The Deubiquitinase OTUD1 Suppresses Secretory Neutrophil Polarization And Ameliorates Immunopathology of Periodontitis

Abstract Tissue‐infiltrating neutrophils (TINs) secrete various signaling molecules to establish paracrine communication within the inflammatory milieu. It is imperative to identify molecular mediators that control this secretory phenotype of TINs. The present study uncovers a secretory neutrophil subset that exhibits increased pro‐inflammatory cytokine production and enhanced migratory capacity which is highly related with periodontal pathogenesis. Further analysis identifies the OTU domain‐containing protein 1 (OTUD1) plays a regulatory role in this secretory neutrophil polarization. In human and mouse periodontitis, the waning of inflammation is correlated with OTUD1 upregulation, whereas severe periodontitis is induced when neutrophil‐intrinsic OTUD1 is depleted. Mechanistically, OTUD1 interacts with SEC23B, a component of the coat protein II complex (COPII). By removing the K63‐linked polyubiquitin chains on SEC23B Lysine 81, the deubiquitinase OTUD1 negatively regulates the COPII secretory machinery and limits protein ER‐to‐Golgi trafficking, thus restricting the surface expression of integrin‐regulated proteins, CD9 and CD47. Accordingly, blockade of protein transport by Brefeldin A (BFA) curbs recruitment of Otud1‐deficient TINs and attenuates inflammation‐induced alveolar bone destruction. The results thus identify OTUD1 signaling as a negative feedback loop that limits the polarization of neutrophils with secretory phenotype and highlight the potential application of BFA in the treatment of periodontal inflammation.


B)
Volcano plots analysis of the differentially expressed genes (DEGs) in neutrophils between patients with periodontitis and healthy control.Red, genes upregulated in neutrophils of patients with periodontitis vs. healthy subjects.Blue, genes downregulated in neutrophils of patients with periodontitis vs. healthy subjects.

C)
Violin plots analysis of the differentially expressed genes in neutrophils between patients with periodontitis and healthy control.H, I) RT-qPCR analysis of indicated pro-inflammatory cytokine mRNA levels in gingiva tissues of WT mice that received bone marrow from WT and Otud1 ─/─ mice after periodontitis induction (n = 6, mean ± s.e.m., *P = 0.0430, ***P = 0.0004, twotailed unpaired Student's t-test).
J) A graphic model of bone marrow transplantation assay.WT and Otud1 ─/─ mice were lethally irradiated and received bone marrow cells from WT mice.Experimental periodontitis induction was performed after bone marrow reconstitution.K) Analysis of CEJ-ABC distance around second molar from WT or Otud1 ─/─ mice received WT mice bone marrow obtained by micro-CT imaging (n = 7; mean ± s.e.m., ns, not significant (P > 0.05), two-tailed unpaired Student's t-test).

M, N)
Protein levels of indicated pro-inflammatory cytokines in mice serum in the context of experimental periodontitis (n = 5, mean ± s.e.m., ns, not significant (P > 0.05), two-tailed unpaired Student's t-test).

Q)
A schematic illustration of the macrophage depletion assay.WT or Otud1 ─/─ mice were intravenously injected with clodronate liposomes to delete macrophages.
Experimental periodontitis induction was then performed.

R)
Representative micro-CT sagittal and 3-D Isosurface images of maxillary alveolar bone surrounding the second molar after periodontitis induction in the context of macrophage depletion.

B)
In vivo ubiquitination assay of SEC23B.HEK293T cells were transfected with indicated plasmids and subjected to immunoprecipitation with anti-FLAG antibody followed by western blot analysis.

C)
In vivo ubiquitination assay of SEC23B.Bone marrow derived neutrophils were isolated with WT and Otud1 −/− mice and treated by LPS (100 ng/ml) for 4 hours.
Neutrophils were lysed, endogenous SEC23B was immunoprecipitated by anti-SEC23B antibody and subsequently analyzed by anti-ubiquitin antibody for the assessment of ubiquitination.

D)
A graphic model of retention using selective hooks (RUSH) system to detect protein transport efficiency.In brief, plasmid expressing Ii-streptavidin and Golgin 84-EGFP-SBP was transfected in HEK293T cells.Golgin 84-EGFP-SBP is retained in ER via its interaction with the hook.This interaction is mediated by streptavidin and streptavidin binding protein (SBP).After biotin added, Golgin 84-EGFP-SBP was released and trafficked to Golgi apparatus.D, E) HEK293T cells were transfected with indicated plasmids and were subjected to immunoprecipitation with anti-FLAG antibody followed analysis by western blot.

G)
H) A graphic model illustrating the role of OTUD1 in modulation of neutrophil secretory phenotype.OTUD1 blocks SEC23B ubiquitination and inhibits its association between COPII components, thereby limiting ER-to-Golgi trafficking and sustaining periodontal immune homeostasis.

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