The Prominent Role of Hematopoietic Peptidyl Arginine Deiminase 4 in Arthritis: Collagen- and Granulocyte Colony-Stimulating Factor-Induced Arthritis Model in C57BL/6 Mice.

OBJECTIVE: Genome-wide association studies have connected PADI4, encoding peptidylarginine deiminase 4 (PAD4), with rheumatoid arthritis (RA). PAD4 promotes neutrophil extracellular trap (NET) formation. This study was undertaken to investigate the origin of PAD4 and the importance of NET formation in a C57BL/6 mouse model of arthritis. METHODS: To permit the effective use of C57BL/6 mice in the collagen-induced arthritis (CIA) model, we introduced the administration of granulocyte colony-stimulating factor (G-CSF) for 4 consecutive days in conjunction with the booster immunization on day 21. Mice with global Padi4 deficiency (Padi4-/- ) and mice with hematopoietic lineage-specific Padi4 deficiency (Padi4Vav1Cre/+ ) were evaluated in the model. RESULTS: G-CSF significantly increased the incidence and severity of CIA. G-CSF-treated mice showed elevated citrullinated histone H3 (Cit-H3) levels in plasma, while vehicle-treated mice did not. Immunofluorescence microscopy revealed deposition of Cit-H3 in synovial tissue in G-CSF-treated mice. Padi4-/- mice developed less severe arthritis and had lower levels of serum interleukin-6 and plasma Cit-H3, lower levels of Cit-H4 in synovial tissue, and less bone erosion on micro-computed tomography than Padi4+/+ mice in the G-CSF-modified CIA model. Similarly, Padi4Vav1Cre/+ mice developed less severe arthritis, compared with Padi4fl/fl mice, and presented the same phenotype as Padi4-/- mice. CONCLUSION: We succeeded in developing an arthritis model suitable for use in C57BL/6 mice that is fully compliant with high animal welfare standards. We observed a >90% incidence of arthritis in male mice and detectable NET markers. This model, with some features consistent with human RA, demonstrates that hematopoietic PAD4 is an important contributor to arthritis development and may prove useful in future RA research.

RhoA/Cdc42 signaling drives cytoplasmic maturation but not endomitosis in megakaryocytes.

Megakaryocytes (MKs), the precursors of blood platelets, are large, polyploid cells residing mainly in the bone marrow. We have previously shown that balanced signaling of the Rho GTPases RhoA and Cdc42 is critical for correct MK localization at bone marrow sinusoids in vivo. Using conditional RhoA/Cdc42 double-knockout (DKO) mice, we reveal here that RhoA/Cdc42 signaling is dispensable for the process of polyploidization in MKs but essential for cytoplasmic MK maturation. Proplatelet formation is virtually abrogated in the absence of RhoA/Cdc42 and leads to severe macrothrombocytopenia in DKO animals. The MK maturation defect is associated with downregulation of myosin light chain 2 (MLC2) and ß1-tubulin, as well as an upregulation of LIM kinase 1 and cofilin-1 at both the mRNA and protein level and can be linked to impaired MKL1/SRF signaling. Our findings demonstrate that MK endomitosis and cytoplasmic maturation are separately regulated processes, and the latter is critically controlled by RhoA/Cdc42.

Extracellular DNA NET-Works With Dire Consequences for Health.

Neutrophils play a central role in innate immune defense. Advances in neutrophil biology have brought to light the capacity of neutrophils to release their decondensed chromatin and form large extracellular DNA networks called neutrophil extracellular traps (NETs). NETs are produced in response to many infectious and noninfectious stimuli and, together with fibrin, block the invasion of pathogens. However, their formation in inflamed blood vessels produces a scaffold that supports thrombosis, generates neo-antigens favoring autoimmunity, and aggravates damage in ischemia/reperfusion injury. NET formation can also be induced by cancer and promotes tumor progression. Formation of NETs within organs can be immediately detrimental, such as in lung alveoli, where they affect respiration, or they can be harmful over longer periods of time. For example, NETs initiate excessive deposition of collagen, resulting in fibrosis, thus likely contributing to heart failure. Here, we summarize the latest knowledge on NET generation and discuss how excessive NET formation mediates propagation of thrombosis and inflammation and, thereby, contributes to various diseases. There are many ways in which NET formation could be averted or NETs neutralized to prevent their detrimental consequences, and we will provide an overview of these possibilities.

Rho GTPases and their downstream effectors in megakaryocyte biology.

Megakaryocytes differentiate from hematopoietic stem cells in the bone marrow. The transition of megakaryocytes to platelets is a complex process. Thereby, megakaryocytes extend proplatelets into sinusoidal blood vessels, where the proplatelets undergo fission to release platelets. Defects in platelet production can lead to a low platelet count (thrombocytopenia) with increased bleeding risk. Rho GTPases comprise a family of small signaling G proteins that have been shown to be master regulators of the cytoskeleton controlling many aspects of intracellular processes. The generation of Pf4-Cre transgenic mice was a major breakthrough that enabled studies in megakaryocyte-/platelet-specific knockout mouse lines and provided new insights into the central regulatory role of Rho GTPases in megakaryocyte maturation and platelet production. In this review, we will summarize major findings on the role of Rho GTPases in megakaryocyte biology with a focus on mouse lines in which knockout strategies have been applied to study the function of the best-characterized members Rac1, Cdc42 and RhoA and their downstream effector proteins.

Increased neutrophil extracellular trap formation promotes thrombosis in myeloproliferative neoplasms.

Thrombosis is a major cause of morbidity and mortality in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), clonal disorders of hematopoiesis characterized by activated Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling. Neutrophil extracellular trap (NET) formation, a component of innate immunity, has been linked to thrombosis. We demonstrate that neutrophils from patients with MPNs are primed for NET formation, an effect blunted by pharmacological inhibition of JAK signaling. Mice with conditional knock-in of Jak2V617F, the most common molecular driver of MPN, have an increased propensity for NET formation and thrombosis. Inhibition of JAK-STAT signaling with the clinically available JAK2 inhibitor ruxolitinib abrogated NET formation and reduced thrombosis in a deep vein stenosis murine model. We further show that expression of PAD4, a protein required for NET formation, is increased in JAK2V617F-expressing neutrophils and that PAD4 is required for Jak2V617F-driven NET formation and thrombosis in vivo. Finally, in a population study of more than 10,000 individuals without a known myeloid disorder, JAK2V617F-positive clonal hematopoiesis was associated with an increased incidence of thrombosis. In aggregate, our results link JAK2V617F expression to NET formation and thrombosis and suggest that JAK2 inhibition may reduce thrombosis in MPNs through cell-intrinsic effects on neutrophil function.

SLAP/SLAP2 prevent excessive platelet (hem)ITAM signaling in thrombosis and ischemic stroke in mice.

Glycoprotein VI and C-type lectin-like receptor 2 are essential platelet activating receptors in hemostasis and thrombo-inflammatory disease, which signal through a (hem)immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway. The adapter molecules Src-like adapter proteins (SLAP and SLAP2) are involved in the regulation of immune cell surface expression and signaling, but their function in platelets is unknown. In this study, we show that platelets expressed both SLAP isoforms and that overexpression of either protein in a heterologous cell line almost completely inhibited glycoprotein VI and C-type lectin-like receptor 2 signaling. In mice, single deficiency of SLAP or SLAP2 had only moderate effects on platelet function, whereas double deficiency of both adapters resulted in markedly increased signal transduction, integrin activation, granule release, aggregation, procoagulant activity, and thrombin generation in response to (hem)ITAM-coupled, but not G protein-coupled, receptor activation. In vivo, constitutive SLAP/SLAP2 knockout mice displayed accelerated occlusive arterial thrombus formation and a dramatically worsened outcome after focal cerebral ischemia. This was attributed to the absence of both adapter proteins in platelets, as demonstrated by adoptive transfer of Slap(-/-)/Slap2(-/-) platelets into wild-type mice. Our results establish SLAP and SLAP2 as critical inhibitors of platelet (hem)ITAM signaling in the setting of arterial thrombosis and ischemic stroke.