Promote Macrophage Proliferation Research Articles

DKK1 loss promotes endometrial fibrosis via autophagy and exosome-mediated macrophage-to-myofibroblast transition

IntroductionIntrauterine adhesions (IUA) manifest as endometrial fibrosis, often causing infertility or recurrent miscarriage; however, their pathogenesis remains unclear.ObjectivesThis study assessed the role of Dickkopf WNT signaling pathway inhibitor 1 (DKK1) and autophagy in endometrial fibrosis, using clinical samples as well as in vitro and in vivo experiments.MethodsImmunohistochemistry, immunofluorescence and western blot were used to determine the localization and expression of DKK1 in endometrium; DKK1 silencing and DKK1 overexpression were used to detect the biological effects of DKK1 silencing or expression in endometrial cells; DKK1 gene knockout mice were used to observe the phenotypes caused by DKK1 gene knockout.ResultsIn patients with IUA, DKK1 and autophagy markers were down-regulated; also, α-SMA and macrophage localization were increased in the endometrium. DKK1 conditional knockout (CKO) mice showed a fibrotic phenotype with decreased autophagy and increased localization of α-SMA and macrophages in the endometrium. In vitro studies showed that DKK1 knockout (KO) suppressed the autophagic flux of endometrial stromal cells. In contrast, ectopic expression of DKK1 showed the opposite phenotype. Mechanistically, we discovered that DKK1 regulates autophagic flux through Wnt/β-catenin and PI3K/AKT/mTOR pathways. Further studies showed that DKK1 KO promoted the secretion of interleukin (IL)-8 in exosomes, thereby promoting macrophage proliferation and metastasis. Also, in DKK1 CKO mice, treatment with autophagy activator rapamycin partially restored the endometrial fibrosis phenotype.ConclusionOur findings indicated that DKK1 was a potential diagnostic marker or therapeutic target for IUA.

Fluorinated hydroxyapatite conditions a favorable osteo-immune microenvironment via triggering metabolic shift from glycolysis to oxidative phosphorylation

BackgroundBiological-derived hydroxyapatite is widely used as a bone substitute for addressing bone defects, but its limited osteoconductive properties necessitate further improvement. The osteo-immunomodulatory properties hold crucial promise in maintaining bone homeostasis, and precise modulation of macrophage polarization is essential in this process. Metabolism serves as a guiding force for immunity, and fluoride modification represents a promising strategy for modulating the osteoimmunological environment by regulating immunometabolism. In this context, we synthesized fluorinated porcine hydroxyapatite (FPHA), and has demonstrated its enhanced biological properties and osteogenic capacity. However, it remains unknown whether and how FPHA affects the immune microenvironment of the bone defects.MethodsFPHA was synthesized and its composition and structural properties were confirmed. Macrophages were cultured with FPHA extract to investigate the effects of FPHA on their polarization and the related osteo-immune microenvironment. Furthermore, total RNA of these macrophages was extracted, and RNA-seq analysis was performed to explore the underlying mechanisms associated with the observed changes in macrophages. The metabolic states were evaluated with a Seahorse analyzer. Additionally, immunohistochemical staining was performed to evaluate the macrophages response after implantation of the novel bone substitutes in critical size calvarial defects in SD rats.ResultsThe incorporation of fluoride ions in FPHA was validated. FPHA promoted macrophage proliferation and enhanced the expression of M2 markers while suppressing the expression of M1 markers. Additionally, FPHA inhibited the expression of inflammatory factors and upregulated the expression of osteogenic factors, thereby enhancing the osteogenic differentiation capacity of the rBMSCs. RNA-seq analysis suggested that the polarization-regulating function of FPHA may be related to changes in cellular metabolism. Further experiments confirmed that FPHA enhanced mitochondrial function and promoted the metabolic shift of macrophages from glycolysis to oxidative phosphorylation. Moreover, in vivo experiments validated the above results in the calvarial defect model in SD rats.ConclusionIn summary, our study reveals that FPHA induces a metabolic shift in macrophages from glycolysis to oxidative phosphorylation. This shift leads to an increased tendency toward M2 polarization in macrophages, consequently creating a favorable osteo-immune microenvironment. These findings provide valuable insights into the impact of incorporating an appropriate concentration of fluoride on immunometabolism and macrophage mitochondrial function, which have important implications for the development of fluoride-modified immunometabolism-based bone regenerative biomaterials and the clinical application of FPHA or other fluoride-containing materials.Graphical . FPHA was successfully prepared through the chemical and thermal process. The immunomodulatory effects of FPHA were investigated through in vitro and in vivo studies, revealing its ability to induce a metabolic shift in macrophages from glycolysis to mitochondrial oxidative phosphorylation (OxPhos). This metabolic remodeling resulted in a notable suppression of M1 macrophage polarization and promotion of M2 macrophage polarization. Furthermore, FPHA was found to enhance osteogenic differentiation and facilitate bone repair. These findings underscore the promising potential of FPHA as a biomaterial for bone regenerative applications, providing valuable insights for the development of bioactive materials with metabolic-immunoregulatory propertiesGraphical

The Role of Copper-Induced M2 Macrophage Polarization in Protecting Cartilage Matrix in Osteoarthritis.

BACKGROUND The pathological mechanism of osteoarthritis is still unclear. The regulation of the immune microenvironment has been of growing interest in the progression and treatment of osteoarthritis. Macrophages with different phenotypes, producing different cytokines, have been linked to the mechanism of cartilage injury in osteoarthritis. Copper ions play a role in the immune response and are involved in the pathological mechanisms of osteoarthritis by affecting the metabolism of the cartilage matrix. Bioactive glass (BG) is an osteogenic material with superior biocompatibility. Here, we report on the regulatory behavior of macrophages using a copper-based composite BG material. MATERIAL AND METHODS Cu-BGC powder was prepared by sol-gel method, and scaffolds were fabricated and characterized using 3D printing. Macrophage cultures grown with Cu-BGC were examined for cell culture and proliferation. The effect of Cu-BGC on the degradation metabolism of chondrocytes, cultured in the environment of inflammatory cytokine IL-1ß, was determined. In addition, the morphology of macrophages, secretion of inflammatory cytokines, and expression of surface markers were examined. RESULTS The results show that Cu-BGC promotes macrophage proliferation at a range of concentrations and increases the secretion of anti-inflammatory cytokines while inhibiting proinflammatory cytokines. At the same time, M2-type cell surface markers are definitely expressed and the morphology of macrophages is altered. In addition, Cu-BGC inhibited the degradation metabolism of chondrocytes in the inflammatory environment induced by IL-1ß. CONCLUSIONS These results suggest that Cu-BGC induced macrophage polarization into an M2 type anti-inflammatory phenotype, and inhibition of immune injury response may play a role in delaying cartilage matrix damage in osteoarthritis.

Vitamin D receptor-deficient keratinocytes-derived exosomal miR-4505 promotes the macrophage polarization towards the M1 phenotype.

The vitamin D receptor (VDR) has a low level of expression in the keratinocytes of patients with psoriasis and plays a role in the development of the disease. Furthermore, the crosstalk between macrophages and psoriatic keratinocytes-derived exosomes is critical for psoriasis progression. However, the effects of VDR-deficient keratinocytes-derived exosomes (Exos-shVDR) on macrophages and their underlying mechanisms remain largely unknown. VDR-deficient keratinocytes were constructed by infecting HaCaT cells with a VDR-targeting lentivirus, mimicking the VDR-deficient state observed in psoriatic keratinocytes. Exosomes were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot. The effect of Exos-shVDR on macrophage proliferation, apoptosis, and M1/M2 polarization was assessed using cell counting kit-8 assay (CCK-8), flow cytometer, real-time quantitative polymerasechain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA). The mechanism underlying the effect of Exos-shVDR on macrophage function was elucidated through data mining, bioinformatics, RT-qPCR, and rescue experiments. Our results revealed that both Exos-shVDR and Exos-shNC exhibited typical exosome characteristics, including a hemispheroid shape with a concave side and particle size ranging from 50 to 100 nm. The levels of expression of VDR were significantly lower in Exos-shVDR than in Exos-shNC. Functional experiments demonstrated that Exos-shVDR significantly promoted macrophage proliferation and polarization towards the M1 phenotype while inhibiting macrophage apoptosis. Moreover, miR-4505 was highly expressed in the skin tissue of patients with psoriasis. Its overexpression significantly increased macrophage proliferation and polarization towards M1 and inhibited apoptosis. Furthermore, the effects of Exos-shVDR on macrophage function occur through miR-4505. Exos-shVDR exacerbates macrophage proliferation, promotes polarization towards the M1 phenotype, and inhibits macrophage apoptosis by increasing the levels of miR-4505. These results indicate that modulation of macrophage function is a potential strategy for developing new drugs for the treatment of psoriasis.

A frog peptide provides new strategies for the intervention against skin wound healing

BackgroundAmphibian derived pro-healing peptides as molecular probes might provide a promising strategy for development of drug candidates and elucidation of cellular and molecular mechanisms of skin wound healing. A novel skin amphibian peptide, OA-RD17, was tested for modulation of cellular and molecular mechanisms associated with skin wound healing.MethodsCell scratch, cell proliferation, trans-well, and colony formation assays were used to explore the pro-healing ability of peptide OA-RD17 and microRNA-632 (miR-632). Then, the therapeutic effects of OA-RD17 and miR-632 were assessed in mice, diabetic patient ex vivo skin wounds and SD rats. Moreover, hematoxylin and eosin (H&E), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and immunofluorescence staining were performed to detect skin wound tissue regeneration, inflammatory factors expression, and macrophage polarization. Finally, RNA sequencing, molecular docking, co-localization, dual luciferase reporter, real-time quantitative reverse transcription PCR (RT-qPCR), and Western blotting were used to explore the mechanism of OA-RD17 and miR-632 on facilitating skin wound healing.ResultsThe non-toxic peptide (OA-RD17) promoted macrophage proliferation and migration by activating MAPK and suppressed inflammation by inhibiting NF-κB. In keratinocytes, OA-RD17 inhibited excessive inflammation, and activated MAPK via the Toll-like receptor 4 (TLR4) to promote proliferation and migration, as well as up-regulate the expression of miR-632, which targeted GSK3β to activate Wnt/β-catenin to boost proliferation and migration in a positive feedback manner. Notably, OA-RD17 promoted transition from the inflammatory to proliferative stage, accelerated epidermal and granulation regeneration, and exhibited therapeutic effects on mouse and diabetic patient ex vivo skin wounds. MiR-632 activated Wnt/β-catenin to promote full-thickness skin wound healing in rats.ConclusionsOA-RD17 exhibited promising therapeutic effects on mice (full-thickness, deep second-degree burns), and ex vivo skin wounds in diabetic patients by regulating macrophages proliferation, migration, and polarization (MAPK, NF-κB), and keratinocytes proliferation and migration (TLR4/MAPK/miR-632/Wnt/β-catenin molecular axis). Moreover, miR-632 also activated Wnt/β-catenin to promote full-thickness skin wound healing in rats. Notably, our results indicate that OA-RD17 and miR-632 are promising pro-healing drug candidates.Graphical

Glycolaldehyde-derived advanced glycation end products promote macrophage proliferation via the JAK-STAT signaling pathway.

Advanced glycation end products (AGEs) are heterogeneous proinflammatory molecules produced by a non-enzymatic glycation reaction between reducing sugars (and their metabolites) and biomolecules with amino groups, such as proteins. Although increases in and the accumulation of AGEs have been implicated in the onset and exacerbation of lifestyle- or age-related diseases, including diabetes, their physiological functions have not yet been elucidated in detail. The present study investigated the cellular responses of the macrophage cell line RAW264.7 stimulated by glycolaldehyde-derived AGEs (Glycol-AGEs) known as representative toxic AGEs. The results obtained showed that Glycol-AGEs significantly promoted the proliferation of RAW264.7 cells at a low concentration range (1-10µg/mL) in a concentration-dependent manner. On the other hand, neither TNF-α production nor cytotoxicity were induced by the same concentrations of Glycol-AGEs. The increases observed in cell proliferation by low concentrations of Glycol-AGEs were also detected in receptor triple knockout (RAGE-TLR4-TLR2 KO) cells as well as in wild-type cells. Increases in cell proliferation were not affected by various kinase inhibitors, including MAP kinase inhibitors, but were significantly suppressed by JAK2 and STAT5 inhibitors. In addition, the expression of some cell cycle-related genes was up-regulated by the stimulation with Glycol-AGEs. These results suggest a novel physiological role for AGEs in the promotion of cell proliferation via the JAK-STAT pathway.

A20 affects macrophage polarization through the NLRP3 inflammasome signaling pathway and promotes breast cancer progression.

Breast cancer is the most common malignant tumor in females, and the majority of patients succumb to metastasis. The present study aimed to investigate the association between tumor necrosis factor alpha-induced protein 3 (A20), NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) and tumor-associated macrophage polarization, and their effects on the proliferation and metastasis of breast cancer cells. The expression of A20 in breast cancer cells was analyzed by reverse transcription-quantitative PCR (RT-qPCR) and western blotting. RT-qPCR and western blotting were also used to confirm the transfection efficiency. The viability, clone formation, migration, invasion and angiogenesis of transfected breast cancer cells were detected by Cell Counting Kit-8, colony formation, wound healing, Transwell and tube formation assays, respectively. Activated macrophages, namely M1 and M2 type macrophages, were observed by double staining immunofluorescence. The levels of M1 and M2 macrophage markers were analyzed by qPCR. The expression of angiogenesis-related proteins and NLRP3 inflammasome activation-associated proteins was detected by western blotting. The results revealed that A20 was highly expressed in breast cancer cells. Interference with A20 inhibited the proliferation, invasion, migration and angiogenesis of breast cancer cells, and inhibited the M2-like polarization of macrophages. Interference with A20 promoted the activation of the NLRP3 inflammasome. The NLRP3 inhibitor MCC950 alleviated the effect of interference with A20 to promote macrophage proliferation and recruitment, as well as M2-like polarization. In conclusion, interference with A20 inhibited macrophage proliferation and M2-like polarization through the NLRP3 inflammasome signaling pathway to inhibit breast cancer progression.

Preparation, Characterization, and Anti-Adhesive Activity of Sulfate Polysaccharide from Caulerpa lentillifera against Helicobacter pylori.

In the gastric mucosa, chronic inflammation due to Helicobacter pylori infection promotes gastrocarcinogenesis. Polysaccharides of Caulerpa lentillifera are well-characterized by broad antimicrobial activity and anti-inflammatory potentials. The present study was undertaken to investigate whether the low molecular sulfate polysaccharides of C. lentillifera (CLCP) exhibit any anti-adhesive activity against H. pylori. After a hot water extraction and purification process, two purified polysaccharide fractions (CLCP-1 and CLCP2) were studied based on structural characterization and bioactivity determination. The results implied that except for the molar ratio, CLCP-1 and CLCP-2 contain high sulfate, mannose, galactose, xylose, glucose levels, and low protein levels. The molecular weight and Fourier transform infrared spectroscopy (FT-IR) assays confirmed that CLCP-1 and CLCP-2 are sulfate polysaccharides with an average molecular weight (Mw) of 963.15 and 648.42 kDa, respectively. In addition, CLCP-1 and CLCP-2 exhibited stronger antibacterial activity against H. pylori. CLCP-1 and CLCP-2 could significantly promote macrophage proliferation and decrease the production of nitric oxide (NO) through downregulated expression of inducible nitric oxide synthase (iNOS). Meanwhile, CLCP-1 and CLCP-2 in this study showed efficiently protected gastric adenocarcinoma (AGS) cells against H. pylori with the inhibition of the IL-8/NF-κB axis. These findings suggested the effect of Caulerpa lentillifera polysaccharides on H. pylori adhesion, a potential supply of nutrients for eradication therapy through the reduction of cell count and inflammation.

Mechanism of pepsin promoting lingual tonsil hypertrophy by stimulating macrophage

Objective: To investigate the possible pathophysiological mechanism of laryngopharyngeal reflux (LPR) in the development of lingual tonsil hypertrophy (LTH). Methods: The lingual tonsil tissues were collected from 73 patients [48 males and 25 females, aged from 24 to 76 (52.86±12.04) years] who underwent surgery for laryngopharyngeal diseases at the Department of Otolaryngology and Head and Neck Surgery, Southern Hospital of Southern Medical University from October 2019 to December 2020, and the lingual tonsil grade (LTG), reflux symptom index (RSI) and reflux finding score (RFS) were assessed. The expression of pepsin in LTH was detected by immunohistochemistry. The coexpression of pepsin and macrophages were detected by immunohistofluorescence. In vitro, cytological experiments and pathway assays were performed on macrophages stimulated by pepsin. Pathway alterations of macrophages in pepsin-positive high-grade LTH were detected by double-fluorescence immunohistochemistry. Data were analyzed by SPSS 20.0 software. Results: There were 44 clinically significant LPRD patients with LTG 3 and 4, and the pepsin positive rate was 88.6% (39/44). While, the pepsin positive rate of LTG 1 and 2 was 48.3% (14/29). LTG was significantly positively correlated with RFS/RSI positive rate(χ2=23.01/19.62, P<0.001/0.001; r=0.54/0.51, P<0.001/0.001) and pepsin tissue staining intensity (H=21.58, P<0.001; r=0.53, P<0.001), respectively. Pepsin and macrophages were clearly colocalized in high grade LTH. In vitro, pepsin promoted macrophage proliferation (P<0.05) and production of IL-6/IL-8 (P<0.05). Pepsin significantly up-regulated the p38/JNK MAPK pathway in macrophages (P<0.05). Pepsin up-regulated the expression of IL-6 and IL-8 of macrophages by activating the p38 MAPK pathway (P<0.05), and up-regulated the expression of IL-8 by activating the JNK pathway (P<0.05). The p38/JNK MAPK pathways were highly expressed in macrophages of pepsin-positive LTH (P<0.05). Conclusions: LPR is an important pathogenic factor in LTH. Macrophages may mediate pepsin-induced inflammation and the pathogenesis of LTH.

Blunt snout bream (Megalobrama amblycephala) MaCSF-1 contributes to proliferation, phagocytosis and immunoregulation of macrophages via MaCSF-1R

CSF-1 and CSF-1R have been well demonstrated in humans, regulating the differentiation, proliferation and survival of the mononuclear phagocyte system. However, the functional study on MaCSF-1 and MaCSF-1R from blunt snout bream (Megalobrama amblycephala) is still unknown. In the present study, we cloned and functionally characterized MaCSF-1 and MaCSF-1R. Multiple sequence alignment and phylogenetic tree analysis showed that both MaCSF-1 and MaCSF-1R were mostly close to the grass carp counterparts. Tissue distribution analysis showed that both MaCSF-1 and MaCSF-1R were widely distributed in all examined tissues, dominantly distributed in spleen, blood and head kidney tissues. Furthermore, confocal microscopy assay and flow cytometry assay showed that MaCSF-1R was the marker on the surface of macrophages. Recombinant MaCSF-1 promoted macrophage proliferation, phagocytosis and the production of IL-10. Through the pull-down experiments and indirect immunofluorescence experiments, the interaction between MaCSF-1 and MaCSF-1R was confirmed. To explore the relationship between MaCSF-1 and its receptor, MaCSF-1R and MaCSF-1R antibody was prepared. Then the MaCSF-1R blockage assay indicated that the role of MaCSF-1 on the macrophages proliferation and phagocytosis was weakened, leading the reduction of IL-10 expression level. In conclusion, MaCSF-1R is the marker on the surface of macrophage membrane; and MaCSF-1 promotes macrophage proliferation, phagocytosis, and significantly increased the expression levels of IL-10 depended on the interacting with MaCSF-1R. This study provides basal data for the biological function of MaCSF-1 and MaCSF-1R, and is valuable for the exploration of MaCSF-1 and MaCSF-1R molecular interactions.

Immunomodulatory activity and active mechanisms of a low molecular polysaccharide isolated from Lanzhou lily bulbs in RAW264.7 macrophages

Exploring immunomodulatory effects of polysaccharides mechanisms is highly significant. In this study, Lanzhou lily (Lilium davidii var. unicolor) polysaccharides (LP) of 6.80 × 103 Da were extracted using the freeze-thaw method. For analytical purposes, structure characterization was conducted using fourier transform infrared spectrometer (FTIR), ultraviolet spectrophotometer (UV–Vis), scanning electron microscope (SEM), and transmission electron microscopy (TEM). Results showed that LP exhibited characteristics of polysaccharide functional groups. Moreover, findings on its immune activity showed that LP can stimulate macrophage RAW264.7, promote macrophage proliferation, secrete nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6). We use RNA-Seq to explore how LP mechanisms effect macrophages. Results revealed that LP likely plays an immunomodulatory role through cytokines secretion, cytokines that effectuate G-protein coupled receptor subunits, and activating downstream immune-related signaling pathways. This study can be used as a reference for the further development of LP while also offering an in-depth understanding of mechanistic immunoregulatory activity.

Mannose Receptor-Mediated Carbon Nanotubes as an Antigen Delivery System to Enhance Immune Response Both In Vitro and In Vivo.

Carbon nanotubes (CNTs) are carbon allotropes consisting of one, two, or more concentric rolled graphene layers. These can intrinsically regulate immunity by activating the innate immune system. Mannose receptors (MR), a subgroup of the C-type lectin superfamily, are abundantly expressed on macrophages and dendritic cells. These play a crucial role in identifying pathogens, presenting antigens, and maintaining internal environmental stability. Utilizing the specific recognition between mannose and antigen-presenting cells (APC) surface mannose receptors, the antigen-carrying capacity of mannose-modified CNTs can be improved. Accordingly, here, we synthesized the mannose-modified carbon nanotubes (M-MWCNT) and evaluated them as an antigen delivery system through a series of in vitro and in vivo experiments. In vitro, M-MWCNT carrying large amounts of OVA were rapidly phagocytized by macrophages and promoted macrophage proliferation to facilitate cytokines (IL-1β, IL-6) secretion. In vivo, in mice, M-MWCNT induced the maturation of dendritic cells and increased the levels of antigen-specific antibodies (IgG, IgG1, IgG2a, IgG2b), and cytokines (IFN-γ, IL-6). Taken together, M-MWCNT could induce both humoral and cellular immune responses and thereby can be utilized as an efficient antigen-targeted delivery system.

Structural characteristics and immune-enhancing activity of fractionated polysaccharides from Athyrium Multidentatum (Doll.) Ching.

Polysaccharides coded as CP were extracted from Athyrium Multidentatum (Doll.) Ching and then fractionated into five fractions (FP-1, FP-2, FP-3, FP-4 and FP-5). A purified polysaccharide designated as FP-3-4 was prepared from FP-3 by Sephadex G-100 column chromatography. Chemical analysis disclosed that CP and these fractions were heteropolysaccharides and mainly composed of glucose, galactose, arabinose, mannose, rhamnose, xylose, fucose, ribose and uronic acid with different molar ratios. They presented different images of SEM. FP-3-4 was highly branched polymers with sixteen types of linkages. The in vitro immunomodulatory results stated that CP and these fractions could promote macrophage proliferation, enhance macrophage phagocytosis and increase the production of NO, TNF-α, IFN-γ, IL-1β, IL-6, IL-10 and IL-2, indicating remarkable immune enhancement activities. RNA sequencing analysis revealed that CP and FP-3 induced macrophage activation mainly through MAPK and alternative NF-κΒ signaling pathways via CD14/TLR4 and Dectin-2 receptors, which were verified by RT-qPCR and western blot.

Preparation, Structural Features and in vitro Immunostimulatory Activity of a Glucomannan From Fresh Dendrobium catenatum Stems.

Dendrobium catenatum polysaccharides (DCPs) have attracted attention due to their multiple physiological activities and health benefits. In this study, a novel water-soluble DCP was obtained from fresh D. catenatum stems through three-phase partitioning and ethanol precipitation at room temperature. Its structural characteristics, rheological property, and in vitro immunostimulatory activity were evaluated. Results demonstrated that DCP was a homogenous polysaccharide with a carbohydrate content of 92.75% and a weight-average molecular weight of 2.21 × 105 Da. This polysaccharide is an O-acetylated glucomannan comprised by glucose, mannose, and galacturonic acid in a molar ratio of 30.2:69.5:0.3 and mainly comprises (1→4)-β-D-mannopyranosyl (Manp), 2-O-acetyl-(1→4)-β-D-Manp, (1→6)-α-D-glucopyranosyl (Glcp), and (1→4)-α-D-Glcp residues. DCP exhibits an extended rigid chain in an aqueous solution and favorable steady shear fluid and dynamic viscoelastic behaviors. In vitro immunostimulating assays indicated that DCP activates RAW264.7 cells, thus markedly promoting macrophage proliferation and phagocytosis and increasing the levels of nitric oxide, interferon-γ, interleukin-6, and interleukin-1β. Moreover, the presence of O-acetyl group and high Mw in DCP might be responsible for its potent immunostimulatory activity in vitro. Therefore, our data suggested that DCP could be developed as a promising immunostimulant in functional food and pharmaceutical industries.

Immunomodulatory activity of Senegalia macrostachya (Reichenb. ex DC.) Kyal. & Boatwr seed polysaccharide fraction through the activation of the MAPK signaling pathway in RAW264.7 macrophages.

Senegalia macrostachya (Reichenb. ex DC.) Kyal. & Boatwr seed (SMS) is a wild legume used as food and medicine in many African countries. In the current study, a novel polysaccharide (SMSP2) was extracted from SMS using hot water and purified with DEAE-52 cellulose. Its structure was characterized, and the immunomodulatory activity and possible molecular mechanism in murine macrophage RAW264.7 were explored. The results revealed that SMSP2 was a uronic acid-rich polysaccharide (51.6%, w/w) with a molecular weight of 52.07 kDa. The neutral sugars were mainly arabinose, xylose, mannose, and galactose at a molar ratio of 1.00 : 0.84 : 0.90 : 0.07. Interestingly, SMSP2 treatment markedly promoted macrophage proliferation and phagocytosis and induced the expression of inflammatory mediators, such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and IL-10. SMSP2-induced macrophage stimulation occurs through the activation of the mitogen-activated protein kinase (MAPK) signaling pathway. Moreover, macrophage surface complement receptor 3 (CR3) might play an important role in SMSP2-induced macrophage activation. This study revealed that SMSP2 is a potent immunomodulator, which could be used as a functional food and a pharmaceutical adjuvant in treating immune-compromising diseases.

Wound healing potential of forest honey for increasing TGF-β1 protein expression in palatoplasty: In-vivo and In-silico studies

Background: With negative side effects of a lengthy healing time and high complication rate, cleft palate surgery is employed to treat cleft palate, which is a common congenital anomaly. Honey is believed to help accelerate wound healing post-treatment. However, its effect on the increase in the transforming growth factor (TGF)-β1 expression after palate surgery is unclear. Objective: This study investigates the effect of forest honey on TGF-β1 protein expression levels in the wound healing palate model. Methods: This study evaluates the TGF-β1 protein expression of a palatoplasty wound model using enzyme-linked immunosorbent assay. For this purpose, a punch biopsy model with 30 Sprague-Dawley rats is treated with forest honey (TG), Aloclair Gel (PC), and distilled water (NC) for 3 days. Analysis of the TGF-β1 expression on day 4 is performed by statistical one-way analysis of variance and post hoc least significance difference, with a 0.05 significant P-value. Online website software helped to predict the effect of honey components on the TGF-β1 expression. Results: Protein levels of treatment group (T), negative control (NC), and positive control (PC) exhibit mean levels of 16.13 ± 1.06883 ng/L, 7.36 ± 0.16543 ng/L, and 15.03 ± 0.34221 ng/L, respectively. The differential expression T group exhibits a 2.19-fold change in TGF-β1 relative to the NC group and a 1.07-fold change in the PC group (P-value of 0.01). TGF expression in the PC group increases in comparison to that in the NC group by 2.04-fold (P-value of 0.01). In-silico analysis revealed that genistein promotes macrophage proliferation and activation via the increase in the TGF-β1 expression. Conclusion: In summary, forest honey can boost the TGF protein expression via genistein to increase macrophage proliferation and activation.

Toxoplasma gondii Infection Inhibits Histone Crotonylation to Regulate Immune Response of Porcine Alveolar Macrophages

Toxoplasma gondii (T. gondii) is an obligate intracellular parasite that can infect almost all warm-blooded animals, causing serious public health problems. Lysine crotonylation (Kcr) is a newly discovered posttranslational modification (PTM), which is first identified on histones and has been proved relevant to procreation regulation, transcription activation, and cell signaling pathway. However, the biological functions of histone crotonylation have not yet been reported in macrophages infected with T. gondii. As a result, a total of 1,286 Kcr sites distributed in 414 proteins were identified and quantified, demonstrating the existence of crotonylation in porcine alveolar macrophages. According to our results, identified histones were overall downregulated. HDAC2, a histone decrotonylase, was found to be significantly increased, which might be the executor of histone Kcr after parasite infection. In addition, T. gondii infection inhibited the crotonylation of H2B on K12, contributing on the suppression of epigenetic regulation and NF-κB activation. Nevertheless, the reduction of histone crotonylation induced by parasite infection could promote macrophage proliferation via activating PI3K/Akt signaling pathway. The present findings point to a comprehensive understanding of the biological functions of histone crotonylation in porcine alveolar macrophages, thereby providing a certain research basis for the mechanism research on the immune response of host cells against T. gondii infection.

Fractionation, chemical characterization and immunostimulatory activity of β-glucan and galactoglucan from Russula vinosa Lindblad

Water-extracted polysaccharides from Russula vinosa Lindblad (WRP) were separated into three fractions (WRP-1, WRP-2 and WRP-3) by gradient ethanol precipitation and gel chromatography. Structural characterization indicated that WRP-1 was a branched β-(1→3)-glucan and exhibited rigid helical conformation in aqueous solution with Mw of 2,180 kDa and radius of gyration (Rg) of 123.4 nm. The galactoglucan of WRP-2 and WRP-3 were mainly composed of →6)-Galp-(1→ and →4)-Glcp-(1→ terminated by glucose and mannose, presenting much lower Mw (392 and 93.6 kDa) and Rg (57.6 and 42.6 nm), and more incompact flexible conformation than WRP-1. All fractions showed potential immunostimulatory activity by promoting macrophage proliferation, phagocytosis, as well as the release of nitric oxide and cytokines (TNF-α and IL-1β). WRP-1 with unique structure and conformation showed the best immunostimulatory effects among them. This study suggests that WRP could be explored as natural immunostimulator used in the food and pharmaceutical industry.

Tumor-derived adenosine promotes macrophage proliferation in human hepatocellular carcinoma

Macrophages (Mϕ) represent a major component of tumor tissues and play an important role in both tumor progression and therapeutic response. Although tumor Mϕ are generally considered to be derived from circulating monocytes, emerging evidence indicates that tissue Mϕ pools can be maintained by self-renewal. We aimed to elucidate the contribution, phenotype, and regulatory mechanisms of proliferating Mϕ in human hepatocellular carcinoma (HCC). Flow cytometry analyses were performed to examine the presence and phenotype of proliferating Mϕ in fresh HCC tissues. Dual immunofluorescence staining was applied to analyze the prognostic value of proliferating Mϕ. The underlying regulatory mechanisms were examined using human monocyte-derived Mϕ. Tumor-infiltrating Mϕ exhibited a significantly higher proliferative capacity than Mϕ in non-tumor tissues. A higher level of Mϕ proliferation was positively correlated with Mϕ density in the tumor and a poor prognosis in patients with HCC. Proliferating Mϕ were less differentiated (with increased CD206 expression) and were induced by the tumor cell-derived soluble small molecule, adenosine, but not proteins, lipids, or large peptides. Mechanistic studies demonstrated that autocrine granulocyte-macrophage colony-stimulating factor (GM-CSF) released by tumor-stimulated Mϕ could enhance A2A receptor expression on Mϕ and function synergistically with adenosine to elicit Mϕ proliferation in HCC. Local Mϕ proliferation is an important mechanism for Mϕ accumulation in HCC tissues. Tumor-derived adenosine functions synergistically with autocrine GM-CSF released from activated Mϕ, which promotes Mϕ proliferation. Thus, selective modulation of Mϕ accumulation at the source may provide a novel strategy for cancer therapy. Tumor-associated macrophages (TAMs) have been reported to play an essential role in both tumor progression and therapeutic response. A fundamental understanding of the mechanisms that regulate macrophage accumulation in tumors will undoubtedly lead to the development of strategies to target macrophages with high specificity and efficiency. The current study unveils a novel mechanism by which local proliferation is linked to macrophage accumulation in the tumor milieu, identifying potential targets for future immune-based anticancer therapies.

Extracellular-Regulated Protein Kinase 5-Mediated Control of p21 Expression Promotes Macrophage Proliferation Associated with Tumor Growth and Metastasis.

The presence of immunosuppressive macrophages that become activated in the tumor microenvironment constitutes a major factor responsible for tumor growth and malignancy. In line with this knowledge, we report here that macrophage proliferation is a significant feature of advanced stages of cancer. Moreover, we have found that a high proportion of proliferating macrophages in human tumors express ERK5. ERK5 was required for supporting the proliferation of macrophages in tumor grafts in mice. Furthermore, myeloid ERK5 deficiency negatively impacted the proliferation of both resident and infiltrated macrophages in metastatic lung nodules. ERK5 maintained the capacity of macrophages to proliferate by suppressing p21 expression to halt their differentiation program. Collectively, these data provide insight into the mechanism underpinning macrophage proliferation to support malignant tumor development, thereby strengthening the value of ERK5-targeted therapies to restore antitumor immunity through the blockade of protumorigenic macrophage activation. SIGNIFICANCE: These findings offer a new rationale for anti-ERK5 therapy to improve cancer patient outcomes by blocking the proliferative activity of tumor macrophages.