Macrophage-conditioned Medium Research Articles

Immunomodulatory and osteogenic effects of chitosan-based injectable hydrogel with geniposide-loaded mesoporous bioactive glass

The immune response dominated by macrophages plays a pivotal role in the regeneration of bone tissue. In this work, an injectable temperature-responsive hydrogel composed of geniposide-loaded mesoporous bioactive glass, chitosan and β-glycerophosphate (G-M Gel) was prepared, showing robustly networks, uniform pore structure, excellent biocompatibility, immunomodulatory effect and osteogenic potential. In an inflammatory microenvironment elicited by lipopolysaccharide (LPS), the proportion of M1 and M2 macrophages measured by flow cytometry were 33.17 % and 2.07 %, respectively. After G-M Gel treatment, the proportion of M1 macrophages decreased to 14.4 %, while the proportion of M2 macrophages increased significantly to 16.2 %. LPS treated macrophage conditioned medium inhibited the expression of osteogenic related factors (OCN, OPN, Runx2), alkaline phosphatase (ALP) and alizarin red S (ARS) in MC3T3-E1 cells. In contrast, LPS + G-M Gel treated macrophage conditioned medium significantly increased the expression of osteogenic related factors, ALP and ARS. These results demonstrated that G-M Gel can augment bone formation by promoting the polarization of M2 macrophages, showing great potential clinical application of G-M Gel in bone regeneration field.

Overexpressed nicotinamide N‑methyltransferase in endometrial stromal cells induced by macrophages and estradiol contributes to cell proliferation in endometriosis

Endometriosis, an estrogen-dependent chronic inflammatory condition, afflicts reproductive-aged women. However, the underlying pathological mechanisms remain to be elucidated. Nicotinamide N-methyltransferase (NNMT) is a critical enzyme involved in cellular metabolism and methylation regulation. This study investigated the role of NNMT in endometriosis. By analyzing datasets GSE5108, GSE7305, GSE141549, GSE23339, and GSE25628, we identified a significant overexpression of NNMT in the eutopic endometrium and ectopic lesions of endometriosis patients compared to normal endometrium. Furthermore, NNMT was upregulated in collected endometrioma specimens and isolated primary endometrial stromal cells (ESCs) compared to their respective controls. Inhibition of NNMT using JBSNF-000088 attenuated the proliferation, migration, and invasion of ESCs. In vivo, treatment of mouse models of endometriosis with JBSNF-000088 resulted in a marked reduction in lesion weight and quantity. NNMT expression in ESCs was dose-dependently upregulated by 17β-estradiol at concentrations of 1 nM, 10 nM, and 100 nM, an effect that was attenuated by 10 nM progesterone. Additionally, treating HESCs with macrophage-conditioned medium elevated NNMT expression at both mRNA and protein levels. Knockdown of NNMT impeded the proliferation, migration, and invasion of ESCs, which was paralleled by decreased phosphorylation levels of Erb-b2 receptor tyrosine kinase 4 (ERBB4), PI3K, and AKT. Conversely, overexpressing ERBB4 mitigated the NNMT knockdown-induced decline in phosphorylated PI3K and AKT and rescued the proliferation of ESCs. Altogether, these results indicate that the overexpression of NNMT induced by estrogen and macrophage interaction modulates ESC proliferation via the NNMT-ERBB4-PI3K/AKT signaling pathway, as well as promotes cellular migration and invasion, contributing to the development of endometriosis.

Effect and mechanism of Adipokine Omentin-1 on cardiac hypertrophy in heart failure with preserved ejection fraction

Abstract Background Omentin-1 secreted by epicardial adipose tissue can inhibit ventricular remodeling and improve early cardiac function after myocardial infarction. Our previous studies have identified the omentin-1 receptor as integrin receptors αvβ3 and αvβ5. However, the effect of omentin-1 on heart failure with preserved ejection fraction (HFpEF) remains poorly understood. Purpose The present study aims to study the role and mechanism of omentin-1 in cardiac function and myocardial cell structure in HFpEF. Methods and Results The influences of omentin-1 were investigated in HFpEF mice (HFD (60% calories from lard) and L-NAME (0.5 g/L in drinking water)), which were treated in vivo. Echocardiography showed better diastolic function in HFpEF mice treated with omentin-1 (5 ug/kg/h) subcutaneously for 4 weeks, and tissue staining showed alleviated myocardial fibrosis and myocardial hypertrophy. The RNA sequencing analysis revealed variations in the gene expression related to ketone body metabolism in HFpEF mice after omentin-1 treatment (600 ng/ml). The supernatant was obtained from mouse bone marrow macrophages treated with ultrapure endotoxin and ATP and was utilized as the macrophage-conditioned medium for culturing primary cardiomyocytes, which were stimulated with isoproterenol for 48 hours to construct the HFpEF cardiomyocyte model. In the heart tissue of HFpEF mice and HFpEF cardiomyocytes treated with omentin-1, there was a notable increase in the expression of a pivotal enzyme of ketone body metabolism- succinyl-CoA transferase (SCOT), and a decrease in the expression of hypertrophy signal. The myocardial hypertrophy model was established by stimulating the myocardial cell line H9C2 with isoproterenol. Following the inhibition of the integrin receptor and PI3K-AKT pathway, the impacts of omentin-1 on SCOT and myocardial hypertrophy signal expression in hypertrophic cardiomyocytes were eliminated. Conclusion This study provides evidence for the effects of omentin-1 on cardiac function and myocardial structure in HFpEF and proposes that omentin-1 may stimulate the downstream PI3K/AKT pathway via integrin receptors on cardiomyocytes, leading to increased expression of SCOT and improvement of cardiomyocyte hypertrophy.

Direct M2 macrophage co-culture overrides viscoelastic hydrogel mechanics to promote fibroblast activation.

Fibroblast activation drives fibrotic diseases such as pulmonary fibrosis. However, the complex interplay of how tissue mechanics and macrophage signals combine to influence fibroblast activation is not well understood. Here, we use hyaluronic acid hydrogels as a tunable cell culture system to mimic lung tissue stiffness and viscoelasticity. We applied this platform to investigate the influence of macrophage signaling on fibroblast activation. Fibroblasts cultured on stiff (50 kPa) hydrogels mimicking fibrotic tissue exhibit increased activation as measured by spreading as well as type I collagen and cadherin-11 expression compared to fibroblasts cultured on soft (1 kPa) viscoelastic hydrogels mimicking normal tissue. These trends were unchanged in fibroblasts cultured with macrophage-conditioned media. However, fibroblasts directly co-cultured with M2 macrophages show increased activation, even on soft viscoelastic hydrogels that normally suppress activation. Inhibition of interleukin 6 (IL6) signaling does not change activation in fibroblast-only cultures but ameliorates the pro-fibrotic effects of M2 macrophage co-culture. These results underscore the ability of direct M2 macrophage co-culture to override hydrogel viscoelasticity to promote fibroblast activation in an IL6-dependent manner. This work also highlights the utility of using hydrogels to deconstruct complex tissue microenvironments to better understand the interplay between microenvironmental mechanical and cellular cues.

Calenduloside E Ameliorates Inflammatory Responses in Adipose Tissue via Sirtuin 2-NLRP3 Inflammasome Axis.

Obesity-related metabolic diseases are associated with a chronic inflammatory state. Calenduloside E (CE) is a triterpene saponin from sugar beet. In mouse models, CE reduced pro-inflammatory cytokines in white adipose tissue (WAT) and decreased macrophage infiltration of WAT. And CE inhibited pyroptosis in J774A.1 cells and WAT by inhibiting the activation of the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3) inflammasome. Moreover, CE could trigger the activation of Sirtuin 2 (SIRT2), leading to a decrease in the acetylation of NLRP3, particularly at the K24 site. In addition, it has been shown that CE can reduce inflammation in adipocytes that have been induced by macrophage-conditioned medium. However, the selective SIRT2 inhibitor AGK2 hindered the beneficial effects of CE. In summary, CE has the capacity to impede NLRP3-mediated pyroptosis by triggering SIRT2 activity, thus positioning CE as a promising therapeutic avenue for combating obesity-related metabolic disorders.

Metformin enhances osteogenic differentiation of BMSC by modulating macrophage M2 polarization

Bone marrow-derived mesenchymal stem cells (BMSCs) are capable of developing into osteoblastic cell lines in vitro and regenerating bone tissue in vivo, and they are considered to be a reliable source for bone regenerative medicine. In recent years, studies have shown that the immune microenvironment is important for osteogenesis, in which macrophages are an important component of innate immunity and coordinate with stem cells. Metformin (Met), a hypoglycemic drug that exerts a powerful effect on metabolic signaling, has been shown to modulate inflammatory responses and osteogenic activity. However, whether metformin modulates macrophage polarization and subsequently affects osteogenesis remains to be elucidated. In the present study, we investigated the potential immunomodulatory capacity of metformin on macrophage inflammatory responses and phenotypic switching, and the subsequent effects on osteogenic differentiation of BMSCs. Flow cytometry and qPCR were used to study the effects of metformin on macrophage phenotypic regulation. qPCR, ALP, ARS and calcium content measurement and ALP activity assay were used to determine the effects of macrophage-secreted activators on the osteogenic differentiation of BMSCs. Our study demonstrates that metformin can improve the immune microenvironment by modulating macrophage polarization towards an anti-inflammatory phenotype, promoting an increase in a range of anti-inflammatory factors and inhibiting pro-inflammatory factors. This was characterized by increased expression of IL-10 and CD206, Arg-1 and decreased expression of IL-1β, TNF-α, IL-6 and iNOS. In addition, metformin-modulated macrophage-conditioned medium promoted osteogenic differentiation of BMSCs, increased the expression levels of genes (ALP, Runx-2, OCN, and Col-1), enhanced ALP activity, and significantly formed mineralized nodules. In conclusion, our new study elucidates that metformin can promote osteogenic differentiation of BMSCs by modulating macrophage phenotype and thereby.

The effect of compressive force in bone tissue induced by implants of different primary stabilities on macrophage polarization and bone regeneration

Dental implants with different primary stabilities give rise to distinct stress distributions at the implant-bone interface after placement and exert mechanical force on the cells in the bone tissue. This study aimed to investigate whether the mechanical forces in peri-implant bone participate in the body’s immune response and influence macrophage polarization. Therefore, an in vivo rat implantation model with different primary implant stabilities was established. The osteoimmune response and macrophage polarization were investigated, and the osseointegration of the implants was evaluated. In an in vitro experiment, an external compressive force was applied to RAW264.7 cells, and the polarization phenotype was observed. MC3T3-E1 cells were cultured in macrophage-conditioned medium to investigate the regulatory effect of the macrophage-secreted cytokines on the osteogenic differentiation of osteoblasts. In vivo experimental results indicated that the primary stability of implants is positively correlated with the mechanical force. The osteoimmune response was significantly amplified by compressive force generated from implants. This compressive force first induced both M1 and M2 macrophage polarization and then accelerated the progression of the transition to M2 macrophages in the bone repair phase. In vitro, compressive force significantly upregulated the M1 and M2 macrophage polarization. In addition, the suppressive effect of macrophages on the osteogenesis of MC3T3 cells was relieved by cytokines secreted by macrophages under compressive force loading, which promoted their osteogenesis. Overall, these results clarify that compressive force from different primary stabilities is an important influencing factor regulating the osteoimmunne response and macrophage polarization in addition to maintaining the implant.

Macrophage-induced Expression of TonEBP/NFAT5 Is Associated With Gefitinib Resistance and Migration in PC-9 Cells.

Macrophages prevail in the microenvironment of several tumors, including non-small-cell lung cancer (NSCLC), where they secrete pro-tumorigenic factors that contribute to cancer progression. This study investigated the role of macrophages on the resistance of epidermal growth factor receptor (EGFR)-mutated NSCLC cells to tyrosine kinase inhibitors (TKIs). EGFR-mutated cell lines PC-9 and HCC827 were cocultured with macrophages and treated with TKIs (erlotinib and gefitinib). The effects of the macrophage-conditioned medium (macrophage CM) on gefitinib resistance and cell migration were also evaluated. Co-culture with macrophages significantly enhanced the resistance to erlotinib and gefitinib in PC-9 and HCC827 cells compared to that in cells cultured independently. Macrophage CM markedly induced gefitinib resistance in PC-9 cells, with maximum resistance observed at 50% CM concentration. This resistance persisted for up to 48 h post-CM removal. Macrophage CM inhibited gefitinib-induced apoptosis, as evidenced by the decreased expression of cleaved caspase-3, PARP, and BIM. Additionally, macrophage CM increased the migration ability of PC-9 cells, as shown by the wound healing and transwell migration assays. Studies have shown that TonEBP is crucial in cancer metastasis and drug resistance; we found that inhibition of TonEBP/NFAT5 expression reduced gefitinib resistance and migration in macrophage CM-induced PC-9 cells, indicating its role as mediator of these effects. Macrophages contribute to TKI resistance and enhance the migration of EGFR-mutated NSCLC cells through mechanisms involving TonEBP/NFAT5. Therefore, targeting TonEBP/NFAT5 represents a potential therapeutic strategy for overcoming macrophage-induced TKI resistance in NSCLC cells.

Periplocin improves the sensitivity of oxaliplatin-resistant hepatocellular carcinoma cells by inhibiting M2 macrophage polarization.

The aim of this research was to investigate the impact of periplocin (PPLN) on oxaliplatin (OXA) resistance in hepatocellular carcinoma (HCC) cells and offer insights for improving clinical treatment of HCC. The IC50 value of HCC cell lines against OXA was detected by the CCK-8 assay, and an OXA-resistant HepG2 cell line (HepG2/OXA) was constructed. THP-1 cells were induced into M1 or M2 macrophages, and M2 macrophage-conditioned medium (M2-CM) was prepared. M1 and M2 macrophage polarization were detected using RT-qPCR and flow cytometry. CCK-8, EdU staining, clone formation assay, flow cytometry, and western blotting were used to assess the proliferation and apoptosis of HepG2/OXA cells treated with PPLN and M2-CM. Additionally, a nude mouse subcutaneous graft tumor model was constructed. PPLN enhanced the sensitivity of HepG2/OXA cells to OXA, reduced their clone-forming ability, and promoted their apoptosis. Notably, PPLN hindered M0 macrophage polarization to M2 macrophages, while M1 polarization remained unaffected. The proliferation-inhibiting and apoptosis-promoting effects of OXA+PPLN on HepG2/OXA cells were significantly attenuated by the addition of M2-CM, suggesting that PPLN improves the OXA sensitivity of HepG2/OXA cells by hindering M2 macrophage polarization. Furthermore, PPLN inhibited M2 macrophage polarization and improved the OXA sensitivity of HepG2/OXA cells in vivo. In conclusion, PPLN inhibited the proliferation of HepG2/OXA cells, promoted their apoptosis, and inhibited M2 macrophage polarization both in vivo and in vitro, which in turn enhanced the OXA sensitivity of HepG2/OXA cells.

Investigation of the Relationship between Surface Features, Protein Adsorption, and Osteoimmunomodulation: The Case of a Chemically Treated Titanium Alloy.

This paper deals with the combined effects of immune response and osseointegration because of the lack of comprehensive studies on this topic. An antibacterial Ti surface was considered because of the high risk of infection for titanium bone implants. A chemically treated Ti6Al4 V alloy [Ti64(Sr-Ag)] with a microporous and Sr-Ag doped surface was compared to a polished version (Ti64) regarding protein adsorption (albumin and fibronectin) and osteoimmunomodulation. Characterization via fluorescence microscopy and zeta potential showed a continuous fibronectin layer on Ti64(Sr-Ag), even with preadsorbed albumin, while it remained filamentous on Ti64. Macrophages (differentiated from THP-1 monocytes) were cultured on both surfaces, with viability and cytokine release analyzed. Differently from Ti64, Ti64(Sr-Ag) promoted early anti-inflammatory responses and significant downregulation of VEGF. Ti64(Sr-Ag) also enhanced human bone marrow mesenchymal cell differentiation toward osteoblasts, when a macrophage-conditioned medium was used, influencing ALP production. Surface properties in relation to protein adsorption and osteoimmunomodulation were discussed.

N-3 and n-6 Polyunsaturated Fatty Acids Modulate Macrophage-Myocyte Inflammatory Crosstalk and Improve Myocyte Insulin Sensitivity.

In obesity, circulating saturated fatty acids (SFAs) and inflammatory cytokines interfere with skeletal muscle insulin signaling, leading to whole body insulin resistance. Further, obese skeletal muscle is characterized by macrophage infiltration and polarization to the inflammatory M1 phenotype, which is central to the development of local inflammation and insulin resistance. While skeletal muscle-infiltrated macrophage-myocyte crosstalk is exacerbated by SFA, the effects of other fatty acids, such as n-3 and n-6 polyunsaturated fatty acids (PUFAs), are less studied. Thus, the objective of this study was to determine the effects of long-chain n-3 and n-6 PUFAs on macrophage M1 polarization and subsequent effects on myocyte inflammation and metabolic function compared to SFA. Using an in vitro model recapitulating obese skeletal muscle cells, differentiated L6 myocytes were cultured for 24 h with RAW 264.7 macrophage-conditioned media (MCM), followed by insulin stimulation (100 nM, 20 min). MCM was generated by pre-treating macrophages for 24 h with 100 μM palmitic acid (16:0, PA-control), arachidonic acid (20:4n-6, AA), or docosahexaenoic acid (22:6n-3, DHA). Next, macrophage cultures were stimulated with a physiological dose (10 ng/mL) of lipopolysaccharide for an additional 12 h to mimic in vivo obese endotoxin levels. Compared to PA, both AA and DHA reduced mRNA expression and/or secreted protein levels of markers for M1 (TNFα, IL-6, iNOS; p < 0.05) and increased those for M2 (IL-10, TGF-β; p < 0.05) macrophage polarization. In turn, AA- and DHA-derived MCM reduced L6 myocyte-secreted cytokines (TNFα, IL-6; p < 0.05) and chemokines (MCP-1, MIP-1β; p < 0.05). Only AA-derived MCM increased L6-myocyte phosphorylation of Akt (p < 0.05), yet this was inconsistent with improved insulin signaling, as only DHA-derived MCM improved L6 myocyte glucose uptake (p < 0.05). In conclusion, dietary n-3 and n-6 PUFAs may be a useful strategy to modulate macrophage-myocyte inflammatory crosstalk and improve myocyte insulin sensitivity in obesity.

Macrophage-conditioned medium enhances tunneling nanotube formation in breast cancer cells via PKC, Src, NF-κB, and p38 MAPK signaling

Tumor-associated macrophages (TAMs) secrete cytokines, chemokines, and growth factors in the tumor microenvironment (TME) to support cancer progression. Higher TAM infiltration in the breast TME is associated with a poor prognosis. Previous studies have demonstrated the role of macrophages in stimulating long-range intercellular bridges referred to as tunneling nanotubes (TNTs) in cancer cells. Intercellular communication between cancer cells via TNTs promotes cancer growth, invasion, metastasis, and therapy resistance. Given the important role of TNTs and macrophages in cancer, the role of macrophage-induced TNTs in chemotherapy drug doxorubicin resistance is not known. Furthermore, the mechanism of macrophage-mediated TNT formation is elusive. In this study, it is shown that the macrophage-conditioned medium (MΦCM) partially mimicked inflammatory TME, induced an EMT phenotype, and increased migration in MCF-7 breast cancer cells. Additionally, secreted proteins in MΦCM induced TNT formation in MCF-7 cells, which led to increased resistance to doxorubicin. Transcriptomic analysis of MΦCM-treated MCF-7 cells showed enrichment of the NF-κB and focal adhesion pathways, as well as upregulation of genes involved in EMT, extracellular remodeling, and actin cytoskeleton reorganization. Interestingly, inhibitors of PKC, Src, NF-κB, and p38 decreased macrophage-induced TNT formation in MCF-7 cells. These results reveal the novel role of PKC and Src in inducing TNT formation in cancer cells and suggest that inhibition of PKC and Src activity may likely contribute to reduced macrophage-breast cancer cell interaction and the potential therapeutic strategy of cancer.

Hyperbaric Oxygen Therapy as a Novel Approach to Modulating Macrophage Polarization for the Treatment of Glioblastoma.

Glioblastoma multiforme (GBM) is a highly aggressive brain cancer with a poor prognosis despite current treatments. This is partially attributed to the immunosuppressive environment facilitated by tumor-associated macrophages, which predominantly underlie the tumor-promoting M2 phenotype. This study investigated the potential of hyperbaric oxygen (HBO) therapy, traditionally used to treat conditions such as decompression sickness, in modulating the macrophage phenotype toward the tumoricidal M1 state and disrupting the supportive tumor microenvironment. HBO has direct antiproliferative effects on tumor cells and reduces hypoxia, which may impair angiogenesis and tumor growth. This offers a novel approach to GBM treatment by targeting the role of the immune system within the tumor microenvironment. The effects of HBO on macrophage polarization and GBM cell viability and apoptosis were evaluated in this study. We detected that HBO promoted M1 macrophage cytokine expression while decreasing GBM cell viability and increasing apoptosis using GBM cell lines and THP-1-derived macrophage-conditioned media. These findings suggest that HBO therapy can shift macrophage polarization toward a tumoricidal M1 state. This can improve GBM cell survival and offers a potential therapeutic strategy. In conclusion, HBO can shift macrophages from a tumor-promoting M2 phenotype to a tumoricidal M1 phenotype in GBM. This can facilitate apoptosis and, in turn, improve treatment outcomes.

Modulation of macrophage transcript and secretion profiles by Sargassum Serratifolium extract is associated with the suppression of muscle atrophy

Recent research has emphasized the role of macrophage-secreted factors on skeletal muscle metabolism. We studied Sargassum Serratifolium ethanol extract (ESS) in countering lipopolysaccharide (LPS)-induced changes in the macrophage transcriptome and their impact on skeletal muscle. Macrophage-conditioned medium (MCM) from LPS-treated macrophages (LPS-MCM) and ESS-treated macrophages (ESS-MCM) affected C2C12 myotube cells. LPS-MCM upregulated muscle atrophy genes and reduced glucose uptake, while ESS-MCM reversed these effects. RNA sequencing revealed changes in the immune system and cytokine transport pathways in ESS-treated macrophages. Protein analysis in ESS-MCM showed reduced levels of key muscle atrophy-related proteins, TNF-α, IL-6, IL-1, and GDF-15. These proteins play crucial roles in muscle function. These findings highlight the intricate relationship between the macrophage transcriptome and their secreted factors in either impairing or enhancing skeletal muscle function. ESS treatment has the potential to reduce macrophage-derived cytokines, preserving skeletal muscle function.

Intranasal Immunotherapy with M2 Macrophage Secretome Ameliorates Language Impairments and Autistic-like Behavior in Children.

Background/Objectives: The intranasal delivery of various neurotropic substances is considered a new attractive therapeutic approach for treating neuropathologies associated with neuroinflammation and altered regeneration. Specific language impairment (SLI) that arises as a result of damage to the cortical speech zones during the developmental period is one of the most common problems in preschool children, and it is characterized by persistent difficulties in the acquisition, understanding, and use of language. This study's objective is to evaluate the efficacy and safety of intranasal immunotherapy using the M2 macrophage secretome as a rich source of immunoregulatory and neurotrophic factors for the treatment of severe language impairment in children. Methods: Seventy-one children (54 boys and 17 girls, aged 3 to 13 years) were recruited to participate in a clinical trial (NCT04689282) in two medical centers. The children were examined before, 1 month after, and 6 months after the start of therapy. In the vast majority of children (55/71), language impairment was associated with autistic-like symptoms and attention deficit hyperactivity disorder (ADHD). Results: Daily intranasal inhalations of M2 macrophage-conditioned medium (for 30 days) were well tolerated and led to a decrease in the severity of language impairments, autistic-like behavior, and ADHD symptoms. The clinical effect appeared within a month after the first procedure and persisted or intensified during a 6-month follow-up. Two-thirds of the children showed a clear clinical improvement, while the rest had less pronounced improvement. Conclusions: Thus, the use of the M2 macrophage secretome and its intranasal delivery is safe, well tolerated, and clinically effective in children with severe language impairments.

Clinical suitability of intranasal delivery of M2 macrophage soluble factors in patients with post-COVID olfactory disorders

IntroductionSARS-CoV virus showed transneuronal penetration through the olfactory bulb resulting in the rapid intracranial spread. So, olfactory dysfunction is an early marker of COVID-19 infection. However, individuals may develop chronic olfactory impairment for more than six months in 1–10% of cases.ObjectivesThe study’s objective was to evaluate the efficacy and safety of intranasal immunotherapy using bioactive substances produced by M2 macrophages for the treatment of people with long-term post-COVID-19 hyposmia.MethodsSeven individuals with long-term persistent hyposmia (7 to 24 months), associated with PCR-confirmed coronavirus infection were evaluated for olfactory function at baseline, one, and six to twelve months after therapy.ResultsThe intranasal inhalation of M2 macrophage conditioned medium (one time per day for 28-30 days) was well tolerated. Furthermore, olfactometry demonstrated that the patients restored their capacity to perceive (Kruskal-Wallis H test 14.123, p = 0.0009) and recognize odors (H = 11.674, p = 0.0029). In addition, the subjective evaluation of smell significantly improved (H = 11.935, p = 0.0026). At the 6- to 12-month follow-up, the majority of patients (5/7) reported extremely high levels of satisfaction with the outcomes, and the remaining two patients also felt generally positive about the therapy’s success.ConclusionsOverall, our study showed that the use of intranasal inhalations as a method of delivering bioactive factors and the conditioned medium of M2 macrophages as a therapeutic agent are both safe, well tolerated and, according to preliminary data, clinically effective in the treatment of patients with long-term post-COVID-19 hyposmia.Disclosure of InterestNone Declared

Macrophages suppress cardiac reprogramming of fibroblasts in vivo via IFN-mediated intercellular self-stimulating circuit.

Direct conversion of cardiac fibroblasts (CFs) to cardiomyocytes (CMs) in vivo to regenerate heart tissue is an attractive approach. After myocardial infarction (MI), heart repair proceeds with an inflammation stage initiated by monocytes infiltration of the infarct zone establishing an immune microenvironment. However, whether and how the MI microenvironment influences the reprogramming of CFs remains unclear. Here, we found that in comparison with cardiac fibroblasts (CFs) cultured in vitro, CFs that transplanted into infarct region of MI mouse models resisted to cardiac reprogramming. RNA-seq analysis revealed upregulation of interferon (IFN) response genes in transplanted CFs, and subsequent inhibition of the IFN receptors increased reprogramming efficiency in vivo. Macrophage-secreted IFN-β was identified as the dominant upstream signaling factor after MI. CFs treated with macrophage-conditioned medium containing IFN-β displayed reduced reprogramming efficiency, while macrophage depletion or blocking the IFN signaling pathway after MI increased reprogramming efficiency in vivo. Co-IP, BiFC and Cut-tag assays showed that phosphorylated STAT1 downstream of IFN signaling in CFs could interact with the reprogramming factor GATA4 and inhibit the GATA4 chromatin occupancy in cardiac genes. Furthermore, upregulation of IFN-IFNAR-p-STAT1 signaling could stimulate CFs secretion of CCL2/7/12 chemokines, subsequently recruiting IFN-β-secreting macrophages. Together, these immune cells further activate STAT1 phosphorylation, enhancing CCL2/7/12 secretion and immune cell recruitment, ultimately forming a self-reinforcing positive feedback loop between CFs and macrophages via IFN-IFNAR-p-STAT1 that inhibits cardiac reprogramming in vivo. Cumulatively, our findings uncover an intercellular self-stimulating inflammatory circuit as a microenvironmental molecular barrier of in situ cardiac reprogramming that needs to be overcome for regenerative medicine applications.

A modified system using macrophage-conditioned medium revealed that the indirect effects of anti-inflammatory food-derived compounds improve inflammation-induced suppression of UCP-1 mRNA expression in 10T1/2 adipocytes.

Recently, it has been suggested that brown and beige adipocytes may ameliorate obesity because these adipocytes express uncoupling protein-1 (UCP-1), which generates heat by consuming lipid. However, obesity-induced inflammation suppresses the expression of UCP-1. To improve such conditions, food components with anti-inflammatory properties are attracting attention. In this study, we developed a modified system to evaluate only the indirect effects of anti-inflammatory food-derived compounds by optimizing the conventional experimental system using conditioned medium. We validated this new system using 6-shogaol and 6-gingerol, which have been reported to show the anti-inflammatory effects and to increase the basal expression of UCP-1 mRNA. In addition, we found that the acetone extract of Sarcodon aspratus, an edible mushroom, showed anti-inflammatory effects and rescued the inflammation-induced suppression of UCP-1 mRNA expression. These findings indicate that the system with conditioned medium is valuable for evaluation of food-derived compounds with anti-inflammatory effects on the inflammation-induced thermogenic adipocyte dysfunction.

TonEBP/NFAT5 expression is associated with cisplatin resistance and migration in macrophage-induced A549 cells

BackgroundMacrophages promote angiogenesis, metastasis, and drug resistance in several cancers. Similarly, TonEBP/NFAT5 induces metastasis in renal carcinoma and colon cancer cells. However, the role of this transcription factor and that of macrophages in lung cancer cells remains unclear. Therefore, this study investigated the effects of macrophages and TonEBP/NFAT5 expression on cisplatin resistance and migration in A549 lung adenocarcinoma cells.ResultsA549 cells were cultured alone or indirectly co-cultured with THP-1-derived macrophages using a transwell culture chamber. Cisplatin-induced cell death was markedly decreased and migration increased in co-cultured A549 cells. Macrophage-conditioned media (CM) showed a similar effect on drug resistance and migration. Cisplatin-induced apoptosis, DNA fragmentation, and cleaved apoptotic proteins PARP and caspase-3 were markedly reduced in macrophage CM-induced A549 cells. Here, ERK, p38, JNK, and NF-κB activities were increased by macrophage CM. Furthermore, the proteins involved in cisplatin resistance and cancer cell migration were identified using specific inhibitors of each protein. ERK and NF-κB inhibition considerably reduced cisplatin resistance. The increase in macrophage CM-induced migration was partially reduced by treatment with ERK, JNK, and NF-κB inhibitors. TonEBP/NFAT5 expression was increased by macrophages, resulting in increased cisplatin resistance, cell migration, and invasion. Moreover, RNAi-mediated knockdown of TonEBP/NFAT5 reduced cisplatin resistance, migration, and invasion in macrophage CM-induced A549 cells.ConclusionsThese findings demonstrate that paracrine factors secreted from macrophages can change A549 cells, resulting in the induction of drug resistance against cisplatin and migration. In addition, the TonEBP/NFAT5 ratio, increased by macrophages, is an important regulator of the malignant transformation of cells.

Sini decoction alleviates inflammation injury after myocardial infarction through regulating arachidonic acid metabolism

ObjectiveMyocardial inflammation during myocardial infarction (MI) could be inhibited by regulating arachidonic acid (AA) metabolism. Recent studies demonstrated that Sini Decoction (SND) was identified to be an effective prescription for treating heart failure (HF) caused by MI. But the anti-inflammatory mechanism of SND remained unclear. The work was designed to investigate the anti-inflammatory mechanism of SND through the AA metabolism pathway in vitro and in vivo experiments. MethodsAn inflammatory injury model of H9c2 cells was established by lipopolysaccharide (LPS)-stimulated macrophage-conditioned medium (CM). The MI model was built by the ligation of left anterior descending (LAD) branch of coronary artery in rat. Meanwhile, the rats were divided into five groups: sham group, MI group, MI + Celecoxib group, MI + low-dose SND group (SND-L) and MI + high-dose SND group (SND-H). Cardiac function, histopathological changes and serum cytokines were examined four weeks later. Western blot analysis was conducted to verify the key enzymes levels in the AA metabolic pathway, including phospholipase A2 (PLA2), cyclooxygenases (COXs) and lipoxygenases (LOXs). ResultsThese in vivo results demonstrated that SND could improve the cardiac function and pathological changes of rats with MI, and regulate the key inflammatory molecules in the AA metabolism pathway, including sPLA2, COX-1, COX-2, 5-LOX and 15-LOX. In vitro, SND could decrease the release of pro-inflammatory cytokines including TNF-α and IL-6 and inhibit cell apoptosis in CM-induced H9c2 cells. Moreover, SND could protect H9c2 cells from the damage of CM by regulating nuclear factor kappa-B (NF-κB) signal pathway and the expression of COX-2. ConclusionSND may be a drug candidate for anti-inflammatory treatment during MI by regulating the multiple targets in the AA metabolism pathway.