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Complement activation is considered to contribute to the pathogenesis of severe SARS-CoV-2 infection, mainly by generating potent immune effector mechanisms including a strong inflammatory response. Involvement of the lectin complement pathway, a major actor of the innate immune anti-viral defense, has been reported previously. It is initiated by recognition of the viral surface Spike glycoprotein by mannose-binding lectin (MBL), which induces activation of the MBL-associated protease MASP-2 and triggers the proteolytic complement cascade. A role for the viral nucleoprotein (N) has also been reported, through binding to MASP-2, leading to protease overactivation and potentiation of the lectin pathway. In the present study, we reinvestigated the interactions of the SARS-CoV-2 N protein, produced either in bacteria or secreted by mammalian cells, with full-length MASP-2 or its catalytic domain, in either active or proenzyme form. We could not confirm the interaction of the N protein with the catalytic domain of MASP-2 but observed N protein binding to proenzyme MASP-2. We did not find a role of the N protein in MBL-mediated activation of the lectin pathway. Finally, we showed that incubation of the N protein with MASP-2 results in proteolysis of the viral protein, an observation that requires further investigation to understand a potential functional significance in infected patients.

COVID-19 is caused by the human pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has resulted in widespread morbidity and mortality. CD4+ T cells, CD8+ T cells and neutralizing antibodies all contribute to control SARS-CoV-2 infection. However, heterogeneity is a major factor in disease severity and in immune innate and adaptive responses to SARS-CoV-2. We performed a deep analysis by flow cytometry of lymphocyte populations of 125 hospitalized SARS-CoV-2 infected patients on the day of hospital admission. Five clusters of patients were identified using hierarchical classification on the basis of their immunophenotypic profile, with different mortality outcomes. Some characteristics were observed in all the clusters of patients, such as lymphopenia and an elevated level of effector CD8+CCR7- T cells. However, low levels of T cell activation are associated to a better disease outcome; on the other hand, profound CD8+ T-cell lymphopenia, a high level of CD4+ and CD8+ T-cell activation and a high level of CD8+ T-cell senescence are associated with a higher mortality outcome. Furthermore, a cluster of patient was characterized by high B-cell responses with an extremely high level of plasmablasts. Our study points out the prognostic value of lymphocyte parameters such as T-cell activation and senescence and strengthen the interest in treating the patients early in course of the disease with targeted immunomodulatory therapies based on the type of adaptive response of each patient.

Our immune system responds to infectious (PAMPs) and tissue damage (DAMPs) signals. The complement system and alarmin High-Mobility Group Box 1 (HMGB1) are two powerful soluble actors of human host defense and immune surveillance. These systems involve molecular cascades and amplification loops for their signaling or activation. Initially activated as alarm raising systems, their function can be finally switched towards inflammation resolution, where they sustain immune maturation and orchestrate repair mechanisms, opening the way back to homeostasis. However, when getting out of control, these defense systems can become deleterious and trigger serious cellular and tissue damage. Therefore, they can be considered as double-edged swords. The close interaction between the complement and HMGB1 pathways is described here, as well as their traditional and non-canonical roles, their functioning at different locations and their independent and collective impact in different systems both in health and disease. Starting from these systems and interplay at the molecular level (when elucidated), we then provide disease examples to better illustrate the signs and consequences of their roles and interaction, highlighting their importance and possible vicious circles in alarm raising and inflammation, both individually or in combination. Although this integrated view may open new therapeutic strategies, future challenges have to be faced because of the remaining unknowns regarding the molecular mechanisms underlying the fragile molecular balance which can drift towards disease or return to homeostasis, as briefly discussed at the end.

The humoral response is frequently dysfunctioning in autoimmunity with a frequent rise in total serum immunoglobulins, among which are found autoantibodies that may be pathogenic by themselves and/or propagate the inflammatory reaction. The infiltration of autoimmune tissues by antibody-secreting cells (ASCs) constitutes another dysfunction. The known high dependency of ASCs on the microenvironment to survive combined to the high diversity of infiltrated tissues implies that ASCs must adapt. Some tissues even within a single clinical autoimmune entity are devoid of infiltration. The latter means that either the tissue is not permissive or ASCs fail to adapt. The origin of infiltrated ASCs is also variable. Indeed, ASCs may be commonly generated in the secondary lymphoid organ draining the autoimmune tissue, and home at the inflammation site under the guidance of specific chemokines. Alternatively, ASCs may be generated locally, when ectopic germinal centers are formed in the autoimmune tissue. Alloimmune tissues with the example of kidney transplantation will also be discussed own to their high similarity with autoimmune tissues. It should also be noted that antibody production is not the only function of ASCs, since cells with regulatory functions have also been described. This article will review all the phenotypic variations indicative of tissue adaptation described so for at the level of ASC-infiltrating auto/alloimmune tissues. The aim is to potentially define tissue-specific molecular targets in ASCs to improve the specificity of future autoimmune treatments.

Pseudomonas aeruginosa, an opportunistic Gram-negative pathogen, is a leading cause of bacteremia with a high mortality rate. We recently reported that P. aeruginosa forms a persister-like sub-population of evaders in human plasma. Here, using a gain-of-function transposon sequencing (Tn-seq) screen in plasma, we identified and validated previously unknown factors affecting bacterial persistence in plasma. Among them, we identified a small periplasmic protein, named SrgA, whose expression leads to up to a 100-fold increase in resistance to killing. Additionally, mutants in pur and bio genes displayed higher tolerance and persistence, respectively. Analysis of several steps of the complement cascade and exposure to an outer-membrane-impermeable drug, nisin, suggested that the mutants impede membrane attack complex (MAC) activity per se. Electron microscopy combined with energy-dispersive X-ray spectroscopy (EDX) revealed the formation of polyphosphate (polyP) granules upon incubation in plasma of different size in purD and wild-type strains, implying the bacterial response to a stress signal. Indeed, inactivation of ppk genes encoding polyP-generating enzymes lead to significant elimination of persisting bacteria from plasma. Through this study, we shed light on a complex P. aeruginosa response to the plasma conditions and discovered the multifactorial origin of bacterial resilience to MAC-induced killing.

Introduction Suspicion of myelodysplastic syndromes (MDS) is the most common reason for bone marrow aspirate in elderly patients. Peripheral blood neutrophil myeloperoxidase expression quantified by flow cytometric analysis might rule out MDS for up to 35% of patients referred for suspected disease, without requiring bone marrow aspiration. Yet laboratory-developed liquid antibody cocktails have practical limitations, because of lack of standardisation and poor stability. This research project aims to estimate the level of agreement and comparative accuracy between a single-use flow cytometry tube of lyophilised reagents (BD Lyotube Stain 468) and its laboratory-developed liquid reagent counterpart in quantifying peripheral blood neutrophil myeloperoxidase expression, among adult patients referred for suspected MDS.Methods and analysis The MPO-MDS-Develop project is a cross-sectional diagnostic accuracy study of two index tests by comparison with a reference standard in consecutive unselected adult patients conducted at a single university hospital. Flow cytometry analysis of peripheral blood samples will be performed by independent operators blinded to the reference diagnosis, using either Lyotube Stain 468 or laboratory-developed liquid reagent cocktail. The reference diagnosis of MDS will be established by cytomorphological evaluation of bone marrow aspirate by two independent haematopathologists blinded to the index test results. Morphologic assessment will be complemented by bone marrow flow cytometric score, karyotype and targeted next-generation sequencing panel of 43 genes, where relevant. The target sample size is 103 patients.Ethics and dissemination An institutional review board (Comité de Protection des Personnes Sud Est III, Lyon, France) approved the protocol prior to study initiation (reference number: 2020-028-B). Participants will be recruited using an opt-out approach. Efforts will be made to release the primary results within 6 months of study completion.Trial registration number NCT04399018.