Neutrophil Subsets Research Articles

Neutrophils show distinct patterns of activation in the context of infection with different pathogens

Abstract Alternatively activated neutrophils (N2) enhance lung anti-helminth trained macrophage effector function in response to infection with the helminth Nippostrongylus. brasiliensis (Nb) (Fei Chen, et al., NI, 2014, CR, 2022). However, potential differences in activation patterns of neutrophils with different pathogens is not yet well studied. We report here lung neutrophils bear unique gene expression profiles at 2 days after infection with Nb, fungi (Aspergillosis fumigatus (Af)), and bacteria (Staphylococcus aureus (SA). AF and SA triggered genes associated with cell death and autophagy but differed greatly in metabolic activity. In contrast, Nb induced gene expression patterns characteristic of type 2 immunity, xenobiotic pathways, and cell proliferation. Further analysis revealed increased edu incorporation, KI-67 and IL-13 expression in neutrophils in the lung but not in the blood and bone marrow. Furthermore, the proliferating neutrophil subset predominantly expressed IL-13 and was IL-4R dependent. To directly examine neutrophils proliferating in the lung after Nb infection, Nb-primed neutrophils (CD45.2) were transferred to CD45.1 Nb inoculated recipients. At day 10 after inoculation, donor neutrophils were readily detected and were proliferating as measured by ki-67 staining and edu incorporation. These studies suggest that neutrophils exhibit distinct activation states following infection with different pathogens. Unlike neutrophils typically associated with microbial infection, where rapid turnover from cell death typically occurs, neutrophils activated following Nb infection persist and undergo cell cycling within the remodeled lung tissue microenvironment.

Neutrophils expressing major histocompatibility complex class II molecules circulate in blood and milk during mastitis and show high microbicidal activity

Bovine mastitis is mainly caused by bacterial infection and is responsible for important economic losses as well as alterations of the health and welfare of animals. The increase in somatic cell count (SCC) in milk during mastitis is mainly due to the influx of neutrophils, which have a crucial role in the elimination of pathogens. For a long time, these first-line defenders have been viewed as microbe killers, with a limited role in the orchestration of the immune response. However, their role is more complex: we recently characterized a bovine neutrophil subset expressing major histocompatibility complex class II (MHC-II) molecules (MHC-IIpos), usually distributed on antigen-presenting cells, as having regulatory capacities in cattle. In this study, our objective was to evaluate the implication of different neutrophils subsets in the mammary gland immunity during clinical and subclinical mastitis. Using flow cytometry, we analyzed the presence of MHC-IIpos neutrophils in blood and in milk during clinical mastitis at different time points of inflammation (n = 10 infected quarters) and during subclinical mastitis, defined as the presence of bacteria and an SCC >150,000 cells/mL (n = 27 infected quarters). Our results show, for the first time, that in blood and milk, neutrophils are a heterogeneous population and encompass at least 2 subsets distinguishable by their expression of MHC-II. In milk without mastitis, we observed higher production of reactive oxygen species and higher phagocytosis capacity of MHC-IIpos neutrophils compared with their MHC-IIneg counterparts, indicating the high bactericidal capacities of MHC-IIpos neutrophils. MHC-IIpos neutrophils are enriched in milk compared with blood during subclinical mastitis but not during clinical mastitis. Moreover, we observed a positive and highly significant correlation between MHC-IIpos neutrophils and T lymphocytes present in milk during subclinical mastitis. Our experiments involved a total of 47 cows (40 Holstein and 7 Normande cows). To conclude, our study opens the way to the discovery of new biomarkers of mastitis inflammation.

Abstract 5956: Tumor-infiltrating neutrophil progenitors impair homologous DNA repair and promote sensitivity to PARP-inhibition

Abstract Tumor evolution is one of the major mechanisms responsible for acquiring therapy-resistant and more aggressive cancer clones. Whether the tumor microenvironment through immune-mediated mechanisms might promote the development of more aggressive cancer types is crucial for the identification of additional therapeutical opportunities. Here, we identified a novel subset of tumor-infiltrating neutrophils, defined as tumor-associated neutrophil progenitors (NePs). These NePs are enriched in highly proliferative hormone-dependent breast cancers and impair DNA repair capacity. Mechanistically, succinate secreted by tumor-associated NePs inhibits homologous recombination, promoting error-prone DNA repair through non-homologous end-joining regulated by PARP-1. Consequently, breast cancer cells acquire genomic instability, resulting in tumor progression and resistance to endocrine therapies. Selective inhibition of these pathways induces increased tumor cell kill in vitro and in vivo. Intra-tumor NePs score correlates with copy number alterations in highly proliferative hormone-dependent tumors from breast cancer patients. Treatment with PARP-1 inhibitors counteract the pro-tumorigenic effect of these neutrophils and reverses endocrine resistance. Citation Format: Arianna Calcinotto. Tumor-infiltrating neutrophil progenitors impair homologous DNA repair and promote sensitivity to PARP-inhibition. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5956.

NADPH oxidase 2 limits amplification of IL-1β-G-CSF axis and animmature neutrophil subset in murine lung inflammation.

The leukocyte NADPH oxidase 2 (NOX2) regulates inflammation independent of its antimicrobial activity. Inherited defects in NOX2 lead to chronic granulomatous disease (CGD), associated with recurrent bacterial and fungal infections, often with excessive neutrophilic inflammation that results in significant inflammatory burden and tissue damage. We previously showed that excessive leukotriene B4 (LTB4) production by NOX2-deficient mouse neutrophils was a key driver of elevated lung neutrophil infiltration in the initial response to pulmonary challenge with the model fungal particle zymosan. We now identify interleukin-1β (IL-1β) and downstream granulocyte colony-stimulating factor (G-CSF) as critical amplifying signals that augment and sustain neutrophil accrual in CGD mice. Neutrophils, delivered into the lung via LTB4, were the primary source of IL-1β within the airways, and their increased numbers in CGD lungs led to significantly elevated local and plasma G-CSF. Elevated G-CSF simultaneously promoted increased granulopoiesis and mobilized the release of higher numbers of an immature CD101- neutrophil subset from the marrow, which trafficked to the lung and acquired a significantly more proinflammatory transcriptome in CGD mice compared with wild-type mice. Thus, neutrophil-produced IL-1β and downstream G-CSF act sequentially but nonredundantly with LTB4 to deploy neutrophils and amplify inflammation in CGD mice after inhalation of zymosan. NOX2 plays a critical role in dampening multiple components of a feed-forward pipeline for neutrophil recruitment, and these findings highlight NOX2 as a key regulator of neutrophil number, subsets, and function at inflamed sites.

Assessment of neutrophil subsets and immune checkpoint inhibitor expressions on T lymphocytes in liver transplantation: A preliminary study beyond the neutrophil-lymphocyte ratio.

Background: Advanced stages of cirrhosis are characterized by the occurrence of progressive immune alterations known as CAID (Cirrhosis Associated Immune Dysfunction). In advanced cirrhosis, liver transplantation (LT) remains the only curative treatment. Sepsis, shares many similarities with decompensated cirrhosis in terms of immuno-inflammatory response. In both conditions, the neutrophil-lymphocyte ratio (NLR) is associated with poor outcomes. Based on alterations in sepsis, we hypothesized that we could observe in cirrhotic and LT patients more detailed neutrophil and lymphocyte phenotypes. To this end, along with leukocyte count, we assessed immature neutrophils, LOX-1+ MDSC and PD-1 and TIM-3 lymphocyte expressions in cirrhotic patients before transplantation in association with liver disease severity and during the first month after transplantation. Methods: We conducted a prospective monocentric study including cirrhotic patients registered on LT waiting-list. Blood samples were collected at enrolment before LT and for 1month post-LT. In addition to NLR, we assessed by whole blood flow cytometry the absolute count of immature neutrophils and LOX-1+ MDSC as well as the expressions of immune checkpoint receptors PD-1 and TIM-3 on T lymphocytes. Results: We included 15 healthy volunteers (HV) and 28 patients. LT was performed for 13 patients. Pre-LT patients presented with a higher NLR compared to HV and NLR was associated with cirrhosis severity. Increased immature neutrophils and LOX-1+ MDSC counts were observed in the most severe patients. These alterations were mainly associated with acute decompensation of cirrhosis. PD-1 and TIM-3 expressions on T lymphocytes were not different between patients and HV. Post-LT immune alterations were dominated by a transitory but tremendous increase of NLR and immature neutrophils during the first days post-LT. Then, immune checkpoint receptors and LOX-1+ MDSC tended to be overexpressed by the second week after surgery. Conclusion: The present study showed that NLR, immature neutrophils and LOX-1+ MDSC counts along with T lymphocyte count and checkpoint inhibitor expression were altered in cirrhotic patients before and after LT. These data illustrate the potential interest of immune monitoring of cirrhotic patients in the context of LT in order to better define risk of sepsis. For this purpose, larger cohorts of patients are now necessary in order to move forward a more personalised care of LT patients.

The Regulation of Neutrophil Migration in Patients with Sepsis: The Complexity of the Molecular Mechanisms and Their Modulation in Sepsis and the Heterogeneity of Sepsis Patients.

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Common causes include gram-negative and gram-positive bacteria as well as fungi. Neutrophils are among the first cells to arrive at an infection site where they function as important effector cells of the innate immune system and as regulators of the host immune response. The regulation of neutrophil migration is therefore important both for the infection-directed host response and for the development of organ dysfunctions in sepsis. Downregulation of CXCR4/CXCL12 stimulates neutrophil migration from the bone marrow. This is followed by transmigration/extravasation across the endothelial cell barrier at the infection site; this process is directed by adhesion molecules and various chemotactic gradients created by chemotactic cytokines, lipid mediators, bacterial peptides, and peptides from damaged cells. These mechanisms of neutrophil migration are modulated by sepsis, leading to reduced neutrophil migration and even reversed migration that contributes to distant organ failure. The sepsis-induced modulation seems to differ between neutrophil subsets. Furthermore, sepsis patients should be regarded as heterogeneous because neutrophil migration will possibly be further modulated by the infecting microorganisms, antimicrobial treatment, patient age/frailty/sex, other diseases (e.g., hematological malignancies and stem cell transplantation), and the metabolic status. The present review describes molecular mechanisms involved in the regulation of neutrophil migration; how these mechanisms are altered during sepsis; and how bacteria/fungi, antimicrobial treatment, and aging/frailty/comorbidity influence the regulation of neutrophil migration.

Tumor microenvironment remodeling after neoadjuvant immunotherapy in non-small cell lung cancer revealed by single-cell RNA sequencing

BackgroundImmunotherapy has revolutionized cancer treatment, but most patients are refractory to immunotherapy or acquire resistance, with the underlying mechanisms remaining to be explored.MethodsWe characterized the transcriptomes of ~92,000 single cells from 3 pre-treatment and 12 post-treatment patients with non-small cell lung cancer (NSCLC) who received neoadjuvant PD-1 blockade combined with chemotherapy. The 12 post-treatment samples were categorized into two groups based on pathologic response: major pathologic response (MPR; n = 4) and non-MPR (NMPR; n = 8).ResultsDistinct therapy-induced cancer cell transcriptomes were associated with clinical response. Cancer cells from MPR patients exhibited a signature of activated antigen presentation via major histocompatibility complex class II (MHC-II). Further, the transcriptional signatures of FCRL4+FCRL5+ memory B cells and CD16+CX3CR1+ monocytes were enriched in MPR patients and are predictors of immunotherapy response. Cancer cells from NMPR patients exhibited overexpression of estrogen metabolism enzymes and elevated serum estradiol. In all patients, therapy promoted expansion and activation of cytotoxic T cells and CD16+ NK cells, reduction of immunosuppressive Tregs, and activation of memory CD8+T cells into an effector phenotype. Tissue-resident macrophages were expanded after therapy, and tumor-associated macrophages (TAMs) were remodeled into a neutral instead of an anti-tumor phenotype. We revealed the heterogeneity of neutrophils during immunotherapy and identified an aged CCL3+ neutrophil subset was decreased in MPR patients. The aged CCL3+ neutrophils were predicted to interact with SPP1+ TAMs through a positive feedback loop to contribute to a poor therapy response.ConclusionsNeoadjuvant PD-1 blockade combined with chemotherapy led to distinct NSCLC tumor microenvironment transcriptomes that correlated with therapy response. Although limited by a small patient sample size subjected to combination therapy, this study provides novel biomarkers to predict therapy response and suggests potential strategies to overcome immunotherapy resistance.

Time-resolved single-cell transcriptomics reveals the landscape and dynamics of hepatic cells in sepsis-induced acute liver dysfunction

Sepsis-induced acute liver dysfunction often occurs early in sepsis and can exacerbate the pathology by triggering multiple organ dysfunction and increasing lethality. Nevertheless, our understanding of the cellular heterogeneity and dynamic regulation of major nonparenchymal cell lineages remains unclear. Here, single-cell RNA sequencing was used to profile multiple nonparenchymal cell subsets and dissect their crosstalk during sepsis-induced acute liver dysfunction in a clinically relevant polymicrobial sepsis model. The transcriptomes of major liver nonparenchymal cells from control and sepsis mice were analysed. The alterations in the endothelial cell and neutrophil subsets that were closely associated with acute liver dysfunction were validated using multiplex immunofluorescence staining. In addition, the therapeutic efficacy of inhibiting activating transcription factor 4 (ATF4) in sepsis and sepsis-induced acute liver dysfunction was explored. Our results present the dynamic transcriptomic landscape of major nonparenchymal cells at single-cell resolution. We observed significant alterations and heterogeneity in major hepatic nonparenchymal cell subsets during sepsis. Importantly, we identified endothelial cell (CD31+Sele+Glut1+) and neutrophil (Ly6G+Lta4h+Sort1+) subsets that were closely associated with acute liver dysfunction during sepsis progression. Furthermore, we found that ATF4 inhibition alleviated sepsis-induced acute liver dysfunction, prolonging the survival of septic mice. These results elucidate the potential mechanisms and subsequent therapeutic targets for the prevention and treatment of sepsis-induced acute liver dysfunction and other liver-related diseases. Sepsis-induced acute liver dysfunction often occurs early in sepsis and can lead to the death of the patient. Nevertheless, the pathogenesis of sepsis-induced acute liver dysfunction is not yet clear. We identified the major cell types associated with acute liver dysfunction and explored their interactions during sepsis. In addition, we also found that ATF-4 inhibition could be invoked as a potential therapeutic for sepsis-induced acute liver dysfunction.

Apolipoprotein E induces pathogenic senescent-like myeloid cells in prostate cancer.

Tumor cells promote the recruitment of immunosuppressive neutrophils, a subset of myeloid cells driving immune suppression, tumor proliferation, and treatment resistance. Physiologically, neutrophils are known to have a short half-life. Here, we report the identification of a subset of neutrophils that have upregulated expression of cellular senescence markers and persist in the tumor microenvironment. Senescent-like neutrophils express the triggering receptor expressed on myeloid cells 2 (TREM2) and are more immunosuppressive and tumor-promoting than canonical immunosuppressive neutrophils. Genetic and pharmacological elimination of senescent-like neutrophils decreases tumor progression in different mouse models of prostate cancer. Mechanistically, we have found that apolipoprotein E (APOE) secreted by prostate tumor cells binds TREM2 on neutrophils, promoting their senescence. APOE and TREM2 expression increases in prostate cancers and correlates with poor prognosis. Collectively, these results reveal an alternative mechanism of tumor immune evasion and support the development of immune senolytics targeting senescent-like neutrophils for cancer therapy.

T cell immunotherapies engage neutrophils to eliminate tumor antigen escape variants

Cancer immunotherapies, including adoptive Tcell transfer, can be ineffective because tumors evolve to display antigen-loss-variant clones. Therapies that activate multiple branches of the immune system may eliminate escape variants. Here, we show that melanoma-specific CD4+ Tcell therapy in combination with OX40 co-stimulation or CTLA-4 blockade can eradicate melanomas containing antigen escape variants. As expected, early on-target recognition of melanoma antigens by tumor-specific CD4+ Tcells was required. Surprisingly, complete tumor eradication was dependent on neutrophils and partly dependent on inducible nitric oxide synthase. In support of these findings, extensive neutrophil activation was observed in mouse tumors and in biopsies of melanoma patients treated with immune checkpoint blockade. Transcriptomic and flow cytometry analyses revealed a distinct anti-tumorigenic neutrophil subset present in treated mice. Our findings uncover an interplay between Tcells mediating the initial anti-tumor immune response and neutrophils mediating the destruction of tumor antigen loss variants.

Fexofenadine protects against lipopolysaccharide-induced acute lung injury by targeting cytosolic phospholipase A2

ObjectiveAcute lung injury (ALI) causes acute respiratory distress syndrome, with a high mortality rate of 40%, with currently available pharmacological treatments. Cytosolic phospholipase A2 (cPLA2) plays a critical role in the lipopolysaccharide (LPS)-induced pathology of ALI. This study assessed the therapeutic effects of fexofenadine (FFD), an on-market small-molecule drug that can target cPLA2 in LPS-induced ALI. MethodsPrimary macrophages obtained from the bone marrow of wild-type and cPLA2 knockout mice and the alveolar macrophage cell line, MHS were used to test the inhibitory effect of FFD on the cPLA2/ERK/p65 signaling pathway, NF-κB p65 translocation, and cytokine and chemokine production. An LPS-induced ALI mouse model was used to assess the treatment effects of FFD. Flow cytometry detected subsets of macrophages and neutrophils. cPLA2 activity and downstream hydrolysates were detected. Treatment with a cPLA2 inhibitor or NF-κB p65 inhibitor confirmed that FFD functioned through the cPLA2/ERK/p65 signaling pathway by targeting cPLA2. ResultsFFD reduced the infiltration of macrophages and neutrophils, decreased the protein secretion in bronchoalveolar lavage fluid, and reduced the production of TNFα, IL-1β, IL-6, MCP-1, and IL-8 in the lung, bronchoalveolar lavage fluid, and sera of LPS-induced ALI mice. FFD inhibited cPLA2 activity, suppressed the cPLA2/ERK/p65 signaling pathway, inhibited translocation of p65, and decreased the production of cytokines, chemokines, and downstream hydrolysates of cPLA2, arachidonic acid, and leukotriene B4. ConclusionFFD inhibits the cPLA2/ERK/p65 signaling pathway by targeting cPLA2. Therefore, FFD is promising as a therapeutic against cPLA2-involved diseases, particularly ALI.

Abstract 18: A Novel Neutrophil Subpopulation Mediates Neuroprotection In The Late Phases Of Cerebral Ischemia

Cerebral ischemia triggers a powerful inflammatory reaction involving infiltration of peripheral leukocytes. Neutrophils, the first immune cells entering the ischemic brain, contribute to brain injury by limiting tissue perfusion and destabilizing the blood-brain barrier. However, recent evidence indicates that neutrophils can also participate to the repairing process. Nevertheless, the dynamics and complexity of neutrophil subsets are poorly understood. Here, we performed single-cell transcriptomics to obtain a picture of infiltrating neutrophil diversity in the mouse brain 2 (D02) and 14 days (D14) after transient middle cerebral artery occlusion (MCAo) or sham surgery (Sham). Clustering analysis of the transcripts revealed 5 neutrophil populations. In Sham, most neutrophils fell into clusters 1 and 4, characterized by expression of genes associated with neutrophil development such Retnlg and Mmp8 (cluster 1) and Camp, Ltf, Chil3 and Lcn2 (cluster 4). After MCAo, clusters 1 and 4 gradually decreased, whereas cluster 2, identified by type I response genes ( Isg15, Ifit3, Rsad2, Ifit1, Ifi204 ) and cluster 3, characterized by upregulation of chemokines/cytokines ( Ccl3 , Ccl4 , Csf1 ), increased progressively from D02 to D14. Interestingly, we found that the number of neutrophils in the ischemic brain was 5 times higher at D14 than at D02 (Sham, 0.2 ± 0.1 x10 3 ; D02: 3 ± 1.3 x10 3 ; D14: 14.5 ± 3.5x10 3 cells; p=0.02 D02 vs D14; n=10-14/group). Therefore, we next investigated if neutrophil infiltration at D14 has a beneficial or adverse effect on stroke recovery. To this end, 7 days after MCAo, neutrophils depletion was induced by systemic injection of both rat anti-Ly6G (50μg/mouse, i.p) and CXCR2-antagonist SB225002 (2 mg/kg, i.p), once per day, for 7 consecutive days. We found that neutrophil depletion increased brain tissue loss (-18.7 ± 2.9 vs -8 ± 2.6% IgG/vehicle mice; p=0.01; n=9/group). In addition, neutrophil depletion decreased ambulatory distance (p<0.05 vs vehicle; n=11/group) and stereotypic behavior (p<0.05 vs vehicle; n=11/group) without altering open field performance in sham mice. Our study unveils an unrecognized neutrophil subpopulation protecting the brain in the subacute stroke phase, opening new avenues for stroke treatment.

Assessing neutrophil subsets in autoimmune disease: Moving away from relying on density?

Assessing neutrophil subsets in autoimmune disease: Moving away from relying on density?

P090 Low-density neutrophils are higher in individuals with active ulcerative colitis

Abstract Background Introduction: Neutrophils and neutrophil extracellular traps (NETs) play a significant role in the pathogenesis of ulcerative colitis (UC), a chronic and relapsing inflammatory disease of the colon. Low-density neutrophils (LDNs) are a recently identified subset of neutrophils that are more prone to develop NETs and produce more pro-inflammatory cytokines and calprotectin than neutrophils of normal density. Moreover, LDNs show reduced phagocytotic activity towards bacteria. LDNs are subclassified as mature (CD15+CD14-CD10+) and immature (CD15+CD14-CD10-) based on CD10 expression, of which the immature LDNs are most pro-inflammatory. LDNs have been identified in a number of autoimmune diseases, including systemic lupus erythematosus, psoriasis, antineutrophil cytoplasmic antibody-associated vasculitis, and rheumatoid arthritis, but not yet studied in UC. Objective Examine the existence of LDNs in UC patients and their possible association to disease severity. Methods A total of 13 healthy controls and 20 patients with UC were included. Their clinical activity was evaluated with the Truelove-Witts score. LDNs and subtypes were quantified by flow cytometry in the peripheral blood mononuclear cells (PBMC). Results The relative abundance of LDNs was significantly higher in UC patients compared to healthy controls (1.55±1.14 vs. 0.62±0.36 respectively, P<0.05). Moreover, LDNs numbers were higher in UC patients with active disease compared to those in remission (2.67±1.36 vs. 1.09±0.55; P<0.05) (Figure 1). The abundance of immature (CD15+CD14-CD10-) LDNs positively correlated with the Truelove-Witts index (rho=.530, P<0.05) and fecal calprotectin levels (OR=1.2, P<0.05, CI=1-1.36) as illustrated in Figure 2. Figure 1 Figure 2 Conclusion LDNs levels are elevated in PBMCs of patients with UC and pro-inflammatory immature LDNs are associated with disease activity.

Distinct phenotype of neutrophil, monocyte, and eosinophil populations indicates altered myelopoiesis in a subset of patients with multiple myeloma.

Hematologic malignancies, including multiple myeloma (MM), promote systemic immune dysregulation resulting in an alteration and increased plasticity of myeloid cell subsets. To determine the heterogeneity of the myeloid cell compartment in the peripheral blood of patients with MM, we performed a detailed investigation of the phenotype and function of myeloid subpopulations. We report that a subset of MM patients exhibits a specific myeloid cell phenotype indicative of altered myelopoiesis characterized by significant changes in the properties of circulating granulocytic, monocytic, and eosinophilic populations. The subset, referred to as MM2, is defined by a markedly elevated level of CD64 (FcγRI) on the surface of circulating neutrophils. Compared to healthy controls or MM1 patients displaying intermediate levels of CD64, neutrophils from MM2 patients exhibit a less differentiated phenotype, low levels of CD10 and CXC chemokine receptor 2 (CXCR2), increased capacity for the production of mitochondrial reactive oxygen species, and an expansion of CD16neg immature neutrophil subset. Classical and patrolling monocytes from MM2 patients express elevated levels of CD64 and activation markers. MM2 eosinophils display lower levels of C-C Chemokine receptor 3 (CCR3), Toll-like receptor 4 (TLR4, CD284), and tissue factor (TF, CD142). The MM2 (CD64high) phenotype is independent of age, race, sex, and treatment type. Characteristic features of the MM2 (CD64high) phenotype are associated with myeloma-defining events including elevated involved/uninvolved immunoglobulin free light chain (FLC) ratio at diagnosis. Detailed characterization of the altered myeloid phenotype in multiple myeloma will likely facilitate the identification of patients with an increased risk of disease progression and open new avenues for the rational design of novel therapeutic approaches.

TGFβ1+CCR5+ neutrophil subset increases in bone marrow and causes age-related osteoporosis in male mice

TGFβ1 induces age-related bone loss by promoting degradation of TNF receptor-associated factor 3 (TRAF3), levels of which decrease in murine and human bone during aging. We report that a subset of neutrophils (TGFβ1+CCR5+) is the major source of TGFβ1 in murine bone. Their numbers are increased in bone marrow (BM) of aged wild-type mice and adult mice with TRAF3 conditionally deleted in mesenchymal progenitor cells (MPCs), associated with increased expression in BM of the chemokine, CCL5, suggesting that TRAF3 in MPCs limits TGFβ1+CCR5+ neutrophil numbers in BM of young mice. During aging, TGFβ1-induced TRAF3 degradation in MPCs promotes NF-κB-mediated expression of CCL5 by MPCs, associated with higher TGFβ1+CCR5+ neutrophil numbers in BM where they induce bone loss. TGFβ1+CCR5+ neutrophils decreased bone mass in male mice. The FDA-approved CCR5 antagonist, maraviroc, reduced TGFβ1+CCR5+ neutrophil numbers in BM and increased bone mass in aged mice. 15-mon-old mice with TGFβRII specifically deleted in MPCs had lower numbers of TGFβ1+CCR5+ neutrophils in BM and higher bone volume than wild-type littermates. We propose that pharmacologic reduction of TGFβ1+CCR5+ neutrophil numbers in BM could treat or prevent age-related osteoporosis.

Distinct subsets of neutrophils crosstalk with cytokines and metabolites in patients with sepsis

Sepsis is a life-threatening condition caused by a dysregulated host response to infection. Despite continued efforts to understand the pathophysiology of sepsis, no effective therapies are currently available. While singular components of the aberrant immune response have been investigated, comprehensive studies linking different data layers are lacking. Using an integrated systems immunology approach, we evaluated neutrophil phenotypes and concomitant changes in cytokines and metabolites in patients with sepsis. Our findings identify differentially expressed mature and immature neutrophil subsets in patients with sepsis. These subsets correlate with various proteins, metabolites, and lipids, including pentraxin-3, angiopoietin-2, and lysophosphatidylcholines, in patients with sepsis. These results enabled the construction of a statistical model based on weighted multi-omics linear regression analysis for sepsis biomarker identification. These findings could help inform early patient stratification and treatment options, and facilitate further mechanistic studies targeting the trifecta of surface marker expression, cytokines, and metabolites.

CD98 defines a metabolically flexible, proinflammatory subset of low-density neutrophils in systemic lupus erythematosus.

Low-density neutrophils (LDN) are a distinct subset of neutrophils rarely detected in healthy people but appear in the blood of patients with autoimmune diseases, including systemic lupus erythematosus (SLE), and are mobilised in response to granulocyte colony-stimulating factor (G-CSF). The aim of this study was to identify novel mechanisms responsible for the pathogenic capacity of LDN in SLE. Neutrophils were isolated from donors treated with G-CSF, and whole-cell proteomic analysis was performed on LDN and normal-density neutrophils. CD98 is significantly upregulated in LDN from G-CSF donors and defines a subset of LDN within the blood of SLE patients. CD98 is a transmembrane protein that dimerises with L-type amino acid transporters. We show that CD98 is responsible for the increased bioenergetic capacity of LDN. CD98 on LDN mediates the uptake of essential amino acids that are used by mitochondria to produce adenosine triphosphate, especially in the absence of glucose. Inhibition of CD98 reduces the metabolic flexibility of this population, which may limit their pathogenic capacity. CD98+ LDN produce more proinflammatory cytokines and chemokines than their normal density counterparts and are resistant to apoptosis, which may also contribute to tissue inflammation and end organ damage in SLE. CD98 provides a phenotypic marker for LDN that facilitates identification of this population without density-gradient separation and represents a novel therapeutic target to limit its pathogenic capacity.

Olfactomedin-4-Positive Neutrophils in Neonates: Link to Systemic Inflammation and Bronchopulmonary Dysplasia

Introduction: Little is known about the interplay between neutrophil heterogeneity in neonates in health and disease states. Olfactomedin-4 (OLFM4) marks a subset of neutrophils that have been described in adults and pediatric patients but not neonates, and this subset is thought to play a role in modulating the host inflammatory response. Methods: This is a prospective cohort of neonates who were born between June 2020 and December 2021 at the University of Cincinnati Medical Center NICU. Olfactomedin-4-positive (OLFM4+) neutrophils were identified in the peripheral blood using flow cytometry. Results: OLFM4+ neutrophil percentage was not correlated with gestational age or developmental age. Neonates with sepsis had a higher percentage than those without the condition, 66.9% (IQR 24.3–76.9%) versus 21.5% (IQR 10.6–34.7%), respectively, p = 0.0003. At birth, a high percentage of OLFM4+ neutrophils was associated with severe chorioamnionitis at 49.1% (IQR 28.2–61.5%) compared to those without it at 13.7% (IQR 7.7–26.3%), p < 0.0001. Among neonates without sepsis, the percentages of OLFM4+ neutrophils were lower in the BPD/early death group compared to those without BPD, 11.8% (IQR 6.3–29.0%) versus 32.5% (IQR 18.5–46.1%), p = 0.003, and this retained significance in a multiple logistic regression model that included gestational age, birthweight, and race. Conclusion: This is the first study describing OLFM4+ neutrophils in neonates and it shows that this neutrophil subpopulation is not influenced by gestational age but is elevated in inflammatory conditions such as sepsis and severe chorioamnionitis, and lower percentage at birth is associated with developing bronchopulmonary dysplasia.

Phenotypic alteration of low-density granulocytes in people with pulmonary post-acute sequalae of SARS-CoV-2 infection.

Low-density granulocytes (LDGs) are a distinct subset of neutrophils whose increased abundance is associated with the severity of COVID-19. However, the long-term effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on LDG levels and phenotypic alteration remain unexplored. Using participants naïve to SARS-CoV-2 (NP), infected with SARS-CoV-2 with no residual symptoms (NRS), and infected with SARS-CoV-2 with chronic pulmonary symptoms (PPASC), we compared LDG levels and their phenotype by measuring the expression of markers for activation, maturation, and neutrophil extracellular trap (NET) formation using flow cytometry. The number of LDGs was elevated in PPASC compared to NP. Individuals infected with SARS-CoV-2 (NRS and PPASC) demonstrated increased CD10+ and CD16hi subset counts of LDGs compared to NP group. Further characterization of LDGs demonstrated that LDGs from COVID-19 convalescents (PPASC and NRS) displayed increased markers of NET forming ability and aggregation with platelets compared to LDGs from NP, but no differences were observed between PPASC and NRS. Our data from a small cohort study demonstrates that mature neutrophils with a heightened activation phenotype remain in circulation long after initial SARS-CoV-2 infection. Persistent elevation of markers for neutrophil activation and NET formation on LDGs, as well as an enhanced proclivity for platelet-neutrophil aggregation (PNA) formation in COVID-19 convalescent individuals may be associated with PPASC prognosis and development.