N2 Phenotype Research Articles

Neutrophil N1 polarization induced by cardiomyocyte-derived extracellular vesicle miR-9-5p aggravates myocardial ischemia/reperfusion injury

Neutrophil polarization contributes to inflammation and its resolution, but the role of neutrophil polarization in myocardial ischemia/reperfusion (I/R) injury remains unknown. Cardiomyocytes (CMs) participate in cardiac inflammation by secreting extracellular vesicles (EVs). Therefore, we investigated the role of neutrophil polarization in myocardial I/R injury and the mechanism by which CM-derived EVs regulated neutrophil polarization. In the present study, our data showed that N1 neutrophil polarization enlarged cardiac infarct size and exacerbated cardiac dysfunction at the early stage of myocardial I/R. Further, CM-EV-derived miR-9-5p was identified as a mediator inducing neutrophils to the N1 phenotype. Mechanistically, miR-9-5p directly suppressed SOCS5 and SIRT1 expression, resulting in activating JAK2/STAT3 and NF-κB signaling pathways in neutrophils. Importantly, we confirmed that serum EV-derived miR-9-5p levels were independently associated with cardiovascular mortality in patients with ST-segment elevation myocardial infarction undergoing percutaneous coronary intervention. These findings suggest neutrophil polarization is a promising therapeutic target against myocardial I/R-induced inflammation and injury, and serum EV-derived miR-9-5p is a promising prognostic biomarker for cardiovascular mortality in patients with ST-segment elevation myocardial infarction undergoing percutaneous coronary intervention.Graphical

The interaction between neutrophils and atrial myocytes in the occurrence and development of atrial fibrillation

BackgroundAtrial fibrillation (AF) is one of the most prevalent sustained cardiac arrhythmias, strongly associated with neutrophils. However, the underlying mechanism remain unclear. This study aims to explore the interaction between neutrophils and atrial myocytes in the pathogenesis of AF.MethodsPatch-clamp was employed to record the action potential duration (APD) and ion channels in HL-1 cells. Flow cytometry was used to assess the differentiation of neutrophils. The mRNA and protein levels of CACNA1C, CACNA2D, and CACNB2 in HL-1 cells were detected.ResultsHigh-frequency electrical stimulation resulted in a shortening of the APD in HL-1 cells. Flow cytometry demonstrated that neutrophils were polarized into N1 phenotype when cultured with stimulated HL-1 cells medium. Compared to control neutrophils conditioned medium (CM), cocultured with TNF-α knockout neutrophils CM prolonged APD and the L-type Ca (2+) channel (LTCC) of HL-1 cells. Additionally, the expression of CACNA2D, CACNB2 and CACNA1C in HL-1 cells were upregulated. Compared with CACNA1C siRNA-transfected HL-1 cells treated with TNF-α siRNA-transfected neutrophils CM, the APD and LTCC of CACNA1C siRNA-transfected HL-1 cells were shortened in control N1 neutrophil CM. The APD and LTCC of control HL-1 cells were also shortened in control N1 neutrophil CM, but prolonged in TNF-α siRNA-transfected neutrophils CM.ConclusionThese findings suggest that neutrophils were polarized into N1 phenotype in AF, TNF-α released from N1 neutrophils contributes to the pathogenesis of AF, via decreasing the APD and LTCC in atrial myocytes through down-regulation of CACNA1C expression.

Xenogenous implanted dental follicle stem cells promote periodontal regeneration through inducing the N2 phenotype of neutrophils

BackgroundPeriodontal tissue loss is the main reason for tooth mobility and loss caused by periodontal disease. Dental follicle stem cells (DFSCs) have significant therapeutic potential in periodontal regeneration, which maybe mainly depends on their potent immunomodulatory capacity. Consequently, this study aims to elucidate the impact of implanted xenogenous DFSCs on innate immune responses during early and late stages in the periodontal defect repair period.MethodsTo trace and investigate the immunomodulation mechanisms of DFSCs in vivo, DFSCs were engineered (E-DFSCs) using lentiviral vectors expressing CD63-enhanced green fluorescent protein (CD63-EGFP) and β-Actin-mCherry protein (ACTB-mCherry) to exhibit green and red fluorescence. The biological characteristics and functions of E-DFSCs were verified by proliferation, differentiation, and co-culture experiments in vitro. In vivo, the periodontal regeneration capacity of E-DFSCs was detected by implantation of murine periodontal defect model, and the response of innate immune cells was detected at the 1st, 3rd, and 5th days (early stage) and 4th week (late stage) after implantation.ResultsIn vitro assessments showed that E-DFSCs retain similar properties to their non-engineered counterparts but exhibit enhanced macrophage immunomodulation capability. In mice models, four-week micro-CT and histological evaluations indicated that E-DFSCs have equivalent efficiency to DFSCs in periodontal defect regeneration. At the early stage of repair in mice periodontal defect, fluorescence tracking showed that implanted E-DFSCs might primarily activate endogenous cells through direct contact and indirect actions, and most of these cells are myeloperoxidase-positive neutrophils. Additionally, compared with the control group, the neutrophilic infiltration and conversion of N2-type were significantly increased in the E-DFSC group. At the late stage of defect regeneration, more M2-type macrophages, fewer TRAP + osteoclasts, and an upregulated OPG/RANKL ratio were detected in the E-DFSC group compared to the control group, which indicated that immune balance tilts towards healing and bone formation.ConclusionThe xenogenous implanted DFSCs can induce the N2 phenotype of neutrophils in the early stage, which can activate the innate immune mechanism of the host to promote periodontal tissue regeneration.

Repolarizing neutrophils via MnO2 nanoparticle-activated STING pathway enhances Salmonella-mediated tumor immunotherapy

Engineered Salmonella has emerged as a promising microbial immunotherapy against tumors; however, its clinical effectiveness has encountered limitations. In our investigation, we unveil a non-dose-dependent type of behavior regarding Salmonella’s therapeutic impact and reveal the regulatory role of neutrophils in diminishing the efficacy of this. While Salmonella colonization within tumors recruits a substantial neutrophil population, these neutrophils predominantly polarize into the pro-tumor N2 phenotype, elevating PD-L1 expression and fostering an immunosuppressive milieu within the tumor microenvironment. In order to bypass this challenge, we introduce MnO2 nanoparticles engineered to activate the STING pathway. Harnessing the STING pathway to stimulate IFN-β secretion prompts a shift in neutrophil polarization from the N2 to the N1 phenotype. This strategic repolarization remodels the tumor immune microenvironment, making the infiltration and activation of CD8+ T cells possible. Through these orchestrated mechanisms, the combined employment of Salmonella and MnO2 attains the synergistic enhancement of anti-tumor efficacy, achieving the complete inhibition of tumor growth within 20 days and an impressive 80% survival rate within 40 days, with no discernible signs of significant adverse effects. Our study not only unveils the crucial in vivo constraints obstructing microbial immune therapy but also sets out an innovative strategy to augment its efficacy. These findings pave the way for advancements in cell-based immunotherapy centered on leveraging the potential of neutrophils.Graphical

The adhesion-GPCR ADGRF5 fuels breast cancer progression by suppressing the MMP8-mediated antitumorigenic effects

ADGRF5 (GPR116) has been identified as a facilitator of breast cancer cell migration and metastasis, yet the underlying mechanisms remain largely elusive. Our current study reveals that the absence of ADGRF5 in breast cancer cells impairs extracellular matrix (ECM)-associated cell motility and impedes in vivo tumor growth. This correlates with heightened expression of matrix metalloproteinase 8 (MMP8), a well-characterized antitumorigenic MMP, and a shift in the polarization of tumor-associated neutrophils (TANs) towards the antitumor N1 phenotype in the tumor microenvironment (TME). Mechanistically, ADGRF5 inhibits ERK1/2 activity by enhancing RhoA activation, leading to decreased phosphorylation of C/EBPβ at Thr235, hindering its nuclear translocation and subsequent activation. Crucially, two C/EBPβ binding motifs essential for MMP8 transcription are identified within its promoter region. Consequently, ADGRF5 silencing fosters MMP8 expression and CXCL8 secretion, attracting increased infiltration of TANs; simultaneously, MMP8 plays a role in decorin cleavage, which leads to trapped-inactivation of TGF-β in the TME, thereby polarizing TANs towards the antitumor N1 neutrophil phenotype and mitigating TGF-β-enhanced cell motility in breast cancer. Our findings reveal a novel connection between ADGRF5, an adhesion G protein-coupled receptor, and the orchestration of the TME, which dictates malignancy progression. Overall, the data underscore ADGRF5 as a promising therapeutic target for breast cancer intervention.

β-Lapachone promotes the recruitment and polarization of tumor-associated neutrophils (TANs) toward an antitumor (N1) phenotype in NQO1-positive cancers

ABSTRACT NAD(P)H:quinone oxidoreductase 1 (NQO1) is overexpressed in most solid cancers, emerging as a promising target for tumor-selective killing. β-Lapachone (β-Lap), an NQO1 bioactivatable drug, exhibits significant antitumor effects on NQO1-positive cancer cells by inducing immunogenic cell death (ICD) and enhancing tumor immunogenicity. However, the interaction between β-Lap-mediated antitumor immune responses and neutrophils, novel antigen-presenting cells (APCs), remains unknown. This study demonstrates that β-Lap selectively kills NQO1-positive murine tumor cells by significantly increasing intracellular ROS formation and inducing DNA double strand breaks (DSBs), resulting in DNA damage. Treatment with β-Lap efficiently eradicates immunocompetent murine tumors and significantly increases the infiltration of tumor-associated neutrophils (TANs) into the tumor microenvironment (TME), which plays a crucial role in the drug’s therapeutic efficacy. Further, the presence of β-Lap-induced antigen medium leads bone marrow-derived neutrophils (BMNs) to directly kill murine tumor cells, aiding in dendritic cells (DCs) recruitment and significantly enhancing CD8+ T cell proliferation. β-Lap treatment also drives the polarization of TANs toward an antitumor N1 phenotype, characterized by elevated IFN-β expression and reduced TGF-β cytokine expression, along with increased CD95 and CD54 surface markers. β-Lap treatment also induces N1 TAN-mediated T cell cross-priming. The HMGB1/TLR4/MyD88 signaling cascade influences neutrophil infiltration into β-Lap-treated tumors. Blocking this cascade or depleting neutrophil infiltration abolishes the antigen-specific T cell response induced by β-Lap treatment. Overall, this study provides comprehensive insights into the role of tumor-infiltrating neutrophils in the β-Lap-induced antitumor activity against NQO1-positive murine tumors.

CXCL9 Overexpression Predicts Better HCC Response to Anti-PD-1 Therapy and Promotes N1 Polarization of Neutrophils.

Anti-programmed death-1 (PD1) antibodies have changed the treatment landscape for hepatocellular carcinoma (HCC) and exhibit promising treatment efficacy. However, the majority of HCCs still do not respond to anti-PD-1 therapy. We analyzed the expression of CXCL9 in blood samples from patients who received anti-PD-1 therapy and evaluated its correlation with clinicopathological characteristics and treatment outcomes. Based on the results of Cox regression analysis, a nomogram was established for predicting HCC response to anti-PD-1 therapy. qRT‒PCR and multiple immunofluorescence assays were utilized to analyze the proportions of N1-type neutrophils in vitro and in tumor samples, respectively. The nomogram showed good predictive efficacy in the training and validation cohorts and may be useful for guiding clinical treatment of HCC patients. We also found that HCC cell-derived CXCL9 promoted N1 polarization of neutrophils in vitro and that AMG487, a specific CXCR3 inhibitor, significantly blocked this process. Moreover, multiple immunofluorescence (mIF) showed that patients with higher serum CXCL9 levels had greater infiltration of the N1 phenotype of tumor-associated neutrophils (TANs). Our study highlights the critical role of CXCL9 as an effective biomarker of immunotherapy efficacy and in promoting the polarization of N1-type neutrophils; thus, targeting the CXCL9-CXCR3 axis could represent a novel pharmaceutical strategy to enhance immunotherapy for HCC.

HucMSC-Exo Induced N2 Polarization of Neutrophils: Implications for Angiogenesis and Tissue Restoration in Wound Healing.

Neutrophils rapidly accumulate in large numbers at sites of tissue damage, exhibiting not only their well-known bactericidal capabilities but also playing crucial roles in angiogenesis and tissue repair. While exosomes derived from human umbilical cord mesenchymal stem cells (HucMSC-Exo) have emerged as a promising therapeutic tool, their exact mechanisms of action remain partly elusive. We hypothesize that HucMSC-Exo treatment may modulate neutrophil phenotypes, thereby significantly influencing wound healing outcomes. HucMSC-Exo were isolated via ultracentrifugation and subsequently administered through subcutaneous injection into full-thickness cutaneous wounds in mice. To determine the impact of host neutrophils on the healing effects of HucMSC-Exo in skin injuries, strategies including neutrophil depletion and adoptive transfer were employed. Flow cytometry was used to evaluate the proportion of N2 subtype neutrophils in both normal and diabetic wounds, and the effect of HucMSC-Exo on this proportion was assessed. Furthermore, the mitochondrial metabolic reprogramming driven by HucMSC-Exo during N2 polarization was investigated through JC1 staining, ATP quantification, fatty acid uptake assays, and assessment of FAO-related genes (Cpt1b, Acadm, and Acadl). Depleting host neutrophils strikingly dampened prohealing effect of HucMSC-Exo on skin injury, while adoptive transfer of bone marrow neutrophils rescued this process. During normal healing process, some neutrophils expressed N2 markers, in contrast, diabetic wounds exhibited a reduced expression of N2 markers. After treatment with HucMSC-Exo, most neutrophils increased the phosphorylation of STAT6, leading to mitochondrial metabolic reprogramming and thus acquired an N2 phenotype. These N2 neutrophils, polarized by HucMSC-Exo, boosted the release of proangiogenic factors, particularly BV8, a myeloid cell-derived proangiogenic factor, and induced angiogenesis thereby favoring tissue restoration. This research uniquely demonstrates the identification of N2 neutrophils in skin injury and shows that HucMSC-Exo could skew neutrophils toward N2 phenotype, enhancing our insight into how cells react to HucMSC-Exo.

ALDH3A1 upregulation inhibits neutrophils N2 polarization and halts oral cancer growth.

Tumor-associated neutrophils (TANs) are among the most abundant inflammatory cells in tumor microenvironment (TME). Aldehyde dehydrogenase 3A1 (ALDH3A1) is significantly reduced in oral squamous cell carcinoma (OSCC), ALDH3A1 overexpression suppresses tumorigenesis by inhibiting inflammation. This study investigated the relationship and mechanisms underlying the crosstalk between ALDH3A1 and TANs in OSCC. Immunohistochemistry and immunofluorescence were performed to investigate the abundance of TANs and the expression of ALDH3A1. dHL-60 were induced with tumor-conditioned media and recombinant IL-6/IL-8. The expression of key proteins in PI3K/AKT/NF-κB pathway were detected by RT-PCR and western blot. A xenograft model was utilized to examine the effect of ALDH3A1 on tumorigenicity and polarization of TANs. In patients with OSCC, TANs significantly increased and were associated with a worse prognosis. Additionally, ALDH3A1 negatively correlated with TANs infiltration and especially the N2 phenotype which was the prominent part in OSCC. Furthermore, our study demonstrated that tumor-derived IL-8 drives ALDH3A1-mediated TANs N2 polarization in the TME through PI3K/AKT/NF-κB pathway invitro and invivo. Our results indicate that TANs can serve as a prognostic biomarker and ALDH3A1 could be a promising therapeutic target for regulating TANs N2 polarization in antitumor therapy.

Investigation on how mechanical force transducer PIEZO1 controls the polarization of tumor-associated neutrophils and its relationship with colorectal cancer development

Nowadays, during the polarization of neutrophils, the function of the mechanical force transducer PIEZO1 is still unclear. Our research shows that the PIEZO1 gene knockout in tumor-associated neutrophils (TANs) can suppress the polarization of TANs towards the N2 phenotype induced by TGF-β; Activating PIEZO1 by Yoda1 promotes polarization. We discover that cancer cell proliferation is enhanced by coculturing colorectal cancer cells with N2 TANs. On the contrary, when PIEZO1 is knocked out, the cancer development alleviates due to its prevention of TANs polarizing towards N2. Our research hints that mechanical force transducer PIEZO1 might be a new target spot during the treatment of colorectal cancer.

Neutrophils are involved in early bone formation during midpalatal expansion.

Midpalatal expansion (MPE) is routinely employed to treat transverse maxillary arch deficiency. Neutrophils are indispensable for recruiting bone marrow stromal cells (BMSCs) at the initial stage of bone regeneration. This study aimed to explore whether neutrophils participate in MPE and how they function during bone formation under mechanical stretching. The presence and phenotype of neutrophils in the midpalatal suture during expansion were detected by flow cytometry and immunofluorescence staining. The possible mechanism of neutrophil recruitment and polarization was explored invitro by exposing vascular endothelial cells (VECs) to cyclic tensile strain. The number of neutrophils in the distracted suture peaked on Day 3, and N2-type neutrophils significantly increased on Day 5 after force application. The depletion of circulatory neutrophils reduced bone volume by 43.6% after 7-day expansion. The stretched VECs recruited neutrophils via a CXCR2 mechanism invitro, which then promoted BMSC osteogenic differentiation through the VEGFA/VEGFR2 axis. Consistently, these neutrophils showed higher expression of canonical N2 phenotype genes, including CD206 and Arg1. These results suggested that neutrophils participated in early bone formation during MPE. Based on these findings, we propose that stretched VECs recruited and polarized neutrophils, which, in turn, induced BMSC osteogenic differentiation.

Retraction: Berberine maintains the neutrophil N1 phenotype to reverse cancer cell resistance to doxorubicin.

[This retracts the article DOI: 10.3389/fphar.2019.01658.].

Harnessing astaxanthin-loaded diselenium cross-linked apotransferrin nanoparticles for the treatment of secretory otitis media

Secretory otitis media (SOM) is a clinical condition characterized by the accumulation of fluids and oxidative stress in the middle ear, leading to hearing impairment and infection complications. One potential solution for mitigating oxidative stress associated with SOM is the use of antioxidants such as astaxanthin. However, its effectiveness is limited due to its poor bioavailability and rapid oxidation. Herein, we developed a novel diselenium-crosslinked apotransferrin enriched with astaxanthin (AST@dSe-AFT) nanoparticles to augment the transport of astaxanthin across biological membranes, resulting in increased bioavailability and reduced oxidative stress in SOM. Our research demonstrated that AST@dSe-AFT efficiently accumulated in the middle ear, allowing for controlled delivery of astaxanthin in response to reactive oxygen species and reducing oxidative stress. Additionally, AST@dSe-AFT stimulated macrophages to polarize towards M2 phenotype and neutrophils to polarize towards N2 phenotype, thereby facilitating an anti-inflammatory response and tissue restoration. Importantly, AST@dSe-AFT exhibited no toxicity or adverse effects, suggesting its potential for safety and future clinical translation. Our findings suggested that AST@dSe-AFT represents a promising approach for the treatment of secretory otitis media and other oxidative stress-related disorders.

TMIC-42. ACROLEIN PRODUCED BY GLIOMA CELLS UNDER HYPOXIA INHIBITS NEUTROPHIL AKT ACTIVITY AND SUPPRESSES ANTI-TUMORAL ACTIVITIES

Abstract Glioblastoma (GBM), WHO grade 4 glioma, is the most malignant primary brain tumor. The median overall survival of GBM ranges from 12 to 16 months. The tumor microenvironment of GBM is characterized by tissue hypoxia and infiltration of immunosuppressive cells. Neutrophils have been reported to shift from an anti-inflammatory phenotype to a pro-tumoral role (N2 phenotype) during glioma progression. However, the mechanism of this transformation remains unclear. Acrolein (Acr) is a highly-reactive α,β-unsaturated aldehyde produced under hypoxic conditions via lipid peroxidation, resulting in cellular impairment through DNA or protein conjugation. In this study, we found that acrolein produced by U87MG cells under hypoxic conditions suppresses neutrophil superoxide generation, inhibits neutrophil tumor-suppressive activities, and induces N2 marker arginase 1 (Arg-1) expression. Scavenging acrolein with dimercaprol reverses these effects. Further investigation revealed that acrolein inhibits AKT enzymatic activity by forming Acr-AKT conjugates at Cys 310. In a syngeneic subcutaneous mice glioma model, dimercaprol treatment is associated with decreased tumor size, reduced acrolein conjugate expression in the tumor tissue, and restored neutrophil superoxide generation. In addition, we also found that the higher acrolein-DNA adduct expression in tumor tissues is associated with a poor prognosis of glioma patients, and the evaluated acrolein-protein conjugates in the serum of glioma patients are correlated to the functional impairment of neutrophils. These results suggest acrolein polarizes the neutrophils toward the N2 phenotype and contributes to the immunosuppressive microenvironment and glioma progression. Acrolein scavenging may be a novel strategy for glioma treatment.

EXTH-69. REGULATION OF N1/N2 NEUTROPHIL PHENOTYPE BY A MANZAMINE-DERIVED COMPOUND FOR BRAIN TUMOR TREATMENT

Abstract In cancer microenvironment, the presence of neutrophils is associated with tumor proliferation, invasion and immunomodulation. The tumor associated neutrophils (TAN) can be polarized toward N1 (anti-tumor) or N2 (pro-tumor) phenotypes. Therefore, modulating neutrophil phenotype may serve as brain tumor treatment strategy. We use immunosuppressive and normal mice bearing brain tumor cell xenograft to study the role of TANs in tumor progression. A manzamine-derived compound 1-(9’-propyl-3’-carbazole)-1, 2, 3, 4-tetrahydro-β-carboline (PCTC) was used to treat brain tumor mice. In brain tumor tissue, flowcytometry study revealed the amount of pro-tumor N2 TAN is significantly greater than anti-tumor N1 subtype. While PCTC was intracranial administrated into brain tumor, the ratio of N1/N2 is reversed. Immunochemical staining showed increasing neutrophils in tumor at day 1 post treatment, followed by subsequent T cell and macrophage gathering at day 5. Cytokine array results revealed the expression level of CXCL9/CXCL10 were upregulated in PCTC treatment glioma cells and brain tumor tissues. Cell study also demonstrated T cell migration ability was significantly increased when co-cultured with neutrophil and PCTC treated glioma cells. In mice tumor model, NU/NU mouse lack of T cell represented no response to PCTC treatment, while significant tumor shrunken was observed in B6 mice. Compensation study by intravenous inoculation of T cell and PCTC to NU/NU glioma mice showed flavor response to animal survival. In this study, we demonstrated the N1/N2 ratio of TAN can be regulated by PCTC, and be beneficial to glioma treatment. Furthermore, PCTC acts on tumor cells, together with TAN to attract T cell by increase CXCL9/CXCL10. Anti-tumor N1 and subsequent T cell gathering both contribute to tumor cell killing. As a conclusion, neutrophil phenotype can be modulated, and cooperated with T cells to serve as a new glioma treatment strategy.

CD39 and LDHA affects the prognostic role of NLR in metastatic melanoma patients treated with immunotherapy

BackgroundIdentifying response markers is highly needed to guide the treatment strategy in patients with metastatic melanoma.MethodsA retrospective study was carried out in patients with unresectable/metastatic melanoma (stage IIIb–IV), treated with anti-PD-1 in the first line setting, to better explore the role and the timing of neutrophil/lymphocyte ratio (NLR) as potential biomarker of response. The relationship of NLR with inflammation-immune mediators and the underlying negative effect of raising NLR during immunotherapy, have been investigated with transcriptomic gene analysis.ResultsThe results confirmed previous findings that a high baseline NLR is associated with a poorer prognosis and with higher serum level of lactate dehydrogenase (LDH), regardless of the presence of brain metastases. The transcriptomic analysis showed that high baseline NLR is associated with a characteristic gene signature CCNA1, LDHA and IL18R1, which correlates with inflammation and tumorigenesis. Conversely, low baseline NLR is associated with the signature CD3, SH2D1A, ZAP70 and CD45RA, linked to the immune-activation. The genes positively associated with NLR (CD39 (ENTPD1), PTEN, MYD88, MMP9 and LDH) are involved in processes of immunosuppression, inflammation and tumor-promoting activity. Increased expression of CD39 correlated with TGFβ2, a marker of the N2 neutrophils with immunosuppressive activity.ConclusionsThese results suggest that increasing NLR is associated with an increased neutrophil population, with polarization to the N2 phenotype, and this process may be the basis for the negatively prognostic role of NLR.

Matrix mechanics regulate the polarization state of bone marrow-derived neutrophils through the JAK1/STAT3 signaling pathway.

Matrix mechanics regulate essential cell behaviors through mechanotransduction, and as one of its most important elements, substrate stiffness was reported to regulate cell functions such as viability, communication, migration, and differentiation. Neutrophils (Neus) predominate the early inflammatory response and initiate regeneration. The activation of Neus can be regulated by physical cues; however, the functional alterations of Neus by substrate stiffness remain unknown, which is critical in determining the outcomes of engineered tissue mimics. Herein, a three-dimensional (3D) culture system made of hydrogels was developed to explore the effects of varying stiffnesses (1.5, 2.6, and 5.7 kPa) on the states of Neus. Neus showed better cell integrity and viability in the 3D system. Moreover, it was shown that the stiffer matrix tended to induce Neus toward an anti-inflammatory phenotype (N2) with less adhesion molecule expression, less reactive oxygen species (ROS) production, and more anti-inflammatory cytokine secretion. Additionally, the aortic ring assay indicated that Neus cultured in a stiffer matrix significantly increased vascular sprouting. RNA sequencing showed that a stiffer matrix could significantly activate JAK1/STAT3 signaling in Neus and the inhibition of JAK1 ablated the stiffness-dependent increase in the expression of CD182 (an N2 marker). Taken together, these results demonstrate that a stiffer matrix promotes Neus to shift to the N2 phenotype, which was regulated by JAK1/STAT3 pathway. This study lays the groundwork for further research on fabricating engineered tissue mimics, which may provide more treatment options for ischemic diseases and bone defects. STATEMENT OF SIGNIFICANCE.

Upregulation of P2Y14 receptor in neutrophils promotes inflammation after myocardial ischemia/reperfusion injury

BackgroundP2Y14 receptor is expressed in neutrophils and is involved in activation of inflammatory signaling. However, the expression and function of P2Y14 receptor in neutrophils after myocardial infarction/reperfusion (MIR) injury remain to be elucidated. MethodsIn this research, rodent and cellular models of MIR were used to detect the involvement and function of P2Y14 receptor, as well as the regulation of inflammatory signaling via P2Y14 receptor in neutrophils post-MIR. ResultsIn the early stage post MIR, the expression of P2Y14 receptor was upregulated in CD4+Ly-6G+ neutrophils. Additionally, the expression of P2Y14 receptor was highly induced in neutrophils subjected to uridine 5′-diphosphoglucose (UDP-Glu), which is proven to be secreted by cardiomyocytes during ischemia and reperfusion. Our results also showed the beneficial role of P2Y14 receptor antagonist PPTN in counteracting inflammation via promoting polarization of neutrophils to N2 phenotype in the infarct area of the heart tissue after MIR. ConclusionThese findings prove that the P2Y14 receptor is involved in the regulation of inflammation in the infarct area after MIR, and establish a novel signaling pathway concerning the interplay between cardiomyocytes and neutrophils in the heart tissue.

Smad3 is essential for polarization of tumor-associated neutrophils in non-small cell lung carcinoma

Neutrophils are dynamic with their phenotype and function shaped by the microenvironment, such as the N1 antitumor and N2 pro-tumor states within the tumor microenvironment (TME), but its regulation remains undefined. Here we examine TGF-β1/Smad3 signaling in tumor-associated neutrophils (TANs) in non-small cell lung carcinoma (NSCLC) patients. Smad3 activation in N2 TANs is negatively correlate with the N1 population and patient survival. In experimental lung carcinoma, TANs switch from a predominant N2 state in wild-type mice to an N1 state in Smad3-KO mice which associate with enhanced neutrophil infiltration and tumor regression. Neutrophil depletion abrogates the N1 anticancer phenotype in Smad3-KO mice, while adoptive transfer of Smad3-KO neutrophils reproduces this protective effect in wild-type mice. Single-cell analysis uncovers a TAN subset showing a mature N1 phenotype in Smad3-KO TME, whereas wild-type TANs mainly retain an immature N2 state due to Smad3. Mechanistically, TME-induced Smad3 target genes related to cell fate determination to preserve the N2 state of TAN. Importantly, genetic deletion and pharmaceutical inhibition of Smad3 enhance the anticancer capacity of neutrophils against NSCLC via promoting their N1 maturation. Thus, our work suggests that Smad3 signaling in neutrophils may represent a therapeutic target for cancer immunotherapy.

Neutrophil Heterogeneity and its Roles in the Inflammatory Network after Ischemic Stroke.

As the first peripheral immune cells to enter the brain after ischemic stroke, neutrophils are important participants in stroke-related neuroinflammation. Neutrophils are quickly mobilized from the periphery in response to a stroke episode and cross the blood-brain barrier to reach the ischemic brain parenchyma. This process involves the mobilization and activation of neutrophils from peripheral immune organs (including the bone marrow and spleen), their chemotaxis in the peripheral blood, and their infiltration into the brain parenchyma (including disruption of the blood-brain barrier, inflammatory effects on brain tissue, and interactions with other immune cell types). In the past, it was believed that neutrophils aggravated brain injuries through the massive release of proteases, reactive oxygen species, pro-inflammatory factors, and extracellular structures known as neutrophil extracellular traps (NETs). With the failure of early clinical trials targeting neutrophils and uncovering their underlying heterogeneity, our view of their role in ischemic stroke has become more complex and multifaceted. As neutrophils can be divided into N1 and N2 phenotypes in tumors, neutrophils have also been found to have similar phenotypes after ischemic stroke, and play different roles in the development and prognosis of ischemic stroke. N1 neutrophils are dominant during the acute phase of stroke (within three days) and are responsible for the damage to neural structures via the aforementioned mechanisms. However, the proportion of N2 neutrophils gradually increases in later phases, and this has a beneficial effect through the release of anti-inflammatory factors and other neuroprotective mediators. Moreover, the N1 and N2 phenotypes are highly plastic and can be transformed into each other under certain conditions. The pronounced differences in their function and their high degree of plasticity make these neutrophil subpopulations promising targets for the treatment of ischemic stroke.