Relevance of Thymic Stromal Lymphopoietin on the Pathogenesis of Glioblastoma: Role of the Neutrophil.

Glioblastoma multiforme (GBM) is the most predominant and malignant primary brain tumor in adults. Thymic stromal lymphopoietin (TSLP), a cytokine primarily generated by activated epithelial cells, has recently garnered attention in cancer research. This study was aimed to elucidate the significance of TSLP in GBM cells and its interplay with the immune system, particularly focused on granulocyte neutrophils. Our results demonstrate that the tumor produces TSLP when stimulated with epidermal growth factor (EGF) in both the U251 cell line and the GBM biopsy (GBM-b). The relevance of the TSLP function was evaluated using a 3D spheroid model. Spheroids exhibited increased diameter, volume, and proliferation. In addition, TSLP promoted the generation of satellites surrounding the main spheroids and inhibited apoptosis in U251 treated with temozolomide (TMZ). Additionally, the co-culture of polymorphonuclear (PMN) cells from healthy donors with the U251 cell line in the presence of TSLP showed a reduction in apoptosis and an increase in IL-8 production. TSLP directly inhibited apoptosis in PMN from GBM patients (PMN-p). Interestingly, the vascular endothelial growth factor (VEGF) production was elevated in PMN-p compared with PMN from healthy donors. Under these conditions, TSLP also increased VEGF production, in PMN from healthy donors. Moreover, TSLP upregulated programed death-ligand 1 (PDL-1) expression in PMN cultured with U251. On the other hand, according to our results, the analysis of RNA-seq datasets from Illumina HiSeq 2000 sequencing platform performed with TIMER2.0 webserver demonstrated that the combination of TSLP with neutrophils decreases the survival of the patient. In conclusion, our results position TSLP as a possible new growth factor in GBM and indicate its modulation of the tumor microenvironment, particularly through its interaction with PMN.

Human Macrophages Activate Bystander Neutrophils' Metabolism and Effector Functions When Challenged with Mycobacterium tuberculosis.

Neutrophils are dynamic cells, playing a critical role in pathogen clearance; however, neutrophil infiltration into the tissue can act as a double-edged sword. They are one of the primary sources of excessive inflammation during infection, which has been observed in many infectious diseases including pneumonia and active tuberculosis (TB). Neutrophil function is influenced by interactions with other immune cells within the inflammatory lung milieu; however, how these interactions affect neutrophil function is unclear. Our study examined the macrophage-neutrophil axis by assessing the effects of conditioned medium (MΦ-CM) from primary human monocyte-derived macrophages (hMDMs) stimulated with LPS or a whole bacterium (Mycobacterium tuberculosis) on neutrophil function. Stimulated hMDM-derived MΦ-CM boosts neutrophil activation, heightening oxidative and glycolytic metabolism, but diminishes migratory potential. These neutrophils exhibit increased ROS production, elevated NET formation, and heightened CXCL8, IL-13, and IL-6 compared to untreated or unstimulated hMDM-treated neutrophils. Collectively, these data show that MΦ-CM from stimulated hMDMs activates neutrophils, bolsters their energetic profile, increase effector and inflammatory functions, and sequester them at sites of infection by decreasing their migratory capacity. These data may aid in the design of novel immunotherapies for severe pneumonia, active tuberculosis and other diseases driven by pathological inflammation mediated by the macrophage-neutrophil axis.

Sex-Specific Dysconnective Brain Injuries and Neuropsychiatric Conditions such as Autism Spectrum Disorder Caused by Group B Streptococcus-Induced Chorioamnionitis.

Global health efforts have increased against infectious diseases, but issues persist with pathogens like Group B Streptococcus (GBS). Preclinical studies have elaborated on the mechanistic process of GBS-induced chorioamnionitis and its impact on the fetal programming of chronic neuropsychiatric diseases. GBS inoculation in rodents demonstrated the following: (i) silent and self-limited placental infection, similar to human chorioamnionitis; (ii) placental expression of chemokines attracting polymorphonuclear (PMN) cells; (iii) in vitro cytokine production; (iv) PMN infiltration in the placenta (histologic hallmark of human chorioamnionitis), linked to neurobehavioral impairments like cerebral palsy and autism spectrum disorders (ASD); (v) upregulation of interleukin-1ß (IL-1ß) in the placenta and fetal blood, associated with higher ASD risk in humans; (vi) sex-specific effects, with higher IL-1ß release and PMN recruitment in male placenta; (vii) male offspring exhibiting ASD-like traits, while female offspring displayed attention deficit and hyperactivity disorder (ADHD)-like traits; (viii) IL-1 and/or NF-kB blockade alleviate placental and fetal inflammation, as well as subsequent neurobehavioral impairments. These findings offer potential therapeutic avenues, including sex-adapted anti-inflammatory treatment (e.g., blocking IL-1; repurposing of FDA-approved IL-1 receptor antagonist (IL-1Ra) treatment). Blocking the IL-1 pathway offers therapeutic potential to alleviate chorioamnionitis-related disabilities, presenting an opportunity for a human phase II RCT that uses IL-1 blockade added to the classic antibiotic treatment of chorioamnionitis.

Association of dry matter intake, milk yield, and days to first ovulation with cytological endometritis in Holstein cows.

Dry matter intake (DMI, kg/d) is closely related to the magnitude of negative energy and protein balance during the transition period, and the metabolic adaptations to support lactation in dairy cows. Thus, DMI might affect the development of cytological endometritis in the early postpartum period. Difficulty to adapt to these metabolic changes is related to impaired immune function and increased occurrence of reproductive disorders. We aimed to examine the association of pre- and postpartum DMI, body weight (BW), body condition score, milk yield and milk composition, and days to first ovulation with cytological endometritis at 15 (CYT15) and 30 DIM (CYT30). A second objective was to understand the association of vaginal discharge with CYT15 and CYT30 and performance. We conducted a pooled statistical analysis of 5 studies, including data from 280 multiparous Holstein cows. Based on the cutoffs for the percentage of uterine polymorphonuclear cells (PMN), determined by taking the median value of the data set for 15 and 30 DIM, cows were categorized as follows: LOW15 (PMN % at 15 DIM ≤24%; n = 125), HIGH15 (PMN % at 15 DIM >24%; n = 125), LOW30 (PMN % at 30DIM ≤7%; n = 141); and HIGH30 (PMN % at 30DIM >7%; n = 139). Cows in HIGH15 consumed an average of 1.97 ± 0.5 kg/d less DM than cows in LOW15 during prepartum, and 3.01 ± 0.5 kg/d less DM during postpartum. Dry matter intake (as a percentage of BW) was higher for cows in LOW15 during pre- and postpartum than for cows in HIGH15. Moreover, cows in HIGH15 tended to have lower milk yield than cows in LOW15 from the third until the fifth week postpartum. Although DMI was not associated with CYT30, DMI (as a percentage of BW) was lower for cows in LOW30 pre- and postpartum than for cows in HIGH30. There was no association between CYT30 and milk yield. Cows in LOW15 had greater days to first ovulation than cows in HIGH15, while cows in LOW30 also had greater days to first ovulation than cows in HIGH30. Simple regression analyses demonstrated linear associations of increased DMI, particularly postpartum, with decreased uterine PMN percentage and lower vaginal discharge score. Additionally, increased units of vaginal discharge score and increased percentage units of uterine PMN were linearly associated with decreased milk yield. Corroborating with the notion of the ovarian function being associated with uterine inflammatory status, cows in HIGH15 and HIGH30 ovulated on average 3 d before than cows in LOW15 and LOW30, respectively. Cytological endometritis at 15 DIM was associated with lower DMI from 4 wk before calving until 4 wk postpartum and was associated with lower milk yield. The association of vaginal discharge with cytological endometritis was variable and dependent on the day of evaluation.

Elevated cfDNA after exercise is derived primarily from mature polymorphonuclear neutrophils, with a minor contribution of cardiomyocytes.

Strenuous physical exercise causes a massive elevation in the concentration of circulating cell-free DNA (cfDNA), which correlates with effort intensity and duration. The cellular sources and physiological drivers of this phenomenon are unknown. Using methylation patterns of cfDNA and associated histones, we show that cfDNA in exercise originates mostly in extramedullary polymorphonuclear neutrophils. Strikingly, cardiomyocyte cfDNA concentration increases after a marathon, consistent with elevated troponin levels and indicating low-level, delayed cardiac cell death. Physical impact, low oxygen levels, and elevated core body temperature contribute to neutrophil cfDNA release, while muscle contraction, increased heart rate, ß-adrenergic signaling, or steroid treatment fail to cause elevation of cfDNA. Physical training reduces neutrophil cfDNA release after a standard exercise, revealing an inverse relationship between exercise-induced cfDNA release and training level. We speculate that the release of cfDNA from neutrophils in exercise relates to the activation of neutrophils in the context of exercise-induced muscle damage.

Dietary administration of the glycolytic inhibitor 2-deoxy-D-glucose reduces endotoxemia-induced inflammation and oxidative stress: Implications in PAMP-associated acute and chronic pathology.

Pathogen-associated molecular patterns (PAMPs) like bacterial cell wall components and viral nucleic acids are known ligands of innate inflammatory receptors that trigger multiple inflammatory pathways that may result in acute inflammation and oxidative stress-driven tissue and organ toxicity. When dysregulated, this inflammation may lead to acute toxicity and multiorgan failure. Inflammatory events are often driven by high energy demands and macromolecular biosynthesis. Therefore, we proposed that targeting the metabolism of lipopolysaccharide (LPS)-driven inflammatory events, using an energy restriction approach, can be an effective strategy to prevent the acute or chronic detrimental effects of accidental or seasonal bacterial and other pathogenic exposures. In the present study, we investigated the potential of energy restriction mimetic agent (ERMA) 2-deoxy-D-glucose (2-DG) in targeting the metabolism of inflammatory events during LPS-elicited acute inflammatory response. Mice fed with 2-DG as a dietary component in drinking water showed reduced LPS-driven inflammatory processes. Dietary 2-DG reduced LPS-induced lung endothelial damage and oxidative stress by strengthening the antioxidant defense system and limiting the activation and expression of inflammatory proteins, viz., P-Stat-3, NfκΒ, and MAP kinases. This was accompanied by decreased TNF, IL-1ß, and IL-6 levels in peripheral blood and bronchoalveolar lavage fluid (BALF). 2-DG also reduced the infiltration of PMNCs (polymorphonuclear cells) in inflamed tissues. Altered glycolysis and improved mitochondrial activity in 2-DG-treated RAW 264.7 macrophage cells suggested possible impairment of macrophage metabolism and, therefore, activation in macrophages. Taken together, the present study suggests that inclusion of glycolytic inhibitor 2-DG as a part of the diet can be helpful in preventing the severity and poor prognosis associated with inflammatory events during bacterial and other pathogenic exposures.

Centella asiatica extract in carboxymethyl cellulose at its optimal concentration improved wound healing in mice model.

Centella asiatica (C. asiatica) has reported to be one of the traditional herbal remedies, whereas poor water solubility leads to lower bioavailability thereby affecting it remedial efficacy. Therefore, we aimed to evaluate its efficacy through increased bioavailability by using high viscosity Carboxymethyl Cellulose (CMC) as solvent on methanol-based extract on wound healing, in vivo. The preparation was applied as 0.0% (control, CMC alone), 0.25. 0.5 and 1% concentrations of extract of C. asiatica. We evaluated the efficiency of preparations on wound healing progression as progression of wound contraction, tissue proliferation and cells deposition, and relative level of gene expression for genes associated with wound healing. The results showed that 0.5% extract in CMC had significantly higher (P < 0.05) wound contraction than control and other concentrations. The level tissue deposition and the infiltration of polymorphonuclear cells in groups treated with 0.5 % concentration preparation were higher than that other treatments and control. Similarly, the relative level of gene expression in 0.5% concentration treated group were statistically significantly higher (P < 0.05) than that of control. It is believed that the lower concentration of the extract would have lessor effect on wound healing, whereas higher concertation would be interfering the optimal inflammatory tissue deposition; and there by negatively affecting wound healing. The results indicated that C. asiatica can be optimally used at 0.5 % of extract in CMC for wound healing as indicated by speeding the progression of wound closure and by increasing the expression of collagen II and III together with reducing the expression of TGFß1. However, higher concentrations of the crude extract of C. asiatica could paradoxically resulting in undesired effects. It is recommended that further evaluation should be performed on wider scale and the economic feasibility evaluation should be performed.

What is the role of leukopenia in the assessment of septic arthritis?

Background: It is not well-understood how leukopenia affects the synovial white blood cell (WBC) and percent neutrophils (%PMNs) in the setting of septic arthritis. We sought to determine 1. Do synovial WBC and %PMNs differ between patients with culture positive septic arthritis with or without leukopenia? And 2. Are traditional thresholds of synovial fluid studies for accurately diagnosing septic arthritis still applicable in the leukopenic patient population? Methods: A retrospective cohort study was performed at a single institution of 79 non-leukopenic and 11 leukopenic patients diagnosed with culture-positive septic arthritis. Demographic data, serum laboratory values, synovial laboratory values, and culture results were recorded. Significant differences in synovial laboratory values were evaluated using the Wilcoxon-Mann-Whitney test. Results are reported as median, interquartile range, and p values. Results: There was a significant difference in synovial WBC in leukopenic patients compared to non-leukopenic patients with culture positive septic arthritis (p = 0.01). No significant difference was found in the synovial %PMNs between two cohorts (p = 0.33). Conclusion: Leukopenic patients with culture positive septic arthritis have significantly lower synovial WBCs compared to non-leukopenic patients. Traditional thresholds for synovial WBC are not reliable for excluding diagnosis of septic arthritis in leukopenic patients.

Spontaneous Regression of Cancer: Revealing Granulocytes and Oxidative Stress as the Crucial Double-edge Sword.

BACKGROUND: It is commonly believed that cancer development is irreversible, organ-specific as well as systemic malignant disorder, often associated with harmful oxidative stress and inflammation. However, there are also well-documented cases of spontaneous cancer regression, the causative mechanisms of which are not understood. It is known that inflammation is a negative pathophysiological process that may support the development of cancer, but it is also believed that the immune system as well as oxidative stress play important roles in prevention of cancer development and defense against tumor progression. Hence, in animal models spontaneous regression of cancer could be mediated by rapid inflammatory response of granulocytes, acting against cancer mostly as innate immune response. In addition, the administration of granulocytes at the site of solid tumors can lead to tumor regression or can slow down tumor growth and extend the overall survival of animals. In both cases, similar to the radiotherapy, surgery and various chemotherapies, oxidative stress occurs generating lipid peroxidation product 4-hydroxynonenal (4-HNE). This "second messenger of free radicals" acts as growth regulating signaling molecule that exerts relatively selective cytotoxicity against cancer cells. CONCLUSIONS: We hypothesize that abundant inflammation and metabolic changes caused by cancer and oxidative stress producing of 4-HNE may be crucial mechanisms for spontaneous cancer regression.

The effect of subclinical endometritis on reproductive performance in postpartum Bos indicus multiparous beef cows.

The objective was to investigate the prevalence of subclinical endometritis (SE) in postpartum Bos indicus multiparous beef cows using different polymorphonuclear cells (PMN) threshold ratios, and to evaluate the impacts of SE on their reproductive performance. A total of 689 postpartum Nellore cows (45.2 ± 7.8 days postpartum) were submitted to an estrus synchronization protocol + timed-artificial insemination (TAI). Endometrial cytology samples were collected by cytobrush before the beginning of the protocol. Cows were considered positive for SE if: (PMN) ≥ 3% (PMN3), PMN ≥ 5% (PMN5) and PMN ≥ 7% (PMN7). Pregnancy diagnosis was performed at 30 and 100 d after TAI. The prevalence of cows categorized as positive for SE increased (P < 0.05) as the threshold was lowered (PMN7 = 3.9%; PMN5 = 5.5%; PMN3 = 9.1%). Positive SE cows had similar (P > 0.41) BCS, days postpartum, and expression of estrus at TAI compared with negative SE cows. Positive SE cows had decreased (P = 0.04) pregnancy rates compared to negative SE cows in the PMN5 threshold (26.3 ± 8.7% vs 44.5 ± 4.1%); however, no difference (P > 0.45) was observed between positive and negative SE cows in the PMN3 and PMN7 thresholds. Embryonic mortality between days 30 and 100 was not affected by SE (P > 0.16). In conclusion, the prevalence of SE varies based on the PMN threshold used, whereas SE at the beginning of the TAI protocol decreased the pregnancy rates in postpartum Bos indicus beef cows when the PMN5 was used.

Cytological endometritis diagnosis in primiparous versus multiparous dairy cows.

Endometritis is a uterine disease of dairy cows causing substantial negative effects on reproductive performance and inflicting considerable economic losses. It is typically diagnosed by endometrial cytology evaluation and commonly named cytological endometritis (CEM). In most previous studies, cows were defined as CEM positive if the proportion of polymorphonuclear cells (%PMN) in their endometrial cytology was above a pre-set threshold. Thresholds were established based on CEM diagnosis in association with reproductive performance, typically analyzed by a single reproductive parameter and calculated for all cows together. Our objective was to examine whether primiparous and multiparous cows should optimally be diagnosed for CEM by different %PMN thresholds and sampling timings, using a combination of several reproductive performance parameters. Two endometrial cytobrush cytology samples were collected from Holstein-Friesian dairy cows (n = 415; 269 multiparous; 146 primiparous), at 30-40 d in milk (DIM) and 60-70 DIM, and %PMN were evaluated microscopically (blindly; Diff-Quick stain, Medi-Market). The %PMN thresholds were set at ≥1% to ≥10%, ≥15%, and ≥20%, and accordingly, for each of the thresholds, several reproductive performance parameters were compared between CEM-positive versus CEM-negative cows. Upon application of several analytic approaches, our results indicated that optimal CEM diagnosis should be performed by different criteria in primiparous and multiparous cows: in primiparous cows at 30-40 DIM, using a threshold of ≥7%PMN, and in multiparous cows at 60-70 DIM, using a threshold of ≥4%PMN. Such a diagnostic approach provides a comprehensive view of the reproductive prognosis of CEM-positive primiparous and multiparous cows, which is pertinent information for researchers, veterinarians, and farmers.

DC-SIGN Mediates the Interaction Between Neutrophils and Leishmania amazonensis-Infected Dendritic Cells to Promote DC Maturation and Parasite Elimination.

Background: Leishmaniasis is a neglected arthropod-borne disease that affects millions of people worldwide. Successful Leishmania infections require the mitigation of immune cell functions leading to parasite survival and proliferation. A large body of evidence highlights the involvement of neutrophils (PMNs) and dendritic cells (DCs) in the establishment of immunological responses against these parasites. However, few studies, contemplate to what extent these cells interact synergistically to constrain Leishmania infection. Objective: We sought to investigate how PMNs and infected DCs interact in an in vitro model of Leishmania amazonensis infection. Material and Methods: Briefly, human PMNs and DCs were purified from the peripheral blood of healthy donors. Next, PMNs were activated with fibronectin and subsequently co-cultured with L. amazonensis-infected DCs. Results: We observed that L. amazonensis-infected DC exhibited lower rates of infection when co-cultivated with either resting or activated PMNs. Surprisingly, we found that the release of neutrophil enzymes was not involved in Leishmania killing. Next, we showed that the interaction between PMNs and infected-DCs was intermediated by DC-SIGN, further suggesting that parasite elimination occurs in a contact-dependent manner. Furthermore, we also observed that TNFα and ROS production was dependent on DC-SIGN-mediated contact, as well as parasite elimination is dependent on TNFα production in the co-culture. Finally, we observed that direct contact between PMNs and DCs are required to restore the expression of DC maturation molecules during L. amazonensis infection. Conclusion: Our findings suggest that the engagement of direct contact between PMNs and L. amazonensis-infected DC via DC-SIGN is required for the production of inflammatory mediators with subsequent parasite elimination and DC maturation.

Effect of quercetin on formation of porcine neutrophil extracellular trap.

Neutrophil extracellular trap (NET) formation is an immune response to the invasion of external microorganisms. Quercetin, a member of the flavonoid family found in fruits and vegetables, has been examined in multiple biological contexts. The objective of this study was to examine the effect of quercetin on porcine NET formation. We measured NET formation by peripheral blood polymorphonuclear cells (PMNs) using propidium iodide (PI) dye. The amount of tumor necrosis factor (TNF)-α in culture supernatants was quantified by ELISA, and TNF-α mRNA expression was measured by RT-PCR. Direct treatment of PMNs with quercetin did not affect NET formation; however, NET formation was inhibited by exposure to culture supernatant from peripheral blood mononuclear cells (PBMCs) treated with quercetin. By contrast, culture supernatant from PBMCs treated with lipopolysaccharide (LPS) induced high levels of NET formation of PMNs, and this effect was reduced by co-treatment with LPS and quercetin. In addition, treatment of PMNs with recombinant porcine (rp) TNF-α induced high levels of NET formation. PBMCs treated with LPS increased higher levels of TNF-α mRNA and protein, but this effect was weakened when they were co-treated with quercetin. These findings indicated that quercetin inhibits NET formation of PMNs by suppressing production of TNF-α from LPS-stimulated PBMCs. These results suggest that quercetin exerts an anti-inflammatory effect, mediated by down-regulation of TNF-α production from LPS-stimulated PBMCs, which inhibits NET formation in PMNs.

Lack of detectable short-term effects of a single dose of ivermectin on the human immune system.

BACKGROUND: Ivermectin is widely used in human and animal medicine to treat and prevent parasite nematode infections. It has been suggested that its mode of action requires the host immune system, as it is difficult to reproduce its clinical efficacy in vitro. We therefore studied the effects of a single dose of ivermectin (Stromectol®-0.15 mg/kg) on cytokine levels and immune cell gene expression in human volunteers. This dose reduces bloodstream microfilariae rapidly and for several months when given in mass drug administration programmes. METHODS: Healthy volunteers with no travel history to endemic regions were given 3-4 tablets, depending on their weight, of either ivermectin or a placebo. Blood samples were drawn immediately prior to administration, 4 h and 24 h afterwards, and complete blood counts performed. Serum levels of 41 cytokines and chemokines were measured using Luminex® and expression levels of 770 myeloid-cell-related genes determined using the NanoString nCounter®. Cytokine levels at 4 h and 24 h post-treatment were compared to the levels pre-treatment using simple t tests to determine if any individual results required further investigation, taking p = < 0.05 as the level of significance. NanoString data were analysed on the proprietary software, nSolver™. RESULTS: No significant differences were observed in complete blood counts or cytokine levels at either time point between people given ivermectin versus placebo. Only three genes showed a significant change in expression in peripheral blood mononuclear cells 4 h after ivermectin was given; there were no significant changes 24 h after drug administration or in polymorphonuclear cells at either time point. Leukocytes isolated from those participants given ivermectin showed no difference in their ability to kill Brugia malayi microfilariae in vitro. CONCLUSIONS: Overall, our data do not support a direct effect of ivermectin, when given at the dose used in current filarial elimination programmes, on the human immune system. Trial registration ClinicalTrials.gov NCT03459794 Registered 9th March 2018, Retrospectively registered https://clinicaltrials.gov/ct2/show/NCT03459794?term=NCT03459794&draw=2&rank=1 .

Effects of Peptide-Induced Immune Tolerance on Murine Lupus.

The regulation of autoimmunity and the molecular mechanisms by which different immune cells, including T cells, polymorphonuclear leukocytes (PMN-granulocytes), and B cells suppress autoimmune diseases is complex. We have shown previously that BWF1 lupus mice are protected from autoimmunity after i.v. injection or oral administration of tolerogenic doses of pCons, an artificial synthetic peptide based on sequences containing MHC class I and MHC class II determinants in the VH region of a J558-encoded BWF1 anti-DNA Ab. Several T cell subsets can transfer this tolerance. In this study, we determined the potential roles of granulocytes, B cells and regulatory T cells altered by pCons treatment in the BWF1 (NZB/NZW) mouse model of lupus. Immunophenotyping studies indicated that pCons treatment of BWF1 mice significantly increased CD4+FoxP3+ T cells, reduced the percent of B cells expressing CD19+CD5+ but increased the percent of CD19+CD1d+ regulatory B cells and increased the ability of the whole B cell population to suppress IgG anti-DNA production in vitro. pCons treatment significantly decreased the expression of CTLA-4 (cytotoxic T-lymphocyte-associated protein-4) in CD8+ T cells. In addition, peptide administration modified granulocytes so they became suppressive. We co-cultured sorted naïve B cells from mice making anti-DNA Ab (supported by addition of sorted naive CD4+ and CD8+ T cells from young auto-antibody-negative BWF1 mice) with sorted B cells or granulocytes from tolerized mice. Both tolerized granulocytes and tolerized B cells significantly suppressed the production of anti-DNA in vitro. In granulocytes from tolerized mice compared to saline-treated littermate controls, real-time PCR analysis indicated that expression of interferon-induced TNFAIP2 increased more than 2-fold while Ptdss2 and GATA1 mRNA were up-regulated more than 10-fold. In contrast, expression of these genes was significantly down-regulated in tolerized B cells. Further, another IFN-induced protein, Bcl2, was reduced in tolerized B cells as determined by Western blot analyses. In contrast, expression of FoxP3 was significantly increased in tolerized B cells. Together, these data suggest that B cells and granulocytes are altered toward suppressive functions by in vivo tolerization of BWF1 mice with pCons and it is possible these cell types participate in the clinical benefits seen in vivo.

Declining neutrophil production despite increasing G-CSF levels is associated with chronic inflammation in elderly rhesus macaques.

Aging is characterized by a loss of bone marrow hematopoietic tissue, systemic chronic inflammation, and higher susceptibility to infectious and noninfectious diseases. We previously reported the tightly regulated kinetics and massive daily production of neutrophils during homeostasis in adult rhesus macaques aged 3 to 19 yr (equivalent to approximately 10 to 70 yr of age in humans). In the current study, we observed an earlier release of recently dividing neutrophils from bone marrow and greater in-group variability of neutrophil kinetics based on in vivo BrdU labeling in a group of older rhesus macaques of 20-26 yr of age. Comparing neutrophil numbers and circulating cytokine levels in rhesus macaques spanning 2 to 26 yr of age, we found a negative correlation between age and blood neutrophil counts and a positive correlation between age and plasma G-CSF levels. Hierarchic clustering analysis also identified strong associations between G-CSF with the proinflammatory cytokines, IL-1ß and MIP-1α. Furthermore, neutrophils from older macaques expressed less myeloperoxidase and comprised higher frequencies of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) compared to the young adult macaques. In summary, we observed an earlier release from bone marrow and a reduced production of neutrophils despite the increased levels of plasma G-CSF, especially in the elderly rhesus macaques. This lower neutrophil production capacity associated with increased production of proinflammatory cytokines as well as an earlier release of less mature neutrophils and PMN-MDSCs may contribute to the chronic inflammation and greater susceptibility to infectious and noninfectious diseases during aging.

Ambiguities in Neutrophil Extracellular Traps. Ongoing Concepts and Potential Biomarkers for Rheumatoid Arthritis: A Narrative Review.

OBJECTIVE: This study aims to provide consolidation of current research findings as well as the most important concepts regarding neutrophil extracellular traps (NETs) in rheumatoid arthritis. DATA SOURCES: Relevant publications from 2004 to 2018 were identified using PubMed, Web of Science, Scopus, and eLibrary databases. Primary search terms used were "neutrophil extracellular traps" or "NETs" in combination with "rheumatoid arthritis". DATA SYNTHESIS: NETs are distinctive structures promoting capture and non-phagocytic cleavage of foreign substances. NETs usually consist of thin chromatin fibers decorated with various molecules of granular, cytosolic, and cytoskeletal origin. NETosis can develop in two ways: either with neutrophil death or when the viability of the cell prolongs. ROS generation and pronounced protein citrullination are essential during the initial phase of NETs formation. NETosis is considered to have certain immunological consequences, including DAMPs-mediated signalling, proinflammatory cytokine secretion, and contact of extensively modified self and foreign epitopes with antigen-presenting cells. There are several putative pathogenetic links between NETosis, citrullination, neoepitope formation, and production of anticitrullined autoantibodies that can strongly influence rheumatoid arthritis progression. NET-induced vascular injury in rheumatoid arthritis can arise directly from NETs and indirectly through enhanced thrombosis and atherosclerosis. CONCLUSION: NETs are currently estimated as a possible influential factor of rheumatoid arthritis initiation and/or progression, especially in the context of vascular involvement. NETs can also serve as a source of novel antigenic biomarkers for the diagnosis of rheumatoid arthritis.

Gene expression in bovine endometrial cells and blood-derived neutrophils stimulated by uterine secretions.

Uterine epithelial cells (UEC) and migrated polymorphonuclear cells (PMN) play important roles in the uterine defence against microbial infection. The aims of the present study were to investigate i) whether undiluted uterine secretions modulate the expression of genes associated with the innate immune response in UEC and PMN in vitro, ii) whether these changes differ between the two cell populations and iii) whether uterine secretions from cows with subclinical endometritis produce a different response to those from unaffected cows. Therefore, undiluted uterine secretions, cytobrush and biopsy samples were collected from bovine uteri at a local abattoir. All cows had calved at least 3 months prior to sample collection. Subclinical endometritis was diagnosed by cytology (≥5% polymorphonuclear neutrophils) and histology. The uteri were thereby retrospectively categorised as endometritis-positive (E-pos; n = 14), if either the cytology or the histology results were positive, or endometritis-negative (E-neg; n = 17), if both diagnostics were negative. Cultured UEC responded to secretions from E-pos and E-neg cows with an increased gene expression of CXC ligand (CXCL) 8 and interleukin (IL) 6 compared to incubation with control medium alone. PMN expressed significantly higher mRNA levels of CXCL5, CXCL8 and IL1B in response to supernatant from UEC incubated with secretions from both groups (E-pos and E-neg) compared to those incubated with control medium alone. Gene expression of IL10 in uterine epithelial cells remained comparable to the control in cells exposed to E-pos secretions and was 3.6 times lower in those exposed to E-neg secretions. These results demonstrate that the expression of genes associated with the innate immune response in UEC and indirectly also PMN is affected by uterine secretions in vitro. Depending on the target gene, these changes differ between the two cell populations. UEC exposed to uterine secretions from cows without subclinical endometritis produce lower levels of IL10 compared to those exposed to secretions from affected cows or control medium alone. Therefore, the model established in this study can be used as a valuable tool to further understand the contributions of the two cell populations to the coordinated immune response in the uterus.

Brucella abortus-infected platelets modulate the activation of neutrophils.

Brucellosis is a contagious disease caused by bacteria of the genus Brucella. Platelets (PLTs) have been widely involved in the modulation of the immune response. We have previously reported the modulation of Brucella abortus-mediated infection of monocytes. As a result, PLTs cooperate with monocytes and increase their inflammatory capacity, promoting the resolution of the infection. Extending these results, in this study we demonstrate that patients with brucellosis present slightly elevated levels of complexes between PLTs and both monocytes and neutrophils. We then assessed whether PLTs were capable of modulating functional aspects of neutrophils. The presence of PLTs throughout neutrophil infection increased the production of interleukin-8, CD11b surface expression and reactive oxygen species formation, whereas it decreased the expression of CD62L, indicating an activated status of these cells. We next analyzed whether this modulation was mediated by released factors. To discriminate between these options, neutrophils were treated with supernatants collected from B. abortus-infected PLTs. Our results show that CD11b expression was induced by soluble factors of PLTs but direct contact between cell populations was needed to enhance the respiratory burst. Additionally, B. abortus-infected PLTs recruit polymorphonuclear (PMN) cells to the site of infection. Finally, the presence of PLTs did not modify the initial invasion of PMN cells by B. abortus but improved the control of the infection at extended times. Altogether, our results demonstrate that PLTs interact with neutrophils and promote a proinflammatory phenotype which could also contribute to the resolution of the infection.