p53 activation during ribosome biogenesis regulates normal erythroid differentiation.

The role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q- syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis showed that ribosome biogenesis is abruptly interrupted by the decline in ribosomal DNA transcription and the collapse of ribosomal protein neosynthesis. Its premature arrest by the RNA Pol I inhibitor CX-5461 targeted the proliferation of immature erythroblasts. p53 was activated spontaneously or in response to CX-5461, concomitant to ribosome biogenesis arrest, and drove a transcriptional program in which genes involved in cell cycle-arrested, negative regulation of apoptosis, and DNA damage response were upregulated. RNA Pol I transcriptional stress resulted in nucleolar disruption and activation of the ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation is crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold downregulation of ribosome biogenesis could be prematurely reached and, together with pathological p53 activation, prevents a normal expansion of erythroid progenitors.

A major role of TGF-beta1 in the homing capacities of murine hematopoietic stem cell/progenitors.

Transforming growth factor-beta1 (TGF-beta1) is a pleiotropic cytokine with major in vitro effects on hematopoietic stem cells (HSCs) and lymphocyte development. Little is known about hematopoiesis from mice with constitutive TGF-beta1 inactivation largely because of important embryonic lethality and development of a lethal inflammatory disorder in TGF-beta1(-/-) pups, making these studies difficult. Here, we show that no sign of the inflammatory disorder was detectable in 8- to 10-day-old TGF-beta1(-/-) neonates as judged by both the number of T-activated and T-regulator cells in secondary lymphoid organs and the level of inflammatory cytokines in sera. After T-cell depletion, the inflammatory disease was not transplantable in recipient mice. Bone marrow cells from 8- to 10-day-old TGF-beta1(-/-) neonates showed strikingly impaired short- and long-term reconstitutive activity associated with a parallel decreased in vivo homing capacity of lineage negative (Lin(-)) cells. In addition an in vitro-reduced survival of immature progenitors (Lin(-) Kit(+) Sca(+)) was observed. Similar defects were found in liver cells from TGF-beta1(-/-) embryos on day 14 after vaginal plug. These data indicate that TGF-beta1 is a critical regulator for in vivo homeostasis of the HSCs, especially for their homing potential.

Persistent but dysfunctional mucosal SARS-CoV-2-specific IgA and low lung IL-1ß associate with COVID-19 fatal outcome: A cross-sectional analysis.

The role of the mucosal pulmonary antibody response in coronavirus disease 2019 (COVID-19) outcome remains unclear. Here, we found that in bronchoalveolar lavage (BAL) samples from 48 patients with severe COVID-19-infected with the ancestral Wuhan virus, mucosal IgG and IgA specific for S1, receptor-binding domain (RBD), S2, and nucleocapsid protein (NP) emerged in BAL containing viruses early in infection and persist after virus elimination, with more IgA than IgG for all antigens tested. Furthermore, spike-IgA and spike-IgG immune complexes were detected in BAL, especially when the lung virus has been cleared. BAL IgG and IgA recognized the four main RBD variants. BAL neutralizing titers were higher early in COVID-19 when virus replicates in the lung than later in infection after viral clearance. Patients with fatal COVID-19, in contrast to survivors, developed higher levels of mucosal spike-specific IgA than IgG but lost neutralizing activities over time and had reduced IL-1ß in the lung. Altogether, mucosal spike and NP-specific IgG and S1-specific IgA persisting after lung severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) clearance and low pulmonary IL-1ß correlate with COVID-19 fatal outcome. Thus, mucosal SARS-CoV-2-specific antibodies may have adverse functions in addition to protective neutralization. Highlights: Mucosal pulmonary antibody response in COVID-19 outcome remains unclear. We show that in severe COVID-19 patients, mucosal pulmonary non-neutralizing SARS-CoV-2 IgA persit after viral clearance in the lung. Furthermore, low lung IL-1ß correlate with fatal COVID-19. Altogether, mucosal IgA may exert harmful functions beside protective neutralization.

Congenital thrombocytopenia and cytochrome C mutation: a matter of birth and death.

Thrombocytopenia (TP) Cargeeg is a unique autosomal dominant disorder, affecting a seven-generation family, caused by cytochrome C (CYCS) mutation that dysregulates platelet formation. The CYCS mutation in this disorder is a glycine 41 replacement by serine, which yields a cytochrome C variant with enhanced apoptotic pathway activity in vitro. The deregulated apoptosis in this disorder affects megakaryocytes (MK) during platelet formation, leading to early and ectopic platelet release in the bone marrow (BM). Notably, the family has no other phenotypic indication of abnormal apoptosis, implying that cytochrome C activity is not a critical regulator of physiological apoptosis in most cells. The pathophysiology of this unique inherited TP, with unaltered platelet survival and normal MK content in the BM, has implications for physiological and pathological mechanisms altering MK apoptosis, with implications for other unexplained thrombocytopenic disorders.

Exposure of human megakaryocytes to high shear rates accelerates platelet production.

Platelets originate from megakaryocytes (MKs) by cytoplasmic elongation into proplatelets. Direct platelet release is not seen in bone marrow hematopoietic islands. It was suggested that proplatelet fragmentation into platelets can occur intravascularly, yet evidence of its dependence on hydrodynamic forces is missing. Therefore, we investigated whether platelet production from MKs could be up-regulated by circulatory forces. Human mature MKs were perfused at a high shear rate on von Willebrand factor. Cells were observed in real time by videomicroscopy, and by confocal and electron microscopy after fixation. Dramatic cellular modifications followed exposure to high shear rates: 30% to 45% adherent MKs were converted into proplatelets and released platelets within 20 minutes, contrary to static conditions that required several hours, often without platelet release. Tubulin was present in elongated proplatelets and platelets, thus ruling out membrane tethers. By using inhibitors, we demonstrated the fundamental roles of microtubule assembly and MK receptor GPIb. Secretory granules were present along the proplatelet shafts and in shed platelets, as shown by P-selectin labeling. Platelets generated in vitro were functional since they responded to thrombin by P-selectin expression and cytoskeletal reorganization. In conclusion, MK exposure to high shear rates promotes platelet production via GPIb, depending on microtubule assembly and elongation.

Increased expression of urokinase plasminogen activator in Quebec platelet disorder is linked to megakaryocyte differentiation.

Quebec platelet disorder (QPD) is an inherited bleeding disorder associated with increased urokinase plasminogen activator (uPA) in platelets but not in plasma, intraplatelet plasmin generation, and alpha-granule protein degradation. These abnormalities led us to investigate uPA expression by QPD CD34(+) progenitors, cultured megakaryocytes, and platelets, and whether uPA was stored in QPD alpha-granules. Although QPD CD34(+) progenitors expressed normal amounts of uPA, their differentiation into megakaryocytes abnormally increased expression of the uPA gene but not the flanking genes for vinculin or calcium/calmodulin-dependent protein kinase IIgamma on chromosome 10. The increased uPA production by cultured QPD megakaryocytes mirrored their production of alpha-granule proteins, which was normal. uPA was localized to QPD alpha-granules and it showed extensive colocalization with alpha-granule proteins in both cultured QPD megakaryocytes and platelets, and with plasminogen in QPD platelets. In QPD megakaryocytes, cultured without or with plasma as a source of plasminogen, alpha-granule proteins were stored undegraded and this was associated with much less uPA-plasminogen colocalization than in QPD platelets. Our studies indicate that the overexpression of uPA in QPD emerges with megakaryocyte differentiation, without altering the expression of flanking genes, and that uPA is costored with alpha-granule proteins prior to their proteolysis in QPD.

Platelets and viruses: an ambivalent relationship.

Thrombocytopenia is a frequent complication of viral infections providing evidence that interaction of platelets with viruses is an important pathophysiological phenomenon. Multiple mechanisms are involved depending on the nature of the viruses involved. These include immunological platelet destruction, inappropriate platelet activation and consumption, and impaired megakaryopoiesis. Viruses bind platelets through specific receptors and identified ligands, which lead to mutual alterations of both the platelet host and the viral aggressor. We have shown that HIV-1 viruses are internalized specifically in platelets and megakaryocytes, where they can be either sheltered, unaltered (with potential transfer of the viruses into target organs), or come in contact with platelet secretory products leading to virus destruction and facilitated platelet clearance. In this issue, we have reviewed the various pathways that platelets use in order to interact with viruses, HIV and others. This review also shows that more work is still needed to precisely identify platelet roles in viral infections, and to answer the challenge of viral safety in platelet transfusion.

COVID-19 Lung Pathogenesis in SARS-CoV-2 Autopsy Cases.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major public health issue. COVID-19 is considered an airway/multi-systemic disease, and demise has been associated with an uncontrolled immune response and a cytokine storm in response to the virus. However, the lung pathology, immune response, and tissue damage associated with COVID-19 demise are poorly described and understood due to safety concerns. Using post-mortem lung tissues from uninfected and COVID-19 deadly cases as well as an unbiased combined analysis of histology, multi-viral and host markers staining, correlative microscopy, confocal, and image analysis, we identified three distinct phenotypes of COVID-19-induced lung damage. First, a COVID-19-induced hemorrhage characterized by minimal immune infiltration and large thrombus; Second, a COVID-19-induced immune infiltration with excessive immune cell infiltration but no hemorrhagic events. The third phenotype correspond to the combination of the two previous ones. We observed the loss of alveolar wall integrity, detachment of lung tissue pieces, fibroblast proliferation, and extensive fibrosis in all three phenotypes. Although lung tissues studied were from lethal COVID-19, a strong immune response was observed in all cases analyzed with significant B cell and poor T cell infiltrations, suggesting an exhausted or compromised immune cellular response in these patients. Overall, our data show that SARS-CoV-2-induced lung damage is highly heterogeneous. These individual differences need to be considered to understand the acute and long-term COVID-19 consequences.

Pannexin-1 channel opening is critical for COVID-19 pathogenesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly rampaged worldwide, causing a pandemic of coronavirus disease (COVID -19), but the biology of SARS-CoV-2 remains under investigation. We demonstrate that both SARS-CoV-2 spike protein and human coronavirus 229E (hCoV-229E) or its purified S protein, one of the main viruses responsible for the common cold, induce the transient opening of Pannexin-1 (Panx-1) channels in human lung epithelial cells. However, the Panx-1 channel opening induced by SARS-CoV-2 is greater and more prolonged than hCoV-229E/S protein, resulting in an enhanced ATP, PGE2, and IL-1ß release. Analysis of lung lavages and tissues indicate that Panx-1 mRNA expression is associated with increased ATP, PGE2, and IL-1ß levels. Panx-1 channel opening induced by SARS-CoV-2 spike protein is angiotensin-converting enzyme 2 (ACE-2), endocytosis, and furin dependent. Overall, we demonstrated that Panx-1 channel is a critical contributor to SARS-CoV-2 infection and should be considered as an alternative therapy.

Platelets from HIV-infected individuals on antiretroviral drug therapy with poor CD4+ T cell recovery can harbor replication-competent HIV despite viral suppression.

In addition to hemostasis, human platelets have several immune functions and interact with infectious pathogens including HIV in vitro. Here, we report that platelets from HIV-infected individuals on combined antiretroviral drug therapy (ART) with low blood CD4+ T cell counts (<350 cells/µl) contained replication-competent HIV despite viral suppression. In vitro, human platelets harboring HIV propagated the virus to macrophages, a process that could be prevented with the biologic abciximab, an anti-integrin αIIb/ß3 Fab. Furthermore, in our cohort, 88% of HIV-infected individuals on ART with viral suppression and with platelets containing HIV were poor immunological responders with CD4+ T cell counts remaining below <350 cells/µl for more than one year. Our study suggests that platelets may be transient carriers of HIV and may provide an alternative pathway for HIV dissemination in HIV-infected individuals on ART with viral suppression and poor CD4+ T cell recovery.