Monocyte-derived M2 Macrophages Research Articles

P0059 Mechanistic understanding of MB310: a consortium of gut commensal bacteria for the treatment of Ulcerative Colitis

Abstract Background Microbiotica is developing MB310, a live biotherapeutic product consisting of 8 bacterial strains for the treatment of ulcerative colitis (UC). These were selected based on association with clinical responses in UC patients in a fecal microbiota transplantation (FMT) clinical study. To understand how MB310 drives clinical benefit, we tested their impact on epithelial barrier repair, innate immune cells, and CD4+ T lymphocytes, which are key to the pathology of UC. We also investigated the effects of MB310 bacterial metabolites on the induction of regulatory T (Treg) cells that can counteract UC-associated inflammation. Methods Effects on barrier function were assessed by measuring electric impedance of Caco-2 cells in the presence or absence of lipopolysaccharide (LPS). Effects on human dendritic cells (DCs) and M1 macrophages were assessed by determining their cytokine production profile. The effects of MB310 or its metabolites on CD4+ T cells were assessed by measuring their expression of the Treg markers Foxp3 and interleukin (IL)-10 following polarization with MB310-treated DCs. Finally, the induced Treg cells were functionally validated in a T cell suppression assay. Results Investigating barrier effects, we observed that five MB310 strains individually increased the integrity of a gut epithelial cell monolayer, and 2 strains repaired the monolayer following damage by LPS challenge. Furthermore, primary human monocyte-derived M1 macrophages and LPS-activated DCs showed an increase in the release of the anti-inflammatory cytokine, IL-10 in comparison to the pro-inflammatory cytokine tumor necrosis factor (TNF), following exposure to all MB310 strains. The MB310-treated DCs were able to enhance IL-10 production by T cells. Investigating the mechanisms of Treg induction, we revealed that the bacterial metabolites from two MB310 strains induced DCs to polarize two distinct subsets of actively suppressive Treg cells: Foxp3+ Tregs and IL-10-producing Tregs. Another MB310 species induced Foxp3+ suppressive Treg cells through direct effect on CD4+ T cells. Conclusion The accumulated data suggest that the strains of MB310 interact with the host to oppose key UC disease pathologies by improving gut epithelial barrier integrity; promoting an anti-inflammatory profile of innate immune cells; and inducing Treg cells. Moreover, the metabolites of some MB310 strains lead to induction of different types of functionally suppressive Treg cells. This highlights that multifaceted effects of MB310 that can circumvent the challenge of UC disease heterogeneity, proposing it as a potential therapeutic for a wide spectrum of UC patients. COMPOSER-1 is an ongoing phase 1b trial testing MB310 in UC patients.

Quantitative proteomics reveals differential extracellular vesicle cargo from M1 and M2 monocyte-derived human macrophages.

Extracellular vesicles (EVs) mediate intercellular communication by carrying molecular cargo that facilitate diverse physiological processes. Macrophages, playing central roles in immune responses, release EVs that modulate various cellular functions. Given the distinct roles of M1 and M2 macrophage states, understanding the proteomic profiles of their EVs is important for elucidation of EV-mediated signalling and identifying potential biomarkers for diseases involving macrophage polarisation. We employed quantitative proteomics combined with bioinformatics to characterise the proteomic profile of EVs released by M1 and M2 monocyte-derived macrophages. We identified 1,731 proteins in M1/M2 EVs, 132 of which were significantly differentially between M1 and M2. Proteomic data, together with pathway analysis, found that M1/M2 macrophage EV cargo relate to cellular source, and may play roles in shaping immune responses, with M1 EV cargo associated with promotion of pro-inflammatory and antiviral functions, while M2 EV cargo associated with immune regulation and tissue repair. M1 EV cargo was associated with cytokine/chemokine signalling pathways, DNA damage, methylation, and oxidative stress. M2 EV cargo were associated with macrophage alternative-activation signalling pathways, antigen presentation, and lipid metabolism. We also report that macrophage EVs carry metallothioneins, and other related proteins involved in response to metals and oxidative stress.

Alamandine, a protective component of the renin-angiotensin system, reduces cellular proliferation and interleukin-6 secretion in human macrophages through MasR–MrgDR heteromerization

Alamandine (ALA) exerts protective effects similar to angiotensin (Ang) (1–7) through Mas-related G protein-coupled receptor type D receptor (MrgDR) activation, distinct from Mas receptor (MasR). ALA induces anti-inflammatory effects in mice but its impact in human macrophages remains unclear. We aimed to investigate the anti-inflammatory effects of ALA in human macrophages. Interleukin (IL)-6 and IL-1β were measured by ELISA in human THP-1 macrophages and human monocyte-derived macrophages exposed to lipopolysaccharide (LPS). Consequences of MasR–MrgDR heteromerization were investigated in transfected HEK293T cells. ALA decreased IL-6 and IL-1β secretion in LPS-activated THP-1 macrophages. The ALA-induced decrease in IL-6 but not in IL-1β was prevented by MasR blockade and MasR downregulation, suggesting MasR–MrgDR interaction. In human monocyte-derived M1 macrophages, ALA decreased IL-1β secretion independently of MasR. MasR–MrgDR interaction was confirmed in THP-1 macrophages, human monocyte-derived macrophages, and transfected HEK293T cells. MasR and MrgDR formed a constitutive heteromer that was not influenced by ALA. ALA promoted Akt and ERK1/2 activation only in cells expressing MasR–MrgDR heteromers, and this effect was prevented by MasR blockade. While Ang-(1–7) reduced cellular proliferation in MasR −but not MrgDR- expressing cells, ALA antiproliferative effect was elicited in cells expressing MasR–MrgDR heteromers. ALA also induced an antiproliferative response in THP-1 cells and this effect was abolished by MasR blockade, reinforcing MasR–MrgDR interaction. MasR–MrgDR heteromerization is crucial for ALA-induced anti-inflammatory and antiproliferative responses in human macrophages. This study broaden our knowledge of the protective axis of the RAS, thus enabling novel therapeutic approaches in inflammatory-associated diseases.

Abstract 6873: Prognostic impact of squamous cell carcinoma antigen as a mediator of anti-inflammatory macrophage polarization in tumor microenvironment

Abstract Squamous cell carcinoma antigen 1 (SCCA1) is a potential biomarker and prognostic indicator for many epithelial cancers including cervix, head and neck, breast, and lung. Cancer patients with high serum levels of SCCA commonly experience treatment resistance and recurrence. We analyze SCCA expression in correlation with tumor microenvironment from 66 human cervical tumor biopsies and find that high serum SCCA is associated with increased macrophage infiltration in tumors and poor overall survival. In a single cell RNAseq analysis of 8 cervical tumors, patients with high SCCA1 show increased immunosuppressive genes (C1QA, C1QC, APOE) and reduced MHC molecules (HLA-DR, HLA-DQA1) in their tumor associated macrophages (TAMs), corresponding to decreased expression of T-cell cytotoxic-associated genes (IFNG, GZMB, PRF1). Thus, we aim to understand the impact of elevated SCCA1 on macrophages and its role in shaping the immune response. Co-culture of peritoneal macrophages isolated from SCCA1-treated mice with T cells from syngeneic wild-type mice, showed significant suppression in T cell proliferation. Downregulation of antigen presentation molecules was also observed SCCA1-treated macrophages and resulted in impaired ability to induce T cell activation by peptide-pulsed macrophages. Compared with treatment-naïve mice, SCCA1-treated macrophages showed increased anti-inflammatory cytokine CCL22, IL10 and immunosuppression regulators ARG1, PD-L1 expression. Moreover, human monocyte-derived M2 macrophages treated with SCCA1 during the differentiation process demonstrated inhibition toward CD3/28-induced T cell proliferation, accompanied by enhanced expression of cytokines, IL10 and CCL18. We further found that targeting SCCA1 by small interfering RNA knockdown significantly reduced tumor growth and macrophage infiltration, resulting in improved T-cell antitumor immunity. Lastly, analysis of cervical cancer patients with chemoradiation therapy showed that patients with high SCCA are associated with increased exhaustion (LAG3, CTLA4) and decreased antitumor gene signature (GZMB, IFNG) during treatment. In summary, SCCA expression promotes immunosuppressive macrophage polarization and function, contributing to T cell exhaustion phenotype and poor treatment response. Citation Format: Liyun Chen, Victoria Shi, Eric Liu, Jasmine Yang, Abhay Singh, Matthew Inkman, Jin Zhang, Julie Schwarz, Stephanie Markovina. Prognostic impact of squamous cell carcinoma antigen as a mediator of anti-inflammatory macrophage polarization in tumor microenvironment [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 6873.

Macrophage phenotype is determinant for fibrosis development in keloid disease

Keloid refers to a fibroproliferative disorder characterized by an accumulation of extracellular matrix (ECM) components at the dermis level, overgrowth beyond initial wound, and formation of tumor-like nodule areas. Treating keloid is still an unmet clinical need and the lack of an efficient therapy is clearly related to limited knowledge about keloid etiology, despite the growing interest of the scientific community in this pathology. In past decades, keloids were often studied in vitro through the sole prism of fibroblasts considered as the major effector of ECM deposition. Nevertheless, development of keloids results from cross-interactions of keloid fibroblasts (KFs) and their surrounding microenvironment, including immune cells such as macrophages. Our study aimed to evaluate the effect of M1 and M2 monocyte-derived macrophages on KFs in vitro. We focused on the effects of the macrophage secretome on fibrosis-related criteria in KFs, including proliferation, migration, differentiation, and ECM synthesis. First, we demonstrated that M2-like macrophages enhanced the fibrogenic profile of KFs in culture. Then, we surprisingly founded that M1-like macrophages can have an anti-fibrogenic effect on KFs, even in a pro-fibrotic environment. These results demonstrate, for the first time, that M1 and M2 macrophage subsets differentially impact the fibrotic fate of KFs in vitro, and suggest that restoring the M1/M2 balance to favor M1 in keloids could be an efficient therapeutic lever to prevent or treat keloid fibrosis.

Fecal Supernatants from Patients with Crohn's Disease Induce Inflammatory Alterations in M2 Macrophages and Fibroblasts.

Intestinal macrophages and fibroblasts act as microenvironmental sentinels mediating inflammation and disease progression in Crohn's disease (CD). We aimed to establish the effects of fecal supernatants (FSs) from patients with CD on macrophage and fibroblast phenotype and function. FS were obtained by ultracentrifugation, and the metabolites were analyzed. Monocyte-derived M2 macrophages and fibroblasts were conditioned with FS, and secreted proteins, surface proteins and gene expression were analyzed. M2 macrophage efferocytosis was evaluated. Patients with CD (n = 15) had a skewed fecal metabolite profile compared to healthy subjects (HS, n = 10). FS from CD patients (CD-FS) induced an anti-inflammatory response in M2 macrophages with higher expression of IL-10, IL1RA and CD206 as compared to healthy FS (HS-FS) while the efferocytotic capacity was unaltered. CD-FS did not affect extracellular matrix production from fibroblasts, but increased expression of the pro-inflammatory proteins IL-6 and MCP-1. Conditioned media from M2 macrophages treated with CD-FS modulated gene expression in fibroblasts for TGFβ superfamily members and reduced IL-4 expression compared to HS-FS. We show that M2 macrophages and fibroblasts react abnormally to the fecal microenvironment of CD patients, resulting in altered protein expression related to inflammation but not fibrosis. This implies that the gut microbiota and its metabolites have an important role in the generation and/or perpetuation of inflammation in CD.

Inhibition of MET Tyrosine Kinase Provides a Strategy for Targeting the Inflammatory Tumor Microenvironment

Background: In acute myeloid leukemia (AML), age-associated inflammation may contribute to leukemia progression and remodel the tumor microenvironment (TME). Macrophages, crucial components of the TME, play a critical role in modulating T cell immune response against leukemia cells. However, macrophage polarization to an M2 phenotype, known as tumor-associated macrophages (TAMs), have an immunosuppressive action and interplay with leukemia cells by promoting their survival, immune evasion, and chemotherapy resistance. MET, a hepatocyte growth factor (HGF) receptor, has been demonstrated to contribute to shifting macrophages to TAMs. Hence, we hypothesize that inhibiting Met and, consequently, HGF action and signaling could be a potential strategy to target the inflammatory TME and improve AML clinical outcomes. Aims: Here,we analyze the MET gene expression in leukemia patients mononuclear cells and the impact on clinical outcomes and we also assess the contribution of a specific MET inhibitor, B2000 (Brigham University), to leukemia progression by investigating the in vitro, ex vivo and in vivo effects with focus on the inflammatory TME. Method s: MET mRNA expression data were obtained from TCGA AML study, performed in cells isolated from 290 AML patients (median age 61 years [range:18-87]). Human peripheral blood and bone marrow (BM) mononuclear cells (BMNC/PBMC) (n=10) were obtained from healthy donors (HD). HD-PBMC were used for the isolation of monocytes. Human leukemia cell lines (OCI-AML3, MOLM-13) and BMNC from HD and AML patients (n=10) were treated with B2000 and apoptosis rates were evaluated. To obtain monocyte-derived macrophages (MDMs), monocytes were stimulated with GM-CSF/LPS/IFN-y or M-CSF/IL-4, for M1 or M2 polarization, respectively, and treated with B2000 in monocultures and cocultures. For in viv o analyses, BALB/c mice received a single intraperitoneal injection of lipopolysaccharide (LPS) to provoke an inflammatory shock and were treated with B2000. The analysis of the immune response and the characterization of inflammatory cells was performed by flow cytometry. Results: High MET expression in FAB M1, M3 and M7 AML patients ( P<.0001) was observed. B2000 was effective in killing leukemia cell lines ( P<.0001) and primary BMNCs from AML patients ( P<.005) without affecting HD-BMNC. HD-PBMC-derived monocytes treated with B2000 had increased classical monocyte population (CD14highCD16-) ( P<.001). B2000 also caused a significant apoptosis increase in leukemia cells cocultured with monocytes, without modulating the monocytes population ( P<.0001). To confirm whether monocytes could acquire a tumoricidal phenotype, monocytes were pre-treated with B2000 and, then, cocultured with leukemia cells. B2000-programmed monocytes enhanced leukemia cell apoptosis ( P<.001). The analysis of MDMs revealed a shorter population of M2 MDMs (HLA-DR-CD206+) and increased population of M1 MDMs (HLA-DR+CD80+) in B2000-treated MDMs when compared with the vehicle treatment ( P<.0001). To validate these findings, we evaluated B2000 action in the inflammatory TME by co-culturing leukemia cells with MDMs and results showed that apoptosis was increased in leukemia cells in the presence of B2000 compound ( P<.01). The in vivo analysis in an inflammatory mice model showed increased frequency of M1 monocytes (CD11b+F4/80+CD80+) and decreased M2 (CD11b+F4/80+CD206+) in B2000-treated ( P<.05). Accordingly, B2000 restored lymphocyte B ( P<.05) and T ( P<.05) levels in the BM, spleen and peripheral blood, decreasing the inflammatory burden. Conclusion: We demonstrate the pre-clinical efficacy of a MET inhibitor, B2000, in enhancing anti-tumor immunity in vitro, ex vivo and in vivo by promoting M1 macrophage polarization and tumoricidal phenotype, contributing to inhibition of leukemia cell expansion and inducing the specific leukemia apoptosis. B2000 also acted on restoring lymphocyte levels and, consequently, reducing inflammatory stress. Our findings highlight an important role of this novel MET-inhibitor onto the inflammatory TME, representing an important agent against hematological neoplasms with a novel immunomodulatory effect in directly reducing leukemia cell proliferation as well as promoting TAMs anti-tumoral effector functions. Funding: FAPESP #2017/21801-2, #2019/25247-5, #2021/05320-0; CNPq #303405/2018-0.

P-316 Involvement of collective cell migration in endometrial invasiveness in uterine adenomyosis and the role of M2 macrophages

Abstract Study question How do endometrial cells manage to invade surrounding myometrium to establish adenomyotic lesions and what is the role of macrophages in this process? Summary answer M2 macrophage-mediated invasiveness and resistance of endometrial cells to physiological cell death appear to contribute to the pathogenesis of adenomyosis. What is known already Despite the high prevalence and debilitating symptoms of uterine adenomyosis, its pathogenesis has not yet been elucidated. To date, the most acceptable theory to explain disease development suggests invasion of the myometrium by eutopic endometrial cells. Recent data suggest aberrant infiltration of immune cells but not platelets in adenomyosis, eventually leading to enhanced cell motility via collective cell migration (CCM) and resistance to physiological cell death during disease pathogenesis. Study design, size, duration A retrospective immunohistochemistry-based study was conducted on formalin-fixed paraffin-embedded uterine tissue from 17 women (8 adenomyosis patients and 9 healthy subjects). Fresh endometrial biopsies were retrieved from 16 women (6 adenomyosis patients and 10 healthy subjects) for in vitro culture of epithelial and stromal cells, and co-culture with THP-1 monocyte-derived M2 macrophages. Participants/materials, setting, methods Double immunofluorescence was performed to detect macrophages of M1 and M2 phenotypes. Invasiveness of endometrial cells upon interaction with macrophages was assessed by in vitro invasion assays and quantitative PCR for genes involved in cell motility and epithelial-mesenchymal transition (EMT). Mechanisms of disease progression were investigated by immunohistochemistry against E-cadherin, N-cadherin and matrix metalloproteinase 9 (MMP9). Caspase 3 and microtubule-associated protein light chain 3 beta (LC3B) were studied as markers of apoptosis and autophagy respectively. Main results and the role of chance Only M2 macrophages were found to accumulate in adenomyosis, in higher numbers in both endometrium and ectopic lesions from adenomyosis patients than healthy controls. Co-culture with M2 macrophages significantly increased invasion capacity in endometrial epithelial and stromal cells from both adenomyosis patients and healthy controls. Epithelial cells from adenomyosis patients were nonetheless more invasive than their healthy counterparts in the presence of M2 macrophages. No gene expression differences pointing to EMT were noted, either between co-cultured and control cells, or between adenomyotic and healthy cells, refuting the implication of this mechanism in adenomyosis invasiveness. E- and N-cadherin protein expression did not differ significantly between eutopic endometrium from adenomyosis subjects and healthy tissue, but N-cadherin expression was stronger in ectopic lesions. In adenomyosis, both E- and N-cadherin were more extensively expressed in basal (also known as invasive) glands than functional glands, suggesting maintenance of intercellular junctions and subsequent CCM. Extracellular matrix-degrading enzyme MMP9 was more abundantly expressed in eutopic stroma from adenomyosis patients compared to healthy tissue. Lower levels of caspase 3 were detected in both endometrium and lesions from adenomyosis patients, while LC3B was found significantly decreased in adenomyotic stroma compared to that of healthy subjects. Limitations, reasons for caution A limitation of the study is the 2D nature of in vitro cultures, which cannot fully reconstitute in vivo cell arrangement and cell-cell interactions, nor allow to observe CCM in real time. Wider implications of the findings M2 macrophages accumulating in endometrium and ectopic lesions from adenomyosis patients boost invasion capacity of endometrial epithelial and stromal cells, as shown by in vitro co-culture experiments. These findings indicate that M2 macrophages play a key role in disease pathogenesis and could be targeted to develop novel therapeutic options. Trial registration number not applicable

Conversion of M1 Macrophages to Foam Cells: Transcriptome Differences Determined by Sex.

M1 macrophages involved in pro-inflammatory processes can be induced by low-density lipoproteins (LDL), giving rise to foam cells. In the atheroma plaque, it has been identified that males present more advanced lesions associated with infiltration. Therefore, our study aims to investigate sex-related changes in the transcriptome of M1 macrophages during the internalization process of LDL particles. Peripheral blood mononuclear cells (PBMCs) from healthy male and female subjects were separated using Hystopaque, and monocytes were isolated from PBMCs using a positive selection of CD14+ cells. Cells were stimulated with LDL 10 µg/mL, and the transcriptional profile of M1 macrophages performed during LDL internalization was determined using a Clariom D platform array. Chromosome Y influences the immune system and inflammatory responses in males expressing 43% of transcripts in response to LDL treatment. Males and females share 15 transcripts, where most correspond to non-coding elements involved in oxidative stress and endothelial damage. During LDL internalization, male monocyte-derived M1 macrophages display more marked proinflammatory gene expression. In contrast, female M1 macrophages display a more significant number of markers associated with cell damage.

Multifaceted Defects in Monocytes in Different Phases of Chronic Hepatitis B Virus Infection: Lack of Restoration after Antiviral Therapy.

Monocytes play an important role in the control of microbial infection, but monocyte biology during chronic hepatitis B virus (HBV) infection (CHI) remains inadequately studied. We investigated the frequency, phenotype, and functions of monocyte subsets in different phases of CHI, namely, immune tolerance (IT), hepatitis B early antigen (HBeAg)-positive/HBeAg-negative chronic hepatitis B (EP-/EN-CHB, respectively), and inactive carrier (IC), identified factors responsible for their functional alterations, and determined the impact of antiviral therapy on these cells. Flow cytometric analysis indicated that HLA-DR+ CD14++ CD16- classical monocytes were significantly reduced while HLA-DR+ CD14++ CD16+ intermediate and HLA-DR+ CD14+ CD16++ nonclassical monocytes were expanded in IT and EP-/EN-CHB compared with those in IC and healthy controls (HC). In comparison to IC/HC, monocytes in IT and CHB exhibited diminished expression of Toll-like receptor 2 (TLR-2)/TLR-4/TLR-9 and cytokines interleukin-12 (IL-12)/tumor necrosis factor alpha (TNF-α)/IL-6 but produced higher levels of IL-10/transforming growth factor β (TGF-β). Further, monocytes in CHB/IT showed impaired phagocytosis and oxidative response relative to those in IC/HC. In vitro assays indicated that high titers of hepatitis B surface antigen (HBsAg) present in IT/CHB and of IL-4 in CHB triggered the functional defects in monocytes via induction of β-catenin. Additionally, monocyte-derived M1 macrophages of CHB/IT produced fewer proinflammatory and more anti-inflammatory cytokines than those of IC/HC, while in CHB/IT, the monocytes skewed the differentiation of CD4+ T cells more toward regulatory T cells and a Th2 phenotype. Moreover, monocytes in CHB and IT overexpressed chemokine receptor CCR2, which coincided with increased intrahepatic accumulation of β-catenin+ CD14+ cells. One year of tenofovir therapy failed to normalize monocyte functions or reduce serum HBsAg/IL-4 levels. Taken together, monocytes are functionally perturbed mostly in IT and EP-/EN-CHB phases. Targeting intramonocytic β-catenin or reducing HBsAg/IL-4 levels might restore monocyte function and facilitate viral clearance. IMPORTANCE Chronic HBV infection (CHI) is a major cause of end-stage liver disease for which pharmacological treatments currently available are inadequate. Chronically HBV-infected patients fail to mount an efficient immune response to the virus, impeding viral clearance and recovery from hepatitis. Monocytes represent a central part of innate immunity, but a comprehensive understanding on monocyte involvement in CHI is still lacking. We here report a multitude of defects in monocytes in chronically HBV-infected patients that include alteration in subset distribution, Toll-like receptor expression, cytokine production, phagocytic activity, oxidative response, migratory ability, polarization of monocyte-derived macrophages, and monocyte-T-cell interaction. We demonstrated that high levels of hepatitis B virus surface antigen and IL-4 potentiate these defects in monocytes via β-catenin induction while therapy with the nucleotide analog tenofovir fails to restore monocyte function. Our findings add to the continuing effort to devise new immunotherapeutic strategies that could reverse the immune defects in CHI.

Functional differences between primary monocyte-derived and THP-1 macrophages and their response to LCPUFAs

BackgroundIn immune cell models, macrophages are one of the most frequently used cell types. THP-1 cells are often used as model to study macrophage function, however they may act differently from primary human monocyte derived macrophages (MDMs). MethodsIn this study, we investigated the intrinsic baseline differences between the human macrophage cell line THP-1 and human primary MDMs. Additionally, we studied the difference in response to treatment with long-chain polyunsaturated fatty acids (LCPUFAs): well-described immunomodulators. ResultsAlthough the amount of cells that phagocytose were similar between the cell types, primary MDMs consumed significantly more E. coli bioparticles compared to THP-1 macrophages. In M1 macrophages, IL-12 secretion was almost fifty times higher by primary MDMs compared to THP-1 macrophages, thereby increasing the IL-12/IL-10 ratio. Despite this, the IL-12 secretion by THP-1 M1 macrophages was higher that the secretion of IL-10, thereby showing that it is still a suitable M1 type. Cytokine profiles differed between primary MDMs and THP-1 M1 and M2 macrophages. In response to LCPUFAs, primary M1 MDMs and THP-1 M1 macrophages were alike. Interestingly, primary M2 MDMs secreted less IL-10 and CCL22 when treated with LCPUFAs, whereas THP-1 M2 macrophages secreted more IL-10 when treated with LCPUFAs and showed no difference in CCL22 secretion. ConclusionsIn conclusion, in an M1 setting, both THP-1 and primary MDMs are suitable models. However, when interested in M2 models, the model choice highly depends on the research question.

Phagocytosis of platelets opsonized with differently glycosylated anti-HLA hIgG1 by monocyte-derived macrophages

Immune-mediated platelet refractoriness (PR) remains a significant problem in the setting of platelet transfusion and is predominantly caused by the presence of alloantibodies directed against class I human leukocyte antigens (HLA). Opsonization of donor platelets with these alloantibodies can result in rapid clearance after transfusion via multiple mechanisms, including antibody dependent cellular phagocytosis (ADCP). Interestingly, not all alloimmunized patients develop PR to unmatched platelet transfusions, suggesting variation in HLA-specific IgG responses between patients. Previously, we observed that the glycosylation profile of anti-HLA antibodies was highly variable between PR patients, especially with respect to Fc galactosylation, sialylation and fucosylation. In the current study, we investigated the effect of different Fc glycosylation patterns, with known effects on complement deposition and FcγR binding, on phagocytosis of opsonized platelets by monocyte-derived human macrophages. We found that the phagocytosis of antibody- and complement-opsonized platelets, by monocyte derived M1 macrophages, was unaffected by these qualitative IgG-glycan differences.

Metabolic Reprogramming Mediates Macrophage Polarization After Myocardial Infarction

BackgroundMacrophages play a critical role in left ventricular (LV) remodeling after myocardial infarction (MI), and limiting macrophage‐mediated inflammatory responses while enhancing reparative roles is a promising therapeutic strategy. Monocyte‐derived M1 macrophages mediate the early inflammatory response, while M2 macrophages mediate the later wound healing and scar formation phase. Metabolic reprogramming from glycolysis to mitochondrial oxidative phosphorylation mediates M1 to M2 polarization. We hypothesized that metabolic reprogramming occurs in macrophages over the course of post‐MI remodeling, and that blocking macrophage glycolysis attenuates post‐MI inflammation.MethodsMI was induced in adult (16‐20 week old) male C57BL/6J mice by permanent ligation of the left coronary artery for 1, 3, or 7 days, and cardiac function was assessed by echocardiography. Macrophages were extracted from the LV infarct area by immunomagnetic sorting. Macrophage metabolic flux was assessed by Extracellular Flux Analysis (Seahorse); upon glucose administration, glycolysis was measured by extracellular acidification rate (ECAR) while glucose oxidation was measured by oxygen consumption (OCR). Leukocyte subtypes (neutrophils, monocytes, resident macrophages) were assessed by flow cytometry. Real‐time PCR was used to assess gene expression by relative mRNA levels. To specifically block macrophage glycolysis, LysMCre mice were crossed with mice floxed for Slc2a1, the major macrophage glucose transporter, to generate macrophage‐Slc2a1 null mice.ResultsMI led to LV contractile dysfunction (decreased ejection fraction; days 1, 3, and 7 versus day 0), thinning of the infarct wall (days 1, 3, and 7) and non‐infarct wall (day 7), and dilation (increased internal diameter at diastole and end‐diastolic volume; day 7). At day 1, increased neutrophils (Ly6G+) and monocytes (Ly6Chi) were observed in the infarcted LV; at day 7, neutrophils and monocytes returned to baseline levels and the majority of macrophages were resident M2 subtype (Ly6Clow versus day 1). Day 1 macrophages showed elevated glycolysis (ECAR versus day 0) and increased mRNA levels of the M1 marker Il1b (versus day 3 and 7); day 3 macrophages showed increased glycolysis (ECAR versus day 0) and decreased glucose oxidation (versus day 0); day 7 macrophages showed decreased glycolysis (ECAR versus day 1 and 3) and increased glucose oxidation (OCR versus day 1 and 3), and increased Slc2a1 mRNA, Krebs cycle genes (Pdha1, Idh1/2, Sdha/bmRNA), pentose phosphate pathway genes (G6pd2/x, Pgd, Rpia, Taldo1 mRNA), and Il10 (M2 marker) mRNA. In macrophage‐Slc2a1 null mice, macrophage glycolysis was decreased and glucose oxidation increased at day 3, and infarct levels of Il1b were decreased compared to floxed controls.ConclusionsOur results indicate that time‐dependent macrophage metabolic reprogramming occurs over the MI remodeling process, and that blocking macrophage glycolysis may be a promising strategy for limiting post‐MI inflammation and adverse remodeling.

Galectin-3 S-glutathionylation regulates its effect on adipocyte insulin signaling

Protein-S-glutathionylation promotes redox signaling in physiological and oxidative distress conditions. Galectin-3 (Gal-3) promotes insulin resistance by down-regulating adipocyte insulin signaling, however, its S-glutathionylation and significance is not known. In this context, we report reversible S-glutathionylation of Gal-3. Site-directed mutagenesis established Gal-3 Cys187 as the putative S-glutathionylation site. Glutathionylated Gal-3 prevents Gal-3(WT)-Insulin Receptor interaction and facilitates insulin-induced murine adipocyte p-IRS1(tyr895) and p-AKT(ser473) signaling and glucose uptake in a Gal-3 Cys187 glutathionylation dependent manner in murine adipocytes, as assessed by Western blotting and 2-NBDG uptake assay respectively. Pre-glutathionylated Gal-3 at Cys187 resisted irreversible oxidation by H2O2. M2 macrophages showed enhanced Gal-3 S-glutathionylation when compared to M1 phenotype. Serum and stromal vascular fraction (SVF) isolated from control mice showed increased Gal-3 S-glutathionylation as compared to db/db mice. A significant increase in Gal-3 S-glutathionylation was observed in metformin-treated db/db mice when compared to db/db mice alone. Similar to murine, enhanced Gal-3 S-glutathionylation is observed in primary human monocyte derived M2 macrophages when compared to the M1 macrophage phenotype and Gal-3 regulates primary human adipocyte insulin signaling in a glutathionylation dependent manner. Collectively, we identified Gal-3 S-glutathionylation as a protective phenomenon, which relieves its inhibitory effect on adipocyte insulin signaling.

Differential Internalization of Thrombin-Derived Host Defense Peptides into Monocytes and Macrophages

Proteolytic cleavage of thrombin generates C-terminal host defense peptides exerting multiple immunomodulatory effects in response to bacterial stimuli. Previously, we reported that thrombin-derived C-terminal peptides (TCPs) are internalized in monocytes and macrophages in a time- and temperature-dependent manner. In this study, we investigated which endocytosis pathways are responsible for the internalization of TCPs. Using confocal microscopy and flow cytometry, we show that both clathrin-dependent and clathrin-independent pathways are involved in the internalization of the prototypic TCP GKY25 in RAW264.7 and human monocyte-derived M1 macrophages, whereas the uptake of GKY25 in monocytic THP-1 cells is mainly dynamin-dependent. Internalized GKY25 was transported to endosomes and finally lysosomes, where it remained detectable for up to 10 h. Comparison of GKY25 uptake with that of the natural occurring TCPs HVF18 and FYT21 indicates that the pathway of TCP endocytosis is not only cell type-dependent but also depends on the length and composition of the peptide as well as the presence of LPS and bacteria. Finally, using neutron reflectometry, we show that the observed differences between HVF18 and the other 2 TCPs may be explained partially by differences in membrane insertion. Taken together, we show that TCPs are differentially internalized into monocytes and macrophages.

Plasminogen Activating Inhibitor-1 Might Predict the Efficacy of Anti-PD1 Antibody in Advanced Melanoma Patients.

Plasminogen activating inhibitor-1 (PAI-1) plays crucial roles in the development of various cancers, including melanomas. Indeed, various pro-tumorigenic functions of PAI-1 in cancer progression and metastasis have been widely reported. Among them, PAI-1 is also reported as a key regulator of PD-L1 expression on melanoma cells through endocytosis, leading to abrogating the efficacy of anti-PD1 antibodies (Abs). These findings suggested that PAI-1 expression might predict the efficacy of anti-PD1 Abs. In this report, the expression and production of PAI-1 in melanoma patients were evaluated, and the immunomodulatory effects of PAI-1 on tumor-associated macrophages were investigated in vitro. Immunohistochemical staining of PAI-1 showed that PAI-1 expression on melanoma cells was significantly decreased in responders compared to non-responders. Moreover, baseline serum levels of PAI-1 were significantly decreased in responders compared to non-responders. Notably, PAI-1 decreased the production of various chemokines from monocyte-derived M2 macrophages in vitro, suggesting that PAI-1 might decrease tumor-infiltrating lymphocytes to hamper the anti-tumor effects of anti-PD1 Abs. These results suggest that baseline serum levels of PAI-1 may be useful as a biomarker for identifying patients with advanced cutaneous melanoma most likely to benefit from anti-melanoma immunotherapy.

Histamine Increases Th2 Cytokine-Induced CCL18 Expression in Human M2 Macrophages.

The chemokine CCL18 is produced in cells of the myelomonocytic lineage and represents one of the most highly expressed chemokines in lesional skin and serum of atopic dermatitis patients. We investigated the role of histamine in CCL18 production in human monocyte-derived M2 macrophages differentiated in the presence of M-CSF and activated with IL-4, IL-13 or with IL-10. Since expression and regulation of histamine H1 receptor (H1R), H2R and H4R by IL-4 and IL-13 on human M2 macrophages were described, we analyzed expression of the histamine receptors in response to IL-10 stimulation by quantitative RT-PCR. IL-10 upregulated H2R and downregulated H4R mRNA expression by trend in M2 macrophages. IL-10, but in a more pronounced manner, IL-4 and IL-13, also upregulated CCL18. Histamine increased the cytokine-induced upregulation of CCL18 mRNA expression by stimulating the H2R. This effect was stronger in IL-10-stimulated M2 macrophages where the upregulation of CCL18 was confirmed at the protein level by ELISA using selective histamine receptor agonist and antagonists. The histamine-induced CCL18 upregulation in IL-10-activated M2 macrophages was almost similar in cells obtained from atopic dermatitis patients compared to cells from healthy control persons. In summary, our data stress a new function of histamine showing upregulation of the Th2 cells attracting chemokine CCL18 in human, activated M2 macrophages. This may have an impact on the course of atopic dermatitis and for the development of new therapeutic interventions.

Macrophages Control the Bioavailability of Vitamin D and Vitamin D-Regulated T Cell Responses.

The active form of vitamin D3 (1,25(OH)2D3) has a great impact on T cell effector function. Thus, 1,25(OH)2D3 promotes T helper 2 (Th2) and regulatory T (Treg) cell function and concomitantly inhibits Th1 and Th17 cell function. Thus, it is believed that vitamin D exerts anti-inflammatory effects. However, vitamin D binding protein (DBP) strongly binds both 1,25(OH)2D3 and the precursor 25(OH)D3, leaving only a minor fraction of vitamin D in the free, bioavailable form. Accordingly, DBP in physiological concentrations would be expected to block the effect of vitamin D on T cells and dendritic cells. In the present study, we show that pro-inflammatory, monocyte-derived M1 macrophages express very high levels of the 25(OH)D-1α-hydroxylase CYP27B1 that enables them to convert 25(OH)D3 into 1,25(OH)2D3 even in the presence of physiological concentrations of DBP. Co-cultivation of M1 macrophages with T cells allows them to overcome the sequestering of 25(OH)D3 by DBP and to produce sufficient levels of 1,25(OH)2D3 to affect T cell effector function. This study suggests that in highly inflammatory conditions, M1 macrophages can produce sufficient levels of 1,25(OH)2D3 to modify T cell responses and thereby reduce T cell-mediated inflammation via a vitamin D-mediated negative feed-back loop.

Cladribine inhibits secretion of pro-inflammatory cytokines and phagocytosis in human monocyte-derived M1 macrophages in-vitro

Cladribine (Cd) is a purine nucleoside analogue which in an oral formulation is approved for treatment of patients with multiple sclerosis (MS). It is known to mediate the effect through a short-term selective reduction of lymphocytes with minimal effect on the innate immune system. However, a few studies have emerged, that also demonstrate a beneficial immunomodulatory effect of cladribine on monocyte-derived cells. As cladribine crosses the blood–brain barrier this effect could have clinical meaningful impact in the treatment of MS, where recruitment of innate cells such as M1 macrophages play a role in plaque development. Here, we investigated the in-vitro effect on monocyte differentiation into M1 and M2 macrophages and dendritic cells as well as the effect on activation of M1 macrophages. In our experiments, cladribine in therapeutic relevant in-vitro concentrations, did not lead to apoptosis in differentiated M1, M2 macrophages or DCs and did not interfere with the phenotype of these differentiated cells. In M1 macrophages, cladribine reduced the secretion of IL-6 and TNF-α observed after activation with LPS. Similar, cladribine reduced the phagocytic capacity of LPS activated M1 macrophages but did not affect unactivated cells. We conclude, that such reduction of inflammatory potential as well as reduced M1 phagocytic activity, e.g. within an MS plaque, could be an additional clinical meaningful effect of cladribine in the treatment of MS while at the same time it would leave M1 macrophages intact for the protection against infections.

Possible roles of CXCL13/CXCR5 axis in the development of bullous pemphigoid.

CXCL13 recruits CXCR5+ follicular helper T (Tfh) cells in inflammatory lesions to develop secondary lymphoid organs. Tfh cells activate B cells to produce antibodies during humoral immune responses. Indeed, as previous reports suggested, CXCR5+ cell numbers were increased in the peripheral blood of bullous pemphigoid (BP) patients when compared with healthy donors, and the ratio of CXCR5+ cells was positively correlated with the anti-BP180-NC16A titers. From the above findings, in this report, we hypothesized that a chemokine related to CXCR5+ cells, namely CXCL13, may play a role in the development of BP. We performed immunohistochemical staining of CXCR5, CXCL13, LL37, CXCL10 and CCL20 for 10 cases of BP and 10 cases of pemphigus vulgaris (PV), and quantitatively analyzed the staining by digital microscopy. Moreover, we investigated the CXCL10 and CXCL13 production in BP and PV patients by enzyme-linked immunosorbent assay. The immunomodulatory effects of LL37 on the production of T-helper 17-related chemokines were evaluated using monocyte-derived M2 macrophages. Immunohistochemical staining and digital microscopic analysis showed that the ratios of CXCR5+ , CXCL13+ and LL37+ cells in the dermis were significantly higher in BP patients than in PV patients. Notably, the ratio of CXCL13+ cells was positively correlated with the anti-BP180-NC16A titers. Moreover, the serum levels of CXCL13 were positively correlated with the anti-BP180-NC16A titers. Furthermore, CD163+ M2 macrophages stimulated by LL37 in vitro produced CXCL10 and CCL20. In the lesional skin of BP, CD163+ macrophages CXCL10 and CCL20 were produced. The serum levels of CXCL10 were negatively correlated with the anti-BP180-NC16A titers. The present study results indicate that the mechanism of the development of BP may involve the CXCL13/CXCR5-mediated migration of Tfh cells.