Pro-inflammatory Macrophage Polarization Research Articles

Manganese immunotherapy for treating osteosarcoma: Glycosylating 1V209 anchored MnO2 nanosheets prompt pro-inflammatory macrophage polarization

Current immunotherapy has not met the curative expectations in the treatment of osteosarcoma. As the most abundant immune cells in the tumor microenvironment (TME), macrophages are closely related to the progression of osteosarcoma. Recently, bivalent manganese ions (Mn2+) have been found to present great potential for immunotherapy in natural killer cells and dendritic cells. However, the effect of Mn2+ on macrophage polarization remains entirely unclear, especially in the TME. Toll-like receptor 7 (TLR7) agonists can initiate innate immune responses but are limited in clinical application for their hydrophobicity. Herein, we prepare BSA-templated MnO2 nanosheets (BMs) and conjugate 1V209, a small molecule TLR7 agonist, with a sugar group to improve hydrophilicity and immunostimulatory effects. Sugar-conjugated 1V209 (SV) is further anchored onto BMs to construct SV@BMs. In our study, SV@BMs can react with glutathione (GSH) to release Mn2+ and produce plenty of reactive oxygen species (ROS) via Mn2+-mediated Fenton-like reactions in macrophages. ROS generated by pro-inflammatory macrophages can induce apoptosis of tumor cells. Mn2+ and co-released SV together promote pro-inflammatory macrophage polarization, which may depend on the activation of the NF-κB signal pathway. In vivo, the application of SV@BMs significantly activates local and systematic immunity, eliminates tumor cells and reduces lung metastases.

Xuanfei Baidu formula alleviates impaired mitochondrial dynamics and activated NLRP3 inflammasome by repressing NF-κB and MAPK pathways in LPS-induced ALI and inflammation models

BackgroundXuanfei Baidu Formula (XBF) is an effective traditional Chinese medicine (TCM) remedy for treating coronavirus disease 2019 (COVID-19) in China. This herbal medicine has shown effects in reducing clinical symptoms and shortening the average length of hospital stay for COVID-19 patients. Previous studies have demonstrated that XBF alleviates acute lung injury (ALI) by regulating macrophage-mediated immune inflammation, but the mechanisms of action remain elusive. PurposeThis study aimed to evaluate the lung-protective and anti-inflammatory effects of XBF and its underlying mechanisms. MethodsHere, XBF's effects were investigated in an ALI mouse model induced by inhalation of atomized lipopolysaccharide (LPS). Besides, the LPS-induced inflammation model in RAW264.7 cells was used to clarify the underlying mechanisms of XBF against ALI. ResultsOur results showed that XBF treatment alleviated LPS-induced lung injury, as evidenced by reduced histopathological changes, pulmonary alveoli permeability, fibrosis, and apoptosis in the lung tissues. In addition, inflammation was alleviated as shown by decreased levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β in serum and bronchoalveolar lavage fluid (BALF), and reduced white blood cell (WBC) count in BALF. Furthermore, consistent with the in vivo assay, XBF inhibited LPS-induced inflammatory cytokines release and pro-inflammatory polarization in RAW264.7 cells. Mechanistically, XBF increased mitochondrial fusion by upregulating Mfn1 and attenuated NLRP3 inflammasome activation by repressing Casp11, respectively, to inhibit NF-κB and MAPK pathways, thus repressing pro-inflammatory macrophage polarization. ConclusionIn this study, we demonstrate that XBF exerts anti-ALI and -inflammatory effects by recovering mitochondrial dynamics and reducing inflammasome activation, providing a biological illustration of the clinical efficacy of XBF in treating COVID-19 patients.

Macrophage-specific deletion of neuropilin-2 inhibits inflammatory signalling and attenuates atherosclerotic plaque development in apolipoprotein e-deficient mice

Abstract Background Atherosclerosis is the underlying pathology in most cases of ischaemic heart disease, which itself remains the leading cause of mortality worldwide. Atherosclerosis is a multifaceted vascular disease characterised by arterial wall inflammation resulting from the subendothelial retention, and modification, of lipoproteins by macrophages. Macrophages exhibit a high degree of plasticity, and undergo polarisation to a pro-inflammatory phenotype in response to pro-inflammatory cytokines and other environmental stimuli, making them an attractive therapeutic target. Neuropilin-2 (NRP2) is a cell-surface co-receptor with essential roles in angiogenesis and axonal guidance, and is also expressed by macrophages. However, little is known about the role of NRP2 in macrophage function or atherosclerosis. Purpose To investigate whether NRP2 is important for macrophage involvement in atherosclerosis, we characterised the effects of conditional NRP2 deletion on macrophage function and atherosclerotic plaque development. Methods We generated mice with macrophage-specific (LysM-Cre) deletion of Nrp2, and lineage tracing via EYFP transgene expression, on the pro-atherogenic Apolipoprotein E-deficient background (Nrp2-KOMac, Apoe−/−, EYFP). These mice were fed a high-fat diet (HFD) for 16 weeks before their aortas were stained with Oil-red-O to assess plaque coverage. Plaque content was then characterised by sectioning and staining the aortic roots. The role of NRP2 in modulating pro-inflammatory macrophage polarisation and signalling, was assessed using functional assays and transcriptome analysis in Nrp2-KO bone marrow-derived macrophages (BMDMs). Results HFD-induced plaque development was significantly reduced in Nrp2-KOMac, Apoe−/−, EYFP mice, with 17.4% total aortic plaque coverage in Nrp2-KOMac, Apoe−/−, EYFP mice, compared to 24.9% in WTApoe−/−, EYFP mice (p=0.0021, n=10). Plaques from Nrp2-KOMac, Apoe−/−, EYFP mice also displayed features consistent with increased plaque stability, including reduced necrotic core area and plaque lipid content, and increased cap thickness. NRP2 was significantly upregulated upon M-CSF-mediated differentiation of bone marrow progenitors into BMDMs, and further upregulated by pro-inflammatory polarisation using LPS and IFN-gamma. Furthermore, upregulation of Il1β, Tnfα and Il6, induced by LPS and IFN-gamma, was significantly reduced in Nrp2-KO BMDMs, and MCP-1 induced migration was also decreased in these cells. Transcriptome analysis revealed that NF-kappaB signalling pathway genes, and genes regulating monocyte chemotaxis, were downregulated in Nrp2-KO BMDMs. Conclusion Macrophage-derived NRP2 is pro-atherogenic, likely resulting from its role in positively regulating pro-inflammatory signalling and macrophage migration. Targeting NRP2 expressed on the surface of macrophages could therefore offer a novel therapeutic approach for reducing the disease burden associated with atherosclerosis. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation (MB PhD Studentship)

Influence of Cu doping on the biocompatibility of zirconia-toughened alumina ceramics for artificial joints

Zirconia-toughened alumina (ZTA) is considered the gold standard material for total hip arthroplasty (THA). However, it still lacks sufficient lubrication and fracture toughness, and it has the risk of wearing, squeaking, and fracturing. Cu doping is considered to address these issues. To further promote the application of Cu doping, its effect on biocompatibility needs to be studied. In this study, Cu-doped ZTA (Cu-ZTA) ceramics with different Cu contents were prepared using a fast hot pressed sintering (FHPS) equipment and their biocompatibility was systematically studied. The results demonstrate that, depending on the Cu content, Cu doping reduces the stability of the tetragonal zirconia, resulting in a tetragonal-to-monoclinic phase transition of zirconia, thereby creating pores on the surface of the ZTA. The biocompatibility of the Cu-ZTA with different Cu contents was analyzed based on osteoblasts and macrophages. The results suggested that Cu doping increased the adhesion of osteoblasts and slightly improved their viability. In contrast, Cu doping influenced the morphology of macrophages, causing the shrinkage and extension of their pseudopodia. Cu doping also induced the pro-inflammatory polarization of macrophages and stimulated the secretion of pro-inflammatory cytokines (TNF-α and IL-6). Therefore, Cu doping can improve the cytocompatibility of osteoblasts with ZTA ceramic surfaces, however, the enhanced effect of Cu-doped ZTA ceramics on the early inflammatory response requires special attention and further study.

Beta-Glucan from S. cerevisiae Protected AOM-Induced Colon Cancer in cGAS-Deficient Mice Partly through Dectin-1-Manipulated Macrophage Cell Energy.

Although the impacts of Saccharomyces cerevisiae on cancers are mentioned, data on its use in mice with cyclic GMP-AMP synthase deficiency (cGAS-/-) are even rarer. Here, 12 weeks of oral administration of S. cerevisiae protected cGAS-/- mice from azoxymethane (AOM)-induced colon cancers, partly through dysbiosis attenuation (fecal microbiome analysis). In parallel, a daily intralesional injection of a whole glucan particle (WGP; the beta-glucan extracted from S. cerevisiae) attenuated the growth of subcutaneous tumor using MC38 (murine colon cancer cell line) in cGAS-/- mice. Interestingly, the incubation of fluorescent-stained MC38 with several subtypes of macrophages, including M1 (using Lipopolysaccharide; LPS), M2 (IL-4), and tumor-associated macrophages (TAM; using MC38 supernatant activation), could not further reduce the tumor burdens (fluorescent intensity) compared with M0 (control culture media). However, WGP enhanced tumoricidal activities (fluorescent intensity), the genes of M1 pro-inflammatory macrophage polarization (IL-1β and iNOS), and Dectin-1 expression and increased cell energy status (extracellular flux analysis) in M0, M2, and TAM. In M1, WGP could not increase tumoricidal activities, Dectin-1, and glycolysis activity, despite the upregulated IL-1β. In conclusion, S. cerevisiae inhibited the growth of colon cancers through dysbiosis attenuation and macrophage energy activation, partly through Dectin-1 stimulation. Our data support the use of S. cerevisiae for colon cancer protection.

Phenotypic and Transcriptional Changes of Pulmonary Immune Responses in Dogs Following Canine Distemper Virus Infection.

Canine distemper virus (CDV), a morbillivirus within the family Paramyxoviridae, is a highly contagious infectious agent causing a multisystemic, devastating disease in a broad range of host species, characterized by severe immunosuppression, encephalitis and pneumonia. The present study aimed at investigating pulmonary immune responses of CDV-infected dogs in situ using immunohistochemistry and whole transcriptome analyses by bulk RNA sequencing. Spatiotemporal analysis of phenotypic changes revealed pulmonary immune responses primarily driven by MHC-II+, Iba-1+ and CD204+ innate immune cells during acute and subacute infection phases, which paralleled pathologic lesion development and coincided with high viral loads in CDV-infected lungs. CD20+ B cell numbers initially declined, followed by lymphoid repopulation in the advanced disease phase. Transcriptome analysis demonstrated an increased expression of transcripts related to innate immunity, antiviral defense mechanisms, type I interferon responses and regulation of cell death in the lung of CDV-infected dogs. Molecular analyses also revealed disturbed cytokine responses with a pro-inflammatory M1 macrophage polarization and impaired mucociliary defense in CDV-infected lungs. The exploratory study provides detailed data on CDV-related pulmonary immune responses, expanding the list of immunologic parameters potentially leading to viral elimination and virus-induced pulmonary immunopathology in canine distemper.

Metformin attenuates osteoarthritis by targeting chondrocytes, synovial macrophages and adipocytes.

To investigate the therapeutic effect and mechanism of metformin on knee OA in normal diet (ND) mice or high-fat diet (HFD)-induced obese mice. Destabilization of the medial meniscus surgery was performed in ND mice or HFD mice, and metformin was administrated in drinking water or not. The changes of OA joint structure, infiltration and polarization of synovial macrophages and circulating and local levels of leptin and adiponectin were evaluated. In vitro, the effects of metformin on chondrocytes and macrophages, and of conditioned mediums derived from mouse abdominal fat on murine chondrogenic cell line ATDC5 and murine macrophage cell line RAW264.7, were detected. Metformin showed protective effects on OA, characterized by reductions on OARSI score [2.00, 95% CI (1.15, 2.86) for ND mice and 3.17, 95% CI (2.37, 3.96) for HFD mice] and synovitis score [1.17, 95% CI (0.27, 2.06) for ND mice and 2.50, 95% CI (1.49, 3.51) for HFD mice] after 10 weeks of treatment, and the effects were more significant in HFD mice than in ND mice. Mechanistically, in addition to decreasing apoptosis and matrix-degrading enzymes expression in chondrocytes as well as infiltration and pro-inflammatory differentiation of synovial macrophages, metformin reduced leptin secretion by adipose tissue in HFD mice. Metformin protects against knee OA which could be through reducing apoptosis and catabolism of chondrocytes, and suppressing infiltration and pro-inflammatory polarization of synovial macrophages. For obese mice, metformin has a greater protective effect in knee OA additionally through reducing leptin secretion from adipose tissue.

Iron dyshomeostasis and time-course changes in iron-uptake systems and ferritin level in relation to pro-inflammatory microglia polarization in sepsis-induced encephalopathy.

Encephalopathy is a frequent and lethal consequence of sepsis. Recently, a growing body of evidence has provided important insights into the role of iron dyshomeostasis in the context of inflammation. The molecular mechanisms underlying iron dyshomeostasis and its relationship with macrophage phenotypes are largely unknown. Here, we aimed to characterize the changes in iron-transporter and storage proteins and the microglia phenotype that occur during the course of sepsis, as well as their relationship with sepsis-induced encephalopathy. We used a cecal ligation and puncture (CLP) murine model that closely resembles sepsis-induced encephalopathy. Rats were subjected to CLP or sham laparotomy, then were neurologically assessed at 6 h, 24 h, and 3 days after sepsis induction. The serum and brain were collected for subsequent biochemical, histological, and immunohistochemical assessment. Here, an iron excess was observed at time points that followed the pro-inflammatory macrophage polarization in CLP-induced encephalopathy. Our results revealed that the upregulation of non-transferrin-bound iron uptake (NTBI) and ferritin reduction appeared to be partially responsible for the excess free iron detected within the brain tissues. We further demonstrated that the microglia were shifted toward the pro-inflammatory phenotype, leading to persistent neuro-inflammation and neuronal damage after CLP. Taken together, these findings led us to conclude that sepsis increased the susceptibility of the brain to the iron burden via the upregulation of NTBI and the reduction of ferritin, which was concomitantly and correlatively associated with dominance of pro-inflammatory microglia and could explain the neurological dysfunction observed during sepsis.

14-Deoxygarcinol improves insulin sensitivity in high-fat diet-induced obese mice via mitigating NF-κB/Sirtuin 2-NLRP3-mediated adipose tissue remodeling.

Interleukin (IL)-1β is a culprit of adipose tissue inflammation, which in turn causes systematic inflammation and insulin resistance in obese individuals. IL-1β is mainly produced in monocytes and macrophages and marginally in adipocytes, through cleavage of the inactive pro-IL-1β precursor by caspase-1, which is activated via the NLRP3 inflammasome complex. The nuclear factor-κB (NF-κB) transcription factor is the master regulator of inflammatory responses. Brindle berry (Garcinia cambogia) has been widely used as health products for treating obesity and related metabolic disorders, but its active principles remain unclear. We previously found a series of polyisoprenylated benzophenones from brindle berry with anti-inflammatory activities. In this study we investigated whether 14-deoxygarcinol (DOG), a major polyisoprenylated benzophenone from brindle berry, alleviated adipose tissue inflammation and insulin sensitivity in high-fat diet fed mice. The mice were administered DOG (2.5, 5 mg · kg-1 · d-1, i.p.) for 4 weeks. We showed that DOG injection dose-dependently improved insulin resistance and hyperlipidemia, but not adiposity in high-fat diet-fed mice. We found that DOG injection significantly alleviated adipose tissue inflammation via preventing macrophage infiltration and pro-inflammatory polarization of macrophages, and adipose tissue fibrosis via reducing the abnormal deposition of extracellular matrix. In LPS plus nigericin-stimulated THP-1 macrophages, DOG (1.25, 2.5, 5 μM) dose-dependently suppressed the activation of NLRP3 inflammasome and NF-κB signaling pathway. We demonstrated that DOG bound to and activated the deacetylase Sirtuin 2, which in turn deacetylated and inactivated NLRP3 inflammasome to reduce IL-1β secretion. Moreover, DOG (1.25, 2.5, 5 μM) dose-dependently mitigated inflammatory responses in macrophage conditioned media-treated adipocytes and suppressed macrophage migration toward adipocytes. Taken together, DOG might be a drug candidate to treat metabolic disorders through modulation of adipose tissue remodeling.

LNA-anti-miR-150 alleviates renal interstitial fibrosis by reducing pro-inflammatory M1/M2 macrophage polarization.

Renal interstitial fibrosis (RIF) is a common pathological feature contributing to chronic injury and maladaptive repair following acute kidney injury. Currently, there is no effective therapy for RIF. We have reported that locked nuclear acid (LNA)-anti-miR-150 antagonizes pro-fibrotic pathways in human renal tubular cells by regulating the suppressor of cytokine signal 1 (SOCS1)/Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. In the present study, we aimed to clarify whether LNA-anti-miR-150 attenuates folic acid-induced RIF mice by regulating this pathway and by reducing pro-inflammatory M1/M2 macrophage polarization. We found that renal miR-150 was upregulated in folic acid-induced RIF mice at day 30 after injection. LNA-anti-miR-150 alleviated the degree of RIF, as shown by periodic acid–Schiff and Masson staining and by the expression of pro-fibrotic proteins, including alpha-smooth muscle actin and fibronectin. In RIF mice, SOCS1 was downregulated, and p-JAK1 and p-STAT1 were upregulated. LNA-anti-miR-150 reversed the changes in renal SOCS1, p-JAK1, and p-STAT1 expression. In addition, renal infiltration of total macrophages, pro-inflammatory M1 and M2 macrophages as well as their secreted cytokines were increased in RIF mice compared to control mice. Importantly, in folic acid-induced RIF mice, LNA-anti-miR-150 attenuated the renal infiltration of total macrophages and pro-inflammatory subsets, including M1 macrophages expressing CD11c and M2 macrophages expressing CD206. We conclude that the anti-renal fibrotic role of LNA-anti-miR-150 in folic acid-induced RIF mice may be mediated by reducing pro-inflammatory M1 and M2 macrophage polarization via the SOCS1/JAK1/STAT1 pathway.

Functional skewing of TRIM21-SIRT5 interplay dictates IL-1β production in DSS-induced colitis.

Macrophage polarization determines the production of pro- or anti-inflammatory cytokines in response to various bacterial and virus infections. Here, we report that pro-inflammatory macrophage polarization induced by lipopolysaccharide (LPS) skews the TRIM21-SIRT5 interplay toward TRIM21 activation and SIRT5 degradation, resulting in an enhancement of interleukin (IL)-1β production in vitro and in vivo. Mechanistically, LPS challenge enhances the interaction between TRIM21 and SIRT5 to promote SIRT5 ubiquitination and degradation, while reducing the binding of SIRT5 to HAUSP, a deubiquitinating enzyme that stabilizes SIRT5. In a feedback loop, SIRT5 degradation sustains the acetylation of TRIM21 at Lys351, thereby increasing its E3 ligase activity in LPS-activated macrophages. Thus, we identify a functional balance between TRIM21 and SIRT5 that is tilted toward SIRT5 suppression in response to LPS stimulation, thereby enhancing IL-1β production during inflammation.

Andrographolide sodium bisulfite ameliorates dextran sulfate sodium-induced colitis and liver injury in mice via inhibiting macrophage proinflammatory polarization from the gut-liver axis

Ulcerative colitis (UC), an inflammatory disease, is widely thought to be associated with colonic barrier damage and inflammatory response. With the destruction of the colonic barrier, lipopolysaccharide (LPS) enters the liver through the portal vein and causes liver injury. Liver injury in turn exacerbates UC to form a vicious cycle, so the treatment of liver injury cannot be ignored. Andrographolide (Andro) has a protective effect against colitis and liver injury, but with low bioavailability. Andrographolide sodium bisulfite (ASB), a water-soluble sulfonate of Andro, has better bioavailability, whether it has a better curative effect against UC and liver injury is rarely reported. Hence, we investigated the protective effect and potential mechanism of ASB against dextran sulfate sodium (DSS)-induced UC and liver injury in mice. The results showed that treatment with ASB significantly relieved the clinical symptoms of UC and liver injury by reducing disease activity index, inhibiting gut-derived LPS leakage, and improving colonic and hepatic injury, and its curative effect was better than Andro. Moreover, ASB effectively decreased the YAP-mediated colonic inflammation and TLR4/MyD88/NF-κB-mediated pro-inflammatory factor release in the liver. Both colonic and hepatic inflammation were associated with macrophage proinflammatory polarization, but they were significantly inhibited by ASB. ASB showed good safety in the treatment of UC and liver injury and has no nephrotoxicity as previously described. In conclusion, ASB has an effective protective effect on DSS-induced UC and liver injury, mainly by suppressing macrophage proinflammatory polarization from the gut-liver axis.

P1249: INHIBITION OF THE PENTOSE-PHOSPHATE-PATHWAY (PPP) MEDIATES MACROPHAGE REPROGRAMMING FROM SUPPORTIVE BYSTANDER FUNCTION TOWARDS ANTIBODY-DEPENDENT LYMPHOMA CELL CLEARANCE AND LYMPHOMA SUPPRESSION

Background: Treatment response in lymphoma depends on the interaction and functional status of macrophages as important effector cells for tumor cell clearance by antibody-dependent cellular phagocytosis (ADCP). In the tumor microenvironment (TME) there is a limited supply of nutrients which implicates metabolic reprograming of immune cells such as changed phagocytic capacity of macrophages. Aims: We aimed to identify metabolic pathways regulating macrophages in the TME to improve macrophage effector cell function in lymphoma therapy. Methods: We addressed macrophage lymphoma cell co-cultures using macrophage cell lines, primary murine macrophages and human monocyte-derived macrophages. For pathway inhibition specific compounds as well as shRNA-mediated knockdown were used for functional read-out of ADCP and viability in lymphoma co-culture. Macrophage reprogramming was assessed by microscopy, FACS-immunophenotyping, ELISA, (phospho)-proteomic and metabolomic assessment. C57BL/6 mice and the humanized aggressive lymphoma model hMB were used for in vivo treatment with the pentose phosphate pathway (PPP) inhibitor S3 and analysis of macrophage programming and overall survival. Results: Inhibition of AMP kinase, glycolysis or mitochondrial ATP-production did not increase the phagocytosis rate in ADCP co-cultures. Inhibiting the PPP induced an increased lymphoma cell phagocytosis by macrophages. Under PPP-inhibition, the oxygen consumption and the glycolysis of macrophages was increased and the cells developed a more activated morphology with formation of filopodia, which are needed for phagocytosis initiation. Moreover, a down-regulation of immune-inhibitory surface proteins PD-L1 and SIRP1α was seen and cytokine secretion was shifted from anti-inflammatory IL10 to pro-inflammatory IL6 secretion. PPP-inhibition of macrophages significantly reduced CLL-cell support in vitro abrogating the “nurse-like” effect of macrophages. The increased lymphoma cell clearance and phenotypic alterations of macrophages were also observed in PPP-enzyme knockdown of transketolase (TKT) and 6-phosphogluconatedehydrogenase (6PGD) in macrophages and by inhibiting the PPP in primary murine and human macrophages. In a multiomics assessment of PPP-inhibition on proteome, phosphoproteome and metabolome level, we found protein expression alterations regulating metabolism and immunity. A connection between PPP inhibition and changed immune profile by modulation of the Stat1-Irg1-itaconate axis was identified and validated. In a humanized lymphoma mouse model the addition of PPP-inhibitor S3 led to significant prolonged survival of lymphoma bearing mice and an increased maturation and pro-inflammatory polarization of macrophages. Summary/Conclusion: An increased maturation, activation and repolarization of macrophages towards a pro-inflammatory and phagocytic active phenotype was observed by inhibiting the pentose-phosphate-pathway (PPP). This led to a significant survival benefit in in vivo lymphoma mouse model. We were able to point out the underlying Stat1-Irg1-itaconate signalling axis, connecting metabolic regulation and immune-phenotype of macrophages. We hypothesize the PPP as a key regulator of macrophage function determining the support of malignant B cell growth versus the capacity of lymphoma cell clearance and thereby therapy outcome. By that, we have identified the PPP as a targetable modulator of macrophage polarization to improve efficacy of B cell lymphoma and CLL therapy.

Abstract 1995: β-Hydroxy-β-methylbutyrate promotes immunotherapy response and pro-inflammatory macrophage polarization in a mouse model of pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) continues to represent a critical unmet therapeutic need in the US, and is exacerbated by obesity. Current chemotherapeutic regimens poorly control PDAC progression, and immunotherapy has yet to be widely successful against PDAC. Obesity promotes a potently protumor PDAC microenvironment including suppression of antitumor immune response. The purpose of our studies was to test whether dietary supplementation with β-hydroxy-β-methylbutyrate (HMB), shown to be safe and well tolerated in cancer patients, could offset obesity-mediated exacerbation of PDAC and promote antitumor immunosurveillance. Methods: Lean and obese C57BL/6 mice were treated with HMB alone or in combination with anti-PD1 immunotherapy following subcutaneous injection of Panc02 PDAC cells. Bone marrow-derived macrophages (BMDM) were treated with HMB in vitro with or without lipopolysaccharide (LPS). Tumor/macrophage transcriptomic analysis using Affymetrix arrays followed by gene set enrichment was performed, and tumor microenvironment immune cell composition was determined by mass cytometry. Results: a) HMB cooperates with obesity and immunotherapy to reduce PDAC progression, as indicated by reduced tumor growth with the combination regimen of HMB + anti-PD1 relative to obese or anti-PD1 immunotherapy treated mice without HMB supplementation; b) HMB mitigates PDAC immunosuppression and promotes tumor immune surveillance, as demonstrated by induction of an antitumor immune-related gene expression profile in response to HMB supplementation; c) HMB promotes pro-inflammatory polarization of tumor-associated macrophages and BMDM, as shown by increased CD38 positivity and reduced Arg1 positivity in tumor-associated macrophages from HMB supplemented mice relative to control. Further, BMDM transcriptional response to HMB alone or HMB in combination with LPS paralleled that of BMDM treated with LPS relative to untreated LPS. Conclusion: Overall, our findings suggest HMB is an adjuvant for antitumor immune responses in PDAC. Moreover, HMB promotes immune surveillance in PDAC, synergizing with immunotherapy. Hence, HMB-induced suppression of PDAC tumor growth and promotion of immune surveillance may offer significant synergy with chemotherapies or immunotherapies in PDAC. Funding: This work was supported by R35CA197627 to SDH. Citation Format: Michael F. Coleman, Kristyn A. Liu, Alexander J. Pfeil, Suhas K. Etigunta, Xiaohu Tang, Laura M. Lashinger, Zhengrong Cui, Stephen D. Hursting. β-Hydroxy-β-methylbutyrate promotes immunotherapy response and pro-inflammatory macrophage polarization in a mouse model of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1995.

Abstract LB514: CD8 T cells and macrophage abundances associated with clinical benefit of tislelizumab in various tumor types

Abstract Background: Functionally activated immune cells (ICs) in the tumor microenvironment (TME) are critical to antitumor efficacy. Here, we report association between ICs and the clinical efficacy of tislelizumab (TIS), an anti-programmed cell death protein 1 monoclonal antibody, by examining tumor tissues from various tumor types in three pooled Phase 1/2 studies (NCT02407990, NCT04068519, NCT04004221). Methods:Available baseline tumor tissues from patients (pts) with advanced solid tumors who received TIS were tested with either multiplex-immunohistochemistry (m-IHC) (n=67, Opal automation Multiplex IHC kit) or gene expression profile (n=629, HTG EdgeSeq Precision Immuno-Oncology Panel). High/low cell density/signature scores were defined per median score, respectively. Median overall survival (OS) was estimated by the Kaplan-Meier method and log rank test was used to compare survival curves between pts with different biomarker levels. Results: Pts with a high CD68 density (CD68Hi) (n=34) had a longer OS compared with pts who had a low CD68 density (n=33), with a median OS of 15.0 vs 10.4 months, p=0.11. A weak association was observed between survival and CD8 cell density. When the two cell types were combined as a composite biomarker, pts with high CD8 (CD8Hi) and CD68Hi showed the longest OS (Table). A consistent finding was confirmed in the gene expression population (Table). Further TME analysis revealed that pts with CD8Hi and CD68Hi signature showed most elevated CD8 T cell cytotoxicity (CD8A, GNLY, GZMA, GZMB), T cell trafficking (CXCL9, CXCL10, CCL4, CCL5), MHCI antigen presentation (TAP1, TAP2, HLA.A/B/C) signatures/genes, and enriched expression of pro-inflammatory macrophage polarization pathway (STAT1, SLAMF7/8, ISG15). Conclusion: Co-enrichment of CD8 T cells and macrophages were associated with survival benefit and an immune-activated TME in pts with various tumor types treated with TIS. This observation warrants further investigation. Association between ICs and the clinical efficacy of TIS m-IHC analysis CD8Hi/CD68 Hi (n=24) CD8 Hi/CD68Lo (n=10) CD8 Lo/CD68Hi (n=10) CD8 Lo/CD68Lo (n=23) Median OS, months (95% CI) 15.7 (8.5, NA) 5.1 (0.8, 10.8) 6.3 (1.8, NA) 11.2 (4.0, 17.6) Gene expression analysis CD8Hi/CD68 Hi (n=202) CD8 Hi/CD68Lo (n=113) CD8 Lo/CD68Hi (n=113) CD8 Lo/CD68Lo (n=201) Median OS, months (95% CI) 14.9 (11.2, 19.2) 11.1 (7.1, 13.5) 7.7 (5.6, 11.4) 9.8 (7.4, 11.6) p value* 0.00033 *p value obtained from log-rank test CI, confidence interval; Hi, high density; IC, immune cell; m-IHC, multiplex immunohistochemistry; Lo, low density; NA, not available; OS, overall survival; TIS, tislelizumab Citation Format: Wei Shen, Tengfei Liu, Yang Shi, Jingwen Shi, Dan Wang, Liang Liang, Silu Yang, Xiaopeng Ma, Wei Jin, Pei Zhang, Ruiqi Huang, Yun Zhang, Zhirong Shen. CD8 T cells and macrophage abundances associated with clinical benefit of tislelizumab in various tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB514.

Mushroom Carboxymethylated β-d-Glucan Functions as a Macrophage-Targeting Carrier for Iron Oxide Nanoparticles and an Inducer of Proinflammatory Macrophage Polarization for Immunotherapy.

β-d-glucans have the potential of serving as both macrophage-targeted carriers and immune stimulators via inducing trained immunity in macrophages. In this study, a carboxymethylated β-glucan from mushroom sclerotium of Pleurotus tuber-regium (CMPTR) was combined with iron oxide nanoparticles (IONPs) to form nanocomplexes (CMPTR/IONPs) with particle size around 193 ± 7 nm, which could exert a concerted effect on inducing proinflammatory M1 phenotype macrophages for immunotherapy. This nanocomplex exhibited good stability and low cytotoxicity (over 80% cellular viability of RAW 264.7 and THP-1) and higher cellular uptake by murine macrophages compared with B16F10 cells (p < 0.05). CMPTR/IONPs could convert M2-like bone marrow-derived macrophages into M1 phenotypes with upregulated expression of pro-inflammatory cytokines (IL12 and TNF-α, p < 0.05) and reduced immune-suppressive cytokines (IL10 and TGF-β, p < 0.05). Such polarization was mediated by the combined signaling regulatory factors, including IONP-stimulated IRF5 and CMPTR-triggered TLRs-NF-κB pathways (p < 0.05). Accordingly, CMPTR could have a dual function as a macrophage-targeting carrier for IONPs and an immunostimulant to induce inflammatory M1 macrophage polarization for immunotherapy.

BCG hydrogel promotes CTSS-mediated antigen processing and presentation, thereby suppressing metastasis and prolonging survival in melanoma

BackgroundThe use of intralesional Mycobacterium bovis BCG (intralesional live BCG) for the treatment of metastatic melanoma resulted in regression of directly injected, and occasionally of distal lesions. However, intralesional-BCG is...

Inhibition of Cyclin-Dependent Kinase 9 Downregulates Cytokine Production Without Detrimentally Affecting Human Monocyte-Derived Macrophage Viability.

Cyclin-dependent kinase (CDK) inhibitor drugs (CDKi), such as R-roscovitine and AT7519, induce neutrophil apoptosis in vitro and enhance the resolution of inflammation in a number of in vivo models. This class of compounds are potential novel therapeutic agents that could promote the resolution of acute and chronic inflammatory conditions where neutrophil activation contributes to tissue damage and aberrant tissue repair. In this study we investigated CDKi effects on macrophage pro-inflammatory mediator production and viability. Treatment of human monocyte-derived macrophages (MDMs) with the CDKi AT7519 and R-roscovitine at concentrations that induce neutrophil apoptosis had no significant effect on control or LPS-activated MDM apoptosis and viability, and did not detrimentally affect MDM efferocytosis of apoptotic cells. In addition, enhanced efferocytosis, induced by the glucocorticoid dexamethasone, was also unaffected after a short time treatment with R-roscovitine. Macrophage cytokine responses to inflammatory stimuli are also of importance during inflammation and resolution. As a key target of CDKi, CDK9, is involved in protein transcription via the RNA polymerase II complex, we investigated the effect of CDKi drugs on cytokine production. Our data show that treatment with AT7519 significantly downregulated expression and release of key MDM cytokines IL-6, TNF, IL-10 and IL-1β, as well as markers of pro-inflammatory macrophage polarisation. R-Roscovitine was also able to downregulate inflammatory cytokine protein secretion from MDMs. Using siRNA transfection, we demonstrate that genetic knock-down of CDK9 replicates these findings, reducing expression and release of pro-inflammatory cytokines. Furthermore, overexpression of CDK9 in THP-1 cells can promote a pro-inflammatory phenotype in these cells, suggesting that CDK9 plays an important role in the inflammatory phenotype of macrophages. Overall, this study demonstrates that pharmacological and genetic targeting of CDK9 inhibits an inflammatory phenotype in human MDMs. As such these data indicate that CDK9 may be key to therapeutically targeting pro-inflammatory macrophage functions during chronic inflammation.

Diallyl disulfide ameliorates ethanol-induced liver steatosis and inflammation by maintaining the fatty acid catabolism and regulating the gut-liver axis

Diallyl disulfide (DADS) has been suggested to possess hepatoprotection against alcoholic liver disease (ALD) by a couple of pilot studies, while the underlying mechanisms remain largely unknown. This study aimed to investigate the hepatoprotective effects of DADS against ethanol-induced liver steatosis and early inflammation by using the chronic-plus-binge mice model and cultured J774A.1 macrophages and AML12 hepatocytes. We found that DADS significantly attenuated ethanol-induced elevation of serum aminotransferase activities, accumulation of liver triglyceride, hepatocytes apoptosis, oxidative stress, infiltration of macrophages and neutrophils, and proinflammatory polarization of macrophages in mice livers. In addition, chronic-plus-binge drinking induced apparent intestinal mucosa damage and disturbance of gut microbiota, endotoxemia, and activation of hepatic NF-κB signaling and NLRP3 inflammasome, which was inhibited by DADS. In vitro studies using cocultured AML12/J774A.1 cells showed that DADS suppressed ethanol/LPS–induced cell injury and inflammatory activation of macrophages. Furthermore, DADS ameliorated ethanol-induced decline of peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase 1 (CPT1), and phosphorylated AMP-activated protein kinase (AMPK) protein levels in mice livers and AML12 cells. These results demonstrate that DADS could prevent ethanol-induced liver steatosis and early inflammation by regulating the gut-liver axis and maintaining fatty acid catabolism.

Ccl3 enhances docetaxel chemosensitivity in breast cancer by triggering proinflammatory macrophage polarization

BackgroundAlthough the antitumor efficacy of docetaxel (DTX) has long been attributed to the antimitotic activities, its impact on the tumor microenvironment (TME) has recently gained more attention. Macrophages are a...