Reduced Neutrophil Recruitment Research Articles

Fungal melanin suppresses airway epithelial chemokine secretion through blockade of calcium fluxing

Respiratory infections caused by the human fungal pathogen Aspergillus fumigatus are a major cause of mortality for immunocompromised patients. Exposure to these pathogens occurs through inhalation, although the role of the respiratory epithelium in disease pathogenesis has not been fully defined. Employing a primary human airway epithelial model, we demonstrate that fungal melanins potently block the post-translational secretion of the chemokines CXCL1 and CXCL8 independent of transcription or the requirement of melanin to be phagocytosed, leading to a significant reduction in neutrophil recruitment to the apical airway both in vitro and in vivo. Aspergillus-derived melanin, a major constituent of the fungal cell wall, dampened airway epithelial chemokine secretion in response to fungi, bacteria, and exogenous cytokines. Furthermore, melanin muted pathogen-mediated calcium fluxing and hindered actin filamentation. Taken together, our results reveal a critical role for melanin interaction with airway epithelium in shaping the host response to fungal and bacterial pathogens.

#855 Cyclophilin D in cholesterol crystal-induced acute kidney injury

Abstract Background and Aims Cholesterol crystal embolism (CCE) occurs when cholesterol crystal (CC) clots move from an atherosclerotic plaque in a large artery to a smaller artery, leading to obstruction, ischemia, and organ failure. CCE directly impairs blood flow by provoking a local inflammatory response and triggering arterial thrombus formation, consisting of collagen and fibrin mesh, platelet and neutrophil activation, and extracellular DNA trap formation. Activated platelets form a procoagulant surface that supports adhesion to collagen and fibrin mesh, promoting thrombus growth and the recruitment of immune cells. CCE often targets kidney tissue, leading to thrombotic angiopathy, acute loss of glomerular filtration rate (GFR), and necrosis in tubular cells. Cyclophilin D (CypD) plays a central role in regulating platelet procoagulant activity by triggering calcium bursts and mitochondrial depolarization. While the increased number of procoagulant platelets contributes to thrombosis, CypD-mediated procoagulant pathomechanisms in the kidney during CCE remain unknown. In our study presented here, we hypothesized that CypD deficiency might attenuate procoagulant platelet activity, thereby inhibiting CCE-induced kidney infarction and organ injury. Method 6-week-old wild-type (WT) and CypD-/- mice were administered 10 mg/kg CC through injections into the left kidney arteries in vivo. Kidneys were collected 24 hours later, and kidney function, infarct size, necroinflammation and kidney injury were evaluated using GFR measurements, TTC colorimetric assay, immunohistochemistry, and immunofluorescence microscopy. For in vitro studies, whole blood samples were isolated from WT and CypD-/- mice and perfused on a collagen-coated surface in the presence or absence of CC under arterial shear conditions of 1000s-1. Results After CC injection in mice, CypD deficiency ameliorated immunothrombosis, acute kidney injury, and organ failure. CypD-/- mice were protected from the sudden drop in GFR, i.e., acute kidney injury, which resulted from the reduced number of arteries obstructed by crystal clots. Kidney infarct size consistently decreased in CypD-/- mice. CypD-/- mice displayed reduced neutrophil recruitment to thrombotic microangiopathic lesions, indicating diminished immunothrombosis. Preemptive injection of the CypD inhibitor cyclosporin A could prevent CC-mediated vessel obstruction and infarct size, thereby improving kidney function. Interestingly, WT platelets showed increased platelet adhesion and phosphatidylserine (PS) exposure on a collagen-coated surface in the presence of CC under shear conditions in whole blood, suggesting increased procoagulant activity, which was strongly reduced in CypD-/- platelets. Conclusion During CCE-mediated kidney injury, CypD-mediated platelet activation and consequent procoagulant activity accelerate ischemic tissue necrosis and the recruitment of neutrophils. Inhibition of CypD-mediated signaling could be a potential therapeutic approach to inhibit acute kidney injury in patients with CCE.

Abstract 1385: Crosstalk between hepatocyte and myeloid-specific IL17 signaling in colorectal cancer metastasis

Abstract Advanced colorectal cancer (CRC) is resistant to current immunotherapies and prone to metastasis. Chronic inflammation mediated by cytokines such as interleukin (IL)17 activates the oncogenic pathways in cancer cells, promoting sporadic and inflammation-associated CRC. Increased levels of IL17A in early stages of CRC portends poor prognosis indicating its potential involvement into metastasis. We and others previously showed that IL17A signaling is required for early CRC tumorigenicity in a variety of models and that in early CRC, its ability to signal to epithelial cells is essential. What remains unknown is whether IL17 signaling to other cell types within the tumor microenvironment (TME) is essential to control tumor progression and metastasis. Using a model of colitis-associated cancer (CAC) we found that “late” IL17A neutralization decreases tumor burden, possibly acting through regulation of myeloid cells and chemokine network essential for the control of immunosuppressive TME. Next, using models of colorectal cancer liver metastasis (CRLM) including intraportal cell injection and cecum transplantation of organoids with distinct set of oncogenic mutation we found that IL17 neutralization reduces metastasis. FACS analysis revealed that IL17A promotes the recruitment of tumor-associated macrophages and other key myeloid subsets into the metastatic liver environment. Interestingly, ablation of IL17RC (receptor) in myeloid cells only (using Il17rcf/f -LysMCre mice) led to reduction in metastasis and reshaping of metastatic myeloid cell compartments. Meanwhile, IL17 signaling in hepatocytes was required for early metastasis and pre-metastatic niche formation, as the ablation of IL17RC in hepatocytes (using Il17rcf/f -AlbCre mice) led to a reduction of metastatic numbers but not growth. This coincided with reduced neutrophil recruitment and enhanced activation of cytotoxic innate lymphoid cells and CD8 T lymphocytes, indicating the role of hepatocyte specific IL17 signaling in blunting anti-tumor immunity. Our findings illuminate the role of inflammatory cytokine IL17 into CRC progression and metastasis and imply its distinct cell type specific role within the metastatic liver TME. Citation Format: Katarzyna Chojnacka, Dan Kamen, Katrina M. Reyes, Hana Tomizawa, Sergei I. Grivennikov. Crosstalk between hepatocyte and myeloid-specific IL17 signaling in colorectal cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1385.

CXCL12+ dermal fibroblasts promote neutrophil recruitment and host defense by recognition of IL-17.

The skin provides an essential barrier for host defense through rapid action of multiple resident and recruited cell types, but the complex communication network governing these processes is incompletely understood. To define these cell-cell interactions more clearly, we performed an unbiased network analysis of mouse skin during invasive S. aureus infection and revealed a dominant role for CXCL12+ fibroblast subsets in neutrophil communication. These subsets predominantly reside in the reticular dermis, express adipocyte lineage markers, detect IL-17 and TNFα, and promote robust neutrophil recruitment through NFKBIZ-dependent release of CXCR2 ligands and CXCL12. Targeted deletion of Il17ra in mouse fibroblasts resulted in greatly reduced neutrophil recruitment and increased infection by S. aureus. Analogous human CXCL12+ fibroblast subsets abundantly express neutrophil chemotactic factors in psoriatic skin that are subsequently decreased upon therapeutic targeting of IL-17. These findings show that CXCL12+ dermal immune acting fibroblast subsets play a critical role in cutaneous neutrophil recruitment and host defense.

Colchicine Attenuates Microvascular Obstruction after Myocardial Ischemia-Reperfusion Injury by Inhibiting the Proliferation of Neutrophil in Bone Marrow

Abstract Purpose Complete and rapid recanalization of blood flow by percutaneous coronary intervention (PCI) is the most effective intervention for patients with ST-segment elevation myocardial infarction (STEMI). However, myocardial ischemia/reperfusion (I/R) injury leads to microvascular obstruction (MVO), limiting its efficacy. Colchicine can reduce myocardial I/R injury, but its effect on MVO is unclear. Hence, this study aimed to assess the role and mechanism of colchicine on MVO. Methods Clinical data on STEMI patients with PCI were collected and risk factors related to MVO were analyzed. The rat myocardial I/R model was established to evaluate the MVO by thioflavin S staining. The myocardial I/R model of mice was treated with PBS or colchicine at the reperfusion. The effect of colchicine on cardiomyocyte apoptosis after I/R was evaluated by TUNEL and expression of cleaved caspase-3. ROS levels were detected in H9c2 cells to evaluate the colchicine effect on myocardial oxidative stress. Moreover, the mechanism through which colchicine attenuated MVO was examined using flow cytometry, WB, ELISA, immunohistochemistry, bioinformatics analysis, and immunofluorescence. Results Multivariate analysis showed that elevated neutrophils were associated with extensive MVO. Colchicine could attenuate MVO and reduce neutrophil recruitment and NETs formation after myocardial I/R. In addition, colchicine inhibited cardiomyocyte apoptosis in vivo and ROS levels in vitro. Furthermore, colchicine inhibited neutrophil proliferation in the bone marrow (BM) by inhibiting the S100A8/A9 inflammatory signaling pathway. Conclusions Colchicine attenuated MVO after myocardial I/R injury by inhibiting the proliferation of neutrophils in BM through the neutrophil-derived S100A8/A9 inflammatory signaling pathway.

Abstract B025: Neutrophil depletion enhances the anti-tumor activity of CAR-T cells in an autochthonous model of non-small cell lung cancer

Abstract CAR-T cell therapy has produced durable clinical responses in hematological malignancies, but efficacy in more common solid tumors is limited in part by poor trafficking of CAR-T cells to tumors. Identifying strategies to improve CAR-T cell trafficking to and persistence within tumors, thus, are needed to improve efficacy against solid tumors. We previously adapted the Kras/p53 (KP) autochthonous model of non-small cell lung cancer (NSCLC) to express the CAR target ROR1 to study the mechanisms limiting the activity of ROR1 CAR-T cells. Similar to what we observed in a phase 1 trial, ROR1 CAR-T cells infiltrated KPROR1 tumors poorly and induced limited tumor control. We showed that using immunogenic chemotherapy to induce the production of T cell-recruiting chemokines significantly improved CAR-T cell infiltration into lung tumors. However, despite improved survival, many tumor nodules remained devoid of CAR-T cells, suggesting that additional mechanisms likely limit CAR-T cell access to tumors. Neutrophils have been shown to inhibit T cell infiltration and function within tumors, and high neutrophil infiltrates in NSCLC patients are associated with poor T cell infiltration and response to immunotherapy. To characterize how neutrophils may impact CAR-T cell localization, we treated KPROR1 mice with ROR1 CAR-T cells or left untreated and analyzed the co-localization of CD8+ T cells and Ly6G+ neutrophils within tumors by multiplex IHC. In untreated mice, neutrophils were primarily found around the perimeters of large tumors (>105 mm2) but were absent from small tumors (<105 mm2). Upon treatment with CAR-Ts, CD8+ T cell frequency increased across all tumors, but intratumoral T cell infiltration was significantly higher in small neutrophil-low tumors than in large neutrophil-high tumors, suggesting that neutrophil-high tumors may be less accessible to CAR-T cells. Interestingly, CAR-T treatment induced a significant increase in neutrophils, with CD8+ T cells in closer proximity to neutrophils, suggesting that CAR-Ts may promote neutrophil recruitment. We hypothesized that this increase in neutrophils might further suppress CAR-T activity. To test this, we pre-treated tumor-bearing KPROR1 mice with anti-Gr1 or a C5aR1 inhibitor, both of which have been shown to reduce neutrophil recruitment to tumors, and injected control or ROR1 CAR-T cells 2 days later. Anti-Gr1 and C5aR1 inhibitor treatments continued for 17 days after CAR-T cell injection. Whereas treatment with CAR-T cells alone had minimal effects, co-treatment with either anti-Gr-1 or C5aR1 inhibitor significantly improved survival. Our findings suggest that neutrophils may limit CAR-T infiltration and/or persistence within tumors and that neutrophil depletion can enhance the anti-tumor activity of CAR-T cells. Future work is aimed at understanding the mechanisms by which neutrophils may limit CAR-T activity. Overall, our results suggest that targeting neutrophils may be a promising strategy to overcome the major challenges in CAR-T therapy for solid tumors. Citation Format: Sergio Ortiz-Espinosa, Shivani Srivastava. Neutrophil depletion enhances the anti-tumor activity of CAR-T cells in an autochthonous model of non-small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B025.

Taurine reduction of injury from neutrophil infiltration ameliorates Streptococcus uberis-induced mastitis

Mastitis is a common disease of dairy cows characterized by infiltration of leukocytes, especially neutrophils, resulting in increased permeability of the blood-milk barrier (BMB). Taurine, a functional nutrient, has been shown to have anti-inflammatory and antioxidant effects. Here, we investigated the regulatory effects and mechanisms of taurine on the complex immune network of the mammary gland in Streptococcus uberis (S. uberis) infection. We found that taurine had no direct effect on CXCL2-mediated neutrophil chemotaxis. However, it inhibited MAPK and NF-κB signalings by modulating the activity of TAK1 downstream of TLR2, thereby reducing CXCL2 expression in macrophages to reduce neutrophil recruitment in S. uberis infection. Further, the AMPK/Nrf2 signaling pathway was activated by taurine to help mitigate oxidative damage, apoptosis and disruption of tight junctions in mammary epithelial cells caused by hypochlorous acid, a strong oxidant produced by neutrophils, thus protecting the integrity of the mammary epithelial barrier. Taurine protects the BMB from damage caused by neutrophils via blocking the macrophage-CXCL2-neutrophil signaling axis and increasing the antioxidant capacity of mammary epithelial cells.

Thymoquinone attenuates inflammation in C. Albicans keratitis by activating Nrf2/HO-1 signaling pathway and reducing fungal load

PurposeThis study aims to investigate the anti-inflammatory and antifungal properties of thymoquinone (TQ) and elucidate its mechanism of action in the context of C. albicans keratitis. MethodsVarious methods were employed to identify a safe and effective concentration of TQ with antifungal properties, including the determination of the minimum inhibitory concentration (MIC), the cell counting kit-8 (CCK-8) test, and the Draize experiment. The severity of fungal keratitis (FK) was assessed through clinical ratings and slit-lamp imaging. Fungus burden was determined using plate counting and periodic acid Schiff (PAS) staining. Neutrophil infiltration and activity were investigated through immunofluorescence staining (IFS), myeloperoxidase (MPO) analysis, and hematoxylin and eosin (HE) staining. To explore the anti-inflammatory effects of TQ and its mechanism of action, we employed RT-PCR, ELISA, and western blot techniques. ResultsTQ effectively controlled fungal growth at a concentration of 50 µg/mL while preserving the integrity of mouse corneas. Human corneal epithelial cells (HCECs) remained unaffected by TQ at concentrations ≤ 3.75 µg/mL. Treatment with TQ led to significant improvements in clinical scores, fungal burden, neutrophil infiltration, and the expression of inflammatory factors compared to the DMSO group. Moreover, TQ demonstrated the ability to reduce the levels of inflammatory factors in HCECs stimulated by C. albicans. Additionally, TQ enhanced the expressions of Nrf2 and HO-1 in mouse corneas. The downregulation of cytokines induced by TQ was reversed upon pretreatment with inhibitors of Nrf2 or HO-1. ConclusionTQ exhibits a protective effect in the context of C. albicans keratitis through multiple mechanisms, including inhibition of C. albicans growth, reduction of neutrophil recruitment, activation of the Nrf2/HO-1 pathway, and limitation of the expression of pro-inflammatory factors.

Knockdown of CXCL1 improves ACLF by reducing neutrophil recruitment to attenuate ROS production and hepatocyte apoptosis.

Acute-on-chronic liver failure (ACLF) is an acute decompensated syndrome based on chronic liver disease, while neutrophil recruitment is the most critical early step. C-X-C motif chemokine ligand 1 (CXCL1), a cytokine that recruits neutrophils, was significantly upregulated in both ACLF mice and patients with ACLF. This present study aims to explore the role of CXCL1 in the pathogenesis of ACLF. We established an ACLF mouse model induced by carbon tetrachloride, lipopolysaccharide, and D-galactosamine, and used adeno-associated virus to achieve overexpression and knockdown of Cxcl1. We employed mass cytometry, flow cytometry, multiplex cytokine and chemokine analysis, Western blot, and reactive oxygen species (ROS) detection in mice blood and liver. ACLF patients (n = 10) and healthy controls (n = 5) were included, and their liver samples were stained using multiplex immunohistochemistry techniques. CXCL1 was significantly elevated in both ACLF mice and patients. CXCL1 recruits neutrophils by binding to the C-X-C motif chemokine receptor 2 on the surface of neutrophils, affects ACLF prognosis by generating ROS and mitochondrial depolarization and modulating caspase3-related apoptotic pathways. We found that the knockdown of CXCL1 attenuated the infiltration of neutrophils in the mouse liver, reduced the expression of inflammatory cytokines, and also significantly downregulated ROS production and caspase3-related hepatocyte apoptosis, thereby ameliorating the liver injury of ACLF. CXCL1 is a core player in the mobilization of neutrophils in ACLF, and the knockdown of Cxcl1 improves neutrophil infiltration, reduces ROS levels, and reduces hepatocyte apoptosis, thereby attenuating inflammation and liver injury in ACLF. Our results revealed a previously unknown link between CXCL1-induced neutrophil recruitment and ACLF, providing evidencing for potential therapies targeting ACLF.

NLRP3 is essential for neutrophil polarization and chemotaxis in response to leukotriene B4 gradient

Neutrophil recruitment to sites of infection and inflammation is an essential process in the early innate immune response. Upon activation, a subset of neutrophils rapidly assembles the multiprotein complex known as the NLRP3 inflammasome. The NLRP3 inflammasome forms at the microtubule organizing center, which promotes the formation of interleukin (IL)-1β and IL-18, essential cytokines in the immune response. We recently showed that mice deficient in NLRP3 (NLRP3-/-) have reduced neutrophil recruitment to the peritoneum in a model of thioglycolate-induced peritonitis. Here, we tested the hypothesis that this diminished recruitment could be, in part, the result of defects in neutrophil chemotaxis. We find that NLRP3-/- neutrophils show loss of cell polarization, as well as reduced directionality and velocity of migration toward increasing concentrations of leukotriene B4 (LTB4) in a chemotaxis assay in vitro, which was confirmed through intravital microscopy of neutrophil migration toward a laser-induced burn injury of the liver. Furthermore, pharmacologically blocking NLRP3 inflammasome assembly with MCC950 in vitro reduced directionality but preserved nondirectional movement, indicating that inflammasome assembly is specifically required for polarization and directional chemotaxis, but not cell motility per se. In support of this, pharmacological breakdown of the microtubule cytoskeleton via nocodazole treatment induced cell polarization and restored nondirectional cell migration in NLRP3-deficient neutrophils in the LTB4 gradient. Therefore, NLRP3 inflammasome assembly is required for establishment of cell polarity to guide the directional chemotactic migration of neutrophils.

HRG inhibits liver cancer lung metastasis by suppressing neutrophil extracellular trap formation.

Distant metastasis is a sign of poor prognosis for cancer patients. Extrahepatic liver cancer metastases commonly spread to the lung. Remodelling of the metastatic microenvironment is essential for tumour metastasis. Neutrophil-associated metastatic microenvironment contributes to the early metastatic colonisation of cancer cells in the lung. The lung metastasis models were constructed via treated cancer cells by tail vein injection into mice. And samples of lung were harvested at the indicated time to analyze tumor growth and immune cells in the microenvironment. Tumors and lung metastasis specimens were obtained via surgical operations for research purposes. Neutrophils were obtained from peripheral blood of patients with liver cancer or healthy donors (HD). Hepatocellular carcinoma cells reduce the secretion of histidine-rich glycoprotein (HRG), regulate the recruitment and activation of neutrophils in the metastatic microenvironment and promote the production of neutrophil extracellular traps (NETs), thereby promoting liver cancer lung metastasis. HRG binds to FCγR1 on the neutrophil membrane while inhibiting PI3K and NF-κB activation, thereby reducing IL-8 secretion to reduce neutrophil recruitment. Meanwhile, HRG inhibited IL8-MAPK and NF-κB pathway activation and ROS production, resulting in reduced NETs formation. Our study reveals that liver cancer regulates neutrophil recruitment and NETs formation in the metastatic microenvironment by reducing HRG secretion, thereby promoting tumour lung metastasis. The results of this study will contribute to the development of possible strategies for treating metastases.

Effects of Catalpol from Rehmannia glutinosa Extract on Skin Flaps.

Flaps are commonly used for repairing tissues and wounds in surgery. However, various factors can cause postoperative necrosis in these flaps. Catalpol is a bioactive component in extracts from Rehmannia glutinosa , which has pharmacologic characteristics that may improve flap survival. The experiments were performed in 36 male Sprague-Dawley rats divided into three groups: control, low-dose catalpol, and high-dose catalpol. The flap survival rate, neutrophil density, microvessel density, superoxide dismutase, and malondialdehyde levels were measured; histopathologic analysis was performed 7 days after surgery. Blood flow was measured by laser Doppler flowmetry and lead oxide-gelatin angiography. The levels of vascular endothelial growth factor, toll-like receptor 4, nuclear factor-kappa B, tumor necrosis factor-α, interleukin (IL)-6, nod-like receptor 3, cysteinyl aspartate specific proteinase-1 (caspase-1), IL-1β, and IL-18 were determined by immunohistochemistry. Catalpol treatment increased flap survival, reduced neutrophil recruitment and release, decreased malondialdehyde levels, and increased superoxide dismutase levels; thus, it effectively reduced oxidative stress, up-regulated the expression of vascular endothelial growth factor, and increased microvessel density. Laser Doppler flowmetry and lead oxide-gelatin angiography showed that catalpol treatment improved angiogenesis. Immunohistochemical analyses showed that catalpol inhibited the production of inflammatory factors, such as tumor necrosis factor-α and IL-6, by down-regulating toll-like receptor 4 and nuclear factor-κB. Furthermore, catalpol reduced cell pyroptosis by inhibiting the production of nod-like receptor 3 inflammasomes, thereby down-regulating the release of IL-1β and IL-18. Catalpol can improve the rate of flap survival. The research verified that the Rehmannia extract catalpol, through angiogenesis, inflammatory response, ischemia-reperfusion injury, and pyroptosis-related pathways, effectively improved the flap survival rate, which will provide new ideas for clinical medication.

ALKBH5-mediated m 6A RNA demethylation promotes antibacterial innate defense by intrinsically driving neutrophil generation and recruitment

Abstract Neutrophil generation and effective recruitment into the site of infection are essential for innate immune defense against bacterial infection. These processes that directed by extracellular signals such as growth factors or chemokines have been extensively studied, yet the intrinsic and new mechanism for determining neutrophil generation and recruitment needs further investigation. N6-methyladenosine (m 6A) RNA modification and its demethylase alkB homolog 5 (ALKBH5) are key epigenetic regulators of immunity and inflammation, whereas their role in neutrophil generation and recruitment remains unclear. Here, using cecal ligation and puncture-induced sepsis to model systemic bacterial infection, we found that ALKBH5 is required for antibacterial innate defense by intrinsically inducing neutrophil generation and recruitment. Indeed, Alkbh5-deficient mice exhibited increased mortality, higher bacterial burden and proinflammatory cytokine production than wild-type littermates undergoing sepsis. Meanwhile, ALKBH5 depletion significantly impaired the generation of immature neutrophils in bone marrow and also reduced neutrophil recruitment into the infected site of sepsis mice. Mechanistically, ALKBH5 imprinted generation- and recruitment-promoting transcriptome signatures in both mouse and human neutrophils during bacterial infection, specially modulated the protein expression of SOCS3 and CXCR2 through altering m 6A methylation-mediated decay of their mRNAs. Together, our findings reveal a previously unknown role of ALKBH5 in intrinsically driving neutrophil generation and recruitment, indicating neutrophil m 6A RNA modification as potential target for controlling bacterial infections. Supported by grants from the National Natural Science Foundation of China (81788101, 82071793), the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2021-I2M-1-017), the Beijing Natural Science Foundation (7212069), and the Beijing Nova Program (20220484065).

Arntl deficiency in myeloid cells reduces neutrophil recruitment and delays skeletal muscle repair

After a muscle injury, a process comprising inflammation, repair, and regeneration must occur in a time-sensitive manner for skeletal muscle to be adequately repaired and regenerated. This complex process is assumed to be controlled by various myeloid cell types, including monocytes and macrophages, though the mechanism is not fully understood. Aryl hydrocarbon receptor nuclear translocator-like (Arntl or Bmal1) is a transcription factor that controls the circadian rhythm and has been implicated in regulating myeloid cell functions. In the present study, we generated myeloid cell-specific Arntl conditional knockout (cKO) mice to assess the role of Arntl expressed in myeloid cell populations during the repair process after muscle injury. Myeloid cell-specific Arntl deletion impaired muscle regeneration after cardiotoxin injection. Flow cytometric analyses revealed that, in cKO mice, the numbers of infiltrating neutrophils and Ly6Chi monocytes within the injured site were reduced on days 1 and 2, respectively, after muscle injury. Moreover, neutrophil migration and the numbers of circulating monocytes were significantly reduced in cKO mice, which suggests these effects may account, at least in part, for the impaired regeneration. These findings suggest that Arntl, expressed in the myeloid lineage regulates neutrophil and monocyte recruitment and is therefore required for skeletal muscle regeneration.

A multiscale hybrid model for exploring the effect of Resolvin D1 on macrophage polarization during acute inflammation

Dysregulated inflammation underlies various diseases. Specialized pro-resolving mediators (SPMs) like Resolvin D1 (RvD1) have been shown to resolve inflammation and halt disease progression. Macrophages, key immune cells that drive inflammation, respond to the presence of RvD1 by polarizing to an anti-inflammatory type (M2). However, RvD1’s mechanisms, roles, and utility are not fully understood.This paper introduces a gene-regulatory network (GRN) model that contains pathways for RvD1 and other SPMs and proinflammatory molecules like lipopolysaccharides. We couple this GRN model to a partial differential equation–agent-based hybrid model using a multiscale framework to simulate an acute inflammatory response with and without the presence of RvD1. We calibrate and validate the model using experimental data from two animal models.The model reproduces the dynamics of key immune components and the effects of RvD1 during acute inflammation. Our results suggest RvD1 can drive macrophage polarization through the G protein-coupled receptor 32 (GRP32) pathway. The presence of RvD1 leads to an earlier and increased M2 polarization, reduced neutrophil recruitment, and faster apoptotic neutrophil clearance.These results support a body of literature that suggests that RvD1 is a promising candidate for promoting the resolution of acute inflammation. We conclude that once calibrated and validated on human data, the model can identify critical sources of uncertainty, which could be further elucidated in biological experiments and assessed for clinical use.

Microglial IL-1RA ameliorates brain injury after ischemic stroke by inhibiting astrocytic CXCL1-mediated neutrophil recruitment and microvessel occlusion.

Acute ischemic stroke (AIS), one of the leading causes of mortality worldwide, is characterized by a rapid inflammatory cascade resulting in exacerbation of ischemic brain injury. Microglia are the first immune responders. However, the role of postischemic microglial activity in ischemic brain injury remains far from being fully understood. Here, using the transgenic mouse line CX3 CR1creER :R26iDTR to genetically ablate microglia, we showed that microglial deletion exaggerated ischemic brain injury. Associated with this worse outcome, there were increased neutrophil recruitment, microvessel blockade and blood flow stagnation in the acute phase, accompanied by transcriptional upregulation of chemokine (C-X-C motif) ligand 1 (CXCL1). Our study showed that microglial interleukin-1 receptor antagonist (IL-1RA) suppressed astrocytic CXCL1 expression induced by oxygen and glucose deprivation and inhibited neutrophil migration. Furthermore, neutralizing antibody therapy against CXCL1 or the administration of recombinant IL-1RA protein reduced brain infarct volume and improved motor coordination performance of mice after ischemic stroke. Our study suggests that microglia protect against acute ischemic brain injury by secreting IL-1RA to inhibit astrocytic CXCL1 expression, which reduces neutrophil recruitment and neutrophil-derived microvessel occlusion.

GPR55 contributes to neutrophil recruitment and mechanical pain induction after spinal cord compression in mice

Pain transmission and processing in the nervous system are modulated by various biologically active substances, including lysophospholipids, through direct and indirect actions on the somatosensory pathway. Lysophosphatidylglucoside (LysoPtdGlc) was recently identified as a structurally unique lysophospholipid that exerts biological actions via the G protein-coupled receptor GPR55. Here, we demonstrated that GPR55-knockout (KO) mice show impaired induction of mechanical pain hypersensitivity in a model of spinal cord compression (SCC) without the same change in the models of peripheral tissue inflammation and peripheral nerve injury. Among these models, only SCC recruited peripheral inflammatory cells (neutrophils, monocytes/macrophages, and CD3+ T-cells) in the spinal dorsal horn (SDH), and GPR55-KO blunted these recruitments. Neutrophils were the first cells recruited to the SDH, and their depletion suppressed the induction of SCC-induced mechanical hypersensitivity and inflammatory responses in compressed SDH. Furthermore, we found that PtdGlc was present in the SDH and that intrathecal administration of an inhibitor of secretory phospholipase A2 (an enzyme required for producing LysoPtdGlc from PtdGlc) reduced neutrophil recruitment to compressed SDH and suppressed pain induction. Finally, by screening compounds from a chemical library, we identified auranofin as a clinically used drug with an inhibitory effect on mouse and human GPR55. Systemically administered auranofin to mice with SCC effectively suppressed spinal neutrophil infiltration and pain hypersensitivity. These results suggest that GPR55 signaling contributes to the induction of inflammatory responses and chronic pain after SCC via the recruitment of neutrophils and may provide a new target for reducing pain induction after spinal cord compression, such as spinal canal stenosis.

Research Highlights

Research Highlights

Shikonin Inhibits Fin Regeneration in Zebrafish Larvae.

Shikonin is a naphthoquinone compound extracted from Chinese comfrey for treating cancer. However, there are few reports on its research on vertebrate tissue regeneration. Zebrafish is an ideal model for studying organ regeneration. In this study, we found that 3-dpf of zebrafish larvae exposed to shikonin at concentrations of 0.2, 0.3, and 0.4 mg/L showed increasingly inhibited regeneration of the tail fin. Immunohistochemical staining showed that shikonin exposure from 6 to 12 hpa increased the number of apoptotic cells in the caudal fin wound of larvae and decreased the number of proliferating cells. Shikonin exposure was found to up-regulate oxidative stress, increase ROS levels, and reduce neutrophil recruitment in the early stage of wound repair. Moreover, shikonin exposure caused disordered expression of fin regeneration blastemal-related genes. The use of astaxanthin to down-regulate oxidative stress was found to significantly reduce the inhibition of caudal fin regeneration. Mixed exposure of AMPK inhibitors or fullerenes (C60) with shikonin also showed the similar rescue effect. Collectively, our study showed that shikonin inhibited fin regeneration in zebrafish larvae by the upregulation of oxidative stress level and AMPK signaling pathway. This research provides valuable information on the mechanism of action of shikonin for its safe application.

Lymph-derived chemokines direct early neutrophil infiltration in the lymph nodes upon Staphylococcus aureus skin infection

A large number of neutrophils infiltrate the lymph node (LN) within 4 h after Staphylococcus aureus skin infection (4 h postinfection [hpi]) and prevent systemic S. aureus dissemination. It is not clear how infection in the skin can remotely and effectively recruit neutrophils to the LN. Here, we found that lymphatic vessel occlusion substantially reduced neutrophil recruitment to the LN. Lymphatic vessels effectively transported bacteria and proinflammatory chemokines (i.e., Chemokine [C-X-C motif] motif 1 [CXCL1] and CXCL2) to the LN. However, in the absence of lymph flow, S. aureus alone in the LN was insufficient to recruit neutrophils to the LN at 4 hpi. Instead, lymph flow facilitated the earliest neutrophil recruitment to the LN by delivering chemokines (i.e., CXCL1, CXCL2) from the site of infection. Lymphatic dysfunction is often found during inflammation. During oxazolone (OX)-induced skin inflammation, CXCL1/2 in the LN was reduced after infection. The interrupted LN conduits further disrupted the flow of lymph and impeded its communication with high endothelial venules (HEVs), resulting in impaired neutrophil migration. The impaired neutrophil interaction with bacteria contributed to persistent infection in the LN. Our studies showed that both the flow of lymph from lymphatic vessels to the LN and the distribution of lymph in the LN are critical to ensure optimal neutrophil migration and timely innate immune protection in S. aureus infection.