Macrophage Phenotype Switching Research Articles

Cellular In Vitro Responses Induced by Human Mesenchymal Stem/Stromal Cell-Derived Extracellular Vesicles Obtained from Suspension Culture.

Mesenchymal stem/stromal cells (MSCs) and their extracellular vesicles (MSC-EVs) have been described to have important roles in tissue regeneration, including tissue repair, control of inflammation, enhancing angiogenesis, and regulating extracellular matrix remodeling. MSC-EVs have many advantages for use in regeneration therapies such as facility for dosage, histocompatibility, and low immunogenicity, thus possessing a lower possibility of rejection. In this work, we address the potential activity of MSC-EVs isolated from adipose-derived MSCs (ADMSC-EVs) cultured on cross-linked dextran microcarriers, applied to test the scalability and reproducibility of EV production. Isolated ADMSC-EVs were added into cultured human dermal fibroblasts (NHDF-1), keratinocytes (HaCat), endothelial cells (HUVEC), and THP-1 cell-derived macrophages to evaluate cellular responses (i.e., cell proliferation, cell migration, angiogenesis induction, and macrophage phenotype-switching). ADMSC viability and phenotype were assessed during cell culture and isolated ADMSC-EVs were monitored by nanotracking particle analysis, electron microscopy, and immunophenotyping. We observed an enhancement of HaCat proliferation; NHDF-1 and HaCat migration; endothelial tube formation on HUVEC; and the expression of inflammatory cytokines in THP-1-derived macrophages. The increased expression of TGF-β and IL-1β was observed in M1 macrophages treated with higher doses of ADMSC-EVs. Hence, EVs from microcarrier-cultivated ADMSCs are shown to modulate cell behavior, being able to induce skin tissue related cells to migrate and proliferate as well as stimulate angiogenesis and cause balance between pro- and anti-inflammatory responses in macrophages. Based on these findings, we suggest that the isolation of EVs from ADMSC suspension cultures makes it possible to induce in vitro cellular responses of interest and obtain sufficient particle numbers for the development of in vivo concept tests for tissue regeneration studies.

Impact of cardiac health status on bone marrow cell secretome and myocardial repair

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): University of Fribourg Graduate assistant Background Restoring the damaged myocardium remains a significant clinical challenge. Administration of bioactive factors, exosomes, or cell secretomes has shown promising results in promoting angiogenesis, tissue repair, immunomodulation, and extracellular matrix remodelling, particularly for cardiovascular disease treatments. Bone marrow (BM) cells are an essential source of secretome-based therapies for myocardial infarction (MI). Harnessing the potential of the cellular secretome for therapeutic applications requires a comprehensive understanding of its composition, functions, and mechanisms of action. MI affects the entire hematopoietic hierarchy, from stem cells to myeloid progenitors in BM. The study investigates how the cardiac status healthy versus infarcted affects the BM cell compositions and the properties of their secretomes. Methods Bone marrow cells (BMC) were isolated from healthy male Lewis rats (hBMC) and infarcted one (iBMC) obtained 2 weeks post MI induced by coronary artery ligation and cultured for 2 weeks. Cells composition, particularly macrophages, was characterised using multiplex flow cytometry Cytek Aurora. (CD90, CD29, CD45. CD68, CD206, CD163, Arg1, CD80, CCR7, iNOS). Cell proliferation (Real-time cell analyser and EdU incorporation), metabolic assays (seahorse assays), PCR, MS proteomic, and potency assays for immunomodulatory properties were performed. Results Multiplex flow cytometry showed distinctions in the percentage of cells: CD34+ stem cells and hematopoietic CD45+ cells were significantly upregulated in hBMCs, while CD68+ cells were higher in iBMCs. Cell proliferation assay demonstrated that iBMCs had significantly lower proliferation rates compared to hBMCs. Cell Metabolism was also impacted, iBMCs showed increased basal respiration, ATP production, and maximal respiration compared to hBMCs. An increase in respiratory spare capacity in iBMCs suggests an increased potential for ATP production under increased energy demands. Relative to hBMC, iBMC significantly regulated the expression of genes involved in immune response, inflammation, angiogenesis, and ECM remodelling gm-csf, il-17, vcam1, stat6, mmp9, cam, arg1, mhcII, arg2 were downregulated and tlr2, cxcl10, il-6, timp1, rantes, mcp-1, vegf, il-1ra, il-1beta, mip-1alpha, tlr4, b2m, and mmp3 upregulated. Secreted proteins also varied. Finally, the secretome of iBMC showed an enhanced capacity to induce a phenotype switch of macrophages toward an anti-inflammatory profile. Conclusion Our study shows that MI significantly alters the properties of bone marrow cells, impacting their composition, proliferation, metabolism, gene expression and the immunomodulatory potential of the secretome. These findings highlight the importance of considering donor health status in developing restoring secretome-based therapies for cardiac repair and emphasise the need to incorporate potency assay of the biologics for a standardised therapy and quality control.

GHK-Cu/Pionin-loaded in situ electrospun PVB/PVP smart dressing promotes wound healing via anti-oxidant, anti-inflammatory, antimicrobial, and tissue regenerative effects

Biocompatible, antibacterial, anti-inflammatory, and tissue regenerative smart dressings are in urgent need for infected skin wound repair in clinics. Portable in situ 3D-electrospun technology has the great advantage of direct deposition of required smart dressing on wound surfaces. Here, we fabricated a copper peptide (GHK-Cu) and quaternium-73 (Pionin) dual-drug-loaded polyvinyl butyral (PVB)/polyvinylpyrrolidone (PVP) smart nanofibers dressing using in-situ electrospinning technology for skin wound repair. The physicochemical properties and biological activities of smart dressing were extensively analyzed. In vivo biocompatibility, anti-oxidant, anti-inflammatory, and antimicrobial activities of smart dressing were analyzed during wound healing. Stable and homogeneous PVB/PVP nanofiber smart dressing with uniformly distributed GHK-Cu and Pionin and anabolic biological activities was obtained. Biocompatible smart dressing gave a sustained release of GHK-Cu and Pionin, promoted the migration and adhesion of dermal fibroblasts and keratinocytes, and showed anti-oxidant and anti-apoptotic effects in vitro and in vivo. Smart dressing exerted anti-inflammatory properties via M1 to M2 macrophage phenotype switching and effectively promoted wound healing. Pionin released from smart dressing not only killed but also inhibited the expression and delivery of virulence factor expression and bacteria adhesion. During wound healing, smart dressing promoted the proliferation of cells in the epidermal tissue, epithelial tissue remodeling, granulation tissue growth, neovascularization, and M2 macrophage polarization. Moreover, this smart dressing scavenged excess reactive oxygen species, increased SOD3 and SIRT1 expression, and repaired peroxidized tissue, consequently promoting wound healing. In summary, our results indicate that GHK-Cu/Pionin-loaded in situ electrospun PVB/PVP smart dressing promotes wound healing via anti-oxidant, anti-inflammatory, antimicrobial, and tissue regenerative effects, suggesting clinical application of this smart dressing for complicated skin wound healing.

Programming of macrophage polarization in different stages for accelerating wound healing

Macrophage polarization and phenotypic switching have a significant impact on the immune microenvironment of wounds, making them crucial for wound healing. Creating immune-adapted microenvironment throughout the wound repair process by regulating macrophage polarization supports rapid wound healing. We explored the segmented use of M1-polarizing and M2-polarizing materials at different healing stages and found that this combination can significantly shorten the healing time. The macrophage polarization could be adjusted to M1 phenotype and M2 phenotype by selective modification of chitosan with dicyandiamide or polyethylene glycol, respectively. In a rat full-thickness infected wound model, combining dicyandiamide modified chitosan (DICY-CS) for M1 macrophage polarization in the inflammatory phase and PEGylated chitosan (PEG-CS) for M2 macrophage polarization in the proliferative phase significantly reduced wound healing time. A higher degree of re-epithelialization, collagen deposition and neovascularization were observed in wounds treated with this combination approach compared to the antimicrobial agents Silvadene and DICY-CS or PEG-CS alone. The use of different macrophage polarizing materials separately in different healing stages provided an optimal immune microenvironment and accelerated wound healing. This strategy provides a more comprehensive understanding of the wound healing process. It will be a new insight for the treatment of more complex and difficult-to-heal wounds such as pressure ulcers and diabetic wounds.

Electrical and immune stimulation-based hydrogels synergistically realize scarless wound healing via amplifying endogenous electrophysiological function and promoting Macrophage Phenotype-Switching

Scars are a consequence of the wound healing process, and scarless wound healing remains a substantial clinical challenge. In this study, conductive hydrogels (named sHA/G-GM) were developed to synergistically realize scarless wound healing through electrical and immune stimulation. sHA/G-GM was prepared as high-sulfated hyaluronan (sHA) modified graphene (sHA/G) homodispersing in gelatin methacrylate, and sHA/G was released and absorbed by cytomembranes. In vitro (human scar fibroblasts and mouse primary macrophages) and in vivo (rat wound models and rabbit ear wound models) studies were conducted and demonstrated that sHA/G-GM not only improves conductivity to fibroblast activity regulation but also combines with interleukin-4 to promote macrophage phenotype switching. These results strongly suggest that sHA/G-GM synergistically amplifies endogenous electrophysiological functions and modulates the immune microenvironment to realize scarless wound healing. Therefore, the electrical and immune stimulation-based sHA/G-GM provide an effective approach to synergistically realize scarless wound healing in the future.

P311 Influences Macrophage Polarization

Obesity is a pandemic disease that has become a serious concern worldwide. In United States, recently self-reported obesity rates of adults were escalated to 40%. In the early phase of obesity, excessive macrophage infiltration and disequilibrium of macrophage phenotypes (M1 vs. M2 or vice versa) play a critical role in the microenvironment of adipose tissue. It often leads to chronic inflammation and is a major cause for metabolic disorders including insulin resistance, type-2 diabetes, and cardiovascular diseases. P311 is an intrinsically disordered protein, contributes to adipogenesis as transcription regulator through activation of PPARγ2, master regulator of adipogenesis. However, the P311 role on the macrophage polarization was not known. Here we studied the macrophage polarization in presence of P311 using Raw 264.7 cells. Raw cells were treated with either interleukin-4 (IL4) or lipopolysaccharide (LPS) for macrophage polarization towards M2 and M1 phenotype respectively in presence and absence of P311 overexpression. Macrophage phenotypic switch was assessed using CD206 (M2 marker) and CD86 (M1 marker) expression. The dose and time dependent effects of IL4 and LPS on the expression of P311 and macrophage markers was also studied. A significant increase in endogenous P311 expression observed with IL4 treatment whereas LPS treatment showed the opposite effect. Our findings showed that the P311 overexpression promotes anti-inflammatory macrophage phenotype (M2) as indicated by the significant increase in mRNA and protein expression of CD206 when compared to empty vector (EV, pCMV) transfected control group. Further, P311 overexpression in LPS treated RAW cells significantly increased the expression of CD206, on contrary to the classical M1 polarization of LPS, compared to EV transfected cells. In addition, P311 suppressed the expression of CD86 there by facilitating macrophage switch towards anti-inflammatory phenotype in presence of LPS treatment. IL4 treatment alone increased macrophage activation towards M2 phenotype however IL4 treated P311 overexpressed cells showed further increase in CD206 expression compared to EV transfected cells. Our studies indicate that the P311 expression plays an important role in promoting macrophage polarization toward M2 phenotype. This study was supported by research funding to KB, from NIH/NIGMS #SC1GM141937. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A long-acting FGF21 attenuates metabolic dysfunction-associated steatohepatitis-related fibrosis by modulating NR4A1-mediated Ly6C phenotypic switch in macrophages.

Because of the absence of effective therapies for metabolic dysfunction-associated steatohepatitis (MASH), there is a rising interest in fibroblast growth factor 21 (FGF21) analogues due to their potential anti-fibrotic activities in MASH treatment. PsTag-FGF21, a long-acting FGF21 analogue, has demonstrated promising therapeutic effects in several MASH mouse models. However, its efficacy and mechanism against MASH-related fibrosis remain less well defined, compared with the specific mechanisms through which FGF21 improves glucose and lipid metabolism. The effectiveness of PsTag-FGF21 was evaluated in two MASH-fibrosis models. Co-culture systems involving macrophages and hepatic stellate cells (HSCs) were employed for further assessment. Hepatic macrophages were selectively depleted by administering liposome-encapsulated clodronate via tail vein injections. RNA sequencing and cytokine profiling were conducted to identify key factors involved in macrophage-HSC crosstalk. We first demonstrated the significant attenuation of hepatic fibrosis by PsTag-FGF21 in two MASH-fibrosis models. Furthermore, we highlighted the crucial role of macrophage phenotypic switch in PsTag-FGF21-induced HSC deactivation. FGF21 was demonstrated to regulate macrophages in a PsTag-FGF21-like manner. NR4A1, a nuclear factor which is notably down-regulated in human livers with MASH, was identified as a mediator responsible for PsTag-FGF21-induced phenotypic switch. Transcriptional control over insulin-like growth factor 1, a crucial factor in macrophage-HSC crosstalk, was exerted by the intrinsically disordered region domain of NR4A1. Our results have elucidated the previously unclear mechanisms through which PsTag-FGF21 treats MASH-related fibrosis and identified NR4A1 as a potential therapeutic target for fibrosis.

Copper-based carbon dots modified hydrogel with osteoimmunomodulatory and osteogenesis for bone regeneration.

Biomaterials with dual functions of osteoimmunomodulation and bone repair are very promising in the field of orthopedic materials. For this purpose, we prepared copper-based carbon dots (CuCDs) and doped them into oxychondroitin sulfate/poly-acrylamide hydrogel (OPAM) to obtain a hybrid hydrogel (CuCDs/OPAM). We evaluated its osteoimmunomodulatory and bone repair properties in vitro and in vivo. The obtained CuCDs/OPAM exhibited good rBMSCs-cytocompatibility and anti-inflammatory properties in vitro. It also could effectively promote rBMSCs differentiation and the expression of osteogenic differentiation factors from rBMSCs under an inflammatory environment. Moreover, CuCDs/OPAM could induce macrophage phenotype switching (from M1-type macrophages to M2-type macrophages) in vivo, which is beneficial for anti-inflammatory action and presents good osteoimmunomodulation capability to induce a bone immune microenvironment to promote the differentiation of rBMSCs. In conclusion, CuCDs/OPAM hydrogel has dual functions of osteoimmunomodulatory and bone repair and is a promising bone filling and repair material.

SMYD3 activates the TCA cycle to promote M1-M2 conversion in macrophages

BackgroundSMYD3 refers to a histone lysine methyltransferase from the SMYD family, which acts as a gene transcriptional regulator chiefly through catalysis of the histone subunit 3 at lysine 4 trimethylation (H3K4me3). Great progress has been made that epigenetic modification plays a pivotal role in regulating macrophage polarization. However, the effects of the histone lysine methyltransferase SMYD3 on macrophage polarization and phenotypic switching are unclear. ResultsWe found that LPS/IFN-γ-stimulated macrophages gradually transformed from M1 to M2 in the late stage, and SMYD3 played a key role in this process. As demonstrated by RNA-seq assessment, SMYD3 prominently activated a metabolic pathway known as TCA cycle inside macrophages during M1-M2 conversion. Besides, by modifying H3K4me3 histone, the target genes regulated by SMYD3 were identified via the ChIP-seq assessment, including citrate synthase (CS), succinate dehydrogenase complex subunit C (SDHC) and pyruvate carboxylase (PC). SMYD3 activated the transcriptional activities of the metabolic enzymes CS, SDHC and PC through H3K4me3 by causing the aggregation of citrate, an intramacrophage metabolite, and the depletion of succinate. And additionally, it facilitated the generation of ROS, as well as the expressions of genes associated with mitochondrial respiratory chain complexes. This increased ROS production ultimately induced mitophagy, triggering the M1 to M2 phenotype switch in the macrophages. ConclusionsOur study provides a detailed intrinsic mechanism in the macrophage phenotypic transition process, in short, SMYD3 promotes the M1-M2 conversion of macrophages by activating the TCA cycle through the simultaneous regulation of the transcriptional activities of the metabolic enzymes CS, SDHC and PC.

Transcriptional regulation of macrophage inflammation in the heart failure with preserved ejection fraction: a translational study

Abstract Background/Introduction Inflammatory response is a key player in heart failure with preserved ejection fraction (HFpEF) but the underlying mechanisms are poorly understood (1). Recent studies have shown a critical involvement of cardiac macrophage recruitment and activation in myocardial remodelling (2,3). Purpose here we sought to determine the role of macrophage inflammation in experimental and human HFpEF. Methods We employed a translational approach combining human and murine LV myocardial tissue, FACS analysis of cardiac immune cells, in vitro experiments in primary macrophages and cardiac cells as well as ex vivo functional analyses in LV tissues. Human LV myocardial samples were used to evaluate the pro-inflammatory activity of macrophages in the HFpEF myocardium. Induction of HFpEF in mice was done through a validated mouse model combining metabolic (high-fat diet) and hemodynamic stress (L-NAME). Cardiac function was investigated by high resolution ultrasound imaging (Vevo3100) and the LV myocardial samples were used to study the macrophage polarization and their inflammatory activity. Based on interrogation of available RNA-seq datasets –showing the transcriptional repressor NCOR1 as a relevant signature in macrophages during cardiac inflammation, we generated myeloid cell-specific NCOR1 knockout (Ncor1-KO) transgenic mice to investigate the in vivo effects of NCOR1 deletion on the HFpEF phenotype. In vitro experiments included RAW 264.7 cells polarization through a metabolic stress model via palmitic acid (PA) and the assessment of macrophage phenotype switching in the presence or the absence of NCOR1. To study the crosstalk of macrophages with the surrounding cardiac cells, cardiomyocytes (H9c2) and endothelial cells (HAECS) were exposed to conditioned media from cultured RAW. Afterwards, cellular function and the pro-inflammatory cytokine levels were investigated. Results The expression of macrophage markers in human LV specimens showed an increase in pro-inflammatory macrophages (M1) and a decrease in regulatory macrophages (M2) as compared to age-matched control mice. HFpEF mice showed increased expression of vascular adhesion molecules (ICAM1, ICAM2, E-selectin) and a significant recruitment of M1 macrophages to the myocardium. Of interest, cardiac function and myocardial inflammation (TNF-a, IL6, IL-1b) were significantly reduced in Ncor1-KO mice. In vitro assays showed that stimulation of macrophages with PA induced M1 type switch and increased NCOR1, TNF-a, IL-6, and IL-1b levels, whereas NCOR1 depletion prevented detrimental changes of macrophage secretome (Fig.2). Conclusion NCOR1 is a crucial co-regulator of immune response and macrophage inflammation in the heart. Our findings pave the way for mechanism-based therapies to prevent HFpEF development.

Abstract 16902: Molecular Basis of the Vascular Cell Phenotypic Switch and Wall Remodeling in Abdominal Aortic Aneurysm: NOX4 NADPH Oxidase-Derived Mitochondrial Oxidative Stress and DNA Damage

Vascular cells phenotypic changes and wall remodeling are the hallmarks of abdominal aortic aneurysm (AAA) pathology. Mitochondrial oxidative stress (mtOS) is associated with the vascular smooth muscle cells (SMC) and macrophage phenotypic switch. We previously reported that NOX4 NADPH oxidase expression is increased in cardiovascular cells in response to pathological insult inducing mtOS and dysfunction and vascular disease progression. We tested the hypothesis that NOX4-dependent mtOS and DNA damage induce vascular phenotypic changes and remodeling resulting in AAA progression. In angiotensin II (Ang II)-induced AAA model Apoe -/- / Nox4 Tg mice with mitochondria-targeted overexpression of NOX4 had the highest, whereas Apoe -/- / Nox4 -/- displayed the lowest incidence of AAA, abdominal aorta area and diameter as compared with Apoe -/- mice. The aortic wall ROS levels, oxidative DNA damage and DNA damage response markers, elastin degradation index, MMP2 expression, and macrophage infiltration were significantly higher in AngII-treated Apoe -/- / Nox4 TG mice compared with Apoe -/- or Apoe -/- / Nox4 -/- mice. Spectral flow cytometry analysis of AAA cell suspension from Ang II-infused mice revealed a decrease in contractile SMCs but an increase in macrophage-like SMC populations, as well as increased number of infiltrating pro-inflammatory macrophages in Apoe -/- / Nox4 TG mice. As compared with wild-type or Nox4 -/- mice, Ang II-treated SMC from Nox4 TG mice showed significantly elevated mtOS, DNA damage, dysfunction, as well as activation of cGAS-STING pathway and NLRP3/AIM2 inflammasome expression. Flow cytometry analysis of Ang II-treated Nox4 TG mice SMC contained a lower proportion of cells with contractile markers (ACTA2 + , MYH11 + , TAGLN + ) and a higher proportion of cells with macrophage-like markers (CD68 + , CD11b + , LGALS3 + ). These data suggest that NOX4-dependent mitochondrial DNA damage and activation of DNA-sensing pathways have a causal role in AAA development, leading to macrophage-like SMCs, increased inflammation, and vessel wall remodeling. NOX4 inhibition and mitochondrial function augmentation may be beneficial in preserving aortic wall integrity in AAA-susceptible patients.

Urinary stem cell-derived exocrine circRNA ATG7 regulates the SOCS1/STAT3 signaling pathway through miR-4500, inhibits M1 macrophage polarization, and alleviates the progression of diabetes nephropathy

ObjectiveThe etiopathogenesis of diabetes nephropathy (DN) has not yet been fully clarified. Finding effective treatments to prevent renal failure in DN patients has become the main focus of research in recent years. Circular RNA (circRNA) has been shown to play a momentous role in DN progression. Based on this, we aimed to investigate the potential mechanism by which urine-derived stem cell (USC)-derived exosome circRNA ATG7 (Exo-ATG7) mediates DN progression.MethodsExosomes from USCs were isolated and identified. The DN rat model was established by intraperitoneally injecting 60 mg/kg streptozotocin. The protein expression levels were measured by Western blot and immunofluorescence. HE and Masson staining were used to evaluate renal injury, and the expression of related genes was detected by RT-qPCR.ResultsCircRNA ATG7 was significantly downregulated in the DN rat model, and the extracellular vesicles of USCs improved renal function and reduced inflammation in DN rats. However, after knocking down the USCs-derived exosome circRNA ATG7, improvement and therapeutic effect on renal function in DN rats were lost. In addition, overexpression of ATG7 facilitated the switching of macrophages from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype both in vivo and in vitro. Mechanistically, upregulation of circRNA ATG7 expression can alleviate renal damage in DN rats. Importantly, the USCs-derived exosome circRNA ATG7 promotes macrophage M2 polarization by regulating the SOCS1/STAT3 signaling pathway through miR-4500. In addition, animal experiments also confirmed that after knocking down ATG7 in USC cells, the extracted exosome-treated DN rats could weaken the therapeutic effect of USC exosomes.ConclusionOur research results indicate that USC-derived exosomal circRNA ATG7 facilitates macrophage phenotype switching from M1 to M2 through the SOCS1/STAT3 signaling pathway mediated by miR-4500, thereby inhibiting DN progression.

Allogeneic Adipose-Derived Mesenchymal Stem Cell Transplantation Alleviates Atherosclerotic Plaque by Inhibiting Ox-LDL Uptake, Inflammatory Reaction and Endothelial Damage in Rabbits.

Atherosclerosis (AS) is a chronic inflammatory disease of arteries fueled by lipids. It is a major cause of cardiovascular morbidity and mortality. Mesenchymal stem cells have been used for the treatment of atherosclerotic lesions. Adipose-derived stem cells (ADSCs) have been shown to regulate the activation state of macrophages and exhibit anti-inflammatory capabilities. However, the effect of allogeneic ADSCs in the treatment of AS have not been investigated. In this study, the early treatment effect and preliminary mechanism analysis of allogeneic rabbit ADSCs intravenous transplantation were investigated in a high-fat diet rabbit model. The polarization mechanism of rabbit ADSCs on the macrophage was further analyzed in vitro. Compared with the model group, blood lipid levels declined, the plaque area, oxidized low-density lipoprotein (ox-LDL) uptake, scavenger receptor A1 and cluster of differentiation (CD) 36 levels were all significantly reduced, and the accumulation of inflammatory M1 macrophages, apoptosis, interleukin (IL)-6 and tumor necrosis factor (TNF)-α expression were decreased. The endothelial cells (CD31), M2 macrophages, IL-10 and the transforming growth factor (TGF)-β levels increased. In vitro, ADSCs can promote the M1 macrophage phenotypic switch toward the M2 macrophage through their secreted exosomes, and the main mechanism includes increasing arginase 1 expression and IL-10 secretion, declining inducible nitric oxide synthase (iNOS) expression and TNF-α secretion, and activating the STAT6 pathway. Therefore, allogeneic rabbit ADSC transplantation can transmigrate to the aortic atherosclerotic plaques and show a good effect in lowering blood lipids and alleviating atherosclerotic plaque in the early stage of AS by inhibiting ox-LDL uptake, inflammatory response, and endothelial damage.

Heme-Induced Macrophage Phenotype Switching and Impaired Endogenous Opioid Homeostasis Correlate with Chronic Widespread Pain in HIV.

Chronic widespread pain (CWP) is associated with a high rate of disability and decreased quality of life in people with HIV-1 (PWH). We previously showed that PWH with CWP have increased hemolysis and elevated plasma levels of cell-free heme, which correlate with low endogenous opioid levels in leukocytes. Further, we demonstrated that cell-free heme impairs β-endorphin synthesis/release from leukocytes. However, the cellular mechanisms by which heme dampens β-endorphin production are inconclusive. The current hypothesis is that heme-dependent TLR4 activation and macrophage polarization to the M1 phenotype mediate this phenomenon. Our novel findings showed that PWH with CWP have elevated M1-specific macrophage chemokines (ENA-78, GRO-α, and IP-10) in plasma. In vitro, hemin-induced polarization of M0 and M2 macrophages to the M1 phenotype with low β-endorphins was mitigated by treating cells with the TLR4 inhibitor, TAK-242. Similarly, in vivo phenylhydrazine hydrochloride (PHZ), an inducer of hemolysis, injected into C57Bl/6 mice increased the M1/M2 cell ratio and reduced β-endorphin levels. However, treating these animals with the heme-scavenging protein hemopexin (Hx) or TAK-242 reduced the M1/M2 ratio and increased β-endorphins. Furthermore, Hx attenuated heme-induced mechanical, heat, and cold hypersensitivity, while TAK-242 abrogated hypersensitivity to mechanical and heat stimuli. Overall, these results suggest that heme-mediated TLR4 activation and M1 polarization of macrophages correlate with impaired endogenous opioid homeostasis and hypersensitivity in people with HIV.

Brucella abortus modulates macrophage polarization and inflammatory response by targeting glutaminases through the NF-κB signaling pathway.

The mechanism of Brucella infection regulating macrophage phenotype has not been completely elucidated until now. This study aimed to determine the mechanism of Brucella abortus in the modulation of macrophage phenotype using RAW264.7 cells as a model. RT-qPCR, ELISA and flow cytometry were used to detect the inflammatory factor production and phenotype conversion associated with M1/M2 polarization of macrophages by Brucella abortus infection. Western blot and immunofluorescence were used to analyze the role of nuclear factor kappa B (NF-κB) signaling pathway in regulation of Brucella abortus-induced macrophage polarization. Chromatin immunoprecipitation sequencing (Chip-seq), bioinformatics analysis and luciferase reporter assay were used to screen and validate NF-κB target genes associated with macrophage polarization and further verify its function. The results demonstrate that B. abortus induces a macrophage phenotypic switch and inflammatory response in a time-dependent manner. With the increase of infection time, B. abortus infection-induced M1-type increased first, peaked at 12h, and then decreased, whereas the M2-type decreased first, trough at 12h, and then increased. The trend of intracellular survival of B. abortus was consistent with that of M2 type. When NF-κB was inhibited, M1-type polarization was inhibited and M2-type was promoted, and the intracellular survival of B. abortus increased significantly. Chip-seq and luciferase reporter assay results showed that NF-κB binds to the glutaminase gene (Gls). Gls expression was down-regulated when NF-κB was inhibited. Furthermore, when Gls was inhibited, M1-type polarization was inhibited and M2-type was promoted, the intracellular survival of B. abortus increased significantly. Our data further suggest that NF-κB and its key target gene Gls play an important role in controlling macrophage phenotypic transformation. Taken together, our study demonstrates that B. abortus infection can induce dynamic transformation of M1/M2 phenotype in macrophages. Highlighting NF-κB as a central pathway that regulates M1/M2 phenotypic transition. This is the first to elucidate the molecular mechanism of B. abortus regulation of macrophage phenotype switch and inflammatory response by regulating the key gene Gls, which is regulated by the transcription factor NF-κB.

Sequence Variation of Candida albicans Sap2 Enhances Fungal Pathogenicity via Complement Evasion and Macrophage M2-Like Phenotype Induction.

Candida albicans (C. albicans) is an opportunistic pathogen increasingly causing candidiasis worldwide. This study aims to investigate the pattern of systemic immune responses triggered by C. albicans with disease associated variation of Sap2, identifying the novel evasion strategies utilized by clinical isolates. Specifically, a variation in clinical isolates is identified at nucleotide position 817 (G to T). This homozygous variation causes the 273rd amino acid exchange from valine to leucine, close to the proteolytic activation center of Sap2. The mutant (Sap2-273L) generated from SC5314 (Sap2-273V) background carrying the V273L variation within Sap2displays higher pathogenicity. In comparison to mice infected with Sap2-273V strain, mice infected with Sap2-273L exhibit less complement activation indicated by less serum C3a generation and weaker C3b deposition in the kidney. This inhibitory effect is mainly achieved by Sap2273L -mediated stronger degradation of C3and C3b. Furthermore, mice infected with Sap2-273L strain exhibit more macrophage phenotype switching from M0to M2-like and more TGF-β release which further influences T cell responses, generating an immunosuppressed cellular microenvironment characterized by more Tregs and exhausted T cell formation. In summary, the disease-associated sequence variation of Sap2enhances pathogenicity by complement evasion and M2-like phenotype switching, promoting a more efficient immunosuppressed microenvironment.

Sulforaphane Inhibits Foam Cell Formation and Atherosclerosis via Mechanisms Involving the Modulation of Macrophage Cholesterol Transport and the Related Phenotype

Sulforaphane (SFN), an isothiocyanate, is one of the major dietary phytochemicals found in cruciferous vegetables. Many studies suggest that SFN can protect against cancer and cardiometabolic diseases. Despite the proposed systemic and local vascular protective mechanisms, SFN’s potential to inhibit atherogenesis by targeting macrophages remains unknown. In this study, in high fat diet fed ApoE-deficient (ApoE−/−) mice, oral SFN treatment improved dyslipidemia and inhibited atherosclerotic plaque formation and the unstable phenotype, as demonstrated by reductions in the lesion areas in both the aortic sinus and whole aorta, percentages of necrotic cores, vascular macrophage infiltration and reactive oxygen species (ROS) generation. In THP-1-derived macrophages, preadministration SFN alleviated oxidized low-density lipoprotein (ox-LDL)-induced lipid accumulation, oxidative stress and mitochondrial injury. Moreover, a functional study revealed that peritoneal macrophages isolated from SFN-treated mice exhibited attenuated cholesterol influx and enhanced apolipoprotein A-I (apoA-I)- and high-density lipoprotein (HDL)-mediated cholesterol efflux. Mechanistic analysis revealed that SFN supplementation induced both intralesional and intraperitoneal macrophage phenotypic switching toward high expression of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and ATP-binding cassette subfamily A/G member 1 (ABCA1/G1) and low expression of peroxisome proliferator-activated receptor γ (PPARγ) and cluster of differentiation 36 (CD36), which was further validated by the aortic protein expression. These results suggest that the regulation of macrophages’ cholesterol transport and accumulation may be mainly responsible for SFN’s potential atheroprotective properties, and the regulatory mechanisms might involve upregulating ABCA1/G1 and downregulating CD36 via the modulation of PPARγ and Nrf2.

Human Salivary Histatin-1 Attenuates Osteoarthritis through Promoting M1/M2 Macrophage Transition.

Osteoarthritis (OA) is an inflammation-driven degenerative joint disease. Human salivary peptide histatin-1 (Hst1) shows pro-healing and immunomodulatory properties. but its role in OA treatment is not fully understood. In this study, we investigated the efficacy of Hst1 in the inflammation modulation-mediated attenuation of bone and cartilage damage in OA. Hst1 was intra-articularly injected into a rat knee joint in a monosodium iodoacetate (MIA)-induced OA model. Micro-CT, histological, and immunohistochemical analyses showed that Hst1 significantly attenuates cartilage and bone deconstruction as well as macrophage infiltration. In the lipopolysaccharide-induced air pouch model, Hst1 significantly reduced inflammatory cell infiltration and inflammation. Enzyme-linked immunosorbent assay (ELISA), RT-qPCR, Western blot, immunofluorescence staining, flow cytometry (FCM), metabolic energy analysis, and high-throughput gene sequencing showed that Hst1 significantly triggers M1-to-M2 macrophage phenotype switching, during which it significantly downregulated nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinases (MAPK) signaling pathways. Furthermore, cell migration assay, Alcian blue, Safranin O staining, RT-qPCR, Western blot, and FCM showed that Hst1 not only attenuates M1-macrophage-CM-induced apoptosis and matrix metalloproteinase expression in chondrogenic cells, but it also restores their metabolic activity, migration, and chondrogenic differentiation. These findings show the promising potential of Hst1 in treating OA.

MiR-126 promotes M1 to M2 macrophage phenotype switching via VEGFA and KLF4.

Macrophage polarization and microRNA play crucial roles in the development of atherosclerosis (AS). The M1 macrophage phenotype contributes to the formation of plaques, while the M2 macrophage phenotype resolves inflammation and promotes tissue repair. MiR-126 has been found to play a role in regulating macrophage polarization in the context of AS. However, the exact mechanism of miR-126 requires further research. The foam cell model was established by stimulating THP-1 with oxidized low-density lipoprotein (ox-LDL). We transfected foam cells with miR-126 mimic and its negative control. The transfection of miR-126 was implemented by riboFECT CP transfection kit. The levels of miR-126 and M1/M2 associated genes in foam cells were quantified using reverse transcription-quantitative PCR (RT-qPCR). Additionally, the expressions of CD86+ and CD206+ cells in foam cells were determined by flow cytometry. Western blotting and RT-qPCR were used to determine the protein and mRNA levels of the vascular endothelial growth factor A (VEGFA) and the transcriptional regulator Krüppel-like factor 4 (KLF4), respectively. Additionally, we detected endothelial cell migration after co-culturing endothelial cells and macrophages. MG-132 was used to indirectly activate the expression of VEGFA, and the expression of KLF4 was also evaluated. The activation of apoptosis and production of foam cells were boosted by the addition of ox-LDL. We transfected foam cells with miR-126 mimic and its negative control and observed that miR-126 greatly suppressed foam cell development and inhibited phagocytosis. Moreover, it caused pro-inflammatory M1 macrophages to switch to the anti-inflammatory M2 phenotype. This was reflected by the increase in anti-inflammatory gene expression and the decrease in pro-inflammatory gene expression. Additionally, miR-126 dramatically decreased the expressions of VEGFA and KLF4. The protein-protein interaction network analysis showed a significantly high correlation between miR-126, VEGFA, and KLF4. MiR-126 may also promote EC migration by activating macrophage PPAR γ expression and effectively suppressing macrophage inflammation. MG-132 indirectly activated the expression of VEGFA, and the expression of KLF4 also significantly increased, which indicates a direct or indirect relationship between VEGFA and KLF4. Our study shows that miR-126 can reverse ox-LDL-mediated phagocytosis and apoptosis in macrophages. Consequently, the potential role of miR-126 was manifested in regulating macrophage function and promoting vascular endothelial migration.

Adenosine receptor activation promotes macrophage class switching from LPS-induced acute inflammatory M1 to anti-inflammatory M2 phenotype

Lipopolysaccharide induced monocytes/macrophages exhibit a pro-inflammatory M1 phenotype. Elevated levels of the purine nucleoside adenosine play a major role in this response. The role of adenosine receptor modulation in directing the macrophage phenotype switch from proinflammatory classically activated M1 phenotype to an anti-inflammatory alternatively activated M2 phenotype is investigated in this study. The mouse macrophage cell line RAW 264.7 was used as the experimental model and stimulated with Lipopolysaccharide (LPS) at a dose of 1 μg/ml. Adenosine receptors were activated by treating cells with the receptor agonist NECA (1 μM). Adenosine receptor stimulation in macrophages is found to suppress LPS-induced production of proinflammatory mediators (pro-inflammatory cytokines, Reactive Oxygen Species and nitrite levels). M1 marker CD38 (Cluster of Differentiation 38) and CD83 (Cluster of Differentiation 83) were significantly decreased while M2 markers Th2 cytokines, Arginase, TIMP (Tissue Inhibitor of Metalloproteinases) and CD206 (Cluster of Differentiation 206) exhibited an increase. Hence from our study we observed that activation of adenosine receptors can program the macrophages from a pro-inflammatory classically activated M1 phenotype to an anti-inflammatory alternatively activated M2 phenotype. We report the significance and a time course profile of phenotype switching by receptor activation. Adenosine receptor targeting may be explored as a therapeutic intervention strategy in addressing acute inflammation.