Regulates Neutrophil Migration Research Articles

Toll-like receptor adaptor protein TIRAP has specialized roles in signaling, metabolic control and leukocyte migration upon wounding in zebrafish larvae.

The TIRAP protein is an adaptor protein in TLR signaling which links TLR2 and TLR4 to the adaptor protein Myd88. The transcriptomic profiles of zebrafish larvae from a tirap, myd88 and tlr2 mutant and the corresponding wild type controls under unchallenged developmental conditions revealed a specific involvement of tirap in calcium homeostasis and myosin regulation. Metabolomic profiling showed that the tirap mutation results in lower glucose levels, whereas a tlr2 mutation leads to higher glucose levels. A tail-wounding zebrafish larval model was used to identify the role of tirap in leukocyte migration to tissue wounding. We found that more neutrophils were recruited to the wounded region in the tirap mutant larvae compared to the wild type controls, whereas there was no difference in macrophage recruitment. In contrast, published data show that tlr2 and myd88 mutants recruit fewer neutrophils and macrophages to the wounds. Based on cell tracking analysis, we demonstrate that the neutrophil migration speed is increased in the tirap mutant in contrast to neutrophil behavior in myd88 and tlr2 mutants. In conclusion, we show that tirap plays specialized roles distinct from tlr2 and myd88 in signaling, metabolic control, and in regulating neutrophil migration speed upon wounding.

LECs regulate neutrophil clearance through IL-17RC/CMTM4/NF-κB axis at sites of inflammation or infection

The lymphatic system plays a vital role in the regulation of tissue fluid balance and the immune response to inflammation or infection. The effects of lymphatic endothelial cells (LECs) on the regulation of neutrophil migration have not been well studied. In three murine models: imiquimod-induced skin inflammation, S. aureus-induced skin infection, and ligature-induced periodontitis, we show that numerous neutrophils migrate from inflamed or infected tissues to the draining lymph nodes via lymphatic vessels. Moreover, inflamed or infected tissues express a high level of IL-17A and TNF-α, simultaneously with a significant increase in the release of neutrophil attractors, including CXCL1, CXCL2, CXCL3, and CXCL5. Importantly, in vitro stimulation of LECs with IL-17A plus TNF-α synergistically promoted these chemokine secretion. Mechanistically, tetra-transmembrane protein CMTM4 directly binds to IL-17RC in LECs. IL-17A plus TNF-α stimulates CXC chemokine secretion by promoting NF-κB signaling. In contrast, knockdown of CMTM4 abrogates IL-17A plus TNF-α activated NF-κB signaling pathways. Lastly, the local administration of adeno-associated virus for CMTM4 in Prox1-CreERT2 mice, mediating LEC-specific overexpression of CMTM4, promotes the drainage of neutrophils by LECs and alleviates immune pathological responses. Thus, our findings reveal the vital role of LECs-mediated neutrophil attraction and clearance at sites of inflammation or infection.

Xanthohumol, a prenylated flavonoid from hops (Humulus lupulus L.) exerts multidirectional pro-healing properties towards damaged zebrafish hair cells by regulating the innate immune response

Xanthohumol (XN) is a prominent prenylated flavonoid present in the hop plant (Humulus lupulus L.). Despite undoubted pro-healing properties of hop plant, there is still a need for clinical investigations confirming these effects as well as the underlying molecular mechanisms. The present study was designed to (1) establish the role of XN in non-invasive inflammation induced by chemical damage to zebrafish hair cells, (2) clarify if it influences cell injury severity, neutrophil migration, macrophage activation, cell regeneration, and (3) find out whether it modulates the gene expression profile of chosen immune and stress response markers. All experiments were performed on 3 dpf zebrafish larvae. After fertilization the embryos were transferred to appropriate XN solutions (0.1 μM, 0.3 μM and 0.5 μM). The 40 min 10 μM CuSO4 exposure evoked severe damage to posterior lateral line hair cells triggering a robust acute inflammatory response. Four readouts were selected as the indicators of XN role in the process of inflammation: 1) hair cell death, 2) neutrophil migration towards damaged hair cells, 3) macrophage activation and recruitment to damaged hair cells, 4) hair cell regeneration. The assessments involved in vivo confocal microscopy imaging and qPCR based molecular analysis. It was demonstrated that XN (1) influences death pathway of damaged hair cells by redirecting their severe necrotic phenotype into apoptotic one, (2) impacts the immune response via regulating neutrophil migration, macrophage recruitment and activation (3) modulates gene expression of immune system markers and (4) accelerates hair cell regeneration.

Exploring the role of neutrophil extracellular traps in neuroblastoma: identification of molecular subtypes and prognostic implications.

Cancer cells induce neutrophil extracellular traps (NETs) to promote tumor progression and metastasis. However, only a few studies have focused on the role of NETs in Neuroblastoma (NB). First, based on the expression of NET-related genes, consensus clustering analysis was conducted to cluster NB samples into different subtypes. Differential analysis was performed to identify DEGs between subtypes. Functional items and related pathways of DEGs were identified using enrichment analysis. Univariate Cox analysis and the LASSO algorithm were used to identify biomarkers for prognosis. Furthermore, independent prognostic analysis was performed. Immune infiltration analysis was performed to identify differential immune cells. Finally, the verification of prognostic model genes were taken by the immunohistochemical staining and quantitative real-time PCR. Consensus clustering analysis demonstrated that NB samples were clustered into two subtypes. There were 125 DEGs between the two subtypes of NB. Moreover, the enrichment analysis results showed that the DEGs were mainly associated with 'external side of plasma membrane,' 'immune receptor activity' 'regulation of leukocyte migration' GO items. There were also several GO items related to neutrophils, such as regulation of neutrophil migration and differentiation. KEGG pathways revealed that the DEGs were correlated with in immunity-related activities, including 'Complement and coagulation cascades,' 'Neutrophil extracellular trap formation, 'T cell receptor signaling pathway,' 'PD-L1 expression and PD-1 checkpoint pathway in cancer' and so on. A total of five biomarkers,[Selenoprotein P1 (SEPP1), Fibrinogen-like protein 2 (FGL2), NK cell lectin-like receptor K1 (KLRK1), ATP-binding cassette transporters 6(ABCA6) and Galectins(GAL)], were screened, and a risk model based on the biomarkers was created. Furthermore, a nomogram for forecasting the survival rates of patients with NB was established based on the risk score, age at diagnosis, and MYCN status. Eight differential immune cells (CD8 + T cells, resting mast cells, etc.) were acquired between the two risk subgroups. The expression levels of five prognostic model genes at the protein and mRNA were verified and all results were consistent with the results of our bioinformatics analysis. We initially found that five NET-related genes were significantly differentially expressed in NETs-associated molecular isoforms and two Netrg molecular isoforms were found to be associated with poorer prognosis. This stratification might provide insight into the prediction of prognosis and ideal immunotherapy strategies for patients with NB. However, we also noted that the formation of NETs is a complex biological process involving the regulation of multiple cytokines and cellular interactions. Therefore, the exact roles of these genes and their specific mechanisms in the formation of NETs and the development of NB still need to be further investigated.

Decreased GCF DEL-1 and increased GCF neutrophils with increasing probing pocket depth.

Developmental endothelial locus-1 (DEL-1) plays a role in regulating neutrophil migration within the periodontium. The objective of this study was to evaluate the levels of DEL-1 in saliva and gingival crevicular fluid (GCF), as well as the number of neutrophils in patients with periodontitis. Forty systemically healthy, non-smoking periodontitis patients participated in this study. Clinical periodontal parameters, including the plaque index, probing pocket depth (PPD), clinical attachment level, bleeding on probing, modified sulcular bleeding index, and marginal bone level, were measured. Levels of DEL-1, interleukin (IL)-1β, IL-6, and IL-8 in unstimulated saliva samples, as well as DEL-1 in the GCF of 3 teeth from each participant, were assessed. Neutrophil counts in oral rinse and GCF samples were recorded. Spearman correlation coefficients were used to examine the correlation between protein levels, clinical parameters, and neutrophil quantities. Participants were divided into 2 age groups (those under 50 years and those 50 years or older) in order to investigate potential age-related differences. DEL-1 levels in the GCF showed a negative relationship with PPD (sum). Neutrophils in oral rinse samples were positively correlated with PPD, IL-8, and IL-1β levels. Neutrophils in GCF exhibited a positive correlation with PPD (sum). Salivary DEL-1 levels showed correlations with IL-8 and IL-1β, but not with the clinical parameters of periodontitis. The negative relationship observed between PPD and GCF DEL-1 levels is consistent with the proposed protective role of DEL-1.

Circadian clock1a coordinates neutrophil recruitment via nfe212a/duox-reactive oxygen species pathway in zebrafish

Neutrophil recruitment to inflammatory sites appears to be an evolutionarily conserved strategy to fight against exogenous insults. However, the rhythmic characteristics and underlying mechanisms of neutrophil migration on a 24-h timescale are largely unknown. Using the advantage of invivo imaging of zebrafish, this study explored how the circadian gene clock1a dynamically regulates the rhythmic recruitment of neutrophils to inflammatory challenges. We generated a clock1a mutant and found that neutrophil migration is significantly increased in caudal fin injury and lipopolysaccharide (LPS) injection. Transcriptome sequencing, chromatin immunoprecipitation (ChIP), and dual-luciferase reporting experiments suggest that the clock1a gene regulates neutrophil migration by coordinating the rhythmic expression of nfe212a and duox genes to control the reactive oxygen species (ROS) level. This study ultimately provides a visual model to expand the understanding of the rhythmic mechanisms of neutrophil recruitment on a circadian timescale in a diurnal organism from the perspective of ROS.

β-hydroxybutyrate impairs the directionality of migrating neutrophils through inhibiting the autophagy-dependent degradation of Cdc42 and Rac1 in ketotic cows

Dairy cows have high incidence of ketosis during perinatal. According to our previous studies, elevated ketone bodies (mainly β-hydroxybutyrate, BHB) in the peripheral blood are believed to contribute to the impairment of neutrophils mobility and directionality thereby contributing to the immunosuppression and further infectious disease secondary to ketosis. However, the specific effect of BHB on the directionality of bovine neutrophil need further study and the underlying molecular mechanisms are still unclear. According to the concentration of serum BHB, 40 multiparous cows (within 3 wk postpartum) were selected and divided into the control (n = 20, BHB <0.6 mM) or clinical ketosis (n = 20, BHB >3.0 mM) group. Blood samples were collected for baseline serum characteristics analysis and neutrophil mobility and directionality detection. Platelet activation factor was used as a chemoattractant in cell migration experiments. Our ex-vivo data showed ketotic cows, compared with control cows, were in a negative energy balance state, and their neutrophils had shorter migration distance, lower migration speed, and impaired migration directionality. Neutrophils from control cows were incubated with 3.0 mM BHB for 6 h in vitro. Similarly, BHB stimulation resulted in impaired mobility and directionality of bovine neutrophils. We further specifically studied the underlying molecular mechanism of BHB regulating neutrophil migration directionality in the present study. Cell division control protein 42 homolog (Cdc42) and Ras-related C3 botulinum toxin substrate 1 (Rac1), 2 key markers in the regulation of migration directionality, were found increased after BHB treatment in their total and activated protein levels while decreasing in their transcription level, suggesting that an imbalance of the protein degradation system may be involved. Interestingly, transmission electron microscopy data revealed a decrease in autophagosome number in neutrophils from ketotic cows. Western blotting data showed the accumulation of sequestosome-1 (p62) protein and a decrease in microtubule-associated protein 1 light chain 3-II (LC3-II) protein abundance after BHB treatment, further confirming that the autophagy flux was inhibited in neutrophils from ketotic cows. Additionally, rapamycin (RAPA), a specific autophagy activator, was used with or without BHB treatment in vitro. Accordingly, the BHB-induced impairment of migration directionality but not mobility was relieved by RAPA. Furthermore, as verified by in vivo experiments, compared with the control cows, the protein abundance of total and activated Cdc42 and Rac1 increased and their mRNA abundance decreased in neutrophils from ketotic cows. Overall, the present study revealed that pathological concentration of BHB impairs neutrophil migration directionality through inhibiting the autophagy-mediated degradation of Cdc42 and Rac1. These findings help explain the immunosuppression caused by ketosis.

The Regulation of Neutrophil Migration in Patients with Sepsis: The Complexity of the Molecular Mechanisms and Their Modulation in Sepsis and the Heterogeneity of Sepsis Patients.

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Common causes include gram-negative and gram-positive bacteria as well as fungi. Neutrophils are among the first cells to arrive at an infection site where they function as important effector cells of the innate immune system and as regulators of the host immune response. The regulation of neutrophil migration is therefore important both for the infection-directed host response and for the development of organ dysfunctions in sepsis. Downregulation of CXCR4/CXCL12 stimulates neutrophil migration from the bone marrow. This is followed by transmigration/extravasation across the endothelial cell barrier at the infection site; this process is directed by adhesion molecules and various chemotactic gradients created by chemotactic cytokines, lipid mediators, bacterial peptides, and peptides from damaged cells. These mechanisms of neutrophil migration are modulated by sepsis, leading to reduced neutrophil migration and even reversed migration that contributes to distant organ failure. The sepsis-induced modulation seems to differ between neutrophil subsets. Furthermore, sepsis patients should be regarded as heterogeneous because neutrophil migration will possibly be further modulated by the infecting microorganisms, antimicrobial treatment, patient age/frailty/sex, other diseases (e.g., hematological malignancies and stem cell transplantation), and the metabolic status. The present review describes molecular mechanisms involved in the regulation of neutrophil migration; how these mechanisms are altered during sepsis; and how bacteria/fungi, antimicrobial treatment, and aging/frailty/comorbidity influence the regulation of neutrophil migration.

Dengue Virus Increases the Expression of TREM-1 and CD10 on Human Neutrophils.

Every year, dengue is responsible for 400 million infections worldwide. Inflammation is related to the development of severe forms of dengue. Neutrophils are a heterogeneous cell population with a key role in the immune response. During viral infection, neutrophils are mainly recruited to the infection site; however, their excessive activation is linked to deleterious results. During dengue infection, neutrophils are involved in the pathogenesis through neutrophils extracellular traps production, tumor necrosis factor-alpha, and interleukin-8 secretion. However, other molecules regulate the neutrophil role during viral infection. TREM-1 is expressed on neutrophils and its activation is related to increased production of inflammatory mediators. CD10 is expressed on mature neutrophils and has been associated with the regulation of neutrophil migration and immunosuppression. However, the role of both molecules during viral infection is limited, particularly during dengue infection. Here, we report for the first time that DENV-2 can significantly increase TREM-1 and CD10 expression as well as sTREM-1 production in cultured human neutrophils. Furthermore, we observed that treatment with granulocyte-macrophage colony stimulating factor, a molecule mostly produced in severe cases of dengue, is capable of inducing the overexpression of TREM-1 and CD10 on human neutrophils. These results suggest the participation of neutrophil CD10 and TREM-1 in the pathogenesis of dengue infection.

Nitration of chemokine CXCL8 acts as a natural mechanism to limit acute inflammation

Chemokine CXCL8 is a key facilitator of the human host immune response, mediating neutrophil migration, and activation at the site of infection and injury. The oxidative burst is an important effector mechanism which leads to the generation of reactive nitrogen species (RNS), including peroxynitrite. The current study was performed to determine the potential for nitration to alter the biological properties of CXCL8 and its detection in human disease. Here, we show peroxynitrite nitrates CXCL8 and thereby regulates neutrophil migration and activation. The nitrated chemokine was unable to induce transendothelial neutrophil migration in vitro and failed to promote leukocyte recruitment in vivo. This reduced activity is due to impairment in both G protein-coupled receptor signaling and glycosaminoglycan binding. Using a novel antibody, nitrated CXCL8 was detected in bronchoalveolar lavage samples from patients with pneumonia. These findings were validated by mass spectrometry. Our results provide the first direct evidence of chemokine nitration in human pathophysiology and suggest a natural mechanism that limits acute inflammation.

Syntaphilin Regulates Neutrophil Migration in Cancer.

Pathologically activated neutrophils (PMN) with immunosuppressive activity, which are termed myeloid-derived suppressor cells (PMN-MDSC), play a critical role in regulating tumor progression. These cells have been implicated in promoting tumor metastases by contributing to premetastatic niche formation. This effect was facilitated by enhanced spontaneous migration of PMN from bone marrow to the premetastatic niches during the early-stage of cancer development. The molecular mechanisms underpinning this phenomenon remained unclear. In this study, we found that syntaphilin (SNPH), a cytoskeletal protein previously known for anchoring mitochondria to the microtubule in neurons and tumor cells, could regulate migration of PMN. Expression of SNPH was decreased in PMN from tumor-bearing mice and patients with cancer as compared with PMN from tumor-free mice and healthy donors, respectively. In Snph-knockout (SNPH-KO) mice, spontaneous migration of PMN was increased and the mice showed increased metastasis. Mechanistically, in SNPH-KO mice, the speed and distance travelled by mitochondria in PMN was increased, rates of oxidative phosphorylation and glycolysis were elevated, and generation of adenosine was increased. Thus, our study reveals a molecular mechanism regulating increased migratory activity of PMN during cancer progression and suggests a novel therapeutic targeting opportunity.

The Regulation of Neutrophil Migration by IL-8 Plays an Important Role in Improving the ECM Environment for Anti-Wrinkles

The Regulation of Neutrophil Migration by IL-8 Plays an Important Role in Improving the ECM Environment for Anti-Wrinkles

Abstract 128: Loss Of Gstm1 Augments Kidney Neutrophil Population In Angii-induced Hypertension

Background: Glutathione-S-transferase Mu1( Gstm1 ) gene, a member of Glutathione-S-transferases (GST) superfamily, encodes an enzyme that functions in the detoxification of electrophilic compounds. The common GSTM1 deletion variant in humans is associated with increased risks of chronic kidney disease (CKD) progression and incident end stage kidney failure. We reported that deletion of Gstm1 increases oxidative stress, kidney inflammation and injury in angiotensin II induced hypertension (Ang II-HTN) in mice. At 4 weeks of Ang II-HTN, neutrophils, CD4+T cells, and F4/80+ cells were significantly increased in Gstm1 knockout (KO) kidney. While a link between oxidative stress and immune activation in hypertension has been well documented, the molecular mechanism by which Gstm1 regulates immune cell activation in hypertension is unknown. Methods: To further characterize the inflammatory response in GSTM1 deficiency, Gstm1 KO and wild-type (WT) male mice at ~ 12 weeks of age were subjected to Angiotensin II at 1000 ng/kg per minute for 4 days via a mini osmotic pump. The kidney cells and bone marrow cells were isolated for flow cytometry analysis. Results: At baseline, there were no differences in inflammatory cell populations including neutrophils between WT and Gstm1 KO mice. At Day 4 of Ang II-HTN, Gstm1 KO kidneys exhibited about a 2.3 fold increase in neutrophil population (CD45 + Ly6G + CD11b + ), compared to WT control mice (10 WT and 8 Gstm1 KO mice, p=0.0075). Interestingly, CD45+ bone marrow cells isolated from both Gstm1 KO and WT mice have similar percentile of neutrophil populations ; however, neutrophils in Gstm1 KO bone marrow express relatively higher levels of CXCR2. Unlike at 4 weeks, there was no difference in other inflammatory cells at Day 4. Conclusion: Deletion of Gstm1 increases renal neutrophil population early in Ang II-HTN and CXCR2 expression in bone marrow neutrophils. Gstm1 may play a role in regulating neutrophil migration and recruitment. Further studies are needed to delineate whether GSTM1 deficiency drives kidney injury via early neutrophil migration, which could shed light on potential therapeutic target for CKD progression in those genetically susceptible.

Tas2R signaling enhances mouse neutrophil migration via a ROCK-dependent pathway.

Type-2 bitter taste receptors (Tas2Rs) are a large family of G protein-coupled receptors that are expressed in the oral cavity and serve to detect substances with bitter tastes in foods and medicines. Recent evidence suggests that Tas2Rs are also expressed extraorally, including in immune cells. However, the role of Tas2Rs in immune cells remains controversial. Here, we demonstrate that Tas2R126, Tas2R135, and Tas2R143 are expressed in mouse neutrophils, but not in other immune cells such as macrophages or T and B lymphocytes. Treatment of bone marrow-derived neutrophils from wild-type mice with the Tas2R126/143 agonists arbutin and d-salicin led to enhanced C-X-C motif chemokine ligand 2 (CXCL2)-stimulated migration in vitro, but this response was not observed in neutrophils from Tas2r126/135/143-deficient mice. Enhancement of CXCL2-stimulated migration by Tas2R agonists was accompanied by increased phosphorylation of myosin light chain 2 (MLC2) and was blocked by pretreatment of neutrophils with inhibitors of Rho-associated coiled-coil-containing protein kinase (ROCK), but not by inhibitors of the small GTPase RhoA. Taken together, these results demonstrate that mouse neutrophils express functional Tas2R126/143 and suggest a role for Tas2R126/143-ROCK-MLC2-dependent signaling in the regulation of neutrophil migration.

Deficiency of leukocyte-specific protein 1 (LSP1) alleviates asthmatic inflammation in a mouse model

BackgroundAsthma is a major cause of morbidity and mortality in humans. The mechanisms of asthma are still not fully understood. Leukocyte-specific protein-1 (LSP-1) regulates neutrophil migration during acute lung inflammation. However, its role in asthma remains unknown.MethodsAn OVA-induced mouse asthma model in LSP1-deficient (Lsp1−/−) and wild-type (WT) 129/SvJ mice were used to test the hypothesis that the absence of LSP1 would inhibit airway hyperresponsiveness and lung inflammation.ResultsLight and electron microscopic immunocytochemistry and Western blotting showed that, compared with normal healthy lungs, the levels of LSP1 were increased in lungs of OVA-asthmatic mice. Compared to Lsp1−/− OVA mice, WT OVA mice had higher levels of leukocytes in broncho-alveolar lavage fluid and in the lung tissues (P < 0.05). The levels of OVA-specific IgE but not IgA and IgG1 in the serum of WT OVA mice was higher than that of Lsp1−/− OVA mice (P < 0.05). Deficiency of LSP1 significantly reduced the levels of IL-4, IL-5, IL-6, IL-13, and CXCL1 (P < 0.05) but not total proteins in broncho-alveolar lavage fluid in asthmatic mice. The airway hyper-responsiveness to methacholine in Lsp1−/− OVA mice was improved compared to WT OVA mice (P < 0.05). Histology revealed more inflammation (inflammatory cells, and airway and blood vessel wall thickening) in the lungs of WT OVA mice than in those of Lsp1−/− OVA mice. Finally, immunohistology showed localization of LSP1 protein in normal and asthmatic human lungs especially associated with the vascular endothelium and neutrophils.ConclusionThese data show that LSP1 deficiency reduces airway hyper-responsiveness and lung inflammation, including leukocyte recruitment and cytokine expression, in a mouse model of asthma.

UDP/P2Y6 contributes to enhancing LPS-induced acute lung injury by regulating neutrophil migration

Neutrophils play a prominent role in the inflammatory response and are a critical factor in the pathogenesis of acute lung injury (ALI). Despite a deep understanding of neutrophil accumulation in the pulmonary microvasculature during the process of this disease, the regulatory mechanism of neutrophil recruitment remains unclear. This study aimed to explore the functions and signaling pathways of the purinergic receptor P2Y6 in mediating the innate immune response in ALI. P2Y6-deficient mice, bone marrow chimeras, and neutrophilic chimeras were created in this work to explore the function of P2Y6 in ALI. The results indicated that the extracellular nucleotide UDP was released as a dangerous signal and activated P2Y6 to promote the inflammatory response and pulmonary damage during the process of ALI. P2Y6 deficiency may mitigate deterioration of this disease, including reduced ALI-related inflammatory factor release and immune cell invasion. Bone marrow and neutrophil chimeras and adoptive transfer in mice showed that P2Y6 expression on neutrophils contributed to neutrophil infiltration into lung tissues induced by UDP. Further work indicated that P2Y6 was involved in the neutrophil migration capability through the ErK signaling pathway by mediating the deformation of F-actin filaments and pseudopodia formation during cell recruitment to pulmonary tissue. Here, we provide evidence for the mechanism by which the purinergic receptor P2Y6 contributes to ALI development by regulating neutrophil infiltration into lung tissues. These data indicated that P2Y6 might be a potential therapeutic target for the treatment of this acute severe disease.

Rora Regulates Neutrophil Migration and Activation in Zebrafish.

Neutrophil migration and activation are essential for defense against pathogens. However, this process may also lead to collateral tissue injury. We used microRNA overexpression as a platform and discovered protein-coding genes that regulate neutrophil migration. Here we show that miR-99 decreased the chemotaxis of zebrafish neutrophils and human neutrophil-like cells. In zebrafish neutrophils, miR-99 directly targets the transcriptional factor RAR-related orphan receptor alpha (roraa). Inhibiting RORα, but not the closely related RORγ, reduced chemotaxis of zebrafish and primary human neutrophils without causing cell death, and increased susceptibility of zebrafish to bacterial infection. Expressing a dominant-negative form of Rorα or disrupting the roraa locus specifically in zebrafish neutrophils reduced cell migration. At the transcriptional level, RORα regulates transmembrane signaling receptor activity and protein phosphorylation pathways. Our results, therefore, reveal previously unknown functions of miR-99 and RORα in regulating neutrophil migration and anti-microbial defense.

Assessment of changes in genetic transcriptome in nasal epithelial cells exposed to ozone-aged black carbon and pollen allergen by high-throughput transcriptomics

BackgroundAir pollution may be associated with increased airway responsiveness to allergens in allergic rhinitis (AR). Ozone-aged environmental black carbon (O3BC) is an important constituent of atmospheric particulate matter (PM), for which the mechanisms underlying its effects have not been fully elucidated in AR. The objective of the present study was to determine the O3BC and pollen-induced alterations in the transcriptome in human nasal epithelial cells (hNECs) in vitro.MethodshNECs from nasal epithelial mucosal samples of healthy individuals undergoing nasal surgery (turbinoplasty or septoplasty) were established as air–liquid interface (ALI) cultures and exposed to O3BC, pollen, or a combination of O3BC+ pollen. Changes in cell viability were analyzed by fluorescence and changes in the transcriptome by high-throughput RNA sequencing (RNA-seq). Several differentially expressed genes were verified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Enrichment analysis, based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database, was performed to determine major biological functions and pathways involved.ResultsExposure to ≥ 50 μg/ml O3BC or 25 μg/ml O3BC+ 200 μg /ml pollen significantly decreased cell viability of the hNECs compared to control (p < 0.05) or 25 μg/ml O3BC alone (p < 0.05); whereas exposure to pollen alone did not alter cell viability at any concentration investigated. High-throughput RNA sequencing analysis indicated that there was significant difference in gene expression between pollen or O3BC alone and O3BC+ pollen exposed cells. Exposure to 200 μg/ml O3BC was associated with hypoxia stress response GO terms, whereas exposure to 25 μg/ml O3BC+ 200 μg/ml pollen was associated with inflammatory response GO terms; including regulation of neutrophil migration and chemotaxis, macrophage differentiation and chemotaxis, mast cell activation, and phagocytosis. KEGG pathway analysis indicated the top 10 upstream regulators to be IL1B, CSF1, CCL2, TLR2, LPL, IGF8, SPP1, CXCL8, FCER1G and IL1RN; of which expressions of inflammation-related genes IL1B, CSF1 and FCER1G were significantly increased.ConclusionO3BC and pollen allergen combined exposure may induce innate immune and allergic inflammation in hNECs, and therefore potentially exacerbate the symptoms of AR in affected individuals.

Annexin A1 Attenuates Neutrophil Migration and IL-6 Expression through Fpr2 in a Mouse Model of Streptococcus suis-Induced Meningitis.

Streptococcus suis serotype 2 is a crucial pathogenic cause of bacterial meningitis, a life-threatening disease with neurological sequelae and high rates of mortality. Inflammation triggered by S. suis infection must be precisely regulated to prevent further tissue damage. As a glucocorticoid anti-inflammatory mediator, annexin A1 (AnxA1) mainly acts through formyl peptide receptor 2 (Fpr2) to alleviate inflammation in the peripheral system. In this study, we evaluated the roles of AnxA1 and Fpr2 in a mouse model of S. suis meningitis created via intracisternal infection in Fpr2-deficient (Fpr2-/-) and wild-type (WT) mice. We revealed that Fpr2-/- mice were highly susceptible to S. suis meningitis, displaying increased inflammatory cytokine levels, bacterial dissemination, and neutrophil migration compared with WT mice. Additionally, AnxA1 exerted anti-inflammatory effects through Fpr2, such as attenuation of leukocyte infiltration, inflammatory mediator production, and astrocyte or microglial activation in the brain. Importantly, we found that the antimigratory function of AnxA1 decreases neutrophil adherence to the endothelium through Fpr2. Finally, an in vitro study revealed that AnxA1 potentially suppresses interleukin-6 (IL-6) expression through the Fpr2/p38/COX-2 pathway. These data demonstrated that Fpr2 is an anti-inflammatory receptor that regulates neutrophil migration in mice with S. suis meningitis and identified AnxA1 as a potential therapeutic option.

GM-CSF instigates a dendritic cell–T-cell inflammatory circuit that drives chronic asthma development

Steroid-resistant asthma is often characterized by high levels of neutrophils and mixed TH2/TH17 immune profiles. Indeed, neutrophils are key drivers of chronic lung inflammation in multiple respiratory diseases. Their numbers correlate strongly with disease severity, and their presence is often associated with exacerbation of chronic lung inflammation. What factors drive development of neutrophil-mediated chronic lung disease remains largely unknown, and we sought to study the role of GM-CSF as a potential regulator in chronic asthma. Different experimental animal models of chronic asthma were used in combination with alveolar macrophage-reconstitution of global GM-CSF receptor knockout mice as well as cell-type-specific knockout animals to elucidate the role of GM-CSF signaling in chronic airway inflammation. We identify GM-CSF signaling as a critical factor regulating pulmonary accumulation of neutrophils. We show that although being not required for intrinsically regulating neutrophil migration, GM-CSF controls lung dendritic cell function, which in turn promotes T-cell-dependent recruitment of neutrophils to the airways. We demonstrate that GM-CSF regulates lung dendritic cell antigen uptake, transport, and TH2/TH17 cell priming in an intrinsic fashion, which in turn drives pulmonary granulocyte recruitment and contributes to development of airway hyperresponsiveness in chronic disease. We identify GM-CSF as a potentially novel therapeutic target in chronic lung inflammation, describing a GM-CSF-dependent lung conventional dendritic cell-T-cell-neutrophil axis that drives chronic lung disease.