Neutrophil Migration Research Articles

Integration of microbiomics, metabolomics, and transcriptomics reveals the therapeutic mechanism underlying Fuzheng-Qushi decoction for the treatment of lipopolysaccharide-induced lung injury in mice

Ethnopharmacological relevanceFuzheng-Qushi decoction (FZQS) is a practical Chinese herbal formula for relieving cough and fever. Therefore, the action and specific molecular mechanism of FZQS in the treatment of lung injury with cough and fever as the main symptoms need to be further investigated. Aims of the studyTo elucidate the protective effects of FZQS against lung injury in mice and reveal its potential targets and key biological pathways for the treatment of lung injury based on transcriptomics, microbiomics, and untargeted metabolomics analyses. Materials and methodsLipopolysaccharide (LPS) was used to induce a mouse model of lung injury, followed by the administration of FZQS. ELISA was used to detect IL-1β, IL-6, IL-17A, IL-4, IL-10, and TNF-α, in mouse lung tissues. Macrophage polarization and neutrophil activation were measured by flow cytometry. RNA sequencing (RNA-seq) was applied to screen for differentially expressed genes (DEGs) in lung tissues. RT-qPCR and Western blot assays were utilized to validate key DEGs and target proteins in lung tissues. 16S rRNA sequencing was employed to characterize the gut microbiota of mice. Metabolites in the gut were analyzed using untargeted metabolomics. ResultsFZQS treatment significantly ameliorated lung histopathological damage, decreased pro-inflammatory cytokine levels, and increased anti-inflammatory cytokine levels. M1 macrophage levels in the peripheral blood decreased, M2 macrophage levels increased, and activated neutrophils were inhibited in mice with LPS-induced lung injury. Importantly, transcriptomic analysis showed that FZQS downregulated macrophage and neutrophil activation and migration and adhesion pathways by reversing 51 DEGs, which was further confirmed by RT-qPCR and Western blot analysis. In addition, FZQS modulated the dysbiosis of the gut microbiota by reversing the abundance of Corynebacterium, Facklamia, Staphylococcus, Paenalcaligenes, Lachnoclostridium, norank_f_Muribaculaceae, and unclassified_f_Lachnospiraceae. Meanwhile, metabolomics analysis revealed that FZQS significantly regulated tryptophan metabolism by reducing the levels of 3-Indoleacetonitrile and 5-Hydroxykynurenine. ConclusionFZQS effectively ameliorated LPS-induced lung injury by inhibiting the activation, migration, and adhesion of macrophages and neutrophils and modulating gut microbiota and its metabolites.

Tagetes erecta L.: A traditional medicine effective in inflammatory process treatment

Ethnopharmacological relevanceTagetes erecta L. (Asteraceae), popularly known as Aztec Marigold, is used in folk medicine to treat several ailments including inflammatory processes. Despite its historical use, the specific mechanisms through which it may modulate inflammation, particularly its effects on neutrophils and macrophages activation, have not yet been completely investigated. Aim of the studyThis study aimed to elucidate the anti-inflammatory mechanism of the hydroalcoholic extract obtained from T. erecta flowers, focusing on its role in the regulation of neutrophil and macrophage functions. Material and methodsThe production of TNF, IL-6, CXCL-1, IL-1β, IL-10 (ELISA) and NO (Griess reaction), adhesion molecule expression (CD62L, CD49d and CD18, flow cytometry), and chemotaxis were analyzed in vitro using oyster glycogen-recruited peritoneal neutrophils or macrophages (RAW 264.7) stimulated with lipopolysaccharide (LPS) and treated with the extract (1, 10 or 100 μg/mL). The resolution of inflammation was accessed by efferocytosis assay. The in vivo anti-inflammatory activity was investigated using carrageenan-induced inflammation in the subcutaneous tissue of male Swiss mice orally treated with the T. erecta extract (30, 100 or 300 mg/kg). The leukocyte influx (optical microscopy), secretion of chemical mediators (TNF, IL-6 and IL-1β, ELISA) and protein exudation (Bradford reaction) were quantified in the inflamed exudate. ResultsIn vitro studies demonstrated that the extract inhibited neutrophil chemotaxis and reduced the production and/or release of cytokines (TNF, IL-1β, CXCL1, and IL-6) as well as nitric oxide (NO) by neutrophils and macrophages when stimulated with LPS. Neutrophils treated with LPS and incubated with the extract showed an increase in CD62L expression, which leads to the impairment of neutrophil adhesion. The extract also enhanced efferocytosis of apoptotic neutrophils by macrophages, which was accompanied by increased IL-10 secretion and decreased TNF levels. In vivo studies yielded similar results, showing reduction in neutrophil migration, protein exudation, and cytokine release (TNF, IL-6, and IL-1β). ConclusionsTogether, the data herein obtained shows that T. erecta flower extract has anti-inflammatory effects by regulating inflammatory mediators, limiting neutrophil migration, and promoting efferocytosis. The in vivo results suggest that an herbal medicine made with T. erecta could represent an interesting pharmacological tool for the treatment of acute inflammatory condition.

Effect on neutrophil migration and antimicrobial functions by the Bruton's tyrosine kinase inhibitors tolebrutinib, evobrutinib and fenebrutinib.

Multiple sclerosis (MS) is a neurodegenerative, autoimmune disease that is still incurable. Nowadays, a variety of new drugs are being developed to prevent excessive inflammation and halt neurodegeneration. Among these are the inhibitors of Bruton's tyrosine kinase (BTK). Being indispensable for B cells, this enzyme became an appealing therapeutic target for autoimmune diseases. Recognizing the emerging importance of BTK in myeloid cells, we investigated the impact of upcoming BTK inhibitors on neutrophil functions. Although adaptive immunity in MS has been thoroughly studied, unanswered questions about the pathogenesis can be addressed by studying the effects of candidate MS drugs on innate immune cells such as neutrophils, previously overlooked in MS. In this study, we used three BTK inhibitors (evobrutinib, fenebrutinib and tolebrutinib), and found that they reduce neutrophil activation by the bacterial peptide N-formylmethionyl-leucyl-phenylalanine and the chemokine interleukin 8/CXCL8. Furthermore, they diminished the production of reactive oxygen species and release of neutrophil extracellular traps. Additionally, the production of CXCL8 and interleukin-1β in response to inflammatory stimuli was decreased. Inhibitory effects of the drugs on neutrophil activation were not related to toxicity. Instead, BTK inhibitors prolonged neutrophil survival in an inflammatory environment. Finally, treatment with BTK inhibitors decreased neutrophil migration towards CXCL8 in a Boyden chamber assay but not in a trans endothelial set-up. Also, in vivo CXCL1-induced migration was unaffected by BTK inhibitors. Collectively, this study provides novel insights into the impact of BTK inhibitors on neutrophil functions, thereby holding important implications for autoimmune or hematological diseases where BTK is crucial.

Synovial fibroblasts from children with oligoarticular juvenile idiopathic arthritis induce migration and prolong viability of neutrophils.

Little is known of the processes that trigger neutrophil activation in the joint of patients with oligoarticular juvenile idiopathic arthritis (oJIA), and if synovial fibroblasts (S-Fib) play an important role in the activation. Therefore, we aimed to investigate whether S-Fib derived from oJIA patients drive neutrophil activation. Synovial fluid (SF) was collected from patients with oJIA. S-Fib were isolated from the SF of n = 7 patients through passaging. Subsequently, the S-Fib were primed or not with 20% of pooled SF. Supernatants were used to study migration of neutrophils in a transwell system. Additionally, the influence of S-Fib on neutrophils were studied in co-cultures. Phenotype and viability were assessed by flow cytometry. Neutrophil function was tested through the production of reactive oxygen species (ROS), and supernatants were tested for myeloperoxidase (MPO) release and elastase activity. Supernatants of S-Fib induced neutrophil migration (n = 5, p = 0.0491), which was further pronounced using supernatants from SF-primed S-Fib (p = 0.0063). Additionally, co-culture between SF-primed S-Fib and neutrophils resulted in prolonged viability (n = 5, p = 0.0094), with little effect on activation markers, e.g., CD11b. Conversely, co-culture did not induce functional alterations (n = 4), such as production of ROS (p > 0.1570), release of MPO (p > 0.4934) or elastase activity (p > 0.0904). Finally, supernatant stimulation did not replicate the results of prolonged viability (p = 0.9102), suggesting a role of cell-contact. S-Fib from patients with oJIA induce migration of neutrophils via soluble mediators and, in addition, S-Fib prolong neutrophil viability in a cell-contact dependent manner. These mechanisms could be important for accumulation of neutrophils during arthritis.

Collagen concentration regulates neutrophil extravasation and migration in response to infection in an endothelium dependent manner.

As the body's first line of defense against disease and infection, neutrophils must efficiently navigate to sites of inflammation; however, neutrophil dysregulation contributes to the pathogenesis of numerous diseases that leave people susceptible to infections. Many of these diseases are also associated with changes to the protein composition of the extracellular matrix. While it is known that neutrophils and endothelial cells, which play a key role in neutrophil activation, are sensitive to the mechanical and structural properties of the extracellular matrix, our understanding of how protein composition in the matrix affects the neutrophil response to infection is incomplete. To investigate the effects of extracellular matrix composition on the neutrophil response to infection, we used an infection-on-a-chip microfluidic device that replicates a portion of a blood vessel endothelium surrounded by a model extracellular matrix. Model blood vessels were fabricated by seeding human umbilical vein endothelial cells on 2, 4, or 6 mg/mL type I collagen hydrogels. Primary human neutrophils were loaded into the endothelial lumens and stimulated by adding the bacterial pathogen Pseudomonas aeruginosa to the surrounding matrix. Collagen concentration did not affect the cell density or barrier function of the endothelial lumens. Upon infectious challenge, we found greater neutrophil extravasation into the 4 mg/mL collagen gels compared to the 6 mg/mL collagen gels. We further found that extravasated neutrophils had the highest migration speed and distance in 2mg/mL gels and that these values decreased with increasing collagen concentration. However, these phenomena were not observed in the absence of an endothelial lumen. Lastly, no differences in the percent of extravasated neutrophils producing reactive oxygen species were observed across the various collagen concentrations. Our study suggests that neutrophil extravasation and migration in response to an infectious challenge are regulated by collagen concentration in an endothelial cell-dependent manner. The results demonstrate how the mechanical and structural aspects of the tissue microenvironment affect the neutrophil response to infection. Additionally, these findings underscore the importance of developing and using microphysiological systems for studying the regulatory factors that govern the neutrophil response.

Isolation and characterization of plant-derived exosome-like nanoparticles from Carica papaya L. fruit and their potential as anti-inflammatory agent.

Inflammation is an immune system response that identifies and eliminates foreign material. However, excessive and persistent inflammation could disrupt the healing process. Plant-derived exosome-like nanoparticles (PDENs) are a promising candidate for therapeutic application because they are safe, biodegradable and biocompatible. In this study, papaya PDENs were isolated by a PEG6000-based method and characterized by dynamic light scattering (DLS), transmission Electron Microscopy (TEM), bicinchoninic acid (BCA) assay method, GC-MS analysis, total phenolic content (TPC) analysis, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. For the in vitro test, we conducted internalization analysis, toxicity assessment, determination of nitrite concentration, and assessed the expression of inflammatory cytokine genes using qRT-PCR in RAW 264.7 cells. For the in vivo test, inflammation was induced by caudal fin amputation followed by analysis of macrophage and neutrophil migration in zebrafish (Danio rerio) larvae. The result showed that papaya PDENs can be well isolated using the optimized differential centrifugation method with the addition of 30 ppm pectolyase, 15% PEG, and 0.2 M NaCl, which exhibited cup-shaped and spherical morphological structure with an average diameter of 168.8±9.62 nm. The papaya PDENs storage is stable in aquabidest and 25 mM trehalose solution at -20˚C until the fourth week. TPC estimation of all papaya PDENs ages did not show a significant change, while the DPPH test exhibited a significant change in the second week. The major compounds contained in Papaya PDENs is 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP). Papaya PDENs can be internalized and is non-cytotoxic to RAW 264.7 cells. Moreover, LPS-induced RAW 264.7 cells treated with papaya PDENs showed a decrease in NO production and downregulation mRNA expression of pro-inflammatory cytokine genes (IL-1B and IL-6) and an upregulation in mRNA expression of anti-inflammatory cytokine gene (IL-10). In addition, in vivo tests conducted on zebrafish treated with PDENs papaya showed inhibition of macrophage and neutrophil cell migration. These findings suggest that PDENs papaya possesses anti-inflammatory properties.

Atherosclerosis associated with Chlamydia pneumoniae: Dissecting the etiology

Chlamydia pneumoniae related infections and atherosclerosis are both common entities. Today, the literature presents an enormous amount of data regarding the role of C. pneumoniae in the development and sustainment of atherosclerosis and allowing us to comprehend the molecular mechanisms behind better. The implications of C. pneumoniae in atherogenesis include altered platelet function, hypercoagulability, macrophage dysfunction, vascular smooth muscle proliferation, and increased neutrophilic migration. Therefore, it would not be wrong to implicate that, C. pneumoniae plays important roles in almost every stage of atherogenesis. Furthermore, various serological markers suggestive of active or past C. pneumoniae infection are known to be associated with multiple clinical presentations, such as abdominal aortic aneurysms, subclinical atherosclerosis in the young individuals, aggravated atherosclerosis in heterozygous familial hypercholesterolemia. This review, as a result, aims to provide detailed insights into the pathophysiological mechanisms of atherogenesis associated with C. pneumoniae and its clinical implications.

Curcumin attenuates lupus nephritis by inhibiting neutrophil migration via PI3K/AKT/NF-κB signalling pathway

ObjectiveTo investigate the role of curcumin in the treatment of lupus nephritis (LN) by inhibiting the migration of neutrophils and the underlying mechanism involved.MethodsTwo lupus mouse models, MRL/lpr mice and...

Neutrophil migration participates in the side effect of recombinant human tissue plasminogen activator.

Ischemic stroke remains a challenge in medical research because of the limited treatment options. Recombinant human tissue plasminogen activator (rtPA) is the primary treatment for recanalization. However, nearly 50% of the patients experience complications that result in ineffective reperfusion. The precise factors contributing to ineffective reperfusion remain unclear; however, recent studies have suggested that immune cells, notably neutrophils, may influence the outcome of rtPA thrombolysis via mechanisms such as the formation of neutrophil extracellular traps. This study aimed to explore the nonthrombolytic effects of rtPA on neutrophils and highlight their contribution to ineffective reperfusion. We evaluated the effects of rtPA treatment on middle cerebral artery occlusion in rats. We also assessed neutrophil infiltration and activation after rtPA treatment invitro and invivo in a small cohort of patients with massive cerebral ischemia (MCI). rtPA increased neutrophil infiltration into the brain microvessels and worsened blood-brain barrier damage during ischemia. It also increased the neutrophil counts of the patients with MCI. Neutrophils play a crucial role in promoting ischemic injury and blood-brain barrier disruption, making them potential therapeutic targets.

Broncho-Vaxom Attenuates Lipopolysaccharide-Induced Inflammation in a Mouse Model of Acute Lung Injury.

Acute lung injury (ALI) is a condition associated with acute respiratory failure, resulting in significant morbidity and mortality. It involves cellular changes such as disruption of the alveolar-capillary membrane, excessive neutrophil migration, and release of inflammatory mediators. Broncho-Vaxom® (BV), a lyophilized product containing cell membrane components derived from eight bacteria commonly found in the respiratory tract, is known for its potential to reduce viral and bacterial lung infections. However, the specific effect of BV on ALI has not been clearly defined. This study explored the preventive effects of BV and its underlying mechanisms in a lipopolysaccharide (LPS)-induced ALI mouse model. Oral BV (1 mg/kg) gavage was administered one hour before the intratracheal injection of LPS to evaluate its preventive effect on the ALI model. The pre-administration of BV significantly mitigates inflammatory parameters, including the production of inflammatory mediators, macrophage infiltration, and NF-κB activation in lung tissue, and the increase in inflammatory cells in bronchoalveolar lavage fluid (BALF). Moreover, BV (3 μg/mL) pretreatment reduced the expression of M1 macrophage markers, interleukins (IL-1β, IL-6), tumor necrosis factor α, and cyclooxygenase-2, which are activated by LPS, in both mouse alveolar macrophage MH-S cells and human macrophage THP-1 cells. These findings showed that BV exhibits anti-inflammatory effects by suppressing inflammatory mediators through the NF-κB pathway, suggesting its potential to attenuate bronchial and pulmonary inflammation.

Semaglutide outperforms insulin in restoring neutrophil function against implant-related infection in diabetic and obese mice: experimental research.

Bone and joint infections (BJI) are a significant complication after arthroplasty and fracture fixation, particularly challenging in patients with type 2 diabetes mellitus (T2DM) and obesity. Semaglutide, a glucagon-like peptide-1 receptor agonist (GLP-1RA), have shown efficacy in managing T2DM and obesity. However, its impact on BJI risk and neutrophil function remains unclear. To investigate whether preoperative semaglutide treatment (1) reduces the risk of BJI in diabetic and obese mice undergoing intra-articular implants, and (2) outperforms insulin in restoring neutrophil function to mitigate implant-related infection. A C57BL/6 mouse model of T2DM/obesity was induced using a high-fat diet (HFD) for 12 weeks. Mice received preoperative insulin or semaglutide therapy for 1-28 days. BJI risk was assessed using an intraarticular-implant model challenged with S. aureus or E. coli. Neutrophil function was evaluated through bactericidal activity, superoxide production, and migration ability. Semaglutide treatment led to a significant and sustained reduction in body weight and improved glucose tolerance in HFD mice. Both insulin and semaglutide therapies significantly reduced BJI risk, with semaglutide showing a more pronounced effect over time. Semaglutide therapy also enhanced neutrophil bactericidal activity, superoxide production, and migration ability compared to insulin therapy. Preoperative semaglutide treatment effectively reduces BJI risk and improves neutrophil function in diabetic and obese mouse models. These findings suggest that semaglutide may be a promising pharmacological intervention to mitigate infection risk in orthopedic patients with T2DM or obesity.

Impaired neutrophil migration underpins host susceptibility to infectious colitis

Citrobacter rodentium models infection with enteropathogenic E. coli and ulcerative colitis (UC). While C57BL/6 (C57) mice recover, C3H/HeN (C3H) mice succumb to infection, partially due to increased colonic neutrophil elastase activity, also seen in UC patients; however, the underlying cause was unknown. Here we found that bone marrow, blood and colonic C57 neutrophils expressed CD11bHi and reached the infected colonic lumen where they underwent productive NETosis. In contrast, while the number of C3H neutrophils increased in the bone marrow, blood, and colon, they remained CD11bLo and got trapped in the submucosa, away from C. rodentium, where they underwent harmful NETosis. CD11bLo neutrophils in C3H mice infected with CRi9, which triggers expression of neutrophil chemoattractants, reached the colonisation site resulting in host survival. UC patient neutrophils also displayed decreased levels of the activation/differentiation markers CD16/CXCR4. These results, suggesting that neutrophil malfunction contributes to exacerbated colitis, provide insight for future therapeutic prospects.

Conditions that promote transcellular neutrophil migration in vivo

Circulating leukocytes enter tissue either through endothelial junctions (paracellular) or via a pore through the body of endothelial cells (transcellular). We have previously shown that genetically replacing VE-cadherin with a VE-cadherin-α-catenin (VEC-αC) fusion construct—which binds constitutively to actin—obstructs junctions, and blocks leukocyte extravasation in lung, skin and postcapillary venules of cremaster muscle. However, neutrophil recruitment into the inflamed peritoneal cavity was unimpaired. Investigating reasons for this, here, we visualized neutrophil diapedesis by 3D intravital video microscopy in the cremaster muscle and omentum, the major site of neutrophil recruitment into the peritoneal cavity. We found that 80% of neutrophil-extravasation occurred through HEVs in the omentum, which was unimpaired by VEC-αC. In addition, in larger venules (60–85 µm) of both tissues, less than 15% of neutrophils extravasated transcellularly in WT mice. However, in VEC-α-C mice, transcellular diapedesis increased severalfold in the omentum, but not in the cremaster. In line with this, omental venules expressed higher levels of ICAM-1 and atypical chemokine receptor 1. Furthermore, only in the omentum, VEC-αC expression caused reduced elongation of venular endothelium in flow-direction, suggesting different biomechanical properties. Collectively, VEC-αC does not inhibit paracellular transmigration in all types of venules and can modulate the diapedesis route.

Characteristics of Neutrophil Migration and Function in Acute Inflammation Induced by Zymosan and Carrageenan in the Mice Air Pouch Model.

Deciphering the complex and redundant process of acute inflammation remains challenging. The failure of numerous clinical trials assessing anti-inflammation agents which had promising preclinical effects inevitably questions the validity of current animal models of inflammation. This study aimed to better understand the process of immune inflammatory response and to select more suitable models to evaluate the effect of potential anti-inflammatory drugs. Zymosan and λ-carrageenan are the most used representatives of particulate and soluble irritants that trigger acute inflammation in the air pouch inflammation model. When zymosan was used, the number of exudate cells first increased at 4h-8h, followed by a drop at 12h-24h. While, the changes in number of leukocytes in peripheral blood and proportion of neutrophils in bone marrow have the opposite trend. Meanwhile, neutrophils released neutrophil extracellular traps (NETs) to clean zymosan particles. In contrast, the cell migration response to carrageenan increased during 4h to 24h, no obvious NETs were observed, and the number of leukocytes in peripheral blood increased and the proportion of neutrophils in bone marrow decreased slightly. This study indicated that although both zymosan and carrageenan are sterile irritants, the characteristics of the inflammatory response induced by each other were different. In the acute phase of inflammation, zymosan-stimulated neutrophils were mobilized, recruited, and engulfed, and then died by NETs. Carrageenan stimulated the production of cytokines/chemokines by neutrophils or macrophages, but did not lead to an obvious death by releasing NETs.

Extracellular vesicles released by host epithelial cells during Pseudomonas aeruginosa infection function as homing beacons for neutrophils

BackgroundPseudomonas aeruginosa (PA) is an opportunistic pathogen that can cause sight threatening infections in the eye and fatal infections in the cystic fibrosis airway. Extracellular vesicles (EVs) are released by host cells during infection and by the bacteria themselves; however, there are no studies on the composition and functional role of host-derived EVs during PA infection of the eye or lung. Here we investigated the composition and capacity of EVs released by PA infected epithelial cells to modulate innate immune responses in host cells.MethodsHuman telomerase immortalized corneal epithelial cells (hTCEpi) cells and human telomerase immortalized bronchial epithelial cells (HBECs) were treated with a standard invasive test strain of Pseudomonas aeruginosa, PAO1, for 6 h. Host derived EVs were isolated by qEV size exclusion chromatography. EV proteomic profiles during infection were compared using mass spectrometry and functional studies were carried out using hTCEpi cells, HBECs, differentiated neutrophil-like HL-60 cells, and primary human neutrophils isolated from peripheral blood.ResultsEVs released from PA infected corneal epithelial cells increased pro-inflammatory cytokine production in naïve corneal epithelial cells and induced neutrophil chemotaxis independent of cytokine production. The EVs released from PA infected bronchial epithelial cells were also chemotactic although they failed to induce cytokine secretion from naïve HBECs. At the proteomic level, EVs derived from PA infected corneal epithelial cells exhibited lower complexity compared to bronchial epithelial cells, with the latter having reduced protein expression compared to the non-infected control.ConclusionsThis is the first study to comprehensively profile EVs released by corneal and bronchial epithelial cells during Pseudomonas infection. Together, these findings show that EVs released by PA infected corneal and bronchial epithelial cells function as potent mediators of neutrophil migration, contributing to the exuberant neutrophil response that occurs during infection in these tissues.

A microphysiological system reveals neutrophil contact-dependent attenuation of pancreatic tumor progression by CXCR2 inhibition-based immunotherapy

Cancer cells recruit neutrophils from the bloodstream into the tumor tissue, where these immune cells promote the progression of numerous solid tumors. Studies in mice suggest that blocking neutrophil recruitment to tumors by inhibition of neutrophil chemokine receptor CXCR2 could be a potential immunotherapy for pancreatic cancer. Yet, the mechanisms by which neutrophils promote tumor progression in humans, as well as how CXCR2 inhibition could potentially serve as a cancer therapy, remain elusive. In this study, we developed a human cell-based microphysiological system to quantify neutrophil-tumor spheroid interactions in both “separated” and “contact” scenarios. We found that neutrophils promote the invasion of tumor spheroids through the secretion of soluble factors and direct contact with cancer cells. However, they promote the proliferation of tumor spheroids solely through direct contact. Interestingly, treatment with AZD-5069, a CXCR2 inhibitor, attenuates invasion and proliferation of tumor spheroids by blocking direct contact with neutrophils. Our findings also show that CXCR2 inhibition reduces neutrophil migration toward tumor spheroids. These results shed new light on the tumor-promoting mechanisms of human neutrophils and the tumor-suppressive mechanisms of CXCR2 inhibition in pancreatic cancer and may aid in the design and optimization of novel immunotherapeutic strategies based on neutrophils.

JP4-039, a Mitochondria-Targeted Nitroxide, Mitigates the Effect of Apoptosis and Inflammatory Cell Migration in the Irradiated Mouse Retina.

We hypothesize that the injection of JP4-039, a mitochondria-targeted nitroxide, prior to irradiation of the mouse retina may decrease apoptosis and reduce neutrophil and macrophage migration into the retina. In our study, we aimed to examine the effects of JP4-039 in the mouse retina using fluorescent microscopy, a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and flow cytometry. Forty-five mice and one eye per mouse were used. In Group 1, fluorescent microscopy was used to determine retinal uptake of 10 µL (0.004 mg/µL) of intravitreally injected BODIPY-labeled JP4-039 at 0, 15, and 60 min after injection. In Group 2, the TUNEL assay was performed to investigate the rate of apoptosis after irradiation in addition to JP4-039 injection, compared to controls. In Group 3, flow cytometry was used to determine the extent of inflammatory cell migration into the retina after irradiation in addition to JP4-039 injection, compared to controls. Maximal retinal uptake of JP4-039 was 15 min after intravitreal injection (p < 0.0001). JP4-039-treated eyes had lower levels of retinal apoptosis (35.8 ± 2.5%) than irradiated controls (49.0 ± 2.7%; p = 0.0066) and demonstrated reduced migration of N1 cells (30.7 ± 11.7% vs. 77.7 ± 5.3% controls; p = 0.004) and M1 cells (76.6 ± 4.2 vs. 88.1 ± 3.7% controls, p = 0.04). Pretreatment with intravitreally injected JP4-039 reduced apoptosis and inflammatory cell migration in the irradiated mouse retina, marking the first confirmed effect of this molecule in retinal tissue. Further studies may allow for safety profiling and potential use for patients with radiation retinopathy.

Neutrophil-specific expression of JAK2-V617F or CALRmut induces distinct inflammatory profiles in myeloproliferative neoplasia

BackgroundNeutrophils play a crucial role in inflammation and in the increased thrombotic risk in myeloproliferative neoplasms (MPNs). We have investigated how neutrophil-specific expression of JAK2-V617F or CALRdel re-programs the functions of neutrophils.MethodsLy6G-Cre JAK2-V617F and Ly6G-Cre CALRdel mice were generated. MPN parameters as blood counts, splenomegaly and bone marrow histology were compared to wild-type mice. Megakaryocyte differentiation was investigated using lineage-negative bone marrow cells upon in vitro incubation with TPO/IL-1β. Cytokine concentrations in serum of mice were determined by Mouse Cytokine Array. IL-1α expression in various hematopoietic cell populations was determined by intracellular FACS analysis. RNA-seq to analyse gene expression of inflammatory cytokines was performed in isolated neutrophils from JAK2-V617F and CALR-mutated mice and patients. Bioenergetics of neutrophils were recorded on a Seahorse extracellular flux analyzer. Cell motility of neutrophils was monitored in vitro (time lapse microscopy), and in vivo (two-photon microscopy) upon creating an inflammatory environment. Cell adhesion to integrins, E-selectin and P-selection was investigated in-vitro. Statistical analysis was carried out using GraphPad Prism. Data are shown as mean ± SEM. Unpaired, two-tailed t-tests were applied.ResultsStrikingly, neutrophil-specific expression of JAK2-V617F, but not CALRdel, was sufficient to induce pro-inflammatory cytokines including IL-1 in serum of mice. RNA-seq analysis in neutrophils from JAK2-V617F mice and patients revealed a distinct inflammatory chemokine signature which was not expressed in CALR-mutant neutrophils. In addition, IL-1 response genes were significantly enriched in neutrophils of JAK2-V617F patients as compared to CALR-mutant patients. Thus, JAK2-V617F positive neutrophils, but not CALR-mutant neutrophils, are pathogenic drivers of inflammation in MPN. In line with this, expression of JAK2-V617F or CALRdel elicited a significant difference in the metabolic phenotype of neutrophils, suggesting a stronger inflammatory activity of JAK2-V617F cells. Furthermore, JAK2-V617F, but not CALRdel, induced a VLA4 integrin-mediated adhesive phenotype in neutrophils. This resulted in reduced neutrophil migration in vitro and in an inflamed vessel. This mechanism may contribute to the increased thrombotic risk of JAK2-V617F patients compared to CALR-mutant individuals.ConclusionsTaken together, our findings highlight genotype-specific differences in MPN-neutrophils that have implications for the differential pathophysiology of JAK2-V617F versus CALR-mutant disease.

Activating A1 adenosine receptor signaling boosts early pulmonary neutrophil recruitment in aged mice in response to Streptococcus pneumoniae infection

BackgroundStreptococcus pneumoniae (pneumococcus) is a leading cause of pneumonia in older adults. Successful control of pneumococci requires robust pulmonary neutrophil influx early in infection. However, aging is associated with aberrant neutrophil recruitment and the mechanisms behind that are not understood. Here we explored how neutrophil recruitment following pneumococcal infection changes with age and the host pathways regulating this.ResultsFollowing pneumococcal infection there was a significant delay in early neutrophil recruitment to the lungs of aged mice. Neutrophils from aged mice showed defects in trans-endothelial migration in vitro compared to young controls. To understand the pathways involved, we examined immune modulatory extracellular adenosine (EAD) signaling, that is activated upon cellular damage. Signaling through the lower affinity A2A and A2B adenosine receptors had no effect on neutrophil recruitment to infected lungs. In contrast, inhibition of the high affinity A1 receptor in young mice blunted neutrophil recruitment to the lungs following infection. A1 receptor inhibition decreased expression of CXCR2 on circulating neutrophils, which is required for trans-endothelial migration. Indeed, A1 receptor signaling on neutrophils was required for their ability to migrate across endothelial cells in response to infection. Aging was not associated with defects in EAD production or receptor expression on neutrophils. However, agonism of A1 receptor in aged mice rescued the early defect in neutrophil migration to the lungs and improved control of bacterial burden.ConclusionsThis study suggests age-driven defects in EAD damage signaling can be targeted to rescue the delay in pulmonary neutrophil migration in response to bacterial pneumonia.

Neutrophil Migration Is Mediated by VLA-6 in the Inflamed Adipose Tissue.

Adipose tissue, well known for its endocrine function, plays an immunological role in the body. The inflamed adipose tissue under LPS-induced systemic inflammation is characterized by the dominance of pro-inflammatory immune cells, particularly neutrophils. Although migration of macrophages toward damaged or dead adipocytes to form a crown-like structure in inflamed adipose tissue has been revealed, the neutrophilic interaction with adipocytes or the extracellular matrix remains unknown. Here, we demonstrated the involvement of adhesion molecules, particularly integrin α6β1, of neutrophils in adipocytes or the extracellular matrix of inflamed adipose tissue interaction. These results suggest that disrupting the adhesion between adipose tissue components and neutrophils may govern the accumulation of excessive neutrophils in inflamed tissues, a prerequisite in developing anti-inflammatory therapeutics by inhibiting inflammatory immune cells.