Ratio Of M2 Macrophages Research Articles

NLRP3 inflammasome activation and macrophage distribution in kidney tissues from patients with acute oxalate nephropathy.

The current study was initiated to evaluate renal nucleotide-binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome pathway activation and macrophage subtype distribution and their clinicopathological significance in a cohort of oxalate-induced acute kidney injury. Twelve patients with biopsy-proven acute oxalate nephropathy (AON) from January 2016 to October 2022 were retrospectively enrolled, with estimated glomerular filtration rate (eGFR)-matched 24 patients with acute tubulointerstitial nephritis (ATIN) as disease control. Pathological lesions as well as markers of NLRP3 inflammasome pathway and macrophage phenotype detected by immunohistochemistry staining were semi-quantitatively analyzed. Oxalate depositions were found in 5% to 20% of tubules with a positive correlation with Sirius Red staining in AON specimens (rp = 0.668, p = 0.02). Disruption of tubular basement membrane and inflammatory cell reaction was more prominent in specimens of AON (both p < 0.05) as compared with ATIN specimens. The expressions of NLRP3, caspase-1, and gasdermin D were significantly increased in AON specimens as well (all p < 0.05). Patients with M1/M2 macrophage ratio <1 were found with more chronic tubulointerstitial lesions and presented with lower eGFR at the last follow-up (24.8  10.6 mL/min/1.73 m2 vs. 55.1  21.2 mL/min/1.73 m2, p = 0.02) in the AON group. The NLRP3 inflammasome pathway was activated in the kidneys of AON patients, and the ratio of M1 and M2 macrophages was associated with chronicity of pathological changes, which needs further exploration.

Exosomes derived from cord blood Treg cells promote diabetic wound healing by targeting monocytes

Chronic nonhealing diabetic wounds are a critical clinical challenge. Regulatory T cells (Tregs) are immunosuppressive modulators affecting wound healing progression by controlling the inflammatory response. The current study attempted to investigate whether the exosomes derived from cord blood (CB) Tregs can accelerate the healing process. Exosomes were isolated from CB-Treg cultures using ultracentrifugation and validated with different specific markers of exosomes. The purified CB-Treg-derived exosomes were co-cultured with peripheral blood mononuclear cells (PBMCs) and CD14+ monocytes. The migration-promoting effect of CB-Treg-derived exosomes on fibroblasts and endothelial cells was investigated. We used thermosensitive Pluronic F-127 hydrogel (PF-127) loaded with CB-Treg-derived exosomes in a diabetic wound healing mouse model. CB-Treg-derived exosomes with 30–120 nm diameters revealed exosome-specific markers, such as TSG101, Alix, and CD63. CB-Treg-derived exosomes were mainly bound to the monocytes when co-cultured with PBMCs, and promoted monocyte polarization to the anti-inflammatory phenotype (M2) in vitro. CB-Treg-derived exosomes enhanced the migration of endothelial cells and fibroblasts. Furthermore, CB-Treg-derived exosomes treatment accelerated wound healing by downregulating inflammatory factor levels and upregulating the M2 macrophage ratio in vivo. Our findings indicated that CB-Treg-derived exosomes could be a promising cell-free therapeutic strategy for diabetic wound healing, partly by targeting monocytes.

CircADAMTS6 via stabilizing CAMK2A is involved in smoking-induced emphysema through driving M2 macrophage polarization

Cigarette smoke (CS), an indoor environmental pollutant, is a prominent risk factor for emphysema, which is a pathological feature of chronic obstructive pulmonary disease (COPD). Emerging function of circRNAs in immune responses and disease progression shed new light to explore the pathogenesis of emphysema. In this research, we demonstrated, by single-cell RNA sequencing (scRNAseq), that the ratio of M2 macrophages were increased in lung tissues of humans and mice with smoking-related emphysema. Further, our data showed that circADAMTS6 was associated with cigarette smoke extract (CSE)-induced M2 macrophage polarization. Mechanistically, in macrophages, circADAMTS6 stabilized CAMK2A mRNA via forming a circADAMTS6/IGF2BP2/CAMK2A RNA-protein ternary complex to activate CREB, which drives M2 macrophage polarization and leads to emphysema. In addition, in macrophages of mouse lung tissues, downregulation of circADAMTS6 reversed M2 macrophage polarization, the proteinase/anti-proteinase imbalance, and the elastin degradation, which protecting against CS-induced emphysema. Moreover, for macrophages and in a model with co-cultured lung organoids, the target of circADAMTS6 restored the growth of lung organoids compared to CSE-treated macrophages. Our results also demonstrated that, for smokers and COPD smokers, elevation of circADAMTS6 negatively correlated with lung function. Overall, this study reveals a novel mechanism for circADAMTS6-driven M2 macrophage polarization in smoking-related emphysema and postulates that circADAMTS6 could serve as a diagnostic and therapeutic marker for smoking-related emphysema.

Mitigating Female Aging Kidney Damage from Ischemic Reperfusion Injury Using Renal Progenitor Cells

Acute kidney injury (AKI) is a sudden event that damages the kidney and disrupts normal function by various causes, such as ischemia, exposure to substances, or obstructions. With AKIs affecting 1 out of 5 hospitalized patients and their association with increased risk of chronic kidney disease (CKD), finding an effective treatment for AKIs is of vital importance. One possible treatment for AKIs could be found within the realm of progenitor cell therapy. Progenitor cells are currently used in the treatment of hematological diseases with the burgeoning information on their possible uses for other disorders. The purpose of this study was to determine whether there is a resident population of renal progenitor cells (RPCs) within the kidney and if these cells can be utilized as an effective treatment to mitigate aging AKI by decreasing inflammation through alteration of the M1:M2 macrophage ratio. This study utilized 75-78 weeks old C57BL/6J female mice to determine if RPCs can reduce the damage caused by ischemic reperfusion (IR)-induced AKI. Kidneys were harvested from young female (40 days old) mice and digested in collagenase type 1 to remove connective tissue. Cells were sorted by flow cytometry, positively selected for c-Kit using magnetic nanoparticles, and subsequently stained for Sca1 and Oct4. Aging female mice underwent renal IR injury (IRI) by clamping off the renal artery and vein for 27 minutes. Following reperfusion, the triple-positive cells were suspended in DMEM and injected into the kidney over the course of 8 minutes to reduce the possibility of pressure necrosis. A separate group of mice was injected with saline to serve as control. The mice were monitored for 3 days at which point kidney function was assessed and tissue harvested for biochemical analysis. Our results indicated an increase in inflammatory cytokines IL-17, NF-κB, and IL-6 in IRI mice which were mitigated with RPCs treatment along with an increase in anti-inflammatory marker IL-10. Furthermore, an increase in M1 macrophage markers was measured in IRI, and a trending increase in M2 macrophage markers in IRI animals treated with RPCs. The kidney function of IRI animals treated with RPC showed improvement compared to IRI control. In summary, we found a population of cells positive for three progenitor cell markers: c-Kit, Sca1, Oct4, and treating IRI aging female kidney with these cells reduced inflammatory markers along with an increase in kidney function, suggesting RPCs promote recovery from AKI by reducing inflammation. Grant Support: DK116591. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 2695: A higher percentage of M2 macrophages in non-tumor part predict the tumor recurrence in patients with hepatocellular carcinoma after surgical resection

Abstract The primary hurdle in improving outcomes for hepatocellular carcinoma (HCC) patients after curative treatment is the high early recurrence rate. The existing literature indicates the presence of type 2 macrophages (M2), renowned for their immunosuppressive capabilities, has been implicated in creating a microenvironment conducive to tumor growth and recurrence. However, the intricate interplay involving M2 macrophages and the recurrence of HCC is underexplored. Therefore, we conducted an analysis involving HCC patients who underwent hepatectomy at our hospital between 2013 and 2017. We collected samples from both the tumor and non-tumor regions and subjected them to bulk RNA sequencing for comprehensive molecular profiling. The proportion of immune cells was calculated by CIBERSORTx.In our study, we enrolled fifty patients, with sixty percent presenting with cirrhosis and seventy-three percent having viral hepatitis. Twenty-three patients experienced tumor recurrence, with a median time to recurrence of 12.6 months. First, we found no correlation between five TIMR subtypes defined by TIMELASER (Xue R, Nature, 2022) and recurrence. However, as for the proportions of immune cell, a significantly lower NK/Treg ratio (p= 0.0163) and high within the resected tumor section in recurrent group was found. On the other hand, the ratio of M2 macrophages to total macrophages (M2/M) was significantly higher within the non-tumor region in patients experiencing tumor recurrence after hepatectomy (p=0.0357). Furthermore, the expression of the ACGT2 gene in non-tumor part, which strongly correlates with Treg function, was significantly higher (p=0.0056) in recurrent group. In addition, the innate T cells score was increased in non-tumor pat in recurrent group but with borderline significance (p=0.0628). However, in multivariable analysis, only a higher M2/M ratio within the non-tumor region (HR 3.756, p=0.0072) were identified as independent risk factors for tumor recurrence. We also found that patients with a higher M2/M ratio had a lower percentage of cirrhotic patients (p=0.0049) and less association with TIME-IA (immune activation) subtype of HCC by TIMELASER classification (p=0.0030). In conclusion, a higher M2/M ratio within the non-tumor region predict the tumor recurrence in patients with hepatocellular carcinoma. Citation Format: Po-Ting Lin, Wei-Ting Ku, Tsung-Han Wu, Cheng-Heng Wu, Chan-Keng Yang, Wei Teng, Yung-Chang Lin, Chun-Yen Lin. A higher percentage of M2 macrophages in non-tumor part predict the tumor recurrence in patients with hepatocellular carcinoma after surgical resection [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 2695.

Abstract 2921: Extended treatment with IAP inhibitor xevinapant post radiotherapy improves therapeutic efficacy and promotes antitumor immunity in preclinical models

Abstract Background: Xevinapant is a first-in-class, oral IAP (inhibitor of apoptosis protein) inhibitor designed to restore cancer cell sensitivity to apoptosis induced by radiotherapy (RT) and chemotherapy. In a randomized phase 2 study of patients with unresected locally advanced squamous cell carcinoma of the head and neck, xevinapant + chemoradiotherapy (CRT) significantly improved locoregional control at 18 months, 3-year progression-free survival and nearly doubled 5-year overall survival vs. placebo + CRT (53% vs. 28%). Two phase 3 trials, in which patients receive 6 cycles of xevinapant or placebo monotherapy (3 cycles in combination with standard-of-care (C)RT followed by 3 cycles of monotherapy), are ongoing. Based on the roles of IAPs in apoptosis and tumor immunity, continual dosing of xevinapant post RT may deliver additional therapeutic benefit through modulation of multiple tumor microenvironment (TME) compartments. Methods: The impact on antitumor efficacy of xevinapant dosing duration was evaluated in A1419 (head and neck), LLC (lung) and MC38 (colorectal) syngeneic tumor-bearing mice treated with vehicle control, RT alone (3.6Gy, QD, 5 days), or RT + xevinapant (100 mg/kg, QD, 1, 2, or 4 weeks). To assess treatment-mediated TME modulation in MC38 tumors, RNAseq and FACS-based immune profiling was performed, followed by functional validation with ex vivo ELISpot assays, in vitro immunogenic cell death assays, T cell activation assays, and macrophage polarization and viability assays. To evaluate the effect of xevinapant on RT-induced cancer associated fibroblast (CAF) activation and CAF-modulated immune phenotype, activation assay and cytokine profiling was performed. Results: In the tumor models tested, RT in combination with extended xevinapant dosing markedly improved efficacy and prolonged survival compared to treatment arms of RT with shorter xevinapant dosing durations, RT alone, and vehicle control. RNAseq and FACS analyses suggested a TME with enhanced antitumor immunity, while ELISpot and in vitro T cell assays confirmed that extended xevinapant dosing promoted antigen-specific T cell response and T cell activation. Furthermore, macrophage polarization and viability assays suggested that xevinapant reduced the viability of M2 macrophages which may have contributed to the increased M1:M2 macrophage ratio and the improved antitumor immune response observed in vivo. In addition, via the suppression of RT-mediated CAF activation, xevinapant may promote M1 polarization of macrophages and recruitment of T cells by upregulating GM-CSF and CXCL10 in CAFs. Conclusions: Extended dosing of xevinapant post concurrent RT improved antitumor efficacy and prolonged survival of tumor-bearing mice. Mechanistic investigation suggested that such benefit may be, in part, modulated by xevinapant enhanced antitumor immunity. Citation Format: Tsz-Lun Yeung, Feng Jiang, Hui Huang, Huakui Yu, Li-Ya Chiu, Mathilde Bonnemaison, Bo Marelli, Roberta Ferretti, Ralph Lindemann, Christina Esdar. Extended treatment with IAP inhibitor xevinapant post radiotherapy improves therapeutic efficacy and promotes antitumor immunity in preclinical models [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 2921.

Abstract 167: Modulation of PD-1/PD-L1 by simvastatin in TNBC and macrophages and its impact on the efficacy of immunotherapy

Abstract Introduction: Statins, known as inhibitors of HMG-CoA, not only reduce cholesterol but also exhibit pleiotropic effects which include anti-tumor activities by targeting tumor microenvironments. They inhibit the synthesis of FPP and GGPP, which is important for prenylation of signaling proteins. Although PD-1/PD-L1 inhibitors has been approved for treatment for TNBC patients with PD-L1 positive, the response remains unsatisfactory. Also, it has been reported that statins affect PD-L1 expression in other tumors, but it is still uncertain in TNBC. Therefore, we aimed to identify the effect of statins on the expression of PD-L1 in TNBC and to verify the efficacy of combination therapy with statins and immune checkpoint inhibitors. Methods: Human and mouse TNBC cell lines, mouse macrophage cell line (RAW 264.7), and clinically approved simvastatin were used. Flow cytometry, western blot, proliferation assay, qRT-PCR, immune phenotyping and immunohistochemistry were conducted. A co-culture of macrophages and cancer cells was performed. An orthotopic syngeneic mouse model by injection of EMT6 cells into mammary gland fat pad were produced. Mice were treated with simvastatin(20mg/kg) by oral gavage daily and anti-PD-1(200μg) by intraperitoneal injection two times per week. Results: Among thirteen human TNBC cell lines, we confirmed that TNBC cell lines showed various PD-L1 expression, and especially MDA-MB-231 and HCC38 highly expressed endogenous/constitutive PD-L1. Statins reduced PD-L1 expression and exerted anti-proliferative effects in MDA-MB-231, HCC38, and EMT6 in a dose- and time-dependent manner. Phosphorylation of AKT and STAT3 was reduced when simvastatin was treated. Mice with combination therapy of anti-PD-1 and simvastatin had less effective inhibitory effect on tumor growth than mice with anti-PD-1 monotherapy, which showed the highest tumor suppression effect. In immune phenotyping, tumor-infiltrated T cells did not have significant differences among groups. However, M1:M2 macrophage ratio was highest in anti-PD-1 group but decreased in combination therapy group with simvastatin. To determine the effect of simvastatin on macrophages, RAW264.7 cells were stimulated with MDA-MB-231 conditioned medium, revealing an upregulation of PD-1 expression. Conclusions: Our findings show that simvastatin has an anti-tumor effect, which reduces Pd-L1 expression and kills breast cancer cells, reducing phosphorylation of STAT3 and AKT. However, when combined with PD-1 checkpoint inhibitor, it reduces the anti-tumor effect by increasing PD-1 expression in macrophages and promoting M1 to M2 polarization. Further study is needed to demonstrate the mechanism by which simvastatin regulate polarization of macrophages and to confirm the polarization of macrophages in samples of TNBC patients who had been taking simvastatin. Citation Format: Sangeun Lee, Ju Hee Kim, A Young Park, Jihong Baik, Ji-Jung Jung, Han-Byoel Lee, Wonshik Han. Modulation of PD-1/PD-L1 by simvastatin in TNBC and macrophages and its impact on the efficacy of immunotherapy [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 167.

Th17/Treg balance and macrophage polarization ratio in lower extremity arteriosclerosis obliterans

Objective: To explore the balance of peripheral blood T helper 17 cells/regulatory T cell (Th17/Treg) ratio and the polarization ratio of M1 and M2 macrophages in lower extremity arteriosclerosis obliterans (ASO). Methods: A rat model of lower extremity ASO was established, and blood samples from patients with lower extremity ASO before and after surgery were obtained. ELISA was used to detect interleukin 6 (IL-6), IL-10, and IL-17. Real-time RCR and Western blot analyses were used to detect Foxp3, IL-6, IL-10, and IL-17 expression. Moreover, flow cytometry was applied to detect the Th17/Treg ratio and M1/M2 ratio. Results: Compared with the control group, the iliac artery wall of ASO rats showed significant hyperplasia, and the concentrations of cholesterol and triglyceride were significantly increased (P&lt;0.01), indicating the successful establishment of ASO. Moreover, the levels of IL-6 and IL-17 in ASO rats were pronouncedly increased (P&lt;0.05), while the IL-10 level was significantly decreased (P&lt;0.05). In addition to increased IL-6 and IL-17 levels, the mRNA and protein levels of Foxp3 and IL-10 in ASO rats were significantly decreased compared with the control group. The Th17/Treg and M1/M2 ratios in the ASO group were markedly increased (P&lt;0.05). These alternations were also observed in ASO patients. After endovascular surgery (such as percutaneous transluminal angioplasty and arterial stenting), all these changes were significantly improved (P&lt;0.05). Conclusions: The Th17/Treg and M1/M2 ratios were significantly increased in ASO, and surgery can effectively improve the balance of Th17/Treg, and reduce the ratio of M1/M2, and the expression of inflammatory factors.

Tumor-associated macrophages in canine visceral hemangiosarcoma.

Canine hemangiosarcoma (HSA) is a highly malignant tumor derived from hematopoietic stem cells and commonly occurs in visceral organs or skin. Visceral HSAs are particularly aggressive and progress rapidly despite multimodal treatment. Tumor-associated macrophages (TAMs) play a central role in carcinogenesis, tumor progression, and metastasis in humans and murine models. In this retrospective study, we investigated the prevalence and phenotype of TAMs in privately owned, treatment-naïve dogs with naturally occurring HSA. We used CD204 as a general macrophage marker and CD206 as a marker for M2-polarized macrophages. Formalin-fixed paraffin-embedded tissues from HSAs in the spleen (n = 9), heart (n = 6), and other locations (n = 12) from 17 dogs were sectioned and immunohistochemically labeled with CD204 and CD206 antibodies. The mean number of log(CD204)- and log(CD206)-positive cells and the ratio of log(CD206/CD204)-positive cells were compared with normal surrounding tissues and between tumor locations. There were significantly more macrophages and M2 macrophages, and a higher ratio of M2 macrophages to total macrophages in tumor hot spots (P = .0002, P < .0001, and P = .0002, respectively) and in tumor tissues outside of hot spots (P = .009, P = .002, and P = .007, respectively) than in normal surrounding tissues. There were no significant differences between tumor locations, but there was a trend toward higher numbers of CD204-positive macrophages within the splenic tumors. There was no association between histological parameters or clinical stage and TAM numbers or phenotype. As in humans, TAMs in dogs with HSA have a predominantly M2-skewed phenotype. Dogs with HSA could serve as excellent models to evaluate new TAM-reprogramming therapies.

Combination of local immunogenic cell death-inducing chemotherapy and DNA vaccine increases the survival of glioblastoma-bearing mice

Immunotherapy efficacy as monotherapy is negligible for glioblastoma (GBM). We hypothesized that combining therapeutic vaccination using a plasmid encoding an epitope derived from GBM-associated antigen (pTOP) with local delivery of immunogenic chemotherapy using mitoxantrone-loaded PEGylated PLGA-based nanoparticles (NP-MTX) would improve the survival of GBM-bearing mice by stimulating an antitumor immune response. We first proved that MTX retained its ability to induce cytotoxicity and immunogenic cell death of GBM cells after encapsulation. Intratumoral delivery of MTX or NP-MTX increased the frequency of IFN-γ-secreting CD8 T cells. NP-MTX mixed with free MTX in combination with pTOP DNA vaccine increased the median survival of GL261-bearing mice and increased M1-like macrophages in the brain. The addition of CpG to this combination abolished the survival benefit but led to increased M1 to M2 macrophage ratio and IFN-γ-secreting CD4 T cell frequency. These results highlight the benefits of combination strategies to potentiate immunotherapy and improve GBM outcome.

Mild treadmill exercise inhibits cartilage degeneration via macrophages in an osteoarthritis mouse model

ObjectiveWe previously reported how treadmill exercise can suppress cartilage degeneration. Here, we examined the changes in macrophage dynamics in knee osteoarthritis (OA) during treadmill exercise and the effect of macrophage depletion. DesignOA mouse model, generated via anterior cruciate ligament transection (ACLT), was subjected to treadmill exercise of different intensities to investigate the effects on cartilage and synovium. In addition, clodronate liposomes, which deplete macrophages, were injected intra-articularly into the joint to examine the role of macrophages during treadmill exercise. ResultsCartilage degeneration was delayed by mild exercise, and concomitantly, an increase in anti-inflammatory factors in the synovium was observed, with a decrease in the M1 and increase in M2 macrophage ratio. On the contrary, high-intensity exercise led to the progress of cartilage degeneration and was associated with an increase in the M1 and a decrease in the M2 macrophage ratio. The clodronate liposome-induced reduction of synovial macrophages delayed cartilage degeneration. This phenotype was reversed by simultaneous treadmill exercise. ConclusionsTreadmill exercise, especially at high intensity, was detrimental to articular cartilage, whereas mild exercise reduced cartilage degeneration. Moreover, M2 macrophage response appeared necessary for the chondroprotective effect of treadmill exercise. This study indicates the importance of a more comprehensive analysis of the effects of treadmill exercise, not limited to the mechanical stress added directly to cartilage. Hence, our findings might help determine the type and intensity of prescribed exercise therapy for patients with knee OA.

Abstract 5499: Synergistic effects of the combination of ERK1/2 with EGFR, KRASG12C, CDK4/6, and PD-L1 inhibition for cancer treatment

Abstract Background: Mitogen-activated protein kinase (MAPK) cascade is a key pathway that regulates a wide variety of cellular processes, which is frequently activated in cancers. Many nods of the MAPK pathway are considered as promising anti-tumor drug targets, such as RTKs, KRASG12C, RAF, MEK1/2, and ERK1/2. Multiple inhibitors targeting proteins on MAPK pathway have been developed for cancer treatment. However, the effectiveness of these inhibitors is often limited due to acquired drug resistance via pathway feedback activation, indicating the necessity of combination strategies to help block reactivation or bypass activation of MAPK pathway. ATG-017 is an oral, potent, and highly selective inhibitor of ERK1/2, which is under Phase 1 clinical investigation. This study tested the in vivo antitumor effects induced by the combination of ATG-017, with EGFR inhibitor (Osimertinib), KRASG12C inhibitor (ATG-012), CDK4/6 inhibitor (Abemaciclib) or PD-L1 inhibitor (Atezolizumab), in preclinical tumor models. Methods: The in vivo combinations of the drugs were tested in NCI-H1975, NCI-H358 (non-small cell lung cancer) or EL4 (T cell Lymphoma) CDX mouse models. The tumor bearing mouse were treated with vehicle control, ATG-017 (15 or 25 mg/kg, QD), Osimertinib (1mg/kg, QD), ATG-012 (10mg/kg, QD), Abemaciclib (25mg/kg, QD), Atezolizumab (10mg/kg, BIW) or the combination for 9 to 21 days. The tumor size was measured twice a week and tumor growth inhibition (TGI) was evaluated compared with vehicle group. Tumor tissues were collected for tumor infiltrating lymphocyte (TIL) analysis using Flow cytometry or multiplex IHC. Results: In the NCI-H1975 CDX in vivo study, the monotherapy of ATG-017 and Osimertinib induced TGIs of 61.95% and 79.18%, respectively on day 17 after grouping. The combination of ATG-017 and Osimertinib showed 101.85% TGI. In the NCI-H358 CDX in vivo study, the monotherapy of ATG-017, ATG-012, and combination induced TGI of 44.29%,73.71% and 97.13%, respectively on day 8 after grouping. The monotherapy of ATG-017, Abemaciclib, and the combination induced TGIs of 91.40%,63.10% and 108.15%, respectively on day 20 after grouping. In the EL4 syngeneic T cell lymphoma model, neither ATG-017 nor Atezolizumab showed single agent activity, while the combination induced a significant TGI of 21.86% on day 9 after grouping. The percentage of infiltrating CD8+ T cells, CD8:CD4 ratio and M1:M2 macrophage ratio were found increased in the combination group, suggesting the potential role of ATG-017 plus PD-L1 inhibitor in changing a “cold” tumor towards a “hot” phenotype. Conclusion: Strong synergism has been observed for the combination of ATG-017 with EGFR, KRASG12C, CDK4/6, and PD-L1 inhibition, suggesting promising therapeutic strategies for cancer patients that warrants further investigation. Citation Format: Ya Kong, Peng Chen, Lulu Jiang, Bin Jiang, Jay Mei, Bo Shan, Bing Hou. Synergistic effects of the combination of ERK1/2 with EGFR, KRASG12C, CDK4/6, and PD-L1 inhibition for cancer treatment. [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 5499.

Autophagy ameliorates Pseudomonas aeruginosa-infected diabetic wounds by regulating the toll-like receptor 4/myeloid differentiation factor 88 pathway.

Diabetic foot ulcers (DFUs) are among the most common complications in patients with diabetes and a leading cause of lower extremity amputation. DFUs are exacerbated by prolonged bacterial infection; therefore, there is an urgent need for effective treatments to alleviate the burden associated with this condition. Although autophagy plays a unique role in pathogen phagocytosis and inflammation, its role in diabetic foot infections (DFIs) remains unclear. Pseudomonas aeruginosa (PA) is the most frequently isolated gram-negative bacterium from DFUs. Here, we evaluated the role of autophagy in ameliorating PA infection in wounds in a diabetic rat model and a bone marrow-derived macrophage (BMDM) hyperglycemia model. Both models were pretreated with or without rapamycin (RAPA) and then infected with or without PA. Pretreatment of rats with RAPA significantly enhanced PA phagocytosis, suppressed wound inflammation, reduced the M1:M2 macrophage ratio, and improved wound healing. In vitro investigation of the underlying mechanisms revealed that enhanced autophagy resulted in decreased macrophage secretion of inflammatory factors such as TNF-α, IL-6, and IL-1β but increased that of IL-10 in response to PA infection. Additionally, RAPA treatment significantly enhanced autophagy in macrophages by increasing LC3 and beclin-1 levels, which led to altered macrophage function. Furthermore, RAPA blocked the PA-induced TLR4/MyD88 pathway to regulate macrophage polarisation and inflammatory cytokine production, which was validated by RNA interference and use of the autophagy inhibitor 3-methyladenine (3-MA). These findings suggest enhancing autophagy as a novel therapeutic strategy against PA infection to ultimately improve diabetic wound healing.

Icing after skeletal muscle injury with necrosis in a small fraction of myofibers limits inducible nitric oxide synthase-expressing macrophage invasion and facilitates muscle regeneration.

Growing evidence from animal experiments suggests that icing after skeletal muscle injury is harmful to muscle regeneration. However, these previous experimental models yielded massive necrotic myofibers, whereas muscle injury with necrosis in a small myofiber fraction (<10%) frequently occurs in human sports activities. Although macrophages play a proreparative role during muscle regeneration, they exert a cytotoxic effect on muscle cells through an inducible nitric oxide synthase (iNOS)-mediated mechanism. In this study, we established an animal injury model with necrosis limited to a small myofiber fraction and investigated the effect of icing on muscle regeneration with a focus on macrophage-related events. Icing after muscle injury of this model resulted in an enlarged size of regenerating myofibers compared with those in untreated animals. During the regenerative process, icing attenuated the accumulation of iNOS-expressing macrophages, suppressed iNOS expression in the whole damaged muscle, and limited the expansion of the injured myofiber area. In addition, icing increased the ratio of M2 macrophages within the injured site at an earlier time point than that in untreated animals. Following these phenomena in icing-treated muscle regeneration, an early accumulation of activated satellite cells within the damaged/regenerating area occurred. The expression level of myogenic regulatory factors, such as MyoD and myogenin, was not affected by icing. Taken together, our results suggest that icing after muscle injury with necrosis limited to a small fraction of myofibers facilitates muscle regeneration by attenuating iNOS-expressing macrophage invasion, limiting muscle damage expansion, and accelerating the accumulation of myogenic cells which form regenerating myofibers.

A new immune signature for survival prediction and immune checkpoint molecules in non-small cell lung cancer.

Immune checkpoint blockade (ICB) therapy has brought remarkable clinical benefits to patients with advanced non-small cell lung carcinoma (NSCLC). However, the prognosis remains largely variable. The profiles of immune-related genes for patients with NSCLC were extracted from TCGA database, ImmPort dataset, and IMGT/GENE-DB database. Coexpression modules were constructed using WGCNA and 4 modules were identified. The hub genes of the module with the highest correlations with tumor samples were identified. Then integrative bioinformatics analyses were performed to unveil the hub genes participating in tumor progression and cancer-associated immunology of NSCLC. Cox regression and Lasso regression analyses were conducted to screen prognostic signature and to develop a risk model. Functional analysis showed that immune-related hub genes were involved in the migration, activation, response, and cytokine-cytokine receptor interaction of immune cells. Most of the hub genes had a high frequency of gene amplifications. MASP1 and SEMA5A presented the highest mutation rate. The ratio of M2 macrophages and naïve B cells revealed a strong negative association while the ratio of CD8 T cells and activated CD4 memory T cells showed a strong positive association. Resting mast cells predicted superior overall survival. Interactions including protein-protein, lncRNA and transcription factor interactions were analyzed and 9 genes were selected by LASSO regression analysis to construct and verify a prognostic signature. Unsupervised hub genes clustering resulted in 2 distinct NSCLC subgroups. The TIDE score and the drug sensitivity of gemcitabine, cisplatin, docetaxel, erlotinib and paclitaxel were significantly different between the 2 immune-related hub gene subgroups. These findings suggested that our immune-related genes can provide clinical guidance for the diagnosis and prognosis of different immunophenotypes and facilitate the management of immunotherapy in NSCLC.

JMJD3 downregulates IL4i1 aggravating lipopolysaccharide-induced acute lung injury via H3K27 and H3K4 demethylation.

The pro-inflammation M1 to anti-inflammation M2 macrophage ratio contribute to the severity of lipopolysaccharide (LPS)-induced acute lung injury (ALI). JMJD3 aggravates the inflammatory reaction through affecting epigenetic modification and macrophage's phenotype to deteriorate ALI. To explore the mechanism underlying the upregulation of the macrophage M1/M2 ratio through JMJD3, we developed an ALI mouse model using intratracheal LPS, LPS-stimulated RAW 264.7 cells, and inhibited JMJD3 using GSK-J4. H3K27me3 and H3K4me3 were investigated as JMJD3-mediated epigenetic alteration sites in vivo and in vitro. C/EBPβ and KDM5A were validated as linking factors between H3K27 and H3K4. IL4i1 was investigated as a JMJD3-mediated targeted gene to regulate the macrophage M1/M2 ratio. Chromatin immunoprecipitation was used to evaluate the relationship between H3K27me3 and C/ebpβ, C/EBPβ and Kdm5a, H3K4me3 and Il4i1. Inhibiting JMJD3 with GSK-J4 can relieve inflammation and pathological performance in ALI. JMJD3 can reduce IL4i1 expression to increase the macrophage M1/M2 ratio and aggravated ALI which process was mediated via JMJD3-indcued H3K27me3 and H3K4me3 demethylation, latter H3K4me3 demethylation inhibited IL4i1 transcription. Inhibiting JMJD3 with GSK-J4 can increase IL4i1 expression, subsequently decreasing the expressions of M1 and increasing of M2 in vivo. The over-expression IL4i1 in LPS-stimulated macrophage or inhibiting JMJD3 with GSK-J4 can both reverse the increase of the macrophage M1/M2 ratio in vitro. C/EBPβ and KDM5A were upregulated by LPS simulation, which linked JMJD3-induced H3K27-H3K4 demethylation. JMJD3 inhibited IL4i1 to increase the macrophage M1/M2 phenotype ratio and aggravate LPS-induced ALI. Using GSK-J4 to inhibit JMJD3 may facilitate the treatment of LPS-induced ALI.

Abstract 10174: Intravenous Muse Cell Transplantation Enhances Angiogenesis and Reduces Muscle Fibrosis in a Mouse Hindlimb Ischemia Model

Introduction: Multilineage-differentiating stress enduring (Muse) cells, a unique subpopulation of mesenchymal stem cells (MSCs), have been proposed as a promising cell source for regenerative medicine. However, the therapeutic potential of the Muse cells in ischemic limb disease remains unclear. In this study, we aimed to investigate therapeutic potential of Muse cell transplantation for the treatment of severe limb ischemia. Methods and Results: Muse cells isolated from human MSCs, non-Muse MSCs, or mouse bone marrow-derived mononuclear cells (BM-MNCs) were administered intravenously or intramuscularly to a mouse model of hindlimb ischemia (HLI). When the same number of cells were administered, blood flow recovery of the ischemic limb was significantly enhanced in the mice administered with Muse cells over 2 weeks compared to the others. Intravenous Muse cell administration resulted in comparable blood flow recovery to intramuscular Muse cell administration. Immunohistochemistry analysis revealed that intravenous Muse cell administration increased microvascular density and ameliorated interstitial fibrosis in the ischemic limb. We detected mCherry-labeled Muse cells expressing CD31 and human genomic DNA in the ischemic limb 1 week after the intravenous Muse cell administration. Muse cells produced higher VEGF compared to non-Muse MSCs, and Muse cell administration increased tissue VEGF level and expression of angiogenesis-related genes in the ischemic limb. Moreover, we found that Muse cell administration increased M2 macrophage and gene expression ratio of anti-inflammatory genes to proinflammatory gene, iNOS, in the ischemic limb. Lastly, we administered Muse cell intravenously at day 1 and 3, or high-dose BM-MNCs intramuscularly at day1 after the HLI surgery and found that repetitive Muse cell administration resulted in significantly enhanced blood flow recovery in the ischemic limb compared to high-dose BM-MNCs administration. Conclusions: Using an animal model, we demonstrated that intravenous Muse cell transplantation exerts robust therapeutic effects on limb ischemia injury. Therefore, Muse cell-based therapy may provide a novel therapeutic approach for patients with severe limb ischemia.

HDAC6-dependent deacetylation of TAK1 enhances sIL-6R release to promote macrophage M2 polarization in colon cancer

Histone deacetylase 6 (HDAC6), a member of the HDAC family, has been identified as a potential therapeutic target for tumor therapy, but the function and underlying mechanisms of HDAC6 in colon cancer are incompletely characterized. Our study showed that the infiltration ratio of M2 macrophages was increased in colon cancer tissues with high HDAC6 expression. Similarly, the knockdown of HDAC6 in colon cancer cells inhibited cocultured macrophage M2 polarization in vitro. Analysis of the antibody chip revealed that HDAC6 promoted sIL-6R release to enhance macrophage M2 polarization. Mass spectrometry and immunoprecipitation demonstrated that, mechanistically, HDAC6 interacted with transforming growth factor β–activated kinase 1 (TAK1), deacetylated TAK1 at T178 and promoted TAK1 phosphorylation. TAK1-p38 MAPK signaling could further increase the phosphorylation and activity of ADAM17, which is responsible for shedding of IL-6R. Notably, the expression of phosphorylated TAK1 was positively correlated with HDAC6 expression and macrophage M2 polarization in human colon cancer tissues. Our study revealed a new HDAC6-TAK1-ADAM17 regulatory axis that mediates sIL-6R release and macrophage polarization in colon cancer.

Pioglitazone-incorporated microspheres alleviates cardiac dysfunction through macrophage polarization in a rat myocardial infarction model

Abstract Inflammatory processes are essential for the repair processes after myocardial infarction. Among inflammatory cells involved in this process, macrophages play a critical role through polarization in pro-inflammatory M1 or reparative M2 subtype. Pioglitazone, known as a treatment drug for diabetes mellitus, is reported to regulate macrophage polarization toward reparative M2 subtype. Poly L-lactic-co-glycolic acid (PLGA) has been widely accepted as the delivery carrier in drug delivery system (DDS). Here, we aimed to validate the therapeutic effects and investigate the mechanisms of DDS-based treatment using pioglitazone to left ventricular remodeling after myocardial infarction. Pioglitazone-incorporated PLGA microspheres (PGZ-MS) were prepared and the drug release profile was evaluated. The concentration of interleukin-10 secreted from murine bone marrow derived macrophages incubated with PGZ-MS was quantified in the culture supernatant. To validate the therapeutic potential of PGZ-MS in vivo, Sprague-Dawley rats were subjected to permanent left coronary artery ligation to induce myocardial infarction. Mock-MS (100 μg) or PGZ-MS (100 μg) was injected to the infarct region just after induction (n=9–11/group). Cardiac function and left ventricular size were assessed by echocardiography. At 28-day after surgery, rats were sacrificed, and excised hearts were evaluated histologically. PGZ-MS released 1.8±0.3% of incorporated PGZ within 24 hours and 29.5±1.2% within 14 days indicating sustained release of PGZ in vitro. PGZ-MS augmented Interleukin-10 release from bone marrow derived macrophages, indicating polarization toward reparative M2 subtype. PGZ-MS significantly ameliorated cardiac function after myocardial infarction (fractional shortening: MI vs MI+Mock-MS vs MI+PGZ-MS, 24.4±1.1 vs 24.3±1.6 vs 32.2±1.4%; P=0.0035) with reverse remodeling (wall thickness: MI vs MI+Mock-MS vs MI+PGZ-MS, 0.69±0.12 vs 0.71±0.13 vs 1.06±0.09; P=0.03). Immunohistochemical analyses revealed that PGZ-MS enhanced macrophage polarization toward reparative M2 subtype (ratio of reparative M2 macrophages: 0.39±0.03 vs 0.42±0.02 vs 0.54±0.02; P=0.0004) and attenuated apoptosis of cardiomyocytes in the ischemic border zone (MI vs MI+Mock-MS vs MI+PGZ-MS, 2.6±0.2 vs 1.9±0.2 vs 1.2±0.2 cells/field; P=0.002). In the present study, we confirmed that PGZ-MS could realize sustained release of pioglitazone which resulted in sustained effect of macrophage polarization and attenuated left ventricular remodeling accompanied with the amelioration of cardiac dysfunction and cardiomyocytes apoptosis. The strategy of DDS-based macrophage polarization might serve as a promising strategy in cardiac regenerative therapy for ischemic heart disease in the future. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan

Brazilian green propolis improves gut microbiota dysbiosis and protects against sarcopenic obesity.

Brazilian green propolis is an important honeybee product that is considered beneficial for health. Here, we examined the therapeutic potential of dietary supplementation with propolis against sarcopenic obesity using Db/Db mice. Db/m mice fed a normal diet alone and Db/Db mice fed normal diet alone, or supplemented with different amounts of propolis (0.08, 0.4 and 2%), were examined for effects on sarcopenic obesity. Propolis improved the glucose tolerance (P<0.001), increased the grip strength (P<0.001) and the weight of soleus (P=0.006) and plantaris muscles (P=0.008). Moreover, propolis improved the non-alcoholic fatty liver disease activity score (P<0.001) and decreased the expression of genes related to inflammation, liver fibrosis and fatty acid metabolism. Propolis decreased the accumulation of saturated fatty acids in the liver and increased their excretion in faeces. With regard to the innate immunity, propolis decreased the ratio of M1 macrophages (P=0.008) and Type 1 and 3 innate lymphoid cells to CD45-positive cells (P<0.001) and increased the ratio of M2 macrophages (P=0.002) and ILC2s (P=0.007) in the liver. Additionally, propolis decreased the expression of genes related to muscle atrophy and inflammation and the concentration of saturated fatty acids in the soleus muscle. 16S rRNA phylogenetic sequencing revealed that propolis increased the Bacteroidetes/Firmicutes ratio, and the abundance of Butyricicoccus and Acetivibrio genera. Gut microbiota related to the pentose phosphatase pathway and glycerolipid metabolism was more prevalent after the administration of propolis. This is the first study to demonstrate that propolis can improve sarcopenic obesity by improving dysbiosis due to overeating and provides new insights into diet-microbiota interactions during sarcopenic obesity.