Exploring the Ion Channel TRPV2 and Testicular Macrophages in Mouse Testis

The cation channel TRPV2 is known to be expressed by murine macrophages and is crucially involved in their functionality. Macrophages are frequent cells of the mouse testis, an immune-privileged and steroid-producing organ. TRPV2 expression by testicular macrophages and possible changes associated with age or inflammation have not been investigated yet. Therefore, we studied testes of young adult and old wild-type (WT) and AROM⁺ mice, i.e., transgenic mice overexpressing aromatase. In these animals, inflammatory changes are described in the testis, involving active macrophages, which increase with age. This is associated with impaired spermatogenesis and therefore AROM⁺ mice are a model for male infertility associated with sterile inflammation. In WT animals, testicular TRPV2 expression was mapped to interstitial CD206⁺ and peritubular MHC II⁺ macrophages, with higher levels in CD206⁺ cells. Expression levels of TRPV2 and most macrophage markers did not increase significantly in old mice, with the exception of CD206. As the number of TRPV2⁺ testicular macrophages was relatively small, their possible involvement in testicular functions and in aging in WT mice remains to be further studied. In AROM⁺ testis, TRPV2 was readily detected and levels increased significantly with age, together with macrophage markers and TNF-α. TRPV2 co-localized with F4/80 in macrophages and further studies showed that TRPV2 is mainly expressed by unusual CD206⁺MHC II⁺ macrophages, arising in the testis of these animals. Rescue experiments (aromatase inhibitor treatment and crossing with ERαKO mice) restored the testicular phenotype and also abolished the elevated expression of TRPV2, macrophage and inflammation markers. This suggests that TRPV2⁺ macrophages of the testis are part of an inflammatory cascade initiated by an altered sex hormone balance in AROM⁺ mice. The changes in testis are distinct from the described alterations in other organs of AROM⁺, such as prostate and spleen. When we monitored TRPV2 levels in another immune-privileged organ, namely the brain, we found that levels of TRPV2 were not elevated in AROM⁺ and remained stable during aging. In the adrenal, which similar to the testis produces steroids, we found slight, albeit not significant increases in TRPV2 in both AROM⁺ and WT mice, which were associated with age. Thus, the changes in the testis are specific for this organ.     

C3 Deficiency Leads to Increased Angiogenesis and Elevated Pro-Angiogenic Leukocyte Recruitment in Ischemic Muscle Tissue

The complement system is a potent inflammatory trigger, activator, and chemoattractant for leukocytes, which play a crucial role in promoting angiogenesis. However, little information is available about the influence of the complement system on angiogenesis in ischemic muscle tissue. To address this topic and analyze the impact of the complement system on angiogenesis, we induced muscle ischemia in complement factor C3 deficient (C3−/−) and wildtype control mice by femoral artery ligation (FAL). At 24 h and 7 days after FAL, we isolated the ischemic gastrocnemius muscles and investigated them by means of (immuno-)histological analyses. C3−/− mice showed elevated ischemic damage 7 days after FAL, as evidenced by H&E staining. In addition, angiogenesis was increased in C3−/− mice, as demonstrated by increased capillary/muscle fiber ratio and increased proliferating endothelial cells (CD31⁺/BrdU⁺). Moreover, our results showed that the total number of leukocytes (CD45⁺) was increased in C3−/− mice, which was based on an increased number of neutrophils (MPO⁺), neutrophil extracellular trap formation (MPO⁺/CitH3⁺), and macrophages (CD68⁺) displaying a shift toward an anti-inflammatory and pro-angiogenic M2-like polarized phenotype (CD68⁺/MRC1⁺). In summary, we show that the deficiency of complement factor C3 increased neutrophil and M2-like polarized macrophage accumulation in ischemic muscle tissue, contributing to angiogenesis.     

A Heterotypic Tridimensional Model to Study the Interaction of Macrophages and Glioblastoma In Vitro

Background: Glioblastoma multiforme (GBM) is the most frequent and aggressive primary brain tumor, and macrophages account for 30–40% of its composition. Most of these macrophages derive from bone marrow monocytes playing a crucial role in tumor progression. Unraveling the mechanisms of macrophages-GBM crosstalk in an appropriate model will contribute to the development of specific and more successful therapies. We investigated the interaction of U87MG human GBM cells with primary human CD14⁺ monocytes or the THP-1 cell line with the aim of establishing a physiologically relevant heterotypic culture model. Methods: primary monocytes and THP-1 cells were cultured in the presence of U87MG conditioned media or co-cultured together with previously formed GBM spheroids. Monocyte differentiation was determined by flow cytometry. Results: primary monocytes differentiate to M2 macrophages when incubated with U87MG conditioned media in 2-dimensional culture, as determined by the increased percentage of CD14⁺CD206⁺ and CD64⁺CD206⁺ populations in CD11b⁺ cells. Moreover, the mitochondrial protein p32/gC1qR is expressed in monocytes exposed to U87MG conditioned media. When primary CD14⁺ monocytes or THP-1 cells are added to previously formed GBM spheroids, both invade and establish within them. However, only primary monocytes differentiate and acquire a clear M2 phenotype characterized by the upregulation of CD206, CD163, and MERTK surface markers on the CD11b⁺CD14⁺ population and induce alterations in the sphericity of the cell cultures. Conclusion: our results present a new physiologically relevant model to study GBM/macrophage interactions in a human setting and suggest that both soluble GBM factors, as well as cell-contact dependent signals, are strong inducers of anti-inflammatory macrophages within the tumor niche.     

Preferential Expression of Programmed Death Ligand 1 Protein in Tumor-Associated Macrophages and Its Potential Role in Immunotherapy for Hepatocellular Carcinoma

A predictive biomarker of immune checkpoint inhibitor (ICI)-based treatments in hepatocellular carcinoma (HCC) has not been clearly demonstrated. In this study, we focused on the infiltration and programmed death ligand 1 (PD-L1) expression of tumor-associated macrophages (TAMs) in the tumor microenvironment of HCC. Immunohistochemistry demonstrated that PD-L1 was preferentially expressed on CD68⁺ macrophages in the tumor microenvironment of HCC, suggestive of its expression in TAMs rather than in T cells or tumor cells (P < 0.05). A co-culture experiment using activated T cells and M2 macrophages confirmed a significant increase in T cell functionality after the pretreatment of M2 macrophages with anti-PD-L1. Syngeneic mouse model experiments demonstrated that TAMs expressed PD-L1 and tumors treated with anti-PD-L1 showed smaller diameters than those treated with IgG. In these mice, anti-PD-L1 treatment increased activation markers in intratumoral CD8⁺ T cells and reduced the size of the TAM population. Regarding nivolumab-treated patients, three of eight patients responded to the anti-PD-1 treatment. The percentage of Ki-67-positive CD4⁺ and CD8⁺ T cells was higher in responders than non-responders after nivolumab. Overall, PD-L1 expression on TAMs may be targeted by immune-based HCC treatment, and ICI treatment results in the reinvigoration of exhausted CD8⁺ T cells in HCC.     

The Absence of Extracellular Cold-Inducible RNA-Binding Protein (eCIRP) Promotes Pro-Angiogenic Microenvironmental Conditions and Angiogenesis in Muscle Tissue Ischemia

Extracellular Cold-inducible RNA-binding protein (eCIRP), a damage-associated molecular pattern, is released from cells upon hypoxia and cold-stress. The overall absence of extra- and intracellular CIRP is associated with increased angiogenesis, most likely induced through influencing leukocyte accumulation. The aim of the present study was to specifically characterize the role of eCIRP in ischemia-induced angiogenesis together with the associated leukocyte recruitment. For analyzing eCIRPs impact, we induced muscle ischemia via femoral artery ligation (FAL) in mice in the presence or absence of an anti-CIRP antibody and isolated the gastrocnemius muscle for immunohistological analyses. Upon eCIRP-depletion, mice showed increased capillary/muscle fiber ratio and numbers of proliferating endothelial cells (CD31⁺/CD45⁻/BrdU⁺). This was accompanied by a reduction of total leukocyte count (CD45⁺), neutrophils (MPO⁺), neutrophil extracellular traps (NETs) (MPO⁺CitH3⁺), apoptotic area (ascertained via TUNEL assay), and pro-inflammatory M1-like polarized macrophages (CD68⁺/MRC1⁻) in ischemic muscle tissue. Conversely, the number of regenerative M2-like polarized macrophages (CD68⁺/MRC1⁺) was elevated. Altogether, we observed that eCIRP depletion similarly affected angiogenesis and leukocyte recruitment as described for the overall absence of CIRP. Thus, we propose that eCIRP is mainly responsible for modulating angiogenesis via promoting pro-angiogenic microenvironmental conditions in muscle ischemia.     

Iron Released after Cryo-Thermal Therapy Induced M1 Macrophage Polarization,Promoting the Differentiation of CD4+ T Cells into CTLs

Macrophages play critical roles in both innate and adaptive immunity and are known for their high plasticity in response to various external signals. Macrophages are involved in regulating systematic iron homeostasis and they sequester iron by phagocytotic activity, which triggers M1 macrophage polarization and typically exerts antitumor effects. We previously developed a novel cryo-thermal therapy that can induce the mass release of tumor antigens and damage-associated molecular patterns (DAMPs), promoting M1 macrophage polarization. However, that study did not examine whether iron released after cryo-thermal therapy induced M1 macrophage polarization; this question still needed to be addressed. We hypothesized that cryo-thermal therapy would cause the release of a large quantity of iron to augment M1 macrophage polarization due to the disruption of tumor cells and blood vessels, which would further enhance antitumor immunity. In this study, we investigated iron released in primary tumors, the level of iron in splenic macrophages after cryo-thermal therapy and the effect of iron on macrophage polarization and CD4⁺ T cell differentiation in metastatic 4T1 murine mammary carcinoma. We found that a large amount of iron was released after cryo-thermal therapy and could be taken up by splenic macrophages, which further promoted M1 macrophage polarization by inhibiting ERK phosphorylation. Moreover, iron promoted DC maturation, which was possibly mediated by iron-induced M1 macrophages. In addition, iron-induced M1 macrophages and mature DCs promoted the differentiation of CD4⁺ T cells into the CD4 cytolytic T lymphocytes (CTL) subset and inhibited differentiation into Th2 and Th17 cells. This study explains the role of iron in cryo-thermal therapy-induced antitumor immunity from a new perspective.     

Chromatin Regulator SRG3 Overexpression Protects against LPS/D-GalN-Induced Sepsis by Increasing IL10-Producing Macrophages and Decreasing IFNγ-Producing NK Cells in the Liver

We previously showed that ubiquitous overexpression of the chromatin remodeling factor SWItch3-related gene (SRG3) promotes M2 macrophage differentiation, resulting in anti-inflammatory responses in the experimental autoimmune encephalomyelitis model of multiple sclerosis. Since hepatic macrophages are responsible for sepsis-induced liver injury, we investigated herein the capacity of transgenic SRG3 overexpression (SRG3^β-actin mice) to modulate sepsis in mice exposed to lipopolysaccharide (LPS) plus d-galactosamine (d-GalN). Our results demonstrated that ubiquitous SRG3 overexpression significantly protects mice from LPS/d-GalN-induced lethality mediated by hepatic M1 macrophages. These protective effects of SRG3 overexpression correlated with the phenotypic conversion of hepatic macrophages from an M1 toward an M2 phenotype. Furthermore, SRG3^β-actin mice had decreased numbers and activation of natural killer (NK) cells but not natural killer T (NKT) cells in the liver during sepsis, indicating that SRG3 overexpression might contribute to cross-talk between NK cells and macrophages in the liver. Finally, we demonstrated that NKT cell-deficient CD1d KO/SRG3^β-actin mice are protected from LPS/d-GalN-induced sepsis, indicating that NKT cells are dispensable for SRG3-mediated sepsis suppression. Taken together, our findings provide strong evidence that SRG3 overexpression may serve as a therapeutic approach to control overwhelming inflammatory diseases such as sepsis.     

CD86+/CD206+, Diametrically Polarized Tumor-Associated Macrophages,Predict Hepatocellular Carcinoma Patient Prognosis

Tumor-associated macrophages (TAMs), the most abundant infiltrating immune cells in tumor microenvironment, have distinct functions in hepatocellular carcinoma (HCC) progression. CD68⁺ TAMs represent multiple polarized immune cells mainly containing CD86⁺ antitumoral M1 macrophages and CD206⁺ protumoral M2 macrophages. TAMs expression and density were assessed by immunohistochemical staining of CD68, CD86, and CD206 in tissue microarrays from 253 HCC patients. Clinicopathologic features and prognostic value of these markers were evaluated. We found that CD68⁺ TAMs were not associated with clinicopathologic characteristics and prognosis in HCC. Low presence of CD86⁺ TAMs and high presence of CD206⁺ TAMs were markedly correlated with aggressive tumor phenotypes, such as multiple tumor number and advanced tumor-node-metastasis (TNM) stage; and were associated with poor overall survival (OS) (p = 0.027 and p = 0.024, respectively) and increased time to recurrence (TTR) (p = 0.037 and p = 0.031, respectively). In addition, combined analysis of CD86 and CD206 provided a better indicator for OS (p = 0.011) and TTR (p = 0.024) in HCC than individual analysis of CD86 and CD206. Moreover, CD86⁺/CD206⁺ TAMs predictive model also had significant prognosis value in α-fetoprotein (AFP)-negative patients (OS: p = 0.002, TTR: p = 0.005). Thus, these results suggest that combined analysis of immune biomarkers CD86 and CD206 could be a promising HCC prognostic biomarker.     

Lipoic Acid Attenuates Innate Immune Infiltration and Activation in the Visceral Adipose Tissue of Obese Insulin Resistant Mice

Visceral adipose inflammation mediated by innate and adaptive immune alterations plays a critical role in diet-induced obesity and insulin resistance (IR). The dietary supplement α-lipoic acid (αLA) has been shown to ameliorate inflammatory processes in macrophages, however the relative significance of these effects in the context of visceral adipose inflammation and IR remain unknown. In this study we investigated its effects via both intraperitoneal and oral administration in lean and obese transgenic mice expressing yellow fluorescent protein (YFP) under control of a monocyte specific promoter (c-fms^YFP+). αLA significantly improved indices of insulin-resistance concomitant with a decrease in total (YFP⁺CD11b⁺) and activated (YFP⁺CD11b⁺CD11c⁺) visceral adipose tissue macrophages. Histologically, the visceral adipose tissue of obese mice receiving αLA had fewer “crown-like structures,” a hallmark of adipose inflammation in murine obesity. Monocyte adhesion assessed by intravital microscopy of cremasteric venules was attenuated by αLA. In cultured WT and toll-like receptor 4 (TLR4) null primary mouse macrophages, αLA significantly decreased basal CCR-2, MCP-1 and TNF-α expression levels. LPS treatment resulted in increased TNFα, MCP-1, and IL-6 expression while αLA partially abrogated the LPS effect on MCP-1 and TNFα; Interestingly, CCR-2 was not coordinately regulated. AαLA prevented LPS-induced nuclear factor kappa B (NFκB) activation in the same cultured macrophages. These data suggest that αLA may modulate visceral adipose inflammation, a critical determinant of IR via TLR4 and NF-κB pathways.     

Functional Ambivalence of Dendritic Cells: Tolerogenicity and Immunogenicity

Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APCs) and inducers of T cell-mediated immunity. Although DCs play a central role in promoting adaptive immune responses against growing tumors, they also establish and maintain peripheral tolerance. DC activity depends on the method of induction and/or the presence of immunosuppressive agents. Tolerogenic dendritic cells (tDCs) induce immune tolerance by activating CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells and/or by producing cytokines that inhibit T cell activation. These findings suggest that tDCs may be an effective treatment for autoimmune diseases, inflammatory diseases, and infertility.     

BMP Receptor Inhibition Enhances Tissue Repair in Endoglin Heterozygous Mice

Hereditary hemorrhagic telangiectasia type 1 (HHT1) is a severe vascular disorder caused by mutations in the TGFβ/BMP co-receptor endoglin. Endoglin haploinsufficiency results in vascular malformations and impaired neoangiogenesis. Furthermore, HHT1 patients display an impaired immune response. To date it is not fully understood how endoglin haploinsufficient immune cells contribute to HHT1 pathology. Therefore, we investigated the immune response during tissue repair in Eng+/− mice, a model for HHT1. Eng+/− mice exhibited prolonged infiltration of macrophages after experimentally induced myocardial infarction. Moreover, there was an increased number of inflammatory M1-like macrophages (Ly6C^high/CD206⁻) at the expense of reparative M2-like macrophages (Ly6C^low/CD206⁺). Interestingly, HHT1 patients also showed an increased number of inflammatory macrophages. In vitro analysis revealed that TGFβ-induced differentiation of Eng+/− monocytes into M2-like macrophages was blunted. Inhibiting BMP signaling by treating monocytes with LDN-193189 normalized their differentiation. Finally, LDN treatment improved heart function after MI and enhanced vascularization in both wild type and Eng+/− mice. The beneficial effect of LDN was also observed in the hind limb ischemia model. While blood flow recovery was hampered in vehicle-treated animals, LDN treatment improved tissue perfusion recovery in Eng+/− mice. In conclusion, BMPR kinase inhibition restored HHT1 macrophage imbalance in vitro and improved tissue repair after ischemic injury in Eng+/− mice.     

Lysophosphatidic Acid Mediates Imiquimod-Induced Psoriasis-like Symptoms by Promoting Keratinocyte Proliferation through LPAR1/ROCK2/PI3K/AKT Signaling Pathway

Psoriasis is a chronic inflammatory skin disease. Recently, lysophosphatidic acid (LPA)/LPAR5 signaling has been reported to be involved in both NLRP3 inflammasome activation in macrophages and keratinocyte activation to produce inflammatory cytokines, contributing to psoriasis pathogenesis. However, the effect and molecular mechanisms of LPA/LPAR signaling in keratinocyte proliferation in psoriasis remain unclear. In this study, we investigated the effects of LPAR1/3 inhibition on imiquimod (IMQ)-induced psoriasis-like mice. Treatment with the LPAR1/3 antagonist, ki16425, alleviated skin symptoms in IMQ-induced psoriasis-like mouse models and decreased keratinocyte proliferation in the lesion. It also decreased LPA-induced cell proliferation and cell cycle progression via increased cyclin A2, cyclin D1, cyclin-dependent kinase (CDK)2, and CDK4 expression and decreased p27^Kip1 expression in HaCaT cells. LPAR1 knockdown in HaCaT cells reduced LPA-induced proliferation, suppressed cyclin A2 and CDK2 expression, and restored p27^Kip1 expression. LPA increased Rho-associated protein kinase 2 (ROCK2) expression and PI3K/AKT activation; moreover, the pharmacological inhibition of ROCK2 and PI3K/AKT signaling suppressed LPA-induced cell cycle progression. In conclusion, we demonstrated that LPAR1/3 antagonist alleviates IMQ-induced psoriasis-like symptoms in mice, and in particular, LPAR1 signaling is involved in cell cycle progression via ROCK2/PI3K/AKT pathways in keratinocytes.     

Immunomodulatory Properties of Umbilical Cord Blood-Derived Small Extracellular Vesicles and Their Therapeutic Potential for Inflammatory Skin Disorders

Umbilical cord blood (UCB) has long been seen as a rich source of naïve cells with strong regenerative potential, likely mediated by paracrine signals. More recently, small extracellular vesicles (sEV), such as exosomes, have been shown to play essential roles in cell-to-cell communication, via the transport of numerous molecules, including small RNAs. Often explored for their potential as biomarkers, sEV are now known to have regenerative and immunomodulating characteristics, particularly if isolated from stem cell-rich tissues. In this study, we aim to characterize the immunomodulating properties of umbilical cord blood mononuclear cell-derived sEV (UCB-MNC-sEV) and explore their therapeutic potential for inflammatory skin diseases. UCB-MNC-sEV were shown to shift macrophages toward an anti-inflammatory phenotype, which in turn exert paracrine effects on fibroblasts, despite previous inflammatory stimuli. Additionally, the incubation of PBMC with UCB-MNC-sEV resulted in a reduction of total CD4⁺ and CD8⁺ T-cell proliferation and cytokine release, while specifically supporting the development of regulatory T-cells (Treg), by influencing FOXP3 expression. In a 3D model of psoriatic skin, UCB-MNC-sEV reduced the expression of inflammatory and psoriatic markers IL6, IL8, CXCL10, COX2, S100A7, and DEFB4. In vivo, UCB-MNC-sEV significantly prevented or reversed acanthosis in imiquimod-induced psoriasis, and tendentially increased the number of Treg in skin, without having an overall impact on disease burden. This work provides evidence for the anti-inflammatory and tolerogenic effect of UCB-MNC-sEV, which may be harnessed for the treatment of Th17-driven inflammatory skin diseases, such as psoriasis.     

Distinct Immune Signatures Indicative of Treatment Response and Immune-Related Adverse Events in Melanoma Patients under Immune Checkpoint Inhibitor Therapy

To identify potential early biomarkers of treatment response and immune-related adverse events (irAE), a pilot immune monitoring study was performed in stage IV melanoma patients by flow cytometric analysis of peripheral blood mononuclear cells (PBMC). Overall, 17 patients were treated with either nivolumab or pembrolizumab alone, or with a combination of nivolumab and ipilimumab every three weeks. Of 15 patients for which complete response assessment was available, treatment responders (n = 10) as compared to non-responders (n = 5) were characterized by enhanced PD-1 expression on CD8⁺ T cells immediately before treatment (median ± median absolute deviation/MAD 26.7 ± 10.4% vs. 17.2 ± 5.3%). Responders showed a higher T cell responsiveness after T cell receptor ex vivo stimulation as determined by measurement of programmed cell death 1 (PD-1) expression on CD3⁺ T cells before the second cycle of treatment. The percentage of CD8⁺ effector memory (CD8⁺CD45RA⁻CD45RO⁺CCR7⁻) T cells was higher in responders compared to non-responders before and immediately after the first cycle of treatment (median ± MAD 39.2 ± 7.3% vs. 30.5 ± 4.1% and 37.7 ± 4.6 vs. 24.0 ± 6.4). Immune-related adverse events (irAE) were accompanied by a higher percentage of activated CD4⁺ (CD4⁺CD38⁺HLADR⁺) T cells before the second treatment cycle (median ± MAD 14.9 ± 3.9% vs. 5.3 ± 0.4%). In summary, PBMC immune monitoring of immune-checkpoint inhibition (ICI) treatment in melanoma appears to be a promising approach to identify early markers of treatment response and irAEs.     

Activation of Dendritic Cells in Tonsils Is Associated with CD8 T Cell Responses following Vaccination with Live Attenuated Classical Swine Fever Virus

Classical swine fever (CSF) is a highly contagious disease caused by the classical swine fever virus (CSFV). The live attenuated C-strain vaccine is highly efficacious, initiating protection within several days of delivery. The vaccine strain is detected in the tonsil early after inoculation, yet little is known of the role that tonsillar immune cells might play in initiating protection. Comparing the C-strain vaccine with the pathogenic CSFV Alfort-187 strain, changes in the myeloid cell compartment of the tonsil were observed. CSFV infection led to the emergence of an additional CD163⁺CD14⁺ cell population, which showed the highest levels of Alfort-187 and C-strain infection. There was also an increase in both the frequency and activation status (as shown by increased MHC-II expression) of the tonsillar conventional dendritic cells 1 (cDC1) in pigs inoculated with the C-strain. Notably, the activation of cDC1 cells coincided in time with the induction of a local CSFV-specific IFN-γ⁺ CD8 T cell response in C-strain vaccinated pigs, but not in pigs that received Alfort-187. Moreover, the frequency of CSFV-specific IFN-γ⁺ CD8 T cells was inversely correlated to the viral load in the tonsils of individual animals. Accordingly, we hypothesise that the activation of cDC1 is key in initiating local CSFV-specific CD8 T cell responses which curtail early virus replication and dissemination.     

MicroRNA-328-5p Alleviates Macrophage Lipid Accumulation through the Histone Deacetylase 3/ATP-binding cassette transporter A1 pathway

MicroRNA-328 (miR-328) was reported to protect against atherosclerosis, but its role in foam cell formation remains unknown. The aim of this study was to investigate the effect of miR-328-5p on macrophage lipid accumulation and the underlying mechanisms. The results showed that miR-328-5p expression was robustly decreased in oxidized low-density lipoprotein (ox-LDL)-treated macrophages. Treatment of human acute monocytic leukemia cel (THP-1) macrophage-derived foam cells with a miR-328-5p mimic markedly increased [³H]-cholesterol efflux, inhibited lipid droplet accumulation, and decreased intracellular total cholesterol (TC), free cholesterol (FC) and cholesteryl ester (CE) contents. Upregulation of miR-328-5p also reduced the expression of histone deacetylase 3 (HDAC3) but increased the levels of ATP-binding cassette transporter A1 (ABCA1) in THP-1 macrophage-derived foam cells. Mechanistically, miR-328-5p inhibited HDAC3 expression by directly targeting its 3′UTR, thereby promoting ABCA1 expression and the subsequent cholesterol efflux. Furthermore, miR-328-5p mimic treatment did not affect the uptake of Dil-ox-LDL or the expression of scavenger receptor-A (SR-A), thrombospondin receptor (CD36) and ABCG1. Taken together, these findings suggest that miR-328-5p alleviates macrophage lipid accumulation through the HDAC3/ABCA1 pathway.