Phorbol Myristate Acetate Stimulation Research Articles

CPHEN-017: Comprehensive phenotyping of neutrophil extracellular traps (NETs) on peripheral human neutrophils.

With the recent discovery of their ability to produce neutrophil extracellular traps (NETs), neutrophils are increasingly appreciated as active participants in infection and inflammation. NETs are characterized as large, web-like networks of DNA and proteins extruded from neutrophils, and there is considerable interest in how these structures drive disease in humans. Advancing research in this field is contingent on developing novel tools for quantifying NETosis. To this end, we have developed a 7-marker flow cytometry panel for analyzing NETosis on human peripheral neutrophils following in vitro stimulation, and in fresh circulating neutrophils under inflammatory conditions. This panel was optimized on neutrophils isolated from whole blood and analyzed fresh or in vitro stimulated with phorbol 12-myristate 13-acetate (PMA) or ionomycin, two known NET-inducing agonists. Neutrophils were identified as SSChighFSChighCD15+CD66b+. Neutrophils positive for amine residues and 7-Aminoactinomycin D (7-AAD), our DNA dye of choice, were deemed necrotic (Zombie-NIR+7-AAD+) and were removed from downstream analysis. Exclusion of Zombie-NIR and positivity for 7-AAD (Zombie-NIRdim7-AAD+) was used here as a marker of neutrophil-appendant DNA, a key feature of NETs. The presence of two NET-associated proteins - myeloperoxidase (MPO) and neutrophil elastase (NE) - were utilized to identify neutrophil-appendant NET events (SSChighFSChighCD15+CD66b+Zombie NIRdim7-AAD+MPO+NE+). We also demonstrate that NETotic neutrophils express citrullinated histone H3 (H3cit), are concentration-dependently induced by in vitro PMA and ionomycin stimulation but are disassembled with DNase treatment, and are present in both chronic and acute inflammation. This 7-color flow cytometry panel provides a novel tool for examining NETosis in humans.

Enhancing intracellular delivery of protein kinase C beta II peptide inhibitor using myristic acid-trans-activator of transcription via N-terminus conjugation in isolated rat polymorphonuclear leukocytes

Introduction: Protein kinase C beta II (PKCβII) activation promotes polymorphonuclear (PMN) superoxide (SO) production by phosphorylating serine and threonine amino acid residues on NADPH oxidase (NOX-2). Previous studies have shown that cell permeable myristic acid-conjugated PKCβII inhibitor (myr-PKCβII-; SLNPEWNET) (20 μM) remarkably attenuated phorbol 12-myristate 13-acetate (PMA)-induced PMN SO release when compared to scrambled control peptide (myr-PKCβII- scram; WNPESLNTE), unconjugated peptides and nontreated controls by ~35%. This suggests an enhanced intracellular delivery of PKCβII-. Studies have also shown that SO release is attenuated via trans-activator of transcription-conjugated (Tat; YGRKKRRQRRR) NOX-2 inhibitor (Nox2ds-Tat) (80 μM) by ~37%. There have not been evaluations of Tat- or dual myr-Tat-conjugated PKCβII-. We hypothesize that dual myr-Tat-conjugation would enhance the intracellular delivery of PKCβII- and reduce SO release compared to myr-, Tat-, and myr-Tat-PKCβII- scram. Methods: This study focused on the effects of myr-Tat-PKCβII- on intracellular delivery compared to myr-PKCβII-, Tat -PKCβII-, and myr-Tat-PKCβII- scram, with 0.5% dimethyl sulfoxide (DMSO) vehicle as the control. Sprague-Dawley (SD) male rats (~400g) were anesthetized with isoflurane and given a 0.5% glycogen i.p. injection (16ml), and 16-18hrs later, PMNs were harvested from the peritoneal cavity and incubated for 15min at 37oC with 5μM myr-Tat-PKCβII-, myr-PKCβII-, Tat-PKCβII-, or myr-Tat-PKCβII- scram. PMN SO release was calculated spectrophotometrically by the change in absorbance at 550 nm over 390s via ferricytochrome c reduction after PMA stimulation (100nM). Data were analyzed with ANOVA Fisher’s PLSD post-hoc analysis. Results: Myr-Tat-PKCβII- (n=20, 0.338±0.024, p<0.05) and Myr-PKCβII- (n=8, 0.342±0.041, p<0.05) displayed significant attenuation of SO release by 28% and 27%, respectively, compared to 0.5% DMSO control (n=81, 0.470±0.013). Tat-PKCβII- (n=5, 0.404±0.049) showed attenuated SO release by 14% when compared to the vehicle control. Myr-Tat-PKCβII- scram (n=3, 0.542±0.081) showed an increase in SO release by 15% when compared to the vehicle control. Cell viability was > 85% in all groups. Conclusion: The results of the study suggest that intracellular delivery of PKCβII- cargo using myr-Tat dual conjugation is enhanced when compared to myr-, Tat-conjugated PKCβII-, and myr-Tat-PKCβII- scram. Future studies will utilize western blot analysis and immunohistochemistry to assess the translocation and activity of PKCβII- when conjugated with myr-Tat, myr-, or Tat- peptides. Funding: This study was funded by the National Heart, Lung & Blood Institute (1R43HL160338) SBIR phase I grant that was awarded to Young Therapeutics, LLC. Philadelphia College of Osteopathic Medicine, the Department of Biomedical Sciences, the Division of Research and the Center for Chronic Diseases of Aging. and Young Therapeutics, LLC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The impact of wound-healing assay, phorbol myristate acetate (PMA) stimulation and siRNA-mediated FURIN gene silencing on endogenous retroviral ERVW-1 expression level in U87-MG astrocytoma cells

PurposeHuman endogenous retroviruses (HERVs) are ubiquitous genomic sequences. Normally dormant HERVs, undergo reactivation by environmental factors. This deregulation of HERVs’ transcriptional equilibrium correlates with medical conditions such as multiple sclerosis (MS). Here we sought to explore whether exposing the U-87 MG astrocytoma cells to traumatic injury deregulates the expression of HERV-W family member ERVW-1 encoding syncytin-1. We also examined the expression of FURIN gene that is crucial in syncytin-1 synthesis. Material and methodsScratch assay was used as a model of cells injury in U-87 MG cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot (WB) and migration assay using Boyden chamber were used. Phorbol 12-myristate 13-acetate (PMA) and small interfering RNA (siRNA) were used for cell stimulation and gene expression inhibition, respectively. ResultsResults revealed reduced ERVW-1 expression in cells exposed to injury (p ​< ​0.05) while GFAP gene - a marker of active astrocytes, was upregulated (p ​< ​0.01). These findings were confirmed by both WB and RT-qPCR. Expression of FURIN gene was not altered after injury, but cell stimulation by PMA strongly increased FURIN expression, simultaneously downregulating ERVW-1 (p ​< ​0.01). SiRNA-mediated expression inhibition of ERVW-1 and FURIN influenced the mRNA level for SLC1A5 (ASCT2) - primary syncytin-1 receptor, that was significantly lower. FURIN inhibition by siRNA caused strong upregulation of ERVW-1 expression (p ​< ​0.01). ConclusionResults showed that mechanical impact affects the expression of endogenous retroviruses in U-87 MG astrocytoma cells by scratch assay. Regulation of FURIN, a crucial enzyme in ERVW-1 turnover may support the therapy of some neurological conditions.

Identification and Validation of Hub Genes Related to Neutrophil Extracellular Traps-Mediated Cell Damage During Myocardial Infarction.

Studies have shown that neutrophil-mediated formation of neutrophil extracellular traps (NETs) leads to increased inflammatory response and cellular tissue damage during myocardial infarction (MI). We aimed to identify and validate possible hub genes in the process of NETs-mediated cell damage. We performed an immune cell infiltration analysis of the MI transcriptome dataset based on CIBERSORT and ssGSEA algorithms. Gene expression profiles of NETs formation (GSE178883) were used to analyze the physiological processes of peripheral blood neutrophils after phorbol myristate acetate (PMA) stimulation. Bioinformatics and machine learning algorithms were utilized to find candidate hub genes based on NETs-related genes and transcriptome datasets (GSE66360 and GSE179828). We generated the receiver operating curve (ROC) to evaluate the diagnostic value of hub genes. Next, the correlation between hub genes and immune cells was analyzed using CIBERSORT, ssGSEA and xCell algorithms. Finally, we used quantitative real-time PCR (qRT-PCR) and immunohistochemistry to verify gene expression. Immune cell infiltration analysis revealed that inflammatory cells such as neutrophils were highly expressed in the peripheral blood of patients with MI. Functional analysis of differentially expressed genes (DEGs) in GSE178883 indicated that the potential pathogenesis lies in immune terms. Using weighted gene co-expression network analysis (WGCNA) and machine learning algorithms, we finally identified the seven hub genes (FCAR, IL1B, MMP9, NFIL3, CXCL2, ICAM1, and ZFP36). The qRT-PCR results showed that IL-1B, MMP9, and NFIL3 mRNA expression was up-regulated in the MI group compared to the control. Immunohistochemical results showed high MMP9, IL-1B, and NFIL3 expression in the infarcted area compared to the non-infarcted area and sham-operated groups. We identified seven hub genes associated with NETs-mediated cellular damage during MI. Our results may provide insights into the mechanisms of neutrophil-mediated cell injury during MI.

Engineered nanovesicles from activated neutrophils with enriched bactericidal proteins have molecular debridement ability and promote infectious wound healing.

Bacterial infections pose a considerable threat to skin wounds, particularly in the case of challenging-to-treat diabetic wounds. Systemic antibiotics often struggle to penetrate deep wound tissues and topically applied antibiotics may lead to sensitization, necessitating the development of novel approaches for effectively treating germs in deep wound tissues. Neutrophils, the predominant immune cells in the bloodstream, rapidly release an abundance of molecules via degranulation upon activation, which possess the ability to directly eliminate pathogens. This study was designed to develop novel neutrophil cell engineered nanovesicles (NVs) with high production and explore their bactericidal properties and application in promoting infectious wound healing. Neutrophils were isolated from peripheral blood and activated in vitro via phorbol myristate acetate (PMA) stimulation. Engineered NVs were prepared by sequentially extruding activated neutrophils followed by ultracentrifugation and were compared with neutrophil-derived exosomes in terms of morphology, size distribution and protein contents. The bactericidal effect of NVs in vitro was evaluated using the spread plate technique, LIVE/DEAD backlight bacteria assay and observation of bacterial morphology. The therapeutic effects of NVs in vivo were evaluated using wound contraction area measurements, histopathological examinations, assessments of inflammatory factors and immunochemical staining. Activated neutrophils stimulated with PMA in vitro promptly release a substantial amount of bactericidal proteins. NVs are similar to exosomes in terms of morphology and particle size, but they exhibit a significantly higher enrichment of bactericidal proteins. In vitro, NVs demonstrated a significant bactericidal effect, presumably mediated by the enrichment of bactericidal proteins such as lysozyme. These NVs significantly accelerated wound healing, leading to a marked reduction in bacterial load, downregulation of inflammatory factors and enhanced collagen deposition in a full-thickness infectious skin defect model. We developed engineered NVs derived from activated neutrophils to serve as a novel debridement method targeting bacteria in deep tissues, ultimately promoting infectious wound healing.

Heterozygous Variants in the Clpbgene Associated with Neutropenia Affect Neutrophil Function

Introduction The CLPB gene encodes a caseinolytic peptidase belonging to the ATPase family and acts as a mitochondrial chaperone. While biallelic mutations in CLPB are associated with the 3-methylglutaconic aciduria and severe neutropenia, (MIM number: 616271 3), some heterozygous variants seem to induce neutropenia exclusively. In the current study, we evaluated for the first time a function of neutrophils among neutropenic carriers of CLPB mutation. Methods Among 282 individuals with neutropenia studied with targeted Next-Generation Sequencing (panel of 725 genes related to hematological disorders), three neutropenic probands with heterozygous CLPB amino acid substitutions were identified. Neutrophils from patients and control healthy donors were isolated from peripheral blood with a Pancoll density gradient. The percentages of freshly isolated neutrophils undergoing apoptosis or necrosis were determined by flow cytometry using Annexin V - FITC and Propidium Iodide (PI) staining. Phagocytic capacity was evaluated using pHrodo Green S. aureus BioParticles Conjugates. A Boyden's chamber model was used to determine the level of the neutrophil's mobility and chemotaxis response to 10% human serum. Formation of Neutrophils Extracellular Traps (NETs) and induction of the oxidative burst were studied upon phorbol 12-myristate 13-acetate (PMA) stimulation (25 nM) with SYTOX™ Green Nucleic Acid and flow cytometry with CellROX Green assay, respectively. The intracellular and circulating concentrations of anti-microbial compounds (Myeloperoxidase MPO, Lactotransferrin LTF, Cathelicidin-LL37, and defensins) were measured using commercially available ELISA assays. The results were evaluated with an unpaired t-test using GraphPad Prism 9. Quantitative data presented as medians (25%-75%). Results Genetic analysis identified three heterozygous variants of CLPB gene p.Arg327Trp, p.Arg628Cys, p .Arg629Cys in three different families. Two of these mutations were novel p.Arg327Trp and p .Arg629Cys. Interestingly, the p.Arg327Trp variant was located outside the ATPase domain. In total 7 family members who suffered from neutropenia and were carriers of the CLPB variants were available for neutrophil functional assays (Figure 1). Neutrophils collected from CLPB-deficient individuals have a higher rate of apoptosis as compared to healthy neutrophils: 57(52-66)% vs 23(20-27)%, P&amp;lt;0.0001, and necrosis: 0.56(0.31-1.07)% vs 0.13(0.10-0.21)%, P=0.02. CLPB-deficient neutrophils display reduced mobility in the absence of stimulation: 298(191-413) vs 1011(936-1201) migrating neutrophils, P&amp;lt;0.0001, and after 10% human serum treatment: 2090(1746-2458) vs 3736(3071-4828) migrating neutrophils, P=0.0014. Similarly, reduced phagocytosis of S. aureus bioparticles was observed in CLPB-deficient neutrophils: 1890(1679-2063) vs 3352(2472-4440) Fluorescence Units, respectively, P=0.0029. Upon PMA stimulation, neutrophils from CLPB patients produced less reactive oxygen species compared to controls: 311(175-542)FMI vs 775(766-1150)FMI, P=0.08, and their ability to produce NETs is almost entirely abolished: 0.5(0.21-0.79)ng/µl vs 1.3(1.08-3.05)ng/µl, P=0.0075. Moreover, CLPB-deficient neutrophils have reduced intracellular concentration of myeloperoxidase: 35(31-39)ng/mg vs 55(41-63)ng/mg, P=0.0043 and cathelicidin LL-37: 5.5(5-6.3)ng/µg vs 19.22(14.21-23.28)ng/µg, P=0.0003. The concertation of the LL-37 is also reduced in patients' plasma 1.34(0.84-1.78)ng/ml vs 7.17(5.38-8.03)ng/ml, P&amp;lt;0.0001. Both intracellular and circulating concentrations of LTF and defensins did not differ between study groups. Conclusion Neutrophils from neutropenic carriers of CLPB mutations are functionally impaired, especially concerning their ability for NETs formation and cathelicidin LL-37 production.

Naked Antibody-Based Conditioning Targeting CD47 and CD117 Plus Janus Kinase Inhibition Enables Toxin-Free, Radiation-Free, and Chemotherapy-Free Fully Mismatched Allogeneic Hematopoietic Stem Cell Transplantation

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for numerous malignant and non-malignant hematologic diseases. To enable engraftment of donor hematopoietic stem cells (HSCs), recipients undergo conditioning with chemotherapy and irradiation to make marrow space for incoming donor HSCs and suppress host immune responses that may lead to graft rejection. These cytotoxic conditioning regimens carry risks of severe toxicities which prevents many patients from accessing the lifesaving benefit of allo-HSCT. Previously, we developed fully ablative CD45- and cKit-targeted antibody-drug conjugates (ADC) which, when combined with Janus kinase 1/2 (JAK1/2) inhibitors, enabled fully mismatched allo-HSCT with full donor chimerism and minimal toxicity while providing durable antileukemia activity (Persaud et al (2022), ASH Abstract). However, even ADCs carry potential risks of acute and chronic toxicities that may preclude their common use as HSCT conditioning agents. Moreover, unlike malignant diseases, allo-HSCT for non-malignant diseases does not require the conditioning regimen to provide antitumor benefit and partial donor chimerism may be sufficient for cure, strongly favoring the use of less intense conditioning strategies with the fewest possible toxicities. We hypothesized that naked antibody-based conditioning strategies can achieve the therapeutic benefits of allo-HSCT for non-malignant hematologic diseases, obviating exposure to toxic ADC payloads. METHODS: We combined unconjugated antibodies targeting CD47 and cKit with JAK1/2 inhibition to perform minor histocompatibility antigen (miHA)-mismatched (129/Sv→B6-Ly5.1) and fully mismatched (BALB/c→B6) murine allo-HSCT. In some full mismatch HSCT experiments, recipients included B6-gp91phox -/- mice, which serve as a model of chronic granulomatous disease (CGD) due to their deficiency in a catalytic subunit of NADPH oxidase. Mice received 100 µg anti-CD47 (clone mIAP410; BioXcell) 8 days before HSCT (d-8) as a priming dose to reduce the severity of CD47 blockade-induced anemia. Next, mice received 500 µg anti-cKit (clone ACK2; BioLegend) on d-6 and daily 500 µg doses of anti-CD47 from d-6 through d-2. For JAK1/2 inhibition, mice received oral ruxolitinib (Incyte; 2 mg drug/g chow) starting d-3 or d-1 and continuing until d+28. On d0, recipients were transplanted with 20 x 10 6 whole bone marrow cells from 129/Sv (miHA) or BALB/c (fully mismatched) mice. Peripheral blood donor chimerism was followed monthly by flow cytometry for CD45.2 + (miHA) or H-2D d+ (fully mismatched) cells. Oxidative burst among granulocytes in B6-gp91phox -/- recipients was assessed by dihydrorhodamine 123 (DHR123) staining after phorbol 12-myristate 13-acetate (PMA) stimulation. RESULTS: CD47 and cKit antibodies plus ruxolitinib enabled multilineage engraftment in both miHA and fully mismatched allo-HSCT, including myeloid lineage donor chimerism of 95-99% (Figure 1A). As expected, CD47 blockade induced significant but transient morbidity from anemia during the peritransplant period; notably, a shorter duration of ruxolitinib treatment before HSCT (starting d-1 instead of d-3) improved the tolerability of the regimen without impacting engraftment success. Allo-HSCT of B6-gp91phox -/- mice led to near complete replacement of host granulocytes in peripheral blood with donor granulocytes, whose DHR123 positivity upon PMA stimulation indicated an intact oxidative burst capacity (Figure 1B). CONCLUSIONS: Combination of antibodies targeting CD47 and cKit with JAK1/2 inhibition provides a novel conditioning strategy for allo-HSCT in mouse models, achieving high-level, stable multilineage engraftment without radiation, chemotherapy, or exposure to toxic ADC payloads. The near complete donor chimerism among granulocytes made it particularly well suited to correcting phagocyte oxidase function in the gp91phox -/- model of CGD. Future studies seek to 1) mitigate the peritransplant anemia caused by CD47 blockade using Fc-silenced anti-CD47 reagents, 2) determine the proportions of donor- versus recipient-derived myeloid cells in non-hematopoietic tissues of allo-HSCT recipients, and 3) use murine infection models to evaluate the impact of allo-HSCT on host defense in transplanted B6-gp91phox -/- recipients.

CD138 promotes the accumulation and activation of autoreactive T cells in autoimmune MRL/lpr mice.

Autoreactive T cells, specifically CD138+ (syndecan-1) T cells produced in Fas-deficient systemic lupus erythematosus (SLE) mouse models, were shown to significantly promote the generation of autoantibodies. In the present study, Murphy Roths Large lymphoproliferative (MRL/lpr) lupus mice were used to investigate the role of CD138 protein expression in T cells in the progression of SLE. Measurement of flow cytometry, immunofluorescence and Luminex were performed to determine the effect of CD138 on T cells in MRL/lpr mice. The results demonstrate that CD138+ T cells induce apoptosis via a Fas-dependent pathway. CD138 protein expression in T cells of MRL/lpr mice significantly reduced T cell apoptosis and contributed to the accumulation of T cells and double negative (DN) T cells, whilst simultaneously promoting T cell activation in Fas-deficient lupus mice. CD138 protein expression in DN T cells also significantly increased the protein expression of Fas ligand to enhance the cytotoxicity of DN T cells. Furthermore, phorbol 12-myristate 13-acetate and ionomycin (PI) stimulation reduced CD138 protein expression in CD3+ T cells and prevented CD138+ T cell accumulation by inducing specific apoptosis. PI stimulation also activated T cells in MRL/lpr mice to increase CD69 protein expression. CD69 protein expression in CD138+ T cells significantly increased the frequency of apoptotic CD138+ T cells. In addition, results from the present study demonstrated that CD138- T cells of MRL/lpr lupus mice had an activation defect. CD138 protein expression in T cells significantly reversed the defective activation and activating T cells could significantly reduce CD138 protein expression in CD3+ T cells of MRL/lpr mice. This suggests that CD138 protein expression in CD3+CD138- T cells of MRL/lpr mice may be a consequence of the impaired activation in autoreactive T cells prior to exposure to self-antigens by the immune system. CD138 expression in autoreactive T cells has a central role in promoting the progression and development of autoimmune response in MRL/lpr mice.

The state of mitochondrial membrane potential in peripheral blood leukocytes of patients with chronic obstructive pulmonary disease

Introduction. Mitochondria provide energy homeostasis of the cell by maintaining an optimal transmembrane electrochemical gradient (ΔΨm), which does not allow excessive formation of reactive oxygen species (ROS). However, under conditions of pathology, the normal functioning of mitochondria is disrupted, which can lead to ATP deficiency and/or increased production of ROS.Aim. The aim of this study was to investigate the ΔΨm parameters and their relationship with the expression of TRP channels in peripheral blood leukocytes of patients with chronic obstructive pulmonary disease (COPD).Materials and methods. The study included 23 patients with COPD of varying severity, 8 smokers without signs of bronchial obstruction and 9 healthy volunteers who had never smoked. All subjects underwent spirometry to assess the lung function. ΔΨm was determined by staining the cells with tetramethylrhodamine ethyl ester (TMRE) and measuring the fluorescent signal by flow cytometry, under basal conditions and pro-inflammatory stimulation with phorbol-12-myristate-13-acetate (PMA).Results. We found that COPD patients were characterized by a significant increase in basal ΔΨm of monocytes (161.8 [153.8; 206.8] vs. 129.3 [75.5; 161.8], p=0.03) and lymphocytes (209,7 [184.7; 257.8] vs. 122.5 [67.9; 164.3], p=0.003) as compared with the control group. Stimulation of cells with PMA led to multidirectional changes in ΔΨm, while its increased level was still preserved in COPD. In monocytes of COPD patients, a decrease in ΔΨm in response to PMA stimulation was prevalent (75%), while in the majority (53.9%) of individuals in the control group ΔΨm, on the contrary, increased (p=0.08). In addition, among COPD patients, an increase in ΔΨm in monocytes was accompanied by an enhanced expression of TRPV4, while in the control group, among individuals with positive dynamics of ΔΨm, TRPV4 expression was, on the contrary, reduced.Conclusion. The increased level of ΔΨm in the mononuclears of COPD patients is consistent with previously detected enhanced ROS production, but does not support the assumption about energy deficit in the cells. The revealed differences in the relationship between TRPV4 expression and ΔΨm dynamics may indicate the presence of pathological features in TRP signaling in COPD patients.

Neutrophil extracellular trap formation during surgical procedures: a pilot study

Neutrophils can release neutrophil extracellular traps (NETs) containing DNA fibres and antimicrobial peptides to immobilize invading pathogens. NET formation (NETosis) plays a vital role in inflammation and immune responses. In this study we investigated the impact of surgical trauma on NETosis of neutrophils. Nine patients undergoing “Transcatheter/percutaneous aortic valve implantation” (TAVI/PAVI, mild surgical trauma), and ten undergoing “Aortocoronary bypass” (ACB, severe surgical trauma) were included in our pilot study. Peripheral blood was collected before, end of, and after surgery (24 h and 48 h). Neutrophilic granulocytes were isolated and stimulated in vitro with Phorbol-12-myristate-13-acetate (PMA). NETosis rate was examined by microscopy. In addition, HLA-DR surface expression on circulating monocytes was analysed by flow-cytometry as a prognostic marker of the immune status. Both surgical procedures led to significant down regulation of monocytic HLA-DR surface expression, albeit more pronounced in ACB patients, and there was a similar trend in NETosis regulation over the surgical 24H course. Upon PMA stimulation, no significant difference in NETosis was observed over time in TAVI/PAVI group; however, a decreasing NETosis trend with a significant drop upon ACB surgery was evident. The reduced PMA-induced NETosis in ACB group suggests that the inducibility of neutrophils to form NETs following severe surgical trauma may be compromised. Moreover, the decreased monocytic HLA-DR expression suggests a post-operative immunosuppressed status in all patients, with a bigger impact by ACB, which might be attributed to the extracorporeal circulation or tissue damage occurring during surgery.

Geranylgeranylacetone Ameliorates Skin Inflammation by Regulating and Inducing Thioredoxin via the Thioredoxin Redox System.

Geranylgeranylacetone (GGA) exerts cytoprotective activity against various toxic stressors via the thioredoxin (TRX) redox system; however, its effect on skin inflammation and molecular mechanism on inducing the TRX of GGA is still unknown. We investigated the effects of GGA in a murine irritant contact dermatitis (ICD) model induced by croton oil. Both a topical application and oral administration of GGA induced TRX production and Nrf2 activation. GGA ameliorated ear swelling, neutrophil infiltration, and inhibited the expression of TNF-α, IL-1β, GM-CSF, and 8-OHdG. GGA's cytoprotective effect was stronger orally than topically in mice. In vitro studies also showed that GGA suppressed the expression of NLRP3, TNF-α, IL-1β, and GM-CSF and scavenged ROS in PAM212 cells after phorbol myristate acetate stimulation. Moreover, GGA induced endogenous TRX production and Nrf2 nuclear translocation in PAM212 cells (dependent on the presence of ROS) and activated the PI3K-Akt signaling pathway. GGA significantly downregulated thioredoxin-interacting protein (TXNIP) levels in PAM212 cells treated with or without Nrf2 siRNA. After knocking down Nrf2 in PAM212 cells, the effect of GGA on TRX induction was significantly inhibited. This suggests that GGA suppress ICD by inducing endogenous TRX, which may be regulated by PI3K/Akt/Nrf2 mediation of the TRX redox system.

Association of IL6 and IL10 gene promotor polymorphisms with susceptibility to acute necrotizing encephalopathy.

Acute necrotizing encephalopathy (ANE) is a severe complication of infectious diseases affecting the brain and systemic organs. The main pathogenesis is cytokine storm, in which interleukin-6 (IL-6) and interleukin-10 (IL-10) are candidates for key cytokines. To further elucidate their roles in the etiology and pathogenesis of ANE, we studied polymorphisms in the promotor regions of the IL6 and IL10 genes by genetic and functional analyses. We first conducted a case-control association study of four IL6 and three IL10 polymorphisms. We genotyped 31 Japanese ANE cases and compared the results with those of approximately 200 Japanese controls. For the two polymorphisms showing a possible association, we next studied whether the polymorphisms alter the production of IL-6 or IL-10 by lymphoblasts upon phorbol 12-myristate 13-acetate (PMA) stimulation. The frequencies of IL6 rs1800796G allele and IL10 rs1800871/rs1800872 CC/CC diplotype were significantly higher in ANE cases than in controls. The IL10 CC/CC diplotype was associated with low IL-10 production, whereas the IL6 GG genotype was not associated with IL-6 production. IL10 rs1800871/rs1800872 CC/CC diplotype may predispose Japanese children to ANE by altering IL-10 production in the early phase of infection. Etio-pathogenetic significance of IL6 rs1800796G remains to be elucidated.

TRPA1 activation and Hsp90 inhibition synergistically downregulate macrophage activation and inflammatory responses in vitro

BackgroundTransient receptor potential ankyrin 1 (TRPA1) channels are known to be actively involved in various pathophysiological conditions, including neuronal inflammation, neuropathic pain, and various immunological responses. Heat shock protein 90 (Hsp90), a cytoplasmic molecular chaperone, is well-reported for various cellular and physiological processes. Hsp90 inhibition by various molecules has garnered importance for its therapeutic significance in the downregulation of inflammation and are proposed as anti-cancer drugs. However, the possible role of TRPA1 in the Hsp90-associated modulation of immune responses remains scanty.ResultsHere, we have investigated the role of TRPA1 in regulating the anti-inflammatory effect of Hsp90 inhibition via 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) in lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA) stimulation in RAW 264.7, a mouse macrophage cell lines and PMA differentiated THP-1, a human monocytic cell line similar to macrophages. Activation of TRPA1 with Allyl isothiocyanate (AITC) is observed to execute an anti-inflammatory role via augmenting Hsp90 inhibition-mediated anti-inflammatory responses towards LPS or PMA stimulation in macrophages, whereas inhibition of TRPA1 by 1,2,3,6-Tetrahydro-1,3-dimethyl-N-[4-(1-methylethyl)phenyl]-2,6-dioxo-7 H-purine-7-acetamide,2-(1,3-Dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7 H-purin-7-yl)-N-(4-isopropylphenyl)acetamide (HC-030031) downregulates these developments. LPS or PMA-induced macrophage activation was found to be regulated by TRPA1. The same was confirmed by studying the levels of activation markers (major histocompatibility complex II (MHCII), cluster of differentiation (CD) 80 (CD80), and CD86, pro-inflammatory cytokines (tumor necrosis factor (TNF) and interleukin 6 (IL-6)), NO (nitric oxide) production, differential expression of mitogen-activated protein kinase (MAPK) signaling pathways (p-p38 MAPK, phospho-extracellular signal-regulated kinase 1/2 (p-ERK 1/2), and phosphor-stress-activated protein kinase/c-Jun N-terminal kinase (p-SAPK/JNK)), and induction of apoptosis. Additionally, TRPA1 has been found to be an important contributor to intracellular calcium levels toward Hsp90 inhibition in LPS or PMA-stimulated macrophages.ConclusionThis study indicates a significant role of TRPA1 in Hsp90 inhibition-mediated anti-inflammatory developments in LPS or PMA-stimulated macrophages. Activation of TRPA1 and inhibition of Hsp90 has synergistic roles towards regulating inflammatory responses associated with macrophages. The role of TRPA1 in Hsp90 inhibition-mediated modulation of macrophage responses may provide insights towards designing future novel therapeutic approaches to regulate various inflammatory responses.

An Infant with Cervical Lymphadenitis Secondary to Nontuberculous Mycobacteria Refractory to Conventional Treatment, Responsive to Interferon-Gamma.

To the Editors: The interferon gamma/interleukin-12 positive feedback loop is critical for immunity against nontuberculous mycobacterium and mycobacterium tuberculosis.1 The use of IFN-γ as an effective treatment for these infections has been poorly described, particularly in the pediatric population. Diniz et al2 and Alangari et al3 describe cases of individuals with an underlying impairment in the interferon gamma/interleukin-12 receptor pathway with nontuberculous mycobacterial infections that were responsive to IFN- γ as an adjunctive treatment to their regimen. Our case presents an infant with lymphadenitis secondary to a nontuberculous mycobacterium that was refractory to conventional treatment and responsive to adjunctive IFN-γ. It is suspected that our infant’s response was due to an impairment in the IL-12/IFN-γ receptor signaling pathway. A previously healthy 9-month-old male presented to his pediatrician with an inflamed cervical lymph node. Ultrasonography revealed a complex cystic lesion. He completed a 10-day cefdinir course. Despite antibiotic treatment, the patient showed progression of the lesions and presented to the emergency department. Workup yielded elevated levels of inflammatory markers, with a white blood cell of 25 (ref. 5.5–17.0 k/µL) and a lymphocytic predominance of 61%. Computed tomography imaging revealed 2 possible retropharyngeal abscesses. The patient was started on clindamycin. Incision and drainage of the lesions were completed, and no growth was observed in tissue cultures. Aspirate culture of the retropharyngeal abscess showed 2+ Haemophilus parainfluenzae. Purified protein derivative was weakly positive at 7 mm of induration. He was discharged with amoxicillin, clarithromycin, and rifampicin. Ten days later, the patient’s physical examination was significant for 3 distinct areas of fistulation on the left side of the neck with no active drainage or tenderness (Fig. 1). Immunologic evaluation showed serum immunoglobulins within the age-appropriate reference range, adequate percentage stimulation and median fluorescence intensity of granulocytes on dihydrorhodamine flow with phorbol myristate acetate stimulation and inappropriate titers to Streptococcus pneumoniae with 1/23 ≥1.3, however, he had pending repeat doses as per the U.S. Centers for Disease Control and Prevention schedule. Immunoglobulin and lymphocyte subsets were within the reference range for age. Pathogenic variants impairing IFN-γ/IL-2 receptor signaling include AR IRF8 LOF, AD STAT1 LOF, AR STAT1 LOF, AR IFN-γR1 LOF, AR IFN-γR2 LOF, AR IKBK2 LOF, AR IL-12Rβ1 LOF, AR IRF8 LOF or AR TYK2 LOF. An X-linked pathology affecting IKBKG could not be excluded but was less likely given the absence of typical associated physical features. This does not rule out the presence of pseudogenes that can lead to a range of symptoms associated with NFkB essential modulator operon deficiency.FIGURE 1.: Demonstrating 3 distinct areas of fistulation on the left side of the neck with no active drainage.Given the progression of his lesions while on guideline-directed therapy, off-label use of subcutaneous IFN-γ treatment (Actimmune) was initiated every Mon, Wed, and Fri. (15 mcg/kg). The patient tolerated the medication without adverse effects. One month after initiating treatment, the patient showed significant improvement in both swelling and appetite, with his weight percentile returning to the previous baseline. Our case, in combination with other studies on the topic, shows great promise in utilizing IFN-γ as an adjunctive treatment for patients with mycobacterial infections that are refractory to conventional antimycobacterial treatments, with few adverse effects.

Oxidative stress in peripheral blood leukocytes of patients with chronic obstructive pulmonary disease

Introduction.It is known that oxidative stress causes pathological changes in the tissues and organs of the human body and in many cases it can explain the disorders associated with chronic obstructive pulmonary disease (COPD).Aim.The aim of the study was to investigate the degree of oxidative stress in peripheral blood leukocytes of COPD patients.Materials and methods. We examined 23 patients with COPD of varying severity, 8 smokers without signs of bronchial obstruction and 9 healthy volunteers who had never smoked. All subjects underwent spirometry to assess lung function. The level of oxidative stress was determined using flow cytometry by adding 10 µM 2',7'-dichloro dihydrofluorescein diacetate to isolated peripheral blood leukocytes, in basal conditions and under pro-inflammatory stimulation with 0.1 ng/ml phorbol-12-myristate-13-acetate (PMA).Results.Patients with COPD demonstrated elevated levels of intracellular oxidative stress compared with controls both under basal conditions and during PMA stimulation (p&lt;0.05). When analyzed in subgroups, stimulation led to a significant increase in oxidative stress regardless of cell type (p&lt;0.01). Smokers tend to have elevated measured values, occupying an intermediate position between non-smokers and COPD patients. The absolute increase in the oxidative stress index was higher in the cells of COPD patients; the values of the relative increase were almost identical in the studied groups. The dynamics of oxidative stress in the cells of smokers tended to be more pronounced than in non-smokers.Conclusion.The obtained results indicate that the leukocytes of COPD patients are characterized by a higher level of intracellular oxidative stress. In addition, smoking has been found to be a factor of oxidative stress development in healthy individuals.

Inflammation and Neutrophil Oxidative Burst in a Family with NFKB1 p.R157X LOF and Sterile Necrotizing Fasciitis

Loss-of-function (LOF) mutations in NFKB1, coding for p105, may cause common variable immunodeficiency due to dysregulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κΒ) pathway. Monoallelic LOF variants of NFKB1 can predispose to uncontrolled inflammation including sterile necrotizing fasciitis or pyoderma gangrenosum. In this study, we explored the impact of a heterozygous NFKB1 c.C936T/p.R157X LOF variant on immunity in sterile fasciitis patients and their family members. The p50 or p105 protein levels were reduced in all variant carriers. Interleukin-1β (IL-1β) and interleukin-8 (IL-8) levels were elevated in vitro, potentially contributing to the very high neutrophil counts observed during fasciitis episodes. Phosphorylation of p65/RelA was reduced in p.R157X neutrophils suggesting defective activation of canonical NF-κB. Oxidative burst after NF-κB-independent phorbol 12-myristate 13-acetate (PMA) stimulation was similar in both p.R157X and control neutrophils. Comparable amounts of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex subunits were found in p.R157X and control neutrophils. However, a compromised oxidative burst was observed in p.R157X neutrophils following activation of NF-κB-dependent mechanisms following stimulation of toll-like receptor 2 (TLR2) and Dectin-1. Neutrophil extracellular trap formation was not affected by p.R157X. In summary, the NFKB1 c.C936T/p.R157X LOF variant has an impact on inflammation and neutrophil function and may play a role in the pathogenesis of sterile necrotizing fasciitis.

Effect of Hydrogen Sulfide on Essential Functions of Polymorphonuclear Leukocytes.

Impaired polymorphonuclear leukocyte (PMNL) functions contribute to increased infections and cardiovascular diseases in chronic kidney disease (CKD). Uremic toxins reduce hydrogen sulfide (H2S) levels and the anti-oxidant and anti-inflammatory effects of H2S. Its biosynthesis occurs as a side process of transsulfuration and in the disposal of adenosylhomocysteine, a transmethylation inhibitor and proposed uremic toxin. PMNL chemotaxis was measured by the under-agarose method, phagocytosis, and oxidative burst by flow cytometry in whole blood and apoptosis by determining DNA content by flow cytometry and morphological features by fluorescence microscopy. Sodium hydrogen sulfide (NaHS), diallyl trisulphide (DATS) and diallyl disulphide (DADS), cysteine, and GYY4137 were used as H2S-producing substances. Increased H2S concentrations did not affect chemotaxis and phagocytosis. NaHS primed PMNL oxidative burst activated by phorbol 12-myristate 13-acetate (PMA) or E. coli. Both DATS and cysteine significantly decreased E. coli-activated oxidative burst but had no effect on PMA stimulation. While NaHS, DADS, and cysteine attenuated PMNL apoptosis, GYY4137 decreased their viability. Experiments with signal transduction inhibitors suggest that the intrinsic apoptosis pathway is mainly involved in GYY4137-induced PMNL apoptosis and that GYY4137 and cysteine target signaling downstream of phosphoinositide 3-kinase.

Characterization of myeloperoxidase and its contribution to antimicrobial effect on extracellular traps in flounder (Paralichthys olivaceus).

Myeloperoxidase (MPO) is a cationic leukocyte haloperoxidase and together with other proteins, they possess activities against various microorganisms and are involved in extracellular trap (ET) formation. The present work describes the gene and deduced protein sequences, and functions of MPO in flounder (PoMPO). The PoMPO possesses a 2313 bp open reading frame (ORF) that encodes a protein of 770 amino acids. The highest PoMPO mRNA expression levels were found in the head kidney, followed by peritoneal cells, gill, spleen, skin, muscle, and liver. PoMPO was expressed in MHCII+ and GCSFR+ cells which indicated that PoMPO mainly is expressed in flounder macrophages and granulocytes. Bacterial lipopolysaccharide-stimulated peritoneal leukocytes showed an increased protein level of PoMPO while it seemed that LPS also promoted the migration of MPO+ cells from the head kidney into the peripheral blood and peritoneal cavity. After phorbol 12-myristate 13-acetate (PMA) or bacterial stimulation, flounder leukocytes produced typical ET structures containing DNA with decoration by MPO. The ETs containing DNA and PoMPO effectively inhibited the proliferation of ET-trapped bacteria. Blocking PoMPO with antibodies decreased the enzymatic activity, which attenuated the antibacterial activity of ETs. This study pinpoints the involvement of ETs in flounder innate responses to pathogens.

Generation of reporter mice for detecting the transcriptional activity of nuclear factor of activated T cells.

Nuclear factor of activated T cells (NFAT) is a transcription factor essential for immunological and other biological responses. To develop analyzing system for NFAT activity in vitro and in vivo, we generated reporter mouse lines introduced with NFAT-driven enhanced green fluorescent protein (EGFP) expressing gene construct. Six tandem repeats of -286 to -265 of the human IL2 gene to which NFAT binds in association with its co-transcription factor, activator protein (AP)-1, was conjunct with thymidine kinase minimum promoter and following EGFP coding sequence. Upon introduction of the resulting reporter cassette into C57BL/6 fertilized eggs, the transgenic mice were obtained. Among 7 transgene-positive mice in 110 mice bone, 2 mice showed the designated reporter mouse character. Thus, the EGFP fluorescence of CD4+ and CD8+ T cells in these mice was enhanced by stimulation through CD3 and CD28. Each of phorbol 12-myristate 13-acetate (PMA) and ionomycin (IOM) stimulation weakly but their combined stimulation strongly enhanced EGFP expression. The stimulation-induced EGFP upregulation was also observed following T cell subset differentiation in a different manner. The EGFP induction by PMA + IOM stimulation was more potent than that by CD3/CD28 stimulation in helper T (Th)1, Th2, Th9, and regulatory T cells, while both stimulation conditions displayed the equivalent EGFP induction in Th17 cells. Our NFAT reporter mouse lines are useful for analyzing stimulation-induced transcriptional activation mediated by NFAT in cooperation with AP-1 in T cells.

(+)-Borneol inhibits the generation of reactive oxygen species and neutrophil extracellular traps induced by phorbol-12-myristate-13-acetate.

Background and purpose: Neutrophil extracellular traps (NETs) are special web-like structures that can be generated in both infectious and noninfectious diseases. Previous studies showed that reactive oxygen species (ROS) were crucial in the formation of NETs (NETosis). The purpose of this study is to evaluate the effect of (+)-borneol, an antioxidant, on NETosis. Methods: Human neutrophils were stimulated with phorbol-12-myristate-13-acetate (PMA) to induce NETosis in vitro. Neutrophils treated with (+)-borneol at three different time points (-30min, 0, and 30min) associated with PMA stimulation were used to examine the effect of (+)-borneol on the formation of NETs. The ROS generation of neutrophils was also measured to explore the potential mechanism of the inhibitory effect of (+)-borneol on NETosis. Results: (+)-Borneol pretreatment inhibited NETosis induced by PMA. Immunofluorescence staining visualized and confirmed the inhibitory effect. (+)-Borneol inhibited the burst of ROS in neutrophils caused by PMA. Suppressing NADPH oxidase or protein kinase C (PKC) eliminated the effect of (+)-borneol on NETosis. Moreover, inhibiting Toll-like receptor 2 (TLR2) led to increased NETosis which can be inhibited by (+)-borneol. Conclusion: (+)-Borneol decreases the ROS level in activated neutrophils and inhibits NETosis triggered by PMA stimulation in vitro. (+)-Borneol therapy may be effective in some NET-dependent conditions.