SARS-CoV-2 infection modifies the transcriptome of the megakaryocytes in the bone marrow.

Megakaryocytes, integral to platelet production, predominantly reside in the bone marrow and undergo regulated fragmentation within sinusoid vessels to release platelets into the bloodstream. Inflammatory states and infections influence megakaryocyte transcription, potentially affecting platelet functionality. Notably, COVID-19 has been associated with altered platelet transcriptomes. In this study, we investigated the hypothesis that SARS-CoV-2 infection could impact the transcriptome of bone marrow megakaryocytes. Utilizing spatial transcriptomics to discriminate subpopulations of megakaryocytes based on proximity to bone marrow sinusoids, we identified approximately 19,000 genes in megakaryocytes. Machine learning techniques revealed that the transcriptome of healthy murine bone marrow megakaryocytes exhibited minimal differences based on proximity to sinusoid vessels. Further, at peak SARS-CoV-2 viremia, when the disease primarily affected the lungs, megakaryocytes were not significantly different from those from healthy mice. Conversely, a significant divergence in the megakaryocyte transcriptome was observed during systemic inflammation, although SARS-CoV-2 RNA was never detected in bone marrow and it was no longer detectable in the lungs. Under these conditions, the megakaryocyte transcriptional landscape was enriched in pathways associated with histone modifications, megakaryocyte differentiation, NETosis, and autoimmunity, which could not be explained by cell proximity to sinusoid vessels. Notably, the type-I interferon signature and calprotectin (S100A8/A9) were not induced in megakaryocytes under any condition. However, inflammatory cytokines induced in the blood and lungs of COVID-19 mice were different from those found in the bone marrow, suggesting a discriminating impact of inflammation on this specific subset of cells. Collectively, our data indicate that a new population of bone marrow megakaryocytes may emerge through COVID-19-related pathogenesis.

Platelet extracellular vesicles and their mitochondrial content improve the mitochondrial bioenergetics of cellular immune recipients.

BACKGROUND: Mitochondria play a critical role in the production of cell energy and the regulation of cell death. Therefore, mitochondria orchestrate numerous cell effector functions, including fine-tuning the immune system. While mitochondria are mainly found intracellularly, they can escape the confine of the cell during the process of extracellular vesicle release. Platelets patrol blood vessels to ensure vasculature integrity and to support the immune system. In blood, platelets are the primary source of circulating mitochondria. Activated platelets produce extracellular vesicles, including a subset of mitochondria-containing vesicles. STUDY DESIGN AND METHODS: We characterized mitochondrial functions in platelet-derived extracellular vesicles, and examined whether they could impact the bioenergetics of cellular immune recipients using an extracellular flux analyzer to measure real-time bioenergetics. RESULTS: We validated that extracellular vesicles derived from activated platelets contain the necessary mitochondrial machinery to respirate and generate energy. Moreover, neutrophils and monocytes efficiently captured platelet-derived extracellular vesicles, enhancing their mitochondrial fitness. This process required functional mitochondria from donor platelets, as it was abolished by the inactivation of extracellular mitochondria using mitochondrial poison. DISCUSSION: Together, the data suggest that extracellular mitochondria produced by platelets may support other metabolic functions through transcellular bioenergetics.

Thermoneutrality and severe malaria: investigating the effect of warmer environmental temperatures on the inflammatory response and disease progression.

Introduction: Most studies using murine disease models are conducted at housing temperatures (20 - 22°C) that are sub-optimal (ST) for mice, eliciting changes in metabolism and response to disease. Experiments performed at a thermoneutral temperature (TT; 28 - 31°C) have revealed an altered immune response to pathogens and experimental treatments in murine disease model that have implications for their translation to clinical research. How such conditions affect the inflammatory response to infection with Plasmodium berghei ANKA (PbA) and disease progression is unknown. We hypothesized that changes in environmental temperature modulate immune cells and modify host response to malaria disease. To test this hypothesis, we conducted experiments to determine: (1) the inflammatory response to malarial agents injection in a peritonitis model and (2) disease progression in PbA-infected mice at TT compared to ST. Methods: In one study, acclimatized mice were injected intraperitoneally with native hemozoin (nHZ) or Leishmania at TT (28 - 31°C) or ST, and immune cells, cytokine, and extracellular vesicle (EV) profiles were determined from the peritoneal cavity (PEC) fluid. In another study, PbA-infected mice were monitored until end-point (i.e. experimental malaria score ≥4). Results: We found that Leishmania injection resulted in decreased cell recruitment and higher phagocytosis of nHZ in mice housed at TT. We found 398 upregulated and 293 downregulated proinflammatory genes in mice injected with nHZ, at both temperatures. We report the presence of host-derived EVs never reported before in a murine parasitic murine model at both temperatures. We observed metabolic changes in mice housed at TT, but these did not result to noticeable changes in disease progression compared to ST. Discussion: To our knowledge, these experiments are the first to investigate the effect of thermoneutrality on a malaria murine model. We found important metabolic difference in mice housed at TT. Our results offer insights on how thermoneutrality might impact a severe malaria murine model and directions for more targeted investigations.

SARS-CoV-2 Nsp2 Contributes to Inflammation by Activating NF-κB.

COVID-19 is associated with robust inflammation and partially impaired antiviral responses. The modulation of inflammatory gene expression by SARS-CoV-2 is not completely understood. In this study, we characterized the inflammatory and antiviral responses mounted during SARS-CoV-2 infection. K18-hACE2 mice were infected with a Wuhan-like strain of SARS-CoV-2, and the transcriptional and translational expression interferons (IFNs), cytokines, and chemokines were analyzed in mouse lung homogenates. Our results show that the infection of mice with SARS-CoV-2 induces the expression of several pro-inflammatory CC and CXC chemokines activated through NF-κB but weakly IL1ß and IL18 whose expression are more characteristic of inflammasome formation. We also observed the downregulation of several inflammasome effectors. The modulation of innate response, following expressions of non-structural protein 2 (Nsp2) and SARS-CoV-2 infection, was assessed by measuring IFNß expression and NF-κB modulation in human pulmonary cells. A robust activation of the NF-κB p65 subunit was induced following the infection of human cells with the corresponding NF-κB-driven inflammatory signature. We identified that Nsp2 expression induced the activation of the IFNß promoter through its NF-κB regulatory domain as well as activation of p65 subunit phosphorylation. The present studies suggest that SARS-CoV-2 skews the antiviral response in favor of an NF-κB-driven inflammatory response, a hallmark of acute COVID-19 and for which Nsp2 should be considered an important contributor.

Efficient megakaryopoiesis and platelet production require phospholipid remodeling and PUFA uptake through CD36.

Lipids contribute to hematopoiesis and membrane properties and dynamics, however, little is known about the role of lipids in megakaryopoiesis. Here, a lipidomic analysis of megakaryocyte progenitors, megakaryocytes, and platelets revealed a unique lipidome progressively enriched in polyunsaturated fatty acid (PUFA)-containing phospholipids. In vitro, inhibition of both exogenous fatty acid functionalization and uptake and de novo lipogenesis impaired megakaryocyte differentiation and proplatelet production. In vivo, mice on a high saturated fatty acid diet had significantly lower platelet counts, which was prevented by eating a PUFA-enriched diet. Fatty acid uptake was largely dependent on CD36, and its deletion in mice resulted in thrombocytopenia. Moreover, patients with a CD36 loss-of-function mutation exhibited thrombocytopenia and increased bleeding. Our results suggest that fatty acid uptake and regulation is essential for megakaryocyte maturation and platelet production, and that changes in dietary fatty acids may be a novel and viable target to modulate platelet counts.

Cytokines and Lipid Mediators of Inflammation in Lungs of SARS-CoV-2 Infected Mice.

Coronavirus disease 19 (COVID-19) is the clinical manifestation of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) infection. A hallmark of COVID-19 is a lung inflammation characterized by an abundant leukocyte infiltrate, elevated levels of cytokines/chemokines, lipid mediators of inflammation (LMI) and microthrombotic events. Animal models are useful for understanding the pathophysiological events leading to COVID-19. One such animal model is the K18-ACE2 transgenic mice. Despite their importance in inflammation, the study of LMI in lung of SARS-CoV-2 infected K18-ACE2 mice has yet to be studied to our knowledge. Using tandem mass spectrometry, the lung lipidome at different time points of infection was analyzed. Significantly increased LMI included N-oleoyl-serine, N-linoleoyl-glycine, N-oleoyl-alanine, 1/2-linoleoyl-glycerol, 1/2-docosahexaenoyl-glycerol and 12-hydroxy-eicosapenatenoic acid. The levels of prostaglandin (PG) E1, PGF2α, stearoyl-ethanolamide and linoleoyl-ethanolamide were found to be significantly reduced relative to mock-infected mice. Other LMI were present at similar levels (or undetected) in both uninfected and infected mouse lungs. In parallel to LMI measures, transcriptomic and cytokine/chemokine profiling were performed. Viral replication was robust with maximal lung viral loads detected on days 2-3 post-infection. Lung histology revealed leukocyte infiltration starting on day 3 post-infection, which correlated with the presence of high concentrations of several chemokines/cytokines. At early times post-infection, the plasma of infected mice contained highly elevated concentration of D-dimers suggestive of blood clot formation/dissolution. In support, the presence of blood clots in the lung vasculature was observed during infection. RNA-Seq analysis of lung tissues indicate that SARS-CoV-2 infection results in the progressive modulation of several hundred genes, including several inflammatory mediators and genes related to the interferons. Analysis of the lung lipidome indicated modest, yet significant modulation of a minority of lipids. In summary, our study suggests that SARS-CoV-2 infection in humans and mice share common features, such as elevated levels of chemokines in lungs, leukocyte infiltration and increased levels of circulating D-dimers. However, the K18-ACE2 mouse model highlight major differences in terms of LMI being produced in response to SARS-CoV-2 infection. The potential reasons and impact of these differences on the pathology and therapeutic strategies to be employed to treat severe COVID-19 are discussed.

Platelet activation by SARS-CoV-2 implicates the release of active tissue factor by infected cells.

Platelets are hyperactivated in coronavirus disease 2019 (COVID-19). However, the mechanisms promoting platelet activation by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not well understood. This may be due to inherent challenges in discriminating the contribution of viral vs host components produced by infected cells. This is particularly true for enveloped viruses and extracellular vesicles (EVs), as they are concomitantly released during infection and share biophysical properties. To study this, we evaluated whether SARS-CoV-2 itself or components derived from SARS-CoV-2-infected human lung epithelial cells could activate isolated platelets from healthy donors. Activation was measured by the surface expression of P-selectin and the activated conformation of integrin αIIbß3, degranulation, aggregation under flow conditions, and the release of EVs. We find that neither SARS-CoV-2 nor purified spike activates platelets. In contrast, tissue factor (TF) produced by infected cells was highly potent at activating platelets. This required trace amounts of plasma containing the coagulation factors FX, FII, and FVII. Robust platelet activation involved thrombin and the activation of protease-activated receptor (PAR)-1 and -4 expressed by platelets. Virions and EVs were identified by electron microscopy. Through size-exclusion chromatography, TF activity was found to be associated with a virus or EVs, which were indistinguishable. Increased TF messenger RNA (mRNA) expression and activity were also found in lungs in a murine model of COVID-19 and plasma of severe COVID-19 patients, respectively. In summary, TF activity from SARS-CoV-2-infected cells activates thrombin, which signals to PARs on platelets. Blockade of molecules in this pathway may interfere with platelet activation and the coagulation characteristic of COVID-19.

Live imaging of platelets and neutrophils during antibody-mediated neurovascular thrombosis.

Immune complexes form in systemic disorders such as rheumatological, autoimmune, and allergic diseases or in response to infections or medications. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) adenoviral vector vaccines have been associated with rare yet serious thrombotic complications in the brain due to the formation of immune complexes that activate platelets. There are currently no data visualizing the interplay of platelets with leukocytes and the brain vasculature endothelium in response to immune complexes. This is in part due to the absence of FcγRIIA in mice, a receptor for immune complexes implicated in these thrombotic incidents. Here, we describe and illustrate events at the cellular level that take place in the brain vasculature in response to systemic administration of surrogate immune complexes. We used Ly6gCre+/-::Rosa26-TdT+/-::CD41-YFP+/- mice expressing the FcγRIIA transgene and fluorescence in neutrophils and platelets. Using real-time videomicroscopy to capture high-velocity events in conjunction with unbiased computer-assisted analyses, we provide images and quantifications of the cellular responses downstream of FcγRIIA stimulation. We observed transient and stable platelet-neutrophil interactions, platelets forming thrombi, and neutrophil adhesion to blood vessel walls. This imaging approach in a quadruple transgenic animal model can be used for the study of the pathogenic roles of immune complexes in disease.

Identification of Mitofusin 1 and Complement Component 1q Subcomponent Binding Protein as Mitochondrial Targets in Systemic Lupus Erythematosus.

OBJECTIVE: Mitochondria are organelles that exhibit several bacterial features, such as a double-stranded genome with hypomethylated CpG islands, formylated proteins, and cardiolipin-containing membranes. In systemic lupus erythematosus (SLE), mitochondria and their inner components are released into the extracellular space, potentially eliciting a proinflammatory response from the immune system. While cardiolipin and mitochondrial DNA and RNA are confirmed targets of autoantibodies, other antigenic mitochondrial proteins in SLE remain to be identified. The present study was undertaken to characterize the protein repertoire recognized by antimitochondrial antibodies (AMAs) in patients with SLE. METHODS: Using shotgun proteomic profiling, we identified 1,345 proteins, 431 of which were associated with the mitochondrial proteome. Immunoreactivities to several of these candidate proteins were assessed in serum samples from a local cohort (n = 30 healthy donors and 87 patients with SLE) using enzyme-linked immunosorbent assay, and further analyzed for associations with demographic and disease characteristics. RESULTS: We determined that IgG antibodies to the complement component C1q binding protein were significantly elevated in the patients with SLE (P = 0.049) and were also associated with lupus anticoagulant positivity (P = 0.049). Elevated levels of IgG antibodies against mitochondrial protein mitofusin 1 (MFN-1) were promising predictors of SLE diagnosis in our cohort (adjusted odds ratio 2.99 [95% confidence interval 1.39-6.43], P = 0.0044). Moreover, increased levels of anti-MFN-1 were associated with the presence of antiphospholipids (P = 0.011) and anti-double-stranded DNA (P = 0.0005). CONCLUSION: In this study, we characterized the mitochondrial repertoire targeted by AMAs in the setting of SLE. Our results indicate that autoantibodies can recognize secreted and/or surface proteins of mitochondrial origin. Profiling of the AMA repertoire in large prospective cohorts may improve our knowledge of mitochondrial biomarkers and their usefulness for patient stratification.

The interaction of secreted phospholipase A2-IIA with the microbiota alters its lipidome and promotes inflammation.

Secreted phospholipase A2-IIA (sPLA2-IIA) hydrolyzes phospholipids to liberate lysophospholipids and fatty acids. Given its poor activity toward eukaryotic cell membranes, its role in the generation of proinflammatory lipid mediators is unclear. Conversely, sPLA2-IIA efficiently hydrolyzes bacterial membranes. Here, we show that sPLA2-IIA affects the immune system by acting on the intestinal microbial flora. Using mice overexpressing transgene-driven human sPLA2-IIA, we found that the intestinal microbiota was critical for both induction of an immune phenotype and promotion of inflammatory arthritis. The expression of sPLA2-IIA led to alterations of the intestinal microbiota composition, but housing in a more stringent pathogen-free facility revealed that its expression could affect the immune system in the absence of changes to the composition of this flora. In contrast, untargeted lipidomic analysis focusing on bacteria-derived lipid mediators revealed that sPLA2-IIA could profoundly alter the fecal lipidome. The data suggest that a singular protein, sPLA2-IIA, produces systemic effects on the immune system through its activity on the microbiota and its lipidome.

Platelet EVs contain an active proteasome involved in protein processing for antigen presentation via MHC-I molecules.

In addition to their hemostatic role, platelets play a significant role in immunity. Once activated, platelets release extracellular vesicles (EVs) formed by the budding of their cytoplasmic membranes. Because of their heterogeneity, platelet EVs (PEVs) are thought to perform diverse functions. It is unknown, however, whether the proteasome is transferred from platelets to PEVs or whether its function is retained. We hypothesized that functional protein processing and antigen presentation machinery are transferred to PEVs by activated platelets. Using molecular and functional assays, we found that the active 20S proteasome was enriched in PEVs, along with major histocompatibility complex class I (MHC-I) and lymphocyte costimulatory molecules (CD40L and OX40L). Proteasome-containing PEVs were identified in healthy donor blood, but did not increase in platelet concentrates that caused adverse transfusion reactions. They were augmented, however, after immune complex injections in mice. The complete biodistribution of murine PEVs after injection into mice revealed that they principally reached lymphoid organs, such as spleen and lymph nodes, in addition to the bone marrow, and to a lesser extent, liver and lungs. The PEV proteasome processed exogenous ovalbumin (OVA) and loaded its antigenic peptide onto MHC-I molecules, which promoted OVA-specific CD8+ T-lymphocyte proliferation. These results suggest that PEVs contribute to adaptive immunity through cross-presentation of antigens and have privileged access to immune cells through the lymphatic system, a tissue location that is inaccessible to platelets.

Human Inflammatory Neutrophils Express Genes Encoding Peptidase Inhibitors: Production of Elafin Mediated by NF-κB and CCAAT/Enhancer-Binding Protein ß.

The concept of plasticity of neutrophils is highlighted by studies showing their ability to transdifferentiate into APCs. In this regard, transdifferentiated neutrophils were found at inflammatory sites of autoimmune arthritis (AIA). Exposure of neutrophils to inflammatory stimuli prolongs their survival, thereby favoring the acquisition of pathophysiologically relevant phenotypes and functions. By using microarrays, quantitative RT-PCR, and ELISAs, we showed that long-lived (LL) neutrophils obtained after 48 h of culture in the presence of GM-CSF, TNF, and IL-4 differentially expressed genes related to apoptosis, MHC class II, immune response, and inflammation. The expression of anti-inflammatory genes mainly of peptidase inhibitor families is upregulated in LL neutrophils. Among these, the PI3 gene encoding elafin was the most highly expressed. The de novo production of elafin by LL neutrophils depended on a synergism between GM-CSF and TNF via the activation and cooperativity of C/EBPß and NF-κB pathways, respectively. Elafin concentrations were higher in synovial fluids (SF) of patients with AIA than in SF of osteoarthritis. SF neutrophils produced more elafin than blood counterparts. These results are discussed with respect to implications of neutrophils in chronic inflammation and the potential influence of elafin in AIA.

Platelets release mitochondrial antigens in systemic lupus erythematosus.

The accumulation of DNA and nuclear components in blood and their recognition by autoantibodies play a central role in the pathophysiology of systemic lupus erythematosus (SLE). Despite the efforts, the sources of circulating autoantigens in SLE are still unclear. Here, we show that in SLE, platelets release mitochondrial DNA, the majority of which is associated with the extracellular mitochondrial organelle. Mitochondrial release in patients with SLE correlates with platelet degranulation. This process requires the stimulation of platelet FcγRIIA, a receptor for immune complexes. Because mice lack FcγRIIA and murine platelets are completely devoid of receptor capable of binding IgG-containing immune complexes, we used transgenic mice expressing FcγRIIA for our in vivo investigations. FcγRIIA expression in lupus-prone mice led to the recruitment of platelets in kidneys and to the release of mitochondria in vivo. Using a reporter mouse with red fluorescent protein targeted to the mitochondrion, we confirmed platelets as a source of extracellular mitochondria driven by FcγRIIA and its cosignaling by the fibrinogen receptor α2bß3 in vivo. These findings suggest that platelets might be a key source of mitochondrial antigens in SLE and might be a therapeutic target for treating SLE.

FcγRIIA expression accelerates nephritis and increases platelet activation in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease characterized by deposits of immune complexes (ICs) in organs and tissues. The expression of FcγRIIA by human platelets, which is their unique receptor for immunoglobulin G antibodies, positions them to ideally respond to circulating ICs. Whereas chronic platelet activation and thrombosis are well-recognized features of human SLE, the exact mechanisms underlying platelet activation in SLE remain unknown. Here, we evaluated the involvement of FcγRIIA in the course of SLE and platelet activation. In patients with SLE, levels of ICs are associated with platelet activation. Because FcγRIIA is absent in mice, and murine platelets do not respond to ICs in any existing mouse model of SLE, we introduced the FcγRIIA (FCGR2A) transgene into the NZB/NZWF1 mouse model of SLE. In mice, FcγRIIA expression by bone marrow cells severely aggravated lupus nephritis and accelerated death. Lupus onset initiated major changes to the platelet transcriptome, both in FcγRIIA-expressing and nonexpressing mice, but enrichment for type I interferon response gene changes was specifically observed in the FcγRIIA mice. Moreover, circulating platelets were degranulated and were found to interact with neutrophils in FcγRIIA-expressing lupus mice. FcγRIIA expression in lupus mice also led to thrombosis in lungs and kidneys. The model recapitulates hallmarks of human SLE and can be used to identify contributions of different cellular lineages in the manifestations of SLE. The study further reveals a role for FcγRIIA in nephritis and in platelet activation in SLE.

Platelets Can Associate with SARS-Cov-2 RNA and Are Hyperactivated in COVID-19.

Rationale: In addition to the overwhelming lung inflammation that prevails in COVID-19, hypercoagulation and thrombosis contribute to the lethality of subjects infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Platelets are chiefly implicated in thrombosis. Moreover, they can interact with viruses and are an important source of inflammatory mediators. While a lower platelet count is associated with severity and mortality, little is known about platelet function during COVID-19. Objective: To evaluate the contribution of platelets to inflammation and thrombosis in COVID-19 patients. Methods and Results: Blood was collected from 115 consecutive COVID-19 patients presenting non-severe (n=71) and severe (n=44) respiratory symptoms. We document the presence of SARS-CoV-2 RNA associated with platelets of COVID-19 patients. Exhaustive assessment of cytokines in plasma and in platelets revealed the modulation of platelet-associated cytokine levels in both non-severe and severe COVID-19 patients, pointing to a direct contribution of platelets to the plasmatic cytokine load. Moreover, we demonstrate that platelets release their alpha- and dense-granule contents in both non-severe and severe forms of COVID-19. In comparison to concentrations measured in healthy volunteers, phosphatidylserine-exposing platelet extracellular vesicles were increased in non-severe, but not in severe cases of COVID-19. Levels of D-dimers, a marker of thrombosis, failed to correlate with any measured indicators of platelet activation. Functionally, platelets were hyperactivated in COVID-19 subjects presenting non-severe and severe symptoms, with aggregation occurring at suboptimal thrombin concentrations. Furthermore, platelets adhered more efficiently onto collagen-coated surfaces under flow conditions. Conclusions: Taken together, the data suggest that platelets are at the frontline of COVID-19 pathogenesis, as they release various sets of molecules through the different stages of the disease. Platelets may thus have the potential to contribute to the overwhelming thrombo-inflammation in COVID-19, and the inhibition of pathways related to platelet activation may improve the outcomes during COVID-19.

Platelet-derived extracellular vesicles infiltrate and modify the bone marrow during inflammation.

During inflammation, steady-state hematopoiesis switches to emergency hematopoiesis to repopulate myeloid cells, with a bias toward the megakaryocytic lineage. Soluble inflammatory cues are thought to be largely responsible for these alterations. However, how these plasma factors rapidly alter the bone marrow (BM) is not understood. Inflammation also drives platelet activation, causing the release of platelet-derived extracellular vesicles (PEVs), which package diverse cargo and reprogram target cells. We hypothesized that PEVs infiltrate the BM, providing a direct mode of communication between the plasma and BM environments. We transfused fluorescent, wild-type (MPL+) platelets into recipient cMpl-/-mice before triggering systemic inflammation. Twenty hours postinfusion, we observed significant infiltration of donor platelet-derived particles in the BM, which we tracked immunophenotypically (MPL+ immunohistochemistry staining) and quantified by flow cytometry. To determine if this phenomenon relates to humans, we extensively characterized both megakaryocyte-derived and PEVs generated in vitro and in vivo, and found enrichment of extracellular vesicles in bone marrow compared with autologous peripheral blood. Last, BM from cMpl-/- mice was cultured in the presence or absence of wild-type (MPL+) PEVs. After 72 hours, flow cytometry revealed increased megakaryocytes only in cultures with added PEVs. The majority of CD41+ cells were bound to PEVs, suggesting a PEV-mediated rescue of megakaryopoiesis. In conclusion, we report for the first time that plasma-residing PEVs infiltrate the BM. Further, PEVs interact with BM cells in vivo and in vitro, causing functional reprogramming that may represent a novel model of inflammation-induced hematopoiesis.

Platelets Disseminate Extracellular Vesicles in Lymph in Rheumatoid Arthritis.

OBJECTIVE: The lymphatic system is a circulatory system that unidirectionally drains the interstitial tissue fluid back to blood circulation. Although lymph is utilized by leukocytes for immune surveillance, it remains inaccessible to platelets and erythrocytes. Activated cells release submicron extracellular vesicles (EV) that transport molecules from the donor cell. In rheumatoid arthritis, EV accumulate in the joint where they can interact with numerous cellular lineages. However, whether EV can exit the inflamed tissue to recirculate is unknown. Here, we investigated whether vascular leakage that occurs during inflammation could favor EV access to the lymphatic system. Approach and Results: Using an in vivo model of autoimmune inflammatory arthritis, we show that there is an influx of platelet EV, but not EV from erythrocytes or leukocytes, in joint-draining lymph. In contrast to blood platelet EV, lymph platelet EV lacked mitochondrial organelles and failed to promote coagulation. Platelet EV influx in lymph was consistent with joint vascular leakage and implicated the fibrinogen receptor α2bß3 and platelet-derived serotonin. CONCLUSIONS: These findings show that platelets can disseminate their EV in fluid that is inaccessible to platelets and beyond the joint in this disease.

Autoantibodies in Systemic Lupus Erythematosus Target Mitochondrial RNA.

The mitochondrion supplies energy to the cell and regulates apoptosis. Unlike other mammalian organelles, mitochondria are formed by binary fission and cannot be directly produced by the cell. They contain numerous copies of a compact circular genome that encodes RNA molecules and proteins involved in mitochondrial oxidative phosphorylation. Whereas, mitochondrial DNA (mtDNA) activates the innate immune system if present in the cytosol or the extracellular milieu, it is also the target of circulating autoantibodies in systemic lupus erythematosus (SLE). However, it is not known whether mitochondrial RNA is also recognized by autoantibodies in SLE. In the present study, we evaluated the presence of autoantibodies targeting mitochondrial RNA (AmtRNA) in SLE. We quantified AmtRNA in an inducible model of murine SLE. The AmtRNA were also determined in SLE patients and healthy volunteers. AmtRNA titers were measured in both our induced model of murine SLE and in human SLE, and biostatistical analyses were performed to determine whether the presence and/or levels of AmtRNA were associated with clinical features expressed by SLE patients. Both IgG and IgM classes of AmtRNA were increased in SLE patients (n = 86) compared to healthy controls (n = 30) (p < 0.0001 and p = 0.0493, respectively). AmtRNA IgG levels correlated with anti-mtDNA-IgG titers (rs = 0.54, p < 0.0001) as well as with both IgG and IgM against ß-2-glycoprotein I (anti-ß2GPI; rs = 0.22, p = 0.05), and AmtRNA-IgG antibodies were present at higher levels when patients were positive for autoantibodies to double-stranded-genomic DNA (p < 0.0001). AmtRNA-IgG were able to specifically discriminate SLE patients from healthy controls, and were negatively associated with plaque formation (p = 0.04) and lupus nephritis (p = 0.03). Conversely, AmtRNA-IgM titers correlated with those of anti-ß2GPI-IgM (rs = 0.48, p < 0.0001). AmtRNA-IgM were higher when patients were positive for anticardiolipin antibodies (aCL-IgG: p = 0.01; aCL-IgM: p = 0.002), but AmtRNA-IgM were not associated with any of the clinical manifestations assessed. These findings identify mtRNA as a novel mitochondrial antigen target in SLE, and support the concept that mitochondria may provide an important source of circulating autoantigens in SLE.

Platelet-derived extracellular vesicles convey mitochondrial DAMPs in platelet concentrates and their levels are associated with adverse reactions.

BACKGROUND: Whereas platelet transfusion is a common medical procedure, inflammation still occurs in a fraction of transfused individuals despite the absence of any apparent infectious agents. Platelets can shed membrane vesicles, called extracellular vesicles (EVs), some of which contain mitochondria (mito+EV). With its content of damage-associated molecular pattern (DAMP), the mitochondrion can stimulate the innate immune system. Mitochondrial DNA (mtDNA) is a recognized DAMP detected in the extracellular milieu in numerous inflammatory conditions and in platelet concentrates. We hypothesized that platelet-derived mitochondria encapsulated in EVs may represent a reservoir of mtDNA. STUDY DESIGN AND METHODS: Herein, we explored the implication of mito+EVs in the occurrence of mtDNA quantified in platelet concentrate supernatants that induced or did not induce transfusion adverse reactions. RESULTS: We observed that EVs were abundant in platelet concentrates, and platelet-derived mito+EVs were more abundant in platelet concentrates that induced adverse reactions. A significant correlation (rs = 0.73; p < 0.0001) between platelet-derived mito+EV levels and mtDNA concentrations was found. However, there was a nonsignificant correlation between the levels of EVs without mitochondria and mtDNA concentrations (rs = -0.11; p = 0.5112). The majority of the mtDNA was encapsulated into EVs. CONCLUSION: This study suggests that platelet-derived EVs, such as those that convey mitochondrial DAMPs, may be a useful biomarker for the prediction of potential risk of adverse transfusion reactions. Moreover, our work implies that investigations are necessary to determine whether there is a causal pathogenic role of mitochondrial DAMP encapsulated in EVs as opposed to mtDNA in solution.

Anti-mitochondrial autoantibodies in systemic lupus erythematosus and their association with disease manifestations.

Mitochondria are organelles that govern energy supply and control cell death. Mitochondria also express bacterial features, such as the presence of inner membrane cardiolipin and a circular genome rich in hypomethylated CpG motifs. While mitochondrial extrusion by damaged organs or activated cells is thought to trigger innate immunity, it is unclear whether extracellular mitochondria also stimulate an adaptive immune response. We describe the development of novel assays to detect autoantibodies specific to two distinct components of the mitochondrion: the mitochondrial outer membrane and mitochondrial DNA. Antibodies to these two mitochondrial constituents were increased in both human and murine systemic lupus erythematosus (SLE), compared to controls, and were present at higher levels than in patients with antiphospholipid syndrome or primary biliary cirrhosis. In both bi- and multi-variate regression models, antibodies to mitochondrial DNA, but not whole mitochondria, were associated with increased anti-dsDNA antibodies and lupus nephritis. This study describes new and optimized methods for the assessment of anti-mitochondrial antibodies, and demonstrates their presence in both human and murine SLE. These findings suggest that different mitochondrial components are immunogenic in SLE, and support the concept that extracellular mitochondria may provide an important source of circulating autoantigens in SLE.