Anti-inflammatory effect of Trichospira verticillata via suppression of the NLRP3 inflammasome in neutrophilic asthma.

Trichospira verticillata is an annual herb that belongs to the family Asteraceae. Trichospira verticillata extract (TVE) elicits anti-plasmodial activity; however, there has been no detailed report about its anti-inflammatory effects and molecular mechanisms. In addition, herbal plants exhibit anti-inflammatory effects by suppressing the NLRP3 inflammasome. Therefore, the primary goal of this study was to examine the effects of TVE on NLRP3 inflammasome activation by measuring interleukin-1ß (IL-1ß) secretion. We treated lipopolysaccharides (LPS)-primed J774A.1 and THP-1 cells with TVE, which attenuated NLRP3 inflammasome activation. Notably, TVE did not affect nuclear factor-kappa B (NF-κB) signalling or intracellular reactive oxygen species (ROS) production and potassium efflux, suggesting that it inactivates the NLRP3 inflammasome via other mechanisms. Moreover, TVE suppressed the formation of apoptosis-associated speck-like protein (ASC) speck and oligomerization. Immunoprecipitation data revealed that TVE reduced the binding of NLRP3 to NIMA-related kinase 7 (NEK7), resulting in reduced ASC oligomerization and speck formation. Moreover, TVE alleviated neutrophilic asthma (NA) symptoms in mice. This study demonstrates that TVE modulates the binding of NLPR3 to NEK7, thereby reporting novel insights into the mechanism by which TVE inhibits NLRP3 inflammasome. These findings suggest TVE as a potential therapeutic of NLRP3 inflammasome-mediated diseases, particularly NA.

Integrative analysis of network pharmacology and proteomics reveal the protective effect of Xiaoqinglong Decotion on neutrophilic asthma.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiaoqinglong Decotion (XQLD) is a commonly used Chinese herbal formula in clinical practice, especially for allergic diseases such as asthma. However, its intrinsic mechanism for the treatment of neutrophilic asthma (NA) remains unclear. AIM OF THE STUDY: The aim of this study was to evaluate the efficacy and potential mechanisms of XQLD on NA using network pharmacology and in vivo experiments. MATERIALS AND METHODS: First, the active compounds, potential targets and mechanisms of XQLD against NA were initially elucidated by network pharmacology. Then, OVA/CFA-induced NA mice were treated with XQLD to assess its efficacy. Proteins were then analyzed and quantified using a Tandem Mass Tags approach for differentially expressed proteins (DEPs) to further reveal the mechanisms of NA treatment by XQLD. Finally, the hub genes, critical DEPs and potential pathways were validated. RESULTS: 176 active compounds and 180 targets against NA were identified in XQLD. Protein-protein interaction (PPI) network revealed CXCL10, CX3CR1, TLR7, NCF1 and FABP4 as hub genes. In vivo experiments showed that XQLD attenuated inflammatory infiltrates, airway mucus secretion and remodeling in the lungs of NA mice. Moreover, XQLD significantly alleviated airway neutrophil inflammation in NA mice by decreasing the expression of IL-8, MPO and NE. XQLD also reduced the levels of CXCL10, CX3CR1, TLR7, NCF1 and FABP4, which are closely associated with neutrophil inflammation. Proteomics analysis identified 28 overlapping DEPs in the control, NA and XQLD groups, and we found that XQLD inhibited ferroptosis signal pathway (elevated GPX4 and decreased ASCL3) as well as the expression of ARG1, MMP12 and SPP1, while activating the Rap1 signaling pathway. CONCLUSION: This study revealed that inhibition of ARG1, MMP12 and SPP1 expression as well as ferroptosis pathways, and activation of the Rap1 signaling pathway contribute to the therapeutic effect of XQLD on NA.

Neutrophil Extracellular Traps in Tumors and Potential Use of Traditional Herbal Medicine Formulations for Its Regulation.

Neutrophil extracellular traps (NETs) are extracellular fibers composed of deoxyribonucleic acid (DNA) and decorated proteins produced by neutrophils. Recently, NETs have been associated with the development of many diseases, including tumors. Herein, we reviewed the correlation between NETs and tumors. In addition, we detailed active compounds from traditional herbal medicine formulations that inhibit NETs, related nanodrug delivery systems, and antibodies that serve as "guiding moieties" to ensure targeted delivery to NETs. Furthermore, we discussed the strategies used by pathogenic microorganisms to evade NETs.

Inhibiting Neutrophil Extracellular Traps Protects against Ultraviolet B-Induced Skin Damage: Effects of Hochu-ekki-to and DNase I.

UV-B radiation induces sunburn, and neutrophils are pivotal in this inflammation. In this study, we examined the potential involvement of neutrophil extracellular traps (NETs) in ultraviolet B (UVB)-induced skin inflammation, correlating the skin inflammation-mitigating effects of Hochu-ekki-to on UV-B irradiation and NETs. To elucidate NET distribution in the dorsal skin, male ICR mice, exposed to UVB irradiation, were immunohistologically analyzed to detect citrullinated histone H3 (citH3) and peptidylarginine deiminase 4 (PAD4). Reactive oxygen species (ROS) production in the bloodstream was analyzed. To establish the involvement of NET-released DNA in this inflammatory response, mice were UV-B irradiated following the intraperitoneal administration of DNase I. In vitro experiments were performed to scrutinize the impact of Hochu-ekki-to on A23187-induced NETs in neutrophil-like HL-60 cells. UV-B irradiation induced dorsal skin inflammation, coinciding with a significant increase in citH3 and PAD4 expression. Administration of DNase I attenuated UV-B-induced skin inflammation, whereas Hochu-ekki-to administration considerably suppressed the inflammation, correlating with diminished levels of citH3 and PAD4 in the dorsal skin. UV-B irradiation conspicuously augmented ROS and hydrogen peroxide (H2O2) production in the blood. Hochu-ekki-to significantly inhibited ROS and H2O2 generation. In vitro experiments demonstrated that Hochu-ekki-to notably inhibited A23187-induced NETs in differentiated neutrophil-like cells. Hence, NETs have been implicated in UV-B-induced skin inflammation, and their inhibition reduces cutaneous inflammation. Additionally, Hochu-ekki-to mitigated skin inflammation by impeding neutrophil infiltration and NETs in the dorsal skin of mice.

Incorporation of Pectin into Vaterite Microparticles Prevented Effects of Adsorbed Mucin on Neutrophil Activation.

The application of vaterite microparticles for mucosal delivery depends on their interaction with mucin and immune cells. As we have shown previously, the binding of mucin onto particles enhances the generation of reactive oxygen species by neutrophils. The attenuation of the pro-oxidant effect of the bound mucin through the modification of vaterite could improve its biocompatibility. Hybrid microparticles composed of vaterite and pectin (CCP) were prepared using co-precipitation. In comparison with vaterite (CC), they had a smaller diameter and pores, a greater surface area, and a negative zeta-potential. We aimed to study the cytotoxicity and mucin-dependent neutrophil-activating effect of CCP microparticles. The incorporated pectin did not influence the neutrophil damage according to a lactate dehydrogenase test. The difference in the CC- and CCP-elicited luminol or lucigenin chemiluminescence of neutrophils was insignificant, with no direct pro- or antioxidant effects from the incorporated pectin. Unlike soluble pectin, the CCP particles were ineffective at scavenging radicals in an ABAP-luminol test. The fluorescence of SYTOX Green demonstrated a CCP-stimulated formation of neutrophil extracellular traps (NETs). The pre-treatment of CC and CCP with mucin resulted in a 2.5-times-higher CL response of neutrophils to the CC-mucin than to the CCP-mucin. Thus, the incorporation of pectin into vaterite microspheres enabled an antioxidant effect to be reached when the neutrophils were activated by mucin-treated microparticles, presumably via exposed ligands.

Physiological Concentrations of Calcium Interact with Alginate and Extracellular DNA in the Matrices of Pseudomonas aeruginosa Biofilms to Impede Phagocytosis by Neutrophils.

Biofilms are communities of interacting microbes embedded in a matrix of polymer, protein, and other materials. Biofilms develop distinct mechanical characteristics that depend on their predominant matrix components. These matrix components may be produced by microbes themselves or, for infections in vivo, incorporated from the host environment. Pseudomonas aeruginosa (P. aeruginosa) is a human pathogen that forms robust biofilms that extensively tolerate antibiotics and effectively evade clearance by the immune system. Two of the important bacterial-produced polymers in the matrices of P. aeruginosa biofilms are alginate and extracellular DNA (eDNA), both of which are anionic and therefore have the potential to interact electrostatically with cations. Many physiological sites of infection contain significant concentrations of the calcium ion (Ca2+). In this study, we investigate the structural and mechanical impacts of Ca2+ supplementation in alginate-dominated biofilms grown in vitro, and we evaluate the impact of targeted enzyme treatments on clearance by immune cells. We use multiple-particle tracking microrheology to evaluate the changes in biofilm viscoelasticity caused by treatment with alginate lyase or DNase I. For biofilms grown without Ca2+, we correlate a decrease in relative elasticity with increased phagocytic success. However, we find that growth with Ca2+ supplementation disrupts this correlation except in the case where both enzymes are applied. This suggests that the calcium cation may be impacting the microstructure of the biofilm in nontrivial ways. Indeed, confocal laser scanning fluorescence microscopy and scanning electron microscopy reveal unique Ca2+-dependent eDNA and alginate microstructures. Our results suggest that the presence of Ca2+ drives the formation of structurally and compositionally discrete microdomains within the biofilm through electrostatic interactions with the anionic matrix components eDNA and alginate. Further, we observe that these structures serve a protective function as the dissolution of both components is required to render biofilm bacteria vulnerable to phagocytosis by neutrophils.

Positive association of acetylcholinesterase (AChE) with the neutrophil-to-lymphocyte ratio and HbA1c, and a negative association with hydrogen sulfide (H2S) levels among healthy African Americans, and H2S-inhibition and high-glucose-upregulation of AChE in cultured THP-1 human monocytes.

The incidence of Alzheimer's disease (AD) is higher in people over the age of 65 and in African Americans (AA). Elevated acetylcholinesterase (AChE) activity has been considered a major player in the onset of AD symptoms. As a result, many FDA-approved AD drugs target AChE inhibition to treat AD patients. Hydrogen sulfide (H2S) is a signaling molecule known to downregulate oxidative stress and inflammation. The neutrophil-to-lymphocyte ratio (NLR) in the blood is widely used as a biomarker to monitor inflammation and immunity. This study examined the hypothesis that plasma AChE levels have a negative association with H2S levels and that a positive association exists between levels of NLR, HbA1c, and ROS with the AChE in the peripheral blood. The fasting blood sample was taken from 114 African Americans who had provided written informed consent approved by the IRB. The effect of H2S and high-glucose treatment on AChE activity levels was also investigated in THP-1 human monocytes. There was a significant negative relationship between AChE and the levels of H2S (r = -0.41, p = 0.001); a positive association between the levels of AChE with age (r = 0.26, p = 0.03), NLR (r = 0.23, p = 0.04), ROS (r = 0.23, p = 0.04) and HbA1c levels (r = 0.24, p = 0.04), in AA subjects. No correlation was seen between blood levels of AChE and acetylcholine (ACh). Blood creatinine had a negative correlation (r = -0.23, p = 0.04) with ACh levels. There was a significant effect of H2S on AChE inhibition and of high glucose in upregulating AChE activity in cultured monocytes. This study suggests hyperglycemia and lower H2S status can contribute to an increase in the AChE activity levels. Future clinical studies are needed to examine the potential benefits of supplementation with hydrogen sulfide pro-drugs/compounds in reducing the AChE and the cognitive dysfunctions associated with AD.

Effect of curcumin on inflammatory biomarkers and iron profile in patients with premenstrual syndrome and dysmenorrhea: A randomized controlled trial.

Premenstrual syndrome (PMS) and primary dysmenorrhea are common gynecological problems and inflammation may have a role in their etiology. Curcumin is a polyphenolic natural product for which there is increasing evidence of anti-inflammatory and iron chelation effects. This study assessed the effects of curcumin on inflammatory biomarkers and iron profile in young women with PMS and dysmenorrhea. A sample of 76 patients was included in this triple-blind, placebo-controlled clinical trial. Participants were randomly allocated to curcumin (n = 38) and control groups (n = 38). Each participant received one capsule (500 mg of curcuminoid+ piperine, or placebo) daily, from 7 days before until 3 days after menstruation for three consecutive menstrual cycles. Serum iron, ferritin, total iron-binding capacity (TIBC) and high-sensitivity C-reactive protein (hsCRP), as well as white blood cell, lymphocyte, neutrophil, platelet counts, mean platelet volume (MPV) and red blood cell distribution width (RDW), were quantified. Neutrophil: lymphocyte ratio (NLR), platelet: lymphocyte ratio (PLR), and RDW: platelet ratio (RPR) were also calculated. Curcumin significantly decreased the median (interquartile range) serum levels of hsCRP [from 0.30 mg/L (0.0-1.10) to 0.20 mg/L (0.0-1.3); p = 0.041] compared with placebo, but did not show any difference for neutrophil, RDW, MPV, NLR, PLR and RPR values (p > 0.05). The treatment schedule was well-tolerated, and none of markers of iron metabolism statistically changed after the intervention in the curcumin group (p > 0.05). Curcumin supplementation may have positive effects on serum hsCRP, a marker of inflammation, with no any changes on iron homeostasis in healthy women with PMS and dysmenorrhea.

Cytokine Storm in Acute Viral Respiratory Injury: Role of Qing-Fei-Pai-Du Decoction in Inhibiting the Infiltration of Neutrophils and Macrophages through TAK1/IKK/NF-[Formula: see text]B Pathway.

COVID-19 has posed unprecedented challenges to global public health since its outbreak. The Qing-Fei-Pai-Du decoction (QFPDD), a Chinese herbal formula, is widely used in China to treat COVID-19. It exerts an impressive therapeutic effect by inhibiting the progression from mild to critical disease in the clinic. However, the underlying mechanisms remain obscure. Both SARS-CoV-2 and influenza viruses elicit similar pathological processes. Their severe manifestations, such as acute respiratory distress syndrome (ARDS), multiple organ failure (MOF), and viral sepsis, are correlated with the cytokine storm. During flu infection, QFPDD reduced the lung indexes and downregulated the expressions of MCP-1, TNF-[Formula: see text], IL-6, and IL-1[Formula: see text] in broncho-alveolar lavage fluid (BALF), lungs, or serum samples. The infiltration of neutrophils and inflammatory monocytes in lungs was decreased dramatically, and lung injury was ameliorated in QFPDD-treated flu mice. In addition, QFPDD also inhibited the polarization of M1 macrophages and downregulated the expressions of IL-6, TNF-[Formula: see text], MIP-2, MCP-1, and IP-10, while also upregulating the IL-10 expression. The phosphorylated TAK1, IKK[Formula: see text]/[Formula: see text], and I[Formula: see text]B[Formula: see text] and the subsequent translocation of phosphorylated p65 into the nuclei were decreased by QFPDD. These findings indicated that QFPDD reduces the intensity of the cytokine storm by inhibiting the NF-[Formula: see text]B signaling pathway during severe viral infections, thereby providing theoretical and experimental support for its clinical application in respiratory viral infections.

Baicalin promotes antiviral IFNs production and alleviates type I IFN-induced neutrophil inflammation.

Type I and III interferons (IFNs) both serve as pivotal components of the host antiviral innate immune system. Although they exert similar antiviral effects, type I IFNs can also activate neutrophil inflammation, a function not born by type III IFNs. Baicalin, the main bioactive component of Scutellariae radix, has been shown to exert therapeutic effects on viral diseases due to its anti-viral, anti-inflammatory and immunomulatory activities. There is uncertainty, however, on the association between the antiviral effects of baicalin and the modulation of anti-viral IFNs production and the immunological effects of type I IFNs. Here, a Poly (I:C)-stimulated A549 cell line was established to mimic a viral infection model. Our results demonstrated that baicalin could elevate the expression of type I and III IFNs and their receptors in Poly (I:C)-stimulated A549 cells. Moreover, the potential regulation effects of baicalin for type I IFN-induced neutrophil inflammation was further explored. Results showed that baicalin diminished the production of the pro-inflammatory cytokines (IL-1ß, IL-6, IL-17 and TNF-α), ROS, and neutrophil extracellular traps and suppressed chemotaxis. Collectively, all these data indicated that baicalin had a dual role on IFNs production and effects: (1) Baicalin was able to elevate the expression of type I and III IFNs and their receptors, (2) and it alleviated type I IFN-mediated neutrophil inflammatory response. This meant that baicalin has the potential to act as an eximious immunomodulator, exerting antiviral effects and reducing inflammation.

Abnormal adenosine metabolism of neutrophils inhibits airway inflammation and remodeling in asthma model induced by Aspergillus fumigatus.

BACKGROUND: Neutrophils consume a large amount of energy when performing their functions. Compared with other white blood cells, neutrophils contain few mitochondria and mainly rely on glycolysis and gluconeogenesis to produce ATP. The inflammatory site is hypoxic and nutrient poor. Our aim is to study the role of abnormal adenosine metabolism of neutrophils in the asthmatic airway inflammation microenvironment. METHOD: In this study, an asthma model was established by intratracheal instillation of Aspergillus fumigatus extract in Ecto-5'-Nucleotidase (CD73) gene-knockout and wild-type mice. Multiple analyses from bronchoalveolar lavage fluid (BALF) were used to determine the levels of cytokines and chemokines. Immunohistochemistry was used to detect subcutaneous fibrosis and inflammatory cell infiltration. Finally, adenosine 5'-(α, ß-methylene) diphosphate (APCP), a CD73 inhibitor, was pumped subcutaneously before Aspergillus attack to observe the infiltration of inflammatory cells and subcutaneous fibrosis to clarify its therapeutic effect. RESULT: PAS staining showed that CD73 knockout inhibited pulmonary epithelial cell proliferation and bronchial fibrosis induced by Aspergillus extract. The genetic knockdownof CD73 significantly reduced the production of Th2 cytokines, interleukin (IL)-4, IL-6, IL-13, chemokine (C-C motif) ligand 5 (CCL5), eosinophil chemokine, neutrophil IL-17, and granulocyte colony-stimulating factor (G-CSF). In addition, exogenous adenosine supplementation increased airway inflammation. Finally, the CD73 inhibitor APCP was administered to reduce inflammation and subcutaneous fibrosis. CONCLUSION: Elevated adenosine metabolism plays an inflammatory role in asthma, and CD73 could be a potential therapeutic target for asthma.

Neutrophil extracellular trap production and CCL4L2 expression influence corticosteroid response in asthma.

The association between neutrophil extracellular traps (NETs) and response to inhaled corticosteroids (ICS) in asthma is unclear. To better understand this relationship, we analyzed the blood transcriptomes from children with controlled and uncontrolled asthma in the Taiwanese Consortium of Childhood Asthma Study using weighted gene coexpression network analysis and pathway enrichment methods. We identified 298 uncontrolled asthma-specific differentially expressed genes and one gene module associated with neutrophil-mediated immunity, highlighting a potential role for neutrophils in uncontrolled asthma. We also found that NET abundance was associated with nonresponse to ICS in patients. In a neutrophilic airway inflammation murine model, steroid treatment could not suppress neutrophilic inflammation and airway hyperreactivity. However, NET disruption with deoxyribonuclease I (DNase I) efficiently inhibited airway hyperreactivity and inflammation. Using neutrophil-specific transcriptomic profiles, we found that CCL4L2 was associated with ICS nonresponse in asthma, which was validated in human and murine lung tissue. CCL4L2 expression was also negatively correlated with pulmonary function change after ICS treatment. In summary, steroids fail to suppress neutrophilic airway inflammation, highlighting the potential need to use alternative therapies such as leukotriene receptor antagonists or DNase I that target the neutrophil-associated phenotype. Furthermore, these results highlight CCL4L2 as a potential therapeutic target for individuals with asthma refractory to ICS.

Selenium-rich yeast counteracts the inhibitory effect of nanoaluminum on the formation of porcine neutrophil extracellular traps.

Aluminum is widely used in daily life due to its excellent properties. However, aluminum exposure to the environment severely threatens animal and human health. Conversely, selenium (Se) contributes to maintaining the balance of the immune system. Neutrophils exert immune actions in several ways, including neutrophil extracellular traps (NETs) that localize and capture exogenous substances. Despite the recent investigations on the toxic effects of aluminum and its molecular mechanisms, the immunotoxicity of aluminum nanoparticles on pigs and the antagonistic effect of selenium on aluminum toxicity are poorly understood. Here, we treated porcine peripheral blood neutrophils with zymosan for 3 h to induce NETs formation. Then, we investigated the effect of nanoaluminum on NETs formation in pigs and its possible molecular mechanisms. Microscopy observations revealed that NETs formation was inhibited by nanoaluminum. Using a multifunctional microplate reader, the production of extracellular DNA and the burst of reactive oxygen species (ROS) in porcine neutrophils were inhibited by nanoaluminum. Western blot analyses showed that nanoaluminum caused changes in amounts of cellular selenoproteins. After Se supplementation, the production of porcine NETs, the burst of ROS, and selenoprotein levels were restored. This study indicated that nanoaluminum inhibited the zymosan-induced burst of ROS and release of NETs from porcine neutrophils, possibly through the selenoprotein signaling pathway. In contrast, Se supplementation reduced the toxic effects of nanoaluminum and restored NETs formation.

Local analgesia of electroacupuncture is mediated by the recruitment of neutrophils and released ß-endorphins.

ABSTRACT: The efficacy of acupuncture in treating pain diseases has been recognized in clinical practice, and its mechanism of action has been a hot topic in academic acupuncture research. Previous basic research on acupuncture analgesia has focused mostly on the nervous system, with few studies addressing the immune system as a potential pathway of acupuncture analgesia. In this study, we investigated the effect of electroacupuncture (EA) on the ß-endorphins (ß-END) content, END-containing leukocyte type and number, sympathetic neurotransmitter norepinephrine (NE), and chemokine gene expression in inflamed tissues. To induce inflammatory pain, about 200 µL of complete Frester adjuvant (CFA) was injected into the unilateral medial femoral muscle of adult Wistar rats. Electroacupuncture treatment was performed for 3 days beginning on day 4 after CFA injection, with parameters of 2/100 Hz, 2 mA, and 30 minutes per treatment. The weight-bearing experiment and enzyme-linked immunosorbent assay showed that EA treatment significantly relieved spontaneous pain-like behaviors and increased the level of ß-END in inflamed tissue. Injection of anti-END antibody in inflamed tissue blocked this analgesic effect. Flow cytometry and immunofluorescence staining revealed that the EA-induced increase in ß-END was derived from opioid-containing ICAM-1 + /CD11b + immune cells in inflamed tissue. In addition, EA treatment increased the NE content and expression of ß2 adrenergic receptor (ADR-ß2) in inflammatory tissues and upregulated Cxcl1 and Cxcl6 gene expression levels. These findings provide new evidence for the peripheral analgesic effect of acupuncture treatment by recruiting ß-END-containing ICAM-1 + /CD11b + immune cells and increasing the ß-END content at the site of inflammation.

Feeding reduced levels of trace minerals in proteinate form and selenium-yeast to transition cows: Performance, trace minerals, and antioxidant status, peripheral neutrophil activity, and oocyte quality.

An experiment was conducted to evaluate the effects of inorganic trace minerals (TM) and reduced levels of TM by using proteinate forms of Co, Zn, Mn, and Cu, and Se-yeast in diets of transition cows on performance, TM concentrations in colostrum, plasma, and liver, blood metabolites, antioxidant status, peripheral neutrophil activity, and oocyte quality. Thirty-two Holstein cows (22 multiparous and 10 primiparous cows) were enrolled in this study from 30 d before the expected calving date to 56 DIM. Cows were blocked according to body condition score, parity, and previous milk yield and randomly assigned to one of the following treatments: control (CON), with TM (Zn, Cu, Mn, and Co) supplied in form of sulfate and Se as sodium selenite to meet or exceed requirement estimates of the National Research Council; and proteinate trace minerals (PTM), with TM supplied bound with AA and peptides at 50% of CON levels and inorganic Se replaced with Se-yeast at 100% of CON level. Treatments were supplied until 56 DIM. Eight cows were removed from the study because of early calving (n = 3) or health issues (n = 5); thus, data of 24 cows (16 multiparous and 8 primiparous cows) were used in the statistical analysis. No differences between treatments were detected on nutrient intake or digestibility. Total excretion of purine derivatives was decreased when feeding PTM during the prepartum period. Feeding reduced levels of TM in proteinate form resulted in greater yield of milk (27.7 and 30.9 kg/d for CON and PTM, respectively) and protein (0.890 and 0.976 kg/d) between wk 5 and 8 of lactation. No treatment differences were detected for feed efficiency, milk somatic cell count, and milk urea nitrogen. Cows fed PTM had lower milk fat concentration during the 56 d of evaluation (4.08 and 3.74% for CON and PTM, respectively). Selenium concentration was greater in colostrum of cows fed PTM compared with CON (48.5 and 71.3 µg/L for CON and PTM, respectively), whereas Zn, Cu, and Mn concentrations were not different. Cows fed PTM showed lower liver Cu concentration compared with CON (51.4 and 73.8, respectively). Plasma concentrations of Mn and Zn were lower, but plasma Se concentration tended to be higher with PTM treatment. Feeding PTM resulted in greater blood concentrations of urea-N (16.6 and 18.2 mg/dL for CON and PTM, respectively) and ß-hydroxybutyrate (0.739 and 0.940 mmol/L). Counts of lymphocytes were higher with PTM but counts of monocytes were lower in complete blood cell count. No differences were observed in serum concentrations of superoxide dismutase and glutathione peroxidase. No differences were detected in phagocytosis and oxidative burst potential of neutrophils after incubation with bacteria. Cows fed PTM had fewer viable oocytes per ovum pick-up in comparison with CON (8.00 and 11.6). Feeding PTM to transition cows may sustain performance without altering neutrophil activity despite some alterations in blood TM concentrations. More studies should be performed to evaluate production and fertility measurements when reducing TM dietary levels by using proteinate forms and Se-yeast with larger number of animals.

Simiao Yong'an decoction ameliorates murine collagen-induced arthritis by modulating neutrophil activities: An in vitro and in vivo study.

ETHNOPHARMACOLOGICAL RELEVANCE: Rheumatoid arthritis (RA) is a common systemic autoimmune disease with high morbidity and disability rate. Currently, there is no effective allopathic treatment for RA, and most of the drugs provoke many adverse effects. Simiao Yong'an decoction (SMYAD) is a traditional Chinese prescription for the treatment of sore and gangrene caused by hot poison. With the development of pharmacology and clinical research, SMYAD has remarkable anti-inflammatory properties and has been used for RA treatments for years. AIM OF THE STUDY: This study aimed to investigate the anti-arthritic effect of SMYAD and further explore the immunopharmacological mechanisms. MATERIALS AND METHODS: Arthritis was induced in DBA/1 mice by two-time immunizations. Collagen-induced rheumatoid arthritis (CIA) mice were divided into 4 groups: control, model, methotrexate (MTX), and SMYAD group (n = 6). The administration groups were given MTX (0.5 mg/kg/3 d) and SMYAD (4.5 g/kg/d) by gavage from day 14. The arthritis index (AI) score was evaluated every 3 days after the second immunization. Hematoxylin and eosin (H&E) staining, Safranin-O fast green staining, Trap staining, and Micro-CT were used to measure the histopathology injuries and bone destruction of joints. Granulocyte changes in the spleen, bone marrow, and period blood were analyzed by flow cytometry. The expression of inflammatory cytokines and chemokines in joints were detected by qRT-PCR. SMYAD-containing serum was obtained from SD rats gavaged with SMYAD. Neutrophils were isolated from peripheral blood and bone marrow for the in vitro experiments of transwell cell assay, apoptosis assay, reactive oxygen species (ROS) generation and neutrophil extracellular traps (NETs) formation. RESULTS: SMYAD significantly relieved arthritis severity in CIA mice. The AI score was significantly decreased in the SMYAD group compared with the model group. Additionally, SMYAD alleviated inflammatory infiltration, cartilage damage, osteoclast formation, and bone damage in the ankle joints. In the flow cytometry assay, SMYAD significantly reduced granulocytes number in the spleen and bone marrow, while increased in peripheral blood. Furthermore, compared with the CIA group, SMYAD suppressed the mRNA levels of inflammatory factors including TNF-α, IL-1ß, IL-6 and chemokines CXCL1, CXCL2, and IL-8 in the inflamed joints. In the in vitro studies, 20% SMYAD-containing serum effectively inhibited the migration of neutrophils, promoted neutrophils apoptosis, reduced ROS production and NETs formation. CONCLUSION: Collectively, our results demonstrated that SMYAD effectively restrained arthritis in CIA mice by modulating neutrophil activities.

ATP breakdown in plasma of children limits the antimicrobial effectiveness of their neutrophils.

Neutrophils (PMNs) require extracellular ATP and adenosine (ADO) to fight bacterial infections, which often have life-threatening consequences in pediatric patients. We wondered whether the ATP and ADO levels in the plasma of children change with age and if these changes influence the antimicrobial efficacy of the PMNs of these children. We measured plasma concentrations of ATP and ADO and the activities of the enzymes responsible for the breakdown of these mediators in plasma samples from healthy children and adolescents (n = 45) ranging in age from 0.2 to 15 years. In addition, using blood samples of these individuals, we compared how effective their PMNs were in the phagocytosis of bacteria. In an experimental sepsis model with young (10 days) and adolescent mice (10 weeks), we studied how age influenced the resilience of these animals to bacterial infections and whether addition of ATP could improve the antimicrobial capacity of their PMNs. We found that plasma ATP levels correlated with age and were significantly lower in infants (< 1 year) than in adolescents (12-15 years). In addition, we observed significantly higher plasma ATPase and adenosine deaminase activities in children (< 12 years) when compared to the adolescent population. The activities of these ATP and ADO breakdown processes correlated inversely with age and with the ability of PMNs to phagocytize bacteria. Similar to their human counterparts, young mice also had significantly lower plasma ATP levels when compared to adolescent animals. In addition, we found that mortality of young mice after bacterial infection was significantly higher than that of adolescent mice. Moreover, bacterial phagocytosis by PMNs of young mice was weaker when compared to that of older mice. Finally, we found that ATP supplementation could recover bacterial phagocytosis of young mice to levels similar to those of adolescent mice. Our findings suggest that rapid ATP hydrolysis in the plasma of young children lowers the antimicrobial functions of their PMNs and that this may contribute to the vulnerability of pediatric patients to bacterial infections.

Modulation of Neutrophil Function by Recombinant Human IgG1 Fc Hexamer in the Endogenous K/BxN Mouse Model of Rheumatoid Arthritis.

INTRODUCTION: Neutrophils are a pivotal cell type in the K/BxN mouse model of rheumatoid arthritis and play an essential role in the progression of the arthritis. They are readily activated by immune complexes (ICs) via their FcγRs to release IL-1ß in addition to other cytokines, which are inducing cartilage destruction. Neutrophils also release neutrophil-active chemokines to recruit themselves in an autocrine manner to perpetuate tissue destruction. FcγR-expression on neutrophils is of crucial importance for the recognition of ICs. METHODS: In this study, due to its high avidity for binding to FcγRs, we investigated the potential anti-inflammatory effect of a recombinant IgG1 Fc hexamer (rFc-µTP-L309C) on neutrophils in the K/BxN mouse model of endogenously generated chronic arthritis. 200 mg/kg rFc-µTP-L309C and human serum albumin (HSA), used as controls, were administered subcutaneously every other day. Mouse ankle joints were monitored daily to generate a clinical score. Immunohistology was used to evaluate neutrophil infiltration and TUNEL to assess apoptosis. ELISA was used to measure IL-1ß. RESULTS: Treatment with rFc-µTP-L309C, but not HSA, was able to significantly ameliorate the arthritis in the K/BxN mice. Significant neutrophil infiltration into the ankle joint was found, but treatment with rFc-µTP-L309C resulted in significantly less neutrophil infiltration. There was no significant influence of rFc-µTP-L309C on neutrophil death or apoptosis. Less neutrophil infiltration could not be correlated to chemokine-mediated migration. Significantly less IL-1ß was measured in mice treated with rFc-µTP-L309C. CONCLUSION: In the endogenous K/BxN mouse model of rheumatoid arthritis, amelioration can be explained in part by inhibition of neutrophil infiltration into the joints as well as inhibition of IL-1ß production. Given the observed inhibitory properties on neutrophils, rFc-µTP-L309C may be a potential therapeutic candidate to treat autoimmune and inflammatory conditions in which neutrophils are the predominant cell type involved in pathogenesis.

Epigallocatechin-3-gallate inhibits the formation of neutrophil extracellular traps and suppresses the migration and invasion of colon cancer cells by regulating STAT3/CXCL8 pathway.

Colon cancer is a common malignant tumor of the digestive tract. Tea catechin exerts anti-tumor effects in colon cancer. This work aimed to determine the functions of epigallocatechin-3-gallate (EGCG), one of the main active components of Tea catechins, in the progression of colon cancer. In this work, enzyme-linked immune-sorbent assay, quantitative real-time PCR and western blotting was utilized to examine the levels of IL-1ß, TNF-α, STAT3, p-STAT3 and CXCL8 in colon cancer patients and healthy controls. Compared with healthy controls, the levels of IL-1ß and TNF-α were significantly increased in the peripheral blood of colon cancer patients, and the expression of STAT3, p-STAT3 and CXCL8 was elevated in the neutrophils derived from colon cancer patients. Moreover, neutrophils were treated with phorbol ester (PMA) or DNase I to induce or impede the formation of neutrophil extracellular traps (NETs). Both STAT3 overexpression and PMA treatment promoted the expression of CXCL8, myeloperoxidase (MPO) and citrullinated histone H3 (H3Cit) in the colon cancer-derived neutrophils, indicating that STAT3 overexpression facilitated the formation of NETs. STAT3 deficiency suppressed the formation of NETs, which consistent with the results of DNase I treatment. Transwell assay was utilized to detect the migration and invasion of colon cancer cell line SW480. EGCG treatment suppressed the formation of NETs and the expression of STAT3 and CXCL8 in the colon cancer-derived neutrophils, and then inhibited the migration and invasion of SW480 cells. In conclusion, this work demonstrated that EGCG inhibited the formation of NETs and subsequent suppressed the migration and invasion of colon cancer cells by regulating STAT3/CXCL8 signalling pathway. Thus, this study suggests that EGCG may become a potential drug for colon cancer therapy.

O-GlcNAc-Mediated Regulation of Galectin Expression and Secretion in Human Promyelocytic HL-60 Cells Undergoing Neutrophilic Differentiation.

In this study, we have tested the hypothesis that the expression and secretion of galectins are driven through mechanisms globally impacted by homeostatic regulation involving the post-translational modification of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc). We showed that neutrophilic differentiation of HL-60 cells induced by all-trans retinoic acid (ATRA) and 6-diazo-5-oxo-L-norleucine (DON) was associated with a significant drop of cellular O-GlcNAc levels in serum-contained and serum-free cell culture media. Galectin gene and protein expression profiles in HL-60 cells were specifically modified by ATRA and by inhibitors of O-GlcNAc cycle enzymes, however overall trends for each drug were similar between cells growing in the presence or absence of serum except for LGALS9 and LGALS12. The secretion of four galectins (-1, -3, -9, and -10) by HL-60 cells in a serum-free medium was stimulated by O-GlcNAc-reducing ATRA and DON while O-GlcNAc-elevating thiamet G (O-GlcNAcase inhibitor) failed to change the basal levels of extracellular galectins. Taken together, these results demonstrate that O-GlcNAc homeostasis is essential not only for regulation of galectin expression in cells but also for the secretion of multiple members of this protein family, which can be an important novel aspect of unconventional secretion mechanisms.