Monocyte Differentiation and Heterogeneity: Inter-Subset and Interindividual Differences.

The three subsets of human monocytes, classical, intermediate, and nonclassical, show phenotypic heterogeneity, particularly in their expression of CD14 and CD16. This has enabled researchers to delve into the functions of each subset in the steady state as well as in disease. Studies have revealed that monocyte heterogeneity is multi-dimensional. In addition, that their phenotype and function differ between subsets is well established. However, it is becoming evident that heterogeneity also exists within each subset, between health and disease (current or past) states, and even between individuals. This realisation casts long shadows, impacting how we identify and classify the subsets, the functions we assign to them, and how they are examined for alterations in disease. Perhaps the most fascinating is evidence that, even in relative health, interindividual differences in monocyte subsets exist. It is proposed that the individual's microenvironment could cause long-lasting or irreversible changes to monocyte precursors that echo to monocytes and through to their derived macrophages. Here, we will discuss the types of heterogeneity recognised in monocytes, the implications of these for monocyte research, and most importantly, the relevance of this heterogeneity for health and disease.

SARS-CoV-2 ORF8 Mediates Signals in Macrophages and Monocytes through MyD88 Independently of the IL-17 Receptor.

SARS-CoV-2 has caused an estimated 7 million deaths worldwide to date. A secreted SARS-CoV-2 accessory protein, known as open reading frame 8 (ORF8), elicits inflammatory pulmonary cytokine responses and is associated with disease severity in COVID-19 patients. Recent reports proposed that ORF8 mediates downstream signals in macrophages and monocytes through the IL-17 receptor complex (IL-17RA, IL-17RC). However, generally IL-17 signals are found to be restricted to the nonhematopoietic compartment, thought to be due to rate-limiting expression of IL-17RC. Accordingly, we revisited the capacity of IL-17 and ORF8 to induce cytokine gene expression in mouse and human macrophages and monocytes. In SARS-CoV-2-infected human and mouse lungs, IL17RC mRNA was undetectable in monocyte/macrophage populations. In cultured mouse and human monocytes and macrophages, ORF8 but not IL-17 led to elevated expression of target cytokines. ORF8-induced signaling was fully preserved in the presence of anti-IL-17RA/RC neutralizing Abs and in Il17ra-/- cells. ORF8 signaling was also operative in Il1r1-/- bone marrow-derived macrophages. However, the TLR/IL-1R family adaptor MyD88, which is dispensable for IL-17R signaling, was required for ORF8 activity yet MyD88 is not required for IL-17 signaling. Thus, we conclude that ORF8 transduces inflammatory signaling in monocytes and macrophages via MyD88 independently of the IL-17R.

GWAS and meta-analysis identifies 49 genetic variants underlying critical COVID-19.

Critical illness in COVID-19 is an extreme and clinically homogeneous disease phenotype that we have previously shown1 to be highly efficient for discovery of genetic associations2. Despite the advanced stage of illness at presentation, we have shown that host genetics in patients who are critically ill with COVID-19 can identify immunomodulatory therapies with strong beneficial effects in this group3. Here we analyse 24,202 cases of COVID-19 with critical illness comprising a combination of microarray genotype and whole-genome sequencing data from cases of critical illness in the international GenOMICC (11,440 cases) study, combined with other studies recruiting hospitalized patients with a strong focus on severe and critical disease: ISARIC4C (676 cases) and the SCOURGE consortium (5,934 cases). To put these results in the context of existing work, we conduct a meta-analysis of the new GenOMICC genome-wide association study (GWAS) results with previously published data. We find 49 genome-wide significant associations, of which 16 have not been reported previously. To investigate the therapeutic implications of these findings, we infer the structural consequences of protein-coding variants, and combine our GWAS results with gene expression data using a monocyte transcriptome-wide association study (TWAS) model, as well as gene and protein expression using Mendelian randomization. We identify potentially druggable targets in multiple systems, including inflammatory signalling (JAK1), monocyte-macrophage activation and endothelial permeability (PDE4A), immunometabolism (SLC2A5 and AK5), and host factors required for viral entry and replication (TMPRSS2 and RAB2A).

Elevated circulating monocytes and monocyte activation in COVID-19 convalescent individuals.

Background: Monocytes and macrophages play a pivotal role in inflammation during acute SARS-CoV-2 infection. However, their contribution to the development of post-acute sequelae of SARS-CoV-2 infection (PASC) are not fully elucidated. Methods: A cross-sectional study was conducted comparing plasma cytokine and monocyte levels among three groups: participants with pulmonary PASC (PPASC) with a reduced predicted diffusing capacity for carbon monoxide [DLCOc, <80%; (PG)]; fully recovered from SARS-CoV-2 with no residual symptoms (recovered group, RG); and negative for SARS-CoV-2 (negative group, NG). The expressions of cytokines were measured in plasma of study cohort by Luminex assay. The percentages and numbers of monocyte subsets (classical, intermediate, and non-classical monocytes) and monocyte activation (defined by CD169 expression) were analyzed using flow cytometry analysis of peripheral blood mononuclear cells. Results: Plasma IL-1Ra levels were elevated but FGF levels were reduced in PG compared to NG. Circulating monocytes and three subsets were significantly higher in PG and RG compared to NG. PG and RG exhibited higher levels of CD169+ monocyte counts and higher CD169 expression was detected in intermediate and non-classical monocytes from RG and PG than that found in NG. Further correlation analysis with CD169+ monocyte subsets revealed that CD169+ intermediate monocytes negatively correlated with DLCOc%, and CD169+ non-classical monocytes positively correlated with IL-1α, IL-1ß, MIP-1α, Eotaxin, and IFN-γ. Conclusion: This study present evidence that COVID convalescents exhibit monocyte alteration beyond the acute COVID-19 infection period even in convalescents with no residual symptoms. Further, the results suggest that monocyte alteration and increased activated monocyte subsets may impact pulmonary function in COVID-19 convalescents. This observation will aid in understanding the immunopathologic feature of pulmonary PASC development, resolution, and subsequent therapeutic interventions.

The prognostic impact of monocyte fluorescence, immunosuppressive monocytes and peripheral blood immune cell numbers in HIV-associated Diffuse Large B-cell Lymphoma.

INTRODUCTION: Diffuse large B-cell lymphoma (DLBCL) is a high grade non-Hodgkin lymphoma which is common among immunodeficient people. Derangements of peripheral blood immune cells have been described to have a prognostic impact in DLBCL in high income countries, including a monocytosis, the ratios of lymphocytes to both monocytes (L:M) and neutrophils (N:L), as well as the numbers of regulatory T-cells (Tregs) and immunosuppressive monocytes (HLA-DRlow monos). To date, the impact of these variables has not been assessed in the setting of HIV-associated DLBCL (HIV-DLBCL), which is among the most common malignancies seen in people living with HIV. In this study, we assessed these factors in a cohort of South African patients with DLBCL and a high HIV-seropositivity-rate. In addition, we evaluated the prognostic value of monocyte activation (as reflected by monocyte fluorescence (MO-Y) on a Sysmex haematology analyser). This parameter has to date not been assessed in the setting of DLBCL. METHODS: A full blood count and differential count as well as flow cytometry for HLA-DRlow monocyte and Treg enumeration were performed in patients with incident DLBCL referred to the Chris Hani Baragwanath Academic Hospital in Johannesburg, South Africa between November 2019 and May 2022. Additional clinical and laboratory data were recorded from the patient charts and laboratory information system. RESULTS: Seventy-six patients were included, of whom 81.3% were people living with HIV with a median CD4 count of 148 cells/ul. Most patients had advanced stage disease (74.8%) and were predominantly treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP)-based chemotherapy (without Rituximab). At a median follow-up period of 19 months, the median survival time was 3.5 months, with a 12-month survival rate of 27.0%. All of the immune-cell-related variables (with the exception of the CD4 count) were similar between the people living with HIV and the HIV-negative individuals. In contrast to previous studies, a high monocyte count, the L:M and increased numbers of HLA-DRlow monocytes were not significantly associated with survival in HIV-DLBCL, while a neutrophilia (>8 x 109/L), the N:L (>6:1), high numbers of Tregs (≥5.17% of CD4s) and lymphopenia (<1.3 x 109/L) were. In addition, increased monocyte fluorescence (MO-Y >115.5) was associated with superior outcomes, which we speculate to reflect a more robust antitumour immune response among individuals with high levels of monocyte activation. On Cox Proportional hazard analysis, immune-cell factors independently associated with survival included a CD4 count <150 cells/ul and a neutrophilia. CONCLUSION: The monocyte count, L:M and the number of HLA-DRlow monos are not strong prognostic indicators in HIV-DLBCL, while a low CD4 count and neutrophilia are. Elevation of the MO-Y shows some promise as a potential biomarker of antitumour immunity; further study in this regard would be of interest.

Effects of 5α-dihydrotestosterone on the modulation of monocyte/macrophage response to Staphylococcus aureus: an in vitro study.

BACKGROUND: Staphylococcus aureus (S. aureus) is a pathogen responsible for a wide range of clinical manifestations and potentially fatal conditions. There is a paucity of information on the influence of androgens in the immune response to S. aureus infection. In this study, we evaluated the influence of the hormone 5α-dihydrotestosterone (DHT) on mouse peritoneal macrophages (MPMs) and human peripheral blood monocytes (HPBMs) induced by S. aureus. METHODS: An in vitro model of MPMs from BALB/c sham males, orchiectomised (OQX) males, and females was used. Cells were inoculated with 10 µL of S. aureus, phage-type 80 or sterile saline (control) for 6 h. The MPMs of OQX males and females were pre-treated with 100 µL of 10-2 M DHT for 24 h before inoculation with S. aureus. The concentration of the cytokines TNF-α, IL-1α, IL-6, IL-8, and IL-10; total nitrites (NO-2); and hydrogen peroxide (H2O2) were measured in the supernatant of MPM cultures. In addition, the toll-like receptor 2 (TLR2) and nuclear factor kappa B (NF-kB) genes that are involved in immune responses were analysed. For the in vitro model of HPBMs, nine men and nine women of childbearing age were selected and HPBMs were isolated from samples of the volunteers' peripheral blood. In women, blood was collected during the periovulatory period. The HPBMs were inoculated with S. aureus for 6 h and the supernatant was collected for the analysis of cytokines TNF-α, IL-6, IL-12; and GM-CSF, NO-2, and H2O2. The HPBMs were then removed for the analysis of 84 genes involved in the host's response to bacterial infections by RT-PCR array. GraphPad was used for statistical analysis with a p value < 0.05. RESULTS: Our data demonstrated that MPMs from sham males inoculated with S. aureus displayed higher concentrations of inflammatory cytokines and lower concentrations of IL-10, NO-2, and H2O2 when compared with MPMs from OQX males and females. A similar result was observed in the HPBMs of men when compared with those of women. Previous treatment with DHT in women HPBMs increased the production of pro-inflammatory cytokines and decreased the levels of IL-10, NO-2, and H2O2. The analysis of gene expression showed that DHT increased the activity of the TLR2 and NF-kB pathways in both MPMs and HPBMs. CONCLUSIONS: We found that DHT acts as an inflammatory modulator in the monocyte/macrophage response induced by S. aureus and females exhibit a better immune defence response against this pathogen.

Monocyte distribution width alterations and cytokine storm are modulated by circulating histones.

OBJECTIVES: Extracellular histone levels are associated with the severity of many human pathologies, including sepsis and COVID-19. This study aimed to investigate the role of extracellular histones on monocyte distribution width (MDW), and their effect on the release of cytokines by blood cells. METHODS: Peripheral venous blood was collected from healthy subjects and treated with different doses of a histone mixture (range 0-200 µg/mL) to analyze MDW modifications up-to 3 h and digital microscopy of blood smears. Plasma obtained after 3 h of histone treatment were assayed to evaluate a panel of 24 inflammatory cytokines. RESULTS: MDW values significantly increased in a time- and dose-dependent manner. These findings are associated with the histone-induced modifications of cell volume, cytoplasmic granularity, vacuolization, and nuclear structure of monocytes, promoting their heterogeneity without affecting their count. After 3 h of treatment almost all cytokines significantly increased in a dose-dependent manner. The most relevant response was shown by the significantly increased G-CSF levels, and by the increase of IL-1ß, IL-6, MIP-1ß, and IL-8 at the histone doses of 50, 100, and 200 µg/mL. VEGF, IP-10, GM-CSF, TNF-α, Eotaxin, and IL-2 were also up-regulated, and a lower but significant increase was observed for IL-15, IL-5, IL-17, bFGF, IL-10, IFN-γ, MCP-1, and IL-9. CONCLUSIONS: Circulating histones critically induce functional alterations of monocytes mirrored by MDW, monocyte anisocytosis, and hyperinflammation/cytokine storm in sepsis and COVID-19. MDW and circulating histones may be useful tools to predict higher risks of worst outcomes.

Increased platelet activation and lower platelet-monocyte aggregates in COVID-19 patients with severe pneumonia.

BACKGROUND: The increased procoagulant platelets and platelet activation are associated with thrombosis in COVID-19. In this study, we investigated platelet activation in COVID-19 patients and their association with other disease markers. METHODS: COVID-19 patients were classified into three severity groups: no pneumonia, mild-to-moderate pneumonia, and severe pneumonia. The expression of P-selectin and activated glycoprotein (aGP) IIb/IIIa on the platelet surface and platelet-leukocyte aggregates were measured prospectively on admission days 1, 7, and 10 by flow cytometry. RESULTS: P-selectin expression, platelet-neutrophil, platelet-lymphocyte, and platelet-monocyte aggregates were higher in COVID-19 patients than in uninfected control individuals. In contrast, aGPIIb/IIIa expression was not different between patients and controls. Severe pneumonia patients had lower platelet-monocyte aggregates than patients without pneumonia and patients with mild-to-moderate pneumonia. Platelet-neutrophil and platelet-lymphocyte aggregates were not different among groups. There was no change in platelet-leukocyte aggregates and P-selectin expression on days 1, 7, and 10. aGPIIb/IIIa expression was not different among patient groups. Still, adenosine diphosphate (ADP)-induced aGPIIb/IIIa expression was lower in severe pneumonia than in patients without and with mild-to-moderate pneumonia. Platelet-monocyte aggregates exhibited a weak positive correlation with lymphocyte count and weak negative correlations with interleukin-6, D-dimer, lactate dehydrogenase, and nitrite. CONCLUSION: COVID-19 patients have higher platelet-leukocyte aggregates and P-selectin expression than controls, indicating increased platelet activation. Compared within patient groups, platelet-monocyte aggregates were lower in severe pneumonia patients.

Monocyte migration profiles define disease severity in acute COVID-19 and unique features of long COVID.

BACKGROUND: COVID-19 is associated with a dysregulated immune response but it is unclear how immune dysfunction contributes to the chronic morbidity persisting in many COVID-19 patients during convalescence (long COVID). METHODS: We assessed phenotypical and functional changes of monocytes in COVID-19 patients during hospitalisation and up to 9 months of convalescence following COVID-19, respiratory syncytial virus or influenza A. Patients with progressive fibrosing interstitial lung disease were included as a positive control for severe, ongoing lung injury. RESULTS: Monocyte alterations in acute COVID-19 patients included aberrant expression of leukocyte migration molecules, continuing into convalescence (n=142) and corresponding with specific symptoms of long COVID. Long COVID patients with unresolved lung injury, indicated by sustained shortness of breath and abnormal chest radiology, were defined by high monocyte expression of C-X-C motif chemokine receptor 6 (CXCR6) (p<0.0001) and adhesion molecule P-selectin glycoprotein ligand 1 (p<0.01), alongside preferential migration of monocytes towards the CXCR6 ligand C-X-C motif chemokine ligand 16 (CXCL16) (p<0.05), which is abundantly expressed in the lung. Monocyte CXCR6 and lung CXCL16 were heightened in patients with progressive fibrosing interstitial lung disease (p<0.001), confirming a role for the CXCR6-CXCL16 axis in ongoing lung injury. Conversely, monocytes from long COVID patients with ongoing fatigue exhibited a sustained reduction of the prostaglandin-generating enzyme cyclooxygenase 2 (p<0.01) and CXCR2 expression (p<0.05). These monocyte changes were not present in respiratory syncytial virus or influenza A convalescence. CONCLUSIONS: Our data define unique monocyte signatures that define subgroups of long COVID patients, indicating a key role for monocyte migration in COVID-19 pathophysiology. Targeting these pathways may provide novel therapeutic opportunities in COVID-19 patients with persistent morbidity.

Corticosteroids reduce pathologic interferon responses by downregulating STAT1 in patients with high-risk COVID-19.

We do not yet understand exactly how corticosteroids attenuate hyperinflammatory responses and alleviate high-risk coronavirus disease 2019 (COVID-19). We aimed to reveal the molecular mechanisms of hyperinflammation in COVID-19 and the anti-inflammatory effects of corticosteroids in patients with high-risk COVID-19. We performed single-cell RNA sequencing of peripheral blood mononuclear cells (PBMCs) from three independent COVID-19 cohorts: cohort 1 was used for comparative analysis of high-risk and low-risk COVID-19 (47 PBMC samples from 28 patients), cohort 2 for longitudinal analysis during COVID-19 (57 PBMC samples from 15 patients), and cohort 3 for investigating the effects of corticosteroid treatment in patients with high-risk COVID-19 (55 PBMC samples from 13 patients). PBMC samples from healthy donors (12 PBMC samples from 12 donors) were also included. Cohort 1 revealed a significant increase in the proportion of monocytes expressing the long noncoding RNAs NEAT1 and MALAT1 in high-risk patients. Cohort 2 showed that genes encoding inflammatory chemokines and their receptors were upregulated during aggravation, whereas genes related to angiogenesis were upregulated during improvement. Cohort 3 demonstrated downregulation of interferon-stimulated genes (ISGs), including STAT1, in monocytes after corticosteroid treatment. In particular, unphosphorylated STAT-dependent ISGs enriched in monocytes from lupus patients were selectively downregulated by corticosteroid treatment in patients with high-risk COVID-19. Corticosteroid treatment suppresses pathologic interferon responses in monocytes by downregulating STAT1 in patients with high-risk COVID-19. Our study provides insights into the mechanisms underlying COVID-19 aggravation and improvement and the effects of corticosteroid treatment.

Single cell RNA-seq resolution revealed CCR1+/SELL+/XAF+ CD14 monocytes mediated vascular endothelial cell injuries in Kawasaki disease and COVID-19.

INTRODUCTION: The COVID-19 pandemic provide the opportunities to explore the numerous similarities in clinical symptoms with Kawasaki disease (KD), including severe vasculitis. Despite this, the underlying mechanisms of vascular injury in both KD and COVID-19 remain elusive. To identify these mechanisms, this study employs single-cell RNA sequencing to explore the molecular mechanisms of immune responses in vasculitis, and validate the results through in vitro experiments. METHOD: The single-cell RNA sequencing (scRNA-seq) analysis of peripheral blood mononuclear cells (PBMCs) was carried out to investigate the molecular mechanisms of immune responses in vasculitis in KD and COVID-19. The analysis was performed on PBMCs from six children diagnosed with complete KD, three age-matched KD healthy controls (KHC), six COVID-19 patients (COV), three influenza patients (FLU), and four healthy controls (CHC). The results from the scRNA-seq analysis were validated through flow cytometry and immunofluorescence experiments on additional human samples. Subsequently, monocyte adhesion assays, immunofluorescence, and quantitative polymerase chain reaction (qPCR) were used to analyze the damages to endothelial cells post-interaction with monocytes in HUVEC and THP1 cultures. RESULTS: The scRNA-seq analysis revealed the potential cellular types involved and the alterations in genetic transcriptions in the inflammatory responses. The findings indicated that while the immune cell compositions had been altered in KD and COV patients, and the ratio of CD14+ monocytes were both elevated in KD and COV. While the CD14+ monocytes share a large scale of same differentiated expressed geens between KD and COV. The differential activation of CD14 and CD16 monocytes was found to respond to both endothelial and epithelial dysfunctions. Furthermore, SELL+/CCR1+/XAF1+ CD14 monocytes were seen to enhance the adhesion and damage to endothelial cells. The results also showed that different types of B cells were involved in both KD and COV, while only the activation of T cells was recorded in KD. CONCLUSION: In conclusion, our study demonstrated the role of the innate immune response in the regulation of endothelial dysfunction in both KD and COVID-19. Additionally, our findings indicate that the adaptive immunity activation differs between KD and COVID-19. Our results demonstrate that monocytes in COVID-19 exhibit adhesion to both endothelial cells and alveolar epithelial cells, thus providing insight into the mechanisms and shared phenotypes between KD and COVID-19.

The histone methyltransferase MLL1/KMT2A in monocytes drives coronavirus-associated coagulopathy and inflammation.

Coronavirus-associated coagulopathy (CAC) is a morbid and lethal sequela of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. CAC results from a perturbed balance between coagulation and fibrinolysis and occurs in conjunction with exaggerated activation of monocytes/macrophages (MO/Mφs), and the mechanisms that collectively govern this phenotype seen in CAC remain unclear. Here, using experimental models that use the murine betacoronavirus MHVA59, a well-established model of SARS-CoV-2 infection, we identify that the histone methyltransferase mixed lineage leukemia 1 (MLL1/KMT2A) is an important regulator of MO/Mφ expression of procoagulant and profibrinolytic factors such as tissue factor (F3; TF), urokinase (PLAU), and urokinase receptor (PLAUR) (herein, "coagulopathy-related factors") in noninfected and infected cells. We show that MLL1 concurrently promotes the expression of the proinflammatory cytokines while suppressing the expression of interferon alfa (IFN-α), a well-known inducer of TF and PLAUR. Using in vitro models, we identify MLL1-dependent NF-κB/RelA-mediated transcription of these coagulation-related factors and identify a context-dependent, MLL1-independent role for RelA in the expression of these factors in vivo. As functional correlates for these findings, we demonstrate that the inflammatory, procoagulant, and profibrinolytic phenotypes seen in vivo after coronavirus infection were MLL1-dependent despite blunted Ifna induction in MO/Mφs. Finally, in an analysis of SARS-CoV-2 positive human samples, we identify differential upregulation of MLL1 and coagulopathy-related factor expression and activity in CD14+ MO/Mφs relative to noninfected and healthy controls. We also observed elevated plasma PLAU and TF activity in COVID-positive samples. Collectively, these findings highlight an important role for MO/Mφ MLL1 in promoting CAC and inflammation.

Endotoxin tolerance and low activation of TLR-4/NF-κB axis in monocytes of COVID-19 patients.

Higher endotoxin in the circulation may indicate a compromised state of host immune response against coinfections in severe COVID-19 patients. We evaluated the inflammatory response of monocytes from COVID-19 patients after lipopolysaccharide (LPS) challenge. Whole blood samples of healthy controls, patients with mild COVID-19, and patients with severe COVID-19 were incubated with LPS for 2 h. Severe COVID-19 patients presented higher LPS and sCD14 levels in the plasma than healthy controls and mild COVID-19 patients. In non-stimulated in vitro condition, severe COVID-19 patients presented higher inflammatory cytokines and PGE-2 levels and CD14 + HLA-DRlow monocytes frequency than controls. Moreover, severe COVID-19 patients presented higher NF-κB p65 phosphorylation in CD14 + HLA-DRlow, as well as higher expression of TLR-4 and NF-κB p65 phosphorylation in CD14 + HLA-DRhigh compared to controls. The stimulation of LPS in whole blood of severe COVID-19 patients leads to lower cytokine production but higher PGE-2 levels compared to controls. Endotoxin challenge with both concentrations reduced the frequency of CD14 + HLA-DRlow in severe COVID-19 patients, but the increases in TLR-4 expression and NF-κB p65 phosphorylation were more pronounced in both CD14 + monocytes of healthy controls and mild COVID-19 patients compared to severe COVID-19 group. We conclude that acute SARS-CoV-2 infection is associated with diminished endotoxin response in monocytes. KEY MESSAGES: Severe COVID-19 patients had higher levels of LPS and systemic IL-6 and TNF-α. Severe COVID-19 patients presented higher CD14+HLA-DRlow monocytes. Increased TLR-4/NF-κB axis was identified in monocytes of severe COVID-19. Blunted production of cytokines after whole blood LPS stimulation in severe COVID-19. Lower TLR-4/NF-κB activation in monocytes after LPS stimulation in severe COVID-19.

P2Y12 Inhibition Suppresses Proinflammatory Platelet-Monocyte Interactions.

BACKGROUND: Monocyte-platelet aggregates (MPAs) represent the crossroads between thrombosis and inflammation, and targeting this axis may suppress thromboinflammation. While antiplatelet therapy (APT) reduces platelet-platelet aggregation and thrombosis, its effects on MPA and platelet effector properties on monocytes are uncertain. OBJECTIVES: To analyze the effect of platelets on monocyte activation and APT on MPA and platelet-induced monocyte activation. METHODS: Agonist-stimulated whole blood was incubated in the presence of P-selectin, PSGL1, PAR1, P2Y12, GP IIb/IIIa, and COX-1 inhibitors and assessed for platelet and monocyte activity via flow cytometry. RNA-Seq of monocytes incubated with platelets was used to identify platelet-induced monocyte transcripts and was validated by RT-qPCR in monocyte-PR co-incubation ± APT. RESULTS: Consistent with a proinflammatory platelet effector role, MPAs were increased in patients with COVID-19. RNA-Seq revealed a thromboinflammatory monocyte transcriptome upon incubation with platelets. Monocytes aggregated to platelets expressed higher CD40 and tissue factor than monocytes without platelets (p < 0.05 for each). Inhibition with P-selectin (85% reduction) and PSGL1 (87% reduction) led to a robust decrease in MPA. P2Y12 and PAR1 inhibition lowered MPA formation (30 and 21% reduction, p < 0.05, respectively) and decreased monocyte CD40 and TF expression, while GP IIb/IIIa and COX1 inhibition had no effect. Pretreatment of platelets with P2Y12 inhibitors reduced the expression of platelet-mediated monocyte transcription of proinflammatory SOCS3 and OSM. CONCLUSIONS: Platelets skew monocytes toward a proinflammatory phenotype. Among traditional APTs, P2Y12 inhibition attenuates platelet-induced monocyte activation.

Recombinant GM-CSF for diseases of GM-CSF insufficiency: Correcting dysfunctional mononuclear phagocyte disorders.

Introduction: Endogenous granulocyte-macrophage colony-stimulating factor (GM-CSF), identified by its ability to support differentiation of hematopoietic cells into several types of myeloid cells, is now known to support maturation and maintain the metabolic capacity of mononuclear phagocytes including monocytes, macrophages, and dendritic cells. These cells sense and attack potential pathogens, present antigens to adaptive immune cells, and recruit other immune cells. Recombinant human (rhu) GM-CSF (e.g., sargramostim [glycosylated, yeast-derived rhu GM-CSF]) has immune modulating properties and can restore the normal function of mononuclear phagocytes rendered dysfunctional by deficient or insufficient endogenous GM-CSF. Methods: We reviewed the emerging biologic and cellular effects of GM-CSF. Experts in clinical disease areas caused by deficient or insufficient endogenous GM-CSF examined the role of GM-CSF in mononuclear phagocyte disorders including autoimmune pulmonary alveolar proteinosis (aPAP), diverse infections (including COVID-19), wound healing, and anti-cancer immune checkpoint inhibitor therapy. Results: We discuss emerging data for GM-CSF biology including the positive effects on mitochondrial function and cell metabolism, augmentation of phagocytosis and efferocytosis, and immune cell modulation. We further address how giving exogenous rhu GM-CSF may control or treat mononuclear phagocyte dysfunction disorders caused or exacerbated by GM-CSF deficiency or insufficiency. We discuss how rhu GM-CSF may augment the anti-cancer effects of immune checkpoint inhibitor immunotherapy as well as ameliorate immune-related adverse events. Discussion: We identify research gaps, opportunities, and the concept that rhu GM-CSF, by supporting and restoring the metabolic capacity and function of mononuclear phagocytes, can have significant therapeutic effects. rhu GM-CSF (e.g., sargramostim) might ameliorate multiple diseases of GM-CSF deficiency or insufficiency and address a high unmet medical need.

Transcriptional reprogramming from innate immune functions to a pro-thrombotic signature by monocytes in COVID-19.

Although alterations in myeloid cells have been observed in COVID-19, the specific underlying mechanisms are not completely understood. Here, we examine the function of classical CD14+ monocytes in patients with mild and moderate COVID-19 during the acute phase of infection and in healthy individuals. Monocytes from COVID-19 patients display altered expression of cell surface receptors and a dysfunctional metabolic profile that distinguish them from healthy monocytes. Secondary pathogen sensing ex vivo leads to defects in pro-inflammatory cytokine and type-I IFN production in moderate COVID-19 cases, together with defects in glycolysis. COVID-19 monocytes switch their gene expression profile from canonical innate immune to pro-thrombotic signatures and are functionally pro-thrombotic, both at baseline and following ex vivo stimulation with SARS-CoV-2. Transcriptionally, COVID-19 monocytes are characterized by enrichment of pathways involved in hemostasis, immunothrombosis, platelet aggregation and other accessory pathways to platelet activation and clot formation. These results identify a potential mechanism by which monocyte dysfunction may contribute to COVID-19 pathology.

Dual activation profile of monocytes is associated with protection in Mexican patients during SARS-CoV-2 disease.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been one of the most catastrophic diseases observed in recent years. It has reported nearly 550 million cases worldwide, with more than 6.35 million deaths. In Mexico, an increased incidence and mortality of this disease were observed, where the immune response has been involved in the magnitude and severity. A critical version of the disease is accompanied by hyperinflammatory responses, with cytokine and defective cellular responses. A detailed understanding of the role of molecules and cells in the immune response during COVID-19 disease may help to generate effective protection mechanisms, improving those we already have. Here we analyzed blood samples obtained from patients at the Hospital Regional de Alta Especialidad de Ixtapaluca (HRAEI), Mexico, which were classified according to living guidance for clinical management of COVID-19 by the World Health Organization: asymptomatic, mild, severe, and critical disease. We observed increased interleukin (IL)-6 levels and a T-CD8+ and T-CD4+ cell reduction correlated with the critical disease version. Importantly, here, we described a significant reduction of CD11b+CD45highCD14low monocytes during severe disease, which displayed a non-classical profile, expressing IL-10, transforming growth factor (TGF)-ß, and indoleamine 2,3-dioxygenase (IDO)1 molecule. Moreover, CD11b+CD45highCD14low monocytes obtained from infected one-dose vaccinated patients (Pfizer® vaccine) who suffered minimal symptoms showed simultaneously a dual classical and no-classical profile expressing pro- and anti-inflammatory cytokines. These results suggest that blood monocytes expressing a dual pro- and anti-inflammatory profile might be a predictive marker for protection in the Mexican population during COVID-19 disease. KEY POINTS : • Exacerbated immune response is associated with COVID-19 severe disease. • Dual monocyte activation profile is crucial for predicting protection during COVID-19. • Vaccination is crucial to induce the dual activation profile in monocytes.

Activation mechanisms of monocytes/macrophages in adult-onset Still disease.

Adult onset Still disease (AOSD) is a systemic inflammatory disorder characterized by skin rash, spiking fever, arthritis, sore throat, lymphadenopathy, and hepatosplenomegaly. Although the etiology of this disease has not been fully clarified, both innate and acquired immune responses could contribute to its pathogenesis. Hyperactivation of macrophages and neutrophils along with low activation of natural killer (NK) cells in innate immunity, as well as hyperactivation of Th1 and Th17 cells, whereas low activation of regulatory T cells (Tregs) in acquired immunity are involved in the pathogenic process of AOSD. In innate immunity, activation of monocytes/macrophages might play central roles in the development of AOSD and macrophage activation syndrome (MAS), a severe life-threating complication of AOSD. Regarding the activation mechanisms of monocytes/macrophages in AOSD, in addition to type II interferon (IFN) stimulation, several pathways have recently been identified, such as the pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs)-pattern recognition receptors (PRRs) axis, and neutrophil extracellular traps (NETs)-DNA. These stimulations on monocytes/macrophages cause activation of the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain (NLRP) 3 inflammasomes, which trigger capase-1 activation, resulting in conversion of pro-IL-1ß and pro-IL-18 into mature forms. Thereafter, IL-1ß and IL-18 produced by activated monocytes/macrophages contribute to various clinical features in AOSD. We identified placenta-specific 8 (PLAC8) as a specifically increased molecule in monocytes of active AOSD, which correlated with serum levels of CRP, ferritin, IL-1ß, and IL-18. Interestingly, PLAC8 could suppress the synthesis of pro-IL-1ß and pro-IL-18 via enhanced autophagy; thus, PLAC8 seems to be a regulatory molecule in AOSD. These findings for the activation mechanisms of monocytes/macrophages could shed light on the pathogenesis and development of a novel therapeutic strategy for AOSD.

Highly restricted SARS-CoV-2 receptor expression and resistance to infection by primary human monocytes and monocyte-derived macrophages.

Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV2), which causes the disease COVID-19, has caused an unprecedented global pandemic. Angiotensin-converting enzyme 2 (ACE2) is the major cellular receptor for SARS-CoV2 entry, which is facilitated by viral Spike priming by cellular TMPRSS2. Macrophages play an important role in innate viral defense and are also involved in aberrant immune activation that occurs in COVID-19, and thus direct macrophage infection might contribute to severity of SARS-CoV2 infection. Here, we demonstrate that monocytes and monocyte-derived macrophages (MDM) under in vitro conditions express low-to-undetectable levels of ACE2 and TMPRSS2 and minimal coexpression. Expression of these receptors remained low in MDM induced to different subtypes such as unpolarized, M1 and M2 polarized. Untreated, unpolarized, M1 polarized, and M2 polarized MDM were all resistant to infection with SARS-CoV2 pseudotyped virions. These findings suggest that direct infection of myeloid cells is unlikely to be a major mechanism of SARS-CoV2 pathogenesis. Summary sentence: Monocytes and macrophages express minimal ACE2 and TMPRSS2 and resist SARS-CoV-2 Spike-mediated infection, suggesting direct myeloid cell infection is unlikely a major contributor to pathogenesis.