Hemolysis dictates monocyte differentiation via two distinct pathways in sickle cell disease vaso-occlusion.

Sickle cell disease (SCD) is a hereditary hemoglobinopathy characterized by painful vaso-occlusive crises (VOC) and chronic hemolysis. The mononuclear phagocyte system is pivotal to SCD pathophysiology, but the mechanisms governing monocyte/macrophage differentiation remain unknown. This study examined the influence of hemolysis on circulating monocyte trajectories in SCD. We discovered that hemolysis stimulated CSF-1 production, partly by endothelial cells via Nrf2, promoting classical monocyte (CMo) differentiation into blood patrolling monocytes (PMo) in SCD mice. However, hemolysis also upregulated CCL-2 through IFN-I, inducing CMo transmigration and differentiation into tissue monocyte-derived macrophages. Blocking CMo transmigration by anti-P selectin antibody in SCD mice increased circulating PMo, corroborating that CMo-to-tissue macrophage differentiation occurs at the expense of CMo-to-blood PMo differentiation. We observed a positive correlation between plasma CSF-1/CCL-2 ratios and blood PMo levels in patients with SCD, underscoring the clinical significance of these two opposing factors in monocyte differentiation. Combined treatment with CSF-1 and anti-P selectin antibody more effectively increased PMo numbers and reduced stasis compared with single-agent therapies in SCD mice. Altogether, these data indicate that monocyte fates are regulated by the balance between two heme pathways, Nrf2/CSF-1 and IFN-I/CCL-2, and suggest that the CSF-1/CCL-2 ratio may present a diagnostic and therapeutic target in SCD.

Intermediate monocytes induced by IFN-γ inhibit cancer metastasis by promoting NK cell activation through FOXO1 and interleukin-27.

BACKGROUND: Circulating monocytes are functionally heterogeneous and can be divided into classical (CMo), intermediate (IMo), and non-CMo/patrolling monocyte (PMo) subsets. CMo can differentiate into PMo through IMo. PMos have been shown to inhibit cancer metastasis but the role of IMo is unclear. To date, no strategy has been developed to inhibit cancer metastasis through enhancing PMo/IMo differentiation. METHODS: We screened multiple inflammatory cytokines/chemokines activity of modulating PMo/IMo associated cell markers expression using human monocyte in vitro culture system. We tested our candidate cytokine activity in vivo using multiple mice models. We identified critical key factors and cytokines for our candidate cytokine activity by using gene-knockout mice and neutralization antibodies. RESULTS: We identified IFN-γ as a candidate inflammatory cytokine in the regulation of human IMo/PMo marker expression. Our in vivo data demonstrated that IMo expansion was induced by short-term (3 days) IFN-γ treatment through increasing CMo-IMo differentiation and blocking IMo-PMo differentiation. The IMo induced by IFN-γ (IFN-IMo), but not IFN-γ activated CMo (IFN-CMo), inhibited cancer metastasis by 90%. Surprizing, the effect of IFN-γ is greater in PMo deficiency mice, indicating the effect of IFN-IMo is not mediated through further differentiation into PMo. We also found that IFN-IMos induced by short-term IFN-γ treatment robustly boosted NK cell expansion for threefold and promoted NK differentiation and function through IL-27 and CXCL9. Furthermore, we identified that FOXO1, a key molecule controlling cellular energy metabolism, mediated the effect of IFN-γ induced IL-27 expression, and that NR4A1, a key molecule controlling PMo differentiation and inhibiting cancer metastasis, inhibited the pro-NK cell and anti-metastasis activity of IFN-IMo by suppressing CXCL9 expression. CONCLUSIONS: We have discovered the antimetastasis and pro-NK cell activity of IFN-IMo, identified FOXO1 as a key molecule for IFN-γ driven monocyte differentiation and function, and found NR4A1 as an inhibitory molecule for IFN-IMo activity. Our study has not only shown novel mechanisms for a classical antitumor cytokine but also provided potential target for developing superior monocytic cell therapy against cancer metastasis.

Type I interferon is induced by hemolysis and drives antibody-mediated erythrophagocytosis in sickle cell disease.

Patients with sickle cell disease (SCD) suffer from intravascular hemolysis-associated vascular injury and tissue damage. Classical monocytes (CMo), which are the most abundant of circulating monocytes, are activated in SCD, but the cause and consequences of activation remain incompletely understood. We found a positive correlation between total plasma heme levels and circulating interferon-α (IFN-α) in patients with SCD along with upregulation of the type I IFN (IFN-I) inducible genes in sort-purified SCD patients' CMo by transcriptome analysis. We demonstrated that hemolysis led to IFN-I expression, predominantly by mouse liver monocyte and macrophages (Mⲫ), primarily through Tank kinase binding 1 (TBK1)/IκB kinase-ε (IKKε) but not TLR4. In response to hemolysis-induced IFN-I, mouse CMo migrated to the liver and differentiated into monocyte-derived Mⲫ, increasing their numbers by sixfold with acute hemin treatment. Hemolysis-driven IFN-I activity also led to the induction of Fc receptor CD64 expression on monocyte and Mⲫ populations, enhancing alloantibody-mediated erythrophagocytosis in SCD both in vivo in mice and in in vitro human cultures. Altogether, these data demonstrate IFN-I response to hemolysis as a novel activation pathway in monocytes and Mⲫ in SCD, opening the possibility for development of IFN-I-based diagnostics and therapeutics against alloantibody-mediated erythrophagocytosis.

Hemolysis inhibits humoral B-cell responses and modulates alloimmunization risk in patients with sickle cell disease.

Red blood cell alloimmunization remains a barrier for safe and effective transfusions in sickle cell disease (SCD), but the associated risk factors remain largely unknown. Intravascular hemolysis, a hallmark of SCD, results in the release of heme with potent immunomodulatory activity, although its effect on SCD humoral response, specifically alloimmunization, remains unclear. Here, we found that cell-free heme suppresses human B-cell plasmablast and plasma cell differentiation by inhibiting the DOCK8/STAT3 signaling pathway, which is critical for B-cell activation, as well as by upregulating heme oxygenase 1 (HO-1) through its enzymatic byproducts, carbon monoxide and biliverdin. Whereas nonalloimmunized SCD B cells were inhibited by exogenous heme, B cells from the alloimmunized group were nonresponsive to heme inhibition and readily differentiated into plasma cells. Consistent with a differential B-cell response to hemolysis, we found elevated B-cell basal levels of DOCK8 and higher HO-1-mediated inhibition of activated B cells in nonalloimmunized compared with alloimmunized SCD patients. To overcome the alloimmunized B-cell heme insensitivity, we screened several heme-binding molecules and identified quinine as a potent inhibitor of B-cell activity, reversing the resistance to heme suppression in alloimmunized patients. B-cell inhibition by quinine occurred only in the presence of heme and through HO-1 induction. Altogether, these data suggest that hemolysis can dampen the humoral B-cell response and that B-cell heme responsiveness maybe a determinant of alloimmunization risk in SCD. By restoring B-cell heme sensitivity, quinine may have therapeutic potential to prevent and inhibit alloimmunization in SCD patients.

Serological Assays Estimate Highly Variable SARS-CoV-2 Neutralizing Antibody Activity in Recovered COVID-19 Patients.

The development of neutralizing antibodies (NAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) following infection or vaccination is likely to be critical for the development of sufficient population immunity to drive cessation of the coronavirus disease of 2019 (COVID-19) pandemic. A large number of serologic tests, platforms, and methodologies are being employed to determine seroprevalence in populations to select convalescent plasma samples for therapeutic trials and to guide policies about reopening. However, the tests have substantial variations in sensitivity and specificity, and their ability to quantitatively predict levels of NAbs is unknown. We collected 370 unique donors enrolled in the New York Blood Center Convalescent Plasma Program between April and May of 2020. We measured levels of antibodies in convalescent plasma samples using commercially available SARS-CoV-2 detection tests and in-house enzyme-linked immunosorbent assays (ELISAs) and correlated serological measurements with NAb activity measured using pseudotyped virus particles, which offer the most informative assessment of antiviral activity of patient sera against viral infection. Our data show that a large proportion of convalescent plasma samples have modest antibody levels and that commercially available tests have various degrees of accuracy in predicting NAb activity. We found that the Ortho anti-SARS-CoV-2 total Ig and IgG high-throughput serological assays (HTSAs) and the Abbott SARS-CoV-2 IgG assay quantify levels of antibodies that strongly correlate with the results of NAb assays and are consistent with gold standard ELISA results. These findings provide immediate clinical relevance to serology results that can be equated to NAb activity and could serve as a valuable roadmap to guide the choice and interpretation of serological tests for SARS-CoV-2.

Serological Assays Estimate Highly Variable SARS-CoV-2 Neutralizing Antibody Activity in Recovered COVID19 Patients.

The development of neutralizing antibodies (nAb) against SARS-CoV-2, following infection or vaccination, is likely to be critical for the development of sufficient population immunity to drive cessation of the COVID19 pandemic. A large number of serologic tests, platforms and methodologies are being employed to determine seroprevalence in populations to select convalescent plasmas for therapeutic trials, and to guide policies about reopening. However, tests have substantial variability in sensitivity and specificity, and their ability to quantitatively predict levels of nAb is unknown. We collected 370 unique donors enrolled in the New York Blood Center Convalescent Plasma Program between April and May of 2020. We measured levels of antibodies in convalescent plasma using commercially available SARS-CoV- 2 detection tests and in-house ELISA assays and correlated serological measurements with nAb activity measured using pseudotyped virus particles, which offer the most informative assessment of antiviral activity of patient sera against viral infection. Our data show that a large proportion of convalescent plasma samples have modest antibody levels and that commercially available tests have varying degrees of accuracy in predicting nAb activity. We found the Ortho Anti-SARS-CoV-2 Total Ig and IgG high throughput serological assays (HTSAs), as well as the Abbott SARS-CoV-2 IgG assay, quantify levels of antibodies that strongly correlate with nAb assays and are consistent with gold-standard ELISA assay results. These findings provide immediate clinical relevance to serology results that can be equated to nAb activity and could serve as a valuable 'roadmap' to guide the choice and interpretation of serological tests for SARS-CoV-2.