PGLYRP-1 mediated intracellular peptidoglycan detection promotes mucosal protection

Abstract

Peptidoglycan recognition proteins (PGRPs or PGLYRPs) are implicated in the control of the intestinal microbiota; however, molecular requirements for peptidoglycan (PGN) binding and receptor signaling mechanisms remain poorly understood. We identified PGLYRP-1 as a receptor for the disaccharide motif of lysine N-acetylglucosamine N-acetylmuramic tripeptide (GMTriP-K) with a newly constructed PGN microarray. Surprisingly, PGLYRP-1 was required for innate immune activation of macrophages by GMTriP-K but not N-acetylglucosamine N-acetylmuramic dipeptide (GMDiP) or muramyl dipeptide (MDP). In macrophages, intracellular PGLYRP-1 complexed with NOD2 and GEF-H1, both of which were required for GMTriP-K-regulated gene expression. PGLYRP-1 localized to the endoplasmic reticulum and interacted at the Golgi with NOD2 upon GMTriP-K stimulation. PGLYRP-1 upregulation and its dependent gene expression signatures were induced in both mouse intestinal inflammation and human ulcerative colitis. Importantly, PGLYRP-1 activation by GMTriP-K resulted in innate immune activation and protection of mice from colitis. Our results show that PGLYRPs can function as intracellular PGN pattern recognition receptors for the control of host defense responses in the intestine.

Extended data Fig. 1 |. PGLYRP1 is required for type I interferon induction by GMTriP-K

(a) Hierarchal clustering of MDP induced genes in BMDMs from wild-type, Pglyrp1^−/−, Nod2^−/−, or Arhgef2^−/− mice. (b) BMDMs from wild type or Ifnar1^−/− mice were stimulated with 25 μM GMTriP-K or 25 μM MDP for 18 hours and analyzed for STAT1, pathway activation with the indicated phospho-specific antibodies as indicated. (c) and (d) BMDMs from wild type, Pglyrp1^−/−, Nod2^−/−, and Arhgef2^−/− mice were stimulated with 25 μM GMTriP-K or 25 μM MDP, lysed, and analyzed via western blotting with the indicated antibodies. (e) Wild type or GEF-H1-deficient BMDMs were treated with 100 ng/mL of IFNγ or IFNβ for 18 hours, and cells were lysed and analyzed via western blotting with the indicated antibodies. The results represent three independent experiments, and GAPDH was used as loading control. (f) pPCR analysis of gene expression in BMDMs from indicated mouse strains stimulated with 25 μM GMTriP-K or 25 μM GMDiP stimulation for 18 hours.

Fig. 1 |. PGLYRP-1 specifically binds GMTriP-K

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. c, Microarray binding of PGLYRPs. PGLYRP-1 binds to the disaccharide components of the array, whereas PGLYRP-3 and PGLYRP-4 show no association with the compounds on the array. d, Determination of Apparent Dissociation Constant for PGLYRP-1 to GMTriP-K. e, Alphafold protein structure prediction of human PGLYRP-1. f-I, Docking prediction of GMTriP-K to human PGLYRP-1. j, Docking prediction of PGLYRP-3 and GMTriP-K demonstrating shallow GlcNAc preventing interaction. For synthetic procedures and compound characterization (NMRs, HRMS), please see the SI. For (c), each condition was screened in at least biological triplicate and technical replicated (see SI for raw image files and other biological replicate binding data).

Fig. 2 |. PGLYRP-1 is required for GMTriP-K-mediated transcriptional regulation in the NOD2/GEF-H1 pathway

a, RNA-seq and volcano blot analysis of gene expression in BMDMs from wild type and Pglyrp1−/− mice after 18 hours of stimulation with 25 μM GMTriP-K. b, Volcano blot analysis of gene expression in BMDMs from wild type and NOD2−/− mice after 18 hours of stimulation with 25 μM GMTriP-K. c, Volcano blot analysis of gene expression in BMDMs from wild type mice after 18 hours 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, pPCR 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 Pylyrp-1−/− mice for 18 hours. 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. Experiments were repeated at least twice.

Fig. 3 |. PGLYRP-1 localizes to the ER and Golgi

a-k, Representative confocal microscopy images of HEK 293T cells expressing untagged (UT) or Flag-tagged PGLYRP-1, orange fluorescent protein (OFP)-tagged Sec61β and GFP-tagged NOD2. Cells were transfected with PGLYRP-1-UT (a-d) or PGLYRP-1-Flag (e-k) and Sec61β-OFP, and then probed with PGLYRP-1 and GM130 antibodies. PGLYRP-1, 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 PGLYRP-1-UT and NOD2-GFP, and then stained for PGLYRP-1 and GM130. NOD2-GFP, PGLYRP-1 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 (bars indicate 2μm in a-d, l-r and 5μm in e-k, s-y). Each imaged cell typically represents 12 independent experiments with 10–12 cells analyzed for each condition.

Fig. 4 |. PGLYRP-1 interacts with NOD2 and GEF-H1 in ER- and Golgi-associated compartments

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 PGLYRP-1 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.

Fig. 5 |. PGLYRP-1 regulates immune responses in TNBS induced colitis

a, Weight development of wild type and Pglyrp1−/− mice during TNBS-induced colitis. Immunostaining of PGLYRP-1 and macrophages (F4/80) in colon sections of wild type and Pglyrp1−/− mice before (b) after TNBS induced colitis (c) and TNBS colitis induction after 3 days of GMTriP-K pretreatment (d) at day 9 (bars indicate 25μm). (e) 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. (f) qPCR analysis of gene expression in BMDMs from wild-type or Pglyrp1−/− mice after stimulation with GMTriP-K in the presence of the indicated amount of PGLYRP-1.