Increased Macrophage Phagocytosis Research Articles

IMMU-27. INHIBITION OF THE EXTRACELLULAR MATRIX PROTEIN EFEMP1/FIBULIN-3 REDUCES IMMUNOSUPPRESIVE SIGNALING IN TUMOR STEM CELLS AND INCREASES MACROPHAGE ACTIVATION AGAINST GLIOBLASTOMA

Abstract Glioblastoma tumors remain a formidable challenge for immune-based treatments because of their molecular heterogeneity, poor immunogenicity, and growth in the largely isolated and immunosuppresive neural environment. As the tumor grows, GBM cells change the composition and architecture of the neural extracellular matrix (ECM), affecting the mobility, survival, and function of immune cells such as tumor-associated microglia and infiltrated macrophages (TAMs). We have previously described the unique expression of the ECM protein EFEMP1/fibulin-3 in GBM compared to normal brain and demonstrated that this secreted protein promotes growth and resistance of the GBM stem cell (GSC) population. Here, we describe a novel immunomodulatory role of fibulin-3 and the immuno-boosting effects of targeting this ECM protein. Mice carrying fibulin-3-deficient intracranial tumors showed increased myeloid infiltration and reduced expression of TAM pro-tumoral markers (Arginase, CD206) compared to controls. The opposite was observed in orthotopic tumors overexpressing fibulin-3. In silico dataset analysis revealed positive correlation of fibulin-3 with an immunosuppresive signature in GBM, which was validated in GBM stem cells (GSCs). Accordingly, expression of fibulin-3 by GSCs promoted anti-inflammatory polarization of co-cultured macrophages. We further demonstrated that fibulin-3 regulated the expression of immunosuppresive signals (CSF-1, TGFbeta) and the innate immune checkpoint CD47 in GSCs via autocrine activation of NF-kB signaling. Immunosuppresive signals in GSCs were downregulated by knockdown of fibulin-3 or inhibition of this protein with an anti-fibulin-3 antibody (mAb428.2). Genetic or antibody-mediated targeting of fibulin-3 in GSCs co-cultured with macrophages (U937 and THP1 lines) increased macrophage phagocytosis and reduced the viability of the tumor cells. Furthermore, local delivery of mAb428.2 in mice carrying intracranial GBM resulted in increased infiltration of TAMs expressing pro-inflammatory markers and reducing tumor viability. Our findings show that anti-fibulin-3 approaches, which impact the tumor ECM, can diminish immunosuppression in GBM and boost innate immune responses against the tumor.

Abstract A051: Bitter agonists increase macrophage phagocytosis of head and neck squamous cell carcinoma cells

Abstract Head and neck squamous cell carcinoma (HNSCC) is the 7th most commonly diagnosed cancer worldwide and is expected to increase in incidence by 30% by 2030. The HNSCC microenvironment is characterized by substantial macrophage infiltration. However, tumor-associated macrophages often cultivate an immunosuppressive environment that aids tumor progression. Previous work has shown that macrophages express bitter taste receptors (T2Rs), a class of GPCRs that can be activated with bitter compounds. Previous work has also demonstrated that stimulation of T2Rs in macrophages with bitter agonists, such as denatonium benzoate, increases phagocytosis of bacteria, but the impact of bitterants on macrophage phagocytosis of tumor cells is unknown. The goal of this study was to assess the impact of bitter compounds on macrophage phagocytosis of HNSCC cells in vitro. Human peripheral blood mononuclear cells obtained from healthy donors were used to generate monocyte-derived macrophages (MDMs). To investigate the effects of bitter agonist stimulation on macrophage phagocytosis, CFSE-labeled MDMs and DAPI-labeled HNSCC cell line SCC47, were co-stimulated with denatonium benzoate, and macrophage phagocytosis was quantified. This revealed that treatment with denatonium benzoate led to a 70% increase in macrophage phagocytosis compared to controls. Additional analysis revealed a 1.8-fold increase in F-actin in MDMs in response to denatonium benzoate measured via CellMask™ Actin staining, and that macrophages had generated F-actin positive tunneling nanotubes (TNTs). Further characterization with live cell imaging showed that these TNTs were being utilized as a means of mitochondrial transfer as observed by MitoTracker™ and CellMask™ Actin staining. Furthermore, there was a 2.5-fold decrease in mitochondrial ATP production in MDMs stimulated with denatonium benzoate, possibly indicating metabolic reprogramming. Timelapse imaging of MDMs demonstrated a reduction in mitochondrial ATP production, with a concomitant increase in F-actin production. These observations were confirmed in Phorbol 12-myristate 13-acetate differentiated THP-1 cells. Taken together, these results indicate that bitter taste receptor stimulation may contribute to a more activated macrophage phenotype denoted by a decrease in mitochondrial ATP production, increased F-actin staining, and increased phagocytosis of tumor cells. Citation Format: Brianna L Hill, Sarah M Sywanycz, Zoey A Miller, Robert J Lee, Ryan M Carey. Bitter agonists increase macrophage phagocytosis of head and neck squamous cell carcinoma cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A051.

Combinatorial macrophage induced innate immunotherapy against Ewing sarcoma: Turning “Two Keys” simultaneously

BackgroundMacrophages play important roles in phagocytosing tumor cells. However, tumors escape macrophage phagocytosis in part through the expression of anti-phagocytic signals, most commonly CD47. In Ewing sarcoma (ES), we found that tumor cells utilize dual mechanisms to evade macrophage clearance by simultaneously over-expressing CD47 and down-regulating cell surface calreticulin (csCRT), the pro-phagocytic signal. Here, we investigate the combination of a CD47 blockade (magrolimab, MAG) to inhibit the anti-phagocytic signal and a chemotherapy regimen (doxorubicin, DOX) to enhance the pro-phagocytic signal to induce macrophage phagocytosis of ES cells in vitro and inhibit tumor growth and metastasis in vivo.MethodsMacrophages were derived from human peripheral blood monocytes by granulocyte–macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). Flow cytometry- and microscopy-based in-vitro phagocytosis assays were performed to evaluate macrophage phagocytosis of ES cells. Annexin-V assay was performed to evaluate apoptosis. CD47 was knocked out by CRISPR/Cas9 approach. ES cell-based and patient-derived-xenograft (PDX)-based mouse models were utilized to assess the effects of MAG and/or DOX on ES tumor development and animal survival. RNA-Seq combined with CIBERSORTx analysis was utilized to identify changes in tumor cell transcriptome and tumor infiltrating immune cell profiling in MAG and/or DOX treated xenograft tumors.ResultsWe found that MAG significantly increased macrophage phagocytosis of ES cells in vitro (p < 0.01) and had significant effect on reducing tumor burden (p < 0.01) and increasing survival in NSG mouse model (p < 0.001). The csCRT level on ES cells was significantly enhanced by DOX in a dose- and time-dependent manner (p < 0.01). Importantly, DOX combined with MAG significantly enhanced macrophage phagocytosis of ES cells in vitro (p < 0.01) and significantly decreased tumor burden (p < 0.01) and lung metastasis (p < 0.0001) and extended animal survival in vivo in two different mouse models of ES (p < 0.0001). Furthermore, we identified CD38, CD209, CD163 and CD206 as potential markers for ES-phagocytic macrophages. Moreover, we found increased M2 macrophage infiltration and decreased expression of Cd209 in the tumor microenvironment of MAG and DOX combinatorial therapy treated tumors.ConclusionsBy turning “two keys” simultaneously to reactivate macrophage phagocytic activity, our data demonstrated an effective and highly translatable alternative therapeutic approach utilizing innate (tumor associated macrophages) immunotherapy against high-risk metastatic ES.

Assessment of immunomodulatory effects of five commonly used parabens on human THP-1 derived macrophages: Implications for ecological and human health impacts

Parabens are widely used as broad-spectrum anti-microbials and preservatives in food, cosmetics, pharmaceuticals, and personal care products. Studies suggest that the utilization of parabens has substantially increased over the past years, particularly during the global pandemic of coronavirus disease 2019 (COVID-19). Although parabens are generally recognized as safe by the U.S. FDA, some concerns have been raised regarding the potential health effects of parabens associated with immunotoxicity. Herein, we comprehensively investigated several key characteristics of immunotoxicants of five commonly used parabens (methyl-, ethyl-, propyl-, butyl-, and benzyl parabens) in human THP-1 derived macrophages, which are effector cells serving as a first line of host defense against pathogens and tumor immunosurveillance. The results indicate parabens, at concentrations found in humans and biota, significantly dampened macrophage chemotaxis and secretion of major pro-inflammatory cytokines (TNF-α and IL-6) and anti-inflammatory cytokine (IL-10), corroborating the mRNA expression profile. Furthermore, some parabens were found to markedly alter macrophage adhesion and cell surface expression of costimulatory molecules, CD80+ and CD86+, and significantly increase macrophage phagocytosis. Collectively, these findings heighten awareness of potential immunotoxicity posed by paraben exposure at biologically relevant concentrations, providing implications for human health and ecological risks associated with immune dysfunctions.

Enhanced treatment of breast cancer brain metastases with oncolytic virus expressing anti-CD47 antibody and temozolomide

Limited therapeutic options are available for patients with breast cancer brain metastases (BCBM), and thus there is an urgent need for novel treatment approaches. We previously engineered an effective oncolytic herpes simplex virus-1 (oHSV) expressing a full-length anti-CD47 monoclonal antibody (mAb) with a human IgG1 scaffold (OV-αCD47-G1) that was used to treat both ovarian cancer and glioblastoma. Here, we demonstrate that the combination of OV-αCD47-G1 and temozolomide (TMZ) improve outcomes in preclinical models of BCBM. The combination of TMZ with OV-αCD47-G1 synergistically increased macrophage phagocytosis against breast tumor cells and led to greater activation of NK cell cytotoxicity. Additionally, the combination of OV-αCD47-G1 with TMZ significantly prolonged the survival of tumor-bearing mice when compared to TMZ or OV-αCD47-G1 alone. Combination treatment with the mouse counterpart of OV-αCD47-G1, termed OV-A4-IgG2b, also enhanced mouse macrophage phagocytosis, NK cell cytotoxicity, and survival in an immunocompetent model of mice bearing BCBM compared to TMZ or OV-A4-IgG2b alone. Collectively, these results suggest that OV-αCD47-G1 combined with TMZ should be explored in patients with BCBM.

Proinflammatory polarization strongly reduces human macrophage in vitro phagocytosis of tumor cells in response to CD47 blockade.

Antibody-based CD47 blockade aims to activate macrophage phagocytosis of tumor cells. However, macrophages possess a high degree of phenotype heterogeneity that likely influences phagocytic capacity. In murine models, proinflammatory (M1) activation increases macrophage phagocytosis of tumor cells, but in human models, results have been conflicting. Here, we investigated the effects of proinflammatory polarization on the phagocytic response of human monocyte-derived macrophages in an in vitro model. Using both flow cytometry-based and fluorescence live-cell imaging-based phagocytosis assays, we observed that mouse monoclonal anti-CD47 antibody (B6H12) induced monocyte-derived macrophage phagocytosis of cancer cells in vitro. Proinflammatory (M1) macrophage polarization with IFN-γ+LPS resulted in a severe reduction in phagocytic response to CD47 blockade. This reduction coincided with increased expression of the antiphagocytic membrane proteins LILRB1 and Siglec-10 but was not rescued by combination blockade of the corresponding ligands. However, matrix metalloproteinase inhibitors (TAPI-0 or GM6001) partly restored response to CD47 blockade in a dose-dependent manner. In summary, these data suggest that proinflammatory (M1) activation reduces phagocytic response to CD47 blockade in human monocyte-derived macrophages.

Abstract 741: Distinct functional effects of ILT family members in mediating the activity of an ILT2/ILT4/ILT5 cross-reactive monoclonal antibody in releasing the suppression on innate and adaptive anti-tumor immunity

Abstract The expression of ILT (LILRB) family members and their major ligand HLA-G within solid tumors are associated with immune suppression and poor patient survival. Blocking multiple ILT family members might be more potent than targeting individual members by overcoming compensatory resistances within tumor microenvironment. Based on this hypothesis, we developed a monoclonal antibody (mAb) LITCHI 7 which reacts to three major ILT family members, ILT2, ILT4 and ILT5, and blocks ligand binding to these receptors. LITCHI 7 exhibits a broad binding capability to a wide range of human myeloid and T cell subsets that express ILT2, ILT4, and/or ILT5. Binding to monocyte-derived dendritic cells (moDC), LITCHI 7 released the inhibition on activating Fcγ receptor. On LPS-treated macrophages, LITCHI 7 enhanced inflammatory macrophage differentiation. The effect is primarily mediated by blocking ILT4 since ILT4 mono-specific mAb is as potent as cross-reactive LITHI 7 in modulating myeloid differentiation although both ILT2 and ILT5 are highly expressed by different myeloid subsets. The result was further confirmed by CRISPR Cas9 knock down of ILT2 or ILT5. In the absence of either ILT2 or ILT5, the accumulation of CD14+ monocytes and inflammatory macrophages were not changed during differentiation. While different ILT family members are generally enriched on myeloid cells, ILT2 is also expressed on NK cells and effector memory CD45RA+ T cells (Temra). Blocking ILT2 by LITCHI 7 enhanced TCR signaling when co-culturing the MART-1 specific T cell reporter line with MART-1/HLA-G positive melanoma cells. The effect of LITCHI 7 is mediated by ILT2 blockade since knocking out ILT2, but not ILT4 and ILT5, reduced accumulation of Temra in activated PBMC. To assess whether immune modulation of LITCHI 7 can be translated into anti-tumor activity by blocking different ILT family members, tumor killing assays were performed using innate and adaptive immune effector cells. LITCHI7 enhanced NK cell-mediated cytotoxicity on HLA-G+ tumor cells by blocking ILT2. Both ILT2 and ILT4 blockade play a role in macrophage phagocytosis on lymphoma cells since LITCHI 7 as well as individual anti-ILT2 or ILT-4 mAbs increased macrophage phagocytosis to a similar level. Finally, LITCHI 7 could boost in vitro T cell-mediated killing of CD19+ lymphoma by CD19 specific CAR-T cells in the presence of CD33+ myeloid cells. In summary, LITCHI 7 can enhance both innate and adaptive anti-tumor immunity through ILT2 or ILT4 blockade directly on effector cells or indirectly through myeloid cell reprogramming. As clinical trials are ongoing by targeting ILT2 and ILT4, blocking multiple ILT family members through a single cross-reactive mAb may provide broad anti-tumor effects and advantages when combining with additional oncology treatments. Citation Format: Shiming Ye, Dong Zhang, Brenal Singh, Jaishree Bankoti, Diane Cohen, Siusze Tan, Donghee Choi, Jonathan Hickson, Min-Zu Wu, Urvi Kolhatkar, Mikhail Binnewies, Wei Jin, Jason Hall, John Engelhardt, Alexander Shoemaker. Distinct functional effects of ILT family members in mediating the activity of an ILT2/ILT4/ILT5 cross-reactive monoclonal antibody in releasing the suppression on innate and adaptive 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 741.

CXCR2 inhibition in G-MDSCs enhances CD47 blockade for melanoma tumor cell clearance

The use of colony-stimulating factor-1 receptor (CSF1R) inhibitors has been widely explored as a strategy for cancer immunotherapy due to their robust depletion of tumor-associated macrophages (TAMs). While CSF1R blockade effectively eliminates TAMs from the solid tumor microenvironment, its clinical efficacy is limited. Here, we use an inducible CSF1R knockout model to investigate the persistence of tumor progression in the absence of TAMs. We find increased frequencies of granulocytic myeloid-derived suppressor cells (G-MDSCs) in the bone marrow, throughout circulation, and in the tumor following CSF1R deletion and loss of TAMs. We find that G-MDSCs are capable of suppressing macrophage phagocytosis, and the elimination of G-MDSCs through CXCR2 inhibition increases macrophage capacity for tumor cell clearance. Further, we find that combination therapy of CXCR2 inhibition and CD47 blockade synergize to elicit a significant anti-tumor response. These findings reveal G-MDSCs as key drivers of tumor immunosuppression and demonstrate their inhibition as a potent strategy to increase macrophage phagocytosis and enhance the anti-tumor efficacy of CD47 blockade in B16-F10 melanoma.

Equine β-defensin 1 regulates cytokine expression and phagocytosis in S. aureus-infected mouse monocyte macrophages via the Paxillin-FAK-PI3K pathway

β-defensin-1 (BD-1) is a rich source of disulfide bonds and antibacterial peptides that exhibit direct bactericidal function. The expression of BD-1 is primarily induced by external stimulation and is known to correlate with TLR-mediated inflammation, suggesting its association with innate immune responses. Equine β-defensin-1 (eBD-1) belongs to the BD-1 family. Our previous study demonstrated that eBD-1 enhances cytokine expression and promotes macrophage phagocytosis of S. aureus, although the underlying mechanism remains unknown. In this study, we utilized a PI-3K inhibitor (PKI-402) to treat eBD-1 -treated S. aureus-infected macrophages in vitro. Our results revealed that PKI-402 decreased the expression of eBD-1-promoted TNF-α, IL-6, CXCL10, CD40, RANTES, and p65 mRNA. To further investigate the relationship between eBD-1 and phagocytosis, we examined the expression of paxillin and FcγRIII (CD16 receptor) using western blot and immunofluorescence techniques. Our findings demonstrated that eBD-1 enhanced CD16 and paxillin expression in S. aureus -infected macrophages. Considering the correlation between paxillin expression and focal adhesion kinase (FAK), we transfected FAK siRNA into macrophages and evaluated paxillin expression using western blot analysis. Additionally, we quantified the number of S. aureus phagocytosed by macrophages. The results indicated a reduction in both paxillin expression and the number of S. aureus phagocytosed by macrophages upon FAK siRNA treatment. Our study showed the eBD-1 promotes cytokine mRNA expression in S. aureus-infected macrophages regulated by PI-3K-NF-κB pathway, and it increases macrophage phagocytosis of S. aureus associated with the FAK-paxillin signaling pathway.

EGFR-Induced and c-Src-Mediated CD47 Phosphorylation Inhibits TRIM21-Dependent Polyubiquitylation and Degradation of CD47 to Promote Tumor Immune Evasion.

Tumor cells often overexpress immune checkpoint proteins, including CD47, for immune evasion. However, whether or how oncogenic activation of receptor tyrosine kinases, which are crucial drivers in tumor development, regulates CD47 expression is unknown. Here, it is demonstrated that epidermal growth factor receptor (EGFR) activation induces CD47 expression by increasing the binding of c-Src to CD47, leading to c-Src-mediated CD47 Y288 phosphorylation. This phosphorylation inhibits the interaction between the ubiquitin E3 ligase TRIM21 and CD47, thereby abrogating TRIM21-mediated CD47 K99/102 polyubiquitylation and CD47 degradation. Knock-in expression of CD47 Y288F reduces CD47 expression, increases macrophage phagocytosis of tumor cells, and inhibits brain tumor growth in mice. In contrast, knock-in expression of CD47 K99/102R elicits the opposite effects compared to CD47 Y288F expression. Importantly, CD47-SIRPα blockade with an anti-CD47 antibody treatment significantly enhances EGFR-targeted cancer therapy. In addition, CD47 expression levels in human glioblastoma (GBM) specimens correlate with EGFR and c-Src activation and aggravation of human GBM. These findings elucidate a novel mechanism underlying CD47 upregulation in EGFR-activated tumor cells and underscore the role of the EGFR-c-Src-TRIM21-CD47 signaling axis in tumor evasion and the potential to improve the current cancer therapy with a combination of CD47 blockade with EGFR-targeted remedy.

Blocking Serum Amyloid-P Component from Binding to Macrophages and Augmenting Fungal Functional Amyloid Increases Macrophage Phagocytosis of Candida albicans.

Candida-macrophage interactions are important immune defense responses associated with disseminated and deep-seated candidiasis in humans. Cells of Candida spp. express functional amyloids on their surfaces during the pathogenesis of disseminated candidiasis. These amyloids become decorated with serum amyloid P-component (SAP) that binds to Candida cells and macrophages and downregulates the cellular and cytokine response to the fungi. In this report, further characterization of the interactions of SAP and fungal functional amyloid are demonstrated. Blocking the binding of SAP to macrophage FcγR1 receptors increases phagocytosis of yeast cells; seeding a pro-amyloid-forming peptide on the yeast cell surface also increases phagocytosis of yeasts by macrophages; and, lastly, miridesap, a small palindromic molecule, prevents binding of SAP to yeasts and removes SAP that is bound to C. albicans thus, potentially increasing phagocytosis of yeasts by macrophages. Some, or all, of these interventions may be useful in boosting the host immune response to disseminated candidiasis.

A Sampling of Highlights from the Literature.

Lactic acid is a metabolic checkpoint for Treg cells (from Jynto via Wikimedia Commons)The intratumoral ratio of CD8+ T cells to regulatory T (Treg) cells impacts responsiveness to PD-1 blockade. Kumagai et al. find the ratio of CD8+ T cells to Treg cells is low in highly glycolytic tumors, including MYC-amplified tumors and liver metastases, and that expression of PD-1 is lower in CD8+ T cells than Treg cells. This effect is mediated by tumor-derived lactic acid and leads to resistance to PD-1 blockade in mouse models of Myc-overexpressing colon cancer and intrahepatic tumors. Blocking lactic-acid production or its uptake by Treg cells overcomes this resistance, suggesting potential new targets for cancer immunotherapy.Kumagai S, …, Nishikawa H.Cancer Cell2022 Feb 14;40:201–18.e9.Pancreatic cancer can be protected from CAR T cells by N-glycans (from Neelima Shah and Edna Cukierman via NCI Visuals Online)Chimeric antigen receptor (CAR) T-cell therapy has limited efficacy against solid tumors. Greco et al. identify expression of N-linked glycans as a targetable barrier to killing of solid tumors by CAR T cells. Knockout of mannoside acetyl-glucosaminyltransferase 5 and exposure to 2-deoxy-D-glucose (2DG), both of which disrupt N-glycan expression, render T3M-4 human pancreatic cancer cells sensitive to killing by CD44v6-targeted CAR T cells in vitro. Treatment with 2DG enhances tumor-cell killing by CAR T cells in xenograft models of pancreatic, ovarian, bladder, and lung cancer, suggesting a new approach to improve CAR T-cell therapy for solid tumors.Greco B, …, Casucci M.Sci Transl Med2022 Jan 19;14:eabg3072.Blocking “don’t eat me” signals increases macrophage phagocytosis (from QuidsUp)Modulation of macrophage phagocytosis is a tumor escape mechanism. Li et al. find a role for signaling lymphocytic activation molecule (SLAM) family receptors (SFR) in limiting macrophage phagocytosis by hematopoietic tumors. Specifically, SLAMF3 and SLAMF4 are identified as “don’t eat me” receptors on macrophages. Deficiency in SFRs, with and without CD47 deficiency, increases phagocytosis of hematopoietic cells (i.e., “self”) and malignant cells, highlighting a novel signaling pathway that can modulate the function of macrophages in the tumor microenvironment.Li D, …, Dong Z.Sci Immunol2022 Jan 21;7:eabj5501.HLA-A*03 is associated with poor response to immune checkpoint blockade (from Pdeitiker via Wikimedia Commons)Human leukocyte antigens (HLA) play a vital role in immunity, and thus, could impact immunotherapy efficacy. Using multiple large-scale datasets, Naranbhai et al. identify HLA-A*03 as a biomarker of response to immunotherapy but not response to other types of cancer treatment. Expression of this HLA associates with worse progression-free survival and lower responses to immune checkpoint inhibitors, which was validated in several independent cohorts of patients with various tumor types.Naranbhai V, …, Carrington M. Lancet Oncol 2022 Jan 1;23:172–84.Understanding of the NSCLC immune landscape continues to deepen (from Librepath via Wikimedia Commons)Deepening understanding of the immune landscape of non–small cell lung cancer (NSCLC) can help identify biomarkers of response to immunotherapy. Using single-cell RNA sequencing and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), Leader et al. find a strong correlation between PDCD1+CXCL13+ activated T cells, IgG+ plasma cells, and SPP1+ macrophages; they refer to these cells collectively as the lung cancer activation module (LCAM). The LCAMhi phenotype correlates with tumor mutational burden, TP53 mutations, and response to anti–PD-L1 therapy but not chemotherapy, suggesting that it could be a new biomarker of NSCLC response to immunotherapy.Leader AM, …, Merad M. Cancer Cell 2021 Dec 13;39:1594–609.e12.TRIB3 regulates CD8+ T-cell exclusion in the tumor microenvironment (from Max Pixel)Colorectal cancer resistance to immune checkpoint therapy is partly attributable to a lack of tumor-infiltrating T cells. Shang et al. report that TRIB3 expression in tumor cells correlates with CD8+ T-cell exclusion in mouse models of colorectal cancer and human samples. TRIB3 expression inhibits expression of the T cell–recruiting chemokine CXCL10 by activating STAT3, which inhibits transcription of STAT1 and subsequently CXCL10. Inhibition of the P300 acetylase, which acetylates TRIB3 and prevents E3 ligase–mediated degradation, improves CD8+ T-cell infiltration and sensitizes colorectal tumors to checkpoint blockade in vivo, suggesting a therapeutic strategy to improve immunotherapy efficacy in immunologically “cold” colorectal tumors.Shang S, …, Hua F. Sci Transl Med 2022 Jan 5;14:eabf0992.

Multi-Walled Carbon Nano-Tubes (MWCNTs) Accelerate Leukaemia Development in Mice: Implications for Drug Development in Humans

Biomedical applications of nano-technology including carbon nano-tubes (CNTs) are of considerable recent interest. However, relatively little attention is given to potential biological effects including cancer-induction or acceleration resulting from long-term exposures. We investigated whether and how multi-walled carbon nano-tubes (MWCNTs) affected leukaemia development in a mouse model. We found injections of MWCNTs accelerated murine leukaemia virus (MLV)-induced leukaemia development in normal mice with the magnitude of acceleration correlated with extent of MWCNT exposure. In contrast, injection of MWCNTs to immune-deficient mice caused no acceleration. Co-treatment of normal mice with MWCNTs and rapamycin reduced or eliminated accelerated leukaemia development. We found exposure to MWCNTs induced phagocytosis by macrophages and production of pro-inflammatory cytokines in normal mice and in cultured RAW264.7 cells. Methoxy-polyethylene glycol amine modified MWCNTs (PEG-MWCNTs) did not accelerate leukaemia development in normal mice, an effect correlated with reduced macrophage phagocytosis and reduced production of pro-inflammatory cytokines. We also found increased levels of pro-inflammatory cytokines induced by exposure to MWCNTs in normal mice increased intra-cellular reactive oxide species (ROS) levels resulting in increased cell proliferation and enhanced survival of the leukaemia cells. Our data indicate exposure to MWCNTs accelerates leukaemia development in a virus-induced leukaemia model. This effect is associated with activation of macrophages and increased levels of pro-inflammatory cytokines resulting in increased intra-cellar ROS levels. Our data have implications for the application of nano-technology to drug development in humans. For example, some MWCNTs are of the same size as asbestos fibers proved to cause mesothelioma in mice and humans. Also, increased macrophage phagocytosis and production of pro-inflammatory cytokines are reported in these settings. Our data also suggest interventions which might reduce a theoretical increased cancer-risk or acceleration of cancer development resulting from MWCNT exposures. DisclosuresNo relevant conflicts of interest to declare.

Anti-CD47 antibody synergizes with cisplatin against laryngeal cancer by enhancing phagocytic ability of macrophages.

Cisplatin is mainly used in late-stage or recurrent laryngeal cancer patients. However, the effect of the chemotherapy is limited due to cisplatin resistance. Therefore, we explored the synergized role of immunosuppressive mediator with cisplatin in laryngeal cancer. Cancer cells isolated from tissues of patients with laryngeal cancer were treated with cisplatin to screen the potential immunosuppressive mediator, whose synergized effects with cisplatin were explored both in vivo and in vitro. CD47 was selected for its high expression in cisplatin-treated laryngeal cancer cells. Blocking CD47 expression using its neutralizing antibody (aCD47) synergized with cisplatin to increase macrophage phagocytosis in a co-culture system of human epithelial type 2 (Hep-2) cancer cells with tumor-associated macrophages (TAMs). Moreover, aCD47 together with cisplatin prevented tumor growth by inhibiting proliferation of cancer cells and the secretion of proinflammatory cytokines, as well as by inducing the apoptosis of cancer cells and phagocytosis of TAMs in a Hep-2-implanted mouse tumor model. aCD47 synergized with cisplatin against laryngeal cancer by enhancing the phagocytic ability of TAMs, and the combined therapy of cisplatin and aCD47 might serve as a novel therapeutic strategy against laryngeal cancer.

Ethanol Extract of Spondias pinnata Leaves Reduce Parasite Number and Increase Macrophage Phagocytosis Capacity of Mice Infected by Plasmodium berghei

BACKGROUND: Currently, there is no vaccine against malaria in humans, the development of resistance to anti-malarial drugs, causing the need to find new alternatives to overcome malaria infections. This study aimed to determine effect of Spondias pinnata in increasing cellular immunity, especially phagocytosis activity of peritoneal macrophages against Plasmodium berghei infection.METHODS: This was an experimental study with two stages of research, each stage requires 36 Balb/c mice, aged 2 months and weight 20-25 grams. After one week of acclimatization, the mice were put into 6 different groups, each group consisted of 6 mice. The negative control was a group of mice given distilled water for 14 days then infected by P. berghei in the 15th day. Meanwhile, T1, T2, T3, T4 and T5 groups were given S. pinnata leaves ethanol extract with dose of 25, 50, 100, 200 and 400 mg/kg body weight (BW)/day, respectively, and then infected by P. berghei in the 15th day.RESULTS: The results showed that the lowest parasitemia and the highest capacity of macrophage to phagocytose latex was found in treatment group T3 that received 50 mg/kg BW of S. pinnata leaves ethanol extract. Based on analysis of the Pearson correlation test, there was a significant correlation between percent phagocytosis and parasitemia (p&lt;0.05).CONCLUSION: Ethanol extract of S. pinnata leaves lower the parasite number of P. berghei in Balb/c mice and increase the capacity of macrophage to phagocytose latex. However, the mechanisms of how S. pinnata leaves extract in activating phagocytosis capacity and reducing parasitemia still need further investigation.KEYWORDS: phagocytosis, Plasmodium berghei, parasite number, Spondias pinnata

Demethoxycurcumin Promotes Macrophage Cell Population and Phagocytosis in WEHI-3 Cell-generated Leukemia BALB/c Mice In Vivo.

Demethoxycurcumin (DMC), one of the components of curcuminoids, has antitumor activities in many human cancer cells and is known to induce apoptosis in human leukemia cells. However, there are no reports showing the effects of DMC on the immune response in leukemia mice in vivo. Herein, we evaluated the impact of DMC on immune responses in WEHI-3-generated leukemia mice in vivo. Fifty male BALB/c mice were separated randomly into five groups. Group I is normal mice, and groups II-V mice of generated leukemia by WEHI-3 cells. Group II-V mice were intraperitoneally injected with dimethyl sulfoxide (DMSO, as the positive control), 15, 30, and 60 mg/kg of DMC, respectively, every two days for 14 days. The body weight, blood, peritoneal fluid, liver, and spleen were individually analyzed. DMC did not significantly affect animal appearance and body weight. It decreased liver and spleen weight at a high dose. DMC did not affect the cluster of differentiation 3 (CD3) and CD19 cell populations but induced decrease of CD11b at 30 mg/kg treatment. However, DMC at low dose significantly increased the cluster of macrophage (Mac-3) cell populations, but at high dose it decreased them. DMC increased macrophage phagocytosis from peripheral blood mononuclear cells at 15 mg/kg treatment and peritoneal cavity at 15, 30 and 60 mg/kg of DMC treatments. DMC did not significantly affect the cytotoxic activity of natural killer (NK) cells. Furthermore, DMC decreased B and T cell proliferation at high doses. DMC elevated macrophage phagocytosis in leukemia mice in vivo.

Structural characterization and immunomodulatory activity of a heterogalactan from Panax ginseng flowers

A neutral polysaccharide fraction (WGFPN) was isolated from Panax ginseng flowers. Monosaccharide composition and HPSEC-MALLS-RI (high-performance size exclusion chromatography coupled with multi-angle laser light scattering detector and refractive index detector) analyses showed WGFPN was a heterogalactan with a molecular weight of 11.0 kDa. Methylation, 1D/2D NMR (nuclear magnetic resonance) spectra and enzymatic hydrolysis methods were used to characterize the structure of WGFPN. It possessed a less branched (1 → 4)-β-D-galactan and a significantly branched (1 → 6)-β-D-galactan. The side chains of (1 → 6)-β-D-galactan were branched with α-L-1,5-Araf and t-α-L-Araf residues at O-3. Trace amount of 1,4-linked Glcp, terminal Galp, terminal Glcp and terminal Manp residues might attached to the 1,6-linked galactan through O-3 or 1,4-linked galactan through O-6 as side chains. WGFPN could activate RAW264.7 macrophages through increasing macrophage phagocytosis, releasing NO and secreting TNF-α, IL-6, IFN-γ and IL-1β in vitro. Moreover, WGFPN could enhance the immunity of cyclophosphamide (CTX)-induced immunosuppressed mice in vivo. Hence, WGFPN might be a potential natural immunomodulatory agent.

The Annexin A1/FPR2 pathway controls the inflammatory response and bacterial dissemination in experimental pneumococcal pneumonia.

Streptococcus pneumoniae is a major cause of community-acquired pneumonia leading to high mortality rates. Inflammation triggered by pneumococcal infection is necessary for bacterial clearance but must be spatially and temporally regulated to prevent further tissue damage and bacterial dissemination. Annexin A1 (AnxA1) mainly acts through Formyl Peptide Receptor 2 (FPR2) inducing the resolution of inflammation. Here, we have evaluated the role of AnxA1 and FPR2 during pneumococcal pneumonia in mice. For that, AnxA1, Fpr2/3 knockout (KO) mice and wild-type (WT) controls were infected intranasally with S pneumoniae. AnxA1 and Fpr2/3 KO mice were highly susceptible to infection, displaying uncontrolled inflammation, increased bacterial dissemination, and pulmonary dysfunction compared to WT animals. Mechanistically, the absence of AnxA1 resulted in the loss of lung barrier integrity and increased neutrophil activation upon S pneumoniae stimulation. Importantly, treatment of WT or AnxA1 KO-infected mice with Ac2-26 decreased inflammation, lung damage, and bacterial burden in the airways by increasing macrophage phagocytosis. Conversely, Ac2-26 peptide was ineffective to afford protection in Fpr2/3 KO mice during infection. Altogether, these findings show that AnxA1, via FPR2, controls inflammation and bacterial dissemination during pneumococcal pneumonia by promoting host defenses, suggesting AnxA1-based peptides as a novel therapeutic strategy to control pneumococcal pneumonia.

Eicosanoid pathway on host resistance and inflammation during Mycobacterium tuberculosis infection is comprised by LTB4 reduction but not PGE2 increment

The functions of eicosanoids, a family of potent biologically active lipid mediators, are not restricted to inflammatory responses and they also act as mediators of the pathogenesis process. However, the role of eicosanoids in tuberculosis remains controversial. To investigate the specific role of LTB4 in Mycobacterium tuberculosis (Mtb) infection, we used 5-lipoxygenase-deficient (5-LO−/−) mice and WT (sv129) mice inoculated intranasally with LTB4 (encapsulated in PLGA microspheres). We showed that deficiency of the 5-LO pathway was related to resistance to Mtb infection. LTB4 inoculation increased susceptibility to Mtb in 5-LO−/− mice but not in WT mice, resulting in worsening of lung inflammation and tissue damage. In infected WT mice, most supplementary LTB4 was metabolized to the inactive form 12-oxo-LTB4 in the lung. A high amount of PGE2 was detected during Mtb infection, and pharmacological inhibition of COX-2 induced a significant reduction of bacterial load and an improved innate immune response in the lungs, independently of baseline LTB4 levels. COX-2 inhibition with celecoxib significantly reduced PGE2 levels, enhanced IFN-γ production and NO release, and increased macrophage phagocytosis of Mtb. The results suggest that a balance between PGE2/LTB4 is essential in the pathogenesis process of tuberculosis to prevent severe inflammation. Moreover, optimal levels of PGE2 are required to induce an effective innate response in the early phase of Mtb infection. Thus, pharmacological modulation of eicosanoid production may provide an important host-directed therapy in tuberculosis.

Ginsenoside Rg3 promotes Fc gamma receptor-mediated phagocytosis of bacteria by macrophages via an extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase-dependent mechanism

Ginsenoside Rg3 is a steroidal saponin abundant in Korean red ginseng that has high anti-inflammatory activity. Rg3 exerts an immunomodulatory effect in acute inflammatory conditions such as bacterial infections. In this study, we determined the effect of Rg3 on bacterial uptake by macrophages and the related intracellular signaling pathways. Rg3 increased macrophage phagocytosis of IgG-opsonized Escherichia coli and IgG-opsonized beads (IgGbeads), but not of non-opsonized beads. Rg3 also enhanced the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (p38 MAPK), but not that of Akt. The inclusion of IgGbeads in macrophage cultures also increased the phosphorylation of ERK1/2 and p38, but co-culture of macrophages with non-opsonized beads did not affect the phosphorylation of ERK1/2 and p38. The Rg3-induced promotion of phagocytosis was inhibited by PD98059, an ERK1/2 inhibitor, and SB203580, a p38 inhibitor. PD98059 inhibited Rg3-induced p38 MAPK phosphorylation, but SB203580 did not suppress ERK1/2 phosphorylation. Culture of macrophages with Rg3 increased actin polymerization, and this effect was inhibited by SB203580 and PD98059. The Rg3-induced increase in phagocytosis was also inhibited by NSC23766, a Rac1 inhibitor and CASIN, a Cdc42 inhibitor. Intraperitoneal injection of Rg3 increased the phosphorylation of ERK1/2 and p38 as well as the phagocytosis of bacteria by lung cells. These results demonstrate that ginsenoside Rg3 enhances macrophage phagocytosis of bacteria by activating the ERK1/2 and p38 MAPK pathways.