M2 macrophages, but not M1 macrophages, support megakaryopoiesis by upregulating PI3K-AKT pathway activity.

Dysfunctional megakaryopoiesis hampers platelet production, which is closely associated with thrombocytopenia (PT). Macrophages (MФs) are crucial cellular components in the bone marrow (BM) microenvironment. However, the specific effects of M1 MФs or M2 MФs on regulating megakaryocytes (MKs) are largely unknown. In the current study, aberrant BM-M1/M2 MФ polarization, characterized by increased M1 MФs and decreased M2 MФs and accompanied by impaired megakaryopoiesis-supporting abilities, was found in patients with PT post-allotransplant. RNA-seq and western blot analysis showed that the PI3K-AKT pathway was downregulated in the BM MФs of PT patients. Moreover, in vitro treatment with PI3K-AKT activators restored the impaired megakaryopoiesis-supporting ability of MФs from PT patients. Furthermore, we found M1 MФs suppress, whereas M2 MФs support MK maturation and platelet formation in humans. Chemical inhibition of PI3K-AKT pathway reduced megakaryopoiesis-supporting ability of M2 MФs, as indicated by decreased MK count, colony-forming unit number, high-ploidy distribution, and platelet count. Importantly, genetic knockdown of the PI3K-AKT pathway impaired the megakaryopoiesis-supporting ability of MФs both in vitro and in a MФ-specific PI3K-knockdown murine model, indicating a critical role of PI3K-AKT pathway in regulating the megakaryopoiesis-supporting ability of M2 MФs. Furthermore, our preliminary data indicated that TGF-ß released by M2 MФs may facilitate megakaryopoiesis through upregulation of the JAK2/STAT5 and MAPK/ERK pathways in MKs. Taken together, our data reveal that M1 and M2 MФs have opposing effects on MKs in a PI3K-AKT pathway-dependent manner, which may lead to new insights into the pathogenesis of thrombocytopenia and provide a potential therapeutic strategy to promote megakaryopoiesis.

Cytokines in the immunity and immunopathogenesis in leishmaniases.

Cytokines are key mediators of immune responses to autoantigens, tumor antigens and foreign antigens including pathogens and transplant antigens. The cytokines are produced by a variety of immune and non-immune cells and are dynamically regulated. Remarkably, during toxic and septic shock syndromes, anaphylactic shock and in certain viral infections supra-physiologic levels of cytokine storms are produced culminating in multi-organ failure and death. However, Leishmania infection is a chronic parasitic infection with alternate outcomes- healing or non-healing. Leishmania invades macrophages and inflicts the complex of diseases called Leishmaniases. Depending on the species of Leishmania and the organs affected, the diseases are categorized into Cutaneous Leishmaniasis (CL), Muco-cutaneous Leishmaniasis (MCL) and Visceral Leishmaniasis (VL). After successful chemotherapy of VL, a dermal manifestation- termed post-kalazar dermal leishmaniasis (PKDL)- of the same infection occurs in some patients. The operational frameworks for different cytokines have been laid to discuss how these immune mediators control each of these forms of leishmaniases. One of these frameworks is the regulation of monocytopoiesis including the role of macrophages subsets and thrombopoiesis in leishmaniases. Macrophage metabolism is linked to different cytokines and is thereby associated with the manifestation of the resistance or susceptibility to Leishmania infection and of drug resistance. The chemokine-regulated immune cell movements present the landscape of infection and pathogenesis. T cells subsets- the IFN-γ-secreting Ly6C + T cells and the regulatory T cell subsets- provide the initial skewing of Th cell subset and regulation of effector Th subsets, respectively, eventually deciding the outcome of infection.

A proof of evidence supporting abnormal immunothrombosis in severe COVID-19: naked megakaryocyte nuclei increase in the bone marrow and lungs of critically ill patients.

Coronavirus disease 2019 (COVID-19) is a global public health emergency with many clinical facets, and new knowledge about its pathogenetic mechanisms is deemed necessary; among these, there are certainly coagulation disorders. In the history of medicine, autopsies and tissue sampling have played a fundamental role in order to understand the pathogenesis of emerging diseases, including infectious ones; compared to the past, histopathology can be now expanded by innovative techniques and modern technologies. For the first time in worldwide literature, we provide a detailed postmortem and biopsy report on the marked increase, up to 1 order of magnitude, of naked megakaryocyte nuclei in the bone marrow and lungs from serious COVID-19 patients. Most likely related to high interleukin-6 serum levels stimulating megakaryocytopoiesis, this phenomenon concurs to explain well the pulmonary abnormal immunothrombosis in these critically ill patients, all without molecular or electron microscopy signs of megakaryocyte infection.

The Coagulation and Immune Systems Are Directly Linked through the Activation of Interleukin-1α by Thrombin.

Ancient organisms have a combined coagulation and immune system, and although links between inflammation and hemostasis exist in mammals, they are indirect and slower to act. Here we investigated direct links between mammalian immune and coagulation systems by examining cytokine proproteins for potential thrombin protease consensus sites. We found that interleukin (IL)-1α is directly activated by thrombin. Thrombin cleaved pro-IL-1α at a site perfectly conserved across disparate species, indicating functional importance. Surface pro-IL-1α on macrophages and activated platelets was cleaved and activated by thrombin, while tissue factor, a potent thrombin activator, colocalized with pro-IL-1α in the epidermis. Mice bearing a mutation in the IL-1α thrombin cleavage site (R114Q) exhibited defects in efficient wound healing and rapid thrombopoiesis after acute platelet loss. Thrombin-cleaved IL-1α was detected in humans during sepsis, pointing to the relevance of this pathway for normal physiology and the pathogenesis of inflammatory and thrombotic diseases.

Pathophysiology of Autoimmune Thrombocytopenia: Current Insight with a Focus on Thrombopoiesis.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by a significant reduction in the number of circulating platelets which is frequently associated with bleeding. The total count of platelets in the body is finely regulated by the balance between platelet production and destruction. Although the pathogenesis of ITP is still not completely elucidated, it is largely recognized that the low platelet count observed in ITP patients is due to alterations of both mechanisms. An abnormal proliferation of autoreactive T cells leading to the breakdown of immune tolerance to platelet antigens is suggested to be responsible for the up-regulated proliferation of autoantibody producing B cells. Consequently, the immune response induces enhanced T cell-mediated cytotoxicity and antibody-mediated platelet destruction through phagocytosis, complement activation and apoptosis. An additional contribution to the pathophysiology of ITP is given by alterations of thrombopoiesis caused by platelet-reactive autoantibodies or cytotoxic T cells leading to impaired megakaryocyte differentiation and platelet production. All these processes involved in ITP pathophysiology account for the complexity and heterogeneity in the clinical manifestation and therapy responsiveness of this disorder. For this reason, a better understanding of the different underlying mechanisms in ITP is necessary to develop more efficient therapeutic treatments in the future. In this review, we will provide an update on the pathophysiology of ITP with a particular focus on the impact of impaired thrombopoiesis.

Transcription factor Etv6 regulates functional differentiation of cross-presenting classical dendritic cells.

An IRF8-dependent subset of conventional dendritic cells (cDCs), termed cDC1, effectively cross-primes CD8+ T cells and facilitates tumor-specific T cell responses. Etv6 is an ETS family transcription factor that controls hematopoietic stem and progenitor cell (HSPC) function and thrombopoiesis. We report that like HSPCs, cDCs express Etv6, but not its antagonist, ETS1, whereas interferon-producing plasmacytoid dendritic cells (pDCs) express both factors. Deletion of Etv6 in the bone marrow impaired the generation of cDC1-like cells in vitro and abolished the expression of signature marker CD8α on cDC1 in vivo. Moreover, Etv6-deficient primary cDC1 showed a partial reduction of cDC-specific and cDC1-specific gene expression and chromatin signatures and an aberrant up-regulation of pDC-specific signatures. Accordingly, DC-specific Etv6 deletion impaired CD8+ T cell cross-priming and the generation of tumor antigen-specific CD8+ T cells. Thus, Etv6 optimizes the resolution of cDC1 and pDC expression programs and the functional fitness of cDC1, thereby facilitating T cell cross-priming and tumor-specific responses.

Emerging drugs for immune thrombocytopenia (ITP).

INTRODUCTION: Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by autoantibody production against platelets, increased platelet destruction, and, in some cases, impaired thrombopoiesis. The majority of affected patients have significant bleeding risks due to low platelet counts and require treatment. The etiology of ITP is an immunological labyrinth. Currently available treatment options are usually not only nonspecific, but are also associated with some risks. Areas covered: Several useful drugs for the treatment of ITP are currently available. Furthermore, ongoing trials with new drugs and preclinical development of additional drugs may help to improve and determine their value. Expert opinion: ITP is a heterogeneous complex requiring individualized treatment. None of the available drugs are specific, nor are they invariably safe and effective. Thus, the need for specific therapy is evident.

Low-level light treatment ameliorates immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an immune-mediated acquired bleeding disorder characterized by abnormally low platelet counts. We reported here the ability of low-level light treatment (LLLT) to alleviate ITP in mice. The treatment is based on noninvasive whole body illumination 30 min a day for a few consecutive days by near infrared light (830 nm) transmitted by an array of light-emitting diodes (LEDs). LLLT significantly lifted the nadir of platelet counts and restored tail bleeding time when applied to two passive ITP models induced by anti-CD41 antibody. The anti-platelet antibody hindered megakaryocyte differentiation from the progenitors, impaired proplatelet and platelet formation, and induced apoptosis of platelets. These adverse effects of anti-CD41 antibody were all mitigated by LLLT to varying degrees, owing to its ability to enhance mitochondrial biogenesis and activity in megakaryocytes and preserve mitochondrial functions in platelets in the presence of the antibody. The observations argue not only for contribution of mitochondrial stress to the pathology of ITP, but also clinical potentials of LLLT as a safe, simple, and cost-effective modality of ITP.

Enhanced Reconstitution of Human Erythropoiesis and Thrombopoiesis in an Immunodeficient Mouse Model with Kit(Wv) Mutations.

In human-to-mouse xenograft models, reconstitution of human hematopoiesis is usually B-lymphoid dominant. Here we show that the introduction of homozygous Kit(Wv) mutations into C57BL/6.Rag2(null)Il2rg(null) mice with NOD-Sirpa (BRGS) strongly promoted human multi-lineage reconstitution. After xenotransplantation of human CD34(+)CD38(-) cord blood cells, these newly generated C57BL/6.Rag2(null)Il2rg(null)NOD-Sirpa Kit(Wv/Wv) (BRGSK(Wv/Wv)) mice showed significantly higher levels of human cell chimerism and long-term multi-lineage reconstitution compared with BRGS mice. Strikingly, this mouse displayed a robust reconstitution of human erythropoiesis and thrombopoiesis with terminal maturation in the bone marrow. Furthermore, depletion of host macrophages by clodronate administration resulted in the presence of human erythrocytes and platelets in the circulation. Thus, attenuation of mouse KIT signaling greatly enhances the multi-lineage differentiation of human hematopoietic stem and progenitor cells (HSPCs) in mouse bone marrow, presumably by outcompeting mouse HSPCs to occupy suitable microenvironments. The BRGSK(Wv/Wv) mouse model is a useful tool to study human multi-lineage hematopoiesis.

Lnk/Sh2b3 controls the production and function of dendritic cells and regulates the induction of IFN-γ-producing T cells.

Dendritic cells (DCs) are proficient APCs that play crucial roles in the immune responses to various Ags and pathogens and polarize Th cell immune responses. Lnk/SH2B adaptor protein 3 (Sh2b3) is an intracellular adaptor protein that regulates B lymphopoiesis, megakaryopoiesis, and expansion of hematopoietic stem cells by constraining cytokine signals. Recent genome-wide association studies have revealed a link between polymorphism in this adaptor protein and autoimmune diseases, including type 1 diabetes and celiac disease. We found that Lnk/Sh2b3 was also expressed in DCs and investigated its role in the production and function of DC lineage cells. In Lnk(-/-) mice, DC numbers were increased in the spleen and lymph nodes, and growth responses of bone marrow-derived DCs to GM-CSF were augmented. Mature DCs from Lnk(-/-) mice were hypersensitive and showed enhanced responses to IL-15 and GM-CSF. Compared to normal DCs, Lnk(-/-) DCs had enhanced abilities to support the differentiation of IFN-γ-producing Th1 cells from naive CD4(+) T cells. This was due to their elevated expression of IL-12Rß1 and increased production of IFN-γ. Lnk(-/-) DCs supported the appearance of IFN-γ-producing T cells even under conditions in which normal DCs supported induction of regulatory T cells. These results indicated that Lnk/Sh2b3 plays a regulatory role in the expansion of DCs and might influence inflammatory immune responses in peripheral lymphoid tissues.

B cells facilitate platelet production mediated by cytokines in patients with essential thrombocythaemia.

We investigated the role of activated B cells in thrombopoiesis through the production of interleukin (IL)-1beta and IL-6 in patients with essential thrombocythaemia. The number of B cells did not differ between essential thrombocythaemia patients, irrespective of the presence of Janus activated kinase-2 V617F mutation or wild type, and age-matched healthy adults. However, the number of IL-1beta/IL-6-producing B cells was significantly higher in essential thrombocythaemia patients than that in healthy controls. The relatively high level of IL-1beta/IL-6 production by B cells was associated with serum B cell-activating factor and expression of Toll-like receptor 4 on B cells. A high level of B cell-activating factor was present in essential thrombocythaemia patients with both Janus activated kinase-2 genotypes. Incubation with B cell-activating factor enhanced the expression of Toll-like receptor 4 on B cells. IL-1beta and IL-6 production was not stimulated by B cell-activating factor alone; Toll-like receptor 4 was activated by lipopolysaccharide or patients' sera to produce IL-1beta and IL-6 in B cells. Moreover, essential thrombocythaemia patient B cells facilitated megakaryocyte differentiation when co-cultured with CD34+ haematopoietic stem cells. Antibody neutralisation of IL-1beta and IL-6 attenuated megakaryocyte differentiation. These data suggest that B cells play a crucial role in thrombopoiesis in essential thrombocythaemia patients.

Impaired proplatelet formation in immune thrombocytopenia: a novel mechanism contributing to decreased platelet count.

The pathophysiological mechanisms contributing to the decreased platelet count in immune thrombocytopenia (ITP) are not entirely understood. Here, we investigated the key step of proplatelet formation (PPF) by studying the effect of ITP plasma in thrombopoiesis. Normal cord blood-derived mature megakaryocytes were cultured in the presence of recalcified plasma from ITP patients, and PPF was evaluated by microscopic analysis. Patient samples induced a dose-dependent inhibition in PPF, as well as decreased complexity of proplatelet architecture. Although slightly increased, plasma-induced megakaryocyte apoptosis was not related to PPF impairment. Purified IgG reproduced the inhibitory effect, while platelet-adsorbed plasma induced its reversion, suggesting the involvement of auto-antibodies in the inhibition of thrombopoiesis. Impaired PPF, induced by ITP plasmas bearing anti-GPIIb-IIIa antibodies, was related to their ability to interfere with the normal function of this integrin, as assessed by megakaryocyte PAC-1 binding and ß3 integrin phosphorylation while the presence of anti-glycoprotein Ia-IIa auto-antibodies was associated with loss of normal inhibition of PPF induced by type I collagen. In conclusion, abnormal thrombopoiesis comprising decreased PPF and morphological changes in proplatelet structure are induced by patient samples, unveiling new mechanisms contributing to decreased platelet count in ITP.

Las trombopoyetinas en el tratamiento de la púrpura trombocitopénica inmunitaria y otras trombocitopenias / Thrombopoietines in the treatment of the thrombocytopenic purpura and other thrombocytopenics

La purpura trombocitopénica inmunitaria y las trombocitopenias secundarias representan condiciones patológicas graves cuyo tratamiento plantea diversos grados de dificultad. La aproximación terapéutica convencional ha sido la administración de esteroides, la esplenectomía y el uso de inmunoglobulina intravenosa u otros tipos de anticuerpos (e.g., anti-D). La mejor comprensión de la fisiología y fisiopatología de la trombopoyesis aunado a los avances en biología molecular ha permitido el desarrollo de una nueva aproximación terapéutica, la aplicación de las trombopoyetinas sintéticas o no inmunogénicas. Dentro de este grupo resaltan dos compuestos: el romiplostin (una proteína de fusión) y el eltrombopag (un compuesto sintético de bajo peso molecular). Ambas se encuentran disponibles comercialmente. Los estudios clínicos indican que estos medicamentos tienen un efecto satisfactorio en el tratamiento de las trombocitopenias, particularmente en los casos refractarios a los tratamientos convencionales.

Immune thrombocytopenic purpura and the secondary thrombocytopenias are conditions potentially severe with diverse degrees of treatment difficulties. Steroids administration, splenectomy and the use of intravenous immunoglobulin and other antibodies (e.g., anti-D) had been the conventional therapy. The better understanding of the thrombopoiesis physiology and physiopathology togetter with the biology advances have permitted the development of a new terapheutic approach: the use of synthetic or nonimmunogenic thrombopoietines. Among this group highlights composites: romiplostim (a fusion protein) and eltrombopag (a synthetic composite with low molecular wheigt). Both are already available and produce a satisfactory effect particularly in nonrespondent cases to the conventional treatment.

CD4+CD25+Foxp3+ regulatory T cells in the pathophysiology of immune thrombocytopenia.

Regulatory T cells characterized by CD4, CD25, and transcription factor forkhead box P3, called Tregs, are a subpopulation of CD4(+) T cells specialized for immune suppression. Tregs contribute to maintenance of peripheral immune tolerance, and their defects are thought to play a role in the pathogenesis of various autoimmune diseases. Immune thrombocytopenia (ITP) is an autoimmune disease characterized by increased platelet destruction and reduced platelet production, resulting in decreased platelet count. Recently, a series of studies in adults and children with ITP have found that the frequency of Tregs is reduced in circulation, bone marrow, and spleen, and Treg function is impaired. Treg dysregulation is improved after platelet count is recovered by treatment with dexamethasone, rituximab, or thrombopoietin receptor agonists. In addition, a critical role of Tregs in preventing the anti-platelet autoimmune response has been demonstrated in mice deficient in functional Tregs. Thrombocytopenia observed in Treg-deficient mice is mediated through production of IgG anti-platelet autoantibodies, which is analogous to human ITP. Further studies evaluating mechanisms of Treg dysregulation in ITP patients are necessary to elucidate the pathogenesis of ITP and develop novel therapeutic strategies that suppress anti-platelet autoimmune response.

Deletion of Crry and DAF on murine platelets stimulates thrombopoiesis and increases factor H-dependent resistance of peripheral platelets to complement attack.

Complement receptor 1-related gene/protein y (Crry) and decay-accelerating factor (DAF) are two murine membrane C3 complement regulators with overlapping functions. Crry deletion is embryonically lethal whereas DAF-deficient mice are generally healthy. Crry(-/-)DAF(-/-) mice were viable on a C3(-/-) background, but platelets from such mice were rapidly destroyed when transfused into C3-sufficient mice. In this study, we used the cre-lox system to delete platelet Crry in DAF(-/-) mice and studied Crry/DAF-deficient platelet development in vivo. Rather than displaying thrombocytopenia, Pf4-Cre(+)-Crry(flox/flox) mice had normal platelet counts and their peripheral platelets were resistant to complement attack. However, chimera mice generated with Pf4-Cre(+)-Crry(flox/flox) bone marrows showed platelets from C3(-/-) but not C3(+/+) recipients to be sensitive to complement activation, suggesting that circulating platelets in Pf4-Cre(+)-Crry(flox/flox) mice were naturally selected in a complement-sufficient environment. Notably, Pf4-Cre(+)-Crry(flox/flox) mouse platelets became complement susceptible when factor H function was blocked. Examination of Pf4-Cre(+)-Crry(flox/flox) mouse bone marrows revealed exceedingly active thrombopoiesis. Thus, under in vivo conditions, Crry/DAF deficiency on platelets led to abnormal platelet turnover, but peripheral platelet count was compensated for by increased thrombopoiesis. Selective survival of Crry/DAF-deficient platelets aided by factor H protection and compensatory thrombopoiesis demonstrates the cooperation between membrane and fluid phase complement inhibitors and the body's ability to adaptively respond to complement regulator deficiencies.

Positioning new treatments in the management of immune thrombocytopenia.

Immune thrombocytopenia (ITP) is a syndrome characterized by low platelet counts and an increased risk of bleeding. For most children, ITP is a self-limiting disease; however, for some children and most adults, thrombocytopenia can become chronic. Newer therapies for ITP include rituximab and thrombopoietin (TPO) receptor agonists. Rituximab is a useful second-line therapy and may be splenectomy-sparing. Thrombopoeitin receptor agonists have demonstrated large treatment effects with respect to increasing platelet levels; however, they require maintenance dosing. This review summarizes how these new agents might be positioned in the management of patients with chronic ITP.

What is the potential for thrombopoietic agents in acute leukemia?

In the 16 years since thrombopoietin was identified and cloned, much has been learned about its biochemistry, how it is regulated, and its involvement in a wide range of functions in a variety of cell lineages. The first generation of recombinant human thrombopoietins, rHuTPO and pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), were shown to increase platelet counts in patients with immune thrombocytopenia, in platelet apheresis donors, and in patients receiving nonmyeloablative chemotherapy. Their effects in patients with acute myeloid leukemia (AML) showed no benefit at a wide range of doses and schedules. The two second-generation TPO mimetics approved by the US Food and Drug Administration (FDA) for the treatment of ITP, romiplostim and eltrombopag, are now being studied in a number of thrombocytopenic disorders including those due to chemotherapy and hepatitis C. Since romiplostim is comparable to the first-generation recombinant thrombopoietins, it may not be beneficial in AML treatment; however, given its novel mechanism of action, eltrombopag may be a TPO potentiator and if given at the proper time during chemotherapy, may enable AML patients to recover platelet counts sooner.