Altered Macrophage Polarization Research Articles

F1 fraction isolated from Mesobuthus eupeus scorpion venom induces macrophage polarization toward M1 phenotype and exerts anti-tumoral effects on the CT26 tumor cell line

Scorpion venoms identified as agents with anti-tumor and anti-angiogenic features. Tumor microenvironment (TME) plays a pivotal role in the process of tumorigenesis, tumor development, and polarization of M2 phenotype tumor associated macrophages (TAMs). M2 polarized cells are associated with tumor growth, invasion, and metastasis. The fractionation process was performed by gel filtration chromatography on a Sephadex G50 column. To elucidate whether scorpion venom can alter macrophage polarization, we treated interleukin (IL)-4-polarized M2 cells with isolated fractions from Mesobuthus eupeus. Next, we evaluated the cytokine production and specific markers expression for M2 and M1 phenotype using enzyme linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR), respectively. The phagocytic capacity of macrophages was also assessed. In addition, the migration assay and MTT analysis were performed to investigate the effects of reprogrammed macrophages on the CT-26 colon cancer cells. The results indicated that F1 fraction of venom significantly upregulated the levels and expression of M1-associated cytokines and markers, including tumor necrosis factor-alpha (TNF-α) (p < 0.001), IL-1 (p < 0.01), interferon regulatory factor 5 (IRF5) (p < 0.0001), induced nitric oxide synthase (iNOS) (p < 0.0001), and CD86 (p < 0.0001), and downregulated M2-related markers, including transforming growth factor-beta (TGF-β) (p < 0.05), IL-10 (p < 0.05), Fizz1 (p < 0.0001), arginase-1 (Arg-1) (p < 0.0001), and CD206 (p < 0.001). The macrophage phagocytic capacity was enhanced after treatment with F1 fraction (p < 0.01). Moreover, incubation of CT-26 cell line with conditioned media of F1-treated macrophages suppressed migration (p < 0.0001) and proliferation (p < 0.01) of tumor cells. In conclusion, our findings demonstrated the potential of Mesobuthus eupeus venom in M2-to-M1 macrophage polarization as a promising therapeutic approach against proliferation and metastasis of colon cancer cells.

CD4+ and CD8+ T cells reduce inflammation and promote bone healing in response to titanium implants

T cells are adaptive immune cells essential in pathogenic response, cancer, and autoimmune disorders. During the integration of biomaterials with host tissue, T cells modify the local inflammatory environment by releasing cytokines that promote inflammatory resolution following implantation. T cells are vital for the modulation of innate immune cells, recruitment and proliferation of mesenchymal stem cells (MSCs), and formation of functional tissue around the biomaterial implant. We have demonstrated that deficiency of αβ T cells promotes macrophage polarization towards a pro-inflammatory phenotype and attenuates MSC recruitment and proliferation in vitro and in vivo. The goal of this study was to understand how CD4+ and CD8+ T cells, subsets of the αβ T cell family, impact the inflammatory response to titanium (Ti) biomaterials. Deficiency of either CD4+ or CD8+ T cells increased the proportion of pro-inflammatory macrophages, lowered anti-inflammatory macrophages, and diminished MSC recruitment in vitro and in vivo. In addition, new bone formation at the implantation site was significantly reduced in T cell-deficient mice compared to T cell-competent mice. Deficiency of CD4+ T cells exacerbated these effects compared to CD8+ T cell deficiency. Our results show the importance of CD4+ and CD8+ T cells in modulating the inflammatory response and promoting new bone formation in response to modified Ti implants. Statement of significanceCD4+ and CD8+ T cells are essential in modulating the peri-implant microenvironment during the inflammatory response to biomaterial implantation. This study shows that deficiency of either CD4+ or CD8+ T cell subsets altered macrophage polarization and reduced MSC recruitment and proliferation at the implantation site.

Abstract 3015: HD-PTP mediates tumor suppression via modulation of anti-tumor immunity

Abstract The role of circulating extracellular vesicles in cancer biology is an area of intense research interest. Here, we reveal a tumor suppressor mechanism of HD-PTP (His Domain Protein Tyrosine Phosphatase, encoded by PTPN23), a protein associated with ESCRT (endosomal sorting complex required for transport), a complex that controls endosome sorting and exosome secretion. While loss of HD-PTP is known to be tumorigenic in mice and is frequently observed in human cancers including Triple Negative Breast Cancer (TNBC), the relevant pathways have remained obscure. We find that inhibition of HD-PTP in vivo promotes tumor progression selectively in a syngeneic, immune-competent TNBC model but not in a matched immune-deficient model. Through systematic profiling of the exosomal proteome in HD-PTP proficient and deficient cells, we identify PD-L1 as a key exosomal cargo selectively enriched due to loss of HD-PTP. Mechanistically, we show that inhibition of HD-PTP results in PD-L1 stabilization, its co-localization with the exosomal marker CD63, the enrichment of endosomal PD-L1 and subsequent increase in ExoPD-L1 release. Exosomes produced post HD-PTP loss inhibits antigen-dependent T cell activity in vitro, and HD-PTP inhibition in a syngeneic TNBC model promotes non-cell-autonomous, exosome- and PD-L1-dependent tumor progression, associated with reduced T cell infiltration and altered macrophage polarization. Consistent with these findings, low HD-PTP levels in human solid tumors are associated with decreased tumor infiltrating lymphocytes, poor response to immune checkpoint inhibition and inferior overall survival. Taken together, our studies unveil a novel tumor suppressor pathway controlling PD-L1 trafficking and ExoPD-L1 secretion, highlighting the critical roles of HD-PTP and ESCRT in anti-tumor immunity. Citation Format: Chenxu Guo, Sheng Sun, Nayana Thimmiah, Agustina Maccio, Siang Koh, Leif Ellisen. HD-PTP mediates tumor suppression via modulation of anti-tumor immunity [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 3015.

HSPA9 reduction exacerbates symptoms and cell death in DSS-Induced inflammatory colitis

Inflammatory bowel disease (IBD) is a chronic inflammatory condition that is influenced by various factors, including environmental factors, immune responses, and genetic elements. Among the factors that influence IBD progression, macrophages play a significant role in generating inflammatory mediators, and an increase in the number of activated macrophages contributes to cellular damage, thereby exacerbating the overall inflammatory conditions. HSPA9, a member of the heat shock protein 70 family, plays a crucial role in regulating mitochondrial processes and responding to oxidative stress. HSPA9 deficiency disrupts mitochondrial dynamics, increasing mitochondrial fission and the production of reactive oxygen species. Based on the known functions of HSPA9, we considered the possibility that HSPA9 reduction may contribute to the exacerbation of colitis and investigated its relevance. In a dextran sodium sulfate-induced colitis mouse model, the downregulated HSPA9 exacerbates colitis symptoms, including increased immune cell infiltration, elevated proinflammatory cytokines, decreased tight junctions, and altered macrophage polarization. Moreover, along with the increased mitochondrial fission, we found that the reduction in HSPA9 significantly affected the superoxide dismutase 1 levels and contributed to cellular death. These findings enhance our understanding of the intricate mechanisms underlying colitis and contribute to the development of novel therapeutic approaches for this challenging condition.

HTRA2/OMI-Mediated Mitochondrial Quality Control Alters Macrophage Polarization Affecting Systemic Chronic Inflammation.

Systemic chronic inflammation (SCI) due to intrinsic immune over-activation is an important factor in the development of many noninfectious chronic diseases, such as neurodegenerative diseases and diabetes mellitus. Among these immune responses, macrophages are extensively involved in the regulation of inflammatory responses by virtue of their polarization plasticity; thus, dysregulation of macrophage polarization direction is one of the potential causes of the generation and maintenance of SCI. High-temperature demand protein A2 (HtrA2/Omi) is an important regulator of mitochondrial quality control, not only participating in the degradation of mis-accumulated proteins in the mitochondrial unfolded protein response (UPRmt) to maintain normal mitochondrial function through its enzymatic activity, but also participating in the regulation of mitochondrial dynamics-related protein interactions to maintain mitochondrial morphology. Recent studies have also reported the involvement of HtrA2/Omi as a novel inflammatory mediator in the regulation of the inflammatory response. HtrA2/Omi regulates the inflammatory response in BMDM by controlling TRAF2 stabilization in a collagen-induced arthritis mouse model; the lack of HtrA2 ameliorates pro-inflammatory cytokine expression in macrophages. In this review, we summarize the mechanisms by which HtrA2/Omi proteins are involved in macrophage polarization remodeling by influencing macrophage energy metabolism reprogramming through the regulation of inflammatory signaling pathways and mitochondrial quality control, elucidating the roles played by HtrA2/Omi proteins in inflammatory responses. In conclusion, interfering with HtrA2/Omi may become an important entry point for regulating macrophage polarization, providing new research space for developing HtrA2/Omi-based therapies for SCI.

Three‐dimensional printed biomimetic multilayer scaffolds coordinated with sleep‐related small extracellular vesicles: A strategy for extracellular matrix homeostasis and macrophage polarization to enhance osteochondral regeneration

AbstractCartilage defects resulting from injury or degeneration are a common clinical problem, and due to its avascular nature, articular cartilage has poor self‐healing capacity. Three‐dimensional (3D) bioprinting has attracted great attention in tissue engineering. Melatonin (MT), a hormone mainly secreted at night, plays an important role in tissue repair. Small extracellular vesicles (sEV) are considered ideal drug delivery vehicles and MT‐sEV (sleep‐related sEV) have the potential ability to promote cartilage regeneration. Here, biomimetic multilayer scaffolds were fabricated using 3D bioprinting. A double network hydrogel, composed of methacrylated hyaluronic acid and gelatin methacryloyl (HG), was prepared. MT‐sEV and HG hydrogel were used to create a cartilage layer. A bone layer was formed using poly(ε‐caprolactone) and hydroxyapatite ultralong nanowires. Additionally, two bioinks were alternately printed at the interface layer. The results of RNA sequencing revealed the potential regulatory mechanisms. MT‐sEV showed promotional effects on cell migration, proliferation, chondrogenic differentiation, and extracellular matrix (ECM) deposition. Moreover, MT‐sEV altered macrophage polarization and regulated the expression of inflammatory cytokines. In vivo experiments demonstrated that the biomimetic multilayer scaffolds promoted cartilage regeneration. These experiments demonstrated the ability of MT‐sEV to regulate the immune microenvironment and promote the secretion of ECM, providing a promising strategy for cartilage regeneration.

Small extracellular vesicles from hypoxia-preconditioned bone marrow mesenchymal stem cells attenuate spinal cord injury via miR-146a-5p-mediated regulation of macrophage polarization.

JOURNAL/nrgr/04.03/01300535-202410000-00027/figure1/v/2024-02-06T055622Z/r/image-tiff Spinal cord injury is a disabling condition with limited treatment options. Multiple studies have provided evidence suggesting that small extracellular vesicles (SEVs) secreted by bone marrow mesenchymal stem cells (MSCs) help mediate the beneficial effects conferred by MSC transplantation following spinal cord injury. Strikingly, hypoxia-preconditioned bone marrow mesenchymal stem cell-derived SEVs (HSEVs) exhibit increased therapeutic potency. We thus explored the role of HSEVs in macrophage immune regulation after spinal cord injury in rats and their significance in spinal cord repair. SEVs or HSEVs were isolated from bone marrow MSC supernatants by density gradient ultracentrifugation. HSEV administration to rats via tail vein injection after spinal cord injury reduced the lesion area and attenuated spinal cord inflammation. HSEVs regulate macrophage polarization towards the M2 phenotype in vivo and in vitro. MicroRNA sequencing and bioinformatics analyses of SEVs and HSEVs revealed that miR-146a-5p is a potent mediator of macrophage polarization that targets interleukin-1 receptor-associated kinase 1. Reducing miR-146a-5p expression in HSEVs partially attenuated macrophage polarization. Our data suggest that HSEVs attenuate spinal cord inflammation and injury in rats by transporting miR-146a-5p, which alters macrophage polarization. This study provides new insights into the application of HSEVs as a therapeutic tool for spinal cord injury.

Diosgenin alters LPS-induced macrophage polarization by activating PPARγ/NF-κB signaling pathway

Diosgenin (DG) is a steroidal saponin derived from plants, and it exhibits anti-inflammatory properties. In this study, we employed an in vitro model of P.g.-LPS-stimulated mouse macrophage cell line RAW264.7 to investigate the anti-inflammatory effects and mechanism of DG under the condition of altered polarization of macrophages. The RAW264.7 cells were subjected to pre-treatment with DG with or without P.g.-LPS. In cultured macrophages, DG inhibited P.g.-LPS-induced pro-inflammatory M1 macrophages, and increased anti-inflammatory M2 macrophages. Notably, DG reduced the expression of phosphorylation levels of NF-κB p65 and IκB while increasing the expression of PPARγ. Further studies revealed that PPARγ inhibitor GW9662 or PPARγ siRNA reversed the inhibitory effect of DG on M1 phenotype. Collectively, the anti-inflammatory mechanism of DG is related to altering macrophage polarization by activating PPARγ and inhibiting NF-κB signaling pathways.

Exercise-mediated macrophage polarization modulates the targeted therapeutic effect of NAFLD: a review.

This review aims to explore the exercise-mediated hepatic macrophage polarization mechanism and its effect on improving and regulating non-alcoholic fatty liver disease (NAFLD) by analyzing the pathogenesis of NAFLD and the cause of the influence of hepatic macrophage polarization. In addition to exploring the varied effects of different exercise types on macrophage polarization regulation in NAFLD, to provide a direction and basis for the treatment of NAFLD. The research methodology involved a comprehensive search of the PubMed database using specific keywords such as "NAFLD", "macrophage polarization", and "exercise", to retrieve relevant literature published. (1) The main factors inducing NAFLD were high-fat diet, obesity, insulin resistance (IR), changes in gut microbiota, and genetic variation in susceptibility. (2) Drug treatment, nutrient induction, microfactor induction, physiological environment induction, and other factors can induce the polarization of hepatic macrophages and affect NAFLD. (3) Different intensities, types, and frequencies of exercise have different effects on polarization macrophages, and may also differently effects improving liver inflammation, fibrosis, and NAFLD. Curently, regular moderate-intensity aerobic exercise is the most effective therapy for treating NAFLD. Approaches to ameliorate NAFLD with exercise involve strategies to alter macrophage polarization by inhibiting M1 or driving M2 activation. However, research on the different types of exercise-mediated macrophage polarization mechanisms and differences in therapeutic effects is not yet sufficient. Future research is necessary to explore the exact mechanisms and differences in the effects of different exercises on the treatment of NAFLD.

Abstract P185: Hydralazine Treatment Does Not Fully Reduce Proinflammatory Immune Cells In The Ovaries Of Mice With Salt-sensitive Hypertension

Salt-sensitive hypertension (SSHTN) is associated with altered macrophage polarization, inflammation, and inflammation-associated lymphangiogenesis in the ovaries of mice, and this is associated with reproductive dysfunction. However, it is unknown whether high pressure or salt or both are involved in these adverse effects. We hypothesized that lowering of blood pressure will not reduce proinflammatory immune cells and inflammation in the ovaries of mice with SSHTN. Female C57BL6/J mice were divided into 3 groups: Control (C) mice received tap water and normal diet; SSHTN mice received L-NAME (0.5 mg/mL) in the drinking water for 2 weeks, followed by a 2-week washout period, and a subsequent 3-week 4% salt diet; and the third group were SSHTN and received hydralazine (250 mg/L) in their drinking water (HYD) during the last 3 weeks of salt diet. There was a significant increase in systolic blood pressure in SSHTN mice (143±2 mm Hg) when compared to control (99±2 mm Hg) but was reduced significantly in HYD mice (119±1 mm Hg). Flow cytometry analysis revealed a significant increase in M1 macrophages in the ovaries of SSHTN and HYD mice when compared to C mice (% of CD45 cells: C: 25%±2, SSHTN: 37.8%±2, HYD: 36.2%±3; p&lt;0.05). There was an increase in CD4+TNFa+IFNg+ Th1 cells in the ovaries of SSHTN and HYD mice when compared to C mice (C: 0.33%±0.06, SSHTN: 0.8%±0.07, HYD: 0.7%±0.1; p&lt;0.05), whereas CD4+ T cells (C: 26.5%±2, SSHTN: 51.5%±4, HYD: 14.7%±3; p&lt;0.05) and CD4+IL17a+ Th17 cells (C: 2.9%±0.2, SSHTN: 5.4%±1, HYD: 3.1%±0.6; p&lt;0.05) significantly increased in SSHTN mice but were reduced in HYD mice. Increased gene expression of IL-17 and iNOS was observed in the ovaries of SSHTN and HYD mice. Together, these data demonstrate that both high blood pressure and salt exert their effect on immune cells differentially contributing to the ovarian dysfunction in SSHTN mice. Further studies are needed to investigate whether manipulating macrophage polarization could reverse these adverse effects in the ovaries of mice with SSHTN.

Abstract P184: Inducing Macrophages Towards A Regulatory Phenotype Reduces Blood Pressure And Testicular Proinflammatory Immune Cells In Hypertensive Mice

Our laboratory has previously reported that salt-sensitive hypertension (SSHTN) induces proinflammatory macrophages, inflammation, and inflammation-associated lymphangiogenesis in the testes of mice. However, it is unknown whether altered macrophage polarization causes these changes leading to testicular dysfunction. We hypothesized that inducing macrophages towards a regulatory phenotype will lower blood pressure and reverse inflammation in the testes that is associated with SSHTN. Control (C) male C57BL6/J mice received tap water and normal diet. SSHTN mice were provided L-NAME (0.5 mg/mL) in the drinking water for 2 weeks, followed by a 2-week washout period, and a subsequent 3-week 4% high salt diet and mice in the third group received AVE0991 (0.58 nmol/g) through i.p injections (AVE) in the last 3 weeks. SSHTN mice had increased systolic blood pressure (146±2 mm Hg) when compared to C mice (96±2 mm Hg), but the blood pressure was reduced in AVE mice (136±1 mm Hg; p&lt;0.05). Flow cytometry analysis revealed a significant reduction in M2 macrophages in the testes of SSHTN mice when compared to C mice but were increased in AVE mice when compared to SSHTN mice (% of CD45 cells: C: 19.3%±2, SSHTN: 7.7%±2, AVE: 15.7%±3; p&lt;0.05). There was an increase in CD45+ F4/80- CD11c+ dendritic cells (C:3.3%±1, SSHTN:6.7%±1, AVE: 3.1%±1; p&lt;0.05), CD4+ IL17+ Th17 cells (C:5.8%±1, SSHTN:28.5%±3, AVE: 10.6%±3; p&lt;0.05), CD4+ TNFa+ Th1 cells (C:11.8%±2, SSHTN:38.8%±8, AVE: 15.2%±3; p&lt;0.05), and gamma-delta T cells (C:2.9%±1, SSHTN:8.9%±2, AVE: 4.9%±1; p&lt;0.05) in the testes of SSHTN mice when compared to C mice, but were decreased significantly in AVE mice. These data demonstrate that alteration in testicular macrophage phenotypes is a contributing factor to testicular inflammation resulting in reproductive dysfunction in SSHTN mice. Therapeutic strategies may be developed by targeting macrophage imbalance to improve testicular function that is associated with SSHTN.

SCARB1 in extracellular vesicles promotes NPC metastasis by co-regulating M1 and M2 macrophage function

Distant metastasis is currently the main factor affecting the prognosis of nasopharyngeal carcinoma (NPC), and understanding the mechanisms of metastasis and identifying reliable therapeutic targets are critical for improving prognosis and achieving clinical translation. Macrophages, as important immune cells in the tumor microenvironment (TME), have been shown to regulate metastasis. And extracellular vesicles (EVs) secreted by stromal cells and tumor cells play the important role in intercellular communication in the tumor microenvironment. However, the role of NPC-EVs on macrophages and their function in regulating macrophages to affect metastasis has not been fully clarified. In this study, we report that NPC-EVs can be uptake by macrophages and alter macrophage polarization, for the first time, we identified the genes implicated in these regulatory functions: SCARB1, HAAO, and CYP1B1. Moreover, we found that SCARB1 was positively associated with metastasis and poor prognosis of NPC. Interestingly, we found that SCARB1-rich EVs promoted M1 macrophages ferroptosis to decrease M1 macrophages infiltration by upregulating the HAAO level while decreasing phagocytosis of M2 macrophages by upregulating the CYP1B1 level. Finally, we identified the SCARB1-binding gene KLF9, which is involved in the transcription of HAAO and CYP1B1. Our findings showed that SCARB1-EVs promoted metastasis by co-regulating M1 and M2 macrophage function. The related mechanism will provide a new therapeutic strategy to help patients with NPC improve their prognosis.

Integrin β6 deficiency protects mice from experimental colitis and colitis-associated carcinoma by altering macrophage polarization.

Given the key role of integrins in maintaining intestinal homeostasis, anti-integrin biologics in inflammatory bowel disease (IBD) are being investigated in full swing. However, the unsatisfactory efficacy and safety of current anti-integrin biologics in clinical trials limit their widespread use in clinic. Therefore, it is particularly important to find a target that is highly and specifically expressed in the intestinal epithelium of patients with IBD. The function of integrin αvβ6 in IBD and colitis-associated carcinoma (CAC) with the underlying mechanisms has been less studied. In the present study, we detected the level of integrin β6 within inflammation including colitis tissues in human and mouse. To investigate the role of integrin β6 in IBD and CAC, integrin β6 deficient mice were hence generated based on the construction of colitis and CAC model. We noted that integrin β6 was significantly upregulated in inflammatory epithelium of patients with IBD. Integrin β6 deletion not only reduced infiltration of pro-inflammatory cytokines, but also attenuated disruption of tight junctions between colonic epithelial cells. Meanwhile, lack of integrin β6 affected macrophage infiltration in mice with colitis. This study further revealed that lack of integrin β6 could inhibit tumorigenesis and tumor progression in CAC model by influencing macrophage polarization, which was also involved in attenuating the degree of intestinal symptoms and inflammatory responses in mice suffering from colitis. The present research provides a potentially new perspective and option for the treatment of IBD and CAC.

Auriculotherapy Modulates Macrophage Polarization to Reduce Inflammatory Response in a Rat Model of Acne.

The inflammatory response is an important part of the pathogenesis of acne vulgaris. Auriculotherapy has been shown to have a good therapeutic effect on this disease. The aim of this study was to explore the mechanism underlying the anti-inflammatory effect of auriculotherapy in the treatment of acne vulgaris. Propionibacterium acnes was injected subcutaneously into the ears of rats to establish an animal model of acne. The auriculotherapy intervention in rats consisted of auricular bloodletting therapy (ABT), auricular point sticking (APS), or a combination of both (ABPS). The anti-inflammatory effects of auriculotherapy were evaluated by measuring changes in ear thickness, local body surface microcirculation in the ear, and serum inflammatory factors in rats. The polarization of macrophages was analyzed by flow cytometry, and the expression of TLR2/NF-κB signaling pathway in the target tissues was analyzed using western blot. ABT, APS, and ABPS all reduced the erythema of ear acne, decreased microcirculation in localized ear acne, and decreased serum levels of TNF-α and IL-1β in rats. Meanwhile, the three interventions reduced M1-type macrophages and increased M2-type macrophages; only APS could reduce the expression of TLR2/NF-κB signaling pathway. ABT, APS, and ABPS can improve the inflammatory symptoms of acne and reduce inflammatory cytokines. APS may exert anti-inflammatory effects by altering macrophage polarization and decreasing TLR2/NF-κB expression.

Pexidartinib synergize PD-1 antibody through inhibiting treg infiltration by reducing TAM-derived CCL22 in lung adenocarcinoma.

There is a crosstalk between Tumor-associated macrophages (TAM) and tumor-infiltrating T cells in tumor environment. TAM could inhibit the activity of cytotoxic T cells; TAM could also regulate the composition of T cells in tumor immune environment. The combination therapy for TAM and tumor infiltrated T cells has been widely noticed, but the crosstalk between TAM and tumor infiltrated T cells remains unclear in the process of combination therapy. We treated lung adenocarcinoma tumor models with pexidartinib, which targets macrophage colony stimulating factor receptor (M-CSFR) and c-kit tyrosine kinase, to inhibited TAM. Pexidartinib inhibited the ratio of macrophages in the tumor and also altered macrophage polarization. In addition to reprogram TAM, pexidartinib also changed the composition of tumor-invasive T cells. After pexidartinib treatment, the total number of T cells, CD8+ T cells and Treg cells were all decreased, the ratio of CD8+T/Treg increased significantly. According to the analysis of cytokines and chemokines during the treatment of pexidartinib, CCL22, as a chemokine for Treg recruitment, significantly decreased after the treatment of pexidartinib. Base on the above observation, the combination of pexidartinib and PD-1 antibody were used in the treatment of lung adenocarcinoma subcutaneous tumor model, the combination therapy has significantly improved the efficacy of tumor treatment compared with the monotherapy. Meanwhile, compared with pexidartinib monotherapy, the combination treatment further switches the polarization status of tumor-associated macrophages. In summary, our results showed that the combination of pexidartinib and PD-1 antibody showed a synergy and significantly improved the anti-tumor efficacy, through pexidartinib increasing CD8T/Treg ratio by reducing TAM-derived CCL22.

Abstract P2-20-08: Expression of ACVRL1 in a subset of recruited tumor-associated macrophages drives resistance to PD-1 therapy in human breast cancer

Abstract The activin-like kinase receptor 1 (ALK1, encoded by ACVRL1) is a member of the transforming growth factor β (TGF-β) superfamily. Expression of ALK1 has been historically associated with endothelial cells, thus serving as a potential target for antiangiogenic therapy in cancer. Despite promising preclinical results with a ligand trap (RAP-041/dalantercept), clinical trials did not show additional benefit of dalantercept when combined or contrasted with the standard of care. This outcome raises the question about patient selection and optimal combination partner in the clinical setting. Moreover, from the biological perspective, how ALK1 signaling shapes the features of a tumor is still largely uncharted. Based on our previous observations, we confirmed that the genetic network correlated with ACVRL1 expression is associated with immune processes in human breast cancer. Next, we validated that inhibition of ALK1 altered the immune landscape in experimental primary breast cancer (MMTV-PyMT model), providing a rationale for a combined regimen with immunotherapy. After developing and validating a murine model of adjuvant therapy with RAP-041 (syngeneic E0771 and 4T1 cell lines), we discovered that ALK1 blockade potentiated immune checkpoint inhibitors. Based on bulk RNA-sequencing and a multicolor FACS approach, addition of RAP-041 to immunotherapy significantly reduced the number of intratumoral macrophages compared with either treatment alone. Notably, these changes were coupled with systemic alterations in peripheral blood cell composition, with the combined administration suppressing circulating monocytes. Interestingly, ALK1 signaling appears to be a pivotal contributor to this phenotype, as fewer circulating monocytes were also detected in the RAP-041 cohort. Based on our in vitro characterization, we revealed that ALK1 expression is not limited to endothelial cells, as different myeloid populations readily expressed Acvrl1, suggesting a direct functional role of ALK1. Stimulation of ALK1 did not alter macrophage polarization, even though different levels of mediators downstream of ALK1 characterized the M1/M2 states. Furthermore, apoptosis and necrosis did not seem to be affected upon activation of ALK1, while the effect on the cell cycle awaits confirmation. In vitro genetic modulation of ALK1 activity is ongoing, with co-culture systems, transendothelial adhesion and migration being currently assessed. Inspection of several human sc-RNA-sequencing datasets confirmed the existence of ACVRL1-positive monocytes in human PBMCs from healthy individuals as well as in human solid cancers, including breast cancer. Focusing on the immune compartment, ACVRL1 expression characterized recruited tumor-associated macrophages (TAMs) and their cycling counterpart. In light of this feature, longitudinal follow-up of patients exposed to chemotherapy and/or anti-PD-1/PD-L1 revealed that expression of ACVRL1 decreased following exposure to chemotherapy. Notably, high ACVRL1 expression and an increased proportion of ACVRL1-positive TAMs were found at progression with anti-PD-1 treatment. In agreement with our preclinical data, ACVRL1-positive cells were depleted from circulation upon recurrence, indicating a migration from blood to tumors. Finally, survival analysis determined that ACVRL1hi signature in macrophages correlated with a significantly lower survival in a 5-year follow-up in breast cancer. In conclusion, our work sheds light on the value of ALK1 as a promising dual antiangiogenic and immunomodulatory target for precision medicine in cancer. Citation Format: Mehrnaz Safaee Talkhoncheh, Jonas Sjölund, Paulina Bolivar Balbas, Ewa Kurzejamska, Sara Larsson, Eugenia Cordero, Clara R. Oudenaarden, Jessica Pantaleo, Göran Jonsson, Charlotte Rolny, Matteo Bocci, Kristian Pietras. Expression of ACVRL1 in a subset of recruited tumor-associated macrophages drives resistance to PD-1 therapy in human breast cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-08.

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.

Expression of Trichinella spiralis serpin Tsp_01570 in Pichia pastoris: a first insight of its biomodulatory activity.

Serpins represent one of the most diverse families of serine protease inhibitors. Despite their complexity, they are virtually found in all organisms and play an important role in homeostasis processes such as blood coagulation, inflammation, fibrinolysis, immune responses, chromatin condensation, tumor suppression, and apoptosis. There has recently been particular interest in studying serpin functions in infection and inflammation, especially since more serpins from parasites have been identified and characterized. Among helminths, Trichinella spiralis is one of the few parasites with an extremely strong ability to induce host immune suppression. Previous studies show that serpins are present in Trichinella and are expressed differentially at different parasite stages. More interesting, there is evidence of a recombinant serpin from Trichinella pseudospiralis that alters macrophage polarization in vitro. This finding could be relevant to comprehend the modulation process of the immune response. We expressed Tsp_01570, a putative serpin gene from Trichinella spiralis, in the eukaryotic system Pichia pastoris SMD1168H and evaluated its presence at different parasite stages, finding the serine protease inhibitor in the crude extract of adult worms. The effect of recombinant serpin on THP-1 cells was tested by quantification of IL-12p40, TNF-α, IL-4, and IL-10 cytokines released by ELISA. We also evaluated the expression of the M1 markers, CCR7 and CD86, and the M2 markers, CD163 and CD206, by immunofluorescence staining. This study represents the first insight in elucidating the importance of serpin Tsp_01570 as a potential molecular modulator.

Distinct macrophage polarization in acute and chronic gout

AbstractMacrophage polarization mediates the development of inflammatory diseases. However, the polarization status at various stages of gout is not fully understood. Our study aimed to define the evolution of macrophage polarization in acute and chronic gout. Normal human synovium and synovium with tophi were collected for immunofluorescence (IF). Rat gouty joints were collected for joint thickness assessment and pathological evaluation. Tissue mRNA expression of inducible nitric oxide synthase (iNOS) and arginase-1 (Arg-1) were evaluated. Mouse peritoneal macrophages and THP-1 derived macrophages were stimulated by monosodium urate (MSU) crystals and were collected for detection of interleukin (IL) -1β and IL-37 levels and iNOS/Arg-1 ratio. Arg-1 and IL-37 were highly expressed in normal synovium and synovium with tophi. In rat gouty joints, the inflammatory cell counts and ankle thickness began to increase at 2 h, peaked at 24 h, and was decreased spontaneously. An increase in macrophages preceded the neutrophils infiltration. Infiltration of M1 was positively related with the severity of arthritis. M2 appeared in an early stage (at 2 h) of inflammation. The number of M1 macrophages was comparable to that of M2 from 2 to 12 h and exceeded M2 number at 18 h and 24 h. The ratios of M2/M1 reversed at 48 h and remained reversed until 120 h. In mice gouty joints, iNOS/Arg-1 mRNA ratio was significantly higher than the that in control group at 8 h. The proportion of neutrophils and M1-macrophages reached peak at 4 h in mice model with peritoneal gout. Concentration of IL-1β and ratio of iNOS/Arg-1 were increased at 6 h, peaked at 48 h, and were then decreased at 72 h in vitro, while the concentration of IL-37 peaked at 2 h and then decreased. In summary, altered macrophage polarization was observed in various stages of gouty inflammation. Macrophages in acute gout were polarized into M1 at early stage and into M2 at later stage while the macrophages in chronic gout mainly were only polarized towards M2. The number of M1 rose with the progression of inflammation. Early increase of M2 was observed, which might be generated directly from M0.

Prophylactic administration of human amniotic fluid stem cells suppresses inflammation-induced preterm birth via macrophage polarization.

Ascending inflammation from the vagina is a major cause of preterm birth. Currently, this condition-especially when uncontrolled-has no effective treatment. Human amniotic fluid stem cells (hAFSCs) are mesenchymal stem cells known to exert potent anti-inflammatory effects in animal models of perinatal diseases, such as periventricular leukomalacia, myelomeningocele, and neonatal sepsis. However, hAFSC therapy for inflammation-induced preterm birth has not been tested. In order to determine the therapeutic effect of hAFSC transplantation, we employed a preterm mouse model of ascending infection; this model was constructed by administering lipopolysaccharide to pregnant mice. We investigated the preterm birth rate and evaluated the inflammation of tissues, which is related to progressive infections, such as those involving the cervix, placenta, and lavage cells, using real-time qPCR. Further, we tracked the fluorescence of fluorescently labeled hAFSCs using an in vivo imaging system, and hAFSC aggregation was evaluated using immunohistochemistry analysis. We also investigated the presence of multiple types of peritoneal macrophages via flow cytometry analysis. Finally, we performed sphere culturing and co-culturing to determine the therapeutic effects of hAFSCs, such as their anti-inflammatory effects and their potential to alter macrophage polarization. We found that hAFSC administration to the peritoneal cavity significantly reduced inflammation-induced preterm birth in the mouse model. The treatment also significantly suppressed inflammation of the placenta and cervix. Transplanted hAFSCs may have aggregated with peritoneal macrophages, switching them from an inflammatory to an anti-inflammatory type. This property has been reported in vivo previously, but here, we examined the effect in vitro. Our findings support the hypothesis that hAFSCs suppress inflammation and reduce preterm birth by switching macrophage polarity. This study is the first to demonstrate that hAFSCs are effective in the treatment and prevention of inflammation-induced preterm birth.