Journal: bioRxiv

Article Title: Borrelia Burgdorferi binds Serum Amyloid A and Modulates Subcutaneous Adipose Tissue Immune Signaling

doi: 10.64898/2026.01.29.702514

Figure Lengend Snippet: SAA2-bound spirochetes were stained for the presence of attached His-tagged protein (SAA2) detected by flow cytometry using an anti-His-tag fluorescent (PE/Cy5) conjugated antibody. (A) Histogram plots showing human SAA (hSAA2 and hSAA1), relative to non-stained controls (grey) and positive control protein (brown) known to bind Borrelia (Peptidoglycan Recognition Protein 1 (PGLYRP1) – Ref 39). The dashed line represents 40 µg/mL of hSAA2. The solid line represents 10µg/mL hSAA2. (B) Streptococcus pneumoniae incubated with 40µg/mL recombinant hSAA2 showing no increase in fluorescence compared to control. (C) Repeat binding assays showing histograms representing SAA2 binding multiple isolates of Bb sensu stricto (B31, CA8, HP19, CT-1, NT-1).

Article Snippet: Human PGLYRP1 (R&D Systems 2590-PGB) [ ] was used as a positive control and allowed to bind to spirochetes as stated above at 40μg/mL.

Techniques: Staining, Flow Cytometry, Positive Control, Incubation, Recombinant, Fluorescence, Control, Binding Assay

Journal: bioRxiv

Article Title: Borrelia Burgdorferi binds Serum Amyloid A and Modulates Subcutaneous Adipose Tissue Immune Signaling

doi: 10.64898/2026.01.29.702514

Figure Lengend Snippet: (A) Experimental workflow diagram of flow cytometry binding assay. Spirochetes cultured at 37°C for 24h were allowed to bind recombinant His-tagged mSAA2. Anti-His tag fluorescent (PE/Cy5) primary conjugated antibodies were bound to SAA-spirochete complexes to label protein attached to spirochetes. Spirochetes were washed and used for flow cytometry analysis compared to unstained spirochete complexes. (B) Histogram plots indicating murine SAA2 (mSAA2) binds SAA2 relative to staining control ( Bb + mSAA2) and positive control protein peptidoglycan recognition protein-1 (PGLYRP1 ref 39). (C) Binding of mSAA2 to Bb sensu stricto strains B31, CA8, HP19, CT-1, NT-1.

Article Snippet: Human PGLYRP1 (R&D Systems 2590-PGB) [ ] was used as a positive control and allowed to bind to spirochetes as stated above at 40μg/mL.

Techniques: Flow Cytometry, Binding Assay, Cell Culture, Recombinant, Staining, Control, Positive Control

Journal: bioRxiv

Article Title: Borrelia Burgdorferi binds Serum Amyloid A and Modulates Subcutaneous Adipose Tissue Immune Signaling

doi: 10.64898/2026.01.29.702514

Figure Lengend Snippet: (A) DiD stained THP-1 differentiated macrophages were incubated with CFSE-labeled N40 spirochetes for 0.5h, 1h, 2h, or 3h at a multiplicity of infection equal to 10 (MOI:10), and hSAA2 at 1 ug/mL and 10 ug/mL. Graphs represent percentages of DiD+CFSE+ double positive populations of cells quantified by flow cytometry. Statistical significance was determined a two-way ANOVA followed by post hoc Tukey test to correct for multiple comparisons. (B) Immunofluorescence of DiD-labeled differentiated THP-1 macrophages and CFSE-labeled Bb at 2h post co-culture at 10µg/mL. Abbreviations: NHS: Normal human serum. PGLYRP1: peptidoglycan recognition protein 1, DiD(red)= cell membrane, Bb (green) = Borrelia Burgdorferi , DAPI (blue) = nuclei.

Article Snippet: Human PGLYRP1 (R&D Systems 2590-PGB) [ ] was used as a positive control and allowed to bind to spirochetes as stated above at 40μg/mL.

Techniques: Staining, Incubation, Labeling, Infection, Flow Cytometry, Immunofluorescence, Co-Culture Assay, Membrane

Journal: Nature Communications

Article Title: PGLYRP1-mediated intracellular peptidoglycan detection promotes intestinal mucosal protection

doi: 10.1038/s41467-025-57126-9

Figure Lengend Snippet: a Peptidoglycan fragment library: GMTriP-K (1) and MDP (2); synthetic peptidoglycan fragments (1a-b, 2a-h and 3a-d) were all prepared with amine linkage points for attachment to the array surface via NHS-chemistry. b General workflow for the printing, incubation, and analysis of the PGN small fragment microarray (Created in BioRender. Putnik, R. (2025) https://BioRender.com/x50o125 ). c Microarray binding of PGLYRPs. PGLYRP1 binds to the disaccharide components of the array, whereas PGLYRP3 and PGLYRP4 show no association with the compounds on the array. d Determination of Apparent Dissociation Constant for PGLYRP1 to GMTriP-K (3 independent experiments were carried out with each experiment done in technical replicates). e Alphafold protein structure prediction of human PGLYRP1. f – i Docking prediction of GMTriP-K to human PGLYRP1. j Docking-prediction of PGLYRP3 and GMTriP-K demonstrating shallow GlcNAc preventing interaction. For synthetic procedures and compound characterization (NMRs, HRMS), please see the . For ( c ), each condition was screened in at least biological triplicate and technical replicated (see for raw image files and other biologically replicate binding data). Data are presented as mean ± SEM. All experiments were repeated twice and yielded consistent results.

Article Snippet: 25 μM GMTriP-K, GMDiP, or MDP (Invivogen; Cat# tlrl-mdp), 100 ng/mL IFNγ (Biolegend #575304), 100 ng/mL IFNβ (Biolegend #581302), or 500 ng/mL PGLYRP1 (R&D system #2590-PGB) were used to stimulate BMDM cells for indicated duration up to 18 h. For heat inactivation, Listeria monocytogenes (Microbiologics Cat#01249 UV-V) were suspended in PBS, pH 7.4, and centrifuged at 10,000 × g for 10 min.

Techniques: Incubation, Microarray, Binding Assay

Journal: Nature Communications

Article Title: PGLYRP1-mediated intracellular peptidoglycan detection promotes intestinal mucosal protection

doi: 10.1038/s41467-025-57126-9

Figure Lengend Snippet: a RNA-seq and volcano plot analysis of gene expression in BMDMs from wild type and Pglyrp1 -/- mice after 18 h of stimulation with 25 µM GMTriP-K. b Volcano plot analysis of gene expression in BMDMs from wild type and NOD2 -/- mice after 18 h of stimulation with 25 µM GMTriP-K. c Volcano plot analysis of gene expression in BMDMs from wild type mice after 18 h of stimulation with 25 µM GMTriP-K or 25 µM MDP. d Hierarchal cluster analysis of genes induced by either stimulus in wild type or Pglyrp1 -/- BMDMs. e Hierarchical cluster analysis of genes induced by GMTriP-K in BMDMs from wild type, Pglyrp1 -/-, Nod2-/- , or Arhgef2 -/- mice. f qPCR analysis of gene expression in BMDMs from indicated mouse strains stimulated with 25 µM GMTriP-K or 25 µM GMDiP stimulation on BMDMs from wild type and Pylyrp1-/- mice for 18 h ( n = 3). g , h Gene set enrichment analysis of the differentially expressed genes induced by indicated stimulus in wild type BMDM. g Top 25 enriched signaling pathways for the differentially expressed gene sets. h Gene set enrichment analysis summarized in mountain plots representing significantly enriched (left) or depleted (right) of genes for the indicated gene sets and collections. Data are presented as mean ± SEM with indicated P values analyzed by one-way ANOVA. All experiments were repeated twice and yielded consistent results.

Article Snippet: 25 μM GMTriP-K, GMDiP, or MDP (Invivogen; Cat# tlrl-mdp), 100 ng/mL IFNγ (Biolegend #575304), 100 ng/mL IFNβ (Biolegend #581302), or 500 ng/mL PGLYRP1 (R&D system #2590-PGB) were used to stimulate BMDM cells for indicated duration up to 18 h. For heat inactivation, Listeria monocytogenes (Microbiologics Cat#01249 UV-V) were suspended in PBS, pH 7.4, and centrifuged at 10,000 × g for 10 min.

Techniques: RNA Sequencing, Gene Expression, Protein-Protein interactions

Journal: Nature Communications

Article Title: PGLYRP1-mediated intracellular peptidoglycan detection promotes intestinal mucosal protection

doi: 10.1038/s41467-025-57126-9

Figure Lengend Snippet: a Hierarchal clustering of MDP induced genes in BMDMs from wild-type, Pglyrp1 -/- , Nod2 -/- , or Arhgef2 -/- mice. b , c BMDMs from wild type, Pglyrp1 -/ , Nod2 -/- , or Arhgef2 -/- mice were stimulated with 25 μM GMTriP-K or 25 μM MDP and analyzed via western blotting with the indicated antibodies. d BMDMs from wild type or Ifnar1 -/- mice were stimulated with 25 μM GMTriP-K or 25 μM MDP for 18 h and analyzed for STAT1 pathway activation with phospho-specific antibodies as indicated. e Wild type or GEF-H1-deficient BMDMs were treated with 100 ng/mL of IFNγ or IFNβ for 18 h and analyzed via western blotting with the indicated antibodies. f BMDMs from wild type, Pglyrp1 -/ , and Nod2 -/- mice were stimulated with 25 μM GMTriP-K or 25 μM MDP, lysed, and analyzed via western blotting with the indicated antibodies. g qPCR analysis of gene expression in BMDMs from indicated mouse strains stimulated with 25 µM GMTriP-K or 25 µM GMDiP for 18 h. Data are presented as mean ± SEM with indicated P values analyzed by one-way ANOVA. All experiments were repeated twice and yielded consistent results.

Article Snippet: 25 μM GMTriP-K, GMDiP, or MDP (Invivogen; Cat# tlrl-mdp), 100 ng/mL IFNγ (Biolegend #575304), 100 ng/mL IFNβ (Biolegend #581302), or 500 ng/mL PGLYRP1 (R&D system #2590-PGB) were used to stimulate BMDM cells for indicated duration up to 18 h. For heat inactivation, Listeria monocytogenes (Microbiologics Cat#01249 UV-V) were suspended in PBS, pH 7.4, and centrifuged at 10,000 × g for 10 min.

Techniques: Western Blot, Activation Assay, Gene Expression

Journal: Nature Communications

Article Title: PGLYRP1-mediated intracellular peptidoglycan detection promotes intestinal mucosal protection

doi: 10.1038/s41467-025-57126-9

Figure Lengend Snippet: a – k Representative confocal microscopy images of HEK 293T cells expressing untagged (UT) or Flag-tagged PGLYRP1, orange fluorescent protein (OFP)-tagged SEC61β and GFP-tagged NOD2. Cells were transfected with PGLYRP1-UT ( a – d ) or PGLYRP1-Flag ( e – k ) and SEC61β-OFP and then probed with PGLYRP1 and GM130 antibodies. PGLYRP1, SEC61β-OFP, and GM130 were detected in Alexa Fluor 488 (green pseudo-color), mOrange (red), and Alexa Fluor 647 (blue) channels, respectively. l – y Cells were transfected with PGLYRP1-UT and NOD2-GFP and stained for PGLYRP1 and GM130. NOD2-GFP, PGLYRP1, and GM130 were detected in EGFP (green pseudo-color), Rhodamine Red-X (red), and Alexa Fluor 647 (blue) channels, respectively. All channels were scanned sequentially, and images are pseudo-colored independent of channel wavelengths with nuclear counterstain in greyscale (Scale bars indicate 2 μm in ( a – d, l – r ) and 5 μm in ( e – k , s – y )). Typically, 10-12 cells were analyzed for each condition. Data are presented as mean ± SEM Pearson’s coefficients were calculated in Imaris 10.1.1 image analysis software. All experiments were repeated twice and yielded consistent results.

Article Snippet: 25 μM GMTriP-K, GMDiP, or MDP (Invivogen; Cat# tlrl-mdp), 100 ng/mL IFNγ (Biolegend #575304), 100 ng/mL IFNβ (Biolegend #581302), or 500 ng/mL PGLYRP1 (R&D system #2590-PGB) were used to stimulate BMDM cells for indicated duration up to 18 h. For heat inactivation, Listeria monocytogenes (Microbiologics Cat#01249 UV-V) were suspended in PBS, pH 7.4, and centrifuged at 10,000 × g for 10 min.

Techniques: Confocal Microscopy, Expressing, Transfection, Staining, Software

Journal: Nature Communications

Article Title: PGLYRP1-mediated intracellular peptidoglycan detection promotes intestinal mucosal protection

doi: 10.1038/s41467-025-57126-9

Figure Lengend Snippet: a Western blot analysis of protein expression in BMDMs from wildtype, Arhgef2-/- , Pglyrp1-/- , and Nod2-/- mice. b Time course of protein interactions with GEF-H1 after GMTriP-K stimulation of BMDMs. c Analysis of protein interactions with PGLYRP1 or d NOD2 after GMTriP-K stimulation of BMDMs. e Input assessment of protein expression for the IPs in ( c , d ). f Assessment of protein interactions with GEF-H1 after stimulation with indicated immune stimuli. Representative experiments of at least three repeats are shown.

Article Snippet: 25 μM GMTriP-K, GMDiP, or MDP (Invivogen; Cat# tlrl-mdp), 100 ng/mL IFNγ (Biolegend #575304), 100 ng/mL IFNβ (Biolegend #581302), or 500 ng/mL PGLYRP1 (R&D system #2590-PGB) were used to stimulate BMDM cells for indicated duration up to 18 h. For heat inactivation, Listeria monocytogenes (Microbiologics Cat#01249 UV-V) were suspended in PBS, pH 7.4, and centrifuged at 10,000 × g for 10 min.

Techniques: Western Blot, Expressing

Journal: Nature Communications

Article Title: PGLYRP1-mediated intracellular peptidoglycan detection promotes intestinal mucosal protection

doi: 10.1038/s41467-025-57126-9

Figure Lengend Snippet: a Weight development of wild type and Pglyrp1 -/- mice during TNBS-induced colitis ( n = 4 per group). Immunofluorescence staining of PGLYRP1 and macrophages (F4/80) in colon sections of wild type and Pglyrp1 -/- mice before ( b ), after TNBS induced colitis ( c , d ) TNBS colitis induction after 3 days of i.p. pretreatment with 100 μg of GMTriP-K in 50 μL PBS at day 9. e Quantitation of F4/80 + macrophages and percentage of PGLYRP1 expressing F4/80 + macrophages during TNBS colitis. Macrophages were counted in 4 independent regions per section and quantitated using Imaris 10.1.1 image analysis software. f Staining for PGLYRP1 and SEC61β in the colon of wild type mice with TNBS colitis. g qPCR analysis of gene expression in mesenteric lymph nodes of wild type and Pglyrp1 -/- mice on day 9 of the TNBS and GMTPriP-K treatment protocol ( n = 3–5). Scale bars represent 20 μm in ( b , c , d) and 2 μm in ( g ). Data are presented as mean ± SEM with indicated P values analyzed by one-way ANOVA. All experiments were repeated twice and yielded consistent results.

Article Snippet: 25 μM GMTriP-K, GMDiP, or MDP (Invivogen; Cat# tlrl-mdp), 100 ng/mL IFNγ (Biolegend #575304), 100 ng/mL IFNβ (Biolegend #581302), or 500 ng/mL PGLYRP1 (R&D system #2590-PGB) were used to stimulate BMDM cells for indicated duration up to 18 h. For heat inactivation, Listeria monocytogenes (Microbiologics Cat#01249 UV-V) were suspended in PBS, pH 7.4, and centrifuged at 10,000 × g for 10 min.

Techniques: Immunofluorescence, Staining, Quantitation Assay, Expressing, Software, Gene Expression

Journal: Nature Communications

Article Title: PGLYRP1-mediated intracellular peptidoglycan detection promotes intestinal mucosal protection

doi: 10.1038/s41467-025-57126-9

Figure Lengend Snippet: a Immunofluorescence staining of PGLYRP1 and macrophages (CD68) in tissue sections of healthy human colon and active ulcerative colitis (arrow indicates immune cells that crossed the epithelial barrier, cr, Crypt.). b Quantitation of CD68 + macrophages and percentage of PGLYRP1 expressing CD68 + macrophages in the normal colon and ulcerative colitis lamina propria. Macrophages were counted in 4 independent regions per sample and quantitated using Imaris 10.1.1 image analysis software. c Hierarchal cluster analysis of genes that were significantly regulated in active, inactive ulcerative colitis and healthy control colons. d Volcano plot analysis of gene expression comparing gene expression in healthy control and active ulcerative colitis. e qPCR analysis of expression of indicated genes in healthy control ( n = 6), inactive ( n = 8), and active ulcerative colitis ( n = 6) samples. Scale bars represent 25 μm. Data are presented as mean ± SEM with indicated P values analyzed by one-way ANOVA. Student’s test was used to compare control and UC samples. All experiments were repeated at least twice and yielded consistent results.

Article Snippet: 25 μM GMTriP-K, GMDiP, or MDP (Invivogen; Cat# tlrl-mdp), 100 ng/mL IFNγ (Biolegend #575304), 100 ng/mL IFNβ (Biolegend #581302), or 500 ng/mL PGLYRP1 (R&D system #2590-PGB) were used to stimulate BMDM cells for indicated duration up to 18 h. For heat inactivation, Listeria monocytogenes (Microbiologics Cat#01249 UV-V) were suspended in PBS, pH 7.4, and centrifuged at 10,000 × g for 10 min.

Techniques: Immunofluorescence, Staining, Quantitation Assay, Expressing, Software, Control, Gene Expression

Journal: Nature Communications

Article Title: PGLYRP1-mediated intracellular peptidoglycan detection promotes intestinal mucosal protection

doi: 10.1038/s41467-025-57126-9

Figure Lengend Snippet: PGLYRP1 is required for detection of GMTriP-K. PGLYRP1 localizes to the endoplasmic reticulum and Golgi and interacts with NOD2 and GEF-H1 to induce the expression of immune regulators that regulate intestinal inflammation (Created in BioRender. Reinecker, H. (2025) https://BioRender.com/i55t156 ).

Article Snippet: 25 μM GMTriP-K, GMDiP, or MDP (Invivogen; Cat# tlrl-mdp), 100 ng/mL IFNγ (Biolegend #575304), 100 ng/mL IFNβ (Biolegend #581302), or 500 ng/mL PGLYRP1 (R&D system #2590-PGB) were used to stimulate BMDM cells for indicated duration up to 18 h. For heat inactivation, Listeria monocytogenes (Microbiologics Cat#01249 UV-V) were suspended in PBS, pH 7.4, and centrifuged at 10,000 × g for 10 min.

Techniques: Expressing

Journal: Nature Communications

Article Title: PGLYRP1-mediated intracellular peptidoglycan detection promotes intestinal mucosal protection

doi: 10.1038/s41467-025-57126-9

Figure Lengend Snippet: a Peptidoglycan fragment library: GMTriP-K (1) and MDP (2); synthetic peptidoglycan fragments (1a-b, 2a-h and 3a-d) were all prepared with amine linkage points for attachment to the array surface via NHS-chemistry. b General workflow for the printing, incubation, and analysis of the PGN small fragment microarray (Created in BioRender. Putnik, R. (2025) https://BioRender.com/x50o125 ). c Microarray binding of PGLYRPs. PGLYRP1 binds to the disaccharide components of the array, whereas PGLYRP3 and PGLYRP4 show no association with the compounds on the array. d Determination of Apparent Dissociation Constant for PGLYRP1 to GMTriP-K (3 independent experiments were carried out with each experiment done in technical replicates). e Alphafold protein structure prediction of human PGLYRP1. f – i Docking prediction of GMTriP-K to human PGLYRP1. j Docking-prediction of PGLYRP3 and GMTriP-K demonstrating shallow GlcNAc preventing interaction. For synthetic procedures and compound characterization (NMRs, HRMS), please see the . For ( c ), each condition was screened in at least biological triplicate and technical replicated (see for raw image files and other biologically replicate binding data). Data are presented as mean ± SEM. All experiments were repeated twice and yielded consistent results.

Article Snippet: Recombinant human PGLYRP1 (R&D Systems, Minneapolis, MN) was applied to the subarrays at various concentrations (typically 10 μg/mL) in PBST, and the slide was sealed with adhesive film and incubated at RT for 18 h. Arrays were washed thoroughly with PBST and patted dry.

Techniques: Incubation, Microarray, Binding Assay

Journal: Nature Communications

Article Title: PGLYRP1-mediated intracellular peptidoglycan detection promotes intestinal mucosal protection

doi: 10.1038/s41467-025-57126-9

Figure Lengend Snippet: a RNA-seq and volcano plot analysis of gene expression in BMDMs from wild type and Pglyrp1 -/- mice after 18 h of stimulation with 25 µM GMTriP-K. b Volcano plot analysis of gene expression in BMDMs from wild type and NOD2 -/- mice after 18 h of stimulation with 25 µM GMTriP-K. c Volcano plot analysis of gene expression in BMDMs from wild type mice after 18 h of stimulation with 25 µM GMTriP-K or 25 µM MDP. d Hierarchal cluster analysis of genes induced by either stimulus in wild type or Pglyrp1 -/- BMDMs. e Hierarchical cluster analysis of genes induced by GMTriP-K in BMDMs from wild type, Pglyrp1 -/-, Nod2-/- , or Arhgef2 -/- mice. f qPCR analysis of gene expression in BMDMs from indicated mouse strains stimulated with 25 µM GMTriP-K or 25 µM GMDiP stimulation on BMDMs from wild type and Pylyrp1-/- mice for 18 h ( n = 3). g , h Gene set enrichment analysis of the differentially expressed genes induced by indicated stimulus in wild type BMDM. g Top 25 enriched signaling pathways for the differentially expressed gene sets. h Gene set enrichment analysis summarized in mountain plots representing significantly enriched (left) or depleted (right) of genes for the indicated gene sets and collections. Data are presented as mean ± SEM with indicated P values analyzed by one-way ANOVA. All experiments were repeated twice and yielded consistent results.

Article Snippet: Recombinant human PGLYRP1 (R&D Systems, Minneapolis, MN) was applied to the subarrays at various concentrations (typically 10 μg/mL) in PBST, and the slide was sealed with adhesive film and incubated at RT for 18 h. Arrays were washed thoroughly with PBST and patted dry.

Techniques: RNA Sequencing, Gene Expression, Protein-Protein interactions

Journal: Nature Communications

Article Title: PGLYRP1-mediated intracellular peptidoglycan detection promotes intestinal mucosal protection

doi: 10.1038/s41467-025-57126-9

Figure Lengend Snippet: a Hierarchal clustering of MDP induced genes in BMDMs from wild-type, Pglyrp1 -/- , Nod2 -/- , or Arhgef2 -/- mice. b , c BMDMs from wild type, Pglyrp1 -/ , Nod2 -/- , or Arhgef2 -/- mice were stimulated with 25 μM GMTriP-K or 25 μM MDP and analyzed via western blotting with the indicated antibodies. d BMDMs from wild type or Ifnar1 -/- mice were stimulated with 25 μM GMTriP-K or 25 μM MDP for 18 h and analyzed for STAT1 pathway activation with phospho-specific antibodies as indicated. e Wild type or GEF-H1-deficient BMDMs were treated with 100 ng/mL of IFNγ or IFNβ for 18 h and analyzed via western blotting with the indicated antibodies. f BMDMs from wild type, Pglyrp1 -/ , and Nod2 -/- mice were stimulated with 25 μM GMTriP-K or 25 μM MDP, lysed, and analyzed via western blotting with the indicated antibodies. g qPCR analysis of gene expression in BMDMs from indicated mouse strains stimulated with 25 µM GMTriP-K or 25 µM GMDiP for 18 h. Data are presented as mean ± SEM with indicated P values analyzed by one-way ANOVA. All experiments were repeated twice and yielded consistent results.

Article Snippet: Recombinant human PGLYRP1 (R&D Systems, Minneapolis, MN) was applied to the subarrays at various concentrations (typically 10 μg/mL) in PBST, and the slide was sealed with adhesive film and incubated at RT for 18 h. Arrays were washed thoroughly with PBST and patted dry.

Techniques: Western Blot, Activation Assay, Gene Expression

Journal: Nature Communications

Article Title: PGLYRP1-mediated intracellular peptidoglycan detection promotes intestinal mucosal protection

doi: 10.1038/s41467-025-57126-9

Figure Lengend Snippet: a – k Representative confocal microscopy images of HEK 293T cells expressing untagged (UT) or Flag-tagged PGLYRP1, orange fluorescent protein (OFP)-tagged SEC61β and GFP-tagged NOD2. Cells were transfected with PGLYRP1-UT ( a – d ) or PGLYRP1-Flag ( e – k ) and SEC61β-OFP and then probed with PGLYRP1 and GM130 antibodies. PGLYRP1, SEC61β-OFP, and GM130 were detected in Alexa Fluor 488 (green pseudo-color), mOrange (red), and Alexa Fluor 647 (blue) channels, respectively. l – y Cells were transfected with PGLYRP1-UT and NOD2-GFP and stained for PGLYRP1 and GM130. NOD2-GFP, PGLYRP1, and GM130 were detected in EGFP (green pseudo-color), Rhodamine Red-X (red), and Alexa Fluor 647 (blue) channels, respectively. All channels were scanned sequentially, and images are pseudo-colored independent of channel wavelengths with nuclear counterstain in greyscale (Scale bars indicate 2 μm in ( a – d, l – r ) and 5 μm in ( e – k , s – y )). Typically, 10-12 cells were analyzed for each condition. Data are presented as mean ± SEM Pearson’s coefficients were calculated in Imaris 10.1.1 image analysis software. All experiments were repeated twice and yielded consistent results.

Article Snippet: Recombinant human PGLYRP1 (R&D Systems, Minneapolis, MN) was applied to the subarrays at various concentrations (typically 10 μg/mL) in PBST, and the slide was sealed with adhesive film and incubated at RT for 18 h. Arrays were washed thoroughly with PBST and patted dry.

Techniques: Confocal Microscopy, Expressing, Transfection, Staining, Software

Journal: Nature Communications

Article Title: PGLYRP1-mediated intracellular peptidoglycan detection promotes intestinal mucosal protection

doi: 10.1038/s41467-025-57126-9

Figure Lengend Snippet: a Western blot analysis of protein expression in BMDMs from wildtype, Arhgef2-/- , Pglyrp1-/- , and Nod2-/- mice. b Time course of protein interactions with GEF-H1 after GMTriP-K stimulation of BMDMs. c Analysis of protein interactions with PGLYRP1 or d NOD2 after GMTriP-K stimulation of BMDMs. e Input assessment of protein expression for the IPs in ( c , d ). f Assessment of protein interactions with GEF-H1 after stimulation with indicated immune stimuli. Representative experiments of at least three repeats are shown.

Article Snippet: Recombinant human PGLYRP1 (R&D Systems, Minneapolis, MN) was applied to the subarrays at various concentrations (typically 10 μg/mL) in PBST, and the slide was sealed with adhesive film and incubated at RT for 18 h. Arrays were washed thoroughly with PBST and patted dry.

Techniques: Western Blot, Expressing

Journal: Nature Communications

Article Title: PGLYRP1-mediated intracellular peptidoglycan detection promotes intestinal mucosal protection

doi: 10.1038/s41467-025-57126-9

Figure Lengend Snippet: a Weight development of wild type and Pglyrp1 -/- mice during TNBS-induced colitis ( n = 4 per group). Immunofluorescence staining of PGLYRP1 and macrophages (F4/80) in colon sections of wild type and Pglyrp1 -/- mice before ( b ), after TNBS induced colitis ( c , d ) TNBS colitis induction after 3 days of i.p. pretreatment with 100 μg of GMTriP-K in 50 μL PBS at day 9. e Quantitation of F4/80 + macrophages and percentage of PGLYRP1 expressing F4/80 + macrophages during TNBS colitis. Macrophages were counted in 4 independent regions per section and quantitated using Imaris 10.1.1 image analysis software. f Staining for PGLYRP1 and SEC61β in the colon of wild type mice with TNBS colitis. g qPCR analysis of gene expression in mesenteric lymph nodes of wild type and Pglyrp1 -/- mice on day 9 of the TNBS and GMTPriP-K treatment protocol ( n = 3–5). Scale bars represent 20 μm in ( b , c , d) and 2 μm in ( g ). Data are presented as mean ± SEM with indicated P values analyzed by one-way ANOVA. All experiments were repeated twice and yielded consistent results.

Article Snippet: Recombinant human PGLYRP1 (R&D Systems, Minneapolis, MN) was applied to the subarrays at various concentrations (typically 10 μg/mL) in PBST, and the slide was sealed with adhesive film and incubated at RT for 18 h. Arrays were washed thoroughly with PBST and patted dry.

Techniques: Immunofluorescence, Staining, Quantitation Assay, Expressing, Software, Gene Expression

Journal: Nature Communications

Article Title: PGLYRP1-mediated intracellular peptidoglycan detection promotes intestinal mucosal protection

doi: 10.1038/s41467-025-57126-9

Figure Lengend Snippet: a Immunofluorescence staining of PGLYRP1 and macrophages (CD68) in tissue sections of healthy human colon and active ulcerative colitis (arrow indicates immune cells that crossed the epithelial barrier, cr, Crypt.). b Quantitation of CD68 + macrophages and percentage of PGLYRP1 expressing CD68 + macrophages in the normal colon and ulcerative colitis lamina propria. Macrophages were counted in 4 independent regions per sample and quantitated using Imaris 10.1.1 image analysis software. c Hierarchal cluster analysis of genes that were significantly regulated in active, inactive ulcerative colitis and healthy control colons. d Volcano plot analysis of gene expression comparing gene expression in healthy control and active ulcerative colitis. e qPCR analysis of expression of indicated genes in healthy control ( n = 6), inactive ( n = 8), and active ulcerative colitis ( n = 6) samples. Scale bars represent 25 μm. Data are presented as mean ± SEM with indicated P values analyzed by one-way ANOVA. Student’s test was used to compare control and UC samples. All experiments were repeated at least twice and yielded consistent results.

Article Snippet: Recombinant human PGLYRP1 (R&D Systems, Minneapolis, MN) was applied to the subarrays at various concentrations (typically 10 μg/mL) in PBST, and the slide was sealed with adhesive film and incubated at RT for 18 h. Arrays were washed thoroughly with PBST and patted dry.

Techniques: Immunofluorescence, Staining, Quantitation Assay, Expressing, Software, Control, Gene Expression

Journal: Nature Communications

Article Title: PGLYRP1-mediated intracellular peptidoglycan detection promotes intestinal mucosal protection

doi: 10.1038/s41467-025-57126-9

Figure Lengend Snippet: PGLYRP1 is required for detection of GMTriP-K. PGLYRP1 localizes to the endoplasmic reticulum and Golgi and interacts with NOD2 and GEF-H1 to induce the expression of immune regulators that regulate intestinal inflammation (Created in BioRender. Reinecker, H. (2025) https://BioRender.com/i55t156 ).

Article Snippet: Recombinant human PGLYRP1 (R&D Systems, Minneapolis, MN) was applied to the subarrays at various concentrations (typically 10 μg/mL) in PBST, and the slide was sealed with adhesive film and incubated at RT for 18 h. Arrays were washed thoroughly with PBST and patted dry.

Techniques: Expressing