Biological Activity of Hybrid Vaterite-Pectin Microparticles Towards Bacteria E. coli and Human Neutrophils.

Interaction of microbiota with hybrid vaterite-pectin microparticles as an attractive multifunctional vehicle for mucosal delivery should not provoke inflammation. Our purpose was to study the reaction of bacteria E. coli strain Mg1655 and isolate SharL from a patient with Crohn disease on the cultivation with hybrid microparticles and vaterite, and the subsequent activation of neutrophils. Vaterite-pectin microparticles enhanced leakage of ATP from bacteria. For E. coli Mg1655, the concentration of DNA decreased, while intracellular ATP increased. For E. coli SharL, the intracellular ATP decreased with simultaneous growth of DNA. Bacteria and microparticles together did not enhance activation of neutrophils in comparison with the particles per se in the medium without serum and in comparison with bacteria in the medium supplemented with serum; microparticles did not reduce functional activity of neutrophils.

Cathepsin C inhibition reduces neutrophil serine protease activity and improves activated neutrophil-mediated disorders.

Cathepsin C (CatC) is an enzyme which regulates the maturation of neutrophil serine proteases (NSPs) essential for neutrophil activation. Activated neutrophils are key players in the innate immune system, and are also implicated in the etiology of various inflammatory diseases. This study aims to demonstrate a therapeutic potential for CatC inhibitors against disorders in which activated neutrophil-derived neutrophil extracellular traps (NETs) play a significant role. We demonstrate that a CatC inhibitor, MOD06051, dose-dependently suppresses the cellular activity of NSPs, including neutrophil elastase (NE), in vitro. Neutrophils derived from MOD06051-administered rats exhibit significantly lower NE activity and NET-forming ability than controls. Furthermore, MOD06051 dose-dependently ameliorates vasculitis and significantly decreases NETs when administered to a rat model of myeloperoxidase (MPO)-antineutrophil cytoplasmic antibody-associated vasculitis (AAV). These findings suggest that CatC inhibition is a promising strategy to reduce neutrophil activation and improve activated neutrophil-mediated diseases such as MPO-AAV.

Multi-omics landscapes reveal heterogeneity in long COVID patients characterized with enhanced neutrophil activity.

BACKGROUND: Omicron variant impacts populations with its rapid contagiousness, and part of patients suffered from persistent symptoms termed as long COVID. The molecular and immune mechanisms of this currently dominant global variant leading to long COVID remain unclear, due to long COVID heterogeneity across populations. METHODS: We recruited 66 participants in total, 22 out of 66 were healthy control without COVID-19 infection history, and 22 complaining about long COVID symptoms 6 months after first infection of Omicron, referred as long COVID (LC) Group. The left ones were defined as non-long COVID (NLC) Group. We profiled them via plasma neutralizing antibody titer, SARS-CoV-2 viral load, transcriptomic and proteomics screening, and machine learning. RESULTS: No serum residual SARS-CoV-2 was observed in the participants 6 months post COVID-19 infection. No significant difference in neutralizing antibody titers was found between the long COVID (LC) Group and the non-long COVID (NLC) Group. Transcriptomic and proteomic profiling allow the stratification of long COVID into neutrophil function upregulated (NU-LC) and downregulated types (ND-LC). The NU-LC, identifiable through a refined set of 5 blood gene markers (ABCA13, CEACAM6, CRISP3, CTSG and BPI), displays evidence of relatively higher neutrophil counts and function of degranulation than the ND-LC at 6 months after infection, while recovered at 12 months post COVID-19. CONCLUSION: The transcriptomic and proteomic profiling revealed heterogeneity among long COVID patients. We discovered a subgroup of long COVID population characterized by neutrophil activation, which might associate with the development of psychiatric symptoms and indicate a higher inflammatory state. Meanwhile, a cluster of 5 genes was manually curated as the most potent discriminators of NU-LC from long COVID population. This study can serve as a foundational exploration of the heterogeneity in the pathogenesis of long COVID and assist in therapeutic targeting and detailed epidemiological investigation of long COVID.

Collagen concentration regulates neutrophil extravasation and migration in response to infection in an endothelium dependent manner.

Introduction: As the body's first line of defense against disease and infection, neutrophils must efficiently navigate to sites of inflammation; however, neutrophil dysregulation contributes to the pathogenesis of numerous diseases that leave people susceptible to infections. Many of these diseases are also associated with changes to the protein composition of the extracellular matrix. While it is known that neutrophils and endothelial cells, which play a key role in neutrophil activation, are sensitive to the mechanical and structural properties of the extracellular matrix, our understanding of how protein composition in the matrix affects the neutrophil response to infection is incomplete. Methods: To investigate the effects of extracellular matrix composition on the neutrophil response to infection, we used an infection-on-a-chip microfluidic device that replicates a portion of a blood vessel endothelium surrounded by a model extracellular matrix. Model blood vessels were fabricated by seeding human umbilical vein endothelial cells on 2, 4, or 6 mg/mL type I collagen hydrogels. Primary human neutrophils were loaded into the endothelial lumens and stimulated by adding the bacterial pathogen Pseudomonas aeruginosa to the surrounding matrix. Results: Collagen concentration did not affect the cell density or barrier function of the endothelial lumens. Upon infectious challenge, we found greater neutrophil extravasation into the 4 mg/mL collagen gels compared to the 6 mg/mL collagen gels. We further found that extravasated neutrophils had the highest migration speed and distance in 2mg/mL gels and that these values decreased with increasing collagen concentration. However, these phenomena were not observed in the absence of an endothelial lumen. Lastly, no differences in the percent of extravasated neutrophils producing reactive oxygen species were observed across the various collagen concentrations. Discussion: Our study suggests that neutrophil extravasation and migration in response to an infectious challenge are regulated by collagen concentration in an endothelial cell-dependent manner. The results demonstrate how the mechanical and structural aspects of the tissue microenvironment affect the neutrophil response to infection. Additionally, these findings underscore the importance of developing and using microphysiological systems for studying the regulatory factors that govern the neutrophil response.

Activation of neutrophils excels the therapeutic potential of Mycobacterium indicus pranii and heat-induced promastigotes against antimony-resistant Leishmania donovani infection.

Repurposing drugs and adjuvants is an attractive choice of present therapy that reduces the substantial costs, chances of failure, and systemic toxicity. Mycobacterium indicus pranii was originally developed as a leprosy vaccine but later has been found effective against Leishmania donovani infection. To extend our earlier study, here we reported the immunotherapeutic modulation of the splenic and circulatory neutrophils in favour of hosts as neutrophils actually serve as the pro-parasitic portable shelter to extend the Leishmania infection specifically during the early entry into the hosts' circulation. We targeted to disrupt this early pro-parasitic incidence by the therapeutic combination of M. indicus pranii and heat-induced promastigotes against antimony-resistant L. donovani infection. The combination therapy induced the functional expansion of CD11b+Ly6CintLy6Ghi neutrophils both in the post-infected spleen, and also in the circulation of post-treated animals followed by the immediate Leishmania infection. More importantly, the enhanced expression of MHC-II, phagocytic uptake of the parasites by the circulatory neutrophils as well as the oxidative burst were induced that limited the chances of the very early establishment of the infection. The enhanced expression of pro-inflammatory cytokines, like IL-1α and TNF-α indicated resistance to the parasite-mediated takeover of the neutrophils, as these cytokines are critical for the activation of T cell-mediated immunity and host-protective responses. Additionally, the induction of essential transcription factors and cytokines for early granulocytic lineage commitment suggests that the strategy not only contributed to the peripheral activation of the neutrophils but also promoted granulopoiesis in the bone marrow.

Maturing neutrophils of lower density associate with thrombocytopenia in Puumala orthohantavirus-caused hemorrhagic fever with renal syndrome.

Puumala orthohantavirus-caused hemorrhagic fever with renal syndrome (PUUV-HFRS) is characterized by strong neutrophil activation. Neutrophils are the most abundant immune cell type in the circulation and are specially equipped to rapidly respond to infections. They are more heterogenous than previously appreciated, with specific neutrophil subsets recently implicated in inflammation and immunosuppression. Furthermore, neutrophils can be divided based on their density to either low-density granulocytes (LDGs) or "normal density" polymorphonuclear cell (PMN) fractions. In the current study we aimed to identify and characterize the different neutrophil subsets in the circulation of PUUV-HFRS patients. PMNs exhibited an activation of antiviral pathways, while circulating LDGs were increased in frequency following acute PUUV-HFRS. Furthermore, cell surface marker expression analysis revealed that PUUV-associated LDGs are primarily immature and most likely reflect an increased neutrophil production from the bone marrow. Interestingly, both the frequency of LDGs and the presence of a "left shift" in blood associated with the extent of thrombocytopenia, one of the hallmarks of severe HFRS, suggesting that maturing neutrophils could play a role in disease pathogenesis. These results imply that elevated circulating LDGs might be a general finding in acute viral infections. However, in contrast to the COVID-19 associated LDGs described previously, the secretome of PUUV LDGs did not show significant immunosuppressive ability, which suggests inherent biological differences in the LDG responses that can be dependent on the causative virus or differing infection kinetics.

A narrative review of chemokine receptors CXCR1 and CXCR2 and their role in acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a severe form of acute respiratory failure characterised by extensive inflammatory injury to the alveolocapillary barrier leading to alveolar oedema, impaired gas exchange and, ultimately, hypoxaemia necessitating the use of supplemental oxygen combined with some degree of positive airway pressure. Although much heterogeneity exists regarding the aetiology, localisation and endotypic characterisation of ARDS, what remains largely undisputed is the role of the innate immune system, and in particular of neutrophils, in precipitating and propagating lung injury. Activated neutrophils, recruited to the lung through chemokine gradients, promote injury by releasing oxidants, proteases and neutrophil extracellular traps, which ultimately cause platelet aggregation, microvascular thrombosis and cellular death. Among various neutrophilic chemoattractants, interleukin-8/C-X-C motif ligand 8 and related chemokines, collectively called ELR+ chemokines, acting on neutrophils through the G protein-coupled receptors CXCR1 and CXCR2, are pivotal in orchestrating the neutrophil activation status and chemotaxis in the inflamed lung. This allows efficient elimination of infectious agents while at the same time minimising collateral damage to host tissue. Therefore, understanding how CXCR1 and CXCR2 receptors are regulated is important if we hope to effectively target them for therapeutic use in ARDS. In the following narrative review, we provide an overview of the role of ELR+ chemokines in acute lung injury (ALI) and ARDS, we summarise the relevant regulatory pathways of their cognisant receptors CXCR1/2 and highlight current preclinical and clinical evidence on the therapeutic role of CXCR1 and CXCR2 inhibition in animal models of ALI, as well as in ARDS patients.

Adenosine Triphosphate Release From Influenza-Infected Lungs Enhances Neutrophil Activation and Promotes Disease Progression.

BACKGROUND: Adenosine triphosphate (ATP) enhances neutrophil responses, but little is known about the role of ATP in influenza infections. METHODS: We used a mouse influenza model to study if ATP release is associated with neutrophil activation and disease progression. RESULTS: Influenza infection increased pulmonary ATP levels 5-fold and plasma ATP levels 3-fold vs healthy mice. Adding ATP at those concentrations to blood from healthy mice primed neutrophils and enhanced CD11b and CD63 expression, CD62L shedding, and reactive oxygen species production in response to formyl peptide receptor stimulation. Influenza infection also primed neutrophils in vivo, resulting in formyl peptide receptor-induced CD11b expression and CD62L shedding up to 3 times higher than that of uninfected mice. In infected mice, large numbers of neutrophils entered the lungs. These cells were significantly more activated than the peripheral neutrophils of infected mice and pulmonary neutrophils of healthy mice. Plasma ATP levels of infected mice and influenza disease progression corresponded with the numbers and activation level of their pulmonary neutrophils. CONCLUSIONS: Findings suggest that ATP release from the lungs of infected mice promotes influenza disease progression by priming peripheral neutrophils, which become strongly activated and cause pulmonary tissue damage after their recruitment to the lungs.

Neutrophil extracellular traps characterize caseating granulomas.

Tuberculosis (TB) remains one of the top 10 causes of death worldwide and still poses a serious challenge to public health. Recent attention to neutrophils has uncovered unexplored areas demanding further investigation. Therefore, the aim of this study was to determine neutrophil activation and circulatory neutrophil extracellular trap (NET) formation in various types of TB. Sera from TB patients (n = 91) and healthy controls (NHD; n = 38) were analyzed for NE-DNA and MPO-DNA complexes, cell-free DNA (cfDNA), and protease activity (elastase). We show that these NET parameters were increased in TB sera. Importantly, NET formation and NE activity were elevated in TB patients with extensive tissue damage when compared to those with minor damage and in patients with relapse, compared to new cases. We discuss the importance of balancing NET formation to prevent tissue damage or even relapse and argue to analyze circulating NET parameters to monitor the risk of disease relapse. To investigate the tissues for NETs and to find the source of the circulating NET degradation products, we collected sections of granulomas in lung and lymph node biopsies. Samples from other diseases with granulomas, including sarcoidosis (SARC) and apical periodontitis (AP), served as controls. Whereas NET formation characterizes the caseating granulomas, both caseating and non-caseating granulomas harbor DNA with unusual conformation. As TB is associated with hypercoagulation and thromboembolism, we further imaged the pulmonary vessels of TB patients and detected vascular occlusions with neutrophil aggregates. This highlights the dual role of neutrophils in the pathology of TB.

Elevated neutrophil extracellular traps in systemic sclerosis-associated vasculopathy and suppression by a synthetic prostacyclin analog.

OBJECTIVES: Neutrophils and neutrophil extracellular traps (NETs) contribute to the vascular complications of multiple diseases, but their role in systemic sclerosis (SSc) is understudied. We sought to test the hypothesis that NETs are implicated in SSc vasculopathy and that treatment with prostacyclin analogs may ameliorate SSc vasculopathy not only through vasodilation but also by inhibiting NET release. METHODS: Blood from 125 patients with SSc (87 diffuse cutaneous SSc and 38 limited cutaneous SSc) was collected at a single academic medical center. Vascular complications such as digital ulcers, pulmonary artery hypertension, and scleroderma renal crisis were recorded. The association between circulating NETs and vascular complications was determined using in vitro and ex vivo assays. The impact of the synthetic prostacyclin analog epoprostenol on NET release was determined. RESULTS: Neutrophil activation and NET release were elevated in patients with SSc-associated vascular complications compared to matched patients without vascular complications. Neutrophil activation and NETs positively correlated with soluble E-selectin and VCAM-1, circulating markers of vascular injury. Treatment of patients with digital ischemia with a synthetic prostacyclin analog boosted neutrophil cyclic AMP, which was associated with the blunting of NET release and reduced NETs in circulation. CONCLUSION: Our study demonstrates an association between NETs and vascular complications in SSc. We also identified the potential for an additional therapeutic benefit of synthetic prostacyclin analogs, namely to reduce neutrophil hyperactivity and NET release in SSc patients.

The dynamic molecular characteristics of neutrophils are associated with disease progression in dengue patients.

Dengue, the most prevalent mosquito-borne disease worldwide, poses a significant health burden. This study integrates clinical data and transcriptomic datasets from different phases of dengue to investigate distinctive and shared cellular and molecular features. Clinical data from 29 dengue patients were collected and analyzed alongside a public transcriptomic data set (GSE28405) to perform differential gene expression analysis, functional enrichment, immune landscape assessment, and development of machine learning model. Neutropenia was observed in 54.79% of dengue patients, particularly during the defervescence phase (65.79%) in clinical cohorts. Bioinformatics analyses corroborated a significant reduction in neutrophil immune infiltration in dengue patients. Receiver operating characteristic curve analysis demonstrated that dynamic changes in neutrophil infiltration levels could predict disease progression, especially during the defervescence phase, with the area under the curve of 0.96. Three neutrophil-associated biomarkers-DHRS12, Transforming growth factor alpha, and ZDHHC19-were identified as promising for diagnosing and predicting dengue progression. In addition, the activation of neutrophil extracellular traps was significantly enhanced and linked to FcγR-mediated signaling pathways and Toll-like receptor signaling pathways. Neutrophil activation and depletion play a critical role in dengue's immune response. The identified biomarkers and their associated pathways offer potential for improved diagnosis and understanding of dengue pathogenesis and progression.

Rapid high-throughput method for investigating physiological regulation of neutrophil extracellular trap formation.

BACKGROUND: Neutrophils, the most abundant white blood cells in humans, play pivotal roles in innate immunity, rapidly migrating to sites of infection and inflammation to phagocytose, neutralize, and eliminate invading pathogens. Neutrophil extracellular trap (NET) formation is increasingly recognized as an essential rapid innate immune response, but when dysregulated, it contributes to pathogenesis of sepsis and immunothrombotic disease. OBJECTIVES: Current NETosis models are limited, routinely employing nonphysiological triggers that can bypass natural NET regulatory pathways. Models utilizing isolated neutrophils and immortalized cell lines do not reflect the complex biology underlying neutrophil activation and NETosis that occurs in whole blood. To our knowledge, we report the first human ex vivo model utilizing naturally occurring molecules to induce NETosis in whole blood. This approach could be used for drug screening and, importantly, inadvertent activators of NETosis. METHODS: Here we describe a novel, high-throughput ex vivo whole blood-induced NETosis model using combinatorial pooling of native NETosis-inducing factors in a more biologically relevant Synthetic-Sepsis model. RESULTS: We found different combinations of factors evoked distinct neutrophil responses in the rate of NET generation and/or magnitude of NETosis. Despite interdonor variability, similar sets of proinflammatory molecules induced consistent responses across donors. We found that at least 3 biological triggers were necessary to induce NETosis in our system including either tumor necrosis factor-α or lymphotoxin-α. CONCLUSION: These findings emphasize the importance of investigating neutrophil physiology in a biologically relevant context to enable a better understanding of disease pathology, risk factors, and therapeutic targets, potentially providing novel strategies for disease intervention and treatment.

HBeAg induces neutrophils activation impairing NK cells function in patients with chronic hepatitis B.

BACKGROUND: The role of neutrophils in hepatitis B virus (HBV) infection has been a subject of debate due to their involvement in antiviral responses and immune regulation. This study aimed to elucidate the neutrophil characteristics in patients with chronic hepatitis B (CHB). METHODS: Through flow cytometry and ribonucleic acid-sequencing analysis, the phenotypes and counts of neutrophils were analyzed in patients with CHB. Moreover, the effects of HBeAg on neutrophils and the corresponding pattern recognition receptors were identified. Simultaneously, the cross-talk between neutrophils and natural killer (NK) cells was investigated. RESULTS: Neutrophils were activated in patients with CHB, characterized by higher expression levels of programmed death-ligand 1 (PD-L1), cluster of differentiation 86, and interleukin-8, and lower levels of CXC motif chemokine receptor (CXCR) 1 and CXCR2. Hepatitis B e antigen (HBeAg) partially induces neutrophil activation through the Toll-like receptor 2 (TLR2). A consistent upregulation of the TLR2 and HBeAg expression was observed in patients with CHB. Notably, the genes encoding molecules pivotal for NK-cell function upon NK receptor engagement enriched in neutrophils after HBeAg activation. The HBeAg-activated neutrophils demonstrated the ability to decrease the production of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) in NK cells, while the PD-1 and PD-L1 pathways partially mediated the immunosuppression. CONCLUSIONS: The immunosuppression of neutrophils induced by HBeAg suggests a novel pathogenic mechanism contributing to immune tolerance in patients with CHB.

naRNA-LL37 composite DAMPs define sterile NETs as self-propagating drivers of inflammation.

Neutrophil extracellular traps (NETs) are a key antimicrobial feature of cellular innate immunity mediated by polymorphonuclear neutrophils (PMNs). NETs counteract microbes but are also linked to inflammation in atherosclerosis, arthritis, or psoriasis by unknown mechanisms. Here, we report that NET-associated RNA (naRNA) stimulates further NET formation in naive PMNs via a unique TLR8-NLRP3 inflammasome-dependent pathway. Keratinocytes respond to naRNA with expression of psoriasis-related genes (e.g., IL17, IL36) via atypical NOD2-RIPK signaling. In vivo, naRNA drives temporary skin inflammation, which is drastically ameliorated by genetic ablation of RNA sensing. Unexpectedly, the naRNA-LL37 'composite damage-associated molecular pattern (DAMP)' is pre-stored in resting neutrophil granules, defining sterile NETs as inflammatory webs that amplify neutrophil activation. However, the activity of the naRNA-LL37 DAMP is transient and hence supposedly self-limiting under physiological conditions. Collectively, upon dysregulated NET release like in psoriasis, naRNA sensing may represent both a potential cause of disease and a new intervention target.

Neutrophil-activating secretome characterizes palbociclib-induced senescence of breast cancer cells.

Senescent cells have a profound impact on the surrounding microenvironment through the secretion of numerous bioactive molecules and inflammatory factors. The induction of therapy-induced senescence by anticancer drugs is known, but how senescent tumor cells influence the tumor immune landscape, particularly neutrophil activity, is still unclear. In this study, we investigate the induction of cellular senescence in breast cancer cells and the subsequent immunomodulatory effects on neutrophils using the CDK4/6 inhibitor palbociclib, which is approved for the treatment of breast cancer and is under intense investigation for additional malignancies. Our research demonstrates that palbociclib induces a reversible form of senescence endowed with an inflammatory secretome capable of recruiting and activating neutrophils, in part through the action of interleukin-8 and acute-phase serum amyloid A1. The activation of neutrophils is accompanied by the release of neutrophil extracellular trap and the phagocytic removal of senescent tumor cells. These findings may be relevant for the success of cancer therapy as neutrophils, and neutrophil-driven inflammation can differently affect tumor progression. Our results reveal that neutrophils, as already demonstrated for macrophages and natural killer cells, can be recruited and engaged by senescent tumor cells to participate in their clearance. Understanding the interplay between senescent cells and neutrophils may lead to innovative strategies to cope with chronic or tumor-associated inflammation.

Whole blood transcriptomics reveals the enrichment of neutrophil activation pathways during erythema nodosum leprosum reaction.

Introduction: Patients with the multibacillary form of leprosy can develop reactional episodes of acute inflammation, known as erythema nodosum leprosum (ENL), which are characterized by the appearance of painful cutaneous nodules and systemic symptoms. Neutrophils have been recognized to play a role in the pathogenesis of ENL, and recent global transcriptomic analysis revealed neutrophil-related processes as a signature of ENL skin lesions. Methods: In this study, we expanded this analysis to the blood compartment, comparing whole blood transcriptomics of patients with non-reactional lepromatous leprosy at diagnosis (LL, n=7) and patients with ENL before administration of anti-reactional treatment (ENL, n=15). Furthermore, a follow-up study was performed with patients experiencing an ENL episode at the time of diagnosis and after 7 days of thalidomide treatment (THAL, n=10). Validation in an independent cohort (ENL=8; LL=7) was performed by RT-qPCR. Results: An enrichment of neutrophil activation and degranulation-related genes was observed in the ENL group, with the gene for the neutrophil activation marker CD177 being the most enriched gene of ENL episode when compared to its expression in the LL group. A more pro-inflammatory transcriptome was also observed, with increased expression of genes related to innate immunity. Validation in an independent cohort indicated that S100A8 expression could discriminate ENL from LL. Supernatants of blood cells stimulated in vitro with Mycobacterium leprae sonicate showed higher levels of CD177 compared to the level of untreated cells, indicating that the leprosy bacillus can activate neutrophils expressing CD177. Of note, suggestive higher CD177 protein levels were found in the sera of patients with severe/moderate ENL episodes when compared with patients with mild episodes and LL patients, highlighting CD177 as a potential systemic marker of ENL severity that deserves future confirmation. Furthermore, a follow-up study was performed with patients at the time of ENL diagnosis and after 7 days of thalidomide treatment (THAL, n=10). Enrichment of neutrophil pathways was sustained in the transcriptomic profile of patients undergoing treatment; however, important immune targets that might be relevant to the effect of thalidomide at a systemic level, particularly NLRP6 and IL5RA, were revealed. Discussion: In conclusion, our study reinforces the key role played by neutrophils in ENL pathogenesis and shed lights on potential diagnostic candidates and novel therapeutic targets that could benefit patients with leprosy.

Extracellular adenosine triphosphate skews the T helper cell balance and enhances neutrophil activation in mice with food allergies.

Exposure to food allergens elicits fast changes in the intestinal microenvironment, which guides the development of allergic reactions. Investigating the key information about these changes may help in better understanding food allergies. In this research, we explored the relationship between a food allergy and extracellular adenosine triphosphate (ATP), a danger molecule that has been proved to regulate the onset of allergic asthma and dermatitis but has not been studied in food allergies, by developing a unique animal model through allergen-containing diet feeding. After consuming an allergen-containing diet for 7 days, the allergic mice exhibited severe enteritis with elevated luminal ATP levels. The dysregulated luminal ATP worsened food-induced enteritis by enhancing Th17 cell responses and increasing mucosal neutrophil accumulation. In vitro experiments demonstrated that ATP intervention facilitated Th17 cell differentiation and neutrophil activation. In addition, the diet-induced allergy showed noticeable gut dysbiosis, characterized by decreased microbial diversity and increased diet-specific microbiota signatures. As the first, we show that food-induced enteritis is associated with an elevated concentration of luminal ATP. The dysregulated extracellular ATP exacerbated the enteritis of mice to a food challenge by manipulating intestinal Th17 cells and neutrophils.

Formyl peptide enhances cancer immunotherapy by activating antitumoral neutrophils, and T cells.

Neutrophils are heterogeneous and plastic, with the ability to polarize from antitumour to protumour phenotype and modulate tumour microenvironment components. While some advances have been made, the neutrophil-targeting therapy remains underexplored. Activation of formyl peptide receptors (FPRs) by formylated peptides is needed for local control of infection through the recruitment of activated neutrophils while the potential contribution of antitumour activity remains underexplored. Here, we demonstrate that neutrophils can be harnessed to suppress tumour growth through the action of the formyl peptide (FP) on the formyl peptide receptor (FPR). Mechanistically, FP efficiently recruits neutrophils to produce reactive oxygen species production (ROS), resulting in the direct killing of tumours. Antitumour functions disappeared when neutrophils were depleted by anti-Ly6G antibodies. Interestingly, extensive T-cell activation was observed in mouse tumours treated with FP, showing the potential to alter the immune suppressed tumour microenvironment (TME) and further sensitize mice to anti-PD1 therapy. Transcriptomic and flow cytometry analyses revealed the mechanisms of FP-sensitized anti-PD1 therapy, mainly including stimulated neutrophils and an altered immune-suppressed tumour microenvironment. Collectively, these data establish FP as an effective combination partner for sensitizing anti-PD1 therapy by stimulating tumour-infiltrated neutrophils.

A Pilot Study to Investigate Peripheral Low-Level Chronic LPS Injection as a Model of Neutrophil Activation in the Periphery and Brain in Mice.

Lipopolysaccharide-induced (LPS) inflammation is used as model to understand the role of inflammation in brain diseases. However, no studies have assessed the ability of peripheral low-level chronic LPS to induce neutrophil activation in the periphery and brain. Subclinical levels of LPS were injected intraperitoneally into mice to investigate its impacts on neutrophil frequency and activation. Neutrophil activation, as measured by CD11b expression, was higher in LPS-injected mice compared to saline-injected mice after 4 weeks but not 8 weeks of injections. Neutrophil frequency and activation increased in the periphery 4-12 h and 4-8 h after the fourth and final injection, respectively. Increased levels of G-CSF, TNFa, IL-6, and CXCL2 were observed in the plasma along with increased neutrophil elastase, a marker of neutrophil extracellular traps, peaking 4 h following the final injection. Neutrophil activation was increased in the brain of LPS-injected mice when compared to saline-injected mice 4-8 h after the final injection. These results indicate that subclinical levels of peripheral LPS induces neutrophil activation in the periphery and brain. This model of chronic low-level systemic inflammation could be used to understand how neutrophils may act as mediators of the periphery-brain axis of inflammation with age and/or in mouse models of neurodegenerative or neuroinflammatory disease.