Effects of temporal IFNγ exposure on macrophage phenotype and secretory profile: exploring GMP-Compliant production of a novel subtype of regulatory macrophages (MregIFNγ0) for potential cell therapeutic applications.

BACKGROUND: Macrophages are involved in tissue homeostasis, angiogenesis and immunomodulation. Proangiogenic and anti-inflammatory macrophages (regulatory macrophages, Mreg) can be differentiated in-vitro from CD14+ monocytes by using a defined cell culture medium and a stimulus of IFNγ. AIM OF THE STUDY: To scrutinize the potential impact of temporal IFNγ exposure on macrophage differentiation as such exposure may lead to the emergence of a distinct and novel macrophage subtype. METHODS: Differentiation of human CD14+ monocytes to Mreg was performed using a GMP compliant protocol and administration of IFNγ on day 6. Monocytes from the same donor were in parallel differentiated to MregIFNγ0 using the identical protocol but with administration of IFNγ on day 0. Cell characterization was performed using brightfield microscopy, automated and metabolic cell analysis, transmission electron microscopy, flow cytometry, qPCR and secretome profiling. RESULTS: Mreg and MregIFNγ0 showed no differences in cell size and volume. However, phenotypically MregIFNγ0 exhibited fewer intracellular vesicles/vacuoles but larger pseudopodia-like extensions. MregIFNγ0 revealed reduced expression of IDO and PD-L1 (P < 0.01 for both). They were positive for CD80, CD14, CD16 and CD38 (P < 0.0001vs. Mreg for all), while the majority of MregIFNγ0 did not express CD206, CD56, and CD103 on their cell surface (P < 0.01 vs. Mreg for all). In terms of their secretomes, MregIFNγ0 differed significantly from Mreg. MregIFNγ0 media exhibited reduced levels of ENA-78, Osteopontin and Serpin E1, while the amounts of MIG (CXCL9) and IP10 were increased. CONCLUSION: Exposing CD14+ monocytes to an alternatively timed IFNγ stimulation results in a novel macrophage subtype which possess additional M1-like features (MregIFNγ0). MregIFNγ0 may therefore have the potential to serve as cellular therapeutics for clinical applications beyond those covered by M2-like Mreg, including immunomodulation and tumor treatment.

Gut microbially produced tryptophan metabolite melatonin ameliorates osteoporosis via modulating SCFA and TMAO metabolism.

Osteoporosis (OP) is a severe global health issue that has significant implications for productivity and human lifespan. Gut microbiota dysbiosis has been demonstrated to be closely associated with OP progression. Melatonin (MLT) is an important endogenous hormone that modulates bone metabolism, maintains bone homeostasis, and improves OP progression. Multiple studies indicated that MLT participates in the regulation of intestinal microbiota and gut barrier function. However, the promising effects of gut microbiota-derived MLT in OP remain unclear. Here, we found that OP resulted in intestinal tryptophan disorder and decreased the production of gut microbiota-derived MLT, while administration with MLT could mitigate OP-related clinical symptoms and reverse gut microbiota dysbiosis, including the diversity of intestinal microbiota, the relative abundance of many probiotics such as Allobaculum and Parasutterella, and metabolic function of intestinal flora such as amino acid metabolism, nucleotide metabolism, and energy metabolism. Notably, MLT significantly increased the production of short-chain fatty acids and decreased trimethylamine N-oxide-related metabolites. Importantly, MLT could modulate the dynamic balance of M1/M2 macrophages, reduce the serum levels of pro-inflammatory cytokines, and restore gut-barrier function. Taken together, our results highlighted the important roles of gut microbially derived MLT in OP progression via the "gut-bone" axis associated with SCFA metabolism, which may provide novel insight into the development of MLT as a promising drug for treating OP.

Tolerogenic probiotics Lactobacillus delbrueckii and Lactobacillus rhamnosus promote anti-inflammatory profile of macrophages-derived monocytes of newly diagnosed patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is known as an autoimmune disorder that is characterized by the breakdown of self-tolerance, resulting in disease onset and progression. Macrophages have been implicated as a factor in the development of SLE through faulty phagocytosis of dead cells or an imbalanced M1/M2 ratio. The study aimed to investigate the immunomodulatory effects of Lactobacillus delbrueckii and Lactobacillus rhamnosus on M1 and M2 macrophages in new case lupus patients. For this purpose, blood monocytes were collected from lupus patients and healthy people and were cultured for 5 days to produce macrophages. For 48 h, the macrophages were then cocultured with either probiotics or lipopolysaccharides (LPS). Flow cytometry and real-time polymerase chain reaction were then used to analyze the expression of cluster of differentiation (CD) 14, CD80, and human leukocyte antigen - DR (HLADR) markers, as well as cytokine expression (interleukin [IL]1-ß, IL-12, tumor necrosis factor α [TNF-α], IL-10, and transforming growth factor beta [TGF-ß]). The results indicated three distinct macrophage populations, M0, M1, and M2. In both control and patient-derived macrophage-derived monocytes (MDMs), the probiotic groups showed a decrease in CD14, CD80, and HLADR expression compared to the LPS group. This decrease was particularly evident in M0 and M2 macrophages from lupus patients and M1 macrophages from healthy subjects. In addition, the probiotic groups showed increased levels of IL-10 and TGF-ß and decreased levels of IL-12, IL1-ß, and TNF-α in MDMs from both healthy and lupus subjects compared to the LPS groups. Although there was a higher expression of pro-inflammatory cytokines in lupus patients, there was a higher expression of anti-inflammatory cytokines in healthy subjects. In general, L. delbrueckii and L. rhamnosus could induce anti-inflammatory effects on MDMs from both healthy and lupus subjects.

Inflammasome Coordinates Senescent Chronic Wound Induced by Thalassophryne nattereri Venom.

Thalassophryne nattereri toadfish (niquim) envenomation, common in the hands and feet of bathers and fishermen in the north and northeast regions of Brazil, is characterized by local symptoms such as immediate edema and intense pain. These symptoms progress to necrosis that lasts for an extended period of time, with delayed healing. Wound healing is a complex process characterized by the interdependent role of keratinocytes, fibroblasts, and endothelial and innate cells such as neutrophils and macrophages. Macrophages and neutrophils are actively recruited to clear debris during the inflammatory phase of wound repair, promoting the production of pro-inflammatory mediators, and in the late stage, macrophages promote tissue repair. Our hypothesis is that injury caused by T. nattereri venom (VTn) leads to senescent wounds. In this study, we provide valuable information about the mechanism(s) behind the dysregulated inflammation in wound healing induced by VTn. We demonstrate in mouse paws injected with the venom the installation of γH2AX/p16Ink4a-dependent senescence with persistent neutrophilic inflammation in the proliferation and remodeling phases. VTn induced an imbalance of M1/M2 macrophages by maintaining a high number of TNF-α-producing M1 macrophages in the wound but without the ability to eliminate the persistent neutrophils. Chronic neutrophilic inflammation and senescence were mediated by cytokines such as IL-1α and IL-1ß in a caspase-1- and caspase-11-dependent manner. In addition, previous blocking with anti-IL-1α and anti-IL-ß neutralizing antibodies and caspase-1 (Ac YVAD-CMK) and caspase-11 (Wedelolactone) inhibitors was essential to control the pro-inflammatory activity of M1 macrophages induced by VTn injection, skewing towards an anti-inflammatory state, and was sufficient to block neutrophil recruitment and senescence.

The Role of Immune Mechanisms in Abdominal Aortic Aneurysm: Could It be a Promising Therapeutic Strategy?

Abdominal aortic aneurysm (AAA) is an enlargement of the aorta greater than 50% in diameter. Although up to 80% of cases result in mortality if the aneurysm ruptures, patients are often diagnosed too late, as most cases are asymptomatic. The current treatment for AAA is still surgery as there are currently no effective drug treatments. Knowledge of the pathophysiological mechanisms is essential for the development of new preventive and therapeutic approaches. However, the molecular mechanisms are complex and remain unclear. Apoptosis of vascular smooth muscle cells, the major cellular component of the aorta, and degeneration of the extracellular matrix, the skeleton of the aortic wall, are hallmarks of AAA pathology. Inflammation, mainly through macrophage cells, has been recognized as a central factor in the development of AAA. Macrophage cells also orchestrate other pathways and immune cells involved in this process. Macrophages do not exist as pure populations at aneurysm sites. M1 macrophages are pro-inflammatory and weaken the aortic wall during AAA development. M2 macrophages, in contrast, are involved in anti-inflammatory reactions and aorta tissue repair. The balancing effect on AAA progression makes M1/M2 macrophages therapeutic targets to control inflammation and destruction of the aortic wall. An early diagnosis is also important to allow for early interventions. This review article, based on the available data, aims to evaluate the role of an immunotherapeutic approach in controlling AAA development by briefly discussing the immunological mechanisms.

Cytokine Profiles and the Effect of Intravitreal Aflibercept Treatment on Experimental Choroidal Neovascularization.

PURPOSE: The purpose of our study was to investigate the profiles of inflammatory cytokines and the macrophage polarization gene in a choroidal neovascularization (CNV) mouse model before and after intravitreal aflibercept treatment. METHODS: The CNV mouse model was conducted by laser photocoagulation. A total of 58 cytokines were measured by the multiplex mouse cytokine antibody array. The macrophage polarization genes were tested by reverse transcription polymerase chain reaction. The relationship between the cytokines and the CNV lesion area was analyzed by correlation. RESULTS: MIP-1a on day 3 after laser photocoagulation, MCP-5 and Fas-L on day 7, and IL-15 and IL-7 on day 14 were significantly upregulated (p < 0.001, fold change >10.0). After the intravitreal aflibercept treatment, GM-CSF and MCP-1 on day 3 and TIMP-1 on days 7 and 14 were the most significantly upregulated cytokines (p < 0.001, fold change >10.0). MIP-1 on day 3, IL-13 and Fas-L on day 7, and Fas-L on day 14 were the most significantly downregulated cytokines after intravitreal aflibercept treatment (p < 0.001, fold change >5.0). M2 polarization and VEGFA genes were significantly increased in the CNV formation, whereas aflibercept suppressed M2 polarization and VEGFA genes. IL-7 was negatively related to the CNV lesion area on day 14 after intravitreal aflibercept treatment (r = -0.938, p = 0.006). CONCLUSION: The inflammatory cytokines and the M1/M2 macrophage genes significantly changed in the CNV mouse model. This result suggests that inflammatory cytokines and macrophages play a critical role in the physiopathology of CNV.

Molybdenum and cadmium co-exposure promotes M1 macrophage polarization through oxidative stress-mediated inflammatory response and induces pulmonary fibrosis in Shaoxing ducks (Anas platyrhyncha).

High molybdenum (Mo) and cadmium (Cd) are harmful to the body, but pulmonary toxicity induced by Mo and Cd co-exposure is unknown. To assess the combined impacts of Mo and Cd on fibrosis through M1 polarization in the lung of ducks, 80 healthy 8-day-old Shaoxing ducks (Anas platyrhyncha) were randomly assigned to 4 groups and fed with containing unequal doses of Mo or/and Cd diet. Lung tissues were collected on the 16th week. Results indicated that Mo or/and Cd significantly increased their contents in the lungs, and led to trace elements disorder and histological abnormality, and oxidative stress accompanied by promoting contents of H2 O2 and MDA and decreasing activities of T-SOD, GSH-Px, and CAT, then activated the TLR4/NF-κB/NLRP3 pathway accompanied by upregulating Caspase-1, ASC, IL-18, IL-1ß, TLR4, NF-κB, and NLRP3 expression levels, and disrupted M1/M2 balance to divert toward M1, which evoked the TGF-ß/Smad2/3-mediated fibrosis by elevating TGF-ß1, Smad2, Smad3, COL1A1, α-SMA, and MMP2 expression levels, and decreasing Smad7 and TIMP2 expression levels. The changes of the combined group were most obvious. To sum up, the research demonstrated that Mo or/and Cd may cause macrophages to polarize toward M1 by oxidative stress-mediated the TLR4/NF-κB/NLRP3 pathway, then result in fibrosis through the TGF-ß1/Smad2/3 pathway in duck lungs. Mo and Cd may worsen lung damage.

Role of Sodium-Glucose Co-Transporter 2 Inhibitors in the Regulation of Inflammatory Processes in Animal Models.

Sodium-glucose co-transporter 2 inhibitors, also known as gliflozins, were developed as a novel class of anti-diabetic agents that promote glycosuria through the prevention of glucose reabsorption in the proximal tubule by sodium-glucose co-transporter 2. Beyond the regulation of glucose homeostasis, they resulted as being effective in different clinical trials in patients with heart failure, showing a strong cardio-renal protective effect in diabetic, but also in non-diabetic patients, which highlights the possible existence of other mechanisms through which gliflozins could be exerting their action. So far, different gliflozins have been approved for their therapeutic use in T2DM, heart failure, and diabetic kidney disease in different countries, all of them being diseases that have in common a deregulation of the inflammatory process associated with the pathology, which perpetuates and worsens the disease. This inflammatory deregulation has been observed in many other diseases, which led the scientific community to have a growing interest in the understanding of the biological processes that lead to or control inflammation deregulation in order to be able to identify potential therapeutic targets that could revert this situation and contribute to the amelioration of the disease. In this line, recent studies showed that gliflozins also act as an anti-inflammatory drug, and have been proposed as a useful strategy to treat other diseases linked to inflammation in addition to cardio-renal diseases, such as diabetes, obesity, atherosclerosis, or non-alcoholic fatty liver disease. In this work, we will review recent studies regarding the role of the main sodium-glucose co-transporter 2 inhibitors in the control of inflammation.

Analysis of the Biological Properties of Blood Plasma Protein with GcMAF Functional Activity.

The main problem related to the studies focusing on group-specific component protein-derived macrophage-activating factor (GcMAF) is the lack of clarity about changes occurring in different types of macrophages and related changes in their properties under the effect of GcMAF in various clinical conditions. We analyzed the antitumor therapeutic properties of GcMAF in a Lewis carcinoma model in two clinical conditions: untreated tumor lesion and tumor resorption after exposure to Karanahan therapy. GcMAF is formed during site-specific deglycosylation of vitamin D3 binding protein (DBP). DBP was obtained from the blood of healthy donors using affinity chromatography on a column with covalently bound actin. GcMAF-related factor (GcMAF-RF) was converted in a mixture with induced lymphocytes through the cellular enzymatic pathway. The obtained GcMAF-RF activates murine peritoneal macrophages (p < 0.05), induces functional properties of dendritic cells (p < 0.05) and promotes in vitro polarization of human M0 macrophages to M1 macrophages (p < 0.01). Treatment of whole blood cells with GcMAF-RF results in active production of both pro- and anti-inflammatory cytokines. It is shown that macrophage activation by GcMAF-RF is inhibited by tumor-secreted factors. In order to identify the specific antitumor effect of GcMAF-RF-activated macrophages, an approach to primary reduction of humoral suppressor activity of the tumor using the Karanahan therapy followed by macrophage activation in the tumor-associated stroma (TAS) was proposed. A prominent additive effect of GcMAF-RF, which enhances the primary immune response activation by the Karanahan therapy, was shown in the model of murine Lewis carcinoma. Inhibition of the suppressive effect of TAS is the main condition required for the manifestation of the antitumor effect of GcMAF-RF. When properly applied in combination with any chemotherapy, significantly reducing the humoral immune response at the advanced tumor site, GcMAF-RF is a promising antitumor therapeutic agent that additively destroys the pro-tumor properties of macrophages of the tumor stroma.

Host sphingolipids: Perspective immune adjuvant for controlling SARS-CoV-2 infection for managing COVID-19 disease.

Sphingolipids are potent bioactive agents involved in the pathogenesis of various respiratory bacterial infections. To date, several sphingolipid derivatives are known, but S1P (Sphingosine-1-phosphate) and Ceramide are the best-studied sphingolipid derivatives in the context of human diseases. These are membrane-bound lipids that influence host-pathogen interactions. Based on these features, we believe that sphingolipids might control SARS-CoV-2 infection in the host. SARS-CoV-2 utilizes the ACE-II receptor (Angiotensin-converting enzyme II receptor) on epithelial cells for its entry and replication. Activation of the ACE-II receptor is indirectly associated with the activation of S1P Receptor 1 signaling which is associated with IL-6 driven fibrosis. This is expected to promote pathological responses during SARS-CoV-2 infection in COVID-19 cases. Given this, mitigating S1P signaling by application of either S1P Lyase (SPL) or S1P analog (Fingolimod / FTY720) seems to be potential approach for controlling these pathological outcomes. However, due to the immunosuppressive nature of FTY720, it can modulate hyper-inflammatory responses and only provide symptomatic relief, which may not be sufficient for controlling the novel COVID-19 infection. Since Th1 effector immune responses are essential for the clearance of infection, we believe that other sphingolipid derivatives like Cermaide-1 Phosphate with antiviral potential and adjuvant immune potential can potentially control SARS-CoV-2 infection in the host by its ability in enhancing autophagy and antigen presentation by DC to promote T cell response which can be helpful in controlling SARS-CoV-2 infection in novel COVID-19 patients.

Crosstalk between Metabolic Disorders and Immune Cells.

Metabolic syndrome results from multiple risk factors that arise from insulin resistance induced by abnormal fat deposition. Chronic inflammation owing to obesity primarily results from the recruitment of pro-inflammatory M1 macrophages into the adipose tissue stroma, as the adipocytes within become hypertrophied. During obesity-induced inflammation in adipose tissue, pro-inflammatory cytokines are produced by macrophages and recruit further pro-inflammatory immune cells into the adipose tissue to boost the immune response. Here, we provide an overview of the biology of macrophages in adipose tissue and the relationship between other immune cells, such as CD4+ T cells, natural killer cells, and innate lymphoid cells, and obesity and type 2 diabetes. Finally, we discuss the link between the human pathology and immune response and metabolism and further highlight potential therapeutic targets for the treatment of metabolic disorders.

Effects of corticosteroids on COPD lung macrophage phenotype and function.

The numbers of macrophages are increased in the lungs of chronic obstructive pulmonary disease (COPD) patients. COPD lung macrophages have reduced ability to phagocytose microbes and efferocytose apoptotic cells. Inhaled corticosteroids (ICSs) are widely used anti-inflammatory drugs in COPD; however, their role beyond suppression of cytokine release has not been explored in COPD macrophages. We have examined the effects of corticosteroids on COPD lung macrophage phenotype and function. Lung macrophages from controls and COPD patients were treated with corticosteroids; effects on gene and protein expression of CD163, CD164, CD206, MERTK, CD64, CD80 and CD86 were studied. We also examined the effect of corticosteroids on the function of CD163, MERTK and cluster of differentiation 64 (CD64). Corticosteroid increased CD163, CD164, CD206 and MERTK expression and reduced CD64, CD80 and CD86 expression. We also observed an increase in the uptake of the haemoglobin-haptoglobin complex (CD163) from 59 up to 81% and an increase in efferocytosis of apoptotic neutrophils (MERTK) from 15 up to 28% following corticosteroid treatment. We observed no effect on bacterial phagocytosis. Corticosteroids alter the phenotype and function of COPD lung macrophages. Our findings suggest mechanisms by which corticosteroids exert therapeutic benefit in COPD, reducing iron available for bacterial growth and enhancing efferocytosis.

Clinical features and monocyte/macrophage subsets characterization in granulomatous vs non-granulomatous Crohn's disease.

Aims: Granuloma, mainly composed of macrophages, is a histological feature of Crohn's disease (CD). However, its significance in CD has not been investigated adequately. Our study aims to address this issue by comparing the clinical manifestations and monocyte/macrophage subtypes between granulomatous and non-granulomatous CD.Materials and methods: Demographics, symptoms, endoscopic manifestations, histopathological features, and Montreal classification of patients with and without granulomas were compared. Flow cytometry was used to determine the phagocytosis and subsets of monocytes. ELISA was used to measure the plasma levels of TNF-α, IL-6, IL-1ß, IL-10, CCL22, and TGF-ß1. Immunohistochemistry was performed to quantify the expression of CD68, CD163 and iNOS.Results: Of the222 CD patients enrolled, granulomas were detected in 90. Compared with non-granulomatous CD patients, those with granulomas had younger age, increased rates of diarrhea and perianal complications, along with higher endoscopic score. Intestinal stenosis and crypt abscess were more frequently observed in granulomatous CD patients. A defective phagocytosis of monocytes was observed in granulomatous CD patients. Meanwhile, higher percentages of intermediate and non-classic monocytes, with a lower percentage of classic monocyte were found in them. Besides, they had higher levels of TGF-ß1 and IL-10, a lower level of TNF-α, an increased ratio of CD163+/CD68+cells, and a decreased ratio of iNOS+/CD68+ cells.Conclusions: Granulomatous CD patients exhibited different manifestations compared with their non-granulomatous counterparts. More aggressive therapy may be needed in granulomatous CD patients. Furthermore, the heterogeneity of monocyte/macrophage subsets and altered plasma cytokine may underlie the difference between those two groups.

Vitamin B Complex Treatment Attenuates Local Inflammation after Peripheral Nerve Injury.

Peripheral nerve injury (PNI) leads to a series of cellular and molecular events necessary for axon regeneration and reinnervation of target tissues, among which inflammation is crucial for the orchestration of all these processes. Macrophage activation underlies the pathogenesis of PNI and is characterized by morphological/phenotype transformation from proinflammatory (M1) to an anti-inflammatory (M2) type with different functions in the inflammatory and reparative process. The aim of this study was to evaluate influence of the vitamin B (B1, B2, B3, B5, B6, and B12) complex on the process of neuroinflammation that is in part regulated by l-type CaV1.2 calcium channels. A controlled transection of the motor branch of the femoral peripheral nerve was used as an experimental model. Animals were sacrificed after 1, 3, 7, and 14 injections of vitamin B complex. Isolated nerves were used for immunofluorescence analysis. Treatment with vitamin B complex decreased expression of proinflammatory and increased expression of anti-inflammatory cytokines, thus contributing to the resolution of neuroinflammation. In parallel, B vitamins decreased the number of M1 macrophages that expressed the CaV1.2 channel, and increased the number of M2 macrophages that expressed this channel, suggesting their role in M1/M2 transition after PNI. In conclusion, B vitamins had the potential for treatment of neuroinflammation and neuroregeneration and thereby might be an effective therapy for PNI in humans.

M1 macrophages decrease in the deciduae from normal pregnancies but not from spontaneous abortions or unexplained recurrent spontaneous abortions.

BACKGROUND/PURPOSE: To investigate the M1/M2 polarity of macrophages in the endometrium among different menstrual cycles, normal and abnormal pregnancies, and unexplained recurrent spontaneous abortions (RSAs). METHODS: Endometrial tissue was obtained from 43 patients undergoing hysterectomy, either in the follicular phase (Group 1, n = 23) or in the luteal phase (Group 2, n = 20). In addition, decidual tissue was obtained from 53 pregnant women during the first trimester, either of normal pregnancies (Group 3, n = 12) or abnormal pregnancies (Group 4: spontaneous abortions, n = 20; Group 5: unexplained RSA, n = 21). Using immunofluorescence to examine the M1 and M2 macrophages in the endometrium and deciduae from cases with different menstrual phases and various pregnancy outcomes, respectively, we endeavored to learn the possible pathophysiology of abortions. RESULTS: M1 macrophages were abundant in the deciduae of spontaneous abortions and unexplained RSA, whereas the frequency of M2 macrophages was significantly higher in the endometrium of luteal phase and normal pregnancies. CONCLUSION: M2 polarization is important for early successful pregnancies in humans.

A droplet-merging platform for comparative functional analysis of m1 and m2 macrophages in response to e. coli-induced stimuli.

Microfluidic droplets are used to isolate cell pairs and prevent crosstalk with neighboring cells, while permitting free motility and interaction within the confined space. Dynamic analysis of cellular heterogeneity in droplets has provided insights in various biological processes. Droplet manipulation methods such as fusion and fission make it possible to precisely regulate the localized environment of a cell in a droplet and deliver reagents as required. Droplet fusion strategies achieved by passive mechanisms preserve cell viability and are easier to fabricate and operate. Here, we present a simple and effective method for the co-encapsulation of polarized M1 and M2 macrophages with Escherichia coli (E. coli) by passive merging in an integrated droplet generation, merging, and docking platform. This approach facilitated live cell profiling of effector immune functions in situ and quantitative functional analysis of macrophage heterogeneity. Biotechnol. Bioeng. 2017;114: 705-709. © 2016 Wiley Periodicals, Inc.

Diabetes during pregnancy influences Hofbauer cells, a subtype of placental macrophages, to acquire a pro-inflammatory phenotype.

Growing evidence indicates that maternal pathophysiological conditions, such as diabetes, influence fetal growth and could program metabolic disease in adulthood. Placental cells, particularly Hofbauer cells (HBCs), which are placental macrophages characterized by an anti-inflammatory profile (M2), can sense the modified maternal environment. The goal of this study was to investigate the direct effect of hyperglycemia on HBCs. We studied, at mRNA and protein levels, some markers of M2 and M1 (pro-inflammatory) macrophages in placentae from control and diabetic patients to assess the balance between pro- and anti-inflammatory macrophages: an imbalance of M2 to M1 macrophages has been observed in humans. We used pregnant rats, receiving a single injection of streptozotocin (STZ), as a model of maternal diabetes. We noticed a M2-to-M1 macrophage unbalance as we observed in human. An in vitro model of isolated rat HBCs was used to identify the direct effects of high glucose. We found that high glucose stimulation activated genes belonging to TLR (Toll-Like Receptor)-dependent inflammatory pathways. Moreover, the HBCs stimulated by high glucose switched their M2 profile towards M1, with increased expression of pro-inflammatory cytokines and markers. We also noticed that the oxidative-stress pathway was activated in response to high glucose driven by Hif-1α. In this study, we demonstrated that diabetes/hyperglycemia affect the anti-inflammatory profile of HBCs, by stimulating these cells to acquire an inflammatory profile leading to adverse consequences for the fetal-placental-maternal axis.

Quercetin Inhibits Neuronal Pyroptosis and Ferroptosis by Modulating Microglial M1/M2 Polarization in Atherosclerosis.

Atherosclerosis (AS) with iron and lipid overload and systemic inflammation is a risk factor for Alzheimer's disease. M1 macrophage/microglia participate in neuronal pyroptosis and recently have been reported to be the ferroptosis-resistant phenotype. Quercetin plays a prominent role in preventing and treating neuroinflammation, but the protective mechanism against neurodegeneration caused by iron deposition is poorly understood. ApoE-/- mice were fed a high-fat diet with or without quercetin treatment. The Morris water maze and novel object recognition tests were conducted to assess spatial learning and memory, and nonspatial recognition memory, respectively. Prussian blue and immunofluorescence staining were performed to assess the iron levels in the whole brain and in microglia, microglia polarization, and the degree of microglia/neuron ferroptosis. In vitro, we further explored the molecular biological alterations associated with microglial polarization, neuronal pyroptosis, and ferroptosis via Western blot, flow cytometry, CCK8, LDH, propidium iodide, and coculture system. We found that quercetin improved brain lesions and spatial learning and memory in AS mice. Iron deposition in the whole brain or microglia was reversed by the quercetin treatment. In the AS group, the colocalization of iNOS with Iba1 was increased, which was reversed by quercetin. However, the colocalization of iNOS with PTGS2/TfR was not increased in the AS group, suggesting a character resisting ferroptosis. Quercetin induced the expression of Arg-1 and decreased the colocalizations of Arg-1 with PTGS2/TfR. In vitro, ox-LDL combined with ferric ammonium citrate treatment (OF) significantly shifted the microglial M1/M2 phenotype balance and increased the levels of free iron, ROS, and lipid peroxides, which was reversed by quercetin. M1 phenotype induced by OF caused neuronal pyroptosis and was promoted to ferroptosis by L-NIL treatment, which contributed to neuronal ferroptosis as well. However, quercetin induced the M1 to M2 phenotype and inhibited M2 macrophages/microglia and neuron pyroptosis or ferroptosis. In summary, quercetin alleviated neuroinflammation by inducing the M1 to M2 phenotype to inhibit neuronal pyroptosis and protected neurons from ferroptosis, which may provide a new idea for neuroinflammation prevention and treatment.

Macrophage Polarization and Osteoclast Differentiation.

Macrophages are a key player to regulate rheumatoid arthritis pathogenesis from onset to remission. They can alter innate functions under microenvironmental conditions. To understand heterogeneous functions of macrophages in rheumatoid arthritis, several activated statuses of macrophages should be mimicked in vitro. Here, we describe basic protocols for macrophage polarization and osteoclast differentiation.

Regulation of tumor antigens-Dependent immunotherapy via the hybrid M1 macrophage/tumor lysates Hydrogel.

Tumor treatment poses a significant obstacle in contemporary healthcare. Using components derived from a patient's own cellular and tissue materials to prepare hydrogels and other therapeutic systems has become a novel therapeutic approach, drawing considerable interest for their applicability in basic research on cancer immunotherapy. These hydrogels can engage with cellular components directly and offer a supportive scaffold, aiding in the normalization of tumor tissues. Additionally, their superior capability for encapsulating targeted anti-tumor medications amplifies treatment effectiveness. Given their origin from a patient's own cells, these hydrogels circumvent the risks of immune rejection by the body and severe side effects typically associated with foreign substance. In this study, we developed a composite hydrogel constructed by the cellular lysates of autologous tumor cells and M1 macrophages. This combination promoted the M2 macrophages polarization to the M1 phenotype. Subsequently, the polarized M1 macrophages infiltrated into the hydrogel and can directly capture tumor antigens. As antigen-presenting cells, M1 macrophages can stimulate the production of antigen-specific T cells to kill tumor cells. This work proposes a dual-benefit research strategy that not only polarizes M2 macrophages but also enhances immune activation, boosting T cell-mediated tumor-killing effects. This approach offers a new therapeutic option for clinical cancer immunotherapy.