Journal of Leukocyte Biology

Some extracellular glycoconjugates have sialic acid as the terminal sugar, and sialidases are enzymes that remove this sugar. Mammals have four sialidases, but their biological functions are unclear. In this report, we show that incubation of human neutrophils with the human sialidase NEU3, but not NEU1, NEU2 or NEU4, inducess human male and female neutrophils to change from a round to a more amoeboid morphology, causes the primed neutrophil markers CD66, CD11B, and CD18 to localize to the cell cortex, and decreases the localization of the unprimed neutrophil markers CD43 and CD62L at the cell cortex. NEU3, but not the other 3 sialidases, also causes human male and female neutrophils to increase their F-actin content. The inhibition of NEU3 by the NEU3 inhibitor 2-acetylpyridine attenuated the NEU3 effect on neutrophil morphology, indicating that the effect of NEU3 is dependent on its enzymatic activity. Together, these results indicate that NEU3 can prime human male and female neutrophils, and that NEU3 is a potential regulator of inflammation.

Pulmonary defense mechanisms are critical for host integrity during the early phase of pneumonia and sepsis. These processes are fundamentally dependent on the activation of neutrophils during the early phase of the innate immune response. Recent work has shown that semaphorin 7A (Sema7A) holds significant impact on platelet activation, yet its role in neutrophil migration and function is not well known. We report here that Sema7A binds to neutrophil PlexinC1, increasing integrins and L-selectin on the neutrophil surface. Sema7A-induced neutrophil activation also prompted neutrophil chemotaxis in vitro and the formation of platelet-neutrophil complexes in vivo. We also observed altered adhesion and transmigration of neutrophils in Sema7A-/- animals in the lung. Sema7A-/- animals also showed altered crawling properties of neutrophils. This resulted in increased number of neutrophils in the interstitial space of Sema7A-/- animals but reduced numbers of neutrophils in the alveolar space during pneumonia-induced pulmonary sepsis. This was associated with significantly worse outcome of Sema7A-/- animals in a model of Klebsiella pneumoniae. Furthermore, we were able to show a correlation between serum levels of Sema7A in patients with ARDS and oxygenation levels. Thus, we show here that Sema7A has an immunomodulatory effect though which might influence patient outcome during pulmonary sepsis. SummarySema7A controls pulmonary immune defense

Anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV) is a life-threatening condition characterized by improper activation of neutrophils and release of neutrophil extracellular traps (NETs) in small vessels. This study aimed to explain the role of NETs in AAV pathogenesis by investigating a link between neutrophil adhesion and NET release. We leveraged an imaging flow cytometry-based assay and three-dimensional culture to demonstrate that neutrophil adhesion is essential for ANCA induced NET formation. We confirmed this requirement for cell adhesion using standard microscopy on ultra-low attachment hydrogel surfaces and demonstrate that this depends on the focal adhesion kinase pathway as determined using inhibitors for multiple targets in this process. ANCA increased expression of {beta}2 integrins on neutrophils, and we confirmed that these integrins were required for NET formation using blocking antibodies. Finally, inhibitors for oxidative burst prevented NET formation, and this oxidative burst was mediated by the focal adhesion pathway. Overall, our findings reveal a central role for neutrophil attachment in NET formation in response to ANCA, helping to explain the restricted localization pattern of vessel damage, and suggesting that targeting neutrophil adhesion factors may be beneficial in preventing pathological damage from NETs during AAV.

Mucosal-associated invariant T (MAIT) cells are innate-like T lymphocytes that are abundant in mucosal tissues and the liver where they can respond rapidly to a broad range of riboflavin producing bacterial and fungal pathogens. Neutrophils, which are recruited early to sites of infection, play a non-redundant role in pathogen clearance and are crucial for controlling infection. The interaction of these two cell types is poorly studied. Here, we investigated both the effect of neutrophils on MAIT cell activation and the effect of activated MAIT cells on neutrophils. We show that neutrophils suppress the activation of MAIT cells by a cell-contact and H2O2 dependent mechanism. Moreover, highly activated MAIT cells were able to produce high levels of TNF that induced neutrophil death. We therefore provide evidence for a negative regulatory feedback mechanism in which neutrophils prevent over-activation of MAIT cells and, in turn, MAIT cells limit neutrophil survival.

Neutrophils have historically been envisioned as a homogenous population of short-lived innate immune cells that migrate to sites of infection, kill pathogens, and die. Recent work, including studies in pneumonia models, has shown that neutrophil transcriptomes reflect the environment from which they were isolated. We used high-parameter spectral flow cytometry to compare and contrast a wide array of surface proteins on neutrophils from different tissues, infections, host age, pathogen virulence, and across multiple time-points of pneumonia. Circulating and airspace neutrophils consistently differed, and surface protein phenotypes unique to each infection setting were identified, revealing tissue-specific and microbe-specific neutrophil plasticity. Phenotypic differences in circulating neutrophils from mice infected with different pathogens (E. coli, S. pneumoniae, S. aureus, and P. aeruginosa) identified, even in the absence of bacteremia. Neutrophil activation state was diminished with less virulent pathogens and host age. In the airspace, VISTA, CD200R, and PD-L1 were selectively high on BAL neutrophils (BALN) during S. pneumoniae infection, and we identified pro-degranulation-like (CD88High VISTAHigh PD-L1+ CD101-) neutrophils in S. pneumoniae and pro-phagocytosis-like (CD101+ CD18Low PD-L1-) neutrophils in E. coli infections. Stimulation of VISTA with its ligand VISG-3 enhanced the neutrophil respiratory burst, degranulation, and killing of S. pneumoniae but not E. coli. We conclude that neutrophil cell surface protein expression depends on anatomic location and infection type, resulting in pathogen-specific neutrophil-mediated immune defense in discreet areas of the pneumonic lung. Graphical AbstractIn brief, Pihl et al. have found that neutrophil cell surface phenotype varies drastically based on tissue, time post-infection, and infection. BALN from early infections have higher activation and maturation statuses, while blood neutrophils are more migration primed, and late infections have more immune-suppressive and altered pathogen killing statuses. Neutrophil phenotype is skewed towards pro-phagocytosis associated marker expression on BALN from E. coli-infected mice while S. pneumoniae results in a pro-degranulation phenotype. In vitro BMN stimulation of VISTA with VSIG-3 results in degranulation, respiratory burst, and pathogen specific killing of S. pneumoniae but not E. coli. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/649365v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@4e4829org.highwire.dtl.DTLVardef@1638108org.highwire.dtl.DTLVardef@1ac413dorg.highwire.dtl.DTLVardef@1ef36b5_HPS_FORMAT_FIGEXP M_FIG C_FIG

The specific granule glycoprotein olfactomedin-4 (Olfm4) marks a constitutive subset of neutrophils in humans, where 1-70% of peripheral blood neutrophils produce Olfm4. The proportion of Olfm4-high (Olfm4-H) neutrophils correlates with the severity of paediatric septic shock and could predict mortality in adult septic shock in previous studies, but it is not known whether and how the Olfm4-H neutrophils contribute to sepsis pathogenesis. The aim of this study was to decipher proteomic differences between the Olfm4-H and Olfm4-low (Olfm4-L) human neutrophil subsets at baseline and in the context of septic shock, hypothesizing that Olfm4 marks a neutrophil subset with a distinct proteomic profile, predisposing it for detrimental processes in sepsis. A novel protocol for the preparation of fixed, antibody-stained and sorted neutrophils for LC-MS/MS analysis of proteome was developed. In neutrophil subsets from healthy blood donors, 47 proteins had significantly higher abundance in the Olfm4-H population, and 62 proteins in the Olfm4-L population. Pathway enrichment analysis showed that the differences concerned proteins related to neutrophil degranulation, with e.g. Rab3d and a subunit of the vacuolar ATPase proton pump being more abundant in the Olfm4-H neutrophils, and the alarmin S100-A7, the major neutrophil chemotactic receptor CXCR1 and the antimicrobial peptide defensin alpha-4 being more abundant in the Olfm4-L neutrophils. The data suggest different preparedness to infection in the subsets. In the limited material analysed here, there was no significant correlation between the severity of sepsis and the proportion of Olfm4-H neutrophils, but an increased concentration of Olfm4 in plasma from septic shock patients as compared to healthy blood donors was observed. Furthermore, in neutrophil subsets isolated from septic shock patients, 28 proteins had significantly higher abundance in the Olfm4-H subset and 38 in the Olfm4-L subset, the latter including e.g. Fc receptor proteins and MHC class I molecules, suggesting distinct immunological responses. This is the first report pointing towards differential functions of the Olfm4-defined neutrophil subpopulations in humans and the data are consistent with the idea of distinct responses in the subsets during infection and inflammation.

Myeloid-derived suppressor cells (MDSCs) are an immunosuppressive cell type found in high abundance in early life. Currently, there has been limited mechanistic understanding of MDSC phagocytosis of bacteria and the corresponding consequences in the context of acute infection. We set out to determine whether human granulocytic MDSCs have phagocytic capacity that is comparable to other professional phagocytes. To investigate these properties, we utilized fluorescent confocal microscopy, flow cytometry, and bacterial burden assays. We demonstrate that human granulocytic MDSCs phagocytose E. coli O1:K1:H7, and subsequently traffic the bacteria into acidic compartments similar to other phagocytes. However, MDSCs were significantly less efficient at bacterial uptake and killing compared to monocytes. This activity is associated with an inflammatory response, but the amount of TNF gene and protein expression was reduced in infected MDSCs compared to monocytes. Interestingly, we also found that MDSCs release DNA (MeDNA) into the extracellular space that resembles neutrophil extracellular traps. We found that MeDNA had some impact on bacterial viability in single cultures, with an increase in bacterial recovery in MDSCs treated with DNAse. However, MeDNA did not impact the ability of monocytes to eliminate bacteria in co-cultures, suggesting that MDSC extracellular DNA does not compromise monocyte function. Overall, our data reveals mechanistic insight into MDSC activity during infection that includes the kinetics and efficiency of bacterial uptake, elimination through trafficking to acidified compartments, and inflammatory contributions relative to primary human monocytes. These results enhance our understanding of MDSC contributions during acute bacterial infection and identify host-directed targets for immune intervention to improve outcomes and reduce susceptibility to infection early in life.

Megakaryocytes (MKs) are precursors to platelets, the second most abundant cells in the peripheral circulation. However, while platelets are known participate in immune responses and play significant roles during infections, the role of MKs within the immune system has not been explored. Here we utilize in vitro techniques to show that both cord blood-derived MKs (CB MKs) and MKs from a human megakaryoblastic leukemia cell line (Meg-01) chemotax towards pathogenic stimuli, phagocytose bacteria, and release chromatin webs in response to bacteria. Moreover, in patients with sepsis, we found that MK counts were significantly higher in the peripheral blood, and CD61+ staining was increased in the kidneys and lungs, correlated with the development of organ dysfunction. Overall, our study suggests that MK cells display basic innate immune cell functions and respond during infections and sepsis.

PurposeNeutrophils express Fc receptors on their surface to trap immune complexes. While the implication of Fc{gamma}RIIa and Fc{gamma}RIIIb have extensively been studied in that context, that of Fc{gamma}RI remains elusive. Recently, aggregated IgGs have been shown to induce rapid Fc{gamma}RI upregulation and reactive oxygen species (ROS) generation, but the biological relevance of this process is still unclear. MethodsBlood samples were obtained from healthy volunteers and patients with lupus. Aggregated IgGs were prepared as a model of immune complex. Fc{gamma}Rs surface expression on circulating leukocytes and on freshly isolated neutrophils was measured by flow cytometry. ROS production was monitored with luminol-based chemiluminescence. ResultsIncubation of blood samples from healthy volunteers with aggregated IgGs rapidly upregulated the surface expression of Fc{gamma}RI, predominantly on neutrophils. Stimulation of isolated neutrophils from healthy donors with aggregated IgGs resulted in the production of ROS in an Fc{gamma}RI-dependent fashion. Cytochalasin B potentiated Fc{gamma}RI expression and ROS production. Positive correlations between neutrophil Fc{gamma}RI and ROS production were observed in resting blood from both healthy volunteers and lupus patients. In the lupus cohort, monocyte Fc{gamma}RI also correlated with ROS production. ConclusionThis study unveils a previously underappreciated role for neutrophil Fc{gamma}RI in ROS production in both healthy individuals and patients with lupus, and identifies Fc{gamma}RI as a potential biomarker of oxidative response.

Neutrophils are rapidly mobilized from the circulation to sites of inflammation. The mechanisms of neutrophil trafficking in the lung are distinct from those in the periphery, in part because the pulmonary capillaries are the primary site of neutrophil emigration rather than postcapillary venules. Since the diameter of a neutrophil is greater than the width of most pulmonary capillary segments, they must deform to transit through this capillary network, even at homeostasis. Resistance to deformation is primarily due to cortical actin that is rapidly assembled when a neutrophil is exposed to a priming or activation stimulus, resulting in neutrophil stiffening and subsequent sequestration within the pulmonary capillary network. In the current study, we use a microfluidic assay to characterize neutrophil transit through model capillary-like channels. Using techniques from single-particle tracking, we analyzed the cumulative distribution of neutrophil transit times and resolve population-based effects. We found that vinculin, an actin-binding adaptor protein, plays an essential role in neutrophil stiffening in response to formyl-Met-Leu-Phe (fMLP). Vinculin-deficient neutrophils lack the development of a population with slow transit through narrow channels that was observed in both wild-type murine bone marrow neutrophils and HoxB8-conditional progenitor-derived neutrophils. Atomic force microscopy studies provide further evidence that vinculin is required for neutrophil stiffening. Consistent with these findings, we observed that neutrophil sequestration in the lungs of mice is attenuated in the absence of vinculin. Together, our studies indicate that vinculin mediates actin-dependent neutrophil stiffening that leads to their sequestration in capillaries.

Tumor-associated neutrophils are found in many types of cancer and are often reported to contribute to negative outcomes. The presence of transforming growth factor-beta (TGF-{beta}) in the tumor microenvironment reportedly contributes to the skewing of neutrophils to a more pro-tumor phenotype. The effects of TGF-{beta} on neutrophil signaling and migration are, however, unclear. We sought to characterize TGF-{beta} signaling in both primary human neutrophils and the neutrophil-like cell line HL-60 and determine whether it directly induces neutrophil migration. We found that TGF-{beta}1 does not induce neutrophil chemotaxis in transwell or underagarose migration assays. TGF-{beta}1 does activate canonical signaling through SMAD3 and noncanonical signaling through ERK1/2 in neutrophils in a time-and dose-dependent manner. Additionally, TGF-{beta}1 present in the tumor-conditioned media (TCM) of invasive breast cancer cells results in SMAD3 activation. We discovered that TCM induces neutrophils to secrete leukotriene B4 (LTB4), which is a lipid mediator important for amplifying the range of neutrophil recruitment. However, TGF-{beta}1 alone does not induce secretion of LTB4. RNA-sequencing revealed that TGF-{beta}1 and TCM alter gene expression in HL-60 cells, including the mRNA levels of the pro-tumor oncostatin M (OSM) and vascular endothelial growth factor A (VEGFA). These new insights into the role and impact of TGF-{beta}1 on neutrophil signaling, migration, and gene expression have significant implications in the understanding of the changes in neutrophils that occur in the tumor microenvironment.

The NPM1 gene is frequently a target of genetic alteration in hematological tumors, particularly of the myeloid lineage. Complete inactivation of Npm1 in the mouse disrupts primitive hematopoiesis and results in embryonic lethality. Npm1 heterozygosity produces features similar to those of MDS that progress to overt leukemia, and specific point mutations of Npm1 lead to bone marrow failure due to loss of hematopoietic stem cells. However, little is known about NPM1s role in mature, differentiated cells. Here we generated a conditional mouse mutant to inactivate Npm1 across the myelomonocytic lineage, and investigated its ability to influence macrophage maturation and function. We found that Npm1 is not required to maintain macrophage viability, while its loss in mature macrophages reduces production of reactive oxygen species, chemotactic properties and phagocytic capacity. Taking advantage of our recently established Npm1D180del mouse model of ribosome dysfunction and hematological disease, we identify cellular translation and rRNA 2-O-methlyation as a crucial element in controlling macrophage function. These analyses demonstrate a role for Npm1 in adult immune cells, and reveal the importance of translation regulation in macrophage function. Statement of significanceMacrophages are a major component of the immune response to various insults including to cancer. Here we show that NPM1, the most frequently mutated gene in acute myeloid leukemia, displays a critical role in macrophage function, and we identify ribosome deregulation as one of the underlying mechanisms.

Efficient clearance of dead cells and pathogens is essential for survival and requires both the innate and adaptive immune systems. Polymorphonuclear leukocytes (PMNs, predominantly consisting of neutrophil granulocytes) constitute an important first line of defense. These highly specialized cells can phagocytose pathogens and clear apoptotic cells through efferocytosis. Beta-2-microglobulin ({beta}2m) serves as the light chain of major histocompatibility complex class I (MHC I) molecules, associating non-covalently with the heavy transmembrane chain that binds and presents antigenic peptides to CD8+ T cells. This represents the canonical role of {beta}2m. {beta}2m is also found in the granules of PMNs and is released into the extracellular space during degranulation. However, a specific function for {beta}2m in the context of PMN function or degranulation has not yet been identified. We now present evidence that {beta}2m is of importance for both phagocytosis of pathogens and efferocytosis of dead cells by PMNs. The addition of exogenous {beta}2m (50 mg/l) to PMNs in the presence of latex beads increased the phagocytic activity from 23% to 31%. Furthermore, both {beta}2m and desLys58-{beta}2m (dK58{beta}2m) enhanced phagocytosis of Gram-negative and -positive bacteria by more than 3.6-fold, though no effect was observed with zymosan bioparticles. Maintaining tissue homeostasis requires the continuous generation of new cells and the efficient clearance of apoptotic cells through efferocytosis. Treatment with {beta}2m or dK58{beta}2m led to a dose-dependent increase in efferocytosis of apoptotic Jurkat cells, reaching up to a two-fold enhancement. This effect was comparable to that obtained by GM-CSF, used as a positive control. In all cases, cytochalasin D blocked {beta}2m-mediated uptake in PMNs. These data demonstrate that {beta}2m can be of importance in phagocytosis of bacterial pathogens as part of the innate immune response and tissue homeostasis by removing dead cells by efferocytosis.

Sepsis is the leading cause of death in adult intensive care units. At present, sepsis diagnosis relies on non-specific clinical features. It could transform clinical care to have objective immune cell biomarkers that could predict sepsis diagnosis and guide treatment. For decades, neutrophil phenotypes have been studied in sepsis, but a diagnostic cell subset has yet to be identified. Here, high dimensional mass cytometry was used to reveal for the first time a specific neutrophil signature of sepsis severity that does not overlap with other inflammatory biomarkers, and that distinguishes patients with sepsis from those with non-infectious inflammatory syndrome. Unsupervised analysis of 42-dimesional mass cytometry data characterized previously unappreciated heterogeneity within the CD64+ immature neutrophils and revealed two new subsets distinguished by CD123 and PD-L1 expression. These immature neutrophils exhibited diminished activation and phagocytosis functions. The proportion of CD123-expressing neutrophils also correlated with clinical severity. Critically, this study showed that these two new neutrophil subsets were specific to sepsis and detectable by routine flow cytometry using seven markers. The demonstration here that a simple blood test distinguishes sepsis from other inflammatory conditions represents a key biological milestone that can be immediately translated into improvements in patient care. One Sentence SummaryCD123+ and/or PD-L1+ immature and dysfunctional neutrophil subsets identified by mass cytometry, define an early human blood signature of sepsis

Sepsis is one of the most common causes of death in intensive care units. The overproduction of proinflammatory mediators during severe sepsis leads to desensitization of CXCR2 on neutrophil, compromising their migration capacity. During early sepsis, kinins are released and bind to bradykinin 1 (BDKRB1) and bradykinin 2 (BDKRB2) receptors, however the involvement of these receptors in sepsis is not yet fully understood. This study demonstrated that the absence of BDKRB2 had no major effects compared to WT mice upon sepsis induction by CLP, suggesting that this receptor plays a minor role under these experimental conditions. In contrast, B1-/- mice showed lower mortality and bacterial recovery compared to WT-CLP mice, which was associated with an increased influx of neutrophils into the peritoneal cavity of CLP-B1-/- mice. WT-CLP mice exhibited increased expression of P110{gamma} and decreased expression of CXCR2 in neutrophils, which was partially reversed in CLP-B1-/- mice. Interestingly, local CXCL1 production was not affected by the absence of BDKRB1. In human neutrophils, LPS induced expression of BDKRB1, and antagonism of this receptor was associated with the restoration of neutrophil recruitment capacity upon stimulation with CXCL8. Furthermore, treatment with a BDKRB1 antagonist in combination with imipenem resulted in a significant improvement in mortality compared to animals treated with the antimicrobial agent alone. Our findings demonstrate that BDKRB1 plays an essential role in exacerbating the inflammatory response and CXCR2 desensitization in neutrophils during CLP-induced severe sepsis, highlighting BDKRB1 as a potential target for sepsis treatment. ImportanceSepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Despite advances in understanding its pathophysiology, sepsis remains a leading cause of mortality in intensive care units nowadays. Here we found that B1 receptor contributes to neutrophil migration failure during severe sepsis. Inhibition of B1 improves neutrophil migration and bacterial clearance, making it a valuable therapeutic candidate for the treatment of sepsis. More importantly, treatment with a BDKRB1 antagonist in combination with imipenem resulted in a significant improvement in mortality compared to animals treated with the antimicrobial agent alone. These results highlight B1 as a potential treatment target for sepsis, offering improved modulation of the inflammatory response and synergy with antibiotics. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/590213v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1f0401aorg.highwire.dtl.DTLVardef@ab8eorg.highwire.dtl.DTLVardef@1ff0c26org.highwire.dtl.DTLVardef@176b5aa_HPS_FORMAT_FIGEXP M_FIG BDKRB1 activation contributes to sepsis-induced hyperinflammation: (A) BDKRB1 activation contributes to sepsis-induced hyperinflammation: (A) BDKRB1 plays an essential role in the pathogenesis of sepsis, partly by mediating impaired neutrophil migration during the disease. It exerts its effects in myeloid cells by controlling the activation of P13K{gamma} and the expression of CXCR2. (B) BDKRB1 antagonist decreases cytokine production and increases neutrophil influx into the peritoneal cavity, resulting in a reduction in bacterial recovery, highlighting DALBK as a potential adjuvant treatment for sepsis C_FIG

Neutrophilic granulocytes are required for antimicrobial defense, but they can also be harmful to the host organism. The current study demonstrates that disordered peptides in the 3-12 kDa size range in the plasma of septic patients alter effector functions of neutrophils from healthy donors. Those peptides stimulated exocytosis, increased extracellular release of reactive oxygen species (ROS), decreased ROS production in phagosomes, and impaired elimination of ROS-sensitive bacteria. Both the concentration of peptides in patients plasma and their effects on healthy cells were proportional to the clinical status of the patients. Proteomic analysis and in silico modeling indicate that multiple proteases generate the toxic peptides, with the greatest number of peptides cleaved by neutrophil elastase. We propose that neutrophils participate in an amplification loop in which proteolytic peptides stimulate extracellular release of proteases, resulting in production of more peptides. The enhanced extracellular ROS release contributes to tissue damage, while reduced intracellular ROS generation impairs elimination of certain bacteria. Breaking of this vicious cycle may offer a potential target for intervention.

Phagocytosis is a conserved biological mechanism that is integral to tissue remodeling, clearance of apoptotic cells, and immune defense in animals. Additionally, it serves as a pivotal means of sustenance for diverse unicellular eukaryotes. In the context of mammals, this crucial role is fulfilled by specific cell types such as macrophages, monocytes, dendritic cells, and neutrophils. It is orchestrated by an array of receptors, kinases, cytoskeletal elements, and enzymes, working collaboratively to enable the recognition, engulfment, and internalization of particles. Despite its profound significance, the intricate mechanisms underpinning the regulation of this phenomenon remains enigmatic. In this study, we present compelling evidence indicating the involvement of ABCF1, a member of the ATP-binding cassette family, in the Fc{gamma}RIIA phagocytic pathway. ABCF1s contribution lies in facilitating downstream signal activation through interactions with Src family members and SYK, pivotal players in this cascade. Additionally, our findings highlight ABCF1s essentiality in the biosynthesis of various SFKs (Src family kinases) and MAPKs (mitogen-activated protein kinases). These molecules collectively oversee the orchestration of phagocytic cup formation, a pivotal step governing the engulfment process within macrophages. Consequently, the regulation of ABCF1 presents a potential avenue for modulating phagocytosis, allowing for the precise modulation of this fundamental process to either enhance or attenuate according to the specific physiological demands.

The complement derived neutrophil chemoattractant C5a, is a potent activator of the neutrophil superoxide anion generating NADPH oxidase. An allosteric modulator specific for the free fatty acid 2 receptor increases the activating potency but not the efficacy of C5a. The allosteric modulator also decreases the inhibitory effect of the C5a receptor antagonist avacopan, suggesting that the NADPH oxidase is activated by two different signaling pathways downstream of the receptor for C5a. While the allosteric modulator affected the C5a-mediated activation of the NADPH oxidase, the C5a-induced rise in the intracellular concentration of free calcium ions was unaffected. The C5a receptor and the free fatty acid receptor belong to the family of G protein-coupled receptors family. Our results show that the activated C5a receptors generate signals that directly activate the NADPH oxidase and allosterically modulated free fatty acid receptors which secondarily generate signals that elicit NADPH oxidase activity. This is in line with an earlier described receptor transactivation model, by which the fatty acid receptor is activated by receptor downstream signals generated by several different neutrophil receptors to which we now add the receptor for C5a. In addition, the fatty acid receptor was higher ranked than the receptor for C5a, in the neutrophil receptor hierarchy. The dual receptor trans-regulatory effects, by which the receptor for C5a activates the fatty acid receptor and by which this receptor reduces the C5a response, represent new regulatory mechanisms of importance for the NADPH oxidase activity in neutrophils.

Neutrophils are crucial to innate immune responses to microbes. The crosslinking of opsonized pathogens by Fc gamma receptors (Fc{gamma}Rs) on neutrophil surfaces mediates multiple antimicrobial functions, including phagocytosis and the production of reactive oxygen species (ROS). Fc{gamma}RIIIb (CD16b) is the most abundant receptor on human neutrophils. It is a GPI-anchored receptor that lacks an intracellular domain. The exact mechanisms by which Fc{gamma}RIIIb transduces signals remain unclear. A rare Fc{gamma}RIIIb-deficient phenotype has been reported in apparently healthy subjects, which is intriguing given the abundance of this receptor on neutrophil surfaces and its crucial role in neutrophil activation by immune complexes. Here, we identified 2 healthy brothers lacking Fc{gamma}RIIIb on neutrophils and characterized their neutrophil activation through Fc{gamma}R crosslinking by immune complexes. Sequencing of the FCGR3B gene revealed mutations in exon 2 resulting in translation loss. In the absence of stimulation, Fc{gamma}RIIIbnull neutrophils showed unaltered levels of Fc{gamma}RIIa, TLR-2, TLR-4 and TLR-6, but significantly higher Fc{gamma}RIIIa and Fc{gamma}RIa. Upon challenge with E. coli immune complexes, increased surface expression of Fc{gamma}RIa, TLR-4, and M integrin (CD11b) was observed exclusively in Fc{gamma}RIIIbnull neutrophils. Antibacterial functions stimulated by immune complexes were significantly lower in Fc{gamma}RIIIbnull neutrophils, including phagocytic capacity and ROS production compared to Fc{gamma}RIIIb-expressing neutrophils. Overall, the absence of Fc{gamma}RIIIb on human neutrophils correlated with impaired antimicrobial functions following stimulation through Fc{gamma}Rs. This study provides new insights into the functional relevance of Fc{gamma}RIIIb and emphasizes the importance of this receptor in neutrophil responses to bacteria.

DEK locates in the nucleus of the cells or the cytoplasmic granules of neutrophils and plays different roles in cellular processes including NETosis, a suicide mechanism of neutrophils. Here we showed that the interaction of rDEK with CXCR2 leads to NETosis, which could be reduced by the CXCR1/CXCR2 inhibitor reparixin. We found that IL-8, IL-6, IL1-{beta}, MPO, and CitH3 were increased whereas DEK was decreased in the serum of COVID-19 patients. Interestingly, reparixin or anti-DEK antibody reduced the NETosis induced by the serums of patients, suggesting that initial cytokine stimulation may further induce the release of DEK. Our results support the use of reparixin as a potential therapeutic strategy in COVID-19 and suggest that DEK-CXCR2 interaction plays a role in NETosis.