Functional plasticity shapes neutrophil response to Leishmania major infection in susceptible and resistant strains of mice.

Neutrophils rapidly infiltrate sites of infection and possess several microbicidal strategies, such as neutrophil extracellular traps release and phagocytosis. Enhanced neutrophil infiltration is associated with higher susceptibility to Leishmania infection, but neutrophil effector response contribution to this phenotype is uncertain. Here, we show that neutrophils from susceptible BALB/c mice (B/c) produce more NETs in response to Leishmania major than those from resistant C57BL/6 mice (B6), which are more phagocytic. The absence of neutrophil elastase contributes to phagocytosis regulation. Microarray analysis shows enrichment of genes involved in NET formation (mpo, pi3kcg, il1b) in B/c, while B6 shows upregulation of genes involved in phagocytosis and cell death (Arhgap12, casp9, mlkl, FasL). scRNA-seq in L. major-infected B6 showed heterogeneity in the pool of intralesional neutrophils, and we identified the N1 subset as the putative subpopulation involved with phagocytosis. In vivo, imaging validates NET formation in infected B/c ears where NETing neutrophils were mainly uninfected cells. NET digestion in vivo augmented parasite lymphatic drainage. Hence, a balance between NET formation and phagocytosis in neutrophils may contribute to the divergent phenotype observed in these mice.

GPCRs overexpression and impaired fMLP-induced functions in neutrophils from chronic kidney disease patients.

Introduction: G-protein coupled receptors (GPCRs) expressed on neutrophils regulate their mobilization from the bone marrow into the blood, their half-live in the circulation, and their pro- and anti-inflammatory activities during inflammation. Chronic kidney disease (CKD) is associated with systemic inflammatory responses, and neutrophilia is a hallmark of CKD onset and progression. Nonetheless, the role of neutrophils in CKD is currently unclear. Methods: Blood and renal tissue were collected from non-dialysis CKD (grade 3 - 5) patients to evaluate GPCR neutrophil expressions and functions in CKD development. Results: CKD patients presented a higher blood neutrophil-to-lymphocyte ratio (NLR), which was inversely correlated with the glomerular filtration rate (eGFR). A higher frequency of neutrophils expressing the senescent GPCR receptor (CXCR4) and activation markers (CD18+CD11b+CD62L+) was detected in CKD patients. Moreover, CKD neutrophils expressed higher amounts of GPCR formyl peptide receptors (FPR) 1 and 2, known as neutrophil pro- and anti-inflammatory receptors, respectively. Cytoskeletal organization, migration, and production of reactive oxygen species (ROS) by CKD neutrophils were impaired in response to the FPR1 agonist (fMLP), despite the higher expression of FPR1. In addition, CKD neutrophils presented enhanced intracellular, but reduced membrane expression of the protein Annexin A1 (AnxA1), and an impaired ability to secrete it into the extracellular compartment. Secreted and phosphorylated AnxA1 is a recognized ligand of FPR2, pivotal in anti-inflammatory and efferocytosis effects. CKD renal tissue presented a low number of neutrophils, which were AnxA1+. Conclusion: Together, these data highlight that CKD neutrophils overexpress GPCRs, which may contribute to an unbalanced aging process in the circulation, migration into inflamed tissues, and efferocytosis.

Chronic ethanol exposure decreases H3K27me3 in the Il6 promoter region of macrophages and generates persistent dysfunction on neutrophils during fungal infection.

OBJECTIVE AND DESIGN: The aim of this study was to investigate the effects of ethanol exposure on epigenetic markers in bone marrow (BM) and their impact on inflammatory response during Aspergillus fumigatus infection. RESULTS: Chronic ethanol exposure decreased H3K27me3 enrichment in the Il6 promoter region while increased H3K4me3 enrichment in Tnf. Chimeric mice were generated by transplanting BM from mice exposed to ethanol or water. Infection of ethanol-chimeric mice culminated in higher clinical scores, although there was no effect on mortality. However, previous chronic exposure to ethanol affects persistently the inflammatory response in lung tissue, demonstrated by increased lung damage, neutrophil accumulation and IL-6, TNF and CXCL2 production in ethanol-chimeric mice, resulting in a decreased neutrophil infiltration into the alveolar space. Neutrophil killing and phagocytosis were also significantly lower. Moreover, BM derived macrophages (BMDM) from ethanol-chimeric mice stimulated with A. fumigatus conidia exhibited higher levels of TNF, CXCL2 and IL-6 release and a higher killing activity. The Il6 promoter of BMDM from ethanol-chimeric mice exhibited a reduction in H3K27me3 enrichment, a finding also observed in BM donors exposed to ethanol. CONCLUSIONS: These evidences demonstrate that prior chronic alcohol exposure of bone-marrow modify immune effector cells functions impairing the inflammatory response during A. fumigatus infection. These findings highlight the persistent impact of chronic ethanol exposure on infectious disease outcomes.

Unexplained fever with consumptive syndrome in the elderly: two cases of VEXAS syndrome with inflammasome dysregulation.

The aim of this study is to investigate the inflammasome dysregulation in peripheral blood leukocytes of VEXAS patients. The constitutive and in vitro triggered activation of inflammasome in PBMC and neutrophils was analyzed in two Brazilian patients with typical UBA1 mutations, and compared with healthy donors. Our findings highlight the constitutive activation of caspase-1 in VEXAS leukocytes, accompanied by increased plasma levels of IL-18. Furthermore, upon stimulation of isolated peripheral blood mononuclear cells (PBMC) and neutrophils, we observed not only the exhaustion of NLRP3 and NLRP1/CARD8 pathways in VEXAS PBMC but also a significant increase in NLRP3-mediated NETs release in VEXAS neutrophils. These findings support previous studies on the contribution of the inflammasome to VEXAS pathogenesis, identifying at least two profoundly affected pathways (NLRP3 and NLRP1/CARD8) in VEXAS peripheral blood.

Notable correlation between serum epidermal growth factor values and inflammatory status in patients with COVID-19.

INTRODUCTION: Despite its crucial role in Epidermal Growth Factor Receptor (EGFR) activation, and the resulting impact on the health-disease process, epidermal growth factor (EGF) is an underexplored molecule in relation to how its serum concentrations relate to other analytes and clinical variables in pathological contexts. OBJECTIVE: To clarify the possible correlation between EGF and clinical and analytical variables in the context of COVID-19. METHODS: Cross-sectional observational and analytical study, in patients with virological and clinical diagnosis of COVID-19, selected by simple random sampling, admitted between August and September 2021. UMELISA-EGF commercial kits were used. RESULTS: Differences in overall EGF values were observed between groups (566.04 vs. 910.53 pg/ml, p = .0430). In COVID-19 patients, no notable correlations were observed for neutrophil, platelet, triglyceride or liver enzyme values (p > .05). Significant correlations were observed with the neutrophil-lymphocyte indicator (r = 0.4711, p = .0128) as well as with the platelet-lymphocyte index (r = 0.4553, p = .0155). Statistical results of multivariate regression analysis suggest NLR (ß = .2232, p = .0353) and PLR (ß = .2117, p = .0411) are predictors of inflammation in patients with COVID-19. CONCLUSIONS: Serum EGF concentrations in COVID-19 correlate positively with prognostic inflammatory markers of severity and could presumably act as an independent risk factor for the development of inflammation in response to new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

MR1 blockade drives differential impact on integrative signatures based on circuits of circulating immune cells and soluble mediators in visceral leishmaniasis.

Introduction: Visceral leishmaniasis (VL) is an important tropical and neglected disease and represents a serious global health problem. The initial interaction between the phagocytes and the parasite is crucial to determine the pathogen's capacity to initiate infection and it shapes the subsequent immune response that will develop. While type-1 T-cells induce IL-6, IL-1ß, TNF-α, and IL-12 production by monocytes/macrophages to fight the infection, type-2 T-cells are associated with a regulatory phenotype (IL-10 and TGF-ß) and successful infection establishment. Recently, our group demonstrated the role of an important Th1/Th17 T-cell population, the mucosal-associated invariant T (MAIT) cells, in VL. MAIT cells can respond to L. infantum by producing TNF-α and IFN-γ upon MR1-dependent activation. Objective and methods: Here, we describe the impact of the MR1-blockage on L. infantum internalization on the functional profile of circulating neutrophils and monocytes as well as the impact of the MR1-blockage on the soluble mediator signatures of in vitro whole blood cultures. Results: Overall, our data showed that VL patients presents higher percentage of activated neutrophils than asymptomatic and non-infected controls. In addition, MR1 blockade led to lower TNF-α and TGF-ß production by non-activated neutrophils from asymptomatic individuals. Moreover, TNF-α and IL-10 production by monocytes was higher in VL patients. In the analysis of soluble mediators produced in vitro, MR1-blockade induced a decrease of IFN-γ and an increase of IL-10, IL-27 and IL-33 in the cell cultures of AS group, a cytokine pattern associated with type 2 deleterious response. Discussion and conclusion: These data corroborate the hypothesis that MR1-restricted responses are associated to a protective role during Leishmania infection.

Leishmania infantum infection modulates messenger RNA, microRNA and long non-coding RNA expression in human neutrophils in vitro.

In the Americas, L. infantum (syn. chagasi) is the main cause of human visceral leishmaniasis. The role of neutrophils as part of the innate response to Leishmania spp. infection is dubious and varies according to the species causing the infection. Global expression of coding RNAs, microRNAs and long non-coding RNAs changes as part of the immune response against pathogens. Changes in mRNA and non-coding RNA expression resulting from infection by Leishmania spp. are widely studied in macrophages, but scarce in neutrophils, the first cell to encounter the trypanosomatid, especially following infection by L. infantum. Herein, we aimed to understand the expression patterns of coding and non-coding transcripts during acute in vitro infection of human neutrophils by L. infantum. We isolated neutrophils from whole blood of healthy male donors (n = 5) and split into groups: 1) infected with L. infantum (MOI = 5:1), and 2) uninfected controls. After 3 hours of exposure of infected group to promastigotes of L. infantum, followed by 17 hours of incubation, total RNA was extracted and total RNA-Seq and miRNA microarray were performed. A total of 212 genes were differentially expressed in neutrophils following RNA-Seq analysis (log2(FC)±0.58, FDR≤0.05). In vitro infection with L. infantum upregulated the expression of 197 and reduced the expression of 92 miRNAs in human neutrophils (FC±2, FDR≤0.01). Lastly, 5 downregulated genes were classified as lncRNA, and of the 10 upregulated genes, there was only 1 lncRNA. Further bioinformatic analysis indicated that changes in the transcriptome and microtranscriptome of neutrophils, following in vitro infection with L. infantum, may impair phagocytosis, apoptosis and decrease nitric oxide production. Our work sheds light on several mechanisms used by L. infantum to control neutrophil-mediated immune response and identifies several targets for future functional studies, aiming at the development of preventive or curative treatments for this prevalent zoonosis.

Pan-immune-inflammation value and survival in patients with breast cancer from a Peruvian reference hospital.

The pan-immune-inflammation value (PIV), calculated as (neutrophil × platelet × monocyte)/lymphocyte count, may be useful for estimating survival in breast cancer patients. To determine the prognostic value of PIV for overall survival in breast cancer patients in Lima, Peru. A retrospective cohort study was conducted. 97 breast cancer patients diagnosed between January 2010 and December 2016 had their medical records analyzed. The primary dependent variable was overall survival, and the key independent variable was the PIV, divided into high (≥ 310) and low (< 310) groups. Patient data included demographics, treatment protocols and other clinical variables. Statistical analysis involved Kaplan-Meier survival curves and Cox proportional hazards modeling. Patients with a PIV ≥ 310 had significantly lower 5-year survival functions (p = 0.004). Similar significant differences in survival were observed for clinical stage III-IV (p = 0.015), hemoglobin levels < 12 mg/Dl (p = 0.007), histological grade (p = 0.019), and nuclear grade (p < 0.001); however, molecular classification did not show a significant survival difference (p = 0.371). The adjusted Hazard Ratios showed that PIV ≥ 310 was significantly associated with poor outcome (5.08, IC95%: 1.52-16.92). While clinical stage and hemoglobin levels were associated with survival in the unadjusted model. These factors did not maintain significance after adjustment. PIV is an independent predictor of reduced survival in Peruvian breast cancer patients.

Celecoxib exhibits antifungal effect against Paracoccidioides brasiliensis both directly and indirectly by activating neutrophil responses.

BACKGROUND: Celecoxib, an anti-inflammatory drug, combined therapies using antimicrobials and immune modulator drugs are being studied. OBJECTIVE: To assess whether Celecoxib has direct in vitro antifungal effect against the Paracoccidioides brasiliensis, the causative agent of Paracoccidioidomycosis-(PCM) and also if it improves the in vivo activity of neutrophils-(PMN) in an experimental murine subcutaneous-(air pouch) model of the disease. METHODS: The antifungal activity of Celecoxib(6 mg/mL) on P. brasiliensis-(Pb18) was evaluated using the microdilution technique. Splenocytes co-cultured with Pb18 and treated with Celecoxib(6 mg/mL) were co-cultured for 24, 48 and 72-hours. Swiss mice were inoculated with Pb18 and treated with Celecoxib(6 mg/kg) in the subcutaneous air pouch. Neutrophils were collected from the air pouch. Mitochondrial activity, reactive oxygen production, catalase, peroxidase, cytokines and chemokines, nitrogen species, total protein, microbicidal activity of PMNs and viable Pb18 cells numbers were analyzed. RESULTS: Celecoxib had no cytotoxic effect on splenocytes co-cultured with Pb18, but had a marked direct antifungal effect, inhibiting fungal growth both in vitro and in vivo. Celecoxib interaction with immune system cells in the air pouch, it leads to activation of PMNs, as confirmed by several parameters (mitochondrial activity, reactive oxygen species, peroxidase, KC and IL-6 increase, killing constant and phagocytosis). Celecoxib was able to reduce IL-4, IL-10 and IL-12 cytokine production. The number of recovered viable Pb18 decreased dramatically. CONCLUSIONS: This is the first report of the direct antifungal activity of Celecoxib against P. brasiliensis. The use of Celecoxib opens a new possibility for future treatment of PCM.

Neutrophilic leukocytosis and erythema nodosum leprosum in leprosy: insights from a retrospective observational study.

Background: Leprosy reactions represent immunologically mediated episodes of acute inflammation that, if not diagnosed and treated promptly, can cause irreversible impairment of nerve function and permanent disabilities. A frequent type of reaction experienced by patients with lepromatous leprosy (LL) and borderline lepromatous leprosy (BL) is erythema nodosum leprosum (ENL), an inflammatory complication that may become chronic or recur in multiple episodes. Although ENL is commonly described as a neutrophil-mediated immune disease, the role of neutrophils is not fully understood. In this study, we assess neutrophilic leukocytosis in a retrospective cohort of patients affected by BL or LL leprosy. Materials and methods: A retrospective observational study was performed using data from 146 patients with BL and LL leprosy diagnosed and treated at the Souza Araújo Outpatient Clinic, Fiocruz, Rio de Janeiro, Brazil. Clinical, demographic, and hematological data were extracted from medical records. Skin biopsy samples obtained from patients for ENL diagnosis were used for histopathological evaluations. Results: Most patients were male (75%) and had a reactional episode (85%), of which 65% were ENL. Multiple episodes were common, 55% of the 80 patients with ENL presented more than 2 episodes (average of 2.6 episodes). In treatment-naive BL/LL patients, the median blood neutrophil counts of patients who developed ENL at some points of their disease course were higher than those who did not experience any reaction (median= 4,567 cells/mm3 vs 3,731 cells/mm3 respectively, p=0.0286). A correlation between the increase in median neutrophil counts and ENL severity was confirmed (6,066 cells/mm3 for mild ENL vs 10,243 cells/mm3 for moderate/severe ENL, p=0.0009). A longitudinal assessment was also performed in 34 patients, confirming the neutrophilic leukocytosis (BL/LL: 4896 cells/mm3 vs ENL: 8408 cells/mm3, p<0.0001). Moreover, increased NLR was associated with a greater neutrophilic infiltration in ENL lesions. Conclusion: We demonstrate that ENL episodes in patients affected by leprosy are associated with elevated blood leukocyte and neutrophil counts and an increased NLR. These findings highlight the significant involvement of neutrophils in the ENL immunological/inflammatory process.

Acute lung injury is prevented by monocyte locomotion inhibitory factor in an experimental severe malaria mouse model.

Acute lung injury caused by severe malaria (SM) is triggered by a dysregulated immune response towards the infection with Plasmodium parasites. Postmortem analysis of human lungs shows diffuse alveolar damage (DAD), the presence of CD8 lymphocytes, neutrophils, and increased expression of Intercellular Adhesion Molecule 1 (ICAM-1). P. berghei ANKA (PbA) infection in C57BL/6 mice reproduces many SM features, including acute lung injury characterized by DAD, CD8+ T lymphocytes and neutrophils in the lung parenchyma, and tissular expression of proinflammatory cytokines and adhesion molecules, such as IFNγ, TNFα, ICAM, and VCAM. Since this is related to a dysregulated immune response, immunomodulatory agents are proposed to reduce the complications of SM. The monocyte locomotion inhibitory factor (MLIF) is an immunomodulatory pentapeptide isolated from axenic cultures of Entamoeba hystolitica. Thus, we evaluated if the MLIF intraperitoneal (i.p.) treatment prevented SM-induced acute lung injury. The peptide prevented SM without a parasiticidal effect, indicating that its protective effect was related to modifications in the immune response. Furthermore, peripheral CD8+ leukocytes and neutrophil proportions were higher in infected treated mice. However, the treatment prevented DAD, CD8+ cell infiltration into the pulmonary tissue and downregulated IFNγ. Moreover, VCAM-1 expression was abrogated. These results indicate that the MLIF treatment downregulated adhesion molecule expression, impeding cell migration and proinflammatory cytokine tissular production, preventing acute lung injury induced by SM. Our findings represent a potential novel strategy to avoid this complication in various events where a dysregulated immune response triggers lung injury.

Short communication: Goat mastitis and the formation of neutrophil extracellular traps (NETs).

Mastitis, an inflammation of the mammary gland affecting milk production and quality in dairy herds, is often associated with Staphylococcus spp. in goats. Neutrophils are crucial in combating infections by migrating into milk and deploying various defense strategies, including the release of neutrophil extracellular traps (NETs) composed of DNA, histones, and bactericidal proteins. This study investigated whether NETs are released by goat neutrophils stimulated in vitro by Staphylococcus aureus and Staphylococcus warneri, two common pathogens of goat mastitis. PMNs were isolated from blood from healthy adult goats. We evaluated goat NET formation by stimulating cells with: phorbol 12-myristate 13-acetate (PMA) as a positive control, cytochalasin for inhibition of actin polymerization, S. aureus, and S. warneri. NET formation was observed in response to chemical stimulation and bacterial presence, effectively trapping pathogens. Variations in NET formation between S. aureus and S. warneri suggest pathogen-specific responses. These findings suggest that the formation of NETs may be an important complementary mechanism in the defense against mastitis in goats. In conclusion, this study unveils a novel defense mechanism in goats, indicating the role of NETs against S. aureus and S. warneri in mastitis.

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.

Combined effects of ambient temperature and food availability on induced innate immune response of a fruit-eating bat (Carollia perspicillata).

Resilience of mammals to anthropogenic climate and land-use changes is associated with the maintenance of adequate responses of several fitness-related traits such as those related to immune functions. Isolated and combined effects of decreased food availability and increased ambient temperature can lead to immunosuppression and greater susceptibility to disease. Our study tested the general hypothesis that decreased food availability, increased ambient temperature and the combined effect of both factors would affect selected physiological and behavioral components associated with the innate immune system of fruit-eating bats (Carollia perspicillata). Physiological (fever, leukocytosis and neutrophil/lymphocyte ratio) and behavioral (food intake) components of the acute phase response, as well as bacterial killing ability of the plasma were assessed after immune challenge with lipopolysaccharide (LPS: 10 mg/kg) in experimental groups kept at different short-term conditions of food availability (ad libitum diet or 50% food-deprived) and ambient temperature (27 and 33°C). Our results indicate that magnitude of increase in body temperature was not affected by food availability, ambient temperature or the interaction of both factors, but the time to reach the highest increase took longer in LPS-injected bats that were kept under food restriction. The magnitude of increased neutrophil/lymphocyte ratio was affected by the interaction between food availability and ambient temperature, but food intake, total white blood cell count and bacterial killing ability were not affected by any factor or interaction. Overall, our results suggest that bacterial killing ability and most components of acute phase response examined are not affected by short-term changes in food availability and ambient temperature within the range evaluated in this study, and that the increase of the neutrophil/lymphocyte ratio when bats are exposed to low food availability and high ambient temperature might represent an enhancement of cellular response to deal with infection.

ß-glucans from Euglena gracilis or Saccharomyces cerevisiae effects on immunity and inflammatory parameters in dogs.

Considering the differences in molecular structure and function, the effects of ß-1,3-glucans from Euglena gracilis and ß-1,3/1,6-glucans from Saccharomyces cerevisiae on immune and inflammatory activities in dogs were compared. Four diets were compared: control without ß-glucans (CON), 0.15 mg/kg BW/day of ß-1,3/1,6-glucans (Β-Y15), 0.15 mg/kg BW/day of ß-1,3-glucans (Β-S15), and 0.30 mg/kg BW/day of ß-1,3-glucans (Β-S30). Thirty-two healthy dogs (eight per diet) were organized in a block design. All animals were fed CON for a 42-day washout period and then sorted into one of four diets for 42 days. Blood and faeces were collected at the beginning and end of the food intake period and analysed for serum and faecal cytokines, ex vivo production of hydrogen peroxide (H2O2) and nitric oxide (NO), phagocytic activity of neutrophils and monocytes, C-reactive protein (CRP), ex vivo production of IgG, and faecal concentrations of IgA and calprotectin. Data were evaluated using analysis of covariance and compared using Tukey's test (P<0.05). Dogs fed Β-Y15 showed higher serum IL-2 than dogs fed Β-S30 (P<0.05). A higher phagocytic index of monocytes was observed in dogs fed the B-S15 diet than in those fed the other diets, and a higher neutrophil phagocytic index was observed for B-S15 and B-Y15 than in dogs fed the CON diet (P<0.05). Monocytes from dogs fed B-S15 and B-S30 produced more NO and less H2O2 than those from the CON and B-Y15 groups (P<0.05). Despite in the reference value, CRP levels were higher in dogs fed B-S15 and B-S30 diets (P<0.05). ß-1,3/1,6-glucan showed cell-mediated activation of the immune system, with increased serum IL-2 and neutrophil phagocytic index, whereas ß-1,3-glucan acted on the immune system by increasing the ex vivo production of NO by monocytes, neutrophil phagocytic index, and serum CRP. Calprotectin and CRP levels did not support inflammation or other health issues related to ß-glucan intake. In conclusion, both ß-glucan sources modulated some immune and inflammatory parameters in dogs, however, different pathways have been suggested for the recognition and action of these molecules, reinforcing the necessity for further mechanistic studies, especially for E. gracilis ß-1,3-glucan.

The Acute Inflammatory Potential of Particles From the Echinococcus granulosus Laminated Layer Is Moderated by Its Calcium Inositol Hexakisphosphate Component.

Cystic echinococcosis is caused by the tissue-dwelling larva (hydatid) of Echinococcus granulosus sensu lato. A salient feature is that this larva is protected by the acellular laminated layer (LL). As the parasite grows, the LL sheds abundant particles that can accumulate in the parasite's vicinity. The potential of LL particles to induce inflammation in vivo has not been specifically analysed. It is not known how each of its two major components, namely highly glycosylated mucins and calcium inositol hexakisphosphate (InsP6) deposits, impacts inflammation induced by the LL as a whole. In this work, we show that LL particles injected intraperitoneally cause infiltration of eosinophils, neutrophils and monocytes/macrophages as well as the disappearance of resident (large peritoneal) macrophages. Strikingly, the absence of calcium InsP6 enhanced the recruitment of all the inflammatory cell types analysed. In contrast, oxidation of the mucin carbohydrates caused decreased recruitment of neutrophils. The carbohydrate-oxidised particles caused cell influx nonetheless, which may be explained by possible receptor-independent effects of LL particles on innate immune cells, as suggested by previous works from our group. In summary, LL particles can induce acute inflammatory cell recruitment partly dependent on its mucin glycans, and this recruitment is attenuated by the calcium InsP6 component.

Analysis of maternal Fc gamma receptor IIIb isoantibodies using immunomagnetic negative selected neutrophils.

BACKGROUND AND OBJECTIVES: The isolation of neutrophils and subsequent detection of anti-human neutrophil antigens (HNA) antibodies are crucial in clinical medicine for the diagnosis of autoimmune neutropenia, neonatal alloimmune neutropenia (NAIN) and transfusion-related acute lung injury (TRALI). This study reports two cases of maternal anti-Fc-gamma-receptor-IIIb (FcγRIIIb) isoimmunization without NAIN symptoms and compares the efficiency of immunomagnetic negative selection (IMNS) with traditional dextran/Ficoll for neutrophil isolation in HNA serological assays. MATERIALS AND METHODS: Investigating two cases of maternal anti-FcγRIIIb isoimmunization, neutrophils from three donors were isolated from 8 mL of whole blood using IMNS and dextran/Ficoll. Serological assays included the granulocyte agglutination and immunofluorescence test, monoclonal antibody immobilization of granulocyte antigens and the LABScreen Multi (One Lambda). IMNS and dextran/Ficoll were compared in terms of cell yield, viability, time, cost and purity. RESULTS: Maternal anti-FcγRIIIb isoantibodies with FCGR3B gene deletion were detected in both cases. Newborns and fathers exhibited specific gene combinations: FCGR3B*02/FCGR3B*02 (Case 1) and FCGR3B*02/FCGR3B*03 (Case 2). IMNS outperformed dextran/Ficoll, yielding four times more neutrophils (average neutrophil counts: 18.5 × 103/µL vs. 4.5 × 103/µL), efficiently removing non-neutrophil cells and reducing processing time (30-40 min vs. 70-90 min), although it incurred a higher cost (2.7 times). CONCLUSION: Two cases of maternal anti-FcγRIIIb isoantibodies, unrelated to NAIN, were identified. Although neutropenia has not been described in these cases, we emphasize the importance of identifying asymptomatic cases with the potential for severe neutropenia. Additionally, IMNS is introduced as a rapid, high-yield, high-purity neutrophil isolation technique, beneficial for serological assays detecting anti-HNA antibodies.

Association of neutrophil extracellular trap levels with Raynaud's phenomenon, glomerulonephritis and disease index score in SLE patients from Brazil.

Neutrophil extracellular traps (NETs) are cell-extruded DNA strands coated with neutrophils' nuclear proteins and enzymes from cytotoxic granules, produced by NETosis, a cell death pathway. They perform an important defensive role in innate immunity, but their increased production and/or inefficient degradation expose new antigens, such as DNA or citrullinated histone peptides, triggering autoimmunity. This study aimed to access possible associations between serum NETs levels with epidemiological, clinical, and serological data from a well-characterized SLE Brazilian patients' cohort. NET levels were evaluated in one hundred seventy serum samples of patients with Systemic Lupus Erythematosus (SLE) using an Immunoassay. Univariate and multivariate binary logistic regression used clinical patients' data as independent variables. Parametric and non-parametric tests compared log10 base serum NET levels transformed between patients' groups. SLE patients were also dichotomized into "High serum NET levels" and "Low serum NET levels" groups. All analyses were performed in R language 4.1.2, and p < 0.05 were considered significant. Increased susceptibility for high serum NET levels was observed in SLE patients with Raynaud's phenomenon (OR = 2.30, 95 % CI = 1.06-5.21 and p = 0.039), independently of any other risk factor. Also, SLE patients with Raynaud's phenomenon presented higher mean NET serum levels (mean = -0.13 vs. -0.51, p = 0.01). In addition, higher mean NET serum levels were associated with glomerulonephritis (mean = -0.45 vs. -0.12, p = 0.03). Ultimately, the SLEDAI index scored higher in the high NETs serum levels group (median = 2.0 vs. 0.0, p = 6 × 10-3). The formation of NETs might be implicated in Raynaud's phenomenon, glomerulonephritis, and disease index score in SLE patients. Our results highlight the importance of serum NET levels as a possible therapeutical target to modulate the clinical course of SLE.

Neutrophils, an emerging new therapeutic platform.

Neutrophils possess unique characteristics that render them indispensable to health, and patients with irregular neutrophil counts or functions suffer from increased morbidity and mortality. As neutrophils are short-lived postmitotic cells, genetic aberrations cannot be corrected directly in neutrophils and must be targeted in their progenitors. Neutrophils are increasingly being contemplated for a range of therapeutic applications, including restoration or modulation of immune function and targeting of solid tumors. This review addresses the state-of-the-art in neutrophil transfusions and their possible applications for infectious disease prevention and treatment. It offers a landscape of the most recent gene therapy approaches to address neutrophil-related genetic diseases. We also discuss how ongoing research could broaden the applicability of neutrophil-based therapies to solid cancer treatments and beyond.

Three Main SCFAs Mitigate Lung Inflammation and Tissue Remodeling Nlrp3-Dependent in Murine HDM-Induced Neutrophilic Asthma.

Neutrophilic asthma is generally defined by poorly controlled symptoms and high levels of neutrophils in the lungs. Short-chain fatty acids (SCFAs) are proposed as nonpharmacological therapy for allergic asthma, but their impact on the neutrophilic asthma lacks evidence. SCFAs regulate immune cell responses and impact the inflammasome NLRP3, a potential pharmacological target for neutrophilic asthma. Here, we explored the capacity of SCFAs to mitigate murine-induced neutrophilic asthma and the contribution of NLRP3 to this asthma. The objective of this study is to analyze whether SCFAs can attenuate lung inflammation and tissue remodeling in murine neutrophilic asthma and NLRP3 contribution to this endotype. Wild-type (WT) C57BL6 mice orotracheally received 10 µg of HDM (house dust mite) in 80 µL of saline on days 0, 6-10. To explore SCFAs, each HDM group received 200 mM acetate, propionate, or butyrate. To explore NLRP3, Nlrp3 KO mice received the same protocol of HDM. On the 14th day, after euthanasia, bronchoalveolar lavage fluid (BALF) and lungs were collected to evaluate cellularity, inflammatory cytokines, and tissue remodeling. HDM group had increased BALF neutrophil influx, TNF-α, IFN-γ, IL-17A, collagen deposition, and mucus secretion compared to control. SCFAs distinctively attenuate lung inflammation. Only features of tissue remodeling were Nlrp3-dependent such as collagen deposition, mucus secretion, active TGF-ß cytokine, and IMs CD206+. SCFAs greatly decreased inflammatory cytokines and tissue remodeling. Only tissue remodeling was dependent on NLRP3. It reveals the potential of SCFAs to act as an additional therapy to mitigate neutrophilic asthma and the NLRP3 contribution to asthma.