Diverse immunological dysregulation, chronic inflammation, and impaired erythropoiesis in long COVID patients with chronic fatigue syndrome.

A substantial number of patients recovering from acute SARS-CoV-2 infection present serious lingering symptoms, often referred to as long COVID (LC). However, a subset of these patients exhibits the most debilitating symptoms characterized by ongoing myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS). We specifically identified and studied ME/CFS patients from two independent LC cohorts, at least 12 months post the onset of acute disease, and compared them to the recovered group (R). ME/CFS patients had relatively increased neutrophils and monocytes but reduced lymphocytes. Selective T cell exhaustion with reduced naïve but increased terminal effector T cells was observed in these patients. LC was associated with elevated levels of plasma pro-inflammatory cytokines, chemokines, Galectin-9 (Gal-9), and artemin (ARTN). A defined threshold of Gal-9 and ARTN concentrations had a strong association with LC. The expansion of immunosuppressive CD71+ erythroid cells (CECs) was noted. These cells may modulate the immune response and contribute to increased ARTN concentration, which correlated with pain and cognitive impairment. Serology revealed an elevation in a variety of autoantibodies in LC. Intriguingly, we found that the frequency of 2B4+CD160+ and TIM3+CD160+ CD8+ T cells completely separated LC patients from the R group. Our further analyses using a multiple regression model revealed that the elevated frequency/levels of CD4 terminal effector, ARTN, CEC, Gal-9, CD8 terminal effector, and MCP1 but lower frequency/levels of TGF-ß and MAIT cells can distinguish LC from the R group. Our findings provide a new paradigm in the pathogenesis of ME/CFS to identify strategies for its prevention and treatment.

Murine Placental Erythroid Cells Are Mainly Represented by CD45+ Immunosuppressive Erythroid Cells and Secrete CXCL1, CCL2, CCL3 and CCL4 Chemokines.

Erythroid cells are emerging players in immunological regulation that have recently been shown to play a crucial role in fetomaternal tolerance in mice. In this work, we set ourselves the goal of discovering additional information about the molecular mechanisms of this process. We used flow cytometry to study placental erythroid cells' composition and BioPlex for the secretome profiling of 23 cytokines at E12.5 and E19.5 in both allogeneic and syngeneic pregnancies. We found that (1) placental erythroid cells are mainly represented by CD45+ erythroid cells; (2) the secretomes of CD71+ placental erythroid cells differ from the ones in syngeneic pregnancy; (3) CCL2, CCL3, CCL4 and CXCL1 chemokines were secreted on each day of embryonic development and in both types of pregnancy studied. We believe that these chemokines lure placental immune cells towards erythroid cells so that erythroid cells can induce anergy in those immune cells via cell-bound ligands such as PD-L1, enzymes such as ARG1, and secreted factors such as TGFß-1.

Murine Bone Marrow Erythroid Cells Have Two Branches of Differentiation Defined by the Presence of CD45 and a Different Immune Transcriptome Than Fetal Liver Erythroid Cells.

Mouse erythropoiesis is a multifaceted process involving the intricate interplay of proliferation, differentiation, and maturation of erythroid cells, leading to significant changes in their transcriptomic and proteomic profiles. While the immunoregulatory role of murine erythroid cells has been recognized historically, modern investigative techniques have been sparingly applied to decipher their functions. To address this gap, our study sought to comprehensively characterize mouse erythroid cells through contemporary transcriptomic and proteomic approaches. By evaluating CD71 and Ter-119 as sorting markers for murine erythroid cells and employing bulk NanoString transcriptomics, we discerned distinctive gene expression profiles between bone marrow and fetal liver-derived erythroid cells. Additionally, leveraging flow cytometry, we assessed the surface expression of CD44, CD45, CD71, and Ter-119 on normal and phenylhydrazine-induced hemolytic anemia mouse bone marrow and splenic erythroid cells. Key findings emerged: firstly, the utilization of CD71 for cell sorting yielded comparatively impure erythroid cell populations compared to Ter-119; secondly, discernible differences in immunoregulatory molecule expression were evident between erythroid cells from mouse bone marrow and fetal liver; thirdly, two discrete branches of mouse erythropoiesis were identified based on CD45 expression: CD45-negative and CD45-positive, which had been altered differently in response to phenylhydrazine. Our deductions underscore (1) Ter-119's superiority over CD71 as a murine erythroid cell sorting marker, (2) the potential of erythroid cells in murine antimicrobial immunity, and (3) the importance of investigating CD45-positive and CD45-negative murine erythroid cells separately and in further detail in future studies.

Neglected Cells: Immunomodulatory Roles of CD71+ Erythroid Cells.

The main role of red blood cells is oxygen-transportation. However, recent studies have unveiled immunomodulatory functions for their immature counterparts, CD71+ erythroid cells, under different physiological and pathological conditions. Here, I provide a perspective on the recent advances in this field to highlight their importance in health and disease.

Extramedullary hematopoiesis contributes to enhanced erythropoiesis during pregnancy via TGF-ß signaling.

Red blood cells are the predominant cellular component in human body, and their numbers increase significantly during pregnancy due to heightened erythropoiesis. CD71+ erythroid cells (CECs) are immature red blood cells, encompassing erythroblasts and reticulocytes, constitute a rare cell population primarily found in the bone marrow, although they are physiologically enriched in the neonatal mouse spleen and human cord blood. Presently, the mechanisms underlying the CECs expansion during pregnancy remain largely unexplored. Additionally, the mechanisms and roles associated with extramedullary hematopoiesis (EMH) of erythroid cells during pregnancy have yet to be fully elucidated. In this study, our objective was to examine the underlying mechanisms of erythroid-biased hematopoiesis during pregnancy. Our findings revealed heightened erythropoiesis and elevated CECs in both human and mouse pregnancies. The increased presence of transforming growth factor (TGF)-ß during pregnancy facilitated the differentiation of CD34+ hematopoietic stem and progenitor cells (HSPCs) into CECs, without impacting HSPCs proliferation, ultimately leading to enhanced erythropoiesis. The observed increase in CECs during pregnancy was primarily attributed to EMH occurring in the spleen. During mouse pregnancy, splenic stromal cells were found to have a significant impact on splenic erythropoiesis through the activation of TGF-ß signaling. Conversely, splenic macrophages were observed to contribute to extramedullary erythropoiesis in a TGF-ß-independent manner. Our results suggest that splenic stromal cells play a crucial role in promoting extramedullary erythropoiesis and the production of CECs during pregnancy, primarily through TGF-ß-dependent mechanisms.

Development and validation of a sensitive flow cytometric method for determining CECs in RBC products.

BACKGROUND AND AIMS: Subpopulations of immature red blood cells (RBCs) named CD71+ erythroid cells (CECs) with different properties may contribute to RBC transfusion outcomes. However, it is challenging to quantify CECs in leukoreduced RCCs and whole blood due to their rarity and fragility. Current flow cytometry methods are not applicable to leukoreduced RCCs as there is limited peripheral blood mononuclear cells (PBMCs) to use for determination of CECs. We have developed and validated a flow cytometry method for quantifying CECs in WB. METHODS: We determined optimal PE-Cy7-CD235a, BV711-CD71, APC-CD45 concentrations and instrument setting by titration. Linearity and level of detection was determined by spiking labelled PBMCs from cord blood units into unlabeled WB. Low, medium, high levels of CECs were used to determine intra- and inter -run precision. RESULTS: Detection of CECs was linear (R2 = 0.98) over the range of concentrations assessed with a limit of detection of 0.005%. The overall CVs for the intra- and inter-run precision were 6.9% and 9.7%. CONCLUSION: We developed a simple, sensitive, and cost-effective flow cytometric method for quantifying the proportion of CECs in non-manipulated WB, which could help understand the impact of RBC products on recipient transfusion outcomes.

CD71+ Erythroid Cell Expansion in Adult Sepsis: Potential Causes and Role in Prognosis and Nosocomial Infection Prediction.

BACKGROUND: Immune suppression contributes to nosocomial infections (NIs) and poor prognosis in sepsis. Recent studies revealed that CD71+ erythroid cells had unappreciated immunosuppressive functions. This study aimed to investigate the values of CD71+ erythroid cells (CECs) in predicting NIs and prognosis among adult septic patients. The potential factors associated with the expansion of CECs were also explored. METHODS: In total, 112 septic patients and 32 critically ill controls were enrolled. The frequencies of CD71+ cells, CD71+CD235a+ cells, and CD45+ CECs were measured by flow cytometry. The associations between CECs and NIs and 30-day mortality were assessed by ROC curve analysis and Cox and competing-risk regression models. Factors associated with the frequency of CECs were identified by linear regression analysis. RESULTS: The percentage of CD71+ cells, CECs, and CD45+ CECs were higher in septic patients than critically ill controls. In septic patients, the percentages of CD71+ cells, CECs, and CD45+ CECs were associated with NI development, while CD71+ cells and CECs were independently associated with 30-day mortality. Linear regression analysis showed that the levels of interleukin (IL)-6 and interferon (IFN)-γ were positively associated with the frequencies of CD71+ cells, CECs, and CD45+ CECs, while IL-10 was negatively associated with them. Additionally, the levels of red blood cells (RBCs) were negatively associated with the percentage of CD45+ CECs. CONCLUSIONS: CECs were expanded in sepsis and can serve as independent predictors of the development of NI and 30-day mortality. Low levels of RBCs and high levels of IL-6 and IFN-γ may contribute to the expansion of CECs in sepsis. TRIAL REGISTRATION: ChiCTR, ChiCTR1900024887. Registered 2 August 2019, http://www.chictr.org.cn/showproj.aspx?proj=38645.

Human Fetal Liver Parenchyma CD71+ Cells Have AIRE and Tissue-Specific Antigen Gene Expression.

Autoimmune regulator (AIRE) is a multifunctional protein that is capable of inducing tissue-specific antigens' (TSAs) gene expression, a key event in the induction of self-tolerance, that is usually expressed and functions in the thymus. However, its expression has been detected outside the thymus and cells expressing the gene have been named extra-thymic AIRE expressing cells (eTACs). Here, we discuss the finding of AIRE and TSAs gene expression in CD71+ cells from human fetal liver parenchyma, which are mostly represented by CD71+ erythroid cells.

Immune Transcriptome and Secretome Differ between Human CD71+ Erythroid Cells from Adult Bone Marrow and Fetal Liver Parenchyma.

CD71+ erythroid cells (CECs) were only known as erythrocyte progenitors not so long ago. In present times, however, they have been shown to be active players in immune regulation, especially in immunosuppression by the means of ROS, arginase-1 and arginase-2 production. Here, we uncover organ-of-origin differences in cytokine gene expression using NanoString and protein production using Bio-Plex between CECs from healthy human adult bone marrow and from human fetal liver parenchyma. Namely, healthy human adult bone marrow CECs both expressed and produced IFN-a, IL-1b, IL-8, IL-18 and MIF mRNA and protein, while human fetal liver parenchymaCECs expressed and produced IFN-a, IL15, IL18 and TNF-b mRNA and protein. We also detected TLR2 and TLR9 gene expression in both varieties of CECs and TLR1 and NOD2 gene expression in human fetal liver parenchymaCECs only. These observations suggest that there might be undiscovered roles in immune response for CECs.

Erythroid precursors and progenitors suppress adaptive immunity and get invaded by SARS-CoV-2.

SARS-CoV-2 infection is associated with lower blood oxygen levels, even in patients without hypoxia requiring hospitalization. This discordance illustrates the need for a more unifying explanation as to whether SARS-CoV-2 directly or indirectly affects erythropoiesis. Here, we show significantly enriched CD71+ erythroid precursors/progenitors in the blood circulation of COVID-19 patients. We found that these cells have distinctive immunosuppressive properties. In agreement, we observed a strong negative correlation between the frequency of these cells with T and B cell proportions in COVID-19 patients. The expansion of these CD71+ erythroid precursors/progenitors was negatively correlated with the hemoglobin levels. A subpopulation of abundant erythroid cells, CD45+ CD71+ cells, co-express ACE2, TMPRSS2, CD147, and CD26, and these can be infected with SARS-CoV-2. In turn, pre-treatment of erythroid cells with dexamethasone significantly diminished ACE2/TMPRSS2 expression and subsequently reduced their infectivity with SARS-CoV-2. This provides a novel insight into the impact of SARS-CoV-2 on erythropoiesis and hypoxia seen in COVID-19 patients.

The role of CD71+ erythroid cells in the regulation of the immune response.

Complex regulation of the immune response is necessary to support effective defense of an organism against hostile invaders and to maintain tolerance to harmless microorganisms and autoantigens. Recent studies revealed previously unappreciated roles of CD71+ erythroid cells (CECs) in regulation of the immune response. CECs physiologically reside in the bone marrow where erythropoiesis takes place. Under stress conditions, CECs are enriched in some organs outside of the bone marrow as a result of extramedullary erythropoiesis. However, the role of CECs goes well beyond the production of erythrocytes. In neonates, increased numbers of CECs contribute to their vulnerability to infectious diseases. On the other side, neonatal CECs suppress activation of immune cells in response to abrupt colonization with commensal microorganisms after delivery. CECs are also enriched in the peripheral blood of pregnant women as well as in the placenta and are responsible for the regulation of feto-maternal tolerance. In patients with cancer, anemia leads to increased frequency of CECs in the peripheral blood contributing to diminished antiviral and antibacterial immunity, as well as to accelerated cancer progression. Moreover, recent studies revealed the role of CECs in HIV and SARS-CoV-2 infections. CECs use a full arsenal of mechanisms to regulate immune response. These cells suppress proinflammatory responses of myeloid cells and T-cell proliferation by the depletion of ʟ-arginine by arginase. Moreover, CECs produce reactive oxygen species to decrease T-cell proliferation. CECs also secrete cytokines, including transforming growth factor ß (TGF-ß), which promotes T-cell differentiation into regulatory T-cells. Here, we comprehensively describe the role of CECs in orchestrating immune response and indicate some therapeutic approaches that might be used to regulate their effector functions in the treatment of human conditions.

Sex Matters: Physiological Abundance of Immuno-Regulatory CD71+ Erythroid Cells Impair Immunity in Females.

Mature erythrocytes are the major metabolic regulators by transporting oxygen throughout the body. However, their precursors and progenitors defined as CD71+ Erythroid Cells (CECs) exhibit a wide range of immunomodulatory properties. Here, we uncover pronounced sexual dimorphism in CECs. We found female but not male mice, both BALB/c and C57BL/6, and human females were enriched with CECs. CECs, mainly their progenitors defined as CD45+CECs expressed higher levels of reactive oxygen species (ROS), PDL-1, VISTA, Arginase II and Arginase I compared to their CD45- counterparts. Consequently, CECs by the depletion of L-arginine suppress T cell activation and proliferation. Expansion of CECs in anemic mice and also post-menstrual cycle in women can result in L-arginine depletion in different microenvironments in vivo (e.g. spleen) resulting in T cell suppression. As proof of concept, we found that anemic female mice and mice adoptively transferred with CECs from anemic mice became more susceptible to Bordetella pertussis infection. These observations highlight the role of sex and anemia-mediated immune suppression in females. Notably, enriched CD45+CECs may explain their higher immunosuppressive properties in female BALB/c mice. Finally, we observed significantly more splenic central macrophages in female mice, which can explain greater extramedullary erythropoiesis and subsequently abundance of CECs in the periphery. Thus, sex-specific differences frequency in the frequency of CECs might be imprinted by differential erythropoiesis niches and hormone-dependent manner.

Tumor Immune Evasion Induced by Dysregulation of Erythroid Progenitor Cells Development.

Cancer cells harness normal cells to facilitate tumor growth and metastasis. Within this complex network of interactions, the establishment and maintenance of immune evasion mechanisms are crucial for cancer progression. The escape from the immune surveillance results from multiple independent mechanisms. Recent studies revealed that besides well-described myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs) or regulatory T-cells (Tregs), erythroid progenitor cells (EPCs) play an important role in the regulation of immune response and tumor progression. EPCs are immature erythroid cells that differentiate into oxygen-transporting red blood cells. They expand in the extramedullary sites, including the spleen, as well as infiltrate tumors. EPCs in cancer produce reactive oxygen species (ROS), transforming growth factor ß (TGF-ß), interleukin-10 (IL-10) and express programmed death-ligand 1 (PD-L1) and potently suppress T-cells. Thus, EPCs regulate antitumor, antiviral, and antimicrobial immunity, leading to immune suppression. Moreover, EPCs promote tumor growth by the secretion of growth factors, including artemin. The expansion of EPCs in cancer is an effect of the dysregulation of erythropoiesis, leading to the differentiation arrest and enrichment of early-stage EPCs. Therefore, anemia treatment, targeting ineffective erythropoiesis, and the promotion of EPC differentiation are promising strategies to reduce cancer-induced immunosuppression and the tumor-promoting effects of EPCs.

CD71+ Erythroid Cells in Human Neonates Exhibit Immunosuppressive Properties and Compromise Immune Response Against Systemic Infection in Neonatal Mice.

Newborns are highly susceptible to infectious diseases. The underlying mechanism of neonatal infection susceptibility has generally been related to their under-developed immune system. Nevertheless, this notion has recently been challenged by the discovery of the physiological abundance of immunosuppressive erythroid precursors CD71+erythroid cells (CECs) in newborn mice and human cord blood. Here, as proof of concept, we show that these cells are also abundant in the peripheral blood of human newborns. Although their frequency appears to be more variable compared to their counterparts in mice, they rapidly decline by 4 weeks of age. However, their proportion remains significantly higher in infants up to six months of age compared to older infants. We found CD45 expressing CECs, as erythroid progenitors, were the prominent source of reactive oxygen species (ROS) production in both humans and mice. Interestingly, a higher proportion of CD45+CECs was observed in the spleen versus bone marrow of neonatal mice, which was associated with a higher ROS production by splenic CECs compared to their siblings in the bone marrow. CECs from human newborns suppressed cytokine production by CD14 monocytes and T cells, which was partially abrogated by apocynin in vitro. Moreover, the depletion of CECs in neonatal mice increased the number of activated effector immune cells in their spleen and liver, which rendered them more resistant to Listeria monocytogenes infection. This was evident by a significant reduction in the bacteria load in the spleen, liver and brain of treated-mice compared to the control group, which enhanced their survival rate. Our finding highlights the immunoregulatory processes mediated by CECs in newborns. Thus, such tightly regulated immune system in newborns/infants may explain one potential mechanism for the asymptomatic or mild COVID-19 infection in this population.

CD71+ Erythroid Cells Exacerbate HIV-1 Susceptibility, Mediate trans-Infection, and Harbor Infective Viral Particles.

CD71+ erythroid cells (CECs) have a wide range of immunomodulatory properties. Here, we show that CECs are expanded in the peripheral blood of HIV patients, with a positive correlation between their frequency and the plasma viral load. CECs from HIV patients and human cord blood/placenta exacerbate HIV-1 infection/replication when cocultured with CD4+ T cells, and that preexposure of CD4+ T cells to CECs enhances their permissibility to HIV infection. However, mature red blood cells (RBCs) do not enhance HIV replication when cocultured with CD4+ T cells. We also found CECs express substantial levels of the NOX2 gene and via a mitochondrial reactive oxygen species (ROS)-dependent mechanism possibly upregulate NF-κB in CD4+ T cells once cocultured, which affects the cell cycle machinery to facilitate HIV-1 replication. The complement receptor-1 (CD35) and the Duffy antigen receptor for chemokines (DARC) as potential HIV target molecules are expressed significantly higher on CECs compared to mature red blood cells. Blocking CD35 or DARC substantially abolishes HIV-1 transmission by RBCs to uninfected CD4+ T cells but not by CECs. In contrast, we observed CECs bind to HIV-1 via CD235a and subsequently transfer the virus to uninfected CD4+ T cells, which can be partially blocked by the anti-CD235a antibody. More importantly, we found that CECs from HIV-infected individuals in the presence of antiretroviral therapy harbor infective viral particles, which mediate HIV-1 trans-infection of CD4+ T cells. Therefore, our findings provide a novel insight into the role of CECs in HIV pathogenesis as potential contributing cells in viral persistence and transmission.IMPORTANCE Immature red blood cells (erythroid precursors or CD71+ erythroid cells) have a wide range of immunomodulatory properties. In this study, we found that these erythroid precursors are abundant in the human cord blood/placental tissues, in the blood of HIV-infected and anemic individuals. We observed that these cells exacerbate HIV-1 replication/infection in target cells and even make HIV target cells more permissible to HIV infection. In addition, we found that HIV gets a free ride by binding on the surface of these cells and thus can travel to different parts of the body. In agreement, we noticed a positive correlation between the plasma viral load and the frequency of these cells in HIV patients. More importantly, we observed that infective HIV particles reside inside these erythroid precursors but not mature red blood cells. Therefore, these cells by harboring HIV can play an important role in HIV pathogenesis.

Mode of delivery by an ulcerative colitis mother in a case of twins: Immunological differences in cord blood and placenta.

AIM: To understand the effects of delivery mode on the immune cells frequency and function in cord blood and placenta. METHODS: We evaluated immunological differences in cord blood and placental tissues for a case of twins one of which delivered vaginally while the other delivered by caesarian section (C-section). Cord blood mononuclear cells were isolated and placenta tissues were processed for cell isolation. Immune phenotyping was performed by flow cytometry methods following staining for T cells, natural killer (NK) cells, monocytes, neutrophils and CD71+ erythroid cells in both cord blood and placenta tissues. In addition, fetal calprotectin of twins was measured 12 wk after birth. RESULTS: We found lower percentages of immune cells (e.g. T cells, monocytes and neutrophils) in the cord blood of C-section delivered compared to vaginally delivered newborn. In contrast, percentages of monocytes and neutrophils were > 2 folds higher in the placental tissues of C-section delivered newborn. More importantly, we observed lower percentages of CD71+ erythroid cells in both cord blood and placental tissues of C-section delivered case. Lower CD71+ erythroid cells were associated with a more pro-inflammatory milieu at the fetomaternal interface reflected by higher expression of inhibitory receptors on CD4+ T cells, higher frequency of monocytes and neutrophils. Furthermore, type of delivery impacted the gene expression profile in CD71+ erythroid cells. Finally, we found that C-section delivered child had > 20-fold higher FCP in his fecal sample at 12 wk of age. CONCLUSION: Mode of delivery impacted immune cells profile in cord blood/placenta. In particular frequency of immunosuppressive CD71+ erythroid cells was reduced in C-section delivered newborn.