Journal of Leukocyte Biology

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

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

Neutrophils were once considered a homogenous population of transcriptionally static, pathogen-killing cells, however, recent models have demonstrated neutrophil functional and transcriptional plasticity. We performed transcriptomic analyses in a murine model of pneumococcal pneumonia to investigate neutrophil plasticity and demonstrate that neutrophils are highly dynamic, leading to three distinct alveolar neutrophil populations - one immature (early bronchoalveolar lavage neutrophils [BALN]) and two mature (late BALN). Early BALNs produce high levels of inflammatory cytokine transcripts, maturing into late BALNs, including a pro-degranulation and phagocytosis population (late-degranulating BALN) or a population specializing in translation machinery and inflammatory cytokine production (late-cytokine producing BALN). Neutrophil metabolism is also regulated in a stepwise manner - tricarboxylic acid (TCA) cycle and respiratory electron transport chain (ETC) genes are downregulated as neutrophils migrate from the vasculature to the interstitium, lipid and carbohydrate metabolism genes are downregulated during migration from interstitium to the airspace. These transitions may be regulated by aspects of the integrated stress response (ISR), as key regulators including Eif2ak2 are upregulated in interstitial neutrophils. Overall, we demonstrate that pneumonic neutrophils are transcriptionally plastic, developing through two distinct transcriptional phenotypes in the airspace, and are metabolically and transcriptionally rewired with potential points of regulation occurring in the interstitial space.

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.

The extravasation of polymorphonuclear neutrophils (PMNs) is a critical component of the innate immune response that involves transendothelial migration (TEM) and interstitial migration. TEM-mediated interactions between PMNs and vascular endothelial cells (VECs) trigger a cascade of biochemical and mechanobiological signals whose effects on interstitial migration are currently unclear. To address this question, we cultured human VECs on a fibronectin-treated transwell insert to model the endothelium and basement membrane, loaded PMN-like differentiated HL60 (dHL-60) cells in the upper chamber of the insert, and collected the PMNs that crossed the membrane-supported monolayer from the lower chamber. The 3D chemotactic migration of the TEM-conditioned PMNs through collagen matrices was then quantified. Data collected from over 50,000 trajectories showed two distinct migratory phenotypes, i.e., a high-persistence phenotype and a low-persistence phenotype. These phenotypes were conserved across treatment conditions, and their existence was confirmed in human primary PMNs. The high-persistence phenotype was characterized by more straight trajectories and faster migration speeds, whereas the low-persistence one exhibited more frequent sharp turns and loitering periods. A key finding of our study is that TEM induced a phenotypic shift in PMNs from high-persistence migration to low-persistence migration. Changes in the relative proportion of high-persistence and low-persistence populations correlated with GRK2 expression levels. Inhibiting GRK2 hindered the TEM-induced shift in migratory phenotype and impaired the phagocytic function of PMNs. Overall, our study suggests that TEM-mediated GRK2 signaling primes PMNs for a migration phenotype better suited for spatial exploration and inflammation resolution. These observations provide novel insight into the biophysical impacts of TEM that priming PMNs is essential to conduct sentinel functions.

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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection resulting in the clinical syndrome COVID-19 is associated with an exaggerated immune response and monocyte infiltrates in the lungs and other peripheral tissues. It is now increasingly recognised that chronic morbidity persists in some patients. We recently demonstrated profound alterations of monocytes in hospitalised COVID-19 patients. It is currently unclear whether these abnormalities resolve or progress following patient discharge. We show here that blood monocytes in convalescent patients at their 12 week follow up, have a greater propensity to produce pro-inflammatory cytokines TNF and IL-6, which was consistently higher in patients with resolution of lung injury as indicated by a normal chest X-ray and no shortness of breath (a key symptom of lung injury). Furthermore, monocytes from convalescent patients also displayed enhanced levels of molecules involved in leucocyte migration, including chemokine receptor CXCR6, adhesion molecule CD31/PECAM and integrins VLA-4 and LFA-1. Expression of migration molecules on monocytes was also consistently higher in convalescent patients with a normal chest X-ray. These data suggest persistent changes in innate immune function following recovery from COVID-19 and indicate that immune modulating therapies targeting monocytes and leucocyte migration may be useful in recovering COVID-19 patients with persistent symptoms.

To date, murine sepsis models have failed to recapitulate human acute respiratory distress syndrome, one of the leading complications of human sepsis. We set out to determine if preexisting T cell memory, which is common in human adults and lacking in laboratory mice, could contribute to lung inflammation in the cecal ligation and puncture (CLP) model of sepsis. After administering an anti-CD3{varepsilon} activating antibody to C57Bl/6 mice to induce a T cell memory repertoire, we compared the pulmonary immune response to CLP in these "Immune-Educated mice" to responses observed in Uneducated control animals. Compared to Uneducated mice, 24 hours after CLP, Immune-Educated mice had higher alveolar inflammatory cytokine and chemokine concentrations and more pulmonary interstitial macrophages. After 48 hours, the proportion of effector CD4 T cells that produced interferon-gamma was greater in Immune- Educated mice. After 72 hours, there were more alveolar macrophages in the lungs of Educated mice. Separately, we performed adoptive transfer of memory CD4 and CD8 T cells from immunized C57Bl/6J to B6.SJL mice and IFN{gamma} blockade at the time of CLP. Interstitial macrophage recruitment 24 hours post-CLP was more pronounced in mice undergoing adoptive transfer of memory T cells compared to mice that did not undergo adoptive transfer. IFN{gamma} blockade resulted in higher absolute numbers of T cells, memory T cells, and innate cells in the lungs of Educated mice 24 hours post-CLP suggesting that IFN{gamma} is necessary for curbing an overactive immune response in these mice. In conclusion, the presence of memory T cells affects the course of CLP- induced lung inflammation and may provide a model that more closely resembles sepsis- associated lung injury. Summary StatementPrior immune memory alters the course of CLP-induced lung inflammation in mice.

Ten-eleven translocation protein 2 (TET2) is commonly mutated in hematologic disorders of bone marrow failure such as myelodysplastic syndrome (MDS), and loss of TET2 is associated with poor prognosis. TET2 loss is also associated with augmented inflammation, as well as impaired innate immune responses. However, many individuals harboring mutations do not develop hematologic disorders, indicating that additional factors, such as inflammation, cooperate with TET2 loss to promote disease progression. Innate immune memory is a phenomenon in which pre-treatment with microbial ligands, including monophosphoryl lipid A (MPLA), improves innate immune function while minimizing inflammation during subsequent infection. Given the previously reported innate immune impairments attributed to TET2 loss, we tested whether MPLA could induce an innate immune response in TET2-deficient mice infected with P. aeruginosa. Moreover, due to the inflammatory role of TET2 loss, we investigated whether MPLA with infection would promote disease-related phenotypes. We found that TET2-deficient mice display impaired infection response to P. aeruginosa which is partially improved with MPLA pretreatment. Assessments of pathogenic clearance functions further showed decreased capacity in TET2-deficient innate immune cells. Moreover, TET2-defcient cells also exhibit impaired differentiation. MPLA pretreatment in infected mice promotes myeloid-biased hematopoiesis at the expense of erythroid- and megakaryocyte-biased hematopoiesis, resembling MDS-like disease progression. Intriguingly, inhibition of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) dampens the effects of TET2 loss on hematopoietic perturbation. Collectively, we find that TET2 loss impairs innate immune memory and infection responses, and MPLA with infection promotes hematopoietic skewing through RIPK1. KEY POINTS- TET2 loss increases susceptibility to infection, but immune response can be improved by MPLA-induced innate immune memory. - MPLA pretreatment and infection promotes aberrant hematopoiesis and myeloid bias in TET2 deficiency that is improved by RIPK1 inhibition.

BackgroundNeutrophils play an important role in the immune system and sense environmental perturbations including pathogens. Upon pathogen detection, neutrophils extrude their chromatin, forming neutrophil extracellular traps (NETs) trapping and removing pathogens. Previous studies have shown that controlled chromatin decondensation occurs during NET formation, reflecting NET inducing pathways, and the cellular environment. While NET inducing stimuli like phorbol 12-myristate 13-acetate (PMA) is commonly used to study NET formation, it bypassing regulatory mechanisms, limiting insights. MethodsWe used the Assay for Transposase-Accessible Chromatin with sequencing (ATAC-Seq) to profile chromatin accessibility in neutrophils stimulated in whole blood with PMA and physiologically relevant inflammatory factors (NFs), including TNF-, GM-CSF, fMLP, C5a, and IL-1{beta}, alone and in combination. Chromatin responses were compared across conditions and integrated with publicly available transcriptomic sepsis cohorts. ResultsWe found that NF stimulation induced stimulus specific chromatin accessibility programs distinct from PMA. Individual NFs increased specific transcription factor (TF) motif enrichments in a stimulus dependent manner, with GM-CSF increasing STATs, TNF- increasing NF-{kappa}B, C5a/fMLP increasing AP-1, and Combined with a cooperative response including CEBP. Integration with sepsis transcriptomic datasets revealed that promoter accessibility changes within NF stimulations correspond to transcriptional states associated with sepsis disease severity, highlighting the upstream regulatory programs linked to clinical outcomes. ConclusionsThese findings demonstrate that NF stimulation in whole blood reveals chromatin accessibility programs in neutrophils that correlate with disease severity in sepsis. This approach provides a framework for linking cytokine driven neutrophil regulation to heterogenous inflammatory states in sepsis and other NET-associated diseases.

Tracking mature neutrophils remains challenging due to the lack of reliable cell surface markers. Although CD101 is a promising candidate for mature neutrophils, its stability under pathological conditions is unclear. Using a CD101-tdTomato reporter mouse model, we confirmed that the reporting system does not alter CD101 expression, and tdTomato fluorescence is predominantly expressed in mature neutrophils across peripheral tissues. Further analysis revealed that CD101+ and tdTomato+ neutrophils display identical characteristics of mature neutrophil, including poly-segmented nuclei, cell size, and key functions under homeostasis. By comparing tdTomato fluorescence with CD101 protein levels, we demonstrate that reduced CD101 expression under pathological states was not attributed to shedding or degradation. Our finding enhances CD101 as a robust and reliable marker of neutrophil maturity, providing a foundation for future applications in spatial transcriptomics and lineage tracing studies to dissect neutrophil heterogeneity and function. Highlights of the studyO_LIIn CD101-tdTomato homozygous mice, tdTomato is predominantly expressed in neutrophils and labels nearly 100% of mature neutrophils, aligning with the phenotype of CD101+ mature neutrophils; C_LIO_LIThe CD101-tdTomato reporting system does not interrupt CD101 expression or neutrophil functions; C_LIO_LICD101 remains a stable and reliable cell surface marker for labeling mature neutrophils, even under pathological conditions. C_LI

Homozygous null mutation of the macrophage colony-stimulating factor receptor (Csf1r) gene in rats leads to the loss of most tissue macrophage populations and has pleiotropic impacts on postnatal growth and organ maturation leading to mortality by 8-12 weeks of age. The phenotype of the Csf1r knockout (Csf1rko) can be reversed by intraperitoneal transfer of wild-type bone marrow cells (BMT) at weaning. Here we used a Csf1r-mApple transgenic reporter, which is expressed in neutrophils and B cells as well as monocytes and macrophages, to track the fate of donor-derived cells. Following BMT into Csf1r recipients, wild-type mApple+ve cells restored IBA1+ tissue macrophage populations in every tissue donor-derived cells also completely replaced recipient macrophages in organs such as spleen, lung and liver that were only partly macrophage-deficient in the Csf1rko. However, monocytes, neutrophils and B cells in bone marrow, blood and lymphoid tissues remained of recipient (mApple-ve) origin. An mApple+ve cell population expanded in the peritoneal cavity and invaded locally in the mesentery, fat pads, omentum and diaphragm. One week after BMT, distal organs contained foci of mApple+ve, IBA1-ve immature progenitors that appeared to proliferate, migrate and differentiate locally. We conclude that rat bone marrow contains progenitor cells that are able to restore and maintain all tissue macrophage populations in a Csf1rko rat directly without contributing to the bone marrow progenitor or blood monocyte populations.

Hematologic side effects are associated with prolonged antibiotic exposure in up to 34% of patients. Neutropenia, reported in 10-15% of patients, increases the risk of sepsis and death. Murine studies have established a link between the intestinal microbiota and normal hematopoiesis. We sought to identify predisposing factors, presence of microbiota-derived metabolites, and changes in intestinal microbiota composition in otherwise healthy pediatric patients who developed neutropenia after prolonged courses of antibiotics. In this multi-center study, patients with infections requiring anticipated antibiotic treatment of two or more weeks were enrolled. Stool samples were obtained at the start and completion of antibiotics and at the time of neutropenia. We identified 10 patients who developed neutropenia on antibiotics and 29 controls matched for age, sex, race, and ethnicity. Clinical data demonstrated no association between neutropenia and type of infection or type of antibiotic used; however intensive care unit admission and length of therapy were associated with neutropenia. Reduced intestinal microbiome richness and decreased abundance of Lachnospiraceae family members correlated with neutropenia. Untargeted stool metabolomic profiling revealed several metabolites that were depleted exclusively in patients with neutropenia, including members of the urea cycle pathway, pyrimidine metabolism and fatty acid metabolism that are known to be produced by Lachnospiraceae. Our study confirms a relationship between intestinal microbiota disruption and abnormal hematopoiesis and identifies taxa and metabolites likely to contribute to microbiota-sustained hematopoiesis. As the microbiome is a key determinant of stem cell transplant and immunotherapy outcomes, these findings are likely to be of broad significance. Key PointsO_LINeutropenia occurred in 17% of patients receiving prolonged antibiotic therapy. C_LIO_LIWe found no association between neutropenia and type of infection or class of antibiotic used. C_LIO_LIDevelopment of neutropenia after prolonged antibiotic treatment was associated with decreased prevalence of Lachnospiraceae and Lachnospiraceae metabolites such as citrulline. C_LI

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.

Neutrophils are the most abundant leukocyte in humans and the principal effectors of the innate immue response. Genetic modification of human neutrophils is challenging due to their short lifespan and tendency to activate in response to even minor perturbation. However, genetic manipulation of haematopoietic progenitor cells and subsequent directed differentation into neutrophils represents a potential avenue to study the contributions of individual genes and pathways to human neutrophil function. Here we present a method of directed granulocytic CD34+ progenitor differentiation into neutrophils capable of key functions such as priming and neutrophil extracellular trap (NET) formation. We further show that differentiating progenitors can be efficiently and stably modified by lentiviral gene delivery and Cas9-gRNP nucleofection to produce potent and activation-free gene knockdown in mature neutrophils, thereby providing new tools for understanding the contribution of neutrophils to health and disease. Using this model we have shown that, contrary to previous reports, CD11b is not required for phagocytosis of serum-opsonised bacterial particles.

Macrophages (M{Phi}s) display remarkable plasticity and the ability to activate diverse responses to a host of intracellular and external stimuli. Despite extensive characterization of M1 M{Phi}s and a broad set of M2 M{Phi}s, comprehensive characterization of metabolic shifts driving M{Phi} activation remains. Herein, we utilized an ex vivo model to produce six M{Phi} functional phenotypes. Isolated CD14+ PBMCs were differentiated into resting M0 M{Phi}s, and then polarized into M1 (IFN-{gamma}/LPS), M2a (IL-4/IL-13), M2b (IC/LPS), M2c (IL-10), and M2d (IL-6/LIF) M{Phi}s. The M{Phi}s were profiled using a bioanalyte matrix of four cell surface markers, [~]50 secreted proteins, [~]800 expressed myeloid genes, and [~]450 identified metabolites relative to M0 M{Phi}s. Signal protein and expressed gene profiles grouped the M{Phi}s into inflammatory (M1 and M2b) and wound resolution (M2a, M2c, and M2d) phenotypes; however, each had a unique metabolic profile. While both M1 and M2b M{Phi}s shared metabotype profiles consistent with an inflammatory signature; key differences were observed in the TCA cycle, FAO and OXPHOS. Additionally, M2a, M2c and M2d M{Phi}s all profiled as tissue repair M{Phi}s; however, metabotype differences were observed in multiple pathways including hexosamine, polyamine, and fatty acid metabolism. These metabolic and other key functional distinctions suggest phagocytic and proliferative functions for M2a M{Phi}s, and angiogenesis and ECM assembly capabilities for M2b, M2c and M2d M{Phi}s. By integrating metabolomics into a systems analysis of M{Phi} phenotypes, we provide the most comprehensive map of M{Phi} diversity to date, along with the global metabolic shifts driving M{Phi} functional plasticity in these phenotypes. Summary SentenceMacrophage functional plasticity of six macrophage phenotypes correlates with unique distinctions in cell-surface marker expression, signal protein secretion, transcriptomics profiles, and metabolic processes.

Activating immune receptors provide mechanisms for phagocytes to elicit important effector functions that promote the killing of microbes. Leukocyte immunoglobulin-like receptor A5 (LILRA5) an orphan immune receptor expressed by human phagocytes and co-localising with FcR{gamma}, remains poorly characterised. To address this, we developed a highly-specific anti-LILRA5 monoclonal antibody that has agonistic properties. Using a specific anti-LILRA5 antibody, we show LILRA5 expression on naive monocytes and neutrophils, and that ligation of LILRA5 stimulates ROS production. While increased LILRA5 transcript copy numbers have been associated with sepsis, we also observed increased levels in patients with systemic infection but without sepsis complications. Ex vivo bacterial infection of whole blood did not alter surface LILRA5 expression, but LPS stimulation changed expression levels. This indicates that surface LILRA5 expression is dynamic and likely regulated posttranscriptionally, changing responses to different stimuli or over time. Soluble (s)LILRA5 was enhanced in sera from sepsis patients and in supernatants of monocytes that were LPS-stimulated, indicative that shedding of LILRA5 from cell surfaces or that expression of sLILRA5 isoforms provides a mechanism to regulate surface LILRA5 expression levels. Finally, we show that altered surface LILRA5 expression influences LILRA5-induced ROS production capacity. We propose that LILRA5 is a dynamically regulated activating receptor expressed on phagocytes that can stimulate ROS production.

The chemokine CXCL4 activates myeloid cells and contributes to the pathogenesis of inflammatory and fibrotic diseases. One mechanism of CXCL4 action is binding of nucleic acids to promote their internalization and activation of endosomal TLRs. However, the signaling pathways and receptors that mediate myeloid cell responses to CXCL4 alone are not well characterized. Here, we report that in primary human monocytes, CXCL4 activated NF-{kappa}B and a TBK1-JNK signaling axis that drive the expression of inflammatory, fibrotic and neutrophil chemokine genes, and also RIPK3-dependent necroptosis. Surprisingly, six distinct lines of evidence targeting TLR4 expression and function suggested a role for TLR4 in CXCL4 responses. However, we were not able to completely dissect the contributions of CXCL4 alone to the observed results versus a contribution from endotoxin contamination. Our findings suggest that the interactions between CXCL4 and TLR4 merit further study.

BackgroundPlatelets play key roles in host immune response during sepsis. Microbial persistence and early dysregulated cytokine response predict poor outcomes in patients with Staphylococcus aureus bacteremia (SAB). Our objective was to determine the relationship between early platelet trends and cytokine response, microbial persistence and 30-day mortality in patients with SAB. MethodsA two-center observational study was conducted in hospitalized patients with monomicrobial SAB. Electronic medical records were reviewed for pertinent demographic, laboratory, and clinical information. Eligible subjects were grouped by platelet count at onset and Day 4 of SAB: normal platelet (NP, [&ge;] 150 x 109/L) and thrombocytopenia (TC, < 150 x 109/L). The groups were compared for clinical characteristics and outcomes. Results812 patients met inclusion criteria. The median age was 59 years with MRSA accounting for 34% of SAB. The most common comorbidities were hypertension followed by diabetes then renal disease. Thrombocytopenia (TC) occurred in 29% of patients at SAB onset: 15% (n = 120) were mild and 14% (n = 114) were moderate-to-severe (MS). Compared to patients with normal platelet (NP) at SAB onset (n = 578), higher proportion of patients with TC had alcohol use disorder (p = 0.015), active malignancy (p = 0.002), liver disease (p < 0.001), in addition to requiring intensive care unit (ICU) level of care during hospital stay (p < 0.001). TC patients had a longer duration of bacteremia (3 days vs 2 days; p = 0.008) and higher risk for 30-day mortality (18% vs 6%; p < 0.001) overall compared to those with NP. Changes in platelet count from SAB onset to Day 4 differed significantly between those with persistent (PB) versus resolving bacteremia (RB). Notably, patients who had NP at SAB onset but developed new onset of TC by Day 4 had a higher risk for 30-day mortality compared to those who maintained NP at Day 4 (20% vs 4%; p < 0.001). Those with recovery of their platelet count from TC to NP by Day 4 (n = 20) had their SAB shortened by one day (2 days vs 3 days; p = 0.413) and trended toward lower risk for 30-day mortality (5% vs 22%; p = 0.068) compared to those with sustained TC by Day 4 (n = 88). ConclusionOur results suggest that platelet dynamics during early course of SAB are associated with dysregulated cytokine response and predictive of 30-day mortality. Future studies should be conducted to assess the impact of utilizing platelet count within the first four days of S. aureus bacteremia to guide clinical interventions and to further investigate S. aureus virulence factors that impair recovery of platelet count in patients with thrombocytopenia. FundingNone Research in contextO_ST_ABSEvidence before this studyC_ST_ABSPersistent S. aureus bacteremia is associated with high mortality and morbidity despite receipt of anti-staphylococcal agents with apparent in vitro activity. We have previously reported that early dysregulated host cytokine response biased towards an anti-inflammatory state (high IL-10/TNF ratio) during clinical course of S. aureus bacteremia is predictive of persistence and mortality. Additionally, we showed that mortality risk increases by 16% for each continued day with S. aureus bacteremia. In recent experimental models, others have shown that platelets play key roles in host immunity and bacterial clearance. Specifically, S. aureus virulence factor, -toxin, induced aberrant platelet aggregation resulting in thrombocytopenia and compromised bacterial clearance from the blood. We performed literature review using PubMed to identify any clinical studies published between January 1, 1996 and April 30, 2021 with the term "thrombocytopenia" AND ("Staphylococcus aureus" OR "S. aureus bacteremia" OR "persistent bacteremia" OR "duration of S. aureus bacteremia" OR "dysregulated immune response"). We identified three related clinical studies published previously: 1) a retrospective study with a main objective of identifying risk factors associated with thrombocytopenia at sepsis onset during S. aureus bacteremia, 2) another retrospective study aiming to evaluate the prognostic impact of thrombocytopenia at onset, Day 3, and Day 7 of methicillin-susceptible S. aureus bacteremia for predicting mortality, and 3) one prospective study that evaluated the association between ICU admission platelet count and host immune response but did not include infections with S. aureus. Added value of this studyTo extend the findings from the few earlier clinical studies by performing a more in-depth analysis of the relationship between platelet count, cytokine response, and outcome of bacteremia caused by both methicillin-sensitive and methicillin-resistant S. aureus in a large patient cohort. We examined early platelet dynamics by accounting for changes in daily platelet count over the course of initial 7 days of bacteremia which revealed that platelet count during the first three days of S. aureus bacteremia was significantly associated with a dysregulated cytokine response and predictive of 30-day mortality. Notably, for every 20 x 109/L drop in platelet count by day 4, the risk of death increased by 25%. Importantly, we observed a significant reduction in the risk of mortality in those with platelet count recovery to normal range by day 4 of bacteremia comparable to those whose platelet count remained normal throughout the course of infection, suggesting a critical window for early therapeutic interventions to mitigate platelet consumption and injury. Implications of all available evidenceOur observations provide strong clinical relevance to the experimental findings implicating the role of S. aureus -toxin in causing platelet injury as well as destruction and sequestration in the microvasculature leading to death in mouse models of bacteremia. The strong link between thrombocytopenia and mortality in S. aureus bacteremia support the need for close monitoring of platelets and a more timely and precise approach to S. aureus bacteremia management that is based on the host-pathogen interface. Considering the distinct patterns of platelet dynamics observed early during the course of S. aureus bacteremia associated with differential mortality risk and the significant improvement in survival among those with platelet recovery by day 4 of bacteremia, follow-up studies should focus on evaluating clinical microbiology procedures that could provide the -toxin phenotype of the infecting strains with prognostic significance to clinicians and investigating therapeutic agents that could mitigate -toxin-mediated insults to platelets to improve treatment outcome.

The annual toll of sepsis is a 33% mortality rate for hospitalized patients with a cost of greater than 60 billion dollars in the U.S. There is a correlation between sepsis mortality rates and time to treatment with broad-spectrum antibiotics. Consequently, antibiotics are prescribed to nearly every patient suspected of bacteremia. However, once determined that broad-spectrum antibiotics are not required, it is unclear how to optimize the de-escalation of the antibiotics. There is an urgent need for methods to distinguish bacteremia from sterile inflammation and to assess antibiotic efficacy. Rho (Rac1 and RhoA) and Ras (Rap1) family GTPases are dynamic nodes of signaling convergence used by immune-activated leukocytes migrating to sites of infection. This study targeted the onset of GTPase activation as a biomarker of infection-induced immune activation in trauma patients. GTP binding assays were performed using a novel GTPase effector trap flow cytometry assay (G-Trap). Here, we demonstrate increased GTP binding to small GTPases (Rac1, Rap1, and RhoA) of resting cells serially exposed to plasma samples from bacteremic trauma patients. Responses to the serial samples showed that GTPase activation was influenced by the concentration of circulating pro- and anti-inflammatory mediators, in tandem with synergy or antagonism from the cytokines and the antibiotic treatment.