β2M Signals Monocytes Through Non-Canonical TGFβ Receptor Signal Transduction.

Rationale: Circulating monocytes can have pro-inflammatory or pro-reparative phenotypes. The endogenous signaling molecules and pathways that regulate monocyte polarization in vivo are poorly understood. We have shown that platelet derived beta-2 microglobulin (β2M) and transforming growth factor beta (TGFβ) have opposing effects on monocytes by inducing inflammatory and reparative phenotypes respectively, but each bind and signal through the same receptor. We now define the signaling pathways involved. Objective: To determine the molecular mechanisms and signal transduction pathways by which β2M and TGFβ regulate monocyte responses both in vitro and in vivo. Methods and Results: Wild-type (WT) and platelet specific β2M knockout (Plt-β2M-/-) mice were treated intravenously with either β2M or TGFβ to increase plasma concentrations to those in cardiovascular diseases. Elevated plasma β2M increased pro-inflammatory monocytes, while increased plasma TGFβ increased pro-reparative monocytes. TGFβ receptor (TGFβR) inhibition blunted monocyte responses to both β2M and TGFβ in vivo. Using imaging flow cytometry, we found that β2M decreased monocyte SMAD2/3 nuclear localization, while TGFβ promoted SMAD nuclear translocation, but decreased noncanonical/ inflammatory (JNK and NFκB nuclear localization). This was confirmed in vitro using both imaging flow cytometry and immunoblots. β2M, but not TGFβ, promoted ubiquitination of SMAD3 and SMAD4, that inhibited their nuclear trafficking. Inhibition of ubiquitin ligase activity blocked noncanonical SMAD-independent monocyte signaling and skewed monocytes towards a pro-reparative monocyte response. Conclusions: Our findings indicate that elevated plasma β2M and TGFβ dichotomously polarize monocytes. Furthermore, these immune molecules share a common receptor, but induce SMAD-dependent canonical signaling (TGFβ) versus non-canonical SMAD-independent signaling (β2M) in a ubiquitin ligase dependent manner. This work has broad implications as β2M is increased in several inflammatory conditions, while TGFβ is increased in fibrotic diseases.pre-print3451 K

Lung Megakaryocytes are Immune Modulatory Cells that Present Antigen to CD4+ T cells.

Although platelets are the cellular mediators of thrombosis, they are also immune cells. Platelets interact both directly and indirectly with immune cells, impacting their activation and differentiation, as well as all phases of the immune response. Megakaryocytes (Mks) are the cell source of circulating platelets, and until recently Mks were typically only considered bone marrow–resident (BM-resident) cells. However, platelet-producing Mks also reside in the lung, and lung Mks express greater levels of immune molecules compared with BM Mks. We therefore sought to define the immune functions of lung Mks. Using single-cell RNA sequencing of BM and lung myeloid-enriched cells, we found that lung Mks, which we term MkL, had gene expression patterns that are similar to antigen-presenting cells. This was confirmed using imaging and conventional flow cytometry. The immune phenotype of Mks was plastic and driven by the tissue immune environment, as evidenced by BM Mks having an MkL-like phenotype under the influence of pathogen receptor challenge and lung-associated immune molecules, such as IL-33. Our in vitro and in vivo assays demonstrated that MkL internalized and processed both antigenic proteins and bacterial pathogens. Furthermore, MkL induced CD4+ T cell activation in an MHC II–dependent manner both in vitro and in vivo. These data indicated that MkL had key immune regulatory roles dictated in part by the tissue environment.pre-print236 K

Genetic and clinical basis for two distinct subtypes of primary Sjögren's syndrome

Objectives Clinical presentation of primary Sjögren’s syndrome (pSS) varies considerably. A shortage of evidence-based objective markers hinders efficient drug development and most clinical trials have failed to reach primary endpoints. Methods We performed a multicentre study to identify patient subgroups based on clinical, immunological and genetic features. Targeted DNA sequencing of 1853 autoimmune-related loci was performed. After quality control, 918 patients with pSS, 1264 controls and 107 045 single nucleotide variants remained for analysis. Replication was performed in 177 patients with pSS and 7672 controls. Results We found strong signals of association with pSS in the HLA region. Principal component analysis of clinical data distinguished two patient subgroups defined by the presence of SSA/SSB antibodies. We observed an unprecedented high risk of pSS for an association in the HLA-DQA1 locus of odds ratio 6.10 (95% CI: 4.93, 7.54, P=2.2×10−62) in the SSA/SSB-positive subgroup, while absent in the antibody negative group. Three independent signals within the MHC were observed. The two most significant variants in MHC class I and II respectively, identified patients with a higher risk of hypergammaglobulinaemia, leukopenia, anaemia, purpura, major salivary gland swelling and lymphadenopathy. Replication confirmed the association with both MHC class I and II signals confined to SSA/SSB antibody positive pSS. Conclusion Two subgroups of patients with pSS with distinct clinical manifestations can be defined by the presence or absence of SSA/SSB antibodies and genetic markers in the HLA locus. These subgroups should be considered in clinical follow-up, drug development and trial outcomes, for the benefit of both subgroups.publishedVersio

Glutamate Receptor Interacting Protein 1 Mediates Platelet Adhesion and Thrombus Formation.

Thrombosis-associated pathologies, such as myocardial infarction and stroke, are major causes of morbidity and mortality worldwide. Because platelets are necessary for hemostasis and thrombosis, platelet directed therapies must balance inhibiting platelet function with bleeding risk. Glutamate receptor interacting protein 1 (GRIP1) is a large scaffolding protein that localizes and organizes interacting proteins in other cells, such as neurons. We have investigated the role of GRIP1 in platelet function to determine its role as a molecular scaffold in thrombus formation. Platelet-specific GRIP1-/- mice were used to determine the role of GRIP1 in platelets. GRIP1-/- mice had normal platelet counts, but a prolonged bleeding time and delayed thrombus formation in a FeCl3-induced vessel injury model. In vitro stimulation of WT and GRIP1-/- platelets with multiple agonists showed no difference in platelet activation. However, in vivo platelet rolling velocity after endothelial stimulation was significantly greater in GRIP1-/- platelets compared to WT platelets, indicating a potential platelet adhesion defect. Mass spectrometry analysis of GRIP1 platelet immunoprecipitation revealed enrichment of GRIP1 binding to GPIb-IX complex proteins. Western blots confirmed the mass spectrometry findings that GRIP1 interacts with GPIbα, GPIbβ, and 14-3-3. Additionally, in resting GRIP1-/- platelets, GPIbα and 14-3-3 have increased interaction compared to WT platelets. GRIP1 interactions with the GPIb-IX binding complex are necessary for normal platelet adhesion to a stimulated endothelium

Proteins highly enriched in mass spectrometry analysis with known roles in platelet function (light gray indicates interact with GPIb complex, darker shading indicates other functional platelet protein interactions).

<p>Proteins highly enriched in mass spectrometry analysis with known roles in platelet function (light gray indicates interact with GPIb complex, darker shading indicates other functional platelet protein interactions).</p

GRIP1^-/- platelets do not have decreased velocity in ionophore-stimulated blood vessels.

<p>A) GRIP1^-/- platelets do not have a decreased velocity in an ionophore-stimulated pinna vessel. WT murine pinna vessels were stimulated with A23187 and the change in fluorescent-labeled WT and GRIP1^-/- platelet velocity determined over time. As a control, platelets were treated with GPIbα blocking antibody (N = 4; *P < 0.05 by students T-test GRIP1^-/- vs WT). B) Representative images of WT and GRIP1^-/- platelets before and 6 minutes after vessel ionophore treatment (smaller, rounder platelets are rolling (arrow), magnification 20X). C) GRIP1^-/- platelets do not have decreased velocity in an ionophore-stimulated mesenteric vessel (N = 4 ± S.E.M; *P < 0.05 by students T-test GRIP1^-/- vs WT). D) Representative images of WT and GRIP1^-/- platelets before and after ionophore treatment in mesenteric arterioles (smaller, rounder platelets are rolling (arrow), magnification 20X). E) GRIP1^-/- platelets have a trend toward reduced vWF adhesion in vitro. Platelets in whole blood from WT and GRIP1^-/- mice were labeled with a fluorescent antibody. Blood was then pumped over a mouse vWF-coated MatTek chamber. Platelet adhesion was determined at multiple time points (N = 4–6 per time point, P < 0.1 at 15 sec).</p

GRIP1 interacts with the GPIb-IX complex.

<p>A) GRIP1 protein interactions in platelets. GRIP1 was immunoprecipitated from WT and GRIP1^-/- (negative control) platelets and the precipitate analyzed by mass spectrometry. Proteins were assigned by Panther into GO functional groups. B) GPIbα and GPIbβ surface expression is similar in WT and GRIP1^-/- platelets. GPIbα and GPIbβ surface expression was determined by flow cytometry (N = 8–13, ± S.E.M; NS by students T-test).</p

Platelets express GRIP1.

<p>A) Human platelets express GRIP1 (representative immunoblot with mouse brain as positive control). B) PF4-Cre⁺ GRIP1^fl/fl mice have reduced platelet GRIP1 expression. Immunoprecipitation of WT and GRIP1^-/- mouse platelets using anti-GRIP1 antibody and immunoblot for GRIP1. C) Representative histogram of GRIP1 expression in WT and GRIP1^-/- mouse platelets by intracellular flow cytometry. D) WT and GRIP1^-/- mouse platelets have similar platelet counts. (± S.E.M., NS by students T-test). E) WT and GRIP1^-/- platelets have similar morphology (representative electron microscopy of resting platelets).</p

<i>In vitro</i> platelet activation is normal in GRIP1^-/- platelets.

<p>A) WT and GRIP1^-/- platelets have similar agonist-induced activation. Washed platelets were incubated with control buffer, 50 ng/mL convulxin, or 0.5 U/mL thrombin and P-selectin expression was determined by flow cytometry (N = 4; ± S.E.M., NS by students T-test between WT and GRIP1^-/-). B) PF4 release from stimulated WT and GRIP1^-/- platelets. Washed platelets were stimulated with thrombin or 2-meADP for 10 min. and PF4 release measured by ELISA (N = 8; ± S.E.M, N.S. by students—test). C) Washed WT and GRIP1^-/- platelets were thrombin-stimulated and ATP release was measured (N = 8; ± S.E.M, N.S. by students T-test). D) Washed WT and GRIP1^-/- platelets have similar GPIIb/IIIa activation. Platelets were stimulated with thrombin or 2-meADP for 10 min. Activated GPIIb/IIIa expression was measured by JON/A antibody binding. (N = 4; ± S.E.M, N.S. by students T-test). E) PRP from WT or GRIP1^-/- mice was incubated with either PE or APC labeled anti-CD9 antibody. PE and APC labeled PRP from mice of the same genotype was then mixed. Control buffer, thrombin or 2-meADP were added and incubated with orbital shaking at 37°C for 15 minutes. Double positive (APC and PE) platelet aggregates were quantified by flow cytometry. GRIP1^-/- platelets stimulated with low dose thrombin had a trend to fewer platelet aggregates compared to WT (N = 6–8). WT and GRIP1^-/- platelets stimulated with 2-meADP had similar aggregation.</p

GRIP1^-/- mice have hemostasis and thrombosis defects.

<p>A) WT and platelet GRIP1^-/- mouse bleeding time. Percentage of patent vessels after 3 mm tail tip amputation (N = 25–37; P < 0.05 for Kaplan-Meier curve). B) Platelet GRIP1^-/- mice have reduced thrombus size. FeCl₃-induced mesenteric artery thrombosis. Fluorescent thrombus burden expressed as percentage of vessel area (N = 11–17; ± S.E.M., *P < 0.05 by students T-test). C) Representative image of thrombus formation in WT and platelet GRIP1^-/- mice 10 min. after injury (dashed lines represent vessel edges, magnification 20X).</p