Neutrophil Adhesion Research Articles

THE USE OF 5-AMINOLEVULINIC ACID GEL IN THE TREATMENT OF DIABETIC FOOT SYNDROME

Background. Diabetes mellitus affects almost all body systems, but the most dramatic complications occur in the lower extremities. Diabetic foot syndrome takes the leading position in the list of complications of diabetes mellitus, leading to early disability and mortality.
 At present, a new technology - photodynamic therapy is being intensively developed worldwide. This technique can be used in many different fields of medicine, including purulent surgery.
 This study aimed to investigate the possibility of using 5-aminolevulinic acid as an photosensitizer in photodynamic therapy in the complex treatment of chronic wounds in patients with ischemic and mixed forms of diabetic foot syndrome.
 Materials and Methods. In 2021, 10 patients with type 2 diabetes mellitus and ischemic and mixed forms of diabetic foot syndrome of both sexes, aged 52 to 75 years, were observed. As a preparation for photodynamic therapy, daily dressings with hydrogel were applied (3-5 days). During this period, limb revascularization was performed, the aim of which was to restore the blood flow to the foot, preferably through the anterior or posterior tibial artery. After the restoration of the main blood flow, photodynamic therapy was performed: after sanitation of the wound surface with physiological NaCl solution, Levuderm gel (6% gel phosphate of 5-aminolaevulinic acid) was applied and covered with occlusive dressing for 2 hours, after which the gel remains were removed and irradiation with Korobov photonic matrices "Barva Flex" with 660 nm wave length was carried out. Energy applied to the wound averaged 30-40 J/cm2. The light power density was between 0.1-1.0 W/cm2, and the exposure time varied according to the wound area.
 Results. After photodynamic therapy on days 2-3, perifocal oedema decreased. By days 5-7, the quality of granulation and the degree of wound epithelialization significantly improved. The wound healing rate on days5-7 of treatment was 1,58±0,44, and on days 10-12 - 4,72±0,63 (p <0,01). The evaluation of the wound healing rate showed good and satisfactory results: By day 32, the wound in the study group was completely epithelialized in the majority of patients (88.9%).
 Conclusions. The method we have developed for treating chronic wounds using photodynamic therapy is pathogenetically feasible and highly effective compared to conventional therapies. Combined light exposure using photosensitizer stimulates the formation of reactive oxygen species, increasing the effect of phagocytic cells, which is manifested in the activation of neutrophil chemotaxis, adhesion, and endocytosis. photodynamic therapy using aminolaevulinic acid as a photosensitizer, enhances molecular mechanisms of intercellular interaction at all stages of primary immunity activation.

Neutrophil phenotypes implicated in the pathophysiology of post-traumatic sepsis.

The disruption of immune homeostasis after trauma is a major cause of post-traumatic organ dysfunction and/or sepsis. Recently, a variety of neutrophil phenotypes with distinct functions have been identified and suggested as involved in various clinical conditions. The association between neutrophil phenotypes and post-traumatic immunodeficiency has also been reported, yet the specific neutrophil phenotypes and their functional significance in post-traumatic sepsis have not been fully clarified. Therefore, we sought to investigate neutrophil phenotypic changes in a murine model, as these may hold prognostic value in post-traumatic sepsis. Third-degree burns affecting 25% of the body surface area were used to establish trauma model, and sepsis was induced 24 h later through cecal ligation and puncture (CLP). The Burn/CLP post-traumatic sepsis model and the Sham/CLP control model were established to assess the immunological status after trauma. Histopathological evaluation was performed on the spleen, liver, kidneys, and lung tissues. Immunological evaluation included the assessment of neutrophil markers using mass cytometry as well as cytokine measurements in serum and ascitic fluid through multiplex analysis using LUMINEX®. The Burn/CLP group had a lower survival rate than the Sham/CLP group. Histopathological examination revealed an impaired immune response and more advanced organ damage in the Burn/CLP group. Furthermore, the Burn/CLP group exhibited higher levels of transforming growth factor-beta 1 in the blood and generally lower levels of cytokines than the Sham/CLP group. CD11b, which is involved in neutrophil adhesion and migration, was highly expressed on neutrophils in the Burn/CLP group. The expression of CD172a, which is related to the inhibition of phagocytosis, was also upregulated on neutrophils in the Burn/CLP group. The expression of sialic acid-binding lg-like lectin F and CD68 also differed between the two groups. Different neutrophil phenotypes were observed between Burn/CLP and Sham/CLP groups, suggesting that neutrophils are implicated in the immune imbalance following trauma. However, further studies are needed to prove the causal relationships between neutrophil phenotypes and outcomes, including survival rate and organ dysfunction.

AVACOPAN: A New Adjunctive Therapy for Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

Avacopan is a small-molecule complement 5a receptor (CD88) antagonist recently approved by the United States Food and Drug Administration as an adjunct therapy in combination with immunosuppressants and corticosteroids for treatment of ANCA-vasculitis. The selective ability of avacopan to inhibit the C5a receptor blocks neutrophil chemoattraction, activation, and adhesion while maintaining other beneficial complement pathways. Therefore, avacopan's unique selective property provides a breakthrough treatment for ANCA- vasculitis given that current therapies of corticosteroid treatment often lead to a decreased quality of life and a possible relapse. Clinical trials prove that avacopan is an excellent adjunctive treatment option, although it is not approved for the primary treatment of ANCA-vasculitis at this time. Initial clinical trials show substantial promise for avacopan, but additional studies with a longer duration will be needed to test for its durability and safety.

Angiotensin-(1-7) alleviates acute lung injury by activating the Mas receptor in neutrophil.

Acute lung injury (ALI) is a major cause of mortality and morbidity in the clinic. None of the current pharmacological interventions has achieved a detectable benefit. The renin-angiotensin system (RAS) is a complex humoral system essentially involved in the regulation of ALI. In the RAS family, angiotensin (Ang)-(1-7) was found to provide protection by counteracting the effects of Ang II in various cardiopulmonary disease models. The downstream receptor of Ang-(1-7) is the G protein-coupled receptor (GPCR) Mas. We hypothesize that the Ang-(1-7)-Mas pathway would protect patients from ALI. To establish a 2-hit ALI model, the mice underwent intratracheal instillation of hydrochloric acid followed by ventilator-induced lung injury (VILI). ALI was evaluated based on lung edema, histology, myeloperoxidase activity, and proinflammatory cytokine production. The effects of the infusion or inhalation of Ang-(1-7) and Mas receptor blocker A779 were examined. The human neutrophils were isolated, and Mas receptor expression was examined. The neutrophil responses to platelet-activating factor (PAF) stimulation were tested by measuring the formation of reactive oxygen species (ROS), neutrophil adhesion, and chemotaxis. Next, in the mouse model, the neutrophils were depleted using an anti-ly6G antibody. The infusion or inhalation of Ang-(1-7) protected mice from ALI as evidenced by decreases in lung edema, the histological lung injury score, myeloperoxidase activity, and proinflammatory cytokine production. Such effects were largely blocked by the Mas receptor blocker A779. Mas receptor expression in the neutrophils was identified at both the messenger ribonucleic acid and protein levels. Ang-(1-7) prevented neutrophil responses to PAF stimulation, including the formation of ROS, neutrophil adhesion, and chemotaxis, while A779 alleviated these effects. The importance of neutrophils in ALI was further confirmed by neutrophil depletion using the anti-ly6G antibody; however, A779 partially reversed the protective role of neutrophil depletion in ALI, indicating the critical role of Ang-(1-7)-Mas signaling in other pulmonary cells. Ang-(1-7)/Mas receptor attenuates the key features of ALI by regulating neutrophil activation. Our study provides new evidence of their role in the pathogenesis of ALI and may lead to the development of a promising therapeutic strategy.

Pleiotropic Effects of Hydroxyurea on Red Blood Cell and Neutrophil Phenotype in Patients with Sickle Cell Disease; Data from the Notice Study

Introduction The pathophysiology of sickle cell disease (SCD) is complex and goes beyond red blood cell (RBC) sickling alone. Neutrophils have been proposed to play an important role in SCD pathophysiology, but their exact contribution remains to be elucidated. Factors such as chronic hemolysis and oxidative stress can influence neutrophil adhesion, activity and aging. We designed a prospective study (Notice) to investigate a broad set of neutrophil characteristics in SCD patients. To correlate treatment effects of hydroxyurea (HU) on neutrophils with those on RBCs in SCD, we also studied RBC sickling and adhesion. Here, we will focus on the effects of hydroxyurea on RBC deformability and neutrophil adhesion, activity and aging. Aim: To assess the effects of HU on RBC deformability and neutrophil phenotypes with respect to adhesion, activation and aging. Methods Blood (EDTA) was collected from SCD patients with HbSS or HbSβ0 in steady-state. A subgroup of patients was included before and during treatment with HU. For measurement of antigen expression by flow cytometry, total leukocyte fraction after lysis of red blood cells (without Percoll gradient) was used and neutrophils were gated based on their forward and side scatter. For in vitro neutrophil adhesion, neutrophils were isolated using a Percoll gradient and lysis buffer, as previously described. (Kuijpers et al. Blood 2007) Neutrophils were labeled with calcein and placed in an uncoated 96-well MaxiSorp plate. After washing, adherent cells were lysed and fluorescence was measured. (Kuijpers et al. J Allergy Clin Immunol 2017). Adhesion of RBCs was measured by flowing whole blood (20x106/ml)over laminin-coated Ibidi slides for 30 minutes with visualization by microscope. Percentage of mean RBC adhesion was normalized to a healthy control (HC). Data were analyzed using SPSS and GraphPad Prism and are presented as mean ±SE or ±SD as appropriate. Conventional statistics were used. Results A total of 49 patients (median age 25.0 (IQR 20.0 - 32.5) years, 39% female) were included. Twenty patients (41%) used a stable dose of HU at baseline. In 10 patients, blood was drawn before and after starting HU treatment. HU treatment improved hemoglobin F and markers of hemolysis and was associated with a decrease in the point of sickling (PoS) of RBCs (from median 24.8 mmHg [IQR 16.0-28.2] to 16.8 [15.0-20.8], P=0.13). Adhesion of RBC was more than 3-fold higher in SCD patients than in HC. At baseline, there was no difference in RBC adhesion between patients with and without HU (P=0.86). After starting HU treatment, RBC adhesion decreased significantly from a median of 318% (normalized to control) to 149% (P=0.016, Figure 1a-b). Adhesion of neutrophils was significantly higher in patients compared to HC (median 18.3% versus 12.6% P <0.0001). No difference was observed between patients with and without HU at baseline. Neutrophil adhesion did not decrease after starting HU treatment (Figure 1c-d). Neutrophil surface expression of CD11b/CD18 was measured as markers of adhesion. While CD11b expression was comparable between SCD patients and HC, CD18 expression was significantly higher in SCD patients (mean MFI 12193 vs 10586, P=0.016; Figure 2b. After treatment with HU both CD11b and CD18 expression decreased, though the differences did not reach statistical significance (P=0.22 and 0.56, respectively). Neutrophils of SCD patients displayed a more aged phenotype compared to neutrophils of HC with lower L-selectin (median MFI 3108 vs 4656, P=0.021) and higher CXCR4 (median MFI 1150 vs 886, P=0.003) expression. Activation marker CD64 was also higher in SCD patients compared to HC (median MFI 1841 vs 1065, P=0.0006). HU treatment seemed to modify all markers more towards a HC phenotype although no statistically significant changes were observed. Conclusion Adhesion of both RBCs and neutrophils is increased in SCD patients compared to HC. HU treatment was associated with significantly improved RBC rheology as shown by decreased PoS and decreased adhesion to laminin. Despite a trend to improvement in expression of adhesion markers (CD11b/CD18), in vitro adhesion of neutrophils did not decrease. These findings could be due to the small sample size of patients starting with HU or by a difference in vitro behaviour of changed composition of neutrophil populations after treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Microglia-Neutrophil Interactions Drive Dry AMD-like Pathology in a Mouse Model.

In dry age-related macular degeneration (AMD), inflammation plays a key role in disease pathogenesis. Innate immune cells such as microglia and neutrophils infiltrate the sub-retinal space (SRS) to induce chronic inflammation and AMD progression. But a major gap in our understanding is how these cells interact with each other in AMD. Here, we report a novel concept of how dynamic interactions between microglia and neutrophils contribute to AMD pathology. Using well-characterized genetically engineered mouse models as tools, we show that in the diseased state, retinal pigmented epithelial (RPE) cells trigger pro-inflammatory (M1) transition in microglia with diminished expression of the homeostatic marker, CX3CR1. Activated microglia localize to the SRS and regulate local neutrophil function, triggering their activation and thereby inducing early RPE changes. Ligand receptor (LR)-loop analysis and cell culture studies revealed that M1 microglia also induce the expression of neutrophil adhesion mediators (integrin β1/α4) through their interaction with CD14 on microglia. Furthermore, microglia-induced neutrophil activation and subsequent neutrophil-mediated RPE alterations were mitigated by inhibiting Akt2 in microglia. These results suggest that the Akt2 pathway in microglia drives M1 microglia-mediated neutrophil activation, thereby triggering early RPE degeneration and is a novel therapeutic target for early AMD, a stage without treatment options.

Inhibitor of apoptosis proteins antagonist SM164 ameliorates experimental MPO-ANCA-associated vasculitis via enhancing fatty acid oxidation in neutrophils.

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of life-threatening autoimmune diseases. Inhibitors of apoptosis proteins (IAPs) are a class of molecules engaged in cell death and inflammation, interventions of which are proven effective in a number of inflammatory diseases. Here we tested whether targeting IAPs could ameliorate AAV and explored the potential mechanism. We collected 19 kidney specimens from patients with myeloperoxidase (MPO)-AAV to investigate the expression of IAPs. The IAP pan-inhibitor SM164 was used to treat the experimental autoimmune vasculitis (EAV) rat model of AAV. RNA sequencing of renal cortex and enrichment analysis were developed to interpret gene expression. Functional experiments were performed to investigate the role of SM164 on neutrophils and endothelial cells. The expression of three IAPs (cIAP1, cIAP2 and XIAP) was upregulated in kidneys of AAV patients compared with normal controls. SM164 dramatically reduced renal injury in EAV rats. Transcriptomic analysis revealed prominent alterations in fatty acid oxidation and respiratory burst following SM164 treatment. Functional studies demonstrated that SM164 inhibited neutrophil activation induced by MPO-ANCA positive IgG or serum from MPO-AAV patients, and such inhibitory effect was abolished by gene silencing or pharmacological inhibition of fatty acid oxidation. SM164 also inhibited the adhesion of neutrophils to endothelial cells with little effect on the endothelial injury induced by serum from MPO-AAV patients. Inhibition of IAPs with SM164 played a protective role in AAV through enhancing intracellular fatty acid oxidation in neutrophils.

Conditioned medium of bone marrow mesenchymal stem cells improves sperm parameters and reduces histological alteration in rat testicular ischaemia/reperfusion model.

Testis ischaemia-reperfusion (I/R) plays a vital role in male infertility. Recent studies have demonstrated that paracrine factors of mesenchymal stem cells exert the transplanted cells' reparative effects. The present experimental study aimed to investigate the effects of conditioned medium (CM) of bone marrow-derived mesenchymal stem cells (BMMSCs). In this study, 21 rats were separated into three groups of 7 animals: sham, I/R and I/R plus CM. Sperm parameters were measured at the end of this study. Moreover, histological parameters were examined. 2-Deoxyuridine 5-triphosphate nick-end labelling (TUNEL) assay was done to assess the apoptotic cells. The count of adhered neutrophils was measured in subtunical venules. Testicular I/R led to a significant reduction in the viability and concentration of sperm and resulted in a significant elevation in the rate of abnormal sperms in comparison with sham. The CM-treated group demonstrated a significant reduction in the rate of abnormal sperm and a significant elevation in the viability and concentration of sperm compared with the I/R group. Based on the morphometric analysis, in the I/R group, epithelial thickness and seminiferous tubule diameter significantly decreased in comparison with sham. A significant reduction was seen between the I/R and sham groups regarding the mean testicular biopsy score (MTBS) value. However, an improvement was observed in the I/R + CM group MTBS value in comparison with the I/R group. TUNEL assay showed that the apoptotic cells in the seminiferous tubules belonging to the I/R group were significantly higher compared with the control. Nevertheless, apoptotic cells were reduced in the I/R + CM group compared with the I/R group. Results of the present study showed that CM of BMMSCs exerts protective effects on the testicular I/R damages.

Nexinhib20 Inhibits Neutrophil Adhesion and β2 Integrin Activation by Antagonizing Rac-1-Guanosine 5'-Triphosphate Interaction.

Neutrophils are critical for mediating inflammatory responses. Inhibiting neutrophil recruitment is an attractive approach for preventing inflammatory injuries, including myocardial ischemia-reperfusion (I/R) injury, which exacerbates cardiomyocyte death after primary percutaneous coronary intervention in acute myocardial infarction. In this study, we found out that a neutrophil exocytosis inhibitor Nexinhib20 inhibits not only exocytosis but also neutrophil adhesion by limiting β2 integrin activation. Using a microfluidic chamber, we found that Nexinhib20 inhibited IL-8-induced β2 integrin-dependent human neutrophil adhesion under flow. Using a dynamic flow cytometry assay, we discovered that Nexinhib20 suppresses intracellular calcium flux and β2 integrin activation after IL-8 stimulation. Western blots of Ras-related C3 botulinum toxin substrate 1 (Rac-1)-GTP pull-down assays confirmed that Nexinhib20 inhibited Rac-1 activation in leukocytes. An in vitro competition assay showed that Nexinhib20 antagonized the binding of Rac-1 and GTP. Using a mouse model of myocardial I/R injury, Nexinhib20 administration after ischemia and before reperfusion significantly decreased neutrophil recruitment and infarct size. Our results highlight the translational potential of Nexinhib20 as a dual-functional neutrophil inhibitory drug to prevent myocardial I/R injury.

Citrullination of histoneH3 in neutrophil via CXCL1 enhances neutrophil adhesion to femoral artery of LDLR−/− mice fed HFD

Abstract Background Vascular inflammation plays an important role in the development of atherosclerosis. Previously we have shown that a high-fat diet (HFD) increased neutrophil adhesion to the vascular intima in wild-type (wt) mice (Osaka M. Sci Rep. 2016). However, the involvement of neutrophils in atherosclerosis-related vascular inflammation is not well known. Purpose This study examined that neutrophil extracellular trap (NETs) or the hypercitrullination of histone H3 in neutrophils enhances neutrophil adhesion to atheroprone-arteries in LDL receptor null (LDLR−/−) mice. Methods We observed leukocyte adhesion in the femoral artery of LDLR−/− mice fed normal chow (NC) or HFD, and determined leukocyte subtype that adhered on vascular endothelium under neutrophil or monocyte depletion using intravital microscopy. Importantly, neutrophil adhesion was examined under the administration of TDFA which inhibits NETs and citrullination of histone H3, in LDLR−/− mice fed HFD. Furthermore, immunohistochemistry for citrullinated histone H3 in peripheral neutrophils of mice was examined. Comprehensive cytokine/chemokine analysis for a plasma of mice was performed to determine the factors citrullinating histone H3 in LDLR−/− mice. Moreover, these mice were treated with a novel specific PPARα agonist, to reduce the elevation of plasma triglyceride levels. Results Leukocyte adhesion in LDLR−/− mice fed HFD significantly increased compared to NC. More interestingly, it significantly enhanced compared to wt mice fed HFD. Furthermore, neutrophil depletion rather than monocyte depletion diminished leukocyte adhesion, suggesting that the leukocyte subtype that adhered in LDLR−/− mice fed HFD was neutrophil. Neutrophil adhesion in these mice significantly was reduced by the administration of TDFA, suggesting a pivotal role for histone H3 citrullination in neutrophil adhesion. Moreover, citrullination of histone H3 in neutrophils from LDLR−/− mice fed HFD but not from those without HFD was significantly enhanced. In addition, comprehensive cytokine/chemokine analysis revealed an increase of CXCL1 in plasma of LDLR−/− mice fed HFD. CXCL1 enhanced neutrophil adhesion to HUVECs, and the adhesion significantly decreased by the treatment of TDFA to neutrophil in vitro non-static adhesion assay. These results showed that CXCL1 enhanced neutrophil adhesion in LDLR−/− mice fed HFD through citrullination. Furthermore, when these mice were treated with PPARα agonist, observed histone citrullination, as well as neutrophil adhesion, was significantly reduced. Conclusion These results suggest that HFD induced histone citrullination in neutrophils in LDLR−/− mice and PPARα agonist plays a role during hypertriglyceridemia-mediated vascular inflammation in atherosclerosis. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Grant-in-Aid for Scientific Research(C)

Microbial gut evaluation in an angolan paediatric population with sickle cell disease.

Sickle cell disease (SCD) is one of the most common genetic conditions worldwide. It can contribute up to 90% of under-5 mortality in sub-Saharan Africa. Clinical manifestations are very heterogeneous, and the intestinal microbiome appears to be crucial in the modulation of inflammation, cell adhesion and induction of aged neutrophils, the main interveners of recurrent vaso-occlusive crisis. Enterocyte injury, increased permeability, altered microbial composition and bacterial overgrowth have all been documented as microbial and pathophysiologic changes in the gut microbiome of SCD patients in recent studies. Our aim was to sequence the bacterial 16S rRNA gene in order to characterize the gut microbiome of Angolan children with SCA and healthy siblings as a control. A total of 72 stool samples were obtained from children between 3 and 14 years old. Our data showed that the two groups exhibit some notable differences in microbiota relative abundance at different classification levels. Children with SCA have a higher number of the phylum Actinobacteria. As for the genus level, Clostridium cluster XI bacteria was more prevalent in the SCA children, whereas the siblings had a higher abundance of Blautia, Aestuariispira, Campylobacter, Helicobacter, Polaribacter and Anaerorhabdus. In this study, we have presented the first microbiota analysis in an Angolan paediatric population with SCD and provided a detailed view of the microbial differences between patients and healthy controls. There is still much to learn before fully relying on the therapeutic approaches for gut modulation, which is why more research in this field is crucial to making this a reality.

KRIT1-mediated regulation of neutrophil adhesion and motility.

Loss of Krev interaction-trapped-1 (KRIT1) expression leads to the development of cerebral cavernous malformations (CCM), a disease in which abnormal blood vessel formation compromises the structure and function of the blood-brain barrier. The role of KRIT1 in regulating endothelial function is well-established. However, several studies have suggested that KRIT1 could also play a role in regulating nonendothelial cell types and, in particular, immune cells. In this study, we generated a mouse model with neutrophil-specific deletion of KRIT1 in order to investigate the effect of KRIT1 deficiency on neutrophil function. Neutrophils isolated from adult Ly6Gtm2621(cre)Arte Krit1flox/flox mice had a reduced ability to attach and spread on the extracellular matrix protein fibronectin and exhibited a subsequent increase in migration. However, adhesion to and migration on ICAM-1 was unchanged. In addition, we used a monomeric, fluorescently-labelled fragment of fibronectin to show that integrin activation is reduced in the absence of KRIT1 expression, though β1 integrin expression appears unchanged. Finally, neutrophil migration in response to lipopolysaccharide-induced inflammation in the lung was decreased, as shown by reduced cell number and myeloperoxidase activity in lavage samples from Krit1PMNKO mice. Altogether, we show that KRIT1 regulates neutrophil adhesion and migration, likely through regulation of integrin activation, which can lead to altered inflammatory responses in vivo.

B-cell receptor associated protein 31 deficiency decreases the expression of adhesion molecule CD11b/CD18 and PSGL-1 in neutrophils to ameliorate acute lung injury.

Acute lung injury (ALI) and its more severe condition acute respiratory distress syndrome (ARDS) are critical life-threatening disorders characterized by an excessive influx of neutrophils into the alveolar space. Neutrophil infiltration is a multi-step process involving the sequential engagement of adhesion molecules. The adhesion molecule CD11b/CD18 acts as an important role in the recruitment of neutrophils to lung tissues in the ALI model. B-cell receptor associated protein 31 (BAP31), an endoplasmic reticulum transmembrane protein, has been reported to regulate the cellular anterograde transport of CD11b/CD18 in human neutrophils. To explore how BAP31 regulates CD11b/CD18 in mouse neutrophils, we constructed myeloid-specific BAP31 knockdown mice in this study. Biological investigations indicated that BAP31 deficiency could significantly alleviated lung injury, as evidenced by the improved histopathological morphology, reduced pulmonary wet/dry weight ratio, inhibited myeloperoxidase level and decreased neutrophil counts in the bronchoalveolar lavage fluid. Further studies clarified that BAP31 deficiency obviously down-regulated the expression of CD11b/CD18 and P-selectin glycoprotein ligand-1 (PSGL-1) by deactivating the nuclear factor kappa B (NF-κB) signaling pathway. Collectively, our results revealed that BAP31 depletion exerted a protective effect on ALI, which was possibly dependent on the attenuation of neutrophil adhesion and infiltration by blocking the expression of adhesion molecules CD11b/CD18 and PSGL-1. These findings implied the potential of BAP31 as an appealing protein to mediate the occurrence of ALI.

Systemic Activation of Neutrophils by Immune Complexes Is Critical to IgA Vasculitis.

In IgA vasculitis (IgAV) perivascular deposition of IgA1 immune complexes (IgA-ICs) is traditionally considered the fundamental trigger for polymorphonuclear neutrophil (PMN)-mediated damage. We propose that IgA-IC deposition, although mandatory, is not sufficient alone for IgAV. Serum IgA-IC levels and IgA-IC binding to PMNs were quantified in IgAV patients and controls. Activation of PMNs was evaluated by neutrophil extracellular trap (NET) release, adherence, and cytotoxicity assays and in a flow system to mirror conditions at postcapillary venules. In vitro results were related to findings in biopsies and a mouse vasculitis model. During acute IgAV flares we observed elevated serum levels of IgA-ICs and increased IgA-IC binding to circulating PMNs. This IgA-IC binding primed PMNs with consequent lowering of the threshold for NETosis, demonstrated by significantly higher release of NETs from PMNs activated in vitro and PMNs from IgAV patients with flares compared with surface IgA-negative PMNs after flares. Blocking of FcαRI abolished these effects, and complement was not essential. In the flow system, marked NETosis only occurred after PMNs had adhered to activated endothelial cells. IgA-IC binding enhanced this PMN tethering and consequent NET-mediated endothelial cell injury. Reflecting these in vitro findings, we visualized NETs in close proximity to endothelial cells and IgA-coated PMNs in tissue sections of IgAV patients. Inhibition of NET formation and knockout of myeloperoxidase in a murine model of IC vasculitis significantly reduced vessel damage in vivo. Binding of IgA-ICs during active IgAV primes PMNs and promotes vessel injury through increased adhesion of PMNs to the endothelium and enhanced NETosis.

Probiotic potential and immunomodulatory properties in Enterococcus faecium GMB24 and Enterococcus hirae SMB16 isolated from goat and sheep milk.

Probiotic attributes of lactic acid bacteria isolated from goat and sheep milk samples were analysed by culturing them on an MRS agar medium. The most potential isolates, GMB24 and SMB16, were identified by biochemical tests which had ability to tolerate different concentrations of acid and bile and phenol resistance. They were further identified as Enterococcus faecium GMB24 and Enterococcus hirae SMB16 by 16S rRNA gene sequencing approach. The probiotic potential of the isolates GMB24 and SMB16 were recorded including antimicrobial activity against pathogenic bacteria viz., Escherichia coli (MTCC118), Staphylococcus aureus (MTCC7443), Pseudomonas aeruginosa (MTCC424), Listeria monocytogens (MTCC657) and Salmonella typhimurium (MTCC733), and antibiotic susceptibility test. The isolates SMB16 and GMB24 exhibited a higher zone of inhibition against P. aeruginosa (19.00 ± 0.57mm) and S. aureus (25.66 ± 0.88mm), respectively. The data from these experiments were used for the principal component analysis (PCA) to assess the survivability of the isolates under different factors. The heatmap generated in this study clustered the bacterial isolates based on their phenotype properties. Further, immunomodulating activities of these probiotic bacteria were tested on neutrophil adhesion test, haemagglutinating antibody titer and delayed-type hypersensitivity. Probiotic E. faecium GMB24 and E. hirae SMB16, at 109 cells/mL doses per day, increased the neutrophil adhesion, haemagglutinating antibody titer and DTH in comparison to the untreated control group. The isolates showed negative test for haemolytic and gelatinase activities and hence were considered safe. E. faecium GMB24 and E. hirae SMB16 were shown to have high probiotic potential and immune-stimulant action.

Apremilast ameliorates acute respiratory distress syndrome by inhibiting neutrophil-induced oxidative stress

BackgroundThe pathogenesis of acute respiratory distress syndrome (ARDS) is attributed to the dysregulation of oxidative stress and neutrophil recruitment. We aimed to investigate the anti-inflammatory effects of apremilast on human neutrophils and assess its efficacy for treating ARDS. MethodsWe analysed superoxide anion generation, integrin expression, and adhesion in activated human neutrophils using spectrophotometry, flow cytometry, and immunofluorescence microscopy. Phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was determined using immunoblotting. A murine lipopolysaccharide (LPS)-induced ARDS model was used to evaluate the therapeutic effects of apremilast. ResultsApremilast significantly decreased superoxide anion production, reactive oxygen species (ROS) generation, cluster of differentiation (CD)11 b expression, and neutrophil adhesion in formyl-l-methionyl-l-leucyl-l-phenylalanine activated human neutrophils. Apremilast elevated cyclic 3′,5′-adenosine monophosphate (cAMP) and protein kinase A (PKA) activity in activated neutrophils. It reduced cellular cAMP-specific phosphodiesterase (PDE) activity and selectively inhibited enzymatic PDE4 activity. The activated cAMP/PKA pathway suppressed the phosphorylation of ERK and JNK as well as Ca2+ mobilization in activated neutrophils. All inhibitory effects of apremilast on activated neutrophils were reversed by a PKA inhibitor. In vivo examinations indicated that apremilast alleviated lung neutrophil infiltration, myeloperoxidase (MPO) activity, pulmonary oedema, and alveolar damage in LPS-induced ARDS. ConclusionApremilast inhibits inflammatory responses after neutrophil activation via cAMP/PKA-dependent inhibition of ERK and JNK activation. Our study revealed apremilast suppresses oxidative stress and chemotaxis by selectively inhibiting PDE4 in neutrophils and thus protects against endotoxin-induced ARDS in mice. Apremilast can be used as an alternative off-label drug in treating acute lung damage.

Extracellular Vesicles in Sickle Cell Disease: A Promising Tool

Sickle cell disease (SCD) is the most common hemoglobinopathy worldwide. It is characterized by an impairment of shear stress-mediated vasodilation, a pro-coagulant, and a pro-adhesive state orchestrated among others by the depletion of the vasodilator nitric oxide, by the increased phosphatidylserine exposure and tissue factor expression, and by the increased interactions of erythrocytes with endothelial cells that mediate the overexpression of adhesion molecules such as VCAM-1, respectively. Extracellular vesicles (EVs) have been shown to be novel actors involved in SCD pathophysiological processes. Medium-sized EVs, also called microparticles, which exhibit increased plasma levels in this pathology, were shown to induce the activation of endothelial cells, thereby increasing neutrophil adhesion, a key process potentially leading to the main complication associated with SCD, vaso-occlusive crises (VOCs). Small-sized EVs, also named exosomes, which have also been reported to be overrepresented in SCD, were shown to potentiate interactions between erythrocytes and platelets, and to trigger endothelial monolayer disruption, two processes also known to favor the occurrence of VOCs. In this review we provide an overview of the current knowledge about EVs concentration and role in SCD.

Histamine promotes adhesion of neutrophils by inhibition of autophagy in dairy cows with subacute ruminal acidosis.

Subacute ruminal acidosis (SARA), a common digestive disease in dairy cows, is accompanied by systemic inflammation and high concentrations of histamine in blood. Histamine-induced neutrophil adhesion may play an important role in the systemic inflammation experienced by cows during SARA. Autophagy, an intracellular degradation system, regulates recycling of membrane-associated integrin and may be involved in histamine-induced adhesion of bovine neutrophils. In the present study, 20 multiparous mid-lactation cows (average body weight 486 ± 24 kg) fitted with ruminal fistula were assigned to a control group (n = 10) or a SARA group (n = 10). We induced SARA by feeding different combinations of wheat-barley pellets and chopped alfalfa hay for 8 wk; SARA was defined as a ruminal pH <5.6 for at least 3 h/d. Blood samples were collected in wk 8. Compared with controls, SARA cows had greater serum concentrations of tumor necrosis factor-α, IL-6, IL-1β, lipopolysaccharide (LPS)-binding protein, haptoglobin, and serum amyloid A. Serum concentrations of these proinflammatory factors had strong positive correlations with the concentration of serum histamine and LPS. In ex vivo adhesion experiments, the number of adherent neutrophils was greater in the SARA group. Additionally, membrane protein abundance of adhesion molecules such as integrin α-L precursor (CD11a) and integrin α-M precursor (CD11b) was greater in neutrophils of the SARA group, confirming enhanced adhesion ability. Neutrophils of SARA cows had greater number of autophagosomes, greater protein abundance of autophagy substrate sequestosome-1 (p62), and higher ratio of microtubule associated proteins 1A/1B light chain 3 (LC3)-II to LC3-I, indicating congestion during the late phase of autophagy flux. For in vitro experiments, neutrophils isolated from control cows were incubated with 0.4 endotoxin units (EU)/mL LPS or 7 μM histamine for 0, 1, 2, and 4 h, respectively. We detected linear and quadratic effects for the number of adherent neutrophils after histamine treatment with a peak response at 2 h, whereas no significant effect was detected after LPS treatment. Membrane protein abundance of CD11a and CD11b was greater after histamine treatment, suggesting that it may have an inhibitory effect on the degradation of adhesion molecules. The greater abundance of p62, higher ratio of LC3-II to LC3-I, and increased co-localization between CD11b and LC3 after histamine treatment indicated that recycling of adhesion molecules and autophagy flux were blocked. These effects were not aggravated further in the presence of chloroquine, a specific inhibitor of the late phase of autophagy flux. Overall, our results revealed that histamine increases adhesion of neutrophils by inhibiting autophagy in dairy cows with SARA.

Decreasing GDF15 Promotes Inflammatory Signals and Neutrophil Infiltration in Psoriasis Models

Psoriasis is driven by the interplay between hyperproliferative keratinocytes and infiltrating inflammatory cells. GDF15, a member of the TGF-β superfamily, has been implicated in cachexia, metabolic control, and cancer invasion. However, the expression and immunomodulatory role of GDF15 in inflammatory diseases has not been clarified. In this study, we report that GDF15 is decreased in the epidermis of patients with psoriasis and in an imiquimod-induced psoriasis-like mouse model. TNF-α suppresses GDF15 expression in keratinocytes by inhibiting the protein level of the transcription factor GATA2. GDF15 deficiency aggravates the development of psoriatic lesions, as evidenced by more severe skin inflammation in imiquimod-treated Gdf15-knockout (Gdf15‒/‒) mice compared with that in wild-type mice. Importantly, GDF15 limited the synthesis of a panel of keratinocyte cytokines and chemokines by inhibiting TAK1/NF-κB activation and directly inhibited neutrophil adhesion and migration by inhibiting the activation of the small GTPase Rap1. Epidermal hyperplasia, infiltration of neutrophils, and transcripts of psoriasis-related markers in imiquimod-induced psoriasiform dermatitis were significantly alleviated by a topical supplement of recombinant murine GDF15. In summary, our study revealed an unexpected role of GDF15 in keratinocyte and neutrophil function in the skin of psoriasis, implying its therapeutic potential in treating psoriasis.

Staphylococcus aureus Causes the Arrest of Neutrophils in the Bloodstream in a Septicemia Model

Staphylococcus aureus induces the expression of VCAM-1, P- and E-selectins on the endothelial cells of the EA.hy926 cell line but, at the same time, causes the significant suppression of the force and work of adhesion between these receptors of endotheliocytes and the receptors of neutrophils in an experimental septicemia model. Adhesion contacts between the receptors of neutrophils and endotheliocytes are statistically significantly suppressed under non-opsonized and opsonized S. aureus treatment, which disrupts the initial stage of transendothelial migration of neutrophils—adhesion. Thus, S. aureus causes the arrest of neutrophils in the bloodstream in an experimental septicemia model.