Neutrophil Chemotaxis Research Articles

Fusobacterium Nucleatum Aggravates Intestinal Barrier Impairment and Colitis Through IL-8 Induced Neutrophil Chemotaxis by Activating Epithelial Cells.

Inflammatory bowel disease (IBD) is affected by interactions between intestinal microbial factors, abnormal inflammation, and an impaired intestinal mucosal barrier. Neutrophils (NE) are key players in IBD. Fusobacterium nucleatum (F. nucleatum) is reported to contribute to IBD progression. However, the relationship between F. nucleatum, abnormal inflammation, and intestinal barrier impairment should be interpreted to understand the role of F. nucleatum in IBD. Dextran sulfate sodium (DSS)-induced colitis model was established and mice were orally administered with F. nucleatum. F. nucleatum colonization was confirmed by fluorescence in situ hybridization (FISH) and PCR. Intestinal barrier impairment was investigated by tight junction protein expression. Immuno-histochemistry (IHC) for Ly6G and flow cytometry detection to measure NE chemotaxis in mouse colon tissues. Caco-2 monolayers were used to evaluate epithelial integrity and permeability in vitro. A transwell model involving caco-2 cells and NE co-culture was used to assess NE chemotaxis. NE chemokines were measured by ELISA. A mouse model of NE exhaustion using an anti-Ly6G antibody was used to identify the role of NEs in F. nucleatum-induced colitis. Transcriptome sequencing and bioinformatics analysis were applied to screen cytokines and signaling pathways. Administration of F. nucleatum aggravated colitis in the DSS model. F. nucleatum infection downregulates ZO-1 and Occludin expression and increases intestinal permeability. Additionally, F. nucleatum-induced NE chemotaxis decreases the integrity and permeability of the caco-2 monolayer. F. nucleatum-induced NE chemotaxis is dependent on IEC-derived interleukin 8 (IL-8) secretion, mediated by the TLR2/ERK signaling pathway. In addition, NE exhaustion in mice inhibited F. nucleatum-induced intestinal barrier impairment and colitis. F. nucleatum improves NE chemotaxis by infecting intestinal epithelial cells (IECs) to secrete IL-8 and aggravate intestinal barrier impairment, contributing to the progression of intestinal inflammation. Examining and eliminating F. nucleatum could be a valuable microbiome-based method for IBD surveillance and prevention.

Acute and chronic rhinosinusitis age characteristics

Children's and adults' rhinosinusitis are two diseases that have both similarities and differences in anatomy, epidemiology, causes, pathogenesis, diagnosis and treatment. At the same rhinosinusitis is one of the most common in otorhinolaryngology's practice, both in children and adults. The of adults paranasal sinuses (PNS) anatomy differs from children's PNS anatomy. Although ostiomeatal complex occlusion is recognized as a major cause of poor ventilation and drainage of the adult paranasal sinuses, it does not have a strong effect on pediatric rhinosinusitis, but adenoids play a key role. Adenoids are bacteria and biofilms reservoirs that cause chronic refractory rhinosinusitis regardless of pharyngeal tonsil size. The prevalence of chronic rhinosinusitis (CRS) is lower in children than in adults. Diagnosis of children's rhinosinusitis is more difficult because nasal cavity endoscopic examination is performed rarely due to the occasional need of general anesthesia during the procedure. Moreover, it's necessary to take into account prevailing etiological role of viruses in ARS at children's age and chronic adenoiditis often accompanies pediatric CRS, which requires attention prescribing medical therapy as the basis of rhinosinusitis treatment. The DysheLORz based on Pelargonium sidoides roots is highly effective and safe for children's and adults ARS and CRS treatment, both as monotherapy and in combination with topical steroids and antibiotics. This herbal medicine immunomodulatory effect is mediated mainly by stimulating the production of TNF-α, IL-1, IL-12 and IFN-γ. It activates macrophages and improves their phagocytic activity. IL-12, together with TNF-α, enhances NK and cytotoxic CD8+ lymphocytes' activity against infected cells. IL-12 effect on Th1 lymphocytes maturation provides a link between innate and adaptive immunity. This is also increasing MCP-1, IP-10 and MIP-1β chemokines synthesis and decreasing MIP-1α, ENA-78, GROα and IL-8 production in PNS and nasal mucosa. This leads to decrease of neutrophils chemotaxis to the inflammation site, and decline of serine proteases concentration (neutrophils main enzymes), that increases mucous membrane epithelial barrier permeability, reducing bacterial infections risk. Additionally, Pelargonium sidoides increases epithelial cells beating cilia frequency and inhibits hemagglutinin and neuraminidase present on influenza virus surface. The drug increases antimicrobial peptides production as defensins, human neutrophil peptides (HNP) and bactericidal permeability-increasing protein (BPI), which is also important for rapid inflammation regression in rhinosinusitis. It causes bacterial adhesion to epithelial cells inhibition, phagocytosis stimulation, nitric oxide (NO) release and oxidative burst. The medicine had a direct effect on Streptococcus pneumoniae, Staphylococcus aureus, Neisseria, Moraxella catarrhalis and Haemophilus influenza. Based on these data, it is possible to explain the high effectiveness and safety of the drugs based on Pelargonium sidoides in ENT organs inflammation treatment, for both adults and children over 1 year old.

Inertial label-free sorting and chemotaxis of polymorphonuclear neutrophil in sepsis patients based on microfluidic technology

Reduced chemotactic migration of polymorphonuclear neutrophil (PMN) in sepsis patients leads to decreased bacterial clearance and accelerates the progression of sepsis disease. Quantification of PMN chemotaxis in sepsis patients can help characterize the immune health of sepsis patients. Microfluidic microarrays have been widely used for cell chemotaxis analysis because of the advantages of low reagent consumption, near-physiological environment, and visualization of the migration process. Currently, the study of PMN chemotaxis using microfluidic chips is mainly limited by the cumbersome cell separation operation and low throughput of microfluidic chips. In this paper, we first designed an inertial cell sorting chip to achieve label-free separation of the two major cell types by using the basic principle that leukocytes (mainly granulocytes, lymphocytes and monocytes) and erythrocytes move to different positions of the spiral microchannel when they move in the spiral microchannel under different strength of inertial force and Dean's resistance. Subsequently, in this paper, we designed a multi-channel cell migration chip and constructed a microfluidic PMN inertial label-free sorting and chemotaxis analysis platform. The inertial cell sorting chip separates leukocyte populations and then injects them into the multi-channel cell migration chip, which can complete the chemotaxis test of PMN to chemotactic peptide (fMLP) within 15 min. The remaining cells, such as monocytes with slow motility and lymphocytes that require pre-activation with proliferative culture, do not undergo significant chemotactic migration. The test results of sepsis patients ( n=6) and healthy volunteers ( n=3) recruited in this study showed that the chemotaxis index (CI) and migration velocity ( v) of PMN from sepsis patients were significantly weaker than those from healthy volunteers. In conclusion, the microfluidic PMN inertial label-free sorting and chemotaxis analysis platform constructed in this paper can be used as a new tool for cell label-free sorting and migration studies.

The inhibition effect of caffeic acid on NOX/ROS-dependent macrophages M1-like polarization contributes to relieve the LPS-induced mice mastitis

The mammary gland is an adipose tissue containing not only adipocytes but also epithelial, endothelial, and immune cells. Epithelial cells and macrophages, as the integral components of the immune system, are on the front line of defense against infection. Our preliminary work proved that caffeic acid (CA) can effectively inhibit the inflammatory cascade of bovine mammary epithelial cells (BMEC) induced by lipopolysaccharide (LPS) and maintain cellular integrity and viability. Here, we investigated the therapeutic effect of CA on LPS-induced mice mastitis and explored its regulatory mechanism on macrophage inflammatory response induced by LPS in vitro. Firstly, the mice mastitis model was established by intramammary injection with 10 μg LPS, after which different CA doses (5, 10, 15 mg/kg) were administered. Then, the pathological section, myeloperoxidase (MPO) activity, proinflammatory factors and chemokines releasement, and redox state of mammary tissues were assessed, confirming CA's effectiveness on mice mastitis. In vitro, we validated the therapeutic relevance of CA in relieving LPS-induced RAW264.7 inflammatory and oxidative stress responses. Moreover, we further provided evidence that CA significantly reduced LPS-induced reactive oxygen species (ROS) generation via NADPH oxidase (NOX), which improved the imbalance relationship between nuclear factor kappa-B (NF-κB) and NF-E2 p45-related factor 2 (Nrf2) and led to a marked weakening of M1 polarization. The NOX-ROS signal inhibited by CA weakened the oxidative burst and neutrophil chemotaxis of macrophages, thus alleviating the immune cascade in mammary gland tissue and reducing the LPS-induced inflammatory damage. Collectively, CA would be a potential candidate or antibacterial synergist for curbing mastitis.

Deficiency of Acute-Phase Serum Amyloid A Exacerbates Sepsis-Induced Mortality and Lung Injury in Mice.

Serum amyloid A (SAA) is a family of proteins, the plasma levels of which may increase >1000-fold in acute inflammatory states. We investigated the role of SAA in sepsis using mice deficient in all three acute-phase SAA isoforms (SAA-TKO). SAA deficiency significantly increased mortality rates in the three experimental sepsis mouse models: cecal ligation and puncture (CLP), cecal slurry (CS) injection, and lipopolysaccharide (LPS) treatments. SAA-TKO mice had exacerbated lung pathology compared to wild-type (WT) mice after CLP. A bulk RNA sequencing performed on lung tissues excised 24 h after CLP indicated significant enrichment in the expression of genes associated with chemokine production, chemokine and cytokine-mediated signaling, neutrophil chemotaxis, and neutrophil migration in SAA-TKO compared to WT mice. Consistently, myeloperoxidase activity and neutrophil counts were significantly increased in the lungs of septic SAA-TKO mice compared to WT mice. The in vitro treatment of HL-60, neutrophil-like cells, with SAA or SAA bound to a high-density lipoprotein (SAA-HDL), significantly decreased cellular transmigration through laminin-coated membranes compared to untreated cells. Thus, SAA potentially prevents neutrophil transmigration into injured lungs, thus reducing exacerbated tissue injury and mortality. In conclusion, we demonstrate for the first time that endogenous SAA plays a protective role in sepsis, including ameliorating lung injury.

Modified Bushen Yiqi Formula mitigates pulmonary inflammation and airway remodeling by inhibiting neutrophils chemotaxis and IL17 signaling pathway in rats with COPD

Ethnopharmacological relevanceChronic obstructive pulmonary disease (COPD) is a major global health concern characterized by pulmonary inflammation and airway remodeling. Traditional Chinese medicine, such as Modified Jiawei Bushen Yiqi Formula (MBYF), has been used as a complementary therapy for COPD in China. Aim of the studyTo investigate the therapeutic potential of MBYF in a rat model of COPD induced by cigarette smoke (CS) exposure and explore the underlying mechanism. Materials and methodsThe COPD rat model was established through 24 weeks of CS exposure, with MBYF administration starting in the 9th week. Pulmonary function, histological analysis, inflammatory cell count and molecular assays were employed to assess the effects of MBYF on airway remodeling, pulmonary inflammation, neutrophils chemotaxis and the IL17 signaling pathway. ResultsMBYF treatment effectively delayed airway remodeling, as evidenced by improved pulmonary function parameters. Histological examination and bronchoalveolar lavage fluid analysis revealed that MBYF mitigated CS-induced pulmonary inflammation by reducing inflammatory cell infiltration. Pharmacological network analysis suggested that MBYF may act through the IL17 signaling pathway to regulate inflammatory responses. RNA-sequencing and molecular assays indicated that MBYF inhibited neutrophils chemotaxis through downregulating the CXCL1/CXCL5/CXCL8-CXCR2 axis, and suppressed IL17A, IL17F and its downstream cytokines, including IL6, TNFα, IL1β, and COX2. Furthermore, MBYF inhibited the activation of NF-κB and MAPKs in the IL17 signaling pathway. ConclusionMBYF exhibits potential as an adjunct or alternative treatment for COPD, effectively mitigating CS-induced pulmonary inflammation and airway remodeling through the inhibition of neutrophil chemotaxis and IL17 signaling pathway.

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<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<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<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.

Single-Cell Sequencing Reveals a Close Correlation between the Number of MDSCs and the Gene Expression Levels of GPX1 and S100A8/S100A9 in Patients with Myelofibrosis

Myelofibrosis is a type of myeloproliferative disorder, and research indicates that the immunosuppressive tumor microenvironment plays a significant role in the pathogenesis of myelofibrosis, with myeloid-derived suppressor cells (MDSCs) being the primary inhibitory cells involved in shaping the tumor immune microenvironment. In this study, we used flow cytometry to detect the number of MDSCs in the bone marrow of myelofibrosis patients and revealed the relationship between MDSCs and gene expression levels through single-cell transcriptome sequencing. We utilized flow cytometry to identify the number of Lin-HLA-DR-CD33+ labeled MDSCs in the bone marrow of 56 myelofibrosis patients and 20 healthy individuals. Additionally, we performed single-cell transcriptome sequencing on samples from two myelofibrosis patients, one showing a favorable response to Lurcitanib treatment and the other showing an ineffective response. The results of flow cytometry revealed a significant increase in the percentage of MDSCs in the bone marrow of myelofibrosis patients (15.16±6.25%) compared to healthy individuals (1.72±0.88%) (p<0.0001) (Fig a, b). Among the two patients subjected to single-cell sequencing, the percentage of MDSCs in their samples was 19.3% and 8.9% (Fig c). Following treatment with different concentrations of decitabine, the MDSCs cell count progressively decreased with increasing treatment time and concentration (Fig d).Subsequently, we identified the major cell types in the two samples through single-cell sequencing, which included T cells, Neutrophils, Monocytes, Erythroblasts, B cells, Platelets, Myelocytes, Hematopoietic Stem Cells（HSCs）, and progenitor Myelocytes (Fig e). The analysis of marker gene expression for each major cell type showed good specificity (Fig f). By comparing the proportions of different cell types between the two samples, it was observed that the high MDSC group had a decrease in the proportions of B cells, T cells, Monocytes, and Platelets, while an increase in the proportions of Neutrophils, Erythroblasts, HSC, Myelocytes, and progenitor Myelocytes (Fig g).Furthermore, we analyzed the highly differentially expressed genes in different cell types and identified significantly enriched pathways. The results showed significant downregulation of the GPX1 gene in monocytes of the high MDSC group, whereas the S100A8/S100A9 genes were significantly upregulated in HSC (Fig h). Enrichment analysis revealed significant enrichment of pathways related to reactive oxygen species metabolism, immune-inflammatory response, etc., in monocytes of the high MDSCs group, and significant enrichment of pathways related to NF-kappaB transcription factor activity regulation, Jak-STATs signaling, mTOR signaling in HSC. Research has shown that the number of MDSCs is elevated in patients with myelofibrosis . Lurcitanib exhibits poor therapeutic efficacy in myelofibrosis patients with high MDSCs counts, whereas decitabine demonstrates more favorable treatment outcomes. Single-cell sequencing results revealed significant downregulation of the GPX1 gene in monocytes of myelofibrosis patients with high MDSCs counts. GPX1 gene encodes glutathione peroxidase 1, which plays a role in reducing oxidative stress and inflammation, and has anti-fibrotic effects.Conversely, in HSC of myelofibrosis patients with high MDSCs counts, the S100A8/S100A9 gene expression is significantly upregulated. This gene can induce neutrophil chemotaxis and adhesion, serving as a danger-associated molecular pattern (DAMP) molecule and stimulating innate immune cells through pattern recognition receptors (e.g., TLR4 and AGER), subsequently activating MAP kinase and NF-kappa-B signaling pathways, leading to inflammation and fibrosis.Thus, it can be inferred that the number of MDSCs in myelofibrosis patients is negatively correlated with GPX1 gene expression and positively correlated with S100A8/S100A9 gene expression. Acknowledgement: This research was funded by the Key R&D Program of Zhejiang, No. 2022C03137; Public Technology Application Research Program of Zhejiang, China, No. LGF21H080003; Zhejiang Medical Association Clinical Medical Research special fund project, No. 2022ZYC-D09. Correspondence to: Dr Jian Huang, Department of Hematology, The First Affiliated Hospital of Zhejiang University School of Medicine. househuang@zju.edu.cn

Role of Glutathione in Neutrophil Chemotaxis in Periodontitis

Periodontitis is a common non-communicable inflammatory disease that leads to the destruction of periodontal tissues and tooth loss. Initiated by the plaque biofilm, there is a strong innate immune response with an abundance of neutrophils in the periodontium of affected individuals. Previous reports have shown that the intracellular concentration of glutathione in peripheral blood neutrophils from periodontitis patients and the chemotactic ability of these cells are compromised. Furthermore, other studies have described that in oxidative stress conditions neutrophil chemotaxis is aberrant and causes the glutathionylation of F-actin, a key player in chemotaxis. In this study, the effects of glutathione-modulating compounds were assessed in neutrophils isolated from healthy donors, showing that the perturbation of glutathione homeostasis decreases the chemotaxis of neutrophils. Following this, the intracellular glutathione status and chemotactic ability of neutrophils isolated from periodontitis patients was compared to that of age- and sex-matched controls. A decrease in glutathione and chemotactic ability were confirmed. Finally, the proteome of these neutrophils was explored, demonstrating a change in the abundance of proteins involved in glutathione homeostasis. Together these data suggest that peripheral blood neutrophils from periodontitis patients are compromised in their ability to cope with oxidative stress and move.

High levels of short-chain fatty acids secreted by Candida albicans hyphae induce neutrophil chemotaxis via free fatty acid receptor 2.

Candida albicans belongs to our commensal mucosal flora and in immune-competent individuals in the absence of epithelial damage, this fungus is well tolerated and controlled by our immune defense. However, C. albicans is an opportunistic microorganism that can cause different forms of infections, ranging from superficial to life-threatening systemic infections. C. albicans is polymorphic and switches between different phenotypes (e.g. from yeast form to hyphal form). C. albicans hyphae are invasive and can grow into tissues to eventually reach circulation. During fungal infections, neutrophils in particular play a critical role for the defense, but how neutrophils are directed toward the invasive forms of fungi is less well understood. We set out to investigate possible neutrophil chemoattractants released by C. albicans into culture supernatants. We found that cell-free culture supernatants from the hyphal form of C. albicans induced both neutrophil chemotaxis and concomitant intracellular calcium transients. Size separation and hydrophobic sorting of supernatants indicated small hydrophilic factors as responsible for the activity. Further analysis showed that the culture supernatants contained high levels of short-chain fatty acids with higher levels from hyphae as compared to yeast. Short-chain fatty acids are known neutrophil chemoattractants acting via the neutrophil free fatty acid receptor 2. In line with this, the calcium signaling in neutrophils induced by hyphae culture supernatants was blocked by a free fatty acid receptor 2 antagonist and potently increased in the presence of a positive allosteric modulator. Our data imply that short-chain fatty acids may act as a recruitment signal whereby neutrophils can detect C. albicans hyphae.

BIOM-09. OPPOSING EFFECTS OF LINKED CX3CR1 GERMLINE VARIANTS ON SURVIVAL AND IMMUNE ACTIVATION IN GLIOMA

Abstract Linkage-disequilibrium connects CX3CR1 V249I and T280M single-nucleotide polymorphisms; V249I occurs as a single-variant (SV) and T280M exclusively as a double-variant (DV) concurrent with V249I. In low-grade gliomas (LGG), we found improved median overall-survival (mOS) in SV (n = 14, mOS 21.03yrs), compared to WT (n = 45, mOS 9.41yrs) and DV (n = 31, mOS 10.49yrs; p = 0.037 Log-rank). In TCGA glioblastoma (GBM), SV (n = 69, mOS 15.65mo) improved survival over DV (n = 106, mOS 11.74mo; p = 0.044 Log-rank). In a separate GBM cohort, bulk RNA-sequencing revealed MMP9 over-expression in DV (n = 15) compared to SV (n = 10; log2FC 2.87, p-adj 0.079). On a single-gene basis, MMP9 expression was decreased in SV compared to DV and WT (n = 33; p = 0.044, Kruskal-Wallis). MMP9 promotes tumor angiogenesis and invasion. SV showed SPP1 over-expression compared to DV (log2FC -1.75, p-adj 0.026), with decreased SPP1 expression in both WT and DV compared to SV (p = 0.004, Kruskal-Wallis). SPP1 is a pro-inflammatory cytokine enriched in glioma-associated macrophages. Gene Ontology revealed enrichment of biological processes: neutrophil migration, granulocyte chemotaxis, and neutrophil chemotaxis in SV (p< 0.05). We single-cell RNA-sequenced IDH mutant gliomas (51777 cells). We subset and re-clustered myeloid-derived cells (microglia/macrophages, 8944 cells), identifying thirteen clusters. Myeloid-derived cells clustered by CX3CR1 status, with 50.8% of SV cells in cluster five and 85.3% of DV cells in clusters six or seven. Cluster five over-expressed IL1B (average-log2FC 3.23), a tumor-suppressive M1 macrophage marker. Clusters six and seven were characterized by IFIT genes – immunomodulatory in myeloid-derived cells (IFIT3 average-log2FC 2.65 and 2.06). Assessing CX3CR1 status in normal population – 74.2% are WT, 10.0% SV, and 15.7% DV. In glioma patients, we identify 59.9% WT, 14.9% SV, and 25.2% DV – an increase of 49% in SV and 61% in DV (p = 1.95e-6, Chi-square). Our findings demonstrate opposing effects of linked CX3CR1 germline variants on glioma survival and indicate enhanced immune activation in SV.

Electroconductive and Immunomodulatory Natural Polymer‐Based Hydrogel Bandages Designed for Peripheral Nerve Regeneration

AbstractSuccessful regeneration of the peripheral nerve relies on the collaborative efforts of neural cells and immune cells. Conductive hydrogels have yielded promising results in supporting axonal growth; however, their inability to regulate the immune response and their poor biological integration with tissues hinder the repair of injured peripheral nerves. Herein, an adhesive conductive immunomodulatory nerve hydrogel bandage is developed for nerve regeneration. The nerve bandage hydrogel is prepared from the bioactive material extracellular matrix (ECM), oxidized polysaccharides, and poly(3,4‐ethylenedioxythiophene)/poly(styrenesulfonate) (PEDOT:PSS) by self‐assembly and dynamic Schiff base cross‐linking. The drug indole‐3‐propionic acid (IPA) is loaded into nerve bandages and contributes to the rapid chemotaxis of neutrophils in the dorsal root ganglia and modulation of the immune system. In addition, the conductive hydrogel bandage exhibits close conformal contact with the injured nerve, forming a stable and tightly coupled electrical bridge with the electroresponsive neural tissue. In summary, the nerve bandage effectively promote nerve regeneration and enable both anatomical and functional recovery of neural tissue while preventing muscle atrophy. This work provides a new strategy for peripheral nerve regeneration and may have critical clinical applications in the future.

Abstract 13994: PKM2 Ablation Increases Pro-Inflammatory Signaling in Mice

Introduction: The predominant isoform of pyruvate kinase (PKM1) promotes oxidative metabolism in the healthy heart. We previously described a hypoxia and ischemia-mediated switch to the alternatively spliced isoform PKM2, known to promote glycolysis. Pkm2 has also been shown to reduce oxidative damage and promote cardiomyocyte cell proliferation after myocardial infarction (MI). PKM2 is also a metabolic regulator of immune cell differentiation. Hypothesis: We hypothesize that PKM2 has a role in limiting inflammatory stress by regulating the migration and activity of immune cells. Methods: PKM2 control and global KO mice were subjected to permanent ligation of the left anterior descending coronary artery to mimic an MI (n=3). Hearts were excised after 3 days and the transcriptomes and gene ontologies (GO) were then assessed by RNA-sequencing and pathway analysis. Circulating C-reactive protein (CRP) was assessed using an ELISA (n=12) and cytokines in plasma and heart tissue were assessed using a multiplex assay (n=3-5). Neutrophil myeloperoxidase secretion and differentiated cardiac macrophages were analyzed by immunofluorescent staining (n=3). Results: GO term enrichment analysis of RNA-seq results showed increased abundance of genes for neutrophil chemotaxis and apoptosis, regulation of cytokine production, and the inflammatory response in PKM2 KO hearts after MI compared to controls. PKM2 KO hearts showed increased neutrophil myeloperoxidase secretion and Ly6G/C staining for neutrophils and monocytes compared to controls. Similarly, M1 macrophages appeared to be more abundant in PKM2 KO hearts compared to controls. IL-6 levels were elevated in PKM2 KO plasma at baseline compared to controls, while CRP was elevated in PKM2 KO plasma across all conditions. IL-6 was also substantially elevated in PKM2 KO hearts after MI compared to controls. Conclusions: Loss of PKM2 increased pro-inflammatory signaling in the heart of KO mice. We saw increased cardiac and plasma IL-6 and CRP, likely related to the polarization of macrophages towards the M1 phenotype, and increased neutrophil activity in PKM2 KO hearts. We suggest that PKM2, induced by a hypoxia-related splicing event, protects the heart against the inflammation that accompanies infarction.

Keratinocyte FABP5-VCP complex mediates recruitment of neutrophils in psoriasis

One of the hallmarks of intractable psoriasis is neutrophil infiltration in skin lesions. However, detailed molecular mechanisms of neutrophil chemotaxis and activation remain unclear. Here, we demonstrate a significant upregulation of epidermal fatty acid binding protein (E-FABP, FABP5) in the skin of human psoriasis and psoriatic mouse models. Genetic deletion of FABP5 in mice by global knockout and keratinocyte conditional (Krt6a-Cre) knockout, but not myeloid cell conditional (LysM-Cre) knockout, attenuates psoriatic symptoms. Immunophenotypic analysis shows that FABP5 deficiency specifically reduces skin recruitment of Ly6G+ neutrophils. Mechanistically, activated keratinocytes produce chemokines and cytokines that trigger neutrophil chemotaxis and activation in an FABP5-dependent manner. Proteomic analysis further identifies that FABP5 interacts with valosin-containing protein (VCP), a key player in NF-κB signaling activation. Silencing of FABP5, VCP, or both inhibits NF-κB/neutrophil chemotaxis signaling. Collectively, these data demonstrate dysregulated FABP5 as a molecular mechanism promoting NF-κB signaling and neutrophil infiltration in psoriasis pathogenesis.

Cisplatin-induced oxPAPC release enhances MDSCs infiltration into LL2 tumour tissues through MCP-1/CCL2 and LTB4/LTB4R pathways.

Lung cancer is the leading global cause of cancer-related death, however, resistance to chemotherapy drugs remains a huge barrier to effective treatment. The elevated recruitment of myeloid derived suppressor cells (MDSCs) to tumour after chemotherapy has been linked to resistance of chemotherapy drugs. Nevertheless, the specific mechanism remains unclear. oxPAPC is a bioactive principal component of minimally modified low-density lipoproteins and regulates inflammatory response. In this work, we found that cisplatin, oxaliplatin and ADM all increased oxPAPC release in tumour. Treating macrophages with oxPAPC in vitro stimulated the secretion of MCP-1 and LTB4, which strongly induced monocytes and neutrophils chemotaxis, respectively. Injection of oxPAPC in vivo significantly upregulated the percentage of MDSCs in tumour microenvironment (TME) of wild-type LL2 tumour-bearing mice, but not CCL2-/- mice and LTB4R-/- mice. Critically, oxPAPC acted as a pro-tumor factor in LL2 tumour model. Indeed, cisplatin increased oxPAPC level in tumour tissues of WT mice, CCL2-/- and LTB4R-/- mice, but caused increased infiltration of Ly6Chigh monocytes and neutrophils only in WT LL2-bearing mice. Collectively, our work demonstrates cisplatin treatment induces an overproduction of oxPAPC and thus recruits MDSCs infiltration to promote the tumour growth through the MCP-1/CCL2 and LTB4/LTB4R pathways, which may restrict the effect of multiple chemotherapy. This provides evidence for a potential strategy to enhance the efficacy of multiple chemotherapeutic drugs in the treatment of lung cancer by targeting oxPAPC.

LRRK2 is involved in the chemotaxis of neutrophils and differentiated HL-60 cells, and the inhibition of LRRK2 kinase activity increases fMLP-induced chemotactic activity

BackgroundNeutrophils depend heavily on glycolysis for energy production under normal conditions. In contrast, neutrophils require energy supplied by mitochondrial oxidative phosphorylation (OXPHOS) during chemotaxis. However, the mechanism by which the energy supply changes from glycolysis to OXPHOS remains unknown. Leucine-rich repeat kinase 2 (LRRK2) is partially present in the outer mitochondrial membrane fraction. Lrrk2-deficient cells show mitochondrial fragmentation and reduced OXPHOS activity. We have previously reported that mitofusin (MFN) 2 is involved in chemotaxis and OXPHOS activation upon chemoattractant N-formyl-Met-Leu-Phe (fMLP) stimulation in differentiated HL-60 (dHL-60) cells. It has been previously reported that LRRK2 binds to MFN2 and partially colocalizes with MFN2 at the mitochondrial membranes. This study investigated the involvement of LRRK2 in chemotaxis and MFN2 activation in neutrophils and dHL-60 cells.MethodsLrrk2 knockout neutrophils and Lrrk2 knockdown dHL-60 cells were used to examine the possible involvement of LRRK2 in chemotaxis. Lrrk2 knockdown dHL-60 cells were used a tetracycline-inducible small hairpin RNA (shRNA) system to minimize the effects of LRRK2 knockdown during cell culture. The relationship between LRRK2 and MFN2 was investigated by measuring the GTP-binding activity of MFN2 in Lrrk2 knockdown dHL-60 cells. The effects of LRRK2 kinase activity on chemotaxis were examined using the LRRK2 kinase inhibitor MLi-2.ResultsfMLP-induced chemotactic activity was reduced in Lrrk2 knockout neutrophils in vitro and in vivo. Lrrk2 knockdown in dHL-60 cells expressing Lrrk2 shRNA also reduced fMLP-induced chemotactic activity. Lrrk2 knockdown dHL-60 cells showed reduced OXPHOS activity and suppressed mitochondrial morphological change, similar to Mfn2 knockdown dHL-60 cells. The amount of LRRK2 in the mitochondrial fraction and the GTP-binding activity of MFN2 increased upon fMLP stimulation, and the MFN2 GTP-binding activity was suppressed in Lrrk2 knockdown dHL-60 cells. Furthermore, the kinase activity of LRRK2 and Ser935 phosphorylation of LRRK2 were reduced upon fMLP stimulation, and LRRK2 kinase inhibition by MLi-2 increased the migration to fMLP.ConclusionsLRRK2 is involved in neutrophil chemotaxis and the GTP-binding activity of MFN2 upon fMLP stimulation. On the other hand, the kinase activity of LRRK2 shows a negative regulatory effect on fMLP-induced chemotactic activity in dHL-60 cells.-J661noP-dBkFUp6aS7aWrVideo

Volatile Composition, Antimicrobial Activity, and In Vitro Innate Immunomodulatory Activity of Echinacea purpurea (L.) Moench Essential Oils.

Echinacea purpurea (L.) Moench is a medicinal plant commonly used for the treatment of upper respiratory tract infections, the common cold, sore throat, migraine, colic, stomach cramps, and toothaches and the promotion of wound healing. Based on the known pharmacological properties of essential oils (EOs), we hypothesized that E. purpurea EOs may contribute to these medicinal properties. In this work, EOs from the flowers of E. purpurea were steam-distilled and analyzed by gas chromatography-mass spectrometry (GC-MS), GC with flame-ionization detection (GC-FID), and chiral GC-MS. The EOs were also evaluated for in vitro antimicrobial and innate immunomodulatory activity. About 87 compounds were identified in five samples of the steam-distilled E. purpurea EO. The major components of the E. purpurea EO were germacrene D (42.0 ± 4.61%), α-phellandrene (10.09 ± 1.59%), β-caryophyllene (5.75 ± 1.72%), γ-curcumene (5.03 ± 1.96%), α-pinene (4.44 ± 1.78%), δ-cadinene (3.31 ± 0.61%), and β-pinene (2.43 ± 0.98%). Eleven chiral compounds were identified in the E. purpurea EO, including α-pinene, sabinene, β-pinene, α-phellandrene, limonene, β-phellandrene, α-copaene, β-elemene, β-caryophyllene, germacrene D, and δ-cadinene. Analysis of E. purpurea EO antimicrobial activity showed that they inhibited the growth of several bacterial species, although the EO did not seem to be effective for Staphylococcus aureus. The E. purpurea EO and its major components induced intracellular calcium mobilization in human neutrophils. Additionally, pretreatment of human neutrophils with the E. purpurea EO or (+)-δ-cadinene suppressed agonist-induced neutrophil calcium mobilization and chemotaxis. Moreover, pharmacophore mapping studies predicted two potential MAPK targets for (+)-δ-cadinene. Our results are consistent with previous reports on the innate immunomodulatory activities of β-caryophyllene, α-phellandrene, and germacrene D. Thus, this study identified δ-cadinene as a novel neutrophil agonist and suggests that δ-cadinene may contribute to the reported immunomodulatory activity of E. purpurea.

Bacterial outer membrane vesicles bound to bacteriophages modulate neutrophil responses to bacterial infection.

Pseudomonas aeruginosa is a major human pathogen, particularly effective at colonizing the airways of patients with cystic fibrosis. Bacteriophages are highly abundant at infection sites, but their impact on mammalian immunity remains unclear. We previously showed that Pf4, a temperate filamentous bacteriophage produced by P. aeruginosa, modifies the innate immune response to P. aeruginosa infections via TLR3 signaling, but the underlying mechanisms remained unclear. Notably, Pf4 is a single-stranded DNA and lysogenic phage, and its production does not typically result in lysis of its bacterial host. We identified previously that internalization of Pf4 by human or murine immune cells triggers maladaptive viral pattern recognition receptors and resulted in bacterial persistence based on the presence of phage RNA. We report now that Pf4 phage dampens inflammatory responses to bacterial endotoxin and that this is mediated in part via bacterial vesicles attached to phage particles. Outer membrane vesicles (OMVs) are produced by Gram-negative bacteria and play a key role in host pathogen interaction. Recently, evidence has emerged that OMVs differentially package small RNAs. In this study, we show that Pf4 are decorated with OMVs that remain affixed to Pf4 despite of purification steps. These phages are endocytosed by human cells and delivered to endosomal vesicles. We demonstrate that short RNAs within the OMVs form hairpin structures that trigger TLR3-dependent type I interferon production and antagonize production of antibacterial cytokines and chemokines. In particular, Pf4 phages inhibit CXCL5, preventing efficient neutrophil chemotaxis in response to endotoxin. Moreover, blocking IFNAR or TLR3 signaling abrogates the effect of Pf4 bound to OMVs on macrophage activation. In a murine acute pneumonia model, mice treated with Pf4 associated with OMVs show significantly less neutrophil infiltration in BAL fluid than mice treated with purified Pf4. These changes in macrophage phenotype are functionally relevant: conditioned media from cells exposed to Pf4 decorated with OMVs are significantly less effective at inducing neutrophil migration in vitro and in vivo. These results suggest that Pf4 phages alter innate immunity to bacterial endotoxin and OMVs, potentially dampening inflammation at sites of bacterial colonization or infection.

Ataluren improves myelopoiesis and neutrophil chemotaxis by restoring ribosome biogenesis and reducing p53 levels in Shwachman-Diamond syndrome cells.

Shwachman-Diamond syndrome (SDS) is characterized by neutropenia, exocrine pancreatic insufficiency and skeletal abnormalities. SDS bone marrow haematopoietic progenitors show increased apoptosis and impairment in granulocytic differentiation. Loss of Shwachman-Bodian-Diamond syndrome (SBDS) expression results in reduced eukaryotic 80S ribosome maturation. Biallelic mutations in the SBDS gene are found in ~90% of SDS patients, ~55% of whom carry the c.183-184TA>CT nonsense mutation. Several translational readthrough-inducing drugs aimed at suppressing nonsense mutations have been developed. One of these, ataluren, has received approval in Europe for the treatment of Duchenne muscular dystrophy. We previously showed that ataluren can restore full-length SBDS protein synthesis in SDS-derived bone marrow cells. Here, we extend our preclinical study to assess the functional restoration of SBDS capabilities in vitro and ex vivo. Ataluren improved 80S ribosome assembly and total protein synthesis in SDS-derived cells, restored myelopoiesis in myeloid progenitors, improved neutrophil chemotaxis in vitro and reduced neutrophil dysplastic markers ex vivo. Ataluren also restored full-length SBDS synthesis in primary osteoblasts, suggesting that its beneficial role may go beyond the myeloid compartment. Altogether, our results strengthened the rationale for a Phase I/II clinical trial of ataluren in SDS patients who harbour the nonsense mutation.

CXCL17 binds efficaciously to glycosaminoglycans with the potential to modulate chemokine signaling.

CXCL17 is a mucosally secreted protein, and the most recently identified human chemokine, an assignment based on protein fold prediction and chemotactic activity for leukocytes. However, these credentials have been the subject of much recent discussion and no experimental evidence has been presented regarding the definitive structure of CXCL17. In this study, we evaluated the structural and chemoattractant credentials of CXCL17 to better characterize this molecule, and gain deeper insights into its functional role as a glycosaminoglycan (GAG) binding protein. In the absence of structural information, in silico modeling techniques assessed the likelihood of CXCL17 adopting a chemokine fold. Recombinant CXCL17 was synthesized in mammalian and prokaryotic systems. Modified Boyden chamber and real-time chemotaxis assays assessed the ability of CXCL17 to promote chemotaxis of murine splenocytes, human neutrophils, and CXCR1 transfectants. The efficacy of CXCL17 binding to GAGs was quantified with solid-phase assays and bio-layer interferometry techniques. All modeling efforts failed to support classification of CXCL17 as a chemokine based on its predicted conformation. Recombinant CXCL17 was observed to dimerize as a function of concentration, a characteristic of several chemokines. Contrary to a previous report, CXCL17 was not chemotactic for murine splenocytes, although it was a low-potency chemoattractant for human neutrophils at micromolar concentrations, several orders of magnitude higher than those required for CXCL8. As anticipated owing to its highly basic nature, CXCL17 bound to GAGs robustly, with key C-terminal motifs implicated in this process. While inactive via CXCR1, CXCL17 was found to inhibit CXCR1-mediated chemotaxis of transfectants to CXCL8 in a dose-dependent manner. In summary, despite finding little evidence for chemokine-like structure and function, CXCL17 readily bound GAGs, and could modulate chemotactic responses to another chemokine in vitro. We postulate that such modulation is a consequence of superior GAG binding, and that C-terminal fragments of CXCL17 may serve as prototypic inhibitors of chemokine function.