Promotes Macrophage Recruitment Research Articles

SRGN promotes macrophage recruitment through CCL3 in osteoarthritis

ABSTRACT Background Osteoarthritis (OA) is a degenerative disease that affects synovial joints and leads to significant pain and disability, particularly in older adults. Infiltration of macrophages plays a key role in the progression of OA. However, the mechanisms underlying macrophage recruitment in OA are not fully understood. Methods The Serglycin (SRGN) expression pattern was analyzed, along with its association with macrophage infiltration in OA, using bioinformatic methods. SRGN expression in chondrocytes was altered by small interfering RNA (siRNA) and plasmids. Conditioned media (CM) was obtained from transfected chondrocytes to establish a co-culture model of chondrocytes and THP-1 derived macrophages. The impact of SRGN on macrophage recruitment was evaluated using a transwell assay. Furthermore, the regulatory effect of SRGN on CCL3 was validated through qPCR, WB, and ELISA experiments. Results In OA patients, the upregulation of SRGN positively correlated with K-L grade and macrophage infiltration. It was found that SRGN expression and secretion were up-regulated in OA and that it can promote macrophage migration in vitro. Further investigation showed that SRGN affects macrophage migration by regulating the expression of CCL3. Conclusion SRGN in chondrocytes plays a role in promoting the recruitment of THP-1 derived macrophages in vitro by regulating production of CCL3.

Analysis of particulate matter-induced alteration of genes and related signaling pathways in the respiratory system

Airborne particulate matter (PM) is a global environmental risk factor threatening human health and is a major cause of cardiovascular and respiratory disease-associated death. Current studies on PM exposure have been limited to large-scale cohort and epidemiological investigations, emphasizing the need for detailed individual-level studies to uncover specific differentially expressed genes and their associated signaling mechanisms. Herein, we revealed that PM exposure significantly upregulated inflammatory and immune responses, such as cytokine-mediated signaling pathways, complement system, and the activation and migration of immune cells in gene set enrichment analysis of our RNA sequencing (RNAseq) data. Remarkably, we discovered that the broad gene expression and signaling pathways mediated by macrophages were predominantly expressed in the respiratory system following PM exposure. Consistent with these observations, individual PMs, classified by aerodynamic size and origin, significantly promoted macrophage recruitment to the lungs in the mouse lung inflammation model. Additionally, we confirmed that RNAseq observations from the respiratory system were reproduced in murine bone marrow-derived macrophages and the alveolar macrophage cell line MH-S after individual PM exposure. Our findings demonstrated that PM exposure augmented broad inflammatory and immune responses in the respiratory system and suggested the reinforcement of global strategies for reducing particulate air pollution to prevent respiratory diseases and their exacerbation.

Abstract PO2-18-05: Blocking soluble TNF to Improve potency of trastuzumab deruxtecan by increasing internalization and antitumor innate immune response in a resistant HER2-positive breast cancer model

Abstract Background Trastuzumab deruxtecan (T-DXd) administration improves response for patients with HER2-positive metastatic breast cancer (HER2+ BC). Unfortunately, 50% of patients relapse after 2 years. T-DXd resistance mechanisms are being explored. For trastuzumab we have shown that mucin 4 (MUC4) expression is an independent predictor of poor response in HER2+BC patients. MUC4 is upregulated by soluble TNF (sTNF) secreted by the tumor, confers resistance to trastuzumab by hiding its epitope on the HER2, reducing its binding and decreasing anti-tumor phagocytic function. In preclinical models of de novo trastuzumab-resistant tumors, combination of a sTNF blocking agent INB03, (DN), with T-DXd decreases tumor growth compared to T-DXd alone. To disclose the underlying mechanism, we studied whether DN improved internalization of T-DXd in tumor cells and modified the innate immune response to enhance T-DXd antitumor effects in a multiple HER2-targeted therapy-resistant model. Methods Nude mice bearing HER2+MUC4+ JIMT-1 tumor, primary resistant to trastuzumab, pertuzumab and lapatinib, were treated with IgG 5 mg/kg, T-DXd 5 mg/kg (T-DXd 5), 2.5 mg/kg (T-DXd 2.5) or 1.25 mg/kg (T-DXd 1.25), DN 10 mg/kg or the combined therapies. T-DXd and IgG were administered i.v. on days 0, 7 and 14. DN was administered i.p. twice a week for 3 weeks. Tumor growth was monitored. Mitotic figures/field (mean) were analyzed in H&E tumor sections. Tumor-infiltrating macrophages were studied by flow cytometry. IFNƔ was determined in tumor extracts by ELISA. Internalization of T-DXd in JIMT-1 cells was studied in a S1 Incucyte along 18h by Fab-rhodo red labeling. Results T-DXd dose-response curves showed inhibition in tumor growth of 83%, 61% and 37% for T-DXd 5, 2.5 and 1.25 mg/kg treatment respectively compared with IgG-treated tumors. DN alone exhibited no antitumor activity. T-DXd+DN increased the antitumor effect by 10%, 33% and 97% for T-DXd dose of 5, 2.5 and 1.25 mg/kg respectively. Tumor growth inhibition of T-DXd 5 was similar to combination of T-DXd1.25+DN. Addition of DN did not have toxicity. DN increases IFNƔ production in the TME of T-DXd treated tumors vs T-DXd alone and promoted macrophage recruitment to the tumor bed and polarization to the antitumor M1-like phenotype. Histopathological analysis of the tumor showed a significant decrease in proliferation in all the combined and in the T-DXd 5mg/kg (2.8-3.7 mitotic figures/field) vs IgG (6.0 mitotic figures/field). In vitro, JIMT-1 cells treated with DN internalized 40% more T-DXd than its vehicle-treated counterparts. Conclusions Neutralizing sTNF with DN enhances T-DXd effect in a multiple HER2 targeted therapy resistant model of MUC4 expressing HER2+ BC. Combination of DN with T-DXd increases tumor response at all dose levels tested. The largest effect was seen at lower doses (T-DXd 1.25+DN), which mimicked the effect of 4 times the dose of T-DXd alone, suggesting significant synergy with DN as dose of T-DXd decreases. Combination of DN with T-DXd in MUC4 expressing HER2+ BC improves response to T-DXd alone by increasing T-DXd internalization and improving anti-tumor innate immune responses in the TME without increasing toxicity. The results suggest this combination should be investigated in clinical trials in patients who have MUC4 expressing tumors when T-DXd is started or become resistant to T-DXd therapy. Citation Format: Sofia Bruni, Florencia Mauro, Sofia Naveiro, Rosalia Cordo-Russo, Agustina Dupont, Mercogliano María Florencia, Roxana Schillaci. Blocking soluble TNF to Improve potency of trastuzumab deruxtecan by increasing internalization and antitumor innate immune response in a resistant HER2-positive breast cancer model [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-18-05.

Bone morphogenetic protein 9 is a candidate prognostic biomarker and host-directed therapy target for sepsis.

Defining next-generation immune therapeutics for the treatment of sepsis will involve biomarker-based therapeutic decision-making. Bone morphogenetic protein 9 (BMP9) is a cytokine in the transforming growth factor-β superfamily. Here, circulating BMP9 concentrations were quantified in two independent cohorts of patients with sepsis. Decreased concentrations of serum BMP9 were observed in the patients with sepsis at the time of admission as compared with healthy controls. Concentrations of BMP9 at the time of admission were also associated with 28-day mortality, because patients with sepsis at a higher risk of death had lower BMP9 concentrations. The mechanism driving the contribution of BMP9 to host immunity was further investigated using in vivo murine sepsis models and in vitro cell models. We found that BMP9 treatment improved outcome in mice with experimental sepsis. BMP9-treated mice exhibited increased macrophage influx into the peritoneal cavity and more efficient bacterial clearance than untreated mice. In vitro, BMP9 promoted macrophage recruitment, phagocytosis, and subsequent bacterial killing. We further found that deletion of the type 1 BMP receptor ALK1 in macrophages abolished BMP9-mediated protection against polymicrobial sepsis in vivo. Further experiments indicated that the regulation of macrophage activation by the BMP9-ALK1 axis was mainly mediated through the suppressor of mother against decapentaplegic 1/5 signaling pathway. Together, these results suggest that BMP9 can both serve as a biomarker for patient stratification with an independent prognostic value and be developed as a host-directed therapy for sepsis.

Sphingosine Kinase 1 Aggravates Liver Fibrosis by Mediating Macrophage Recruitment and Polarization

Sphingosine kinase 1 (SphK1) has distinct roles in the activation of Kupffer cells and hepatic stellate cells in liver fibrosis. Here, we aim to investigate the roles of SphK1 on hepatic macrophage recruitment and polarization in liver fibrosis. Liver fibrosis was induced by carbon tetrachloride in wild-type and SphK1-/- mice to study the recruitment and polarization of macrophages. The effects of SphK1 originated from macrophages or other liver cell types on liver fibrosis were further strengthened by bone marrow transplantation. The direct effects of SphK1 on macrophage polarization were also investigated invitro. Expression analysis of SphK1 and macrophage polarization index was conducted with human liver samples. SphK1 deletion attenuated the recruitment of hepatic macrophages along with reduced M1 and M2 polarization in mice induced by carbon tetrachloride. SphK1 deficiency in endogenous liver cells attenuated macrophage recruitment via C-C motif chemokine ligand 2. Macrophage SphK1 activated the ASK1-JNK1/2-p38 signaling pathway to promote M1 polarization. Furthermore, macrophage SphK1 downregulated small ubiquitin-like modifier-specific peptidase1 to decrease de-SUMOylation of Kruppel-like factor 4 to promote M2 polarization. Finally, we confirmed that SphK1 expression was elevated and positively correlated with macrophage M1 and M2 polarization in human fibrosis livers. Our findings demonstrated that SphK1 aggravated liver fibrosis by promoting macrophage recruitment and M1/M2 polarization. SphK1 in macrophages is a potential therapeutic target for the treatment of liver fibrosis.

RSK4 promotes the macrophage recruitment and M2 polarization in esophageal squamous cell carcinoma

High infiltration of tumor-associated macrophages (TAMs) participates in host immunity and tumor progression in patients with esophageal squamous cell carcinoma (ESCC). Ribosomal s6 kinase 4 (RSK4) has been shown to be aberrantly overexpressed in ESCC. The role of RSK4 in cytokine secretion and its impact on macrophage recruitment and M2 polarization remains unclear. Therefore, a thorough understanding of RSK4 is needed to expand our knowledge of its therapeutic potential. Herein, RSK4 expression in human ESCC tissues and a xenograft mouse model was positively correlated with high infiltration of M0 and M2 macrophages which is positively associated with unfavorable overall survival outcomes and treatment resistance in patients with ESCC. In vitro experiments revealed that RSK4 derived from ESCC cells promoted macrophage recruitment and M2 polarization by enhancingsoluble intercellular adhesion molecule-1 (sICAM-1) secretion via direct and indirect STAT3 phosphorylation. Furthermore, RSK4-induced macrophages enhanced tumor proliferation, migration, and invasion by secreting C-C motif chemokine ligand 22 (CCL22). We further showed that patients with elevated CD68 and CD206 expression had unfavorable overall survival. Collectively, these results demonstrate that RSK4 promotes the macrophage recruitment and M2 polarization by regulating the STAT3/ICAM-1 axis in ESCC, influencing tumor progression primarily in a CCL22-dependent manner. These data also offer valuable insights for developing novel agents for the treatment of ESCC.

Extracellular ATP (eATP) inhibits the progression of endometriosis and enhances the immune function of macrophages

BackgroundExtracellular adenosine triphosphate (eATP) is an important inflammatory mediator that can boost the antitumour immune response, but its role in endometriosis remains unknown. We hypothesized that eATP could inhibit endometriosis cell function both directly and indirectly through macrophages. MethodsPeritoneal and cyst fluid from endometriosis patients and non-endometriosis controls was collected to measure eATP levels. The addition of eATP was performed to explore its effects on endometriotic cell and macrophage functions, including cell proliferation, apoptosis, pyroptosis, mitochondrial membrane potential, phagocytosis, and the production of inflammatory cytokines and reactive oxygen species. A coculture of endometriotic epithelial cells and U937 macrophages was established, followed by P2X7 antagonist and eATP treatment. Endometriosis model eATP-treated rats were used to evaluate in situ cell death and macrophage marker expression. ResultsThe pelvic microenvironment of endometriosis patients shows high eATP levels, which could induce endometriotic epithelial cell apoptosis and pyroptosis and significantly inhibit cell growth via the MAPK/JNK/Akt pathway. eATP treatment ameliorated endometriosis-related macrophage dysfunction and promoted macrophage recruitment. eATP treatment in the presence of macrophages exerted a stronger cytotoxic effect on endometriotic epithelial cells by regulating P2X7. eATP treatment effectively induced cell death in an endometriosis rat model and prominently increased the macrophage number without affecting the eutopic endometrium. ConclusioneATP induces endometriotic epithelial cell death and enhances the immune function of macrophages to inhibit the progression of endometriosis, while eutopic endometrium is not affected. eATP treatment may serve as a nonhormonal therapeutic strategy for endometriosis.

A Crosstalk Between Castration-Resistant Prostate Cancer Cells, M2 Macrophages, and NK Cells: Role of the ATM-PI3K/AKT-PD-L1 Pathway

ABSTRACT Castration-resistant prostate cancer (CRPC) in males is associated with a poor prognosis and a higher risk of treatment-related adverse effects, with high mortality among cancers globally. It is thus imperative to explore novel potential molecules with dual therapeutic and biomarker functions. Based on the recent research findings, the expression levels of ataxia telangiectasia mutant kinase (ATM) in prostate cancer (PC) tissues collected from CRPC patients were higher than hormone-dependent PC patients. Using CRPC cell lines (C4–2 and CWR22Rv1), the transwell chamber experiments revealed ATM promoted macrophage recruitment in CRPC cells in vitro via C-X-C motif chemokine ligand 12 (CXCL12). Further in vitro investigations demonstrated that polarized macrophages prevented NK cell recruitment and reduced the immunocidal activity of NK cells against CRPC cell lines. Moreover, ATM boosted programmed death receptor ligand 1 (PD-L1) expression while inhibiting NK group 2D (NKG2D) ligand expression in selected cell lines via PI3K/AKT signaling pathway. The in vivo investigations revealed ATM induced proliferation of CRPC and macrophage recruitment, while the NK cell recruitment was found to suppress ATM expression and CRPC proliferation. In conclusion, it could be demonstrated that inhibiting ATM increased the susceptibility of CRPC to NK cell inhibitors by dampening the CXCL12 and PI3K/AKT-PD-L1 pathways, thereby offering a novel and individualized treatment protocol for treating CRPC.

Preparation of a Dual-Functional Sulfated Galactofucan Polysaccharide/Poly(vinyl alcohol) Hydrogel to Promote Macrophage Recruitment and Angiogenic Potential in Diabetic Wound Healing.

A diabetic foot ulcer is a common high-risk complication in diabetic patients, but there is still no universal dressing for clinical treatment. In this study, a novel dual-functional sulfated galactofucan polysaccharide/poly(vinyl alcohol) hydrogel (DPH20) is developed during freeze-thaw cycles. Experimental results indicated that DPH20 had a high specific surface area, a dense porous structure, and a good swelling property, which could effectively adsorb the exudates and keep the wound moist. Furthermore, DPH20 exhibited remarkably recruited macrophage capability and accelerated the inflammation stage by improving the expression of the mRNA of CCL2, CCR2, and CCL22 in macrophages. DPH20 could promote cell migration and growth factor release to accelerate tube formation under hyperglycemic conditions in cell models of L929s and HUEVCs, respectively. Significantly, DPH20 accelerates the reconstruction of the full-thickness skin wound by accelerating the recruitment of macrophages, promoting angiogenesis, and releasing the growth factor in the diabetic mouse model. Collectively, DPH20 is a promising multifunctional dressing to reshape the damaged tissue environment and accelerate wound healing. This study provides an efficient strategy to repair and regenerate diabetic skin ulcers.

Protective role of the novel cytokine Metrnl/ interleukin-41 in host immunity defense during sepsis by promoting macrophage recruitment and modulating Treg/Th17 immune cell balance

BackgroundMetrnl play an immunocytokine-like role in several diseases, which is also known as meteorin-like because it is homologous to the neurotrophic factor meteorin (Metrn). Although the expression and function of Metrnl, including neurotrophic, immunomodulatory, and insulin resistance functions in different tissues have been extensively studied, its role in sepsis has remained largely limited. MethodsThe present work analyzed the levels of Metrnl and cytokines in the circulation, such as tumor necrosis factor (TNF-α), interleukin (IL-1)β, IL-6, IL-8, together with IL-10 among septic adult patients. Clinical information was obtained from such patients, including sofa score, procalcitonin(PCT)count, and C-reactive count (CRP) within 24 h when entering the intensive care unit (ICU). We constructed a sepsis model in Metrnl-deficient or normal wild-type mice using cecal ligation and perforation to study its functions in bacterial burden, survival, cytokine/chemokine generation, peritoneal lavage fluid neutrophils, macrophage and lymphocyte recruitment, and Treg/Th17 immune cell balance after CLP-induced sepsis. ResultsThe expression of Metrnl was remarkably elevated in the early phase of sepsis clinically. Its serum content in patients dying of sepsis slightly decreased relative to that in survivors. Furthermore, the concentration of Metrnl in septic cases when entering the ICU independently predicted the 28-day mortality. For septic patients who had low serum Metrnl content (≤ 274.40 pg/mL), the death risk increased by 2.3 folds relative to those who had a high serum content. It is reported that Metrnl is probably insufficient among patients dying of sepsis. Additionally, the content of Metrnl in the serum of septic patients when entering the ICU is markedly and negatively related to the levels of TNF-α, IL-1β, IL-6, IL-8, IL-17, PCT, and Sofa score. Collectively, Metrnl could be a potential therapeutic target for sepsis. A low-lethality non-severe sepsis (NSS) model was constructed, which suggested that Metrnl insufficiency elevated the death rate and reduced bacterial clearance during sepsis. For Metrnl-deficient mice, impaired sepsis immunity defense might be related to decreased macrophage recruitment and Treg/Th17 lymphocyte imbalance. Recombinant Metrnl administered to Metrnl-deficient mice abolished the immunity defense impairment following NSS while protecting the high-lethality severe sepsis (SS) model in wild-type (WT) mice. In addition, Metrnl-induced sepsis prevention was intricately associated with the increased recruitment of peritoneal macrophages and modulation of the Treg/TH17 immune cell balance. Furthermore, CCL3 exposure in Metrnl-deficient mice reduced peritoneal bacterial loads while improving survival during sepsis partially by promoting the recruitment of peritoneal macrophages. Furthermore, Metrnl regulated the polarization of M1 macrophages through the ROS signaling pathway and promoted macrophage phagocytosis, thereby killing Escherichia coli. ConclusionsThe present proof-of-concept work suggests that Metrnl-mediated recruitment of macrophages significantly affects sepsis defense in the host and modulates the Treg/Th17 immune cell balance. Findings in this work shed more light on the development of host-directed treatments that can be used to manipulate host immunity to treat sepsis.

Abstract 763: Minimally invasive colorectal resection is associated with significantly elevated plasma levels of extracellular cold inducible RNA binding protein for a month after colorectal resection & may promote the growth of residual metastases

Abstract Introduction: Cold-Inducible RNA-Binding Protein (CIRP) is similar to a class of stress-induced RNA binding proteins and is mostly found in the nucleus where it regulates transcription. In response to stress, CIRP moves to the cytoplasm where it regulates translation; a fraction of the cytoplasmic CIRP is released extracellularly (eCIRP). eCIRP is a damage-associated molecular pattern (DAMP) molecule that promotes inflammatory responses including TNF-alpha, IL-6, and HMGB production. Lymphocytes, macrophages, and PMN’s are activated after surgery and can generate eCIRP. Elevated eCIRP blood levels may impact macrophage function and induce immune tolerance that may promote tumor growth and progression. This study’s purpose was to measure plasma eCIRP levels before and for a month after MICR for CRC. Methods: CRC patients (pts) in an IRB approved data/tissue bank who underwent MICR for whom plasma was available were selected. Demographic, clinical and pathology data were reviewed. Plasma samples were collected preoperatively (preOp) and at 6 post-operative (Postop) time points (POD 1, 3, 7-13, 14-20, 21-27, 28-41); samples were processed and frozen until used. Late samples were bundled into 7-day blocks and considered as single time points. eCIRP levels were analyzed in duplicate using ELISA. The Wilcoxon paired t-test was used for analysis. Results: 83 CRC pts who underwent MICR were studied (colon 66%, rectal 34%; mean age 65); 26% had Hand assisted (HA) MIS (mean incision length[IL] 10.0±2.9cm) and 69% laparoscopic (LAP, mean IL 6.9±4.1cm) MICR. The cancer stage breakdown was; I, 30%; II, 32%; III, 35% and IV, 3%. The mean PreOp eCIRP level was 896.8± 757.0pg/ml; vs. preop levels increased mean plasma levels (p=<0.001) were noted on POD1 (2549± 2632pg/ml, n= 83), POD3 (1871± 1362pg/ml, n= 77), POD7-13 (1788± 1403pg/ml, n=57), POD14-20 (1473± 738.8pg/ml, n=30), and POD 21-27 (1681± 1375pg/ml, n= 21). With regard to MIS method, greater eCIRP increases were noted in the HA vs the Lap group from POD1-POD20 (p=0.01-0.04). Conclusions: Plasma eCIRP levels are elevated for a month after MICR for CRC (% change range 100-184) and peaked on POD1. The cause of the early increase may be the acute inflammatory response whereas the late elevations may be related to wound healing-related tissue remodeling. Higher mean levels in the HA vs Lap group suggests that the extent of surgical trauma impacts eCIRP levels postop. Previous studies noted increased post-MICR plasma levels of MCP-1, CHI3L1 and IL8 that stimulate macrophage activity. eCIRP, together with those proteins may promote macrophage recruitment and function and might promote the growth of residual tumor deposits postop. Further studies are warranted. Citation Format: Chandana S. K. Herath Mudiyanselage, Xiaohong Yan, Neil Mitra, Hansani N. Angammana, Doaa F. Morrar, Vincent Chia, Yanni Hedjar, Vesna Cekic, Jennifer L. Agnew, Richard L. Whelan. Minimally invasive colorectal resection is associated with significantly elevated plasma levels of extracellular cold inducible RNA binding protein for a month after colorectal resection & may promote the growth of residual metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 763.

WTAP promotes macrophage recruitment and increases VEGF secretion via N6-methyladenosine modification in corneal neovascularization

BackgroundCorneal neovascularization (CNV) can be caused by chemical burns. Macrophages are involved in angiogenesis and lymphangiogenesis during CNV. The aim of this study was to investigate whether Wilms' tumor 1-associated protein (WTAP) is involved in macrophage recruitment and VEGF secretion via N6-methyladenosine (m6A) modification. MethodsA CNV mouse model was established by corneal alkali burn. Tumor necrosis factor alpha (TNF-α) was used to stimulate vascular endothelial cells. m6A immunoprecipitation qPCR was used to determine the enrichment of m6A levels in mRNAs. The H3K9me3 enrichment in the promoter region of CC motif chemokine ligand 2 (CCL2) was detected by chromatin immunoprecipitation assay. The WTAP inhibition in vivo was performed using the adeno-associated virus. ResultsIn the alkali burn corneal tissues, angiogenesis and lymphangiogenesis were promoted as CD31 and LYVE-1 expressions were elevated, and the number of macrophages as well as WTAP expression were increased. Under the TNF-α stimulation, WTAP promoted the recruitment of endothelial cells to macrophages by promoting CCL2 secretion. Mechanistically, WTAP affected the enrichment of H3K9me3 at the CCL2 promoter by regulating the m6A level of SUV39H1 mRNA. The in vivo experiment showed that VEGFA/C/D secretion of macrophages was reduced after WTAP interference. Mechanistically, WTAP regulated the translational efficiency of HIF-1α via m6A modification. ConclusionWTAP affected macrophage recruitment to endothelial cells via regulation of H3K9me3-mediated CCL2 transcription. WTAP also affected macrophage secretion of VEGFA/C/D via m6A-mediated translation regulation of HIF-1α. Both pathways were involved in the WTAP regulation of angiogenesis and lymphangiogenesis during CNV.

Delay modulates the immune response to nerve repair

Effective regeneration after peripheral nerve injury requires macrophage recruitment. We investigated the activation of remodeling pathways within the macrophage population when repair is delayed and identified alteration of key upstream regulators of the inflammatory response. We then targeted one of these regulators, using exogenous IL10 to manipulate the response to injury at the repair site. We demonstrate that this approach alters macrophage polarization, promotes macrophage recruitment, axon extension, neuromuscular junction formation, and increases the number of regenerating motor units reaching their target. We also demonstrate that this approach can rescue the effects of delayed nerve graft.

Mitoxantrone targets both host and bacteria to overcome vancomycin resistance in Enterococcus faecalis.

Antibiotic resistance critically limits treatment options for infection caused by opportunistic pathogens such as enterococci. Here, we investigate the antibiotic and immunological activity of the anticancer agent mitoxantrone (MTX) in vitro and in vivo against vancomycin-resistant Enterococcus faecalis (VRE). We show that, in vitro, MTX is a potent antibiotic against Gram-positive bacteria through induction of reactive oxygen species and DNA damage. MTX also synergizes with vancomycin against VRE, rendering the resistant strains more permeable to MTX. In a murine wound infection model, single-dose MTX treatment effectively reduces VRE numbers, with further reduction when combined with vancomycin. Multiple MTX treatments accelerate wound closure. MTX also promotes macrophage recruitment and proinflammatory cytokine induction at the wound site and augments intracellular bacterial killing in macrophages by up-regulating the expression of lysosomal enzymes. These results show that MTX represents a promising bacterium- and host-targeted therapeutic for overcoming vancomycin resistance.

DNA damage repair kinase DNA‐PK and cGAS synergize to induce cancer‐related inflammation in glioblastoma

Cytosolic DNA promotes inflammatory responses upon detection by the cyclic GMP‐AMP (cGAMP) synthase (cGAS). It has been suggested that cGAS downregulation is an immune escape strategy harnessed by tumor cells. Here, we used glioblastoma cells that show undetectable cGAS levels to address if alternative DNA detection pathways can promote pro‐inflammatory signaling. We show that the DNA‐PK DNA repair complex (i) drives cGAS‐independent IRF3‐mediated type I Interferon responses and (ii) that its catalytic activity is required for cGAS‐dependent cGAMP production and optimal downstream signaling. We further show that the cooperation between DNA‐PK and cGAS favors the expression of chemokines that promote macrophage recruitment in the tumor microenvironment in a glioblastoma model, a process that impairs early tumorigenesis but correlates with poor outcome in glioblastoma patients. Thus, our study supports that cGAS‐dependent signaling is acquired during tumorigenesis and that cGAS and DNA‐PK activities should be analyzed concertedly to predict the impact of strategies aiming to boost tumor immunogenicity.

PGE2 promotes macrophage recruitment and neovascularization in murine wet-type AMD models

Age-related macular degeneration (AMD), a progressive chronic disease of the central retina, is a leading cause of blindness worldwide. Activated macrophages recruited to the injured eyes greatly contribute to the pathogenesis of choroidal neovascularization (CNV) in exudative AMD (wet AMD). This study describes the effects of cyclooxygenase-2 (COX2)/prostaglandin E2 (PGE2) signalling on the macrophage activation and CNV formation of wet AMD. In a mouse model of laser-induced wet AMD, the mice received an intravitreal injection of celecoxib (a selective COX2 inhibitor). Optical coherence tomography (OCT), fundus fluorescein angiography (FFA), choroidal histology of the CNV lesions, and biochemical markers were assessed. The level of PGE2 expression was high in the laser-induced CNV lesions. Macrophage recruitment and CNV development were significantly less after celecoxib treatment. E-prostanoid1 receptor (EP1R)/protein kinase C (PKC) signalling was involved in M2 macrophage activation and interleukin-10 (IL-10) production of bone marrow-derived macrophages (BMDMs) in vitro. In addition, IL-10 was found to induce the proliferation and migration of human choroidal microvascular endothelial cells (HCECs). Thus, the PGE2/EP1R signalling network serves as a potential therapeutic target for CNV of the wet-type AMD.Graphical AXv86vuL_n_YsWmKFp4z_2Video abstract

AIF1 + CSF1R + MSCs, induced by TNF-α, act to generate an inflammatory microenvironment and promote hepatocarcinogenesis.

Increasing evidence suggests that mesenchymal stem cells (MSCs) home to injured local tissues and the tumor microenvironment in the liver. Chronic inflammation is regarded as the major trait of primary liver cancer. However, the characteristics of endogenous MSCs in the inflammatory environment and their role in the occurrence of liver cancer remain obscure. Using single-cell RNA sequencing, we identified a distinct inflammation-associated subset of MSCs, namely AIF1 + CSF1R + MSCs, which existed in the microenvironment before the occurrence of liver cancer. Furthermore, we found that this MSC subgroup is likely to be induced by TNF-α stimulation through the TNFR1/SIRT1 (sirtuin 1) pathway. In a rat primary liver cancer model, we showed that MSCs with high SIRT1 expression (Ad-Sirt1-MSCs) promoted macrophage recruitment and synergistically facilitated liver cancer occurrence by secreting C-C motif chemokine ligand (CCL) 5. Interestingly, depletion of macrophages or knockdown of CCL5 expression in Ad-Sirt1-MSCs attenuated the promotive effect of Ad-Sirt1-MSCs on liver inflammation and hepatocarcinogenesis (HCG). Finally, we demonstrated that SIRT1 up-regulated CCL5 expression through activation of the AKT/HIF1α signaling axis in MSCs. Together, our results show that MSCs, which are mobilized to the injured site, can be educated by macrophages. In turn, the educated MSCs are involved in generating a chronic inflammatory microenvironment and promoting HCG.

Hyaluronic acids mediate the infiltration, migration, and M2 polarization of macrophages: evaluating metabolic molecular phenotypes in gliomas.

Gliomas cause high mortality around the world. The metabolic pattern of the tumor was previously suggested to be associated with the patient's survival outcome and immune activity. Yet, this relationship in glioma remains unknown. This study systematically evaluated the immune landscape in different phenotypes classified by metabolic-related pathways of 3068 glioma samples and 33 glioblastoma single-cell sequencing samples. Machine learning prediction analysis of microarray with R (pamr) was used for validating clustering results. A total of 5842 pan-cancer samples were used for external validation of the metabolic clusters. Cell Counting Kit-8 (CCK8) assay, cell clone assay, EdU assay, wound healing assay, Transwell assay, and co-culture assay were performed to verify the distinction in molecular characteristics among metabolic clusters. Metabolomics and RNA sequencing were performed on HS683 and U251 cells to annotate potential hyaluronic acid (HA)-mediated pathways. Three distinct metabolic phenotypes were identified. Metabolic cluster 1 correlated with a high number of immune infiltrating cells and poor survival of glioma patients. Metabolic clusters were proved with different levels of the macrophage markers CD68 and CD163 by multiplex immunofluorescence staining. Glioma cells from other metabolic clusters also expressed various levels of HA. HA was further found to mediate glioma proliferation, progression, and invasion. Moreover, HA potentially promoted macrophage recruitment and M2 polarization through the IL-1/CHI3L1 and TGF-b/CHI3L1 axes. HA also regulated the expression of PD-L1. This work revealed the significant connection between metabolic patterns, especially HA, and tumor immune infiltration in gliomas.

CC chemokine receptor 7 promotes macrophage recruitment and induces M2-polarization through CC chemokine ligand 19&21 in oral squamous cell carcinoma

PurposeThis study aimed to investigate the impact of CC chemokine receptor 7 (CCR7) on the recruitment and polarization of tumor-associated macrophages (TAMs) in oral squamous cell carcinoma (OSCC).MethodsWe analyzed CCR7 expression pattern, clinicopathological significance, and its association with M2 macrophage infiltration in OSCC by bioinformatic methods. Small interfering RNA (siRNA) was utilized to silence CCR7 in OSCC cells. Conditioned media (CM) was harvested from transfected OSCC cells to establish a co-culture model of THP-1 derived macrophages and OSCC cells. Transwell assay and cell adhesion assay were performed to examine the effect of CCR7 on macrophages recruitment and adhesion. Cytoskeleton was labelled by phalloidin to observe macrophage morphological changes. Moreover, phenotypic alteration of macrophages was measured using quantitative real-time PCR (qRT-PCR), flow cytometry, and immunofluorescence (IF) staining. Ultimately, recombinant human CCL19 and CCL21 were added into the medium of THP-1 derived macrophages to explore their effects on polarization in vitro.ResultsIn OSCC patients, the overexpression of CCR7 positively correlated with lymph node metastasis and M2 macrophage infiltration. Macrophage not only exhibited enhanced migration, invasion and adhesion abilities, but also appeared more spindle and branched in vitro when treated with CM from OSCC cells. However, these phenomena were abrogated with knockdown of CCR7. We also discovered that inhibition of CCR7 in OSCC cells suppressed TAMs polarization to an M2 phenotype. In addition, recombinant human CCL19 and CCL21 promoted macrophage M2-polarization in vitro.ConclusionCCR7 in OSCC cells promoted recruitment and M2-polarization of THP-1 derived macrophages in vitro by regulating production of CCL19 and CCL21.

Substitution of the SERCA2 Cys674 reactive thiol accelerates atherosclerosis by inducing endoplasmic reticulum stress and inflammation.

The cysteine674 (C674) thiol of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 is easily and irreversibly oxidized under atherosclerotic conditions. However, the contribution of the C674 thiol redox status in the development of atherosclerosis remains unclear. Our goal was to elucidate the possible mechanism involved. Heterozygous SERCA2 C674S knock-in mice in which half of the C674 was substituted by serine (S674) were used to mimic the removal of the reactive C674 thiol, which occurs under pathological conditions. Bone marrow-derived macrophages (BMDMs) and cardiac endothelial cells (ECs) were used for intracellular Ca2+ , macrophage adhesion, and protein expression analysis. The whole aorta and aortic root were isolated for histological analysis. Cell culture studies suggest the partial substitution of SERCA2 C674 increased intracellular Ca2+ levels and induced ER stress in both BMDMs and ECs. The release of proinflammatory factors and macrophage adhesion increased in SKI BMDMs. In ECs, overexpression of S674 induced endothelial inflammation and promoted macrophage recruitment. SKI mice developed more severe atherosclerotic plaque and macrophage accumulation. Additionally, 4-phenyl butyric acid, an ER stress inhibitor, suppressed ER stress and inflammatory responses in BMDMs and ECs, and alleviated atherosclerosis in SKI mice. The substitution of SERCA2 C674 thiol accelerates the development of atherosclerosis by inducing ER stress and inflammation. Our findings highlight the importance of SERCA2 C674 redox state in the context of atherosclerosis and open up a novel therapeutic strategy to combat atherosclerosis.