Role of GM-CSF in lung balance and disease.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor originally identified as a stimulus that induces the differentiation of bone marrow progenitor cells into granulocytes and macrophages. GM-CSF is now considered to be a multi-origin and pleiotropic cytokine. GM-CSF receptor signals activate JAK2 and induce nuclear signals through the JAK-STAT, MAPK, PI3K, and other pathways. In addition to promoting the metabolism of pulmonary surfactant and the maturation and differentiation of alveolar macrophages, GM-CSF plays a key role in interstitial lung disease, allergic lung disease, alcoholic lung disease, and pulmonary bacterial, fungal, and viral infections. This article reviews the latest knowledge on the relationship between GM-CSF and lung balance and lung disease, and indicates that there is much more to GM-CSF than its name suggests.

Evaluation of the immunomodulatory effects of interleukin-10 on peripheral blood immune cells of COVID-19 patients: Implication for COVID-19 therapy.

Objective: Several therapies with immune-modulatory functions have been proposed to reduce the overwhelmed inflammation associated with COVID-19. Here we investigated the impact of IL-10 in COVID-19, through the ex-vivo assessment of the effects of exogenous IL-10 on SARS-CoV-2-specific-response using a whole-blood platform. Methods: Two cohorts were evaluated: in "study population A", plasma levels of 27 immune factors were measured by a multiplex (Luminex) assay in 39 hospitalized "COVID-19 patients" and 29 "NO COVID-19 controls" all unvaccinated. In "study population B", 29 COVID-19 patients and 30 NO COVID-19-Vaccinated Controls (NO COVID-19-VCs) were prospectively enrolled for the IL-10 study. Whole-blood was stimulated overnight with SARS-COV-2 antigens and then treated with IL-10. Plasma was collected and used for ELISA and multiplex assay. In parallel, whole-blood was stimulated and used for flow cytometry analysis. Results: Baseline levels of several immune factors, including IL-10, were significantly elevated in COVID-19 patients compared with NO COVID-19 subjects in "study population A". Among them, IL-2, FGF, IFN-γ, and MCP-1 reached their highest levels within the second week of infection and then decreased. To note that, MCP-1 levels remained significantly elevated compared with controls. IL-10, GM-CSF, and IL-6 increased later and showed an increasing trend over time. Moreover, exogenous addition of IL-10 significantly downregulated IFN-γ response and several other immune factors in both COVID-19 patients and NO COVID-19-VCs evaluated by ELISA and a multiplex analysis (Luminex) in "study population B". Importantly, IL-10 did not affect cell survival, but decreased the frequencies of T-cells producing IFN-γ, TNF-α, and IL-2 (p<0.05) and down-modulated HLA-DR expression on CD8+ and NK cells. Conclusion: This study provides important insights into immune modulating effects of IL-10 in COVID-19 and may provide valuable information regarding the further in vivo investigations.

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.

The impact on colostrum oxidative stress, cytokines, and immune cells composition after SARS-CoV-2 infection during pregnancy.

Background: Limited data are available regarding the differences between immunological, biochemical, and cellular contents of human colostrum following maternal infection during pregnancy with coronavirus 2 disease (COVID-19). Objective: To investigate whether maternal COVID-19 infection may affect immunological, biochemical, and cellular contents of human colostrum. Methods: Using a case-control study design, we collected colostrum from 14 lactating women with a previous diagnosis of COVID-19 during pregnancy and 12 without a clear diagnosis during September 2020 to May 2021. Colostrum samples were analysed for some enzymes and non-enzymatic oxidative stress markers (SOD, CAT, GPx, MDA, GSH, GSSG, H2O2, MPO) and for IL-1ß, IL-6, tumour necrosis factor (TNF)-α, protein induced by interferon gamma (IP)-10, IL-8, IFN-λ1, IL12p70, IFN-α2, IFN-λ2/3, granulocyte macrophage colony stimulating factor (GM-CSF), IFN-ß, IL-10 and IFN-γ, along with IgA and IgG for the SARS-CoV-2 S protein. We perform immunophenotyping to assess the frequency of different cell types in the colostrum. Results: Colostrum from the COVID-19 symptomatic group in pregnancy contained reduced levels of H2O2, IFN-α2, and GM-CSF. This group had higher levels of GSH, and both NK cell subtypes CD3-CD56brightCD16-CD27+IFN-γ+ and CD3-CD56dimCD16+CD27- were also increased. Conclusion: The present results reinforce the protective role of colostrum even in the case of mild SARS-Cov-2 infection, in addition to demonstrating how adaptive the composition of colostrum is after infections. It also supports the recommendation to encourage lactating women to continue breastfeeding after COVID-19 illness.

Spectrum of Novel Anti-Central Nervous System Autoantibodies in the Cerebrospinal Fluid of 119 Patients With Schizophreniform and Affective Disorders.

BACKGROUND: Autoimmune psychosis may be caused by well-characterized anti-neuronal autoantibodies, such as those against the NMDA receptor. However, the presence of additional anti-central nervous system (CNS) autoantibodies in these patients has not been systematically assessed. METHODS: Serum and cerebrospinal fluid (CSF) from patients with schizophreniform and affective syndromes were analyzed for immunoglobulin G anti-CNS autoantibodies using tissue-based assays with indirect immunofluorescence on unfixed murine brain tissue as part of an extended routine clinical practice. After an initial assessment of patients with red flags for autoimmune psychosis (n = 30), tissue-based testing was extended to a routine procedure (n = 89). RESULTS: Based on the findings from all 119 patients, anti-CNS immunoglobulin G autoantibodies against brain tissue were detected in 18% (n = 22) of patients (serum 9%, CSF 18%) following five principal patterns: 1) against vascular structures, most likely endothelial cells (serum 3%, CSF 8%); 2) against granule cells in the cerebellum and/or hippocampus (serum 4%, CSF 6%); 3) against myelinated fibers (serum 2%, CSF 2%); 4) against cerebellar Purkinje cells (serum 0%, CSF 2%); and 5) against astrocytes (serum 1%, CSF 1%). The patients with novel anti-CNS autoantibodies showed increased albumin quotients (p = .026) and white matter changes (p = .020) more frequently than those who tested negative for autoantibodies. CONCLUSIONS: The study demonstrates five novel autoantibody-binding patterns on brain tissue of patients with schizophreniform and affective syndromes. CSF yielded positive findings more frequently than serum analysis. The frequency and spectrum of autoantibodies in these patient groups may be broader than previously thought.

GM-CSF-activated human dendritic cells promote type 1 T follicular helper cell polarization in a CD40-dependent manner.

T follicular helper (Tfh) cells regulate humoral responses and present a marked phenotypic and functional diversity. Type 1 Tfh (Tfh1) cells were recently identified and associated with disease severity in infection and autoimmune diseases. The cellular and molecular requirements to induce human Tfh1 differentiation are not known. Here, using single-cell RNA sequencing (scRNAseq) and protein validation, we report that human blood CD1c+ dendritic cells (DCs) activated by GM-CSF (also known as CSF2) drive the differentiation of naive CD4+ T cells into Tfh1 cells. These Tfh1 cells displayed typical Tfh molecular features, including high levels of PD-1 (encoded by PDCD1), CXCR5 and ICOS. They co-expressed BCL6 and TBET (encoded by TBX21), and secreted large amounts of IL-21 and IFN-γ (encoded by IFNG). Mechanistically, GM-CSF triggered the emergence of two DC sub-populations defined by their expression of CD40 and ICOS ligand (ICOS-L), presenting distinct phenotypes, morphologies, transcriptomic signatures and functions. CD40High ICOS-LLow DCs efficiently induced Tfh1 differentiation in a CD40-dependent manner. In patients with mild COVID-19 or latent Mycobacterium tuberculosis infection, Tfh1 cells were positively correlated with a CD40High ICOS-LLow DC signature in scRNAseq of peripheral blood mononuclear cells or blood transcriptomics, respectively. Our study uncovered a novel CD40-dependent Tfh1 axis with potential physiopathological relevance to infection. This article has an associated First Person interview with the first author of the paper.

Huashibaidu formula attenuates sepsis-induced acute lung injury via suppressing cytokine storm: Implications for treatment of COVID-19.

BACKGROUND: Acute lung injury (ALI) is a common complication of sepsis with poor effective interventions. Huashibaidu formula (HSBD) showed good therapeutic effects in treating coronavirus disease 2019 (COVID-19) patients. PURPOSE: This study was designed to investigate the therapeutic potential and precise mechanism of HSBD against sepsis-induced ALI based on network pharmacology and animal experiments. MATERIALS AND METHODS: Network pharmacology was used to predict the possible mechanism of HSBD against sepsis. Next, a sepsis-induced ALI rat model via intraperitoneal lipopolysaccharide (LPS) was constructed to evaluate the level of inflammatory cytokines and the degree of lung injury. The expression of inflammation-related signaling pathways, including TLR4/NF-κB and PI3K/Akt was determined by western blot. RESULTS: Network pharmacology analysis indicated that HSBD might have a therapeutic effect on sepsis mainly by affecting inflammatory and immune responses. Animal experiments demonstrated that HSBD protected the lung tissue from LPS-induced injury, and inhibited the levels of inflammatory cytokines such as interleukin (IL)-1ß, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-γ and tumor necrosis factor (TNF)-α in the serum and IL-1ß, IL-5, IL-6, IL-18, GM-CSF, IFN-γ and TNF-α in the lung tissue. Western blot results revealed that HSBD downregulated the expression of TLR4/NF-κB and upregulated the expression of PI3K/Akt. CONCLUSION: The therapeutic mechanism of HSBD against sepsis-induced ALI mainly involved suppressing cytokine storms and relieving inflammatory symptoms by regulating the expression of TLR4/NF-κB and PI3K/Akt. Our study provides a scientific basis for the mechanistic investigation and clinical application of HSBD in the treatment of sepsis and COVID-19.

Role of Innate and Adaptive Cytokines in the Survival of COVID-19 Patients.

SARS-CoV-2 is a new coronavirus characterized by a high infection and transmission capacity. A significant number of patients develop inadequate immune responses that produce massive releases of cytokines that compromise their survival. Soluble factors are clinically and pathologically relevant in COVID-19 survival but remain only partially characterized. The objective of this work was to simultaneously study 62 circulating soluble factors, including innate and adaptive cytokines and their soluble receptors, chemokines and growth and wound-healing/repair factors, in severe COVID-19 patients who survived compared to those with fatal outcomes. Serum samples were obtained from 286 COVID-19 patients and 40 healthy controls. The 62 circulating soluble factors were quantified using a Luminex Milliplex assay. Results. The patients who survived had decreased levels of the following 30 soluble factors of the 62 studied compared to those with fatal outcomes, therefore, these decreases were observed for cytokines and receptors predominantly produced by the innate immune system-IL-1α, IL-1α, IL-18, IL-15, IL-12p40, IL-6, IL-27, IL-1Ra, IL-1RI, IL-1RII, TNFα, TGFα, IL-10, sRAGE, sTNF-RI and sTNF-RII-for the chemokines IL-8, IP-10, MCP-1, MCP-3, MIG and fractalkine; for the growth factors M-CSF and the soluble receptor sIL2Ra; for the cytokines involved in the adaptive immune system IFNγ, IL-17 and sIL-4R; and for the wound-repair factor FGF2. On the other hand, the patients who survived had elevated levels of the soluble factors TNFß, sCD40L, MDC, RANTES, G-CSF, GM-CSF, EGF, PDGFAA and PDGFABBB compared to those who died. Conclusions. Increases in the circulating levels of the sCD40L cytokine; MDC and RANTES chemokines; the G-CSF and GM-CSF growth factors, EGF, PDGFAA and PDGFABBB; and tissue-repair factors are strongly associated with survival. By contrast, large increases in IL-15, IL-6, IL-18, IL-27 and IL-10; the sIL-1RI, sIL1RII and sTNF-RII receptors; the MCP3, IL-8, MIG and IP-10 chemokines; the M-CSF and sIL-2Ra growth factors; and the wound-healing factor FGF2 favor fatal outcomes of the disease.

Enhanced Severe Acute Respiratory Syndrome Coronavirus 2 Antigen-Specific Systemic Immune Responses in Multisystem Inflammatory Syndrome in Children and Reversal After Recovery.

BACKGROUND: Multisystem inflammatory syndrome in children (MIS-C) presents with inflammation and pathology of multiple organs in the pediatric population in the weeks following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: We characterized the SARS-CoV-2 antigen-specific cytokine and chemokine responses in children with MIS-C, coronavirus disease 2019 (COVID-19), and other infectious diseases. RESULTS: MIS-C is characterized by elevated levels of type 1 (interferon-γ, interleukin [IL] 2), type 2 (IL-4, IL-13), type 17 (IL-17), and other proinflammatory cytokines (IL-1α, IL-6, IL-12p70, IL-18, and granulocyte-macrophage colony-stimulating factor) in comparison to COVID-19 and other infectious diseases following stimulation with SARS-CoV-2-specific antigens. Similarly, upon SARS-CoV-2 antigen stimulation, CCL2, CCL3, and CXCL10 chemokines were significantly elevated in children with MIS-C in comparison to the other 2 groups. Principal component analysis based on these cytokines and chemokines could clearly distinguish MIS-C from both COVID-19 and other infections. In addition, these responses were significantly diminished and normalized 6-9 months after recovery. CONCLUSIONS: Our data suggest that MIS-C is characterized by an enhanced production of cytokines and chemokines that may be associated with disease pathogenesis.

Formation of eicosanoids and other oxylipins in human macrophages.

In this review it is attempted to summarize current studies about formation of eicosanoids and other oxylipins in different human macrophages. There are several reports on M1 and M2 cells, also other phenotypes have been described. The eicosanoids formed in the largest amounts are the COX products TxB₂ and PGE₂. Thus shortlived bioactive TxA₂ is a dominating product both in M1- and in M2-lineages, one exception seems to be M_{GM-CSF, TGFß} cells. 5-LOX products are produced in both M1 and M2 macrophages, as well as in not fully polarized cells of both lineages. M_M-CSF as well as M2 macrophages produced LTC₄ more readily compared to M1 lineage cells. In M_{GM-CSF, TGFß} cells LTB₄ is a major eicosanoid, in line with high expression of LTA₄ hydrolase. Recent reports described increased formation of leukotrienes in macrophages subjected to trained immunity with inflammatory transcriptional reprogramming. Also in macrophages derived from monocytes collected from post-COVID-19 patients. 15-LOX-1 is strongly upregulated in CD206⁺ M2 cells (M2a), differentiated in presence of IL-4. These macrophages also express 15-LOX-2. In incubations with pathogenic E. coli as well as other stimuli 15(S)-HETE and 17(S)-HDHA were major oxylipins formed. Also, the SPM precursor 5,15-diHETE and the SPM RvD5 were produced in considerable amounts, while other SPMs were less abundant. In M2 macrophages incubated with E. coli or S. aureus the cytosolic 15-LOX-1 enzyme accumulated to punctuate structures in a Ca²⁺ dependent manner with a relatively slow time course, leading to formation of mediators from endogenous substrate. Chalcones, flavone-like anti-inflammatory natural products, induced translocation of 15-LOX-1 in M2 cells, with high formation of 15-LOX derived oxylipins.

Increased proinflammatory cytokines in tears correspond with conjunctival SARS-CoV-2 positivity in symptomatic COVID-19 patients.

Tear fluid cytokine levels may serve as biomarkers of innate immune system response against SARS-CoV-2 infection. Therefore, our aim was to analyze panel of selected inflammatory cytokines in tears of COVID-19 patients in relation to presence of SARS-CoV-2 viral load in conjunctival secretions. In this study concentrations of TNF-α, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 p70, GM-CSF, and IFN-γ were determined by a magnetic bead assay in tear film collected from 232 symptomatic COVID-19 patients. SARS-CoV-2 ocular infection was confirmed based on positive conjunctival swab-based RT-PCR testing. Viral RNA in conjunctival sac was detected in 21 patients (9%). No relation between presence and the duration of ophthalmic symptoms and SARS-CoV-2 infection detected in conjunctival secretions was found. The tear film concentrations of IFN-γ, TNF-α, IL-5, IL-8 and GM-CSF were found to be significantly greater among patients with positive conjunctival swab results as compared to the group negative for SARS-CoV-2 in conjunctival sac. Our current data depict a group of inflammatory mediators in human tears, which may play a significant role in ocular pathology of SARS-CoV-2 conjunctival infection.

Effect of a single high dose of vitamin D3 on cytokines, chemokines, and growth factor in patients with moderate to severe COVID-19.

BACKGROUND: The modulating effect of vitamin D on cytokine concentrations in severe coronavirus disease 2019 (COVID-19) remains unknown. OBJECTIVES: We aimed to investigate the effect of a single high dose of vitamin D3 on cytokines, chemokines, and growth factor in hospitalized patients with moderate to severe COVID-19. METHODS: This is a post hoc, ancillary, and exploratory analysis from a multicenter, double-blind, placebo-controlled, randomized clinical trial. Patients with moderate to severe COVID-19 were recruited from 2 hospitals in São Paulo, Brazil. Of 240 randomly assigned patients, 200 were assessed in this study and randomly assigned to receive a single oral dose of 200,000 IU vitamin D3 (n = 101) or placebo (n = 99). The primary outcome was hospital length of stay, which has been published in our previous study. The prespecified secondary outcomes were serum concentrations of IL-1ß, IL-6, IL-10, TNF-α, and 25-hydroxyvitamin D. The post hoc exploratory secondary outcomes were IL-4, IL-12p70, IL-17A, IFN-γ, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-8, IFN-inducible protein-10 (IP-10), macrophage inflammatory protein-1ß (MIP-1ß), monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), and leukocyte count. Generalized estimating equations for repeated measures, with Bonferroni's adjustment, were used for testing all outcomes. RESULTS: The study included 200 patients with a mean ± SD age of 55.5 ± 14.3 y and BMI of 32.2 ± 7.1 kg/m2, of which 109 (54.5%) were male. GM-CSF concentrations showed a significant group-by-time interaction effect (P = 0.04), although the between-group difference at postintervention after Bonferroni's adjustment was not significant. No significant effects were observed for the other outcomes. CONCLUSIONS: The findings do not support the use of a single dose of 200,000 IU vitamin D3, compared with placebo, for the improvement of cytokines, chemokines, and growth factor in hospitalized patients with moderate to severe COVID-19.This trial was registered at clinicaltrials.gov as NCT04449718.

Chronic pharmacological antagonism of the GM-CSF receptor in mice does not replicate the pulmonary alveolar proteinosis phenotype but does alter lung surfactant turnover.

Granulocyte macrophage colony stimulating factor (GM-CSF) is a key participant in, and a clinical target for, the treatment of inflammatory diseases including rheumatoid arthritis (RA). Therapeutic inhibition of GM-CSF signalling using monoclonal antibodies to the α-subunit of the GM-CSF receptor (GMCSFRα) has shown clear benefit in patients with RA, giant cell arteritis (GCAs) and some efficacy in severe SARS-CoV-2 infection. However, GM-CSF autoantibodies are associated with the development of pulmonary alveolar proteinosis (PAP), a rare lung disease characterised by alveolar macrophage (AM) dysfunction and the accumulation of surfactant lipids. We assessed how the anti-GMCSFRα approach might impact surfactant turnover in the airway. Female C57BL/6J mice received a mouse-GMCSFRα blocking antibody (CAM-3003) twice per week for up to 24 weeks. A parallel, comparator cohort of the mouse PAP model, GM-CSF receptor ß subunit (GMCSFRß) knock-out (KO), was maintained up to 16 weeks. We assessed lung tissue histopathology alongside lung phosphatidylcholine (PC) metabolism using stable isotope lipidomics. GMCSFRß KO mice reproduced the histopathological and biochemical features of PAP, accumulating surfactant PC in both broncho-alveolar lavage fluid (BALF) and lavaged lung tissue. The incorporation pattern of methyl-D9-choline showed impaired catabolism and not enhanced synthesis. In contrast, chronic supra-pharmacological CAM-3003 exposure (100 mg/kg) over 24 weeks did not elicit a histopathological PAP phenotype despite some changes in lung PC catabolism. Lack of significant impairment of AM catabolic function supports clinical observations that therapeutic antibodies to this pathway have not been associated with PAP in clinical trials.

The intersection of COVID-19 and autoimmunity.

Acute COVID-19, caused by SARS-CoV-2, is characterized by diverse clinical presentations, ranging from asymptomatic infection to fatal respiratory failure, and often associated with varied longer-term sequelae. Over the past 18 months, it has become apparent that inappropriate immune responses contribute to the pathogenesis of severe COVID-19. Researchers working at the intersection of COVID-19 and autoimmunity recently gathered at an American Autoimmune Related Diseases Association Noel R. Rose Colloquium to address the current state of knowledge regarding two important questions: Does established autoimmunity predispose to severe COVID-19? And, at the same time, can SARS-CoV-2 infection trigger de novo autoimmunity? Indeed, work to date has demonstrated that 10% to 15% of patients with critical COVID-19 pneumonia exhibit autoantibodies against type I interferons, suggesting that preexisting autoimmunity underlies severe disease in some patients. Other studies have identified functional autoantibodies following infection with SARS-CoV-2, such as those that promote thrombosis or antagonize cytokine signaling. These autoantibodies may arise from a predominantly extrafollicular B cell response that is more prone to generating autoantibody-secreting B cells. This Review highlights the current understanding, evolving concepts, and unanswered questions provided by this unique opportunity to determine mechanisms by which a viral infection can be exacerbated by, and even trigger, autoimmunity. The potential role of autoimmunity in post-acute sequelae of COVID-19 is also discussed.

Characterizing the BCR repertoire during lymphocyte reduction and recovery mediated by cyclophosphamide and granulocyte-macrophage colony-stimulating factor.

Leukopenia is a common manifestation of many diseases, including global outbreak SAS-CoV-2 infection. Granulocyte-macrophage colony-stimulating factor (GM -CSF) has been proved to be effective in promoting lymphocyte regeneration, but adverse immunological effects have also emerged. This study aim to investigate the effect of GM -CSF on BCR heavy chain CDR3 repertoire while promoting lymphocyte regeneration. Cyclophosphamide (CTX) and GM -CSF were used to inhibit and stimulate bone marrow hematopoiesis, respectively. High throughput sequencing was applied to detect the characteristics of BCR CDR3 repertoire in controls, CTX group and GM -CSF group. The white blood cells (WBCs) were quickly reduced (P < 0.05) with lymphocytes decreasing causing by CTX, and the WBCs and lymphocytes returned to the level of controls after GM -CSF treatment. The diversity of BCR heavy chain CDR3 repertoire was also significantly decreased in CTX group. Although there is still a big gap from the controls, the diversity was picked up after GM -CSF treatment. The expression of IGHD01-01, IGHD02-14 and IGHJ04-01 with high-frequency usage regularly and significantly changed in three groups, and many genes with low-frequency usage lost in CTX group and did not reappear in GM -CSF group. Moreover, two shared sequences and accounted for the highest proportion in GM -CSF group have been detected in animal model of chronic lymphocytic leukemia. These results revealed that GM -CSF can partially restore changes in the BCR heavy chain CDR3 repertoire while promoting lymphocyte regeneration, but it may also lead to rearrangement, proliferation and activation of abnormal B cells, which can provide a basis for further study on the adverse immunological effects and mechanism of GM -CSF treatment.

Mild and Asymptomatic COVID-19 Convalescents Present Long-Term Endotype of Immunosuppression Associated With Neutrophil Subsets Possessing Regulatory Functions.

The SARS-CoV-2 infection [coronavirus disease 2019 (COVID-19)] is associated with severe lymphopenia and impaired immune response, including expansion of myeloid cells with regulatory functions, e.g., so-called low-density neutrophils, containing granulocytic myeloid-derived suppressor cells (LDNs/PMN-MDSCs). These cells have been described in both infections and cancer and are known for their immunosuppressive activity. In the case of COVID-19, long-term complications have been frequently observed (long-COVID). In this context, we aimed to investigate the immune response of COVID-19 convalescents after a mild or asymptomatic course of disease. We enrolled 13 convalescents who underwent a mild or asymptomatic infection with SARS-CoV-2, confirmed by a positive result of the PCR test, and 13 healthy donors without SARS-CoV-2 infection in the past. Whole blood was used for T-cell subpopulation and LDNs/PMN-MDSCs analysis. LDNs/PMN-MDSCs and normal density neutrophils (NDNs) were sorted out by FACS and used for T-cell proliferation assay with autologous T cells activated with anti-CD3 mAb. Serum samples were used for the detection of anti-SARS-CoV-2 neutralizing IgG and GM-CSF concentration. Our results showed that in convalescents, even 3 months after infection, an elevated level of LDNs/PMN-MDSCs is still maintained in the blood, which correlates negatively with the level of CD8+ and double-negative T cells. Moreover, LDNs/PMN-MDSCs and NDNs showed a tendency for affecting the production of anti-SARS-CoV-2 S1 neutralizing antibodies. Surprisingly, our data showed that in addition to LDNs/PMN-MDSCs, NDNs from convalescents also inhibit proliferation of autologous T cells. Additionally, in the convalescent sera, we detected significantly higher concentrations of GM-CSF, indicating the role of emergency granulopoiesis. We conclude that in mild or asymptomatic COVID-19 convalescents, the neutrophil dysfunction, including propagation of PD-L1-positive LDNs/PMN-MDSCs and NDNs, is responsible for long-term endotype of immunosuppression.

The Gene Signature of Activated M-CSF-Primed Human Monocyte-Derived Macrophages Is IL-10-Dependent.

During inflammatory responses, monocytes are recruited into inflamed tissues, where they become monocyte-derived macrophages and acquire pro-inflammatory and tissue-damaging effects in response to the surrounding environment. In fact, monocyte-derived macrophage subsets are major pathogenic cells in inflammatory pathologies. Strikingly, the transcriptome of pathogenic monocyte-derived macrophage subsets resembles the gene profile of macrophage colony-stimulating factor (M-CSF)-primed monocyte-derived human macrophages (M-MØ). As M-MØ display a characteristic cytokine profile after activation (IL10high TNFlow IL23low IL6low), we sought to determine the transcriptional signature of M-MØ upon exposure to pathogenic stimuli. Activation of M-MØ led to the acquisition of a distinctive transcriptional profile characterized by the induction of a group of genes (Gene set 1) highly expressed by pathogenic monocyte-derived macrophages in COVID-19 and whose presence in tumor-associated macrophages (TAM) correlates with the expression of macrophage-specific markers (CD163, SPI1) and IL10. Indeed, Gene set 1 expression was primarily dependent on ERK/p38 and STAT3 activation, and transcriptional analysis and neutralization experiments revealed that IL-10 is not only required for the expression of a subset of genes within Gene set 1 but also significantly contributes to the idiosyncratic gene signature of activated M-MØ. Our results indicate that activation of M-CSF-dependent monocyte-derived macrophages induces a distinctive gene expression profile, which is partially dependent on IL-10, and identifies a gene set potentially helpful for macrophage-centered therapeutic strategies.

Robust innate responses to SARS-CoV-2 in children resolve faster than in adults without compromising adaptive immunity.

SARS-CoV-2 infection in children is less severe than it is in adults. We perform a longitudinal analysis of the early innate responses in children and adults with mild infection within household clusters. Children display fewer symptoms than adults do, despite similar initial viral load, and mount a robust anti-viral immune signature typical of the SARS-CoV-2 infection and characterized by early interferon gene responses; increases in cytokines, such as CXCL10 and GM-CSF; and changes in blood cell numbers. When compared with adults, the antiviral response resolves faster (within a week of symptoms), monocytes and dendritic cells are more transiently activated, and genes associated with B cell activation appear earlier in children. Nonetheless, these differences do not have major effects on the quality of SARS-CoV-2-specific antibody responses. Our findings reveal that better early control of inflammation as observed in children may be key for rapidly controlling infection and limiting the disease course.

Sargramostim (rhu GM-CSF) as Cancer Therapy (Systematic Review) and An Immunomodulator. A Drug Before Its Time?

BACKGROUND: Sargramostim [recombinant human granulocyte-macrophage colony-stimulating factor (rhu GM-CSF)] was approved by US FDA in 1991 to accelerate bone marrow recovery in diverse settings of bone marrow failure and is designated on the list of FDA Essential Medicines, Medical Countermeasures, and Critical Inputs. Other important biological activities including accelerating tissue repair and modulating host immunity to infection and cancer via the innate and adaptive immune systems are reported in pre-clinical models but incompletely studied in humans. OBJECTIVE: Assess safety and efficacy of sargramostim in cancer and other diverse experimental and clinical settings. METHODS AND RESULTS: We systematically reviewed PubMed, Cochrane and TRIP databases for clinical data on sargramostim in cancer. In a variety of settings, sargramostim after exposure to bone marrow-suppressing agents accelerated hematologic recovery resulting in fewer infections, less therapy-related toxicity and sometimes improved survival. As an immune modulator, sargramostim also enhanced anti-cancer responses in solid cancers when combined with conventional therapies, for example with immune checkpoint inhibitors and monoclonal antibodies. CONCLUSIONS: Sargramostim accelerates hematologic recovery in diverse clinical settings and enhances anti-cancer responses with a favorable safety profile. Uses other than in hematologic recovery are less-well studied; more data are needed on immune-enhancing benefits. We envision significantly expanded use of sargramostim in varied immune settings. Sargramostim has the potential to reverse the immune suppression associated with sepsis, trauma, acute respiratory distress syndrome (ARDS) and COVID-19. Further, sargramostim therapy has been promising in the adjuvant setting with vaccines and for anti-microbial-resistant infections and treating autoimmune pulmonary alveolar proteinosis and gastrointestinal, peripheral arterial and neuro-inflammatory diseases. It also may be useful as an adjuvant in anti-cancer immunotherapy.