Journal of Leukocyte Biology最新文献

Natural killer (NK) cell antibody-dependent cellular cytotoxicity (ADCC) contributes to effective antiviral immunity, yet the relative contribution of NK cell-intrinsic factors and antibodies in mediating these responses remain poorly understood. Here, we combined functional ADCC assays with single-cell transcriptomics of peripheral NK cells from COVID-19 participants. Our analysis revealed distinct transcriptional programs between participants with different ADCC response levels: NK cells from participants with lower ADCC responses upregulated proliferation pathways, while those with high ADCC responses showed enhanced expression of interferon-stimulated genes and NKG2D. Blocking NKG2D significantly reduced NK cell ADCC degranulation and cytokine responses. Paradoxically, greater interferon-mediated NK cell activation was associated with reduced proficiency of participants' antibodies to mediate ADCC, suggesting a regulatory checkpoint mechanism. These findings enhance our understanding of the molecular determinants of ADCC responses and provide novel insights into leveraging these responses for more effective vaccination and therapeutic strategies.

Despite advances in engineered adaptive immune cell therapies, current options for innate immune cell therapies are sparse. In this work, we demonstrate the utility of a neutrophil progenitor-based cell therapy. Murine conditionally-immortalized neutrophil progenitors (NPs) overcome some of the hurdles of alternative cell therapies, such as granulocyte transfusion, by engrafting in the unconditioned host and undergoing substantial expansion in vivo. Here we demonstrate the therapeutic value of NPs using a murine model of the primary immunodeficiency chronic granulomatous disease (CGD). Those with CGD are highly susceptible to infection with Staphylococcus aureus because of genetic mutations that impair neutrophil antimicrobial function. We find that the prophylactic treatment of CGD mice with transfused NPs rescue them from an otherwise lethal S. aureus pulmonary infection. In investigating the mechanisms behind the improved clearance of S. aureus and survival of CGD mice, our data suggests that the antimicrobial function of host CGD neutrophils is rescued by the presence of donor-derived wild-type neutrophils. We also observe that survival is improved to >50% in the CGD model when mice receive NPs post-infection. This work highlights the application of NPs to improving outcomes to acute bacterial infection in CGD, demonstrating the translational potential of conditionally-immortalized myeloid progenitors as a cellular therapy.

Acute myeloid leukemia (AML) is characterized by profound immune dysregulation, yet the mechanisms underlying impaired cytotoxicity remain unclear. By analyzing samples from 20 AML patients and 20 healthy donors, we integrated scRNA-seq (GSE223844), bulk transcriptomes (GSE37642, GSE71014), and TCGA-AML. Single-cell profiling, BayesPrism deconvolution, WGCNA, and multiple machine-learning algorithms were used to define immune alterations and construct a prognostic model, with peripheral blood validation through RT-qPCR and multicolor flow cytometry. We identified 11 immune and progenitor populations showing AML-specific shifts, including depletion of T/NK cells and expansion of stem-like compartments. Regulatory network and ligand-receptor analyses revealed broad immune suppression and disrupted cellular communication. Deconvolution showed extensive transcriptional remodeling of CD8+ T cells, and WGCNA identified a CD8+ T-cell gene module. Nine candidate genes were incorporated into 18 machine-learning models, with ridge regression generating a stable nine-gene prognostic signature. Enrichment analyses indicated activation of TGF-β, TNF receptor, and TCR pathways in high-risk patients. LILRB1 emerged as a central immunosuppressive hub in CD8+ T and NK cells, and experimental validation confirmed its elevated expression alongside impaired cytotoxicity in AML-derived lymphocytes. Overall, LILRB1 serves as a key immune checkpoint driving cytotoxic dysfunction, marking exhausted CD8+ T cells and CD16+ NK cells. The nine-gene signature links CD8+ T-cell impairment to poor prognosis, while NK-cell involvement positions LILRB1 as a promising therapeutic target for restoring anti-leukemic immunity.

Novel leukolectin-proteins (LL-proteins) are present in multiple lower vertebrates. LL-proteins (∼255 AAs) possess five TECPR-domains spanning 4/5 of conserved AA-sequences, and are detected in dermal lectocytes, (primitive and tissue resident) macrophages and some leukocytes. Hatched fish embryos employ secreted LL-proteins for innate immunity-defense. LL-genes display 4 introns, 5 exons and multiple 5'-upstream binding-sites for hematopoietic transcription-factors. This study aims to identify potential LL-proteins in human leukocytes. Western-blots of protein-extracts (resolved by 2D-PAGE) exclusively revealed LL-proteins (MW ̴̴30 kD; pI ̴6.0), but only in PolymorphprepTM-enriched leukocytes, and not in LymphoprepTM-enriched leukocytes. Single-label immuno-fluorescence demonstrated LL-proteins in some PMN-leukocytes. Dual-label immuno-fluorescence corroborated LL-coexpression in some myeloperoxidase-positive neutrophils (denoted lectophils). Intervillous spaces in Caesarean placentas displayed both lectophils and large lectophils without well-defined PMN-nuclei. Some moderately LL-positive placental endothelium appeared to cytodifferentiate with intensified LL-expression to large, non-PMN lectophils for circulation. Functional distinctions between non-lectophilic neutrophils and lectophilic neutrophils await experimental delineation.

Age-related decline in neutrophil function reduces vaccine protection against Streptococcus pneumoniae. In vaccinated hosts, neutrophil activation via complement and Fcγ receptors mediates bacterial uptake and killing. Mechanisms behind age-related changes in signaling of these receptors is unknown. Using neutrophils from young and old mice, we found opsonin-dependent differences in MAPK activation. Neutrophils from old mice had higher basal phosphorylation of MAPK proteins compared to young controls, including a 15-fold increase in phosphorylated ERK1/2, but failed to increase phosphorylation upon infection with antibody-opsonized bacteria. Inhibition of ERK1/2 signaling blunted killing of antibody-opsonized pneumococci by neutrophils from young mice but improved killing in old mice. In young adult human participants, inhibition of ERK1/2 signaling in neutrophils decreased pneumococcal killing, but only in vaccinated hosts, demonstrating the clinical relevance of this pathway. This study demonstrates that balanced activation of ERK1/2 is crucial for neutrophil antimicrobial activity against antibody-opsonized bacteria but is disrupted in old hosts.

Neutrophils release their chromatin with toxic granular proteins as neutrophil extracellular traps (NETs) when activated. Diabetes exacerbates NET formation (NETosis), resulting in tissue damage and diabetic complications such as non-healing wounds. How diabetes predisposes neutrophils to NETosis remains unclear. Herein, we found that pharmacological inhibition or siRNA-knockdown of sirtuin 1 (SIRT1) increased NETosis in neutrophils of healthy humans and mice, unveiling SIRT1 as an endogenous suppressor of NETosis. In contrast, SIRT1 inhibition did not cause further increase in NETosis in neutrophils of diabetic patients and mice, indicative of SIRT1 dysfunction in disease state. Indeed, SIRT1 deacetylase activity was significantly lower in neutrophils of diabetic individuals, accompanied by a concomitant increase in the activity of peptidylarginine deiminase 4 (PAD4), a key enzyme that mediates NETosis. PAD4 was co-detected with SIRT1 immunoprecipitated from neutrophils isolated from healthy subjects and HL-60-derived neutrophils (dHL-60) cultured in basal glucose; such co-immunoprecipitation was absent in neutrophils of subjects with diabetes and dHL-60 cells treated with high glucose, suggesting that hyperglycemia disrupts the SIRT1-PAD4 interaction. SIRT1 activators restored the SIRT1-PAD4 interaction and normalized the exacerbated NETosis and PAD4 activity in diabetes and hyperglycemia, culminating in improved diabetic wound healing. This study reveals a novel regulatory role of SIRT1 on PAD4 activity. Revitalizing SIRT1 can be a new preventive or therapeutic strategy for combating NET-mediated inflammation in diabetes and beyond.

Extravasation control of leukocytes and their functional fates are crucial to maintain tissue homeostasis. Leukocyte lineage origin regulates these processes that blood monocytes derived from PDGFRα+ embryonic progenitors (PDGFRα-lineage) extravasate more efficiently to inflamed skin than non-PDGFRα-lineage cells. Here we elucidated the underlying mechanism and their macrophage identity fates in skin. Spatial confocal imaging showed PDGFRα-lineage monocytes adhered better on venular endothelium than non-PDGFRα-lineage before trans-venular migration dependently on ICAM-1. At endothelial interface, more ICAM-1-aggregates were observed with PDGFRα-lineage monocytes and disruption with a Src-family kinases antagonist reduced adhesion advantage. Extravasated PDGFRα-lineage monocytes exhibited slower β1-integrin-dependent migration to interstitium and remained proximal to hypodermal capillaries. Skin-interstitial fibronectin binds integrin-α5 and activates Src-family kinases to suppress macrophage expression of S1-marking CD163. By accessing nearby vascular matrices, PDGFRα-lineage monocytes mitigated this effect and developed stronger S1-identity. Together, extravasation fate of PDGFRα-lineage monocyte/macrophage reveals the vasculatures foster S1-macrophage identity formation in skin.

Expression of the Csf1r gene in mice is restricted to cells of the mononuclear phagocyte system and placental trophoblasts. A conserved element (Csf1r upstream regulatory element A, CUREA) in the mouse Csf1r locus contains transcription start sites utilised by trophoblasts and osteoclasts and an enhancer essential for expression of multicopy transgenic reporters in most tissue macrophages. Here we describe the impact of deletion of CUREA in the mouse genome, on the background of a knock-in Csf1r-FusionRed reporter. By contrast to the essential function in transgene expression, CUREA deletion had no effect on expression of FusionRed (FRed) or differentiation of blood monocytes or tissue resident macrophages. The deletion reduced Csf1r mRNA in hematopoietic stem cells and committed myeloid progenitors (MPP3) leading to a subtle differentiation delay and had a significant impact on microglial density in the brain and the differentiation of osteoclasts. The expression of FRed in placenta confirmed expression of CSF1R in trophoblasts. 5'RACE analysis demonstrated that the effect of CUREA deletion on Csf1r transcription in placenta was overcome by the use of cryptic upstream transcription start sites. We conclude that CUREA is a regulatory element controlling Csf1r transcription. The function overlaps with other enhancers identified in the locus and is therefore partly redundant.

Emerging evidence demonstrates that innate immune cells can maintain a non-specific memory, not only in response to microbe-associated ligands such as β-glucan, but also synthetic biomaterials and nano- and microparticles. This creates an opportunity to leverage biomaterials which can establish favourable innate immune responses and memory for therapeutic applications. In this study, we identify particle size as a critical physical determinant influencing both acute macrophage activation and long-term innate immune memory. Specifically, biodegradable poly(lactic-co-glycolic acid) particles in the 1-2 µm size range promoted an anti-inflammatory phenotype and enhanced oxidative phosphorylation in bone marrow-derived macrophages, through a process dependent on mTOR signalling. In contrast to the well documented pro-inflammatory innate immune training seen with microbial stimuli such as β-glucans, exposure of macrophages to 1-2um poly(lactic-co-glycolic acid) particles promoted a durable anti-inflammatory reprogramming, marked by elevated IL-10 and IL-1 receptor antagonist secretion upon secondary stimulation, and metabolic re-wiring. Moreover, bone marrow from mice injected with PLGA particles in this size range, were reprogrammed to upregulate IL-1Ra and IL-10 secretion upon a re-stimulation, which persisted up to one week post-injection. These findings uncover how the physicochemical properties of polymeric nanoparticles differentially modulate innate immune cells and regulate the induction of innate training.