M2 Bone Marrow-derived Macrophages Research Articles

Sulforaphane Regulates Macrophage M1/M2 Polarization to Attenuate Macrophage-induced Caco-2 Cell Injury in an Inflammatory Environment.

The imbalance between M1 and M2 macrophage activation is closely associated with the pathogenesis of inflammatory bowel diseases (IBDs). Sulforaphane (SFN) plays an important role in the treatment of inflammatory diseases. To investigate the effect of SFN on macrophage polarization and its underlying regulatory mechanism. Mouse bone marrow-derived macrophages (BMDMs) were treated with SFN and an Nrf2 inhibitor, Brusatol. M1 macrophages were induced by LPS and IFN-γ stimulation, whereas M2 macrophages were induced by stimulation with IL-4 and IL-13. LPS-stimulated BMDMs were co-cultured with Caco-2 cells. Flow cytometry, qRT-PCR, and Western blot were performed to assess macrophage polarization. Cell function was assessed using CCK8 assay, transepithelial electrical resistance (TEER) assay, and biochemical analysis. Higher concentrations of SFN resulted in better intervention effects, with an optimal concentration of 10 μM. SFN decreased the levels of IL-12, IL-6, and TNF-α, as well as the percentages of CD16/32 in M1 BMDMs. At the same time, SFN increased the levels of YM1, Fizz1, and Arg1 as well as the percentages of CD206+ cells in M2 BMDMs. In addition, SFN enhanced the accumulation of Nrf2, NQO1, and HO-1 in M1 BMDMs, and the downregulation of Nrf2 reversed the regulatory effect of SFN on M1/M2 macrophages. LPS-stimulated BMDMs induced Caco-2 cell damage, which was partially alleviated by SFN. Our findings indicate that SFN may act as an Nrf2 agonist to regulate macrophage polarization from M1 to M2. Furthermore, SFN may represent a potential protective ingredient against IBD.

Zerumbone reduces TLR2 stimulation-induced M1 macrophage polarization pattern via upregulation of Nrf-2 expression in murine macrophages

Hyperuricemia contributes significantly to gout arthritis pathogenesis, which promotes urate crystal deposition in the joints and activates joint-resident macrophages and circulating monocytes to initiate a state of inflammatory arthritis. In the joint, macrophages have an immune defense role where the presence of urate crystals results in the inflammatory mediators secretion, inflammatory cells recruitment to the joint, and shift macrophage population toward M1 pro-inflammatory phenotypes. Current treatment modalities of gout arthritis have side effects that limit their use in the elderly. A novel treatment that targets macrophage polarization to re-establish homeostasis may initiate a drug discovery program of novel disease-modifying agents for gout. Zerumbone (Zer) is a sesquiterpenoid bioactive compound found in the rhizome of Zingiberaceae family and possesses anti-inflammatory, antioxidant, and anti-proliferative activity. Our study hypothesized that soluble uric acid (sUA) and Pam3CSK4 (TLR2 agonist) reduce the anti-inflammatory function of murine M2 bone marrow-derived macrophages and change the expression of M2 genetic markers toward M1 phenotypes. We observed that priming of M2 macrophages with sUA and Pam3CSK4 significantly decreased M2 specific markers expression, e.g., Arg-1, Ym-1, and Fizz-1, enhanced mRNA expression of IL-1β, TNF-α, CXCL2, and iNOS and increased oxidative stress in M2 macrophages, as exhibited by a reduction in Nrf2 expression. We also aimed to study the impact of Zer on reducing the pro-inflammatory effect of sUA in TLR2-stimulated M2 macrophages. We noticed that Zer treatment significantly reduced L-1β and TNF-α production following Pam3CSK4 + sUA treatment on M2 macrophages. Furthermore, Zer reduced the caspase-1 activity without altering cytosolic NLRP3 content in challenged M2 BMDMs. We also observed that Zer significantly enhanced M2-associated marker’s expression, e.g., Arg-1, Ym-1, and Fizz-1, and augmented Nrf-2 and other antioxidant proteins, including HMOX1 and srxn1expression following Pam3CSK4 + sUA treatment. We draw the conclusion that Zer is a potentially effective anti-inflammatory treatment for gout arthritis linked to hyperuricemia.

Tumor Treating Fields (TTFields) Delivery to Macrophages Promotes a Pro-Inflammatory Phenotype

<h3>Purpose/Objective(s)</h3> Tumor Treating Fields (TTFields) are electric fields that disrupt cancer cell division. In addition to an established anti-mitotic mechanism of action, cancer cell death following application of TTFields has previously been shown to stimulate anti-tumor immunity and promote dendritic cell maturation. In the current research, the effect of TTFields on the phenotypic regulation of M1 and M2 macrophages was investigated. <h3>Materials/Methods</h3> Bone marrow–derived macrophages (BMDMs) were generated from the femurs and tibias of 5–8-week-old Balb\C mice. TTFields were applied for 24 h at a frequency of 150 kHz to BMDMs stimulated with LPS+IFN-γ (M1 polarization) or IL-4 (M2 polarization). Surface expression of the macrophage biomarker F4/80 and activation markers CD80, major histocompatibility complex class II (MHC II), inducible nitric oxide synthase (iNOS), CD206, and ARG-1 were examined by flow cytometry. To analyze the heterogeneity of the stimulated macrophages, multiplexed secretion assays were employed measuring levels of CXCL1 (KC), IL-18, IL-23, IL-12p70, IL6, TNF-α, IL-12p40, TGF-β1, CCL22 (MDC), IL-10, IL-6, G-CSF, CCL17 (TARC), and IL-1β. <h3>Results</h3> A significant increase in the percentage of cells expressing the pro-inflammatory M1 markers CD80⁺ and MHC II^high was seen following TTFields application to the polarized BMDMs, while expression of the M2 markers CD206 and ARG-1 was significantly decreased. Cell supernatants of M1 and M2 BMDMs treated with TTFields all showed a pro-inflammatory secretion pattern, with increased levels of CXCL1, IL-18, IL-23, IL-12p70, TNF-α, IL-12p40, CCL22, G-CSF, CCL17 and IL-1β. Overall, this indicates that TTFields induced phenotype skewing of M2 polarized BMDMs to the M1 phenotype. <h3>Conclusion</h3> TTFields treatment displays a novel immunoregulatory role and promotes a pro-inflammatory phenotype in vitro. Future investigations will focus on defining the underlying mechanism of this phenotypic skewing.

Anti-inflammaging effects of black soybean and black rice mixture extract by reprogramming of mitochondrial respirations in murine macrophages

• Aging exacerbates the inflammatory responses in both M1 and M2 BMDMs, for which the phenomenon was termed as inflammaging. • Mitochondrial oxygen consumption in BMDMs was increased by black rice and black bean mixture extract (BBME) in aged-mice. • Increment in oxygen consumption by BBME further drove macrophage subsets into M2-like anti-inflammatory phenotypes. Chronic, persistent, low-grade inflammation in the elderly is termed ‘inflammaging’ and is one of the main causes of age-related diseases. Although the detailed molecular mechanisms of inflammaging are poorly understood, its amelioration by modulating macrophage phenotypes is of high interest. As a dietary approach, it was previously reported that black soybean ( Rhynchosia nulubilis ) and black rice ( Oryza sativa L.) contain potent bioactive components that aid in the mitigation of several metabolic diseases. The current study investigated whether dietary intervention of black soybean and black rice mixture extract (BBME) modulates inflammaging through functional and/or phenotypic alteration of mitochondrial respiration in bone marrow-derived macrophages (BMDMs). Administration of BBME not only inhibited the expression of inflammatory mediators, but also enhanced anti-inflammatory cytokines. BBME further regulated mitochondrial respiration in M1/M2 BMDMs. These results suggest BBME as a dietary modulator for prevention or amelioration of diseases related to inflammaging.

2-OR: Exosomes from Adipose Tissue Macrophages Can Mediate Insulin-Sensitizing Effects of Rosiglitazone

Background: The beneficial metabolic effects of the PPARγ agonist rosiglitazone (Rosi) are associated with anti-inflammatory changes in adipose tissue macrophages (ATMs) . One study aim was to characterize the in vivo Rosi-induced phenotypic switch of ATMs using a variety of macrophage markers, including the lipid-associated macrophage markers CD9 and Trem2. As a second aim, since ATM-derived exosomes can exert insulin-sensitizing or desensitizing effects dependent on ATM cell context, we assessed the effects of ATM exosomes from Rosi-treated obese mice on in vitro insulin actions. Research Design and Method: ATMs were isolated from 4 months HFD/obese mice treated for the last month with Rosi (60 mg/kg HFD) compared to HFD or chow-fed controls. Among CD11b+F4/80+ or CD64+ macrophages, we identified pro-inflammatory (“M1-like” CD11c+ and/or CD9+) , anti-inflammatory (“M2-like” CD11c-CD206+) and lipid-associated (CD9+ and/or Trem2+) ATMs by flow cytometry. Bone marrow-derived macrophages (BMDMs) were treated in vitro with µM Rosi for 24h. 3T3-L1 adipocytes, L6 myotubes, and primary hepatocytes were cultured 24h with exosomes isolated from ATMs of Rosi treated obese mice or in vitro Rosi treated BMDMs and then assessed for insulin sensitivity. Results: Rosi treatment reduced adipose tissue inflammation, as seen by reduced numbers of pro-inflammatory (“M1-like”) ATMs and increased the Trem2 signature in “M2-like” ATMs. Importantly, Rosi directly induced Trem2 expression in vitro in BMDMs. Additionally, exosomes isolated from Rosi-treated obese mice or M1 BMDMs increased glucose uptake and insulin signalling (pAKT) in adipocytes and myotubes and reduced glucose production in hepatocytes compared to control exosomes. Conclusion: Our data indicate that Trem2 is a PPARγ target gene. Since exosomes from Rosi treated ATMs or BMDMs enhance cellular insulin action, this could provide a new explanation for the well-known insulin-sensitizing effects of PPARγ agonists. Disclosure T.V.Rohm: None. F.C.G.Reis: None. R.Isaac: None. M.Paszek: None. G.Bandyopadhyay: None. J.M.Olefsky: None. Funding Swiss National Science Foundation (SNF) Early Postdoc.Mobility grant (to T.R.) .

Male Macrophages and Fibroblasts from C57/BL6J Mice Are More Susceptible to Inflammatory Stimuli.

Mounting evidence argues for the significant impact of sex in numerous cardiac pathologies, including myocarditis. Macrophage polarization and activation of cardiac fibroblasts play a key role in myocardial inflammation and remodeling. However, the role of sex in these processes is still poorly understood. In this study, we investigated sex-specific alterations in the polarization of murine bone marrow-derived macrophages (BMMs) and the polarization-related changes in fibroblast activation. Cultured male and female murine BMMs from C57/BL6J mice were polarized into M1 (LPS) and M2 (IL-4/IL-13) macrophages. Furthermore, male and female cardiac fibroblasts from C57/BL6J mice were activated with TNF-α, TGF-β, or conditioned medium from M1 BMMs. We found a significant overexpression of M1 markers (c-fos, NFκB, TNF-α, and IL-1β) and M2 markers (MCP-1 and YM1) in male but not female activated macrophages. In addition, the ROS levels were higher in M1 male BMMs, indicating a stronger polarization. Similarly, the pro-fibrotic markers TGF-β and IL-1β were expressed in activated cardiac male fibroblasts at a significantly higher level than in female fibroblasts. In conclusion, the present study provides strong evidence for the male-specific polarization of BMMs and activation of cardiac fibroblasts in an inflammatory environment. The data show an increased inflammatory response and tissue remodeling in male mice.

M2 macrophages have unique transcriptomes but conditioned media does not promote profibrotic responses in lung fibroblasts or alveolar epithelial cells in vitro.

Macrophages are critical regulators of pulmonary fibrosis. Their plasticity, proximity, and ability to cross talk with structural cells of the lung make them a key cell type of interest in the regulation of lung fibrosis. Macrophages can express a variety of phenotypes, which have been historically represented through an "M1-like" to "M2-like" delineation. In this classification, M1-like macrophages are proinflammatory and have increased phagocytic capacity compared with alternatively activated M2-like macrophages that are profibrotic and are associated with wound healing. Extensive evidence in the field in both patients and animal models aligns pulmonary fibrosis with M2 macrophages. In this study, we performed RNA sequencing (RNAseq) to fully characterize M1- vs. M2-skewed bone marrow-derived macrophages (BMDMs) and investigated the profibrotic abilities of M2 BMDM conditioned media (CM) to promote fibroblast migration and proliferation, alveolar epithelial cell (AEC) apoptosis, and mRNA expression of key fibrotic genes in both fibroblasts and AECs. Although M2 CM-treated fibroblasts had increased migration and M2 CM-treated fibroblasts and AECs had increased expression of profibrotic proteins over M1 CM-treated cells, all differences can be attributed to M2 polarization reagents IL-4 and IL-13 also present in the CM. Collectively, these data suggest that the profibrotic effects associated with M2 macrophage CM in vitro are attributable to effects of polarization cytokines rather than additional factors secreted in response to those polarizing cytokines.

Ceramides and sphingosine-1-phosphate mediate the distinct effects of M1/M2-macrophage infusion on liver recovery after hepatectomy

Post-hepatectomy liver dysfunction is a life-threatening morbidity that lacks efficient therapy. Bioactive lipids involved in macrophage polarization crucially regulate tissue injury and regeneration. Herein, we investigate the key bioactive lipids that mediate the cytotherapeutic potential of polarized-macrophage for post-hepatectomy liver dysfunction. Untargeted lipidomics identified elevation of ceramide (CER) metabolites as signature lipid species relevant to M1/M2 polarization in mouse bone-marrow-derived-macrophages (BMDMs). M1 BMDMs expressed a CER-generation-metabolic pattern, leading to elevation of CER; M2 BMDMs expressed a CER-breakdown-metabolic pattern, resulting in upregulation of sphingosine-1-phosphate (S1P). After infusing M1- or M2-polarized BMDMs into the mouse liver after hepatectomy, we found that M1-BMDM infusion increased M1 polarization and CER accumulation, resulting in exaggeration of hepatocyte apoptosis and liver dysfunction. Conversely, M2-BMDM infusion enhanced M2 polarization and S1P generation, leading to alleviation of liver dysfunction with improved hepatocyte proliferation. Treatment of exogenous CER and S1P or inhibition CER and S1P synthesis by siRNA targeting relevant enzymes further revealed that CER induced apoptosis while S1P promoted proliferation in post-hepatectomy primary hepatocytes. In conclusion, CER and S1P are uncovered as critical lipid mediators for M1- and M2-polarized BMDMs to promote injury and regeneration in the liver after hepatectomy, respectively. Notably, the upregulation of hepatic S1P induced by M2-BMDM infusion may have therapeutic potential for post-hepatectomy liver dysfunction.

MiR-690, an exosomal-derived miRNA from M2-polarized macrophages, improves insulin sensitivity in obese mice

Insulin resistance is a major pathophysiologic defect in type 2 diabetes and obesity, while anti-inflammatory M2-like macrophages are important in maintaining normal metabolic homeostasis. Here, we show that M2 polarized bone marrow-derived macrophages (BMDMs) secrete miRNA-containing exosomes (Exos), which improve glucose tolerance and insulin sensitivity when given to obese mice. Depletion of their miRNA cargo blocks the ability of M2 BMDM Exos to enhance insulin sensitivity. We found that miR-690 is highly expressed in M2 BMDM Exos and functions as an insulin sensitizer both invivo and invitro. Expressing an miR-690 mimic in miRNA-depleted BMDMs generates Exos that recapitulate the effects of M2 BMDM Exos on metabolic phenotypes. Nadk is a bona fide target mRNA of miR-690, and Nadk plays a role in modulating macrophage inflammation and insulin signaling. Taken together, these data suggest miR-690 could be a new therapeutic insulin-sensitizing agent for metabolic disease.

Calcitriol in the Presence of Conditioned Media from Metastatic Breast Cancer Cells Enhances Ex Vivo Polarization of M2 Alternative Murine Bone Marrow-Derived Macrophages.

Simple SummaryIn this study, we stimulated bone marrow-derived macrophages to M0, M1, and M2 subtypes, with or without calcitriol, or with or without 4T1 (metastatic), 67NR (non-metastatic), and Eph4-Ev (normal) cell culture supernatants (CMs) to test their effect on polarization. We showed that calcitriol increased the expression of Cd206 and Spp1 mRNA and CD36, CCL2, and arginase levels for M2 macrophages and decreased Cd80 and Spp1 mRNA and IL-1, IL-6, OPN, and iNOS for M1 macrophages. 4T1 CM influenced the expression of the studied genes and proteins to a greater extent than 67NR and Eph4; the strongest effect was noted for M2 macrophages. We show that calcitriol and 4T1 CM enhance the polarization of M2 macrophages and M2 macrophages differentiated with calcitriol-stimulated migration of 4T1 and 67NR cells. We indicate that the immunosuppressive properties of calcitriol may unfavorably affect the tumor microenvironment, and supplementation with vitamin D in oncological patients may not always bring benefits.In this study, we differentiated murine bone marrow-derived macrophages (BMDMs) into M0, M1, and M2 in the presence or absence of calcitriol. Real-time PCR analysis of gene expression, FACS analysis of surface markers, and chemokine/cytokine production assays were performed. In addition, the effect of the conditioned media (CM) from murine breast cancer 4T1 (metastatic) and 67NR (non-metastatic) and Eph4-Ev (normal) cells with and without calcitriol on the polarization of M1/M2 cells was determined. We found that calcitriol enhanced the differentiation of M2 macrophages, which was manifested by increased expression of Cd206 and Spp1 mRNA and CD36, Arg, and CCL2 in M2 BMDMs and by decreased expression of Cd80 and Spp1 mRNA and IL-1, IL-6, OPN, and iNOS in M1 BMDMs. 4T1 CM showed a higher effect on the gene and protein expression in macrophages than 67NR and Eph4-Ev, with the greatest effect observed on M2 macrophages which increased their differentiation and properties characteristic of alternative macrophages. Moreover, M2 macrophages differentiated with calcitriol-stimulated migration of 4T1 and 67NR cells through fibronectin and collagen type IV, respectively. Overall, our results indicated that vitamin D supplementation may not always be beneficial, especially in relation to cancers causing excessive, pathological activation of the immune system.

MicroRNA-421-3p-abundant small extracellular vesicles derived from M2 bone marrow-derived macrophages attenuate apoptosis and promote motor function recovery via inhibition of mTOR in spinal cord injury

BackgroundSpinal cord injury (SCI) has a very disabling central nervous system impact but currently lacks effective treatment. Bone marrow-derived macrophages (BMDMs) are recruited to the injured area after SCI and participate in the regulation of functional recovery with microglia. Previous studies have shown that M2 microglia-derived small extracellular vesicles (SEVs) have neuroprotective effects, but the effects of M2 BMDM-derived sEVs (M2 BMDM-sEVs) have not been reported in SCI treatment.ResultsIn this study, we investigated the role of M2 BMDM-sEVs in vivo and in vitro for SCI treatment and its mechanism. Our results indicated that M2 BMDM-sEVs promoted functional recovery after SCI and reduced neuronal apoptosis in mice. In addition, M2 BMDM-sEVs targeted mammalian target of rapamycin (mTOR) to enhance the autophagy level of neurons and reduce apoptosis. MicroRNA-421-3P (miR-421-3p) can bind to the 3′ untranslated region (3′UTR) of mTOR. MiR-421-3p mimics significantly reduced the activity of luciferase-mTOR 3′UTR constructs and increased autophagy. At the same time, tail vein injection of inhibitors of SEVs (Inh-sEVs), which were prepared by treatment with an miR-421-3p inhibitor, showed diminished protective autophagy of neuronal cells in vivo.ConclusionsIn conclusion, M2 BMDM-sEVs inhibited the mTOR autophagy pathway by transmitting miR-421-3p, which reduced neuronal apoptosis and promoted functional recovery after SCI, suggesting that M2 BMDM-sEVs may be a potential therapy for SCI.

Different monocyte phenotypes result in proresolving macrophages in conjugated linoleic acid-induced attenuated progression and regression of atherosclerosis.

Monocytes/macrophages drive progression and regression of atherosclerosis. Conjugated linoleic acid (CLA), an anti-inflammatory lipid, mediates atheroprotective effects. We investigated how CLA alters monocyte/macrophage phenotype during attenuated progression and regression of atherosclerosis. Apolipoprotein E knockout (ApoE-/-) mice were fed a high-fat (60%) high-cholesterol (1%) diet (HFHCD) for 2 wk, followed by 6-wk 1% CLA 80:20 supplementation to investigate disease progression. Simultaneously, ApoE-/- mice were fed a 12-wk HFHCD with/without CLA for the final 4 wk to investigate regression. Aortic lesions were quantified by en face staining. Proteomic analysis, real-time quantitative PCR and flow cytometry were used to interrogate monocyte/macrophage phenotypes. CLA supplementation inhibited atherosclerosis progression coincident with decreased proinflammatory and increased anti-inflammatory macrophages. However, CLA-induced regression was associated with increased proinflammatory monocytes resulting in increased proresolving M2 bone marrow-derived macrophages, splenic macrophages, and dendritic cells in lesion-draining lymph nodes. Proteomic analysis confirmed regulation of a proinflammatory bone marrow response, which was abolished upon macrophage differentiation. Thus, in attenuation and regression of atherosclerosis, regardless of the monocyte signature, during monocyte to macrophage differentiation, proresolving macrophages prevail, mediating vascular repair. This study provides novel mechanistic insight into the monocyte/macrophage phenotypes in halted atherosclerosis progression and regression of atherosclerosis.-Bruen, R., Curley, S., Kajani, S., Lynch, G., O'Reilly, M. E., Dillon, E. T., Fitzsimons, S., Mthunzi, L., McGillicuddy, F. C., Belton, O. Different monocyte phenotypes result in proresolving macrophages in conjugated linoleic acid-induced attenuated progression and regression of atherosclerosis.

Abstract 1909: Stromal cell driven proteolysis of versican is limited by epithelial cells in pancreatic ductal adenocarcinoma

Abstract Background: Clinical responses to checkpoint inhibitors in gastrointestinal cancers have largely been limited to MSI-high disease. Proteolysis of an immunosuppressive proteoglycan versican (VCAN) by ADAMTS proteases correlates with enhanced CD8+ T cell infiltration in colorectal cancer independent of mismatch repair status. Here we investigate the potential of this biomarker in pancreatic ductal adenocarcinoma (PDAC) as VCAN is abundant VCAN in this disease. Methods: KPC mice were used to generate mouse PDAC tissue and organotypic spheroids. A human PDAC tissue microarray (TMA) was developed representing normal and neoplastic pancreatic tissues across 131 patients. IHC was used to determine levels of VCAN, an immunostimulatory fragment versikine (Vkine), and CD8+ T cells. Bone marrow-derived macrophages (BMDMs) were derived from BALB/c mice. VCAN-specific ADAMTS protease expression profiles were compared between antitumor (M1) and protumor (M2) mouse macrophages, human pancreatic stellate cells (PSCs) and patient-derived PDAC-organotypic spheroids. Results: IHC analysis of normal murine pancreas demonstrated low VCAN and Vkine staining. VCAN is abundant in metaplastic/neoplastic KPC mouse pancreatic tissues. Rare VCAN proteolytic predominant areas (samples with both low VCAN and high Vkine) in KPC tumors had increased tumor-infiltrating CD8+ T cells/ high-powered field compared to weak areas (17 vs 4, p&amp;lt;0.001). Human PDACs had abundant VCAN expression compared to normal pancreatic tissues (p&amp;lt;0.001, n=118). Vkine was absent from normal tissues and present only at low levels in tumor cores (p&amp;lt;0.001, n=117). No human PDAC VCAN proteolytic-predominant samples were identified. M1 BMDMs demonstrated high relative expression (RE) of VCAN-specific ADAMTS proteases relative to unpolarized M0 BMDMs; however, these levels decreased under KPC-conditioned media (ADAMTS4, 101 vs 5, p=0.003; ADAMTS9, 80 vs 1, p=0.02; ADAMTS15, 103 versus 1.6, p&amp;lt;0.001; ADAMTS20, 881 vs 3.2, p&amp;lt;0.001). Similarly, M2 BMDMs had elevated ADAMTS-levels that were reduced under KPC-conditioned media, (ADAMTS4, 42 vs 0.4, p=0.0029; ADAMTS9, 40 vs 1, p=0.002; ADAMTS15, 23 vs 3, p=0.027; ADAMTS20, 310 vs 0.8, p&amp;lt;0.001; n=3). RE levels of ADAMTS proteases in KPC spheroids was consistently lower than reported above in macrophages. RE of ADAMTS proteases in human PSCs were between 18 and 304. No differences in ADAMTS expression were identified under PDAC-conditioned media in the human PSCs (p=0.3). Conclusions: The VCAN proteolysis predominant phenotype correlates with increased CD8+ T cell infiltration in PDAC though is rarely present in the human disease. PDAC epithelial cells influence the stromal cells to reduce VCAN proteolysis through decreased ADAMTS expression by macrophages. Future studies examining measures to enhance VCAN proteolysis could result in a more immune permissive microenvironment. Citation Format: Philip B. Emmerich, Chelsie Sievers, Hanna Rainiero, Rosabella Pitera, Connor Maloney, Susan Payne, Mitchell Depke, Cheri Pasch, Linda Clipson, Jillian K. Johnson, Kristina Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. Stromal cell driven proteolysis of versican is limited by epithelial cells in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1909.

Abstract 1904: Versican production is driven by both epithelial and stromal cells in pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) recently became the third-deadliest cancer due to resistance to chemotherapy and immunotherapies. The tumor microenvironment (TME) in PDAC is thought to contribute to this resistance, with up to 80% of the tumor bulk consisting of stroma. We investigated the role of two major stromal cell types, pancreatic stellate cells (PSCs) and macrophages, in the production of the immunosuppressive proteoglycan versican (VCAN) as a precursor to the identification of novel mechanisms that may enhance therapeutic response. Methods: B6 KPC mice were utilized as a murine model of PDAC. A human PDAC tissue microarray (TMA) was developed representing normal and neoplastic pancreatic tissue across 131 patients. Immunohistochemistry (IHC) was used to determine levels of VCAN and CD8+ T cells. Bone marrow-derived macrophages (BMDMs) were differentiated from BALB/c mouse femurs and polarized to M1 (antitumor) or M2 (protumor) status. Additionally, PDAC organotypic spheroids were derived from both human and murine tissues whereas PSCs were derived solely from human tissue. Results: IHC analysis of KPC tumors revealed elevated levels of stromal VCAN compared to normal pancreatic tissue (p&amp;lt;0.001, n=20). VCAN accumulation was increased even in the earliest stages of acinar-to-ductal metaplasia in KPC mice. Areas of intense stromal VCAN staining trended toward reduced CD8+ T cell infiltration (4.9 vs 7.3 cells/high power field (hpf), p=0.3). IHC analysis of human PDAC revealed elevated VCAN accumulation across all stages compared to the normal adjacent tissue (n=231, p&amp;lt;0.001). Areas of high VCAN accumulation demonstrated reduced CD8+ T cells compared to areas of low VCAN (0.6 vs 2.9 cells/hpf, p&amp;lt;0.001). To investigate cell types responsible for enhanced VCAN accumulation in the tumor microenvironment, relative expression (RE) of VCAN was compared in vitro to M0 macrophages. Organotypic cancer spheroids demonstrated increased expression of VCAN from KPC mice (RE=49, n=2) and patient-derived PDAC tissue (RE=14, p=0.01, n=3). M1-polarized BMDMs had increased expression of VCAN (RE=24) compared to M2 BMDMs (RE=8) (p&amp;lt;0.001, n=3). Interestingly, M1 BMDMs cultured in PDAC-conditioned media had reduced RE of M1 markers: TNFα (395 vs 37, p=0.03) and iNOS (24723 vs 4813, p=0.003), and increased M2 markers: Arg-1 (127 versus 1049, p=0.02) and YM-1 (0.5 vs 3, p=0.02). PDAC-conditioned media also reduced VCAN expression of M1 BMDMs (24 vs 9, p=0.02). PSCs derived from human PDAC also demonstrated enhanced RE of VCAN compared to negative controls (RE=68, p=0.017) with no significant change in the presence of PDAC conditioned media (p=0.4). Conclusions: The accumulation of VCAN is common in PDAC and correlates with CD8+ T cell exclusion. Epithelial and stromal components are responsible for VCAN production. VCAN deserves further investigation as a target for therapeutic interventions for PDAC. Citation Format: Hanna R. Rainiero, Philip B. Emmerich, Chelsie K. Sievers, Connor J. Maloney, Rosabella T. Pitera, Susan N. Payne, Mitchell G. Depke, Cheri A. Pasch, Linda Clipson, Jillian K. Johnson, Kristina A. Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. Versican production is driven by both epithelial and stromal cells in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1904.

The Notch ligand DNER regulates macrophage IFNγ release in chronic obstructive pulmonary disease.

BackgroundChronic Obstructive Pulmonary Disease (COPD) is the third leading cause of death worldwide with no curative therapy. A non-canonical Notch ligand, DNER, has been recently identified in GWAS to associate with COPD severity, but its function and contribution to COPD is unknown.MethodsDNER localisation was assessed in lung tissue from healthy and COPD patients, and cigarette smoke (CS) exposed mice. Microarray analysis was performed on WT and DNER deficient M1 and M2 bone marrow-derived macrophages (BMDM), and gene set enrichment undertaken. WT and DNER deficient mice were exposed to CS or filtered air for 3 day and 2 months to assess IFNγ-expressing macrophages and emphysema development. Notch and NFKB active subunits were quantified in WT and DNER deficient LPS-treated and untreated BMDM.FindingsImmunofluorescence staining revealed DNER localised to macrophages in lung tissue from COPD patients and mice. Human and murine macrophages showed enhanced DNER expression in response to inflammation. Interestingly, pro-inflammatory DNER deficient BMDMs exhibited impaired NICD1/NFKB dependent IFNγ signalling and reduced nuclear NICD1/NFKB translocation. Furthermore, decreased IFNγ production and Notch1 activation in recruited macrophages from CS exposed DNER deficient mice were observed, protecting against emphysema and lung dysfunction.InterpretationDNER is a novel protein induced in COPD patients and 6 months CS-exposed mice that regulates IFNγ secretion via non-canonical Notch in pro-inflammatory recruited macrophages. These results provide a new pathway involved in COPD immunity that could contribute to the discovery of innovative therapeutic targets.FundingThis work was supported from the Helmholtz Alliance ‘Aging and Metabolic Programming, AMPro’.

Mannose receptor high, M2 dermal macrophages mediate nonhealing Leishmania major infection in a Th1 immune environment.

The origin and functional specialization of dermal macrophages in cutaneous infections have been little studied. In this paper, we show that a strain of Leishmania major (L. major Seidman [LmSd]) that produces nonhealing cutaneous lesions in conventionally resistant C57BL/6 mice was more efficiently taken up by M2-polarized bone marrow (BM)-derived macrophages (BMDMs) in vitro and by mannose receptor (MR)hi dermal macrophages in vivo compared with a healing strain (L. major Friedlin V1). Both in steady and in T helper type 1 (Th1) cell-driven inflammatory states, the MRhi dermal macrophages showed M2 characteristics. The dermal macrophages were radio resistant and not replaced by monocytes or adult BM-derived cells during infection, but were locally maintained by IL-4 and IL-10. Notably, the favored infection of M2 BMDMs by LmSd in vitro was MR dependent, and genetic deletion of MR or selective depletion of MRhi dermal macrophages by anti-CSF-1 receptor antibody reversed the nonhealing phenotype. We conclude that embryonic-derived, MRhi dermal macrophages are permissive for parasite growth even in a strong Th1-immune environment, and the preferential infection of these cells plays a crucial role in the severity of cutaneous disease.

Cytotherapy with M1-polarized macrophages ameliorates liver fibrosis by modulating immune microenvironment in mice

Macrophages play vital roles in chronic liver injury, and have been tested as a tool for cytotherapy in liver fibrosis. However, macrophages possess ontogenic and functional heterogeneities. Some subsets are pro-fibrotic, whereas others are anti-fibrotic. This study aimed to clarify which macrophage subset is efficient for cytotherapy in liver fibrosis and to elucidate the underlying mechanisms. Liver fibrosis was induced in mice by carbon tetrachloride injection or bile duct ligation. Bone-marrow-derived macrophages (BMDMs) were polarized into M0, M1, or M2 macrophages, respectively. BMDMs were infused into mice through the tail vein at different stages of fibrogenesis. Fibrosis progression, hepatic cell populations, and related molecular changes were evaluated. Both M0 and M1 BMDMs significantly ameliorated liver fibrosis, but M1 exhibited stronger therapeutic effects than M0. M2 macrophages were not effective on liver fibrosis. M1 macrophages reduced the number and activation of hepatic stellate cells (HSCs), which could be attributed at least partly to increased HSC apoptosis. M1 macrophages enhanced the recruitment of endogenous macrophages into fibrotic liver, which displayed the phenotype of Ly6Clo restorative macrophages and produced matrix metalloproteinases (MMPs) and hepatic growth factor (HGF) to enhance collagen degradation and hepatocyte proliferation, respectively. M1 macrophages also increased the number of total and activated natural killer (NK) cells in the fibrotic liver, which released TNF-related apoptosis-inducing ligand (TRAIL), inducing HSC apoptosis. M1 macrophages, which modulate the immune microenvironment to recruit and modify the activation of endogenous macrophages and NK cells, are effective for cytotherapy in experimental liver fibrosis. Lay summary: M1 Bone marrow-derived macrophages (BMDMs) exhibit a stronger therapeutic effect by modulating the hepatic microenvironment to recruit and modify the activation of endogenous macrophages and natural killer (NK) cells, which likely lead to hepatic stellate cells (HSCs) apoptosis and hampered fibrogenesis.

Transfusion of CD206+ M2 Macrophages Ameliorates Antibody-Mediated Glomerulonephritis in Mice

Macrophages are multifunctional immune cells that may either drive or modulate disease pathogenesis, depending on the activated phenotype. In this study, we investigated the protective effects of CD206+ M2 macrophages against nephrotoxic serum nephritis in mice. We found that these immunosuppressive macrophages, derived from bone marrow and stimulated with IL-4/IL-13 [CD206+ M2 bone marrow-derived macrophages (M2BMMs)], protected against renal injury, decreased proteinuria, and diminished the infiltration of CD68+ macrophages, neutrophils, and T cells into glomerular tissue. Comparable therapeutic results were obtained with CD206+ M2 cells derived from induced pluripotent stem cells. Notably, CD206+ M2BMMs, which retained an M2 signature, could elicit a switch of M1 to M2 phenotype in co-cultured macrophages. Moreover, these cells were found to induce the production of regulatory T cells in the spleen and renal draining lymph node. Accordingly, mRNA expression of the T helper 1 cytokines tumor necrosis factor-α, interferon-β, interferon-γ, and IL-12 was significantly reduced in kidneys from mice treated with CD206+ M2BMMs. Taken together, the data suggest that CD206+ M2 may have therapeutic potential against antibody-mediated glomerular injury and presents its therapeutic value for the treatment ofcrescentic nephritis in humans.

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay. Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage’s metabolic phenotype and inflammatory status.

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis.

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay. Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage's metabolic phenotype and inflammatory status.