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