (TLR2 cKO CH). IFA of Mptx2 (green), E-Cadherin (blue), and DAPI. E-Cadherin (blue) stains IEC boundaries. Scale bar, 20μm. Cells with Mptx2+ granules were quantified per crypt (right); 10 crypts per mouse. Data are presented as mean ± SEM. **** p < 0.0001, one-way ANOVA.

Article Snippet: C57BL/6J wild type (WT), TLR2 cKO (strain name: B6.129-Tlr2tm1Kir/J; stock number 004650), TLR4 cKO (strain name: B6(Cg)-Tlr4tm1.2Karp/J; stock number: 029015), CD45.1 (strain name: B6.SJL-Ptprca Pepcb/BoyJ; stock number: 002014), GFP (strain name: C57BL/6-Tg(UBC-GFP)30Scha/J; stock number: 004353) and Villin-Cre (strain name: B6.Cg-Tg(Vil1-cre)997Gum/J; stock number: 004586) mice were purchased from the Jackson Laboratory.

Techniques: Flow Cytometry, Two Tailed Test, Irradiation, Transplantation Assay, Labeling, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a ) Two-dimensional Uniform Manifold Approximation and Projection (UMAP) graphical representation of EpCAM + single cell RNA-sequencing (scRNA-seq), colored by graph clustering per different clusters from chimeric (CH) WT and TLR2-KO mice (left) and stacked bar of relative cell subset abundance in chimeric (CH) WT and TLR2 cKO (right). Data are presented as mean ± SEM. n =2 mice per condition. TA, transient amplifying cells; EEC, enteroendocrine. ( b ) Box-and-whisker plot of cell cycle signature score in chimeric (CH) WT vs TLR2 cKO. n =2 mice per condition. Boxes represent median and Interquartile Range (IQR); whiskers extend to 1.5× IQR. Mann-Whitney U test; **** p <0.0001. ( c ) Diffusion pseudotime (dpt) score comparing the differentiation state of chimeric (CH) WT (blue) and TLR2 cKO (orange). Dpt scores showing a right-shift peak in TLR2 cKO cells, indicating accelerated differentiation compared to WT. ( d ) Diffusion map of enterocytes differentiation trajectory colored by cell subset in chimeric (CH) WT and TLR2 cKO (left) and diffusion pseudotime (dpt) score comparing the enterocytes differentiation state of chimeric (CH) WT (blue) and TLR2 cKO (orange) (right). Dpt scores showing a right-shift peak in TLR2 cKO cells, indicating accelerated differentiation compared to WT. ( e ) Dot plot of immature and mature enterocyte cell markers comparing enterocyte subsets of chimeric (CH) WT (blue) vs. TLR2 cKO (orange) mice. The size of the dot indicates the proportion of cells expressing a gene, while the color indicates the mean expression level. ( f ) Volcano plot showing log2fc estimates and the negative log10 of enrichment padj. of top upregulated (right) and downregulated (left) DEG in mature enterocytes of chimeric (CH) WT ( n =2) compared to TLR2 cKO ( n =2) chimeras. Colored points correspond to padj ≤ 0.05 and |log2FoldChange| ≥ 1. Color coding is shown on the top left. ( g ) Distribution of proximal (left) and distal (right) enterocyte signatures in mature enterocytes of chimeric (CH) WT (blue) vs. TLR2 cKO (orange) mice; Significance was determined by Pearson’s r test, **** p <0.0001. ( h ) Scatter plot comparing Pearson correlation coefficients (r) of proximal and distal enterocyte gene signatures in chimeric (CH) WT and TLR2 cKO mature enterocyte cells. The x-axis represents the Pearson r values for proximal enterocyte signature genes, and the y-axis shows Pearson r values for distal enterocyte signature genes. Each point corresponds to a cell within the respective signature. The colors represent different conditions: blue for WT and orange for TLR2 cKO. ( i-j ) Antimicrobial peptides expression in Paneth cells. (i) Dot plot of top DEG in Paneth cells from chimeric (CH) WT vs. TLR2 cKO mice. The size of the dot indicates the proportion of cells expressing a gene, while the color indicates the average relative expression (mean Z score of log2(TPM+1)). (j) Reduction of AMP expressed by Paneth cells in TLR2 cKO. IFA of Lyz1 (green), Mptx2 (red), E-Cadherin, and DAPI co-staining in chimeric (CH) WT (left) and TLR2 cKO (right) mice. (k) Uncharacterized top AMP-encrypted candidates in mature enterocytes derived from DEG upregulated in chimeric WT compared to TLR2 cKO. X-axis: gene list, Y-axis: absolute Torres score.

Article Snippet: C57BL/6J wild type (WT), TLR2 cKO (strain name: B6.129-Tlr2tm1Kir/J; stock number 004650), TLR4 cKO (strain name: B6(Cg)-Tlr4tm1.2Karp/J; stock number: 029015), CD45.1 (strain name: B6.SJL-Ptprca Pepcb/BoyJ; stock number: 002014), GFP (strain name: C57BL/6-Tg(UBC-GFP)30Scha/J; stock number: 004353) and Villin-Cre (strain name: B6.Cg-Tg(Vil1-cre)997Gum/J; stock number: 004586) mice were purchased from the Jackson Laboratory.

Techniques: Single Cell, RNA Sequencing, Whisker Assay, MANN-WHITNEY, Diffusion-based Assay, Expressing, Staining, Derivative Assay

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a ) Epithelial cell subset markers used for cluster annotation. Dot plot of five known markers per epithelial cell subset derived from publicly available data . The size of the dot indicates the proportion of cells expressing a gene, while the color indicates the mean expression level. TA, transit amplifying; E. Immature, immature enterocyte; E. Mature, mature enterocyte; E. Mature T, mature top-villus enterocytes; G/P Progenitors, goblet-Paneth progenitors; EEC, enteroendocrine. ( b ) Box-and-whisker plot of cell cycle signature score in different epithelial clusters from chimeric (CH) WT (blue) vs TLR2 cKO (orange) mice. n =2 per condition. EEC, enteroendocrine. Boxes represent median and Interquartile Range (IQR); whiskers extend to 1.5× IQR; ns (non-significant), * p < 0.05, ** p <0.01, *** p <0.001, Wilcox-test . ( c ) Volcano plot showing log2fc estimates and the negative log10 of enrichment padj. of top upregulated in WT (blue, right) and downregulated (orange, left) DEG in stem cells compared to TLR2 cKO ( n =2 mice per condition). Colored points correspond to padj ≤ 0.05 and |log2FoldChange| ≥ 1. Color coding is shown in the top-right corner. ( d ) Proliferation analysis in SI ileal crypts of WT (left panel), TLR2 cKO (middle panel), and TLR2 cKO chimera (right panel). IFA of Ki67 (white), Olfm4 (green), E-Cadherin (blue), and DAPI. E-cadherin (blue) stains IEC boundaries. Scale bar, 20μm. Ki67 + Cells were quantified per crypt (right); 10 crypts per mouse. Data are presented as mean ± SEM. * p <0.05, one-way ANOVA. ( e ) Apoptosis analysis in SI ileal crypts of positive control (24h post-irradiation, left), chimeric (CH) WT (middle), or TLR2 cKO (right) mice. IFA of Cleaved caspase 3 (CC3) (red), Ki67 (green), E-cadherin (white) and DAPI. E-cadherin (white) stains IEC boundaries. Yellow arrows show CC3+ cells. Scale bar, 20μm. ( f ) Diffusion map of differentiation trajectory to the main epithelial lineages colored by cell subset (left) or by cell cycle state (right) in chimeric (CH) WT and TLR2 cKO. Color coding is shown on the right. TA, transit amplifying; EEC, Enteroendocrine. ( g ) Diffusion pseudotime (dpt) score comparing the enterocyte subsets differentiation state of chimeric (CH) WT (blue) and TLR2 cKO (orange). Dpt scores showing an accelerated differentiation in TLR2 cKO CH. ( h ) Distribution of Antimicrobial humoral immune response mediated by antimicrobial peptide-signature score in Paneth cells from chimeric WT vs. TLR2 cKO; significance was determined by Wilcox-test, **** p <0.0001. ( i ) Top AMP-encrypted candidates in Paneth cells derived from DEG upregulated in WT CH compared to TLR2 cKO CH. X-axis: gene list, Y-axis: absolute Torres score. ( j ) Distribution of proximal (left) and distal (right) Paneth signatures in Paneth cells of chimeric (CH) WT (blue) vs. TLR2 cKO (orange) mice; Significance was determined by Pearson’s r test, ** p <0.01, **** p <0.0001.

Article Snippet: C57BL/6J wild type (WT), TLR2 cKO (strain name: B6.129-Tlr2tm1Kir/J; stock number 004650), TLR4 cKO (strain name: B6(Cg)-Tlr4tm1.2Karp/J; stock number: 029015), CD45.1 (strain name: B6.SJL-Ptprca Pepcb/BoyJ; stock number: 002014), GFP (strain name: C57BL/6-Tg(UBC-GFP)30Scha/J; stock number: 004353) and Villin-Cre (strain name: B6.Cg-Tg(Vil1-cre)997Gum/J; stock number: 004586) mice were purchased from the Jackson Laboratory.

Techniques: Derivative Assay, Expressing, Whisker Assay, Positive Control, Irradiation, Diffusion-based Assay

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a-c ) Epithelial-specific TLR2 deletion (TLR2 ΔIEC ) mouse model. (a) Experimental scheme for the generation of epithelial-specific TLR2 deletion by crossing Vil-Cre to TLR2 fl/fl mouse model (TLR2 ΔIEC ). (b) Representative images of PAS and AB staining of ileum sections from TLR2 fl/fl (upper panel) and TLR2 ΔIEC (lower panel). scale bar: 20 μm. Insets, x5 magnification. (c) AMP (Lyz1 and Mptx2) expressed by Paneth cells staining in TLR2 fl/fl (left) and TLR2 ΔIEC (right) ileal sections. IFA of Lyz1 (green), Mptx2 (red), E-cadherin (white) and DAPI. E-cadherin (white) stains IEC boundaries; scale bar: 20 μm. ( d-g ) Lgr5+-ISC-specific TLR2 deletion (TLR2 ΔISC ) mouse model. (d) Schematic representation of Lgr5+-ISC-specific TLR2 deletion lineage tracing (TLR2 DISC+tdTom ) generation by crossing Lgr5+-ISC-CreER T -TLR2 fl/fl mice to Rosa26-tdTomato mice. TdTomato cells are progeny of TLR2-deleted Lgr5+-ISC after one injection of Tamoxifen (2mg) at 48h (d-f) or 5 days (g). (e) Representative flow cytometry plot of the percentage of TdTomato + EpCAM + cells among EpCAM + cells in TLR2 fl/fl vs. TLR2 ΔISC mice (left panel); quantification of TdTomato + EpCAM + percentages in both groups is depicted on a bar graph (right panel); n =3 mice per TLR2 fl/fl and 2 mice per TLR2 ΔISC . Data are presented as mean ± SEM. * p <0.05, two-tailed Student’s t-test . (f) IFA of tdTomato (red), Ki67 (green), E-cadherin (white), and DAPI of distal SI, 48h after Tamoxifen induction. E-cadherin (white) stains IEC boundaries; scale bar, 20 μm. (left panel). Quantification of tdTom+ cells (mid) and of Ki67+ tdTom+ cells (Right) per crypt. Data are presented as mean ± SEM. 10 fields (tdTom+) or 20 fields (tdTom+ Ki67+) per mouse. **** p < 0.001, two-tailed Student’s t-test . (g) IFA of tdTomato (red), Lyz1 (green), E-cadherin+DAPI (blue) of distal SI, 5 days after Tamoxifen induction; scale bar, 20 μm. (left panel). Quantification of tdTom+ Lyz1+ cells (right panel) per crypt. Data are presented as mean ± SEM. 20 fields per mouse and n =2 mice. * p < 0.05, two-tailed Student’s t-test .

Article Snippet: C57BL/6J wild type (WT), TLR2 cKO (strain name: B6.129-Tlr2tm1Kir/J; stock number 004650), TLR4 cKO (strain name: B6(Cg)-Tlr4tm1.2Karp/J; stock number: 029015), CD45.1 (strain name: B6.SJL-Ptprca Pepcb/BoyJ; stock number: 002014), GFP (strain name: C57BL/6-Tg(UBC-GFP)30Scha/J; stock number: 004353) and Villin-Cre (strain name: B6.Cg-Tg(Vil1-cre)997Gum/J; stock number: 004586) mice were purchased from the Jackson Laboratory.

Techniques: Staining, Injection, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a ) Representative H&E images of SI ileum sections from TLR2 fl/fl and TLR2 ΔIEC ; scale bars, 75μm; yellow arrows show the changes in granular cells at the bottom of the crypt in TLR2 ΔIEC mice. ( b ) Relative quantification (RQ) of Tlr2 expression in TLR2 fl/fl and TLR2 ΔIEC mice using qPCR. Data are presented as mean ± SEM. n =4 mice per group, ** p <0.01, two-tailed Student’s t-test. ( c ) Goblet-Paneth intermediate cells accumulation is observed in TLR2 ΔIEC . The number of Paneth (left) and goblet-Paneth intermediate (right) cells per crypt was quantified (right); 25 crypts per mouse, 2 mice per group; data are mean ± SEM; ** p < 0.01, two-tailed Student’s t-test . ( d ) TUNEL assay analysis of TLR2 ΔISC SI ileal sections shows no signs of cell death after 48h of TLR2 deletion induction. Crypts of positive control (24h post-irradiation (1000 rad), left), TLR2 fl/fl (middle), or TLR2 ΔISC (right) mice. IFA of tdTomato cells (red), BrdU (TUNEL, white), and DAPI (blue). Scale bar, 20μm. ( e ) Relative quantification (RQ) of Tlr2 expression levels in TLR2fl/fl ( n =4 mice) or TLR2 ΔISC ( n =3 mice) using qPCR (right). Data are presented as mean ± SEM; * p <0.05, two-tailed Student’s t-test . ( f ) IFA of tdTomato (red), Ki67 (white), E-cadherin and DAPI (blue) of distal SI TLR2 fl/fl or TLR2 ΔISC , 5 days after Tamoxifen induction; scale bar, 20μm. (left panel). Quantification of Ki67+ tdTomato+ cells (Right) per crypt. Data are presented as mean ± SEM. 20 crypts per mouse, 2 mice per group. * p < 0.05, two-tailed Student’s t-test .

Article Snippet: C57BL/6J wild type (WT), TLR2 cKO (strain name: B6.129-Tlr2tm1Kir/J; stock number 004650), TLR4 cKO (strain name: B6(Cg)-Tlr4tm1.2Karp/J; stock number: 029015), CD45.1 (strain name: B6.SJL-Ptprca Pepcb/BoyJ; stock number: 002014), GFP (strain name: C57BL/6-Tg(UBC-GFP)30Scha/J; stock number: 004353) and Villin-Cre (strain name: B6.Cg-Tg(Vil1-cre)997Gum/J; stock number: 004586) mice were purchased from the Jackson Laboratory.

Techniques: Quantitative Proteomics, Expressing, Two Tailed Test, TUNEL Assay, Positive Control, Irradiation

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a ) Representative flow cytometry plot (left) and quantification (right) of the percentage of CD24 + side-scattered low (SSC low ) cells among EpCAM + cells in WT vs. TLR2 cKO mice (left panel); quantification of CD24 + SSC low percentages in both groups are depicted on a bar graph (right panel); n =3 mice per group. Data are presented as mean ± SEM. ** p <0.01, two-tailed Student’s t-test . ( b ) Relative quantification (RQ) of Dclk1 expression levels in EpCAM + cells from WT or TLR2 cKO using qPCR. Data are presented as mean ± SEM. n =5 mice per group, * p <0.05, two-tailed Student’s t-test . ( c ) Heatmap of the top tuft cell markers (Tuft 1 or 2 subsets) in EpCAM + cells from WT ( n =3) vs. TLR2 cKO ( n =2) mice out of the DEG using bulk RNA-seq. Average relative expression (mean Z score of log 2 (TPM+1), color bar). ( d-e ) elevation in Tuft 1 and 2 in TLR2 deletion. (d) IFA of DCLK1 (red), CD45 (white), E-cadherin (green), and DAPI. E-Cadherin (green) stains IEC boundaries; scale bar, 20 μm. Inset, x2 magnification. (e) The number of Tuft 1 (DCLK1 + CD45 - ) and Tuft 2 (DCLK + CD45 + ) cells per villus-crypt axis was quantified. Data are presented as mean ± SEM. 10 fields per mouse, **** p < 0.0001, two-tailed Student’s t-test . ( f-g ) Pseudotime analysis for tuft cell differentiation trajectory in chimeric (CH) WT or TLR2 cKO at single cell resolution. (f) Diffusion pseudotime ( dpt ) score comparing the tuft cell differentiation state of chimeric (CH) WT (blue) and TLR2 cKO (orange). Dpt scores showing a left-shift peak in TLR2 cKO cells, indicating accelerated differentiation compared to WT. (g) Scatter plot comparing log2 fold-change between chimeric (CH) WT or TLR2 cKO (y-axis) in tuft cells and log2 fold-change between Tuft1 or Tuft2 (x-axis) clusters. Each point represents one gene. The colors represent different conditions. Highlighted genes (blue) represent Tuft-2–specific bacterial-sensing receptors.

Article Snippet: C57BL/6J wild type (WT), TLR2 cKO (strain name: B6.129-Tlr2tm1Kir/J; stock number 004650), TLR4 cKO (strain name: B6(Cg)-Tlr4tm1.2Karp/J; stock number: 029015), CD45.1 (strain name: B6.SJL-Ptprca Pepcb/BoyJ; stock number: 002014), GFP (strain name: C57BL/6-Tg(UBC-GFP)30Scha/J; stock number: 004353) and Villin-Cre (strain name: B6.Cg-Tg(Vil1-cre)997Gum/J; stock number: 004586) mice were purchased from the Jackson Laboratory.

Techniques: Flow Cytometry, Two Tailed Test, Quantitative Proteomics, Expressing, RNA Sequencing, Cell Differentiation, Single Cell, Diffusion-based Assay

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a-d ) The involvement of microbiota in TLR2 activation and Paneth differentiation. (a) Representative flow cytometry plot (left) and quantification (right) of the percentage of Lyz1 + cells among EpCAM + cells in ileal crypts of SPF vs. GF mice. n =6 mice per group; data are presented as mean ± SEM. * p < 0.05, two-tailed Student’s t-test . (b) Representative images of PAS and AB staining of ileum sections from SPF (upper panel) and GF (lower panel) mice; Scale bar, 20μm. Inset, x5 magnification. (c) Relative quantification (RQ) of Mptx2 and Reg3γ expression levels, key Paneth and enterocyte AMPs, in EpCAM + cells of GF ( n =5) vs. SPF ( n =4) mice using qPCR. Data are presented as mean ± SEM; ns (non-significant), * p <0.05, ** p <0.01, two-tailed Student’s t-test . (d) GF colonization experiment. Representative images of PAS and AB staining of ileum sections (Left) from GF (upper panel) or GF colonized with WT bacteria (lower panel); n =4 mice per group, scale bar, 20μm. Inset, x5 magnification. Quantification of Paneth cells per crypt of GF vs GF colonized with WT bacteria (right). 6 fields of view per condition; data are presented as mean ± SEM. **** p < 0.0001, two-tailed Student’s t-test . ( e-g ) Human ileal spheroids in vitro activation assay. (e) Experimental scheme of generating spheroids from human ileum biopsies and activation with TLR2/TLR1 agonist (Pam3Csk4). (f) Relative quantification (RQ) of TLR2 and IL8 expression levels in spheroids generated from ileal biopsies after 4h or 8h of ex vivo activation with TLR2/TLR1 agonist (Pam3Csk4, 1μg/mL) or without (control) using qPCR; n =4 biological repetitions per group. Data are presented as mean ± SEM. ns (non-significant), * p <0.05, ** p <0.01, *** p <0.001, one-way ANOVA. (g) Human Paneth cell expansion following Pam3Csk4 induction. IFA of LYZ1 (red), E-cadherin (blue), and DAPI in fixed differentiated spheroids after 4h, 24h, and 72h of ex vivo activation with TLR2/TLR1 agonist (Pam3Csk4, 1μg/mL) or without (control); scale bar, 50μm. Yellow arrows, LYZ1 + crypts. ( h ) Suggested model for the symbiotic relationship of the Lgr5+-ISC with microbiota mediated by TLR2 signaling.

Article Snippet: C57BL/6J wild type (WT), TLR2 cKO (strain name: B6.129-Tlr2tm1Kir/J; stock number 004650), TLR4 cKO (strain name: B6(Cg)-Tlr4tm1.2Karp/J; stock number: 029015), CD45.1 (strain name: B6.SJL-Ptprca Pepcb/BoyJ; stock number: 002014), GFP (strain name: C57BL/6-Tg(UBC-GFP)30Scha/J; stock number: 004353) and Villin-Cre (strain name: B6.Cg-Tg(Vil1-cre)997Gum/J; stock number: 004586) mice were purchased from the Jackson Laboratory.

Techniques: Activation Assay, Flow Cytometry, Two Tailed Test, Staining, Quantitative Proteomics, Expressing, Bacteria, In Vitro, Generated, Ex Vivo, Control

Journal: bioRxiv

Article Title: TLR2-mediated microbial sensing by intestinal stem cells coordinates epithelial antimicrobial defense

doi: 10.64898/2026.05.04.722581

Figure Lengend Snippet: ( a ) Relative quantification (RQ) of Lgr5 and TLR2-related genes ( Tlr2, Tlr1 and Tnfα ) expression levels in SPF ( n =4 mice) or GF ( n =5 mice) using qPCR (right). Data are presented as mean ± SEM; * p <0.05, **p<0.01, two-tailed Student’s t-test . ( b-c ) GF colonization experiment. (b) Experimental scheme of colonization experiment in GF mice. (c) Representative flow cytometry plots (left) and quantification (right) of the percentage of Lyz1 + cells in ileal crypts of GF (left) or GF colonized with WT microbiota (right). n =4 biological repetitions. Data are presented as mean ± SEM; ns (non-significant), two-tailed Student’s t-test . ( d ) droplet-based epithelial single cell re-analysis of Toll-like receptors (TLRs). Dot plot of 7 human TLRs expressed in different IECs under homeostasis, taken from a publicly available dataset . The dot size indicates the proportion of cells expressing a gene, and the color indicates the mean expression levels. TA, transient amplifying cells.

Article Snippet: C57BL/6J wild type (WT), TLR2 cKO (strain name: B6.129-Tlr2tm1Kir/J; stock number 004650), TLR4 cKO (strain name: B6(Cg)-Tlr4tm1.2Karp/J; stock number: 029015), CD45.1 (strain name: B6.SJL-Ptprca Pepcb/BoyJ; stock number: 002014), GFP (strain name: C57BL/6-Tg(UBC-GFP)30Scha/J; stock number: 004353) and Villin-Cre (strain name: B6.Cg-Tg(Vil1-cre)997Gum/J; stock number: 004586) mice were purchased from the Jackson Laboratory.

Techniques: Quantitative Proteomics, Expressing, Two Tailed Test, Flow Cytometry, Single Cell