Neutrophil Phagocytosis Research Articles

THE SPECTRUM OF IMMUNORESISTANCE DISORDERS AND INFLUENCE OF CYTOTOXICITY AND PATHOGENICITY OF DAMP FRACTIONS ON THE POST-COVID-19 SYNDROME FORMATION AND THE MAIN DОISEASE COMPLICATION IN PATIENTS WITH URGENT SURGICAL PATHOLOGY

Summary. The infectious nature of COVID-19 leads to complications in the immune system. It is relevant to determine the long-term consequences of COVID-19, which can be triggers for the urgent surgical pathology’s development of the thoracic and abdominal organs in patients with certain risk factors associated with characteristic disorders of immunometabolic reactions.
 The aim. To determine the spectrum of immune resistance disorders and to find out the effect of cytotoxicity and pathogenicity of DAMP fractions on the post-COVID-19 syndrome formation and complications of the main disease in patients with urgent surgical pathology.
 Materials and methods. Patients (n = 143) with urgent cardiovascular and abdominal surgical pathology that arose against the background of post-COVID-19 syndrome were examined. We used: spectrophotometry (content of DAMP fractions), immunoturbimetry (concentration of C3 and C4 complement components); luminescent microscopy (phagocytic activity of neutrophils); light microscopy (spontaneous activity of NADPH-oxidase systems of neutrophils); flow cytometry (expression of T lymphocyte activation markers); immunofluorescence (determination of antinuclear antibodies).
 Results. It was found that the predominance of certain DAMP fractions is the cause of the immunopathological patterns formation in the development of post-COVID-19 syndrome. In patients with urgent surgical pathology and the predominance of the DAMP260 oligonucleotide fraction, significant changes in the hemogram, failure of endocytosis and incomplete phagocytosis of neutrophils, increased expression of the T lymphocytes late activation marker (CD3+HLA-DR+) were found. In all examined patients, a high frequency of autoantibodies to nuclear structures (ANA) was found, which depended on the content of DAMP fractions. The maximum repertoire of antibodies was found in patients with the oligonucleotide fraction (DAMP260) predominance.
 Conclusions. In patients with urgent surgical pathology, during the formation of post-COVID-19 syndrome, the chronic immunoinflammatory reaction formation occurs against the background of the activation cascades development of innate and adaptive immunity factors.

Features of immune status in patients with acute coronary syndrome with and without COVID-19, depending on the level of B1 lymphocytes

The aim of our study was to evaluate the blood cell indices and phagocytic activity of neutrophils in persons with acute coronary syndrome, depending on their history of COVID-19 infection.
 The study involved 65 males aged 35 to 65 years with acute coronary syndrome (acute myocardial infarction and unstable angina pectoris). All patients underwent coronary angiography and stenting of the coronary arteries within 3 days from the terms of admission to the hospital. The following clinical examination were carried out: a general blood test by a standardized method on a hematological analyzer Medonic M20 (Sweden). Of immunological indices, the phagocytic activity of neutrophils was assessed. Spontaneous and induced NBT test of neutrophils was determined by light microscopy using light microscopy (Olimpus, Japan). The phagocytic activity of neutrophils was recorded by their ability to absorb latex particles. B1 lymphocytes were determined using flow cytometry.
 All patients, depending on the content of B1 lymphocytes and the presence or absence of COVID-19 in previous history, were divided into 6 groups: patients with COVID-19 and those with reduced (group 1), normal (group 2), or elevated number of B1 lymphocytes (group 3). The patients who did not have COVID-19 were also classified into those with low (group 4), normal (group 5), or elevated B1 lymphocytes (group 6). The numbers of leukocytes in routine blood test were significantly higher, and the average corpuscular volume of hemoglobin was lower in the patients who have undergone COVID-19. Platelet counts were higher in post-COVID-19 patients, being maximal at normal B1 lymphocytes. The largest number of monocytes was recorded in patients with COVID-19 and normal B1 lymphocytes, and the minimal content of monocytes was registered in patients of group 4. The highest number of granulocytes was observed in individuals who did not have COVID-19, with reduced B1 lymphocytes. Thrombocytocrit was the highest in group 2 patients. The activity and intensity of neutrophil phagocytosis was lower in individuals with a history of COVID-19 and elevated B1 lymphocytes. The phagocytic number of neutrophils was minimal in those patients without COVID-19 who had low B1 lymphocytes. The maximal spontaneous HBT activity was recorded in individuals with high B1 lymphocytes and a history of COVID-19, and the minimal values have been recorded in those with low B1 lymphocytes and previous COVID-19. NBT spontaneous index was also the highest in patients of the 3rd group. The minimal NBT-induced activity and index were noted in group 1. The most severe patients were in groups 1 and 2. In group 1, 50% were diagnosed with acute myocardial infarction, stent thrombosis was diagnosed in 2 patients, four patients deceased. Among patients with normal B1 cell contents and a history of COVID-19, 2 patients died, 2 patients had stent thrombosis, 65% had acute myocardial infarction. These groups had higher platelet levels and lower HBT activity, both spontaneous and induced.
 In patients with acute coronary syndrome and prior COVID-19, in comparison with persons without a history of COVID-19, there is an increased number of leukocytes, platelets, a decrease in the activity and intensity of neutrophil phagocytosis, spontaneous and stimulated NBT activity, which was most pronounced in patients with low B1 lymphocytes. The most clinically severe patients were found in the group of people who had undergone COVID-19 and had low B1 lymphocytes.

Dysregulated pulmonary inflammatory responses exacerbate the outcome of secondary aspergillosis following influenza.

Severe influenza is a risk factor for fatal invasive pulmonary aspergillosis; however, the mechanistic basis for the lethality is unclear. Utilizing an influenza-associated pulmonary aspergillosis (IAPA) model, we found that mice infected with influenza A virus followed by Aspergillus fumigatus had 100% mortality when superinfected during the early stages of influenza but survived at later stages. While superinfected mice had dysregulated pulmonary inflammatory responses compared to controls, they had neither increased inflammation nor extensive fungal growth. Although influenza-infected mice had dampened neutrophil recruitment to the lungs following subsequent challenge with A. fumigatus, influenza did not affect the ability of neutrophils to clear the fungi. Our data suggest that the lethality seen in our model of IAPA is multifactorial with dysregulated inflammation being a greater contributor than uncontrollable microbial growth. If confirmed in humans, our findings provide a rationale for clinical studies of adjuvant anti-inflammatory agents in the treatment of IAPA.

The aging process and its relation to periodontal conditions

Periodontal tissue destruction can cause complaints for sufferers. Inflammatory conditions of the gingiva, bleeding gums, and even tooth loss are clinical features of the destruction of the periodontal tissues. Periodontitis is an inflammatory disease involving the periodontal tissues. The prevalence of periodontium destruction increases with aging. Changes in innate and adaptive immunity that occur in the elderly also play a role in the severity of periodontitis. “Inflammaging” is a chronic inflammatory state associated with old age in humans. Periodontitis contributes to inflammaging since periodontitis in the elderly is associated with increased markers of systemic inflammation. Age-related changes also affect neutrophil function, especially antimicrobial activity, so neutrophils may become more pathological. After infiltration into the tissue, neutrophils are equipped with several antimicrobial strategies to reduce the number of antigens. Phagocytosis is the ability of neutrophils to engulf and kill microbes, but neutrophil phagocytosis is weakened in the elderly. Age-related changes affecting neutrophils, macrophages, and T cells appear to promote pathogenic immune responses and contribute to the increased prevalence of periodontal disease in aging individuals. Proper regulation of the host immune response is critical in maintaining periodontal health. This paper aims to describe the aging process and its relation to periodontal conditions.

Identification of biomarkers associated with oxidative stress-related genes in postmenopausal osteoporosis.

Osteoporosis (OP) is a prevalent metabolic disease, with aging and menopause being the major risk factors. Studies have shown that nearly one-third of postmenopausal women suffer from osteoporosis. However, there is a scarcity of research on antioxidant systems for the prevention and treatment of postmenopausal osteoporosis (PM-OP). To address this gap, we performed differential analysis using Limma to identify differentially expressed genes (DEGs) in PM-OP samples. We employed weighted correlation network analysis (WGCNA) to identify oxidative stress (OS)-related genes (OSRGs) highly correlated with PM-OP. The intersection of key modular genes and DEGs yielded differentially expressed OSRGs (DE-OSRGs) specific to PM-OP. We conducted GO and KEGG functional enrichment analyses on these genes. Additionally, we constructed a protein-protein interaction (PPI) network and utilized support vector machine recursive feature elimination (SVM-RFE) and random forest (RF) algorithms to identify signature genes. The diagnostic value of the signature genes was evaluated and validated using ROC curves. GSEA enrichment analysis was employed to explore the potential mechanisms associated with the signature genes. Finally, we constructed a regulatory network involving TF-miRNA-mRNA interactions for the signature genes and verified the biological roles of FOXO3 and DDIT3 in PM-OP and healthy groups using quantitative real-time PCR (qRT-PCR). Our analysis revealed 20 DE-OSRGs specific to PM-OP, obtained by intersecting modular and differential genes. The PPI network identified central genes (DDIT3, MAPK8, CDK2, SIRT1, and FOXO3) with more than 3 nodes. Through integration with machine learning algorithms, we identified DDIT3 and FOXO3 as signature genes. The ROC curve analysis indicated that the AUC value was greater than 0.7, suggesting the potential diagnostic value of these signature genes. Furthermore, GSEA results revealed their involvement in pathways related to the regulation of neutrophil activation, oxidative phosphorylation, MAPK signaling, mitochondrial matrix, and phagocytosis. Lastly, we constructed a regulatory network comprising 27 nodes (22 TFs, 3 miRNAs, and 2 mRNAs) and 28 edges. Additionally, qRT-PCR confirmed the significant up-regulation of FOXO3 and DDIT3 expressions in the PM-OP group compared to the healthy control group. In summary, this study employed bioinformatics analysis to identify OS-related biomarkers (DDIT3 and FOXO3) in PM-OP, providing new biological targets for clinical treatment.

Ways to promote the efficiency of herpetic stomatitis treatment

The treatment of herpetic stomatitis should be etiotropic, pathogenetic, symptomatic, and local. The research determined the effectiveness of inosine pranobex (1000 mg 4 times a day) in herpetic stomatitis treatment. Addition of inosine pranobex to the pharmacotherapy of herpetic stomatitis reduced hospital stay and duration of treatment. The drug suppresses the synthesis of the virus due to the structure of inosinic acid in the polyribosomes of the cell affected by the virus, and also helps to eliminate the deficiency or dysfunction of immunity that occurred in the case of herpetic stomatitis, it affects the activity of T-cytotoxic lymphocytes and natural killers, enhances the action of neutrophil granulocytes, chemotaxis and phagocytosis of monocytes and macrophages, increases the synthesis of interleukins IL-1 and IL-2, affects the number of Ig G immunoglobulins. Applying inosine pranobex also significantly decreased pain syndrome manifestations from the start of the treatment. It contributed to body temperature normalization 2 days earlier which shortened the treatment period at the in-patient department. Applying inosine pranobex in the combination therapy of herpetic stomatitis has shown a potent clinical efficacy and can be an alternatіve to acyclic nucleosides in patients with herpetic stomatitis of the oral mucosa.

DOCK8 is essential for neutrophil mediated clearance of cutaneous S. aureus infection

DOCK8 deficient patients are susceptible to skin infection with Staphylococcus aureus which is normally cleared by neutrophils. We examined the mechanism of this susceptibility in mice. Dock8−/− mice had delayed clearance of S. aureus from skin mechanically injured by tape stripping. The numbers and viability of neutrophils in infected but not in uninfected, tape stripped skin were significantly reduced in Dock8−/− mice compared to WT controls. This is despite comparable numbers of circulating neutrophils, and normal to elevated cutaneous expression of Il17a and IL-17A inducible neutrophil attracting chemokines Cxcl1, Cxcl2 and Cxcl3. DOCK8 deficient neutrophils were significantly more susceptible to cell death upon in vitro exposure to S. aureus and exhibited reduced phagocytosis of S. aureus bioparticles but had a normal respiratory burst. Impaired neutrophil survival in infected skin and defective neutrophil phagocytosis likely underlie the susceptibility to cutaneous S. aureus infection in DOCK8 deficiency.

Disrupting the Dok3-Card9 Interaction with Synthetic Peptides Enhances Antifungal Effector Functions of Human Neutrophils.

Invasive fungal disease is an emerging and serious public health threat globally. The expanding population of susceptible individuals, together with the rapid emergence of multidrug-resistant fungi pathogens, call for the development of novel therapeutic strategies beyond the limited repertoire of licensed antifungal drugs. Card9 is a critical signaling molecule involved in antifungal defense; we have previously identified Dok3 to be a key negative regulator of Card9 activity in neutrophils. In this study, we identified two synthetic peptides derived from the coiled-coil domain of Card9, which can specifically block Dok3-Card9 binding. We showed that these peptides are cell-permeable, non-toxic, and can enhance antifungal cytokine production and the phagocytosis of human neutrophils upon fungal infection. Collectively, these data provide a proof of concept that disrupting the Dok3-Card9 interaction can boost the antifungal effector functions of neutrophils; they further suggest the potential utility of these peptide inhibitors as an immune-based therapeutic to fight fungal infection.

Coronavac inactivated vaccine triggers durable, cross-reactive Fc-mediated phagocytosis activities

ABSTRACT Although humoral responses elicited by infection or vaccine lost the ability to prevent transmission against Omicron, vaccine-induced antibodies may still contribute to disease attenuation through Fc-mediated effector functions. However, Fc effector function elicited by CoronaVac, as the most widely supplied inactivated vaccine globally, has not been characterized. For the first time, our study depicted Fc-mediated phagocytosis activity induced by CoronaVac, including antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent neutrophil phagocytosis (ADNP) activities, and further compared with that from convalescent individuals and CoronaVac recipients with subsequent breakthrough infections. We showed that 2-dose of CoronaVac effectively induced both ADCP and ADNP, but was substantially lower compared to infection, whereas the booster dose further augmented ADCP and ADNP responses, and remained detectable for 52 weeks. Among CoronaVac recipients, ADCP and ADNP responses also demonstrated cross-reactivity against Omicron subvariants, and breakthrough infection could enhance the phagocytic response. Meanwhile, serum samples from vaccinees, convalescent individuals with wildtype infection, BA.2 and BA.5 breakthrough infection demonstrated differential cross-reactive ADCP and ADNP responses against Omicron subvariants, suggesting the different subvariants of spike antigen exposure might alter the cross-reactivity of Fc effector function. Further, ADCP and ADNP responses were strongly correlated with Spike-specific IgG responses and neutralizing activities, indicating coordinated neutralization activity, ADCP and ADNP responses triggered by CoronaVac. Of note, the ADCP and ADNP responses were more durable and cross-reactive than corresponding Spike-specific IgG titers and neutralizing activities. Our study has important implications for optimal boosting vaccine strategies that may induce potent and broad Fc-mediated phagocytic activities.

Dock2 generates characteristic spatiotemporal patterns of Rac activity to regulate neutrophil polarisation, migration and phagocytosis.

Rac-GTPases and their Rac-GEF activators play important roles in neutrophil-mediated host defence. These proteins control the adhesion molecules and cytoskeletal dynamics required for neutrophil recruitment to inflamed and infected organs, and the neutrophil effector responses that kill pathogens. Here, we used live cell TIRF-FRET imaging in neutrophils from Rac-FRET reporter mice with deficiencies in the Rac-GEFs Dock2, Tiam1 or Prex1/Vav1 to evaluate if these proteins activate spatiotemporally distinct pools of Rac, and to correlate patterns of Rac activity with the neutrophil responses they control. All the GEFs were required for neutrophil adhesion, and Prex1/Vav1 were important during spreading and for the velocity of migration during chemotaxis. However, Dock2 emerged as the prominent regulator of neutrophil responses, as this GEF was required for neutrophil polarisation and random migration, for migration velocity during chemokinesis, for the likelihood to migrate and for the speed of migration and of turning during chemotaxis, as well as for rapid particle engulfment during phagocytosis. We identified characteristic spatiotemporal patterns of Rac activity generated by Dock2 which correlate with the importance of the Rac-GEF in these neutrophil responses. We also demonstrate a requirement for Dock2 in neutrophil recruitment during aseptic peritonitis. Collectively, our data provide a first direct comparison of the pools of Rac activity generated by different types of Rac-GEFs, and identify Dock2 as a key regulator of polarisation, migration and phagocytosis in primary neutrophils.

Infectious neutrophil deployment is regulated by resolvin D4

AbstractNeutrophils reside in the bone marrow (BM), ready for deployment to sites of injury/infection, initiating inflammation and its resolution. Here, we report that distal infections signal to the BM via resolvins to regulate granulopoiesis and BM neutrophil deployment. Emergency granulopoiesis during peritonitis evoked changes in BM resolvin D1 (RvD1) and BM RvD4. We found that leukotriene B4 stimulates neutrophil deployment. RvD1 and RvD4 each limited neutrophilic infiltration to infections, and differently regulated BM myeloid populations: RvD1 increased reparative monocytes, and RvD4 regulated granulocytes. RvD4 disengaged emergency granulopoiesis, prevented excess BM neutrophil deployment, and acted on granulocyte progenitors. RvD4 also stimulated exudate neutrophil, monocyte, and macrophage phagocytosis, and enhanced bacterial clearance. This mediator accelerated both neutrophil apoptosis and clearance by macrophages, thus expediting the resolution phase of inflammation. RvD4 stimulated phosphorylation of ERK1/2 and STAT3 in human BM-aspirate–derived granulocytes. RvD4 in the 1 to 100 nM range stimulated whole-blood neutrophil phagocytosis of Escherichia coli. RvD4 increased BM macrophage efferocytosis of neutrophils. Together, these results demonstrate the novel functions of resolvins in granulopoiesis and neutrophil deployment, contributing to the resolution of infectious inflammation.

Immunopathology of early and advanced epididymis lesions caused by Brucella ovis in rams

Ovine brucellosis is an infectious disease that causes alterations in the reproductive tract in ram and abortion in ewes. Their negative economic impact in ovine production warrants a thorough understanding the interactions between B. ovis and the host. Here, epididymis lesions of rams infected by B. ovis were histopathologically staged into early and advanced. Expression by immunohistochemistry of Brucella antigens, inflammatory cell markers (CD3, CD79αcy) and cytokines (IFN-γ, TNF-α, TGF-β1) was assessed in both stages. Early lesions were characterized by epithelial changes, interstitial inflammation, and mild fibrosis; whereas advanced lesions displayed caseous granulomas containing numerous macrophages, multinucleated giant cells, lymphocytes, and plasma cells. Expression of Brucella antigens were observed in both stages. The cellular response in B. ovis lesions were predominantly of T-cells (CD3+) whereas low numbers of B-cells and plasma cells (CD79αcy+) were present in both early and advanced lesions. IFN-γ was expressed by lymphocytes in early lesions suggesting that the adaptive immune response against B. ovis is initiated by Th1 cells, this response was also preserved in advanced stages. Expression of TNF-α was observed in neutrophils of epithelial microabscesses and intraepithelial T-cells of early lesions suggesting a promotion of neutrophil phagocytosis triggered by TNF-α. On the other hand, advanced lesions showed a reduction of TNF-α expression which may permit B. ovis persistence in granulomas. Lastly, TGF-β1 expression (fibroblast, macrophages and less in lymphocytes) were increased with time, suggesting that B. ovis promotes TGF-β1 secretion promoting chronicity of the lesions.

Resolvin D1 reprograms energy metabolism to promote microglia to phagocytize neutrophils after ischemic stroke

Neutrophil aggregation and clearance are important factors affecting neuroinflammatory injury during acute ischemic stroke. Emerging evidence suggests that energy metabolism is essential for microglial functions, especially microglial phagocytosis, which determines the degree of brain injury. Here, we demonstrate that Resolvin D1 (RvD1), a lipid mediator derived from docosahexaenic acid (DHA), promotes the phagocytosis of neutrophils by microglia, thereby reducing neutrophil accumulation in the brain and alleviating neuroinflammation in the ischemic brain. Further studies reveal that RvD1 reprograms energy metabolism from glycolysis to oxidative phosphorylation (OXPHOS), providing sufficient energy for microglial phagocytosis. Moreover, RvD1 enhances microglial glutamine uptake and stimulates glutaminolysis to support OXPHOS to boost ATP production depending on adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) activation. Overall, our results reveal that RvD1 reprograms energy metabolism to promote the microglial phagocytosis of neutrophils after ischemic stroke. These findings may guide perspectives for stroke therapy from modulating microglial immunometabolism.

Phagocytosis and oxidative activity of neutrophils after interaction with uropathogenic <i>Escherichia coli</i> biofilms

Recurrent urinary tract infections (UTIs) are associated primarily with the ability of Escherichia coli to form biofilms. The interaction of neutrophils, factors of innate immunity, with microorganisms in biofilms is difficult compared to planktonic forms due to the lack of direct contact, as well as due to the antiphagocytic action of the extracellular matrix of biofilms. The purpose of this study was evaluation of neutrophils phagocytic and oxidative activity during interaction with biofilms of uropathogenic E. coli (UPEC) DL82 and R44. Peripheral blood neutrophils from healthy men were isolated using ficoll-urographin double gradient, incubated for 1 h with bacterial cells from biofilms or their supernatants, then leukocytes functional activity was evaluated. Phagocytic activity of neutrophils was determined by the degree of bioluminescence inhibition of bioluminescent strain E. coli K12 TG1 lux+ (pXen) upon their absorption by neutrophils. Production of extracellular reactive oxygen species (ROS) was analyzed by the intensity of luminol-dependent chemiluminescence in spontaneous and stimulated by E. coli K12 variants. Significance of differences was determined using Student’s t-test at p < 0.05. It was found that neutrophils interaction with UPEC biofilm cells or supernatants did not affect the phagocytic activity. E. coli DL82 supernatants reduce neutrophils spontaneous ROS production compared to control and biofilm cells. E. coli R44 supernatants with a low virulence potential did not affect ROS production, while biofilm cells stimulated it. When assessing stimulated ROS production, exposure to R44 strain supernatants did not cause a decrease in neutrophils activation in response to an external stimulus (E. coli K12 cells). Preliminary contact of neutrophils with E. coli R44 bacteria resulted in a high and prolonged level of ROS production compared to the control. Neutrophils interaction with DL82 cells resulted in a higher level of ROS compared to supernatants, however a subsequent rapid depletion of neutrophils oxidative potential was observed. Thus, cells and supernatants of UPEC biofilms can determine the activation of neutrophils.

The immunological effects against invasive aspergillosis disease on inbred mice after the dietary intake of honey varieties with the determination of diastase and invertase enzyme activities

Aspergillus fumigatus is a universal pathogenic mold. Invasive aspergillosis disease can affect persons with weakened immune systems. Natural immunostimulators are very important in improving the immune response in contradiction to various diseases. In the current study four varieties of honey samples (nigella, moringa, sidr, and pumpkin honey) which are coded as NS, MO, SI, and PU honey; were used to evaluate diastase and invertase enzyme activities. Eight groups of mice were fed with the four honey varieties, after injecting half of them with invasive aspergillosis infection and comparing results with the positive and negative control of mice groups. Diastase enzyme activity in honey samples ranged from 15.10 ND by Goth Scale in PU Honey to 23.22 ND by Goth Scale in NS Honey. Invertase enzyme activity in honey samples ranged from 21.99 IN in PU Honey to 26.57 IN in MO Honey. All honey varieties activated the cytokine productions such as IL1β and IL6 cytokines. Honey can act as a killing stimulator against invasive aspergillosis infections due to the presence of neutrophil phagocytosis levels in rats. The survival rates among animals treated with the four honey varieties varied from 12.37 in PU Honey Group+Infection to 26.15 in SI Honey Group+Infection. Honey can positively increase innate immune responses and survival rates, and it may be used as a prophylactic or beneficial agent for several diseases.

Chenopodium murale Juice Shows Anti-Fungal Efficacy in Experimental Oral Candidiasis in Immunosuppressed Rats in Relation to Its Chemical Profile.

Chenopodium murale (Syn. Chenopodiastrum murale) (amaranthaceae) is used in the rural Egypt to treat oral ulcers in newborn children. The current study aimed to discover new natural products suitable for treating candidiasis disease with minimal side effects. Characterization of bioactive compounds by LC-QTOF-HR-MS/MS from Chenopodium murale fresh leaves' juice (CMJ) was carried out in order to elucidate their potential anti-fungal and immunomodulatory effects in oral candidiasis in immunosuppressed rats. An oral ulcer candidiasis model was created in three stages: (i) immunosuppression by drinking dexamethasone (0.5 mg/L) for two weeks; (ii) Candida albicans infection (3.00 × 106 viable cell/mL) for one week; and (iii) treatment with CMJ (0.5 and 1.0 g/kg orally) or nystatin (1,000,000 U/L orally) for one week. Two doses of CMJ exhibited antifungal effects, for example, through a significant reduction in CFU/Petri (236.67 ± 37.86 and 4.33 ± 0.58 CFU/Petri), compared to the Candida control (5.86 × 104 ± 1.21 CFU/Petri), p ≤ 0.001. In addition, CMJ significantly induced neutrophil production (32.92% ± 1.29 and 35.68% ± 1.77) compared to the Candida control level of 26.50% ± 2.44. An immunomodulatory effect of CMJ at two doses appeared, with a considerable elevation in INF-γ (103.88 and 115.91%), IL-2 (143.50, 182.33%), and IL-17 (83.97 and 141.95% Pg/mL) compared with the Candida group. LC-MS/MS analysis operated in negative mode was used for tentative identification of secondary (SM) metabolites based on their retention times and fragment ions. A total of 42 phytoconstituents were tentatively identified. Finally, CMJ exhibited a potent antifungal effect. CMJ fought Candida through four strategies: (i) promotion of classical phagocytosis of neutrophils; (ii) activation of T cells that activate IFN-γ, IL-2, and IL-17; (iii) increasing the production of cytotoxic NO and H2O2 that can kill Candida; and (iv) activation of SOD, which converts superoxide to antimicrobial materials. These activities could be due to its active constituents, which are documented as anti-fungal, or due to its richness in flavonoids, especially the active compounds of kaempferol glycosides and aglycone, which have been documented as antifungal. After repetition on another type of small experimental animal, their offspring, and an experimental large animal, this study may lead to clinical trials.

Lung Inflammation Resolution by RvD1 and RvD2 in a Receptor-Dependent Manner.

Inflammation resolution is an active process via specialized pro-resolving mediators (SPMs) to fight invading microbes and repair tissue injury. RvD1 and RvD2 are SPMs produced from DHA during inflammation responses and show a benefit in treating inflammation disorders, but it is not completely understood how they act on vasculature and immune cells in the lung to promote inflammation resolution programs. Here, we studied how RvD1 and RvD2 regulated the interactions between endothelial cells and neutrophils in vitro and in vivo. In an acute lung inflammation (ALI) mouse model, we found that RvD1 and RvD2 resolved lung inflammation via their receptors (ALX/GPR32 or GPR18) and enhanced the macrophage phagocytosis of apoptotic neutrophils, which may be the molecular mechanism of lung inflammation resolution. Interestingly, we observed the higher potency of RvD1 over RvD2, which may be associated with unique downstream signaling pathways. Together, our studies suggest that the targeted delivery of these SPMs into inflammatory sites may be novel strategies with which to treat a wide range of inflammatory diseases.

Methylprednisolone improves the quality of liquid preserved boar spermatozoa in vitro and reduces polymorphonuclear neutrophil chemotaxis and phagocytosis.

After artificial insemination, immune cells such as polymorphonuclear neutrophils will be recruited into the genital tract and induce endometrial inflammation, adversely affecting the spermatozoa. This study aimed to analyze the effect of methylprednisolone (MPS) on boar spermatozoa quality of in vitro liquid preservation and chemotaxis and phagocytosis of polymorphonuclear neutrophils toward boar spermatozoa. Various concentrations of MPS were added to the extender and analyzed for their effects on spermatozoa motility, kinetic parameters, abnormality rate, total antioxidant capacity (T-AOC) levels, H2O2 content, mitochondrial membrane potential and acrosome integrity. Testing of MPS on chemotaxis and phagocytosis of polymorphonuclear neutrophils toward spermatozoa induced by lipopolysaccharide (LPS). The results showed that an extender containing 2 × 10-7 mol/mL MPS was the most effective for preserving boar spermatozoa during in vitro liquid preservation at 17°C. It effectively improved spermatozoa motility, kinetic parameters, T-AOC levels, mitochondrial membrane potential and acrosome integrity, reducing the abnormality rate and H2O2 content. Meanwhile, the chemotaxis and phagocytosis of polymorphonuclear neutrophils toward spermatozoa under LPS induction were inhibited in a concentration-dependent manner. In conclusion, MPS has positive implications for improving in vitro liquid preserved boar spermatozoa quality, inhibiting chemotaxis and phagocytosis of polymorphonuclear neutrophils toward spermatozoa.

Neutrophil Structural and Functional Alterations After High Mechanical Shear Stress Exposure.

Patients on mechanical circulatory support are prone to infections, increasing morbidity and mortality. These circulatory support devices generate high mechanical shear stress (HMSS) that can causes trauma to blood. When leukocytes become damaged, their immune response function may be impaired or weakened, leading to increased infection vulnerability. This study examined neutrophil structural and functional alterations after exposure to 75, 125, and 175 Pa HMSS for 1 second. Human blood was exposed to three levels of HMSS using a blood shearing device. Neutrophil morphological alteration was characterized by examining blood smears. Flow cytometry assays were used to analyze expression levels of CD62L and CD162 receptors, activation level (CD11b), and aggregation (platelet-neutrophil aggregates). Neutrophil phagocytosis and rolling were examined via functional assays. The results show neutrophil structure (morphology and surface receptors) and function (activation, aggregation, phagocytosis, rolling) were significantly altered after HMSS exposure. These alterations include cell membrane damage, loss of surface receptors (CD62L and CD162), initiation of activation and aggregation, upregulation of phagocytic ability and increased rolling speed. The alterations were the most severe after 175 Pa exposure. HMSS caused damage and activation of neutrophils, potentially impairing normal neutrophil function, leading to weakened immune defense and increasing a patient's vulnerability to infections.

Sulforaphane Attenuates Neutrophil ROS Production, MPO Degranulation and Phagocytosis, but Does Not Affect NET Formation Ex Vivo and In Vitro.

Sulforaphane has several effects on the human body, including anti-inflammation, antioxidation, antimicrobial and anti-obesity effects. In this study, we examined the effect of sulforaphane on several neutrophil functions: reactive oxygen species (ROS) production, degranulation, phagocytosis, and neutrophil extracellular trap (NET) formation. We also examined the direct antioxidant effect of sulforaphane. First, we measured neutrophil ROS production induced by zymosan in whole blood in the presence of 0 to 560 µM sulforaphane. Second, we examined the direct antioxidant activity of sulforaphane using a HOCl removal test. In addition, inflammation-related proteins, including an azurophilic granule component, were measured by collecting supernatants following ROS measurements. Finally, neutrophils were isolated from blood, and phagocytosis and NET formation were measured. Sulforaphane reduced neutrophil ROS production in a concentration-dependent manner. The ability of sulforaphane to remove HOCl is stronger than that of ascorbic acid. Sulforaphane at 280 µM significantly reduced the release of myeloperoxidase from azurophilic granules, as well as that of the inflammatory cytokines TNF-α and IL-6. Sulforaphane also suppressed phagocytosis but did not affect NET formation. These results suggest that sulforaphane attenuates neutrophil ROS production, degranulation, and phagocytosis, but does not affect NET formation. Moreover, sulforaphane directly removes ROS, including HOCl.