RESUMO
Clinical manifestations of COVID-19 caused by the new coronavirus SARS-CoV-2 are associated with age1,2. Adults develop respiratory symptoms, which can progress to acute respiratory distress syndrome (ARDS) in the most severe form, while children are largely spared from respiratory illness but can develop a life-threatening multisystem inflammatory syndrome (MIS-C)3-5. Here, we show distinct antibody responses in children and adults after SARS-CoV-2 infection. Adult COVID-19 cohorts had anti-spike (S) IgG, IgM and IgA antibodies, as well as anti-nucleocapsid (N) IgG antibody, while children with and without MIS-C had reduced breadth of anti-SARS-CoV-2-specific antibodies, predominantly generating IgG antibodies specific for the S protein but not the N protein. Moreover, children with and without MIS-C had reduced neutralizing activity as compared to both adult COVID-19 cohorts, indicating a reduced protective serological response. These results suggest a distinct infection course and immune response in children independent of whether they develop MIS-C, with implications for developing age-targeted strategies for testing and protecting the population.
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Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , COVID-19/imunologia , Proteínas do Nucleocapsídeo/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adolescente , Adulto , Idoso , COVID-19/virologia , Criança , Pré-Escolar , Feminino , Humanos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Masculino , Pessoa de Meia-Idade , SARS-CoV-2/fisiologia , Adulto JovemRESUMO
Immune checkpoint inhibitors (ICPi) have revolutionized cancer immunotherapy but also can induce autoimmune hemolytic anemia (AIHA), a severe disease with high mortality. However, the cellular and molecular mechanism(s) of ICPi-AIHA are unclear, other than being initiated through decreased checkpoint inhibition. Herein, we report ICPi-AIHA in a novel mouse model that shows similar characteristics of known human ICPi-AIHA (e.g., autoantibodies, hemolysis, increased mortality). During ICPi-AIHA, there is the simultaneous reduction of two regulatory T cell populations (FoxP3+ and Tr1 Tregs) and an increase in inflammatory TH17 T cells. Moreover, a novel CD39+CD73-FoxP3-CD25- CD4+ T cell subset (i.e., CD39 single positive [CD39SP]) emerges, and early increases in CD39SP predict AIHA development; CD39 is an ectonuclease that breaks down ATP. Additionally, we found that boosting ATPase activity by injecting recombinant apyrase mitigates AIHA development and significant CD39SP reductions, both suggesting a functional role for CD39 and demonstrating a novel therapeutic approach. Importantly, CD39SP are detectable in multiple mouse models developing AIHA and in patients with AIHA, demonstrating applicability to idiopathic and secondary AIHA. Highlighting broader autoimmunity relevance, ICPi-treated NZB mice experienced accelerated onset and severity of lupus, including AIHA. Moreover, ICPi treatment of healthy B6 animals led to detectable CD39SP and development of autoantibodies against multiple autoantigens including those on red blood cells and platelets. Together, our findings elucidate cellular and molecular mechanisms of ICPi-AIHA, leading to novel diagnostic and therapeutic approaches with translational potential for use in humans being treated with ICPi.
RESUMO
Antibodies against red blood cell (RBC) alloantigens can increase morbidity and mortality among transfusion recipients. However, alloimmunization rates can vary dramatically, as some patients never generate alloantibodies after transfusion, whereas others not only become alloimmunized but may also be prone to generating additional alloantibodies after subsequent transfusion. Previous studies suggested that CD4 T-cell responses that drive alloantibody formation recognize the same alloantigen engaged by B cells. However, because RBCs express numerous antigens, both internally and externally, it is possible that CD4 T-cell responses directed against intracellular antigens may facilitate subsequent alloimmunization against a surface RBC antigen. Here, we show that B cells can acquire intracellular antigens from RBCs. Using a mouse model of donor RBCs expressing 2 distinct alloantigens, we demonstrate that immune priming to an intracellular antigen, which would not be detected by any currently used RBC compatibility assays, can directly influence alloantibody formation after exposure to a subsequent distinct surface RBC alloantigen. These findings suggest a previously underappreciated mechanism whereby transfusion recipient responders may exhibit an increased rate of alloimmunization because of prior immune priming toward intracellular antigens.
Assuntos
Transfusão de Eritrócitos , Isoanticorpos , Transfusão de Eritrócitos/efeitos adversos , Eritrócitos , Antígenos , Isoantígenos , ImunizaçãoRESUMO
Antibodies against fetal red blood cell (RBC) antigens can cause hemolytic disease of the fetus and newborn (HDFN). Reductions in HDFN due to anti-RhD antibodies have been achieved through use of Rh immune globulin (RhIg), a polyclonal antibody preparation that causes antibody-mediated immunosuppression (AMIS), thereby preventing maternal immune responses against fetal RBCs. Despite the success of RhIg, it is only effective against 1 alloantigen. The lack of similar interventions that mitigate immune responses toward other RBC alloantigens reflects an incomplete understanding of AMIS mechanisms. AMIS has been previously attributed to rapid antibody-mediated RBC removal, resulting in B-cell ignorance of the RBC alloantigen. However, our data demonstrate that antibody-mediated RBC removal can enhance de novo alloimmunization. In contrast, inclusion of antibodies that possess the ability to rapidly remove the target antigen in the absence of detectable RBC clearance can convert an augmented antibody response to AMIS. These results suggest that the ability of antibodies to remove target antigens from the RBC surface can trigger AMIS in situations in which enhanced immunity may otherwise occur. In doing so, these results hold promise in identifying key antibody characteristics that can drive AMIS, thereby facilitating the design of AMIS approaches toward other RBC antigens to eliminate all forms of HDFN.
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Eritroblastose Fetal , Eritrócitos , Feminino , Recém-Nascido , Humanos , Eritrócitos/metabolismo , Anticorpos , Tolerância Imunológica , Terapia de Imunossupressão , Imunoglobulina rho(D) , Isoantígenos , IsoanticorposRESUMO
BACKGROUND AND OBJECTIVES: Platelet transfusions are increasing with medical advances. Based on FDA criteria, platelet units are assessed by in vitro measures; however, it is not known how platelet processing and storage duration affect function in vivo. Our study's aim was to develop a novel platelet transfusion model stored in mouse plasma that meets FDA criteria adapted to mice, and transfused fresh and stored platelets are detectable in clots in vivo. STUDY DESIGN AND METHODS: Platelet units stored in mouse plasma were prepared using a modified platelet-rich plasma (PRP) collection protocol. Characteristics of fresh and stored units, including pH, cell count, in vitro measures of activity, including activation and aggregation, and post-transfusion recovery (PTR), were determined. Lastly, a tail transection assay was conducted using mice transfused with fresh or stored units, and transfused platelets were identified by confocal imaging. RESULTS: Platelet units had acceptable platelet and white cell counts and were negative for bacterial contamination. Fresh and 1-day stored units had acceptable pH; the platelets were activatable by thrombin and adenosine diphosphate, agreeable with thrombin, had acceptable PTR, and were present in vivo in clots of recipients after tail transection. In contrast, 2-day stored units had clinically unacceptable quality. CONCLUSION: We developed mouse platelets for transfusion analogous to human platelet units using a modified PRP collection protocol with maximum storage of 1 day for an 'old' unit. This provides a powerful tool to test how process modifications and storage conditions affect transfused platelet function in vivo.
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Plaquetas , Preservação de Sangue , Transfusão de Plaquetas , Animais , Camundongos , Transfusão de Plaquetas/métodos , Plaquetas/metabolismo , Plaquetas/citologia , Preservação de Sangue/métodos , Humanos , Plasma Rico em Plaquetas/citologia , Modelos AnimaisRESUMO
RBC transfusion therapy is essential for the treatment of anemia. A serious complication of transfusion is the development of non-ABO alloantibodies to polymorphic RBC Ags; yet, mechanisms of alloantibody formation remain unclear. Storage of mouse RBCs before transfusion increases RBC immunogenicity through an unknown mechanism. We previously reported that sterile, stored mouse RBCs activate splenic dendritic cells (DCs), which are required for alloimmunization. Here we transfused mice with allogeneic RBCs to test whether stored RBCs activate pattern recognition receptors (PRRs) on recipient DCs to induce adaptive immunity. TLRs are a class of PRRs that regulate DC activation, which signal through two adapter molecules: MyD88 and TRIF. We show that the inflammatory cytokine response, DC activation and migration, and the subsequent alloantibody response to transfused RBCs require MyD88 but not TRIF, suggesting that a restricted set of PRRs are responsible for sensing RBCs and triggering alloimmunization.
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Imunidade Adaptativa , Eritrócitos/imunologia , Eritrócitos/metabolismo , Imunidade Inata , Fator 88 de Diferenciação Mieloide/metabolismo , Animais , Biomarcadores , Transfusão de Eritrócitos , Imunofluorescência , Humanos , Isoanticorpos/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/genéticaRESUMO
Although red blood cell (RBC) transfusions save lives, some patients develop clinically-significant alloantibodies against donor blood group antigens, which then have adverse effects in multiple clinical settings. Few effective measures exist to prevent RBC alloimmunization and/or eliminate alloantibodies in sensitized patients. Donor-related factors may influence alloimmunization; thus, there is an unmet clinical need to identify which RBC units are immunogenic. Repeat volunteer blood donors and donors on iron supplements have elevated reticulocyte counts compared to healthy non-donors. Early reticulocytes retain mitochondria and other components, which may act as danger signals in immune responses. Herein, we tested whether reticulocytes in donor RBC units could enhance RBC alloimmunization. Using a murine model, we demonstrate that transfusing donor RBC units with increased reticulocyte frequencies dose-dependently increased RBC alloimmunization rates and alloantibody levels. Transfusing reticulocyte-rich RBC units was associated with increased RBC clearance from the circulation and a robust proinflammatory cytokine response. As compared to previously reported post-transfusion RBC consumption patterns, erythrophagocytosis from reticulocyte-rich units was increasingly performed by splenic B cells. These data suggest that reticulocytes in a donated RBC unit impact the quality of blood transfused, are targeted to a distinct compartment, and may be an underappreciated risk factor for RBC alloimmunization.
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Isoanticorpos , Reticulócitos , Humanos , Camundongos , Animais , Doadores de Sangue , Eritrócitos , Fatores de RiscoRESUMO
BACKGROUND: Transgenic mice expressing RBC specific antigens are widely used in mechanistic studies of RBC alloimmunization. Existing RBC donor strains have random transgene integration, potentially disrupting host elements that can confound biological interpretation. STUDY DESIGN AND METHODS: Integration site and genomic alterations were characterized by both targeted locus amplification and congenic backcrossing in the five most commonly used RBC alloantigen donor strains (KEL-K2hi , KEL-K2med , and KEL-K2lo , and KEL-K1). A targeted transgenic approach was developed to allow RBC specific transgene expression from a safe harbor locus (ROSA26). Alloimmune responses were assessed by transfusing alloantigen expressing RBCs into wild-type recipients and measuring alloantibodies by flow cytometry. RESULTS/FINDINGS: Four of the five analyzed strains had at least one gene disrupted by the transgene integration but none of the disrupted genes are known to be involved in RBC biology. The integration of KEL-K2med potentially altered the immunological properties of RBCs, although the biological significance of the observed changes is unclear. The ROSA26 targeted approach resulted in a single copy of the transgene that maintains RBC specific expression without random disruption of genomic elements. CONCLUSION: These findings provide a detailed characterization of genomic disruption by transgene integration found in commonly used RBC donor strains that is relevant to numerous previous publications as well as future studies. With the possible exception of KEL-K2med , transgene integration is not predicted to affect RBC biology in existing models, and new models can avoid this concern using the described targeted transgenic approach.
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Antígenos de Grupos Sanguíneos , Eritrócitos , Isoanticorpos , Animais , Camundongos , Eritrócitos/imunologia , Isoanticorpos/sangue , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transgenes/genética , Antígenos de Grupos Sanguíneos/genética , Antígenos de Grupos Sanguíneos/imunologiaRESUMO
INTRODUCTION: The impact of blood storage on red blood cell (RBC) alloimmunization remains controversial, with some studies suggesting enhancement of RBC-induced alloantibody production and others failing to observe any impact of storage on alloantibody formation. Since evaluation of storage on RBC alloimmunization in patients has examined antibody formation against a broad range of alloantigens, it remains possible that different clinical outcomes reflect a variable impact of storage on alloimmunization to specific antigens. METHODS: RBCs expressing two distinct model antigens, HEL-OVA-Duffy (HOD) and KEL, separately or together (HOD × KEL), were stored for 0, 8, or 14 days, followed by detection of antigen levels prior to transfusion. Transfused donor RBC survival was assessed within 24 h of transfusion, while IgM and IgG antibody production were assessed 5 and 14 days after transfusion. RESULTS: Stored HOD or KEL RBCs retained similar HEL or KEL antigen levels, respectively, as fresh RBCs, but did exhibit enhanced RBC clearance with increased storage age. Storage enhanced IgG antibody formation against HOD, while the oppositive outcome occurred following transfusion of stored KEL RBCs. The distinct impact of storage on HOD or KEL alloimmunization did not appear to reflect intrinsic differences between HOD or KEL RBCs, as transfusion of stored HOD × KEL RBCs resulted in increased IgG anti-HOD antibody development and reduced IgG anti-KEL antibody formation. CONCLUSIONS: These data demonstrate a dichotomous impact of storage on immunization to distinct RBC antigens, offering a possible explanation for inconsistent clinical experience and the need for additional studies on the relationship between RBC storage and alloimmunization.
Assuntos
Antígenos , Transfusão de Eritrócitos , Camundongos , Animais , Transfusão de Eritrócitos/efeitos adversos , Eritrócitos , Isoantígenos , Isoanticorpos , Imunoglobulina GRESUMO
Sickle cell disease (SCD) is an inherited blood disorder characterized by sickled red blood cells (RBCs), which are more sensitive to haemolysis and can contribute to disease pathophysiology. Although treatment of SCD can include RBC transfusion, patients with SCD have high rates of alloimmunization. We hypothesized that RBCs from patients with SCD have functionally active mitochondria and can elicit a type 1 interferon response. We evaluated blood samples from more than 100 patients with SCD and found elevated frequencies of mitochondria in reticulocytes and mature RBCs, as compared to healthy blood donors. The presence of mitochondria in mature RBCs was confirmed by flow cytometry, electron microscopy, and proteomic analysis. The mitochondria in mature RBCs were metabolically competent, as determined by enzymatic activities and elevated levels of mitochondria-derived metabolites. Metabolically-active mitochondria in RBCs may increase oxidative stress, which could facilitate and/or exacerbate SCD complications. Coculture of mitochondria-positive RBCs with neutrophils induced production of type 1 interferons, which are known to increase RBC alloimmunization rates. These data demonstrate that mitochondria retained in mature RBCs are functional and can elicit immune responses, suggesting that inappropriate retention of mitochondria in RBCs may play an underappreciated role in SCD complications and be an RBC alloimmunization risk factor.
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Anemia Falciforme , Proteômica , Eritrócitos/metabolismo , Hemólise , Humanos , MitocôndriasRESUMO
Polyclonal anti-D (Rh immune globulin [RhIg]) therapy has mitigated hemolytic disease of the newborn over the past half century, although breakthrough anti-D alloimmunization still occurs in some treated females. We hypothesized that antiviral responses may impact the efficacy of immunoprophylaxis therapy in a type 1 interferon (IFN)-dependent manner and tested this hypothesis in a murine model of KEL alloimmunization. Polyclonal anti-KEL immunoprophylaxis (KELIg) was administered to wild-type or knockout mice in the presence or absence of polyinosinic-polycytidilic acid (poly[I:C]), followed by the transfusion of murine red blood cells (RBCs) expressing the human KEL glycoprotein. Anti-KEL alloimmunization, serum cytokines, and consumption of the transfused RBCs were evaluated longitudinally. In some experiments, recipients were treated with type 1 IFN (IFN-α/ß). Recipient treatment with poly(I:C) led to breakthrough anti-KEL alloimmunization despite KELIg administration. Recipient CD4+ T cells were not required for immunoprophylaxis efficacy at baseline, and modulation of the KEL glycoprotein antigen occurred to the same extent in the presence or absence of recipient inflammation. Under conditions where breakthrough anti-KEL alloimmunization occurred, KEL RBC consumption by inflammatory monocytes and serum monocyte chemoattractant protein-1 and interleukin-6 were significantly increased. Poly(I:C) or type I IFN administration was sufficient to cause breakthrough alloimmunization, with poly(I:C) inducing alloimmunization even in the absence of recipient type I IFN receptors. A better understanding of how recipient antiviral responses lead to breakthrough alloimmunization despite immunoprophylaxis may have translational relevance to instances of RhIg failure that occur in humans.
Assuntos
Eritrócitos/efeitos dos fármacos , Eritrócitos/imunologia , Glicoproteínas de Membrana/sangue , Glicoproteínas de Membrana/genética , Metaloendopeptidases/sangue , Metaloendopeptidases/genética , Poli I-C/farmacologia , Animais , Linfócitos T CD4-Positivos/imunologia , Citocinas/sangue , Modelos Animais de Doenças , Eritroblastose Fetal/sangue , Eritroblastose Fetal/imunologia , Eritroblastose Fetal/prevenção & controle , Transfusão de Eritrócitos/efeitos adversos , Feminino , Humanos , Imunização Passiva , Interferon Tipo I/sangue , Isoantígenos/sangue , Isoantígenos/genética , Sistema do Grupo Sanguíneo de Kell/sangue , Sistema do Grupo Sanguíneo de Kell/genética , Glicoproteínas de Membrana/imunologia , Metaloendopeptidases/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fagocitose/imunologia , GravidezRESUMO
BACKGROUND: Alloimmunization can be a significant barrier to red blood cell (RBC) transfusion. While alloantigen matching protocols hold promise in reducing alloantibody formation, transfusion-dependent patients can still experience RBC alloimmunization and associated complications even when matching protocols are employed. As a result, complementary strategies capable of actively preventing alloantibody formation following alloantigen exposure are warranted. STUDY DESIGN AND METHODS: We examined whether pharmacological removal of macrophages using clodronate may provide an additional strategy to actively inhibit RBC alloimmunization using two preclinical models of RBC alloimmunization. To accomplish this, mice were treated with clodronate, followed by transfusion of RBCs expressing the HOD (HEL, OVA, and Duffy) or KEL antigens. On days 5 and 14 post transfusion, anti-HOD or anti-KEL IgM and IgG antibodies were evaluated. RESULTS: Low dose clodronate effectively eliminated key marginal zone macrophage populations from the marginal sinus. Prior treatment with clodronate, but not empty liposomes, also significantly inhibited IgM and IgG anti-HOD alloantibody formation following transfusion of HOD RBCs. Similar exposure to clodronate inhibited IgM and IgG antibody formation following KEL RBC transfusion. CONCLUSIONS: Clodronate can inhibit anti-HOD and anti-KEL antibody formation following RBC transfusion in preclinical models. These results suggest that clodronate may provide an alternative approach to actively inhibit or prevent the development of alloantibodies following RBC transfusion, although future studies will certainly be needed to fully explore this possibility.
Assuntos
Ácido Clodrônico , Isoantígenos , Animais , Ácido Clodrônico/farmacologia , Eritrócitos , Humanos , Imunoglobulina G , Imunoglobulina M , Isoanticorpos , CamundongosRESUMO
FcRn, a non-classical Fc gamma (γ) receptor (FcγR) with near ubiquitous expression, plays key roles in disease pathogenesis and progression though immunoglobulin G (IgG) transport, IgG recycling, and IgG-immune complex clearance. FcRn function can be inhibited using IgG-based and non-IgG-based antagonists, by exploiting the pH-dependent binding affinity of FcRn for the IgG Fc region. FcRn therapeutics have shown promise in murine models and human clinical trials for autoimmune diseases and maternal-fetal immune cytopenias; they appear safe, well-tolerated, and reduce circulating IgG levels. Compared to traditional therapeutics, inhibiting FcRn has fewer adverse side effects and represents a new approach that is less invasive, time-consuming, and costly.
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Doenças Autoimunes/tratamento farmacológico , Púrpura Trombocitopênica Idiopática/tratamento farmacológico , Receptores Fc/antagonistas & inibidores , Animais , Doenças Autoimunes/imunologia , Autoimunidade/efeitos dos fármacos , Desenvolvimento de Medicamentos , Feminino , Feto/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoglobulina G/imunologia , Terapia de Alvo Molecular , Gravidez , Púrpura Trombocitopênica Idiopática/imunologia , Receptores Fc/imunologiaRESUMO
BACKGROUND: Genetically modified mice are used widely to explore mechanisms in most biomedical fields-including transfusion. Concluding that a gene modification is responsible for a phenotypic change assumes no other differences between the gene-modified and wild-type mice besides the targetted gene. STUDY DESIGN AND METHODS: To test the hypothesis that the N-terminus of Band3, which regulates metabolism, affects RBC storage biology, RBCs from mice with a modified N-terminus of Band3 were stored under simulated blood bank conditions. All strains of mice were generated with the same initial embryonic stem cells from 129 mice and each strain was backcrossed with C57BL/6 (B6) mice. Both 24-h recoveries post-transfusion and metabolomics were determined for stored RBCs. Genetic profiles of mice were assessed by a high-resolution SNP array. RESULTS: RBCs from mice with a mutated Band3 N-terminus had increased lipid oxidation and worse 24-h recoveries, "demonstrating" that Band3 regulates oxidative injury during RBC storage. However, SNP analysis demonstrated variable inheritance of 129 genetic elements between strains. Controlled interbreeding experiments demonstrated that the changes in lipid oxidation and some of the decreased 24-hr recovery were caused by inheritance of a region of chromosome 1 of 129 origin, and not due to the modification of Band 3. SNP genotyping of a panel of commonly used commercially available KO mice showed considerable 129 contamination, despite wild-type B6 mice being listed as the correct control. DISCUSSION: Thousands of articles published each year use gene-modified mice, yet genetic background issues are rarely considered. Assessment of such issues are not, but should become, routine norms of murine experimentation.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/genética , Camundongos/genética , Animais , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Pesquisa Biomédica , Preservação de Sangue , Eritrócitos/metabolismo , Patrimônio Genético , Camundongos/metabolismo , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
BACKGROUND: Transfusion of red blood cells (RBCs) stored for longer durations induces hemolysis and inflammatory cytokine production in murine and canine models. Despite immune system activation by stored RBCs, human randomized trials suggest that fresher RBC transfusions do not improve clinical outcomes. We hypothesized that underlying recipient hemolysis may affect cytokine responses to older RBC transfusions. STUDY DESIGN AND METHODS: C57BL/6 mouse cohorts were infused with anti-TER119 antibody to induce hemolysis, rabbit anti-platelet antiserum to induce immune thrombocytopenia (ITP), or appropriate control antibodies. Two days later, mice were transfused with fresh or stored RBCs. Furthermore, in a prospective, randomized, blinded trial, 38 client-owned dogs with primary autoimmune hemolytic anemia (AIHA) and two dogs with ITP, requiring RBC transfusion, were enrolled and randomized to receive fresh (≤7 days) or old (≥21 days) stored RBC transfusions. Monocyte chemoattractant protein (MCP)-1 levels were assessed at defined times after transfusion. RESULTS: Prior immune-mediated hemolysis blunted the MCP-1 response to stored RBC transfusion in mice (361 ± 111 pg/ml vs. 6836 ± 1528 pg/ml in mice with immune hemolysis vs. ITP, respectively; mean ± SD; p < .0001). Although hemolysis markers increased after transfusion of older RBCs, the cytokine response was also muted in dogs with AIHA. No differences in morbidity or mortality were evident comparing dogs randomized to fresh or old RBCs. CONCLUSION: These data suggest that underlying hemolysis blunts inflammatory responses to old RBC transfusions. The canine data support randomized trial results suggesting a lack of clinical benefit with fresh RBC transfusions in subjects with underlying, baseline hemolysis.
Assuntos
Anemia Hemolítica Autoimune , Hemólise , Anemia Hemolítica Autoimune/metabolismo , Anemia Hemolítica Autoimune/terapia , Animais , Citocinas , Cães , Transfusão de Eritrócitos/métodos , Eritrócitos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Estudos Prospectivos , CoelhosRESUMO
BACKGROUND: Failure of humoral tolerance to red blood cell (RBC) antigens may lead to autoimmune hemolytic anemia (AIHA), a severe and sometimes fatal disease. Previous studies have shown that although tolerance is robust in HOD mice, autoantibodies are generated upon adoptive transfer of OTII CD4+ T cells, which are specific for an epitope contained within the HOD antigen. These data imply that antigen-presenting cells (APCs) are presenting RBC-derived autoantigen(s) and are capable of driving T-cell activation. Given that multiple APCs participate in erythrophagocytosis, we used a transgenic approach to determine which cellular subsets were required for autoantigen presentation and subsequent autoreactive T-cell activation. STUDY DESIGN AND METHODS: HOD mice, which express an RBC-specific antigen consisting of hen egg lysozyme, ovalbumin, and human blood group molecule Duffy, were bred with IAbfl/fl and Cre-expressing transgenic animals to generate mice that lack I-Ab expression on particular cell subsets. OTII CD4+ T cell proliferation was assessed in vivo in HOD+ I-Abfl/fl xCre+ mice and in vitro upon coculture with sorted APCs. RESULTS: Analysis of HOD+ I-Abfl/fl xCre+ mice demonstrated that splenic conventional dendritic cells (DCs), but not macrophages or monocytes, were required for autoantigen presentation to OTII CD4+ T cells. Subsequent in vitro coculture experiments revealed that both CD8+ and CD8- DC subsets participate in erythrophagocytosis, present RBC-derived autoantigen and stimulate autoreactive T-cell proliferation. CONCLUSION: These data suggest that if erythrocyte T-cell tolerance fails, DCs are capable of initiating autoimmune responses. As such, targeting DCs may be a fruitful strategy for AIHA therapies.
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Autoantígenos/imunologia , Células Dendríticas/imunologia , Eritrócitos/imunologia , Baço/citologia , Anemia Hemolítica Autoimune/etiologia , Anemia Hemolítica Autoimune/imunologia , Anemia Hemolítica Autoimune/mortalidade , Animais , Autoanticorpos , Autoimunidade , Linfócitos T CD4-Positivos/metabolismo , Eritrócitos/metabolismo , Feminino , Proteínas de Homeodomínio/metabolismo , Humanos , Tolerância Imunológica , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL/imunologia , Monócitos/imunologiaRESUMO
Over 5 million people around the world have tested positive for the beta coronavirus SARS-CoV-2 as of May 29, 2020, a third of which are in the United States alone. These infections are associated with the development of a disease known as COVID-19, which is characterized by several symptoms, including persistent dry cough, shortness of breath, chills, muscle pain, headache, loss of taste or smell, and gastrointestinal distress. COVID-19 has been characterized by elevated mortality (over 100 thousand people have already died in the US alone), mostly due to thromboinflammatory complications that impair lung perfusion and systemic oxygenation in the most severe cases. While the levels of pro-inflammatory cytokines such as interleukin-6 (IL-6) have been associated with the severity of the disease, little is known about the impact of IL-6 levels on the proteome of COVID-19 patients. The present study provides the first proteomics analysis of sera from COVID-19 patients, stratified by circulating levels of IL-6, and correlated to markers of inflammation and renal function. As a function of IL-6 levels, we identified significant dysregulation in serum levels of various coagulation factors, accompanied by increased levels of antifibrinolytic components, including several serine protease inhibitors (SERPINs). These were accompanied by up-regulation of the complement cascade and antimicrobial enzymes, especially in subjects with the highest levels of IL-6, which is consistent with an exacerbation of the acute phase response in these subjects. Although our results are observational, they highlight a clear increase in the levels of inhibitory components of the fibrinolytic cascade in severe COVID-19 disease, providing potential clues related to the etiology of coagulopathic complications in COVID-19 and paving the way for potential therapeutic interventions, such as the use of pro-fibrinolytic agents. Raw data for this study are available through ProteomeXchange with identifier PXD020601.
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Proteínas Sanguíneas/análise , Proteínas do Sistema Complemento/análise , Infecções por Coronavirus , Interleucina-6/sangue , Pandemias , Pneumonia Viral , Proteoma/análise , Adulto , Betacoronavirus , Coagulação Sanguínea/fisiologia , COVID-19 , Infecções por Coronavirus/sangue , Infecções por Coronavirus/fisiopatologia , Feminino , Hemólise , Humanos , Masculino , Pessoa de Meia-Idade , Pneumonia Viral/sangue , Pneumonia Viral/fisiopatologia , Proteômica , SARS-CoV-2RESUMO
The SARS-CoV-2 beta coronavirus is the etiological driver of COVID-19 disease, which is primarily characterized by shortness of breath, persistent dry cough, and fever. Because they transport oxygen, red blood cells (RBCs) may play a role in the severity of hypoxemia in COVID-19 patients. The present study combines state-of-the-art metabolomics, proteomics, and lipidomics approaches to investigate the impact of COVID-19 on RBCs from 23 healthy subjects and 29 molecularly diagnosed COVID-19 patients. RBCs from COVID-19 patients had increased levels of glycolytic intermediates, accompanied by oxidation and fragmentation of ankyrin, spectrin beta, and the N-terminal cytosolic domain of band 3 (AE1). Significantly altered lipid metabolism was also observed, in particular, short- and medium-chain saturated fatty acids, acyl-carnitines, and sphingolipids. Nonetheless, there were no alterations of clinical hematological parameters, such as RBC count, hematocrit, or mean corpuscular hemoglobin concentration, with only minor increases in mean corpuscular volume. Taken together, these results suggest a significant impact of SARS-CoV-2 infection on RBC structural membrane homeostasis at the protein and lipid levels. Increases in RBC glycolytic metabolites are consistent with a theoretically improved capacity of hemoglobin to off-load oxygen as a function of allosteric modulation by high-energy phosphate compounds, perhaps to counteract COVID-19-induced hypoxia. Conversely, because the N-terminus of AE1 stabilizes deoxyhemoglobin and finely tunes oxygen off-loading and metabolic rewiring toward the hexose monophosphate shunt, RBCs from COVID-19 patients may be less capable of responding to environmental variations in hemoglobin oxygen saturation/oxidant stress when traveling from the lungs to peripheral capillaries and vice versa.
Assuntos
Infecções por Coronavirus , Eritrócitos , Lipídeos de Membrana , Pandemias , Pneumonia Viral , Betacoronavirus , COVID-19 , Infecções por Coronavirus/sangue , Infecções por Coronavirus/patologia , Infecções por Coronavirus/fisiopatologia , Eritrócitos/química , Eritrócitos/citologia , Eritrócitos/patologia , Humanos , Lipidômica , Lipídeos de Membrana/análise , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/análise , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Metaboloma/fisiologia , Modelos Moleculares , Pneumonia Viral/sangue , Pneumonia Viral/patologia , Pneumonia Viral/fisiopatologia , Proteoma/análise , Proteoma/química , Proteoma/metabolismo , SARS-CoV-2RESUMO
Autoimmune hemolytic anemia (AIHA) leads to accelerated destruction of autologous red blood cells (RBCs) by autoantibodies. AIHA is a severe and sometimes fatal disease. While there are several therapeutic strategies available, there are currently no licensed treatments for AIHA and few therapeutics result in treatment-free durable remission. The etiology of primary AIHA is unknown; however, secondary AIHA occurs concurrently with lymphoproliferative disorders and infections. Additionally, AIHA is the second most common manifestation of primary immunodeficiency disorders and has been described as a side effect of checkpoint inhibitor therapy. Given the severity of AIHA and the lack of treatment options, understanding the initiation of autoimmunity is imperative. Herein, we utilized a well-described model of RBC biology to dissect how RBC-specific autoreactive T cells become educated against RBC autoantigens. We show that, unlike most autoantigens, T cells do not encounter RBC autoantigens in the thymus. Instead, when they leave the thymus as recent thymic emigrants (RTEs), they retain the ability to positively respond to RBC autoantigens; only after several weeks in circulation do RTEs become nonresponsive. Together, these data suggest that any disruption in this process would lead to breakdown of tolerance and initiation of autoimmunity. Thus, RTEs and this developmental process are potential targets to prevent and treat AIHA.
Assuntos
Autoimunidade , Movimento Celular/imunologia , Eritrócitos/imunologia , Tolerância Imunológica , Linfócitos T/imunologia , Timo/imunologia , Anemia Hemolítica Autoimune/sangue , Anemia Hemolítica Autoimune/diagnóstico , Anemia Hemolítica Autoimune/imunologia , Anemia Hemolítica Autoimune/terapia , Autoantígenos/imunologia , Humanos , Linfócitos T/metabolismoRESUMO
PURPOSE OF REVIEW: Red blood cell (RBC) transfusion is an important treatment for some complications of sickle cell disease (SCD). On the contrary, transfusion may lead to alloimmunization to RBC antigens, with such alloantibodies putting patients at risk for acute or delayed hemolysis, and increasing the difficulty of finding compatible RBCs. Patients with SCD are more susceptible to developing RBC alloantibodies than other multiply transfused patient populations, for reasons that are not completely understood. In this review, we summarize the available data about risk factors and underlying mechanisms associated with RBC alloimmunization in SCD. RECENT FINDINGS: Although RBC antigen matching between blood donors and transfusion recipients can decrease alloimmunization, complete matching at all loci is not feasible. Patients with SCD show evidence of increased inflammation at baseline and in times of illness. Resultant changes to the innate and adaptive immune systems presumably influence the development of RBC alloantibodies as well as RBC autoantibodies. SUMMARY: The inflammation and immune dysregulation associated with SCD may be therapeutic targets for preventing the formation of antibodies and/or for mitigating the dangers of existing RBC alloantibodies. As long as RBC transfusion therapy remains an important treatment for SCD, the quest to improve its safety profile will continue.