Expression Of M2 Markers Research Articles

Upregulation of programmed death ligand-1 in tumor-associated macrophages affects chemotherapeutic response in ovarian cancer cells.

To better understand the mechanism of chemoresistance in ovarian cancer cells, we aimed to investigate the influence of macrophages on the tumor cell response to carboplatin and identify the genes associated with chemoresistance. We mimicked the tumor microenvironment (TME) using a co-culture technique and compared the proliferation of ovarian cells with and without macrophages. We also examined M1 and M2 marker expression and the expression of key TME genes. Post the co-culture, we treated ovarian cancer cells with carboplatin and elucidated the function of programmed death-ligand 1 (PD-L1) in carboplatin chemoresistance. We investigated CD68 and PD-L1 expression in normal and cancerous ovarian tissues using immunohistochemistry (IHC). Finally, we analyzed the association between CD68 or PD-L1 expression and survival outcomes. Inducible nitric oxide synthase (iNOS) was downregulated, while the gene expression of M2 macrophage markers was increased in ovarian cancer cells. PD-L1, vascular endothelial growth factor (VEGF), Interleukin (IL)-6, IL-10, IL-12, signal transducer and activator of transcription 3 (STAT3), B-cell lymphoma 2 (BCL2), multidrug resistance 1 (MDR1), and colony stimulating factor 1 (CSF-1) were upregulated. Notably, PD-L1 was upregulated in both the ovarian cancer cells and macrophages. Ovarian cancer cells co-cultured with macrophages exhibited statistically significant carboplatin resistance compared to single-cultured ovarian cancer cells. PD-L1 silencing induced chemosensitivity in both types of co-cultured ovarian cancer cells. However, IHC results revealed no correlation between PD-L1 expression and patient survival or cancer stage. CD68 expression was significantly increased in cancer cells compared to normal or benign ovarian tumor cells, but it was not associated with the survival outcomes of ovarian cancer patients. Our study demonstrated that ovarian cancer cells interact with macrophages to induce the M2 phenotype. We also established that PD-L1 upregulation in both ovarian cancer cells and macrophages is a key factor for carboplatin chemoresistance.

TRNA-derived small RNA, 5'tiRNA-Gly-CCC, promotes skeletal muscle regeneration through the inflammatory response.

Increasing evidence shows that tRNA-derived small RNAs (tsRNAs) are not only by-products of transfer RNAs, but they participate in numerous cellular metabolic processes. However, the role of tsRNAs in skeletal muscle regeneration remains unknown. Small RNA sequencing revealed the relationship between tsRNAs and skeletal muscle injury. The dynamic expression level of 5'tiRNA-Gly after muscle injury was confirmed by real-time quantitative PCR (q-PCR). In addition, q-PCR, flow cytometry, the 5-ethynyl-2'-deoxyuridine (Edu), cell counting kit-8, western blotting and immunofluorescence were used to explore the biological function of 5'tiRNA-Gly. Bioinformatics analysis and dual-luciferase reporter assay were used to further explore the mechanism of action under the biological function of 5'tiRNA-Gly. Transcriptome analysis revealed that tsRNAs were significantly enriched during inflammatory response immediately after muscle injury. Interestingly, we found that 5'tiRNA-Gly was significantly up-regulated after muscle injury (P<0.0001) and had a strong positive correlation with inflammation in vivo. In vitro experiments showed that 5'tiRNA-Gly promoted the mRNA expression of proinflammatory cytokines (IL-1β, P=0.0468; IL-6, P=0.0369) and the macrophages of M1 markers (TNF-α, P=0.0102; CD80, P=0.0056; MCP-1, P=0.0002). On the contrary, 5'tiRNA-Gly inhibited the mRNA expression of anti-inflammatory cytokines (IL-4, P=0.0009; IL-10, P=0.0007; IL-13, P=0.0008) and the mRNA expression of M2 markers (TGF-β1, P=0.0016; ARG1, P=0.0083). Flow cytometry showed that 5'tiRNA-Gly promoted the percentage of CD86+ macrophages (16%, P=0.011) but inhibited that of CD206+ macrophages (10.5%, P=0.012). Immunofluorescence showed that knockdown of 5'tiRNA-Gly increased the infiltration of M2 macrophages to the skeletal muscles (13.9%, P=0.0023) and inhibited the expression of Pax7 (P=0.0089) in vivo. 5'tiRNA-Gly promoted myoblast the expression of myogenic differentiation marker genes (MyoD, P=0.0002; MyoG, P=0.0037) and myotube formation (21.3%, P=0.0016) but inhibited the positive rate of Edu (27.7%, P=0.0001), cell viability (22.6%, P=0.003) and the number of myoblasts in the G2 phase (26.3%, P=0.0016) in vitro. Mechanistically, we found that the Tgfbr1 gene is a direct target of 5'tiRNA-Gly mediated by AGO1 and AGO3. 5'tiRNA-Gly dysregulated the expression of downstream genes related to inflammatory response, activation of satellite cells and differentiation of myoblasts through the TGF-β signalling pathway by targeting Tgfbr1. These results reveal that 5'tiRNA-Gly potentially regulated skeletal muscle regeneration by inducing inflammation via the TGF-β signalling pathway. The findings of this study uncover a new potential target for skeletal muscle regeneration treatment.

LMT503, A NOVEL THERAPY FOR INFLAMMATORY BOWEL DISEASE THROUGH MACROPHAGE POLARIZATIONS BY METABOLIC REPROGRAMMING OF ACTIVATED MACROPHAGE

Abstract Macrophages are the first line of defense against self-stimuli and non-self-pathogens and adopt diverse activation states by reprogramming their cell metabolism and thereby polarizing their phenotypes in order to drive their pro-inflammatory or pro-resolving responses. As the key gatekeeper of intestinal immune homeostasis, macrophage polarization has an important effect on maintaining tissue homeostasis with inducing resolution, and therefore macrophages phenotype alteration by modulating their cellular metabolism might be a novel therapeutic approach in IBD. LMT503 is an orally available organic small molecule and exerted the macrophage polarization from M1-like inflammatory macrophage to M2-like anti-inflammatory macrophages in LPS-induced bone marrow-derived macrophage in vitro experiment. The expression level of M1-related intracellular functional marker iNOS was suppressed by LMT503, while Arg-1 which is M2-associated marker was increased. LMT503 also performed metabolic reprogramming with decreasing anabolic metabolism but enhancing catabolic metabolism including mitochondrial metabolism. In the therapeutic settings using various IBD in vivo models such as DSS-induced acute/chronic mice models and adoptive T cell transfer mice model, LMT503 exerted macrophage polarization with showing reduced expression level of M1 markers such as iNOS, CD80, and CD86 but increased expression of M2 markers like Arg-1, CD168, and CD206. LMT503 mitigated inflammation with decreased production of inflammatory cytokines (TNF-a, IL-6, IL-1b, IL-18), increased anti-inflammatory cytokines (IL-10, IL-22), and reduced neutrophil infiltrations. In the addition to increased ratio of M2/M1 macrophages, LMT503 increased ratio of Treg/Th17. LMT503 also induced epithelial restitution with increased number of goblet cells, and showed decreased bacterial translocation from gut to mesenteric lymph node. In a chronic IBD mice model, LMT503 showed reduced intestinal fibrotic marker a-SMA which is one of critical complications for patients with long-term IBD. Mode of action through macrophage polarization was proved by using a macrophage-depleted IBD in vivo model, and LMT503 did not improve the disease associate index or inflammation. Also LMT503 showed re-storing immunity in an in vivo study of challenging pathogen to LMT503-treated and washed-out model, which avoids the possibility of an opportunistic infections. In summary, LMT503 was very efficacious in various IBD in vivo models with showing the resolution of intestinal inflammation and mucosal healing through improved epithelial restitution via macrophage polarizations, and this approach can be a very effective and safe therapy for multifaceted IBD. The first in human study with LMT503 will be initiating in 4Q 2022.

TRPA1 as a potential factor and drug target in scleroderma: dermal fibrosis and alternative macrophage activation are attenuated in TRPA1-deficient mice in bleomycin-induced experimental model of scleroderma

BackgroundSystemic sclerosis is a rheumatoid disease best known for its fibrotic skin manifestations called scleroderma. Alternatively activated (M2-type) macrophages are normally involved in the resolution of inflammation and wound healing but also in fibrosing diseases such as scleroderma. TRPA1 is a non-selective cation channel, activation of which causes pain and neurogenic inflammation. In the present study, we investigated the role of TRPA1 in bleomycin-induced skin fibrosis mimicking scleroderma.MethodsWild type and TRPA1-deficient mice were challenged with intradermal bleomycin injections to induce a scleroderma-mimicking disease. Macrophages were investigated in vitro to evaluate the underlying mechanisms.ResultsBleomycin induced dermal thickening and collagen accumulation in wild type mice and that was significantly attenuated in TRPA1-deficient animals. Accordingly, the expression of collagens 1A1, 1A2, and 3A1 as well as pro-fibrotic factors TGF-beta, CTGF, fibronectin-1 and YKL-40, and M2 macrophage markers Arg1 and MRC1 were lower in TRPA1-deficient than wild type mice. Furthermore, bleomycin was discovered to significantly enhance M2-marker expression particularly in the presence of IL-4 in wild type macrophages in vitro, but not in macrophages harvested from TRPA1-deficient mice. IL-4-induced PPARγ-expression in macrophages was increased by bleomycin, providing a possible mechanism behind the phenomenon.ConclusionsIn conclusion, the results indicate that interfering TRPA1 attenuates fibrotic and inflammatory responses in bleomycin-induced scleroderma. Therefore, TRPA1-blocking treatment could potentially alleviate M2 macrophage driven diseases like systemic sclerosis and scleroderma.

1,25-Dihydroxyvitamin D regulates macrophage activation through FBP1/PKR and ameliorates arthritis in TNF-transgenic mice

1,25-Dihydroxyvitamin D (1,25(OH)2D3) has immunomodulatory activity and its deficiency correlates with rheumatoid arthritis (RA) incidence. Whether 1,25(OH)2D3 modulates macrophage activation or protects against RA remains unclear. We demonstrate that 1,25(OH)2D3 suppresses M1 macrophage polarization and CD80, IL-6, CXCL10, IFIT1, IFI44, and double-stranded RNA-dependent protein kinase R (PKR) expression in the macrophages of RA patients. In phorbol 12-myristate 13-acetate-induced THP-1 cells, 1,25(OH)2D3 upregulates fructose-1,6-bisphosphatase 1 (FBP1) expression through direct promoter interaction. FBP1 interacts with PKR and promotes PKR ubiquitination degradation. SiR-FBP1 transfection impairs 1,25(OH)2D3 action and suppresses IL-6, CXCL10, IFIT1, IFI27, and IFI44 expression in macrophages, whereas siR-PKR transfection impairs siR-FBP1 activity in 1,25(OH)2D3-treated macrophages. 1,25(OH)2D3 treatment ameliorates the clinical signs of arthritis in tumor necrosis factor-transgenic mice, inhibits M1 polarization and marker expression, and promotes FBP1 expression in mononuclear cells isolated from swollen joints; thus, 1,25(OH)2D3 suppresses M1 macrophage activation through FBP1/PKR and ameliorates arthritis by restoring the macrophage subtype.

Statins change the cytokine profile in Trypanosoma cruzi-infected U937 macrophages and murine cardiac tissue through Rho-associated kinases inhibition.

Chronic Chagasic cardiomyopathy (CCC), caused by the protozoan Trypanosoma cruzi, is the most severe manifestation of Chagas disease.CCC is characterized by cardiac inflammation and fibrosis caused by a persistent inflammatory response. Following infection, macrophages secrete inflammatory mediators such as IL-1β, IL-6, and TNF-α to control parasitemia. Although this response contains parasite infection, it causes damage to the heart tissue. Thus, the use of immunomodulators is a rational alternative to CCC. Rho-associated kinase (ROCK) 1 and 2 are RhoA-activated serine/threonine kinases that regulate the actomyosin cytoskeleton. Both ROCKs have been implicated in the polarization of macrophages towards an M1 (pro-inflammatory) phenotype. Statins are FDA-approved lipid-lowering drugs that reduce RhoA signaling by inhibiting geranylgeranyl pyrophosphate (GGPP) synthesis. This work aims to identify the effect of statins on U937 macrophage polarization and cardiac tissue inflammation and its relationship with ROCK activity during T. cruzi infection. PMA-induced, wild-type, GFP-, CA-ROCK1- and CA-ROCK2-expressing U937 macrophages were incubated with atorvastatin, or the inhibitors Y-27632, JSH-23, TAK-242, or C3 exoenzyme incubated with or without T. cruzi trypomastigotes for 30 min to evaluate the activity of ROCK and the M1 and M2 cytokine expression and secretion profiling. Also, ROCK activity was determined in T. cruzi-infected, BALB/c mice hearts. In this study, we demonstrate for the first time in macrophages that incubation with T. cruzi leads to ROCK activation via the TLR4 pathway, which triggers NF-κB activation. Inhibition of ROCKs by Y-27632 prevents NF-κB activation and the expression and secretion of M1 markers, as does treatment with atorvastatin. Furthermore, we show that the effect of atorvastatin on the NF-kB pathway and cytokine secretion is mediated by ROCK. Finally, statin treatment decreased ROCK activation and expression, and the pro-inflammatory cytokine production, promoting anti-inflammatory cytokine expression in chronic chagasic mice hearts. These results suggest that the statin modulation of the inflammatory response due to ROCK inhibition is a potential pharmacological strategy to prevent cardiac inflammation in CCC.

Impact of the flame retardant 2,2'4,4'-tetrabromodiphenyl ether (PBDE-47) in THP-1 macrophage-like cell function via small extracellular vesicles.

2,2'4,4'-tetrabromodiphenyl ether (PBDE-47) is one of the most widespread environmental brominated flame-retardant congeners which has also been detected in animal and human tissues. Several studies have reported the effects of PBDEs on different health issues, including neurobehavioral and developmental disorders, reproductive health, and alterations of thyroid function. Much less is known about its immunotoxicity. The aim of our study was to investigate the effects that treatment of THP-1 macrophage-like cells with PBDE-47 could have on the content of small extracellular vesicles' (sEVs) microRNA (miRNA) cargo and their downstream effects on bystander macrophages. To achieve this, we purified sEVs from PBDE-47 treated M(LPS) THP-1 macrophage-like cells (sEVsPBDE+LPS) by means of ultra-centrifugation and characterized their miRNA cargo by microarray analysis detecting the modulation of 18 miRNAs. Furthermore, resting THP-1 derived M(0) macrophage-like cells were cultured with sEVsPBDE+LPS, showing that the treatment reshaped the miRNA profiles of 12 intracellular miRNAs. This dataset was studied in silico, identifying the biological pathways affected by these target genes. This analysis identified 12 pathways all involved in the maturation and polarization of macrophages. Therefore, to evaluate whether sEVsPBDE+LPS can have some immunomodulatory activity, naïve M(0) THP-1 macrophage-like cells cultured with purified sEVsPBDE+LPS were studied for IL-6, TNF-α and TGF-β mRNAs expression and immune stained with the HLA-DR, CD80, CCR7, CD38 and CD209 antigens and analyzed by flow cytometry. This analysis showed that the PBDE-47 treatment does not induce the expression of specific M1 and M2 cytokine markers of differentiation and may have impaired the ability to make immunological synapses and present antigens, down-regulating the expression of HLA-DR and CD209 antigens. Overall, our study supports the model that perturbation of miRNA cargo by PBDE-47 treatment contributes to the rewiring of cellular regulatory pathways capable of inducing perturbation of differentiation markers on naïve resting M(0) THP-1 macrophage-like cells.

1,25-Dihydroxy Vitamin D3 Facilitates the M2 Polarization and β-Amyloid Uptake by Human Microglia in a TREM2-Dependent Manner.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by dementia as the primary clinical symptom. The production and accumulation of aggregated β-amyloid (Aβ) in patient brain tissues is one of the hallmarks of AD pathogenesis. Microglia, brain-resident macrophages, produce inflammatory cytokines in response to Aβ oligomers or fibrils exacerbating Aβ pathology in AD. HMO6 cells were treated with Aβ42 in the presence or absence of 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) to determine its potential immunomodulatory effects, and the expression of pro-/anti-inflammatory cytokines, M1/M2-associated markers, Toll-like receptors (TLRs), and triggering receptor expressed on myeloid cells 2 (TREM2) was examined. 1,25(OH)2D3 was found to suppress Aβ-induced expression of proinflammatory cytokines (TNF-α, IL-1β, and IL-6), M1 markers (CD86 and iNOS), and TLR2/4, whilst increasing the expression of anti-inflammatory cytokines (IL-4, IL-10, and CCL17) and M2 markers (CD206 and Arg-1). Furthermore, 1,25(OH)2D3 promoted TREM2 expression and Aβ uptake by HMO6 cells, and the enhancement of Aβ uptake and M2 polarization was revealed to be TREM2-dependent. The findings of this study suggest that 1,25(OH)2D3 facilitates M2 polarization and Aβ uptake in a TREM2-dependent manner.

EMR1/ADGRE1 Expression in Cancer Cells Upregulated by Tumor-Associated Macrophages Is Related to Poor Prognosis in Colorectal Cancer.

EMR1, a member of the adhesion G protein-coupled receptor family (ADGRE1), is a macrophage marker that is abnormally expressed in cancer cells. However, its clinical significance in colorectal cancer (CRC) is not well-known. In this investigation, EMR1 expression in tumor cells (EMR1-TC) was found in 91 (22.8%) of the 399 CRC samples tested by immunohistochemical staining and showed a significant relationship with lymph node metastasis. Furthermore, EMR1-TC was significantly associated with CD68+ CD163+ tumor-associated macrophages (TAMs), and CRC with a high combined EMR1-TC+CD68+CD163+ score showed worse recurrence-free survival prognosis. In an in vitro co-culture assay of colon cancer cells with myeloid cells, we found that EMR1 expression significantly upregulated in cancer cells was induced by macrophages. In addition, there was increased expression of M2 markers (CD163 and interleukin-6 & 10) in myeloid portion, while that of M1 markers (CD86 and iNOS) remained unchanged. Accordingly, upon treatment with M2 macrophage polarization inhibitors (O-ATP, trametinib, bardoxolone methyl), EMR1 expression reduced significantly, along with M2 markers (CD163 and interleukin-6 & 10). In conclusion, EMR1-TC was a high-risk factor for lymph node metastasis and correlated with poor recurrence free survival, particularly in patients with TAM-rich CRC. Furthermore, EMR1 expression in colon cancer cells may be related to M2 macrophage polarization and vice versa.

Liposome-based nanoparticles impact on regulatory and effector phenotypes of macrophages and T cells in multiple Sclerosis patients

Current available treatments of Multiple Sclerosis (MS) reduce neuroinflammation acting on different targets on the immune system, but potentially lead to severe side effects and have a limited efficacy in slowing the progression of the disease. Here, we evaluated in vitro the immunomodulatory potential of a new class of nanoparticles – liposomes, constituted by a double-layer of phosphatidylserine (PSCho/PS), and double-faced, with an outer layer of phosphatidylserine and an inner layer of phosphatidic acid (PSCho/PA), either alone or in the presence of the myelin basic protein (MBP) peptide (residues 85–99) (PSCho/PS-MBP and PSCho/PA-MBP). Results showed that PSCho/PS are equally and efficiently internalized by pro- and anti-inflammatory macrophages (M1 and M2 respectively), while PSCho/PA were internalized better by M2 than M1. PSCho/PS liposomes were able to inhibit the secretion of innate pro-inflammatory cytokine IL-1β. PSCho/PS liposomes expanded Tregs, reducing Th1 and Th17 cells, while PSCho/PA liposomes were unable to dampen pro-inflammatory T cells and to promote immune-regulatory phenotype (Treg). The ability of PSCho/PS liposomes to up-regulate Treg cells was more pronounced in MS patients with high basal expression of M2 markers. PSCho/PS liposomes were more effective in decreasing Th1 (but not Th17) cells in MS patients with a disease duration >3 months. On the other hand, down-modulation of Th17 cells was evident in MS patients with active, Gadolinium enhancing lesions at MRI and in MS patients with a high basal expression of M1-associated markers in the monocytes. The same findings were observed for the modulation of MBP-driven Th1/Th17/Treg responses.These observations suggest that early MS associate to a hard-wired pro-Th1 phenotype of M1 that is lost later during disease course. On the other hand, acute inflammatory events reflect a temporary decrease of M2 phenotype that however is amenable to restauration upon treatment with PSCho/PS liposomes. Thus, together these data indicate that monocytes/macrophages may play an important regulatory function during MS course and suggest a role for PSCho/PS and PSCho/PS-MBP as new therapeutic tools to dampen the pro-inflammatory immune responses and to promote its regulatory branch.

Macrophage polarity and wound age determination

We investigated the dynamics of the gene expression of M1 and M2 macrophage markers during skin wound healing in mice. Expression of M1-macrophage markers, such as Il12a, Tnf, Il6, Il1b, and Nos2 was upregulated after wounding and peaked at 1 or 3 days after injury, and that of M2-macrophage markers such as Mrc1, Cd163, Ccl17, Arg, and Tgfb1, peaked at 6 days after injury. Consistent with these findings, using triple-color immunofluorescence analysis revealed that F4/80+CD80+ M1 macrophages were more abundant than F4/80+CD206+ M2 macrophages on day 3 in mouse wound specimens, and that M2 macrophages were prominently detected in day 6 wounds. For application in forensic practice, we examined macrophage polarization using human wound specimens. The average ratios of CD68+iNOS+ M1 macrophages to CD68+CD163+ M2 macrophages (M1/M2 ratios) were greater than 2.5 for the wounds aged 2–5 days. Out of 11 wounds aged 1–5 days, five samples had the M1/M2 ratios of > 3.0. These observations propose that the M1/M2 ratios of 3.0 would indicate a wound age of 1–5 days as the forensic opinion. This study showed that M1 and M2 macrophages in human skin wound might be a promising marker for wound age determination.

ALA-PDT regulates macrophage M1 polarization via ERK/MAPK-NLRP3 pathway to promote the early inflammatory response.

Photodynamic therapy (PDT) is a promising new approach to promote wound healing and its effectiveness has been demonstrated in both clinical and animal studies. Macrophages are the key cells in wound healing and inflammatory response. However, the mechanism of action of PDT on macrophages in promoting wound healing is still unclear. In this study, RAW264.7 cells were used. We analyzed the expression levels of macrophage markers arginase 1 (Arg-1), CD206, iNOS, CD86, and inflammatory factors IL-6, TNF-α, andIL-1β by reverse transcription-polymerase chain reaction and Western blot, Milliplex microtubule-associated protein multiplex assay was performed to analyze the expression of inflammatory factors in the supernatant. Live cell Imaging System to observe the dynamic process of macrophage phagocytosis. Western blot was performed to observe the activation of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) and NOD-like receptor protein 3 (NLRP3) inflammasome. 5-Aminolevulinic acid (ALA)-PDT increased the expression of M1 marker iNOS/CD86 and decreased the expression of Arg-1/CD206 in RAW264.7 cells, while, proinflammatory factors IL-6, TNF-α, and IL-1β expression was enhanced and macrophage phagocytosis was increased. We also found that these phenomena were associated with activation of the ERK/MAPK-NLRP3 pathway. ALA-PDT promotes early inflammatory responses by regulating macrophage M1 polarization through the ERK/MAPK-NLRP3 pathway. It also promotes macrophage phagocytosis.

Intranasal interferon-beta alleviates anxiety and depressive-like behaviors by modulating microglia polarization in an Alzheimer's disease model

Alzheimer's disease (AD) patients frequently experience neuropsychiatric symptoms (NPS), which are linked to a lower quality of life and a faster rate of disease progression. A growing body of research indicates that several microglial phenotypes control the inflammatory response and are crucial in the pathophysiology of AD-related NPS. Given the crucial role played by inflammatory mediators produced by microglia in developing of NPS, interferon-beta (IFNβ), a cytokine with anti-inflammatory capabilities, maybe a successful treatment for NPS caused by AD. In this investigation, using a rat model of AD, we examined the impact of intranasal treatment of IFNβ on anxious/depressive-like behavior and microglial M1/M2 polarization. The rat hippocampus was bilaterally injected with lentiviruses harboring mutant human amyloid precursor protein. Rats were given recombinant IFNβ1a (68,000 IU/rat) via the intranasal route, starting on day 23 following viral infection and continuing until day 49. On days 47–49, the elevated plus maze, forced swim, and tail suspension tests were applied to measure anxiety- and depressive-like behavior. Additionally, qPCR was utilized to quantify the expression of M1 markers (CD68, CD86, and CD40) and M2 markers (Ym1, CD206, Arg1, GDNF, BDNF, and SOCS1). Our findings demonstrated that decreased M2 marker expression is accompanied by anxious/depressive-like behavior when the mutant human APP gene is overexpressed in the hippocampus. In the rat model of AD, IFNβ therapy reduces anxious/depressive-like behaviors, at least in part by polarizing microglia towards M2. Therefore, IFNβ may be a viable therapeutic drug for reducing NPS in the context of AD.

Melatonin Suppresses Macrophage M1 Polarization and ROS-Mediated Pyroptosis via Activating ApoE/LDLR Pathway in Influenza A-Induced Acute Lung Injury.

Influenza virus infection is one of the strongest pathogenic factors for the development of acute lung injury (ALI)/ acute respiratory distress syndrome (ARDS). However, the underlying cellular and molecular mechanisms have not been clarified. In this study, we aim to investigate whether melatonin modulates macrophage polarization, oxidative stress, and pyroptosis via activating Apolipoprotein E/low-density lipoprotein receptor (ApoE/LDLR) pathway in influenza A-induced ALI. Here, wild-type (WT) and ApoE-/- mice were instilled intratracheally with influenza A (H3N2) and injected intraperitoneally with melatonin for 7 consecutive days. In vitro, WT and ApoE-/- murine bone marrow-derived macrophages (BMDMs) were pretreated with melatonin before H3N2 stimulation. The results showed that melatonin administration significantly attenuated H3N2-induced pulmonary damage, leukocyte infiltration, and edema; decreased the expression of proinflammatory M1 markers; enhanced anti-inflammatory M2 markers; and switched the polarization of alveolar macrophages (AMs) from M1 to M2 phenotype. Additionally, melatonin inhibited reactive oxygen species- (ROS-) mediated pyroptosis shown by downregulation of malonaldehyde (MDA) and ROS levels as well as inhibition of the NLRP3/GSDMD pathway and lactate dehydrogenase (LDH) release. Strikingly, the ApoE/LDLR pathway was activated when melatonin was applied in H3N2-infected macrophages and mice. ApoE knockout mostly abrogated the protective impacts of melatonin on H3N2-induced ALI and its regulatory ability on macrophage polarization, oxidative stress, and pyroptosis. Furthermore, recombinant ApoE3 (re-ApoE3) inhibited H3N2-induced M1 polarization of BMDMs with upregulation of MT1 and MT2 expression, but re-ApoE2 and re-ApoE4 failed to do this. Melatonin combined with re-ApoE3 played more beneficial protective effects on modulating macrophage polarization, oxidative stress, and pyroptosis in H3N2-infected ApoE-/- BMDMs. Our study indicated that melatonin attenuated influenza A- (H3N2-) induced ALI by inhibiting the M1 polarization of pulmonary macrophages and ROS-mediated pyroptosis via activating the ApoE/LDLR pathway. This study suggested that melatonin-ApoE/LDLR axis may serve as a novel therapeutic strategy for influenza virus-induced ALI.

In vitro phenotypic effects of Lipoxin A4 on M1 and M2 polarized macrophages derived from THP-1.

Lipoxin A4 (LXA4) is a specialized pro-resolving mediator involved in the resolution phase of inflammation that is crucial for the return of tissues to homeostasis, healing, and regenerative processes. LXA4 can modify the microenvironment via its receptor, formyl peptide receptor 2 (FPR2) and thus modulate the inflammatory response. However, the effect of exogeneous LXA4 application on polarized macrophages remains unstudied. The objective of this study was to assess the effect of LXA4 on macrophage activity and on the phenotype modulation of polarized M1 and M2 macrophages derived from THP-1 monocytes. Once differentiated, human macrophages were incubated with interleukin 4 (IL-4) and IL-13 to obtain M2-polarized macrophages or with interferon gamma and lipopolysaccharide for classical macrophage activation. The mRNA and protein expression of M1 and M2 markers confirmed the polarization of THP-1-derived macrophages. LXA4 (0-100nM) did not affect the viability of M1 and M2 macrophages or the phagocytic activity of these cells. Gene expression of FPR2, referred as a receptor for the LXA4, was higher in M1 compared with M2, and was not modified by the LXA4 at the doses used. Moreover, LXA4 exhibited anti-inflammatory properties illustrated by the decreasing in the gene expression of pro-inflammatory cytokines (IL-6, tumor necrosis factor alpha, IL-1β) in M1 and by the increase in the expression of anti-inflammatory cytokines (IL-10) in M2 macrophages. These results provide new insights regarding the potential of LXA4 to regulate the polarization state of macrophages.

Cycloastragenol suppresses M1 and promotes M2 polarization in LPS-stimulated BV-2 cells and ischemic stroke mice

There are two distinct phenotypes of activated microglia, pro-inflammatory M1 and anti-inflammatory M2. Accumulating evidence indicates that shifting the microglial polarization from M1 to M2 is a potential strategy for the treatment of neuroinflammation-associated brain diseases, including ischemic stroke. Cycloastragenol (CAG) is a hydrolysis product of astragaloside IV, the major active component of Astragalus radix. We have previously demonstrated that CAG has anti-inflammatory effect in a mouse model of ischemic stroke. This study investigated the effect of CAG on the phenotype polarization of microglia in lipopolysaccharide (LPS)-stimulated BV-2 mouse microglial cells and ischemic stroke mice. In LPS-treated BV-2 cells, we found that CAG significantly reduced the expression of M1 markers, including pro-inflammatory cytokines and enzymes. In contrast, CAG promoted the expression of M2 markers, including anti-inflammatory cytokines and growth factor. In addition, CAG inhibited the activation of nuclear factor-κB (NF-κB) and enhanced the activation of nuclear factor E2-related factor 2 (Nrf2) and the expression of its downstream heme oxygenase-1 (HO-1). Furthermore, CAG also inhibited levels of M1 markers, promoted those of M2 markers, and enhanced Nrf2 activation and HO-1 expression in ischemic mouse brain. Importantly, the effect of CAG on M2 markers, but not M1 markers, was reversed by Nrf2 siRNA in LPS-stimulated BV-2 cells. Together, our results suggested that CAG promoted microglial M2 and suppressed M1 polarization through activating Nrf2 and inhibiting NF-κB, respectively, in LPS-stimulated BV-2 cells and ischemic mouse brain. CAG is a promising candidate for the treatment of neuroinflammation-related diseases, including ischemic stroke.

CXCL13 chemokine is a novel player in multiple myeloma osteolytic microenvironment, M2 macrophage polarization, and tumor progression

BackgroundWe assessed the mechanism by which multiple myeloma (MM) shapes the bone marrow (BM) microenvironment and affects MΦ polarization.MethodsIn vivo xenograft model of BM-disseminated human myeloma, as well as analysis of MM cell lines, stromal components, and primary samples from patients with MM, was utilized.ResultsAnalysis of the BM from MM-bearing mice inoculated with human CXCR4-expressing RPMI8226 cells revealed a significant increase in M2 MΦ cell numbers (p < 0.01). CXCL13 was one of the most profoundly increased factors upon MM growth with increased levels in the blood of MM-bearing animals. Myeloid cells were the main source of the increased murine CXCL13 detected in MM-infiltrated BM. MM cell lines induced CXCL13 and concurrent expression of M2 markers (MERTK, CD206, CD163) in co-cultured human MΦ in vitro. Interaction with MΦ reciprocally induced CXCL13 expression in MM cell lines. Mechanistically, TGFβ signaling was involved in CXCL13 induction in MM cells, while BTK signaling was implicated in MM-stimulated increase of CXCL13 in MΦ. Recombinant CXCL13 increased RANKL expression and induced TRAP+ osteoclast (OC) formation in vitro, while CXCL13 neutralization blocked these activities. Moreover, mice inoculated with CXCL13-silenced MM cells developed significantly lower BM disease. Reduced tumor load correlated with decreased numbers of M2 MΦ in BM, decreased bone disease, and lower expression of OC-associated genes. Finally, higher levels of CXCL13 were detected in the blood and BM samples of MM patients in comparison with healthy individuals.ConclusionsAltogether, our findings suggest that bidirectional interactions of MΦ with MM tumor cells result in M2 MΦ polarization, CXCL13 induction, and subsequent OC activation, enhancing their ability to support bone resorption and MM progression. CXCL13 may thus serve as a potential novel target in MM.

Oceanimonas sp. BPMS22-derived protein protease inhibitor induces anti-leishmanial immune responses through macrophage M2 to M1 repolarization

The present study aimed to validate the potential of a novel serine protein protease inhibitor (PPI), purified from marine Oceanimonas sp. BPMS22, induced M2 to M1 repolarization of the macrophages to treat visceral leishmaniasis (VL). Peptide mass fingerprint of the purified trypsin digested PPI peptide was obtained using matrix-assisted laser desorption ionization-time of flight combined with tandem mass spectrometry (MALDI-TOF MS/MS) and the sequence was used to construct a 3D protein model by homology modelling. The IC50 of PPI were 25.28 ± 1.675 μg/mL and 0.415 ± 0.015 μg/mL against promastigotes and intracellular amastigotes, respectively, indicating the host-directed therapy using PPI. The PPI enhanced the effector molecule i.e., nitric oxide (NO), and dampened the arginase activity in a dose-dependent manner. In vitro studies revealed that the BPMS22-derived PPI significantly (p < 0.05) decreased the mRNA expressions of M2 markers (FIZZ-1, YM-1, CD206, Arg-1) and increased the mRNA expressions of M1 markers (iNOS, IL-1β, IL-12) in rIL-4 + rIL-10 induced M2 macrophages. Interestingly, the BPMS22-derived PPI also significantly (p < 0.05) decreased the FIZZ-1, YM-1, CD206, and Arg-1; significantly (p < 0.05) increased iNOS, IL-12, and IFN-γ mRNA expression in L. donovani -infected murine macrophages, alongside the decreased parasite load in it. Hence, PPI has the potential to repolarize the cytokines (rIL-4 + rIL-10) pre-stimulated and L. donovani-infected M2 macrophages to M1 phenotype in vitro. A decrease in parasite burden after treatment with PPI indicated the acceleration of the parasite killing by enhancing the macrophage effector functions. Further, in vivo PPI treatment reduced hepatic and splenic Leishman donovan units (LDU) up to 93.34 % and 87.63 %, respectively. This was followed by a surge in pro-inflammatory cytokines and dampening anti-inflammatory cytokines (p < 0.01), which exhibited anti-VL immunity. These observations might open new perspectives on PPI in macrophage repolarization to treat VL.

Synthesis, Formulation and Characterization of Immunotherapeutic Glycosylated Dendrimer/cGAMP Complexes for CD206 Targeted Delivery to M2 Macrophages in Cold Tumors.

Anti-tumor responses can be achieved via the stimulation of the immune system, a therapeutic approach called cancer immunotherapy. Many solid tumor types are characterized by the presence of immune-suppressive tumor-associated macrophage (TAMs) cells within the tumor microenvironment (TME). Moreover, TAM infiltration is strongly associated with poor survival in solid cancer patients and hence a low responsiveness to cancer immunotherapy. Therefore, 2′3′ Cyclic GMP-AMP (2′3′ cGAMP) was employed for its ability to shift macrophages from pro-tumoral M2-like macrophages (TAM) to anti-tumoral M1. However, cGAMP transfection within macrophages is limited by the molecule’s negative charge, poor stability and lack of targeting. To circumvent these barriers, we designed nanocarriers based on poly(amidoamine) dendrimers (PAMAM) grafted with D-glucuronic acid (Glu) for M2 mannose-mediated endocytosis. Two carriers were synthesized based on different dendrimers and complexed with cGAMP at different ratios. Orthogonal techniques were employed for synthesis (NMR, ninhydrin, and gravimetry), size (DLS, NTA, and AF4-DLS), charge (DLS and NTA), complexation (HPLC-UV and AF4-UV) and biocompatibility and toxicity (primary cells and hen egg chorioallantoic membrane model) evaluations in order to evaluate the best cGAMP carrier. The best formulation was selected for its low toxicity, biocompatibility, monodispersed distribution, affinity towards CD206 and ability to increase M1 (STAT1 and NOS2) and decrease M2 marker (MRC1) expression in macrophages.

Engineering Surfaces with Immune Modulating Properties of Mucin Hydrogels.

Hydrogels of cross-linked mucin glycoproteins (Muc-gel) have shown strong immune-modulating properties toward macrophages in vitro, which are translated in vivo by the dampening of the foreign body response to implantation in mice. Beyond mucin hydrogels, other biomaterials such as sensors, electrodes, and other long-term implants would also benefit from such immune-modulating properties. In this work, we aimed to transfer the bioactivity observed for three-dimensional Muc-gels to the surface of two model materials by immobilizing mucin into thin films (Muc-film) using covalent layer-by-layer assembly. We tested how the surface immobilization of mucins affects macrophage responses compared to Muc-gels. We showed that Muc-films on soft polyacrylamide gels mimic Muc-gel in their modulation of macrophage responses with activated gene expression of inflammatory cytokines on day 1 and then dampening them on day 3. Also, the markers of polarized macrophages, M1 and M2, were expressed at the same level for macrophages on Muc-film-coated soft polyacrylamide gels and Muc-gel. In contrast, Muc-film-coated hard polystyrene led to a different macrophage response compared to Muc-gel, having no activated expression of inflammatory cytokines and a different M1 marker expression. This suggested that the substrate mechanical properties and mucin molecular configuration determined by substrate–mucin interactions affect mucin immune-modulating properties. We conclude that mucin immune-modulating properties can be transferred to materials by mucin surface immobilization but will be dependent on the substrate chemical and mechanical properties.