Abnormal Activation Of Macrophages Research Articles

Pro-inflammatory activation of monocytes in patients with immunoinflammatory rheumatic diseases

The pathogenesis of immunoinflammatory rheumatic diseases (IRDs) is based on chronic inflammation, one of the key mechanisms of which may be abnormal activation of macrophages, leading to further disruption of the immune system.The aim – to evaluate the pro-inflammatory activation of circulating monocytes in patients with IRDs.Material and methods. The study included 149 participants: 53 patients with rheumatoid arthritis (RA), 45 – with systemic lupus erythematosus (SLE), 34 – with systemic scleroderma (SSc) and 17 participants without IRD, aged 30 to 65 years. Basal and lipolysaccharide (LPS)-stimulated secretion of monocytes was studied in a primary culture of monocytes obtained by immunomagnetic separation from blood. Quantitative assessment of the cytokines tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β) and the monocyte chemoattractant protein-1 (MCP-1) was carried out in the culture fluid by ELISA. Pro-inflammatory activation of monocytes was calculated as the ratio of LPS-stimulated and basal secretions.Results. It was shown that the basal secretion of all studied cytokines was significantly increased in all groups of patients with IRDs, except for the secretion of IL-1β in the SLE group, compared with the control. LPS-stimulated secretion of TNF-α was increased and MCP-1 was decreased in patients with IRDs compared to the control group; LPS-stimulated IL-1β secretion only in the SSc group was significantly different from the control group. In the RA group, monocyte activation was reduced for all cytokines compared to the control, in the SLE group – for TNF-α and MCP-1, in the SSc group – for MCP-1.Conclusion. The decrease in pro-inflammatory activation of monocytes in patients with IRDs is due to a high level of basal secretion of cytokines, which can lead to disruption of the adequate immune response in these diseases and is an important link in the pathogenesis of chronic inflammation.

Polymyositis concomitant with hepatitis B virus infection: Treatment challenges.

Polymyositis is a chronic autoimmune disease that presents with symmetrical progressive proximal muscle weakness. The cause of this disease due to abnormal activation of macrophages that might be associated with systemic diseases such as other autoimmune diseases, malignancy or viral infections including hepatitis B virus. The aim of this case report was to highlight treatment challenges in a patient with polymyositis concomitant with hepatitis B. A 28-years-old man with history of completed hepatitis B treatment with negative viral load presented with symmetrical progressive weakness on both inferior proximal extremities. The patient complained of pain predominantly in both tights and calves. No dermatological manifestation was observed. Elevated muscle enzymes and liver function were observed. Along with the course of the disease, hepatitis B reactivation was discovered as hepatitis B virus DNA was re-detected. Treatment options of this patient (polymyositis concomitant with hepatitis B viral infection) remain challenging. The main treatment of polymyositis consists of high dose methylprednisolone and this immunosuppressant could worsen the hepatitis B virus infection. The patient was finally treated with combination of mycophenolic acid and methylprednisolone for polymyositis and entecavir for hepatitis B. After one month of treatment, the patient showed a clinical improvement. This case highlights that viral screening must be done prior to starting polymyositis treatment as it could concomitant with viral infections such as hepatitis B. Antiviral prophylaxis must be given 1-2 weeks before immunosuppression starts. Management for both polymyositis and hepatitis B is important with entecavir or tenofovir as the optimal agents against hepatitis B virus.

Secondary Hemophagocytic Lymphohistiocytosis in Adults: A Single Institution Case Series

Introduction: Hemophagocytic lymphohistiocytosis (HLH) is a severe hyperinflammatory syndrome caused by the abnormal activation of macrophages and T cells. The primary form, resulting from lymphocyte-related mutations, is more common in children, while the secondary (acquired) form known as sHLH prevails in adults. sHLH is often triggered by infections or malignancies (Mal-HLH) but can also be induced by autoinflammatory disorders called macrophage activation syndrome (MAS; or MAS-HLH). We present a retrospective case series of adults treated for sHLH at a single institution from 2012 to 2023. Methods: Retrospective data was collected using the EDW database, and 36 patients were identified using ICD 9/10 codes 288.4 and D76.1 for HLH. Subsequently, a manual review of electronic medical records was conducted; criteria for inclusion were age >18 years at the time of diagnosis and meeting HLH-2004 criteria ( Panel-1). Results: Out of the 36 patients reviewed, 11 met the criteria and were included in the analysis. These cases occurred between 2015 and 2023. The median age at diagnosis was 52 (range 18 to 81). The female-to-male ratio in our study was 4:7. Most patients (73%) were white. Additionally, 45% of the patients were immunocompromised. 91% of the patients presented with fever, while only 45% had splenomegaly. Trilineage cytopenias were observed in all patients. In our study, 5/11 had hypofibrinogenemia, and 6/11 had hypertriglyceridemia. Hemophagocytosis was evident in 91% of the patients. Natural Killer (NK) cell activity was measured in 4 patients, and all exhibited low or no NK cell activity. In 10 out of 10 patients, soluble IL-2 receptors were elevated. None of the patients in the study had hepatomegaly, but all had transaminitis and hyperbilirubinemia, and 82% had elevated alkaline phosphatase levels. Elevated lactate levels were observed in 78% of patients, and 27% had a rash. Neurological features were observed in 6 out of 10 patients. Lactate dehydrogenase was elevated in all patients with a median value of 1551 U/L (range 354 - 3689) Presumed etiologies for sHLH included malignancy (4), infection (3), autoimmune disease (3), and unknown cause (1). Treatment was highly individualized based on the underlying trigger and was guided by the HLH-94 protocol ( Panel-2). All patients received high-dose steroids, 6/11 received etoposide, and 2 received intrathecal therapy for neurological involvement. Conclusion: At a median follow-up period of 16 days, five patients had died. The survival rates at 30 days and 1 year were 45% and 27%, respectively. The overall mortality rate was 55%. While mortality rates were 75% in the Mal-HLH arm and 67% in the infectious arm, all patients with MAS-HLH survived while performing the study indicating a better prognosis for patients with this type of HLH.

1574-P: FoxO1 Links Insulin Resistance to Hepatic Inflammation and Catalyzes the Evolution of Benign Steatosis to Nonalcoholic Steatohepatitis

Hepatic inflammation is intertwined with insulin resistance and is a causative factor for the transition of benign steatosis to nonalcoholic steatohepatitis (NASH). We found that FoxO1 was markedly upregulated in hepatic macrophages, coinciding with hepatic inflammation and steatosis in dietary obese mice. We generated myeloid-conditional FoxO1-transgenic and FoxO1-knockout mice, two complementary models for determining the effect of FoxO1 gain- vs. loss-of-function on macrophage activation in response to insulin resistance. We found that myeloid FoxO1 gain-of-function aggravated hepatic inflammation in mice in response to overnutrition or endotoxin. In contrast, myeloid FoxO1 loss-of-function skewed macrophage polarization from pro-inflammatory M1 to anti-inflammatory M2 phenotypes, accompanied by the reduction of macrophage infiltration in liver. This effect contributed to the suppression of hepatic inflammation and improvement of insulin sensitivity. Despite fat-induced obesity, myeloid-conditional FoxO1-deficient mice were protected from developing hyperglycemia, hyperinsulinemia and glucose intolerance. Furthermore, the improvement in hepatic inflammation and insulin resistance translated into a significant beneficial effect on NASH, culminating in the reduction of hepatic steatosis and fibrosis in myeloid FoxO1-deficient mice on NASH-inducing diet. Human FOXO1 expression was upregulated in inflammatory macrophages in liver, correlating with hepatic inflammation, steatosis and fibrosis in patients with NASH. Mechanistically, FoxO1 counteracts Stat6 to skew macrophage polarization from M2 toward M1 signatures with pro-inflammatory profiles. We concluded that myeloid FoxO1 dysregulation is culpable for linking insulin resistance to abnormal macrophage activation. This effect perpetuates hepatic inflammation and catalyzes the evolution of simple steatosis to NASH. Disclosure S.Lee: None. T.Usman: None. G.Chhetri: None. X.Wang: None. H.Dong: None.

Human MIKO-1, a Hybrid Protein That Regulates Macrophage Function, Suppresses Lung Fibrosis in a Mouse Model of Bleomycin-Induced Interstitial Lung Disease.

Although interstitial lung disease (ILD) is a life-threatening pathological condition that causes respiratory failure, the efficiency of current therapies is limited. This study aimed to investigate the effects of human MIKO-1 (hMIKO-1), a hybrid protein that suppresses the abnormal activation of macrophages, on murine macrophage function and its therapeutic effect in a mouse model of bleomycin-induced ILD (BLM-ILD). To this end, the phenotype of thioglycolate-induced murine peritoneal macrophages co-cultured with hMIKO-1 was examined. The mice were assigned to normal, BLM-alone, or BLM + hMIKO-1 groups, and hMIKO-1 (0.1 mg/mouse) was administered intraperitoneally from day 0 to 14. The mice were sacrificed on day 28, and their lungs were evaluated by histological examination, collagen content, and gene expression levels. hMIKO-1 suppressed the polarization of murine macrophages to M2 predominance in vitro. The fibrosis score of lung pathology and lung collagen content of the BLM + hMIKO-1 group were significantly lower than those in the BLM-alone group. The expression levels of TNF-α, IL-6, IL-1β, F4/80, and TIMP-1 in the lungs of the BLM + hMIKO-1 group were significantly lower than those in the BLM-alone group. These findings indicate that hMIKO-1 reduces lung fibrosis and may be a future therapeutic candidate for ILD treatment.

Macrophage Glucose Metabolism Reprogramming and Cardiovascular Complications of Diabetes

Macrophages are the main participants in the pathogenesis of many chronic inflammatory and autoimmune diseases, including diabetes, and cardiovascular complications mediated by abnormal activation of macrophages caused by various reasons are the main causes of morbidity and death in patients with diabetes. Different environmental stimuli can easily lead to glucose metabolism reprogramming in macrophages, resulting in a series of changes in metabolization-related enzymes, metabolites and metabolic pathways. Glucose metabolism reprogramming is closely related to M1 polarization and M2 polarization of macrophages, for example, M1 macrophages tend to anaerobic glycolysis while M2 macrophages tend to aerobic metabolism, and different polarization of macrophages is closely related to various diseases. Therefore, this paper reviews the mechanism and inducing factors of reprogramming of glucose metabolic pathways, as well as the effect of these pathways on macrophage polarization, so as to provide services for clinical research and treatment of the occurrence and development of cardiovascular complications in the context of chronic inflammation of diabetes.

Myeloid FoxO1 depletion attenuates hepatic inflammation and prevents nonalcoholic steatohepatitis.

Hepatic inflammation is culpable for the evolution of asymptomatic steatosis to nonalcoholic steatohepatitis (NASH). Hepatic inflammation results from abnormal macrophage activation. We found that FoxO1 links overnutrition to hepatic inflammation by regulating macrophage polarization and activation. FoxO1 was upregulated in hepatic macrophages, correlating with hepatic inflammation, steatosis, and fibrosis in mice and patients with NASH. Myeloid cell conditional FoxO1 knockout skewed macrophage polarization from proinflammatory M1 to the antiinflammatory M2 phenotype, accompanied by a reduction in macrophage infiltration in liver. These effects mitigated overnutrition-induced hepatic inflammation and insulin resistance, contributing to improved hepatic metabolism and increased energy expenditure in myeloid cell FoxO1–knockout mice on a high-fat diet. When fed a NASH-inducing diet, myeloid cell FoxO1–knockout mice were protected from developing NASH, culminating in a reduction in hepatic inflammation, steatosis, and fibrosis. Mechanistically, FoxO1 counteracts Stat6 to skew macrophage polarization from M2 toward the M1 signature to perpetuate hepatic inflammation in NASH. FoxO1 appears to be a pivotal mediator of macrophage activation in response to overnutrition and a therapeutic target for ameliorating hepatic inflammation to stem the disease progression from benign steatosis to NASH.

α-Mangostin Treats Early-Stage Adjuvant-Induced Arthritis of Rat by Regulating the CAP-SIRT1 Pathway in Macrophages.

BackgroundStudies have found that α-mangostin (MG) can relieve experimental arthritis by activating cholinergic anti-inflammatory pathway (CAP). It affects the polarization of macrophages and the balance of related immune cell subpopulations, but the specific mechanism is still unclear. It has been found that silent information regulator 1 (SIRT1) is closely related to macrophage activity. The purpose of this study is to explore the mechanism of MG intervening in macrophage polarization during treatment of early adjuvant-induced (AIA) rats through the CAP-SIRT1 pathway.MethodsWe investigated the polarization of M1 macrophages and the differentiation of Th1 in AIA rats by flow cytometry. Activity of acetylcholinesterase (AChE) and the level of nicotinic adenine dinucleotide (NAD+) in serum were also detected, and immunohistochemical was used to detect the levels of α7 nicotinic cholinergic receptor (α7nAChR) and SIRT1. Then in macrophages, the molecular mechanism of MG regulating the abnormal activation of macrophages in rats with early AIA through the CAP-SIRT1 pathway was studied.ResultsMG can significantly inhibit the polarization of M1 macrophages and the differentiation of Th1 in AIA rats in the acute phase of inflammation. MG can significantly inhibit the activity of AChE and increase the level of NAD+, thereby further up-regulated the expression levels of α7nAChR and SIRT1. Meanwhile, MG inhibited nuclear factor-κB (NF-κB)-mediated inflammation by activating the CAP-SIRT1 pathway in macrophages.ConclusionIn summary, the stimulation of MG induced CAP activation, which up-regulated SIRT1 signal, and thereby inhibited M1 polarization through the NF-κB pathway, and improved the pathological immune environment of early-stage AIA rats.

Single-cell transcriptome analysis reveals defective decidua stromal niche attributes to recurrent spontaneous abortion.

ObjectivesSuccessful pregnancy involves the homeostasis between maternal decidua and fetoplacental units, whose disruption contributes to compromised pregnancy outcomes, including recurrent spontaneous abortion (RSA). The role of cell heterogeneity of maternal decidua in RSA is yet to be illustrated.Materials and methodsA total of 66,078 single cells from decidua samples isolated from patients with RSA and healthy controls were analysed by unbiased single‐cell RNA sequencing (scRNA‐seq).ResultsOur scRNA‐seq results revealed that stromal cells are the most abundant cell type in decidua during early pregnancy. RSA samples are accompanied by aberrant decidualization and obviously obstructed communication between stromal cells and other cell types, such as abnormal activation of macrophages and NK cells. In addition, the over‐activated TNF superfamily member 12 (TNFSF12, TWEAK) and FASLG in RSA are closely related to stromal cell demise and pregnancy failure.ConclusionsOur research reveals that the cell composition and communications in normal and RSA decidua at early pregnancy and provides insightful information for the pathology of RSA and will pave the way for pregnancy loss prevention.

Acute Epstein‐Barr virus associated haemophagocytosis in an Asian female: What is the diagnosis?

Acute <scp>Epstein‐Barr</scp> virus associated haemophagocytosis in an Asian female: What is the diagnosis?

Contribution of toll-like receptor 2 and nicotinamide adenine dinucleotide phosphate oxidase to the trimethylamine N-oxide-induced inflammatory reactions in U937-derived macrophages.

BACKGROUNDTrimethylamine N-oxide (TMAO) is emerging as a new generation of metabolites related to the activation of inflammatory reactions in the macrophages during atherosclerosis. Stress-activation of cell surface toll-like receptors (TLRs) as well as nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX) is also assumed to be involved in TMAO-induced inflammatory reaction in the macrophages. To elucidate the possible contribution of TLRs and NOX to the mentioned signaling pathway, we aimed to simultaneously evaluate the expression level of TLR2, TLR6, and NOX2 in TMAO-treated macrophages.METHODS2.5 × 106 cells of U937-derived macrophages were treated in triplicates with different concentrations (37.5, 75, 150, and 300 μM) of TMAO for 24 hours. The cells were also treated with tunicamycin (TUN), as a positive control of stress. Normal control group (CTR) cells received no treatment. The viability of treated cells was checked by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole (MTT) assay. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was also used to evaluate the relative expression (fold change) of TLR2, TLR6, and NOX2 at messenger ribonucleic acid (mRNA) levels. One-way analysis of variance (ANOVA) with post-hoc Dunnett’s test was performed to compare every mean with that of the control.RESULTSNo cell death occurred because of treatments. Dose of 300 μM of TMAO significantly increased the relative expression of both TLR2 and NOX2 compared to the CTR cells (P < 0.001 for both). The elevation of TLR6 was not statistically significant in all groups of TMAO-treated cells (P > 0.050).CONCLUSIONOur results provide documentation supporting contribution of TLR2 and NOX2 to previously described inflammatory reactions induced by TMAO in macrophages. In addition, they may clarify the proatherogenic role of TMAO in foam cell formation as well as abnormal activation of macrophages during atherosclerosis.

Abstract LB224: The potential effect of chronically increased endotoxin levels on breast carcinoma progression in obese patients.

Abstract Obesity serves as a risk factor for estrogen-dependent postmenopausal breast cancer (BC). While the exact association occurring between these two disease states remains unknown, obesity-associated inflammation is thought to be the most likely contributing factor. In addition, obesity has been correlated to changes in gut microbiota composition and a subsequent chronic increase in bacterial endotoxin (lipopolysaccharide [LPS] - canonic ligand of toll-like receptor 4 [TLR4]) blood levels. It was recently shown that BC cells intrinsically express TLR4 and such expression has been associated with decreased patient survival as well as increased tumor growth. We hypothesized that obesity-associated endotoxemia may contribute to BC progression by utilizing TLR-dependent mechanisms and exerting cancer-promoting effects directly (on carcinoma cells) and indirectly (triggering abnormal activation of macrophages). Utilizing a chronic metabolic endotoxemia and breast cancer murine model as well as in vitro experimental systems, we found that continuous exposure to low concentrations of LPS not only promotes BC progression in vivo but stimulates BC cell growth in culture through the activation of key breast cancer-promoting signaling pathways (Stat3, Akt, ERK1/2). Obesity has reached epidemic proportions globally, where elucidation of the molecular mechanisms underlying breast tumor-promoting action of obesity has become of vital importance. Improving the understanding of such mechanisms has the potential to reveal improved efficacious therapy regimens and prevention strategies in a rapidly growing population of obese, breast cancer patients. Citation Format: Amichay Meirovitz, Daniela Nahmias, Esther Hermano, Ofra Maimon, Tamar Peretz, Michael Elkin. The potential effect of chronically increased endotoxin levels on breast carcinoma progression in obese patients. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB224.

Murine myeloid cell MCPIP1 suppresses autoimmunity by regulating B-cell expansion and differentiation.

ABSTRACTMyeloid-derived cells, in particular macrophages, are increasingly recognized as critical regulators of the balance of immunity and tolerance. However, whether they initiate autoimmune disease or perpetuate disease progression in terms of epiphenomena remains undefined.Here, we show that depletion of MCPIP1 in macrophages and granulocytes (Mcpip1fl/fl-LysMcre+ C57BL/6 mice) is sufficient to trigger severe autoimmune disease. This was evidenced by the expansion of B cells and plasma cells and spontaneous production of autoantibodies, including anti-dsDNA, anti-Smith and anti-histone antibodies. Consequently, we document evidence of severe skin inflammation, pneumonitis and histopathologic evidence of glomerular IgG deposits alongside mesangioproliferative nephritis in 6-month-old mice. These phenomena are related to systemic autoinflammation, which secondarily induces a set of cytokines such as Baff, Il5, Il9 and Cd40L, affecting adaptive immune responses. Therefore, abnormal macrophage activation is a key factor involved in the loss of immune tolerance.Overall, we demonstrate that deficiency of MCPIP1 solely in myeloid cells triggers systemic lupus-like autoimmunity and that the control of myeloid cell activation is a crucial checkpoint in the development of systemic autoimmunity.

Lithium Modified Mesoporous Hydroxyapatite Nanoparticles Mediate the Polarization of M2 Macrophages to Promote Bone Defect Repair in Glucocorticoid-Induced Osteonecrosis of the Femoral Head

Glucocorticoid (GC)-induced osteonecrosis of femoral head(GI-ONFH) is a chronic inflammatory disease characterized by abnormal activation of macrophages and infiltration of necrotic sites. However, previous studies on osteonecrosis repair have neglected the immunomodulatory effect of bone, which leads to the failure of bone repair. Therefore, in this study, we produced an immunomodulatory bone repair material with lithium (Li)-modified mesoporous hydroxyapatite nanoparticles (Li-mHANP) that has the potential to enhance bone regeneration by manipulating macrophage (M) polarization to create an immunomodulatory microenvironment that promotes bone healing in GIONFH. In order to investigate whether Li incorporation into mHANP can synergistically improve bone formation and repair osteonecrosis by changing the polarization of macrophages, we evaluated Li-mHANP and macrophages in the process of GIONFH bone regeneration through in vitro and in vivo biological tests. We found that, compared with mHANP, Li-mHANP activated the JAK1/STAT6/STAT3 signaling pathway and promoted the transformation of macrophages to M2 phenotype. In addition, Li-mHANP-treated macrophages promote osteogenesis of BMSCs and endothelial cell angiogenesis in vitro. After Li-mHANP nanoparticles were implanted into the model of GIONFH, we found that Li-mHANP mediated the transformation of pro-inflammatory M1 macrophages into pro-tissue repair M2 phenotype, restored the M2/M1 ratio to normal levels, effectively alleviated inflammation, and ultimately promoted osteoblast differentiation and bone regeneration, compared with mHANP without Li modified. In conclusion, these findings suggest that Li-mHANP is an immunomodulatory material that can direct immunoregulatory bone repair related to tissue repair and regeneration and promote bone regeneration of bone defect in the condition of GIONFH. Funding Information: This study was supported by The National Natural Science Foundation of China (grant no. 81974333) and Science and Technology Program of Chengdu (grant no.2019-YF05-01188-SN). Declaration of Interests: The authors declare no competing financial interest. Ethics Approval Statement: All animal operations were approved by the Animal Research Committee of West China Hospital of Sichuan University (Chengdu, China).

Role of Heparanase in Macrophage Activation.

Macrophages represent one of the most diverse immunocyte populations, constantly shifting between various phenotypes/functional states. In addition to execution of vital functions in normal physiological conditions, macrophages represent a key contributing factor in the pathogenesis of some of the most challenging diseases, such as chronic inflammatory disorders, diabetes and its complications, and cancer. Macrophage polarization studies focus primarily on cytokine-mediated mechanisms. However, to explore the full spectrum of macrophage action, additional, non-cytokine pathways responsible for altering macrophage phenotype have to be taken into consideration as well. Heparanase, the only known mammalian endoglycosidase that cleaves heparan sulfate glycosaminoglycans, has been shown to contribute to the altered macrophage phenotypes in vitro and in numerous animal models of inflammatory conditions, occurring either in the presence of microbial products or in the setting of non-infectious "aseptic" inflammation. Here we discuss the involvement of heparanase in shaping macrophage responses and provide information that may help to establish the rationale for heparanase-targeting interventions aimed at preventing abnormal macrophage activation in various disorders.

Review: the Role and Mechanisms of Macrophage Autophagy in Sepsis.

Sepsis is a systemic inflammatory response syndrome caused by infection. The core mechanism underlying sepsis is immune dysfunction, with macrophages, as important cells of the innate immune system, playing an essential role. Autophagy has been shown to be closely related to inflammation and immunity, and autophagy enhancement in sepsis can play a protective role by negatively regulating abnormal macrophage activation, modulating macrophage polarization phenotype, reducing activation of the inflammasome and release of inflammatory factors, and affecting macrophage apoptosis. However, excessive autophagy may also lead to autophagic death of macrophages, which further aggravates the inflammatory response. The mechanisms underlying these functions are relatively complex and remain unclear, but may be related to a variety of signaling pathways such as NF-κB, mTOR, and PI3K/AKT. The administration of drugs to assist in the regulation of macrophage autophagy has become a novel treatment for sepsis. The present review focuses on the role and the potential mechanisms of macrophage autophagy in sepsis.

Serum interleukin-34 levels in patients with systemic sclerosis: Clinical association with interstitial lung disease.

Interleukin (IL)-34 is a hematopoietic cytokine promoting proliferation and differentiation of macrophages. Because abnormal activation of macrophages is involved in the development of systemic sclerosis (SSc), we investigated serum IL-34 levels in patients with SSc. Serum IL-34 levels were significantly increased in diffuse cutaneous SSc compared with limited cutaneous SSc and healthy controls, while there were no significant differences between limited cutaneous SSc and healthy controls. In addition, SSc patients with increased serum IL-34 levels more often had interstitial lung disease (ILD) than those with normal levels. Moreover, in SSc patients, serum IL-34 levels negatively correlated with the percentage of predicted vital capacity, while they positively correlated with ground-glass opacity score and fibrotic score on chest computed tomography. Collectively, increased serum IL-34 levels were associated with greater frequency and severity of ILD in SSc patients. Serum IL-34 levels may be a useful serological marker for SSc-associated ILD.

Trimethylamine-N-oxide, as a risk factor for atherosclerosis, induces stress in J774A.1 murine macrophages

PurposeTrimethylamine N-oxide (TMAO) is a biomarker for kidney problems. It has also been introduced as a risk factor for atherosclerosis. The classic risk factors for atherosclerosis trigger cellular and humeral immunoreaction in macrophages through induction of heat shock protein expressions and increased levels of GRP94 and HSP70 are associated with increased atherosclerosis risk. The present study evaluated the possible effect(s) of TMAO on the expression of GRP94 and HSP70 at protein levels. MethodsJ774A.1 murine macrophages were treated with different micromolar concentrations of TMAO and 4-phenylbutyric acid (PBA), a chemical chaperone, for 8, 18, 24, and 48h intervals. Tunicamycin was also used as a control for induction of endoplasmic reticulum stress. Western blotting was used to evaluate the expression of GRP94 and HSP70 in macrophages at protein levels. ResultTunicamycin greatly increased protein levels of GRP94. Similarly, but to a lesser extent compared to tunicamycin, TMAO also increased GRP94. In 24h treated cells, only 300μM of TMAO, and in cells treated for 48h, all doses of TMAO produced a significant increase in relative HSP70 protein levels compared to the control. PBA failed to induce any changes in GRP94 or HSP70 protein levels. ConclusionGRP94 and HSP70 are stress-inducible heat shock protein, so the elevation in J774A.1 murine macrophages can clearly define cells under stress and elucidate the contribution of stress induced by TMAO that may have a part in the abnormal activation of macrophages involved in foam cell formation.

Interleukin-6 stimulates Akt and p38 MAPK phosphorylation and fibroblast migration in non-diabetic but not diabetic mice.

Persistent inflammatory environment and abnormal macrophage activation are characteristics of chronic diabetic wounds. Here, we attempted to characterize the differences in macrophage activation and temporal variations in cytokine expression in diabetic and non-diabetic wounds, with a focus on interleukin (IL)-6 mRNA expression and the p38 MAPK and PI3K/Akt signaling pathways. Cutaneous wound closure, CD68- and arginase-1 (Arg-1)-expressing macrophages, and cytokine mRNA expression were examined in non-diabetic and streptozotocin-induced type 1 diabetic mice at different time points after injury. The effect of IL-6 on p38 MAPK and Akt phosphorylation was investigated, and an in vitro scratch assay was performed to determine the role of IL-6 in primary skin fibroblast migration. Before injury, mRNA expression levels of the inflammatory markers iNOS, IL-6, and TNF-α were higher in diabetic mice; however, IL-6 expression was significantly lower 6 h post injury in diabetic wounds than that in non-diabetic wounds. Non-diabetic wounds exhibited increased p38 MAPK and Akt phosphorylation; however, no such increase was found in diabetic wounds. In fibroblasts from non-diabetic mice, IL-6 increased the phosphorylation of p38 MAPK and levels of its downstream factor CREB, and also significantly increased Akt phosphorylation and levels of its upstream factor P13K. These effects of IL-6 were not detected in fibroblasts derived from the diabetic mice. In scratch assays, IL-6 stimulated the migration of primary cultured skin fibroblasts from the non-diabetic mice, and the inhibition of p38 MAPK was found to markedly suppress IL-6–stimulated fibroblast migration. These findings underscore the critical differences between diabetic and non-diabetic wounds in terms of macrophage activation, cytokine mRNA expression profile, and involvement of the IL-6-stimulated p38 MAPK–Akt signaling pathway. Aberrant macrophage activation and abnormalities in the cytokine mRNA expression profile during different phases of wound healing should be addressed when designing effective therapeutic modalities for refractory diabetic wounds.

A Three-Dimensional Chondrocyte-Macrophage Coculture System to Probe Inflammation in Experimental Osteoarthritis.

The goal of the present study was to develop a fully three-dimensional (3D) coculture system that would allow for systematic evaluation of the interplay between activated macrophages (AMs) and chondrocytes in osteoarthritic disease progression and treatment. Toward this end, our coculture system was first validated against existing in vitro osteoarthritis models, which have generally cultured healthy normal chondrocytes (NCs)-in two-dimensional (2D) or 3D-with proinflammatory AMs in 2D. In this work, NCs and AMs were both encapsulated within poly(ethylene glycol) diacrylate hydrogels to mimic the native 3D environments of both cell types within the osteoarthritic joint. As with previous studies, increases in matrix metalloproteinases (MMPs) and proinflammatory cytokines associated with the early stages of osteoarthritis were observed during NC-AM coculture, as were decreases in protein-level Aggrecan and collagen II. Thereafter, the coculture system was extended to osteoarthritic chondrocytes (OACs) and AMs to evaluate the potential effects of AMs on pre-existing osteoarthritic phenotypes. OACs in coculture with AMs expressed significantly higher levels of MMP-1, MMP-3, MMP-9, MMP-13, IL-1β, TNF-α, IL-6, IL-8, and IFN-γ compared to OACs in mono-culture, indicating that proinflammatory macrophages may intensify the abnormal matrix degradation and cytokine secretion already associated with OACs. Likewise, AMs cocultured with OACs expressed significantly more IL-1β and VEGF-A compared to AM mono-culture controls, suggesting that OACs may intensify abnormal macrophage activation. Finally, OACs cultured in the presence of nonactivated macrophages produced lower levels of MMP-9 and proinflammatory cytokines IL-1β, TNF-α, and IFN-γ compared to OACs in the OAC-AM system, results that are consistent with anti-inflammatory agents temporarily reducing certain OA symptoms. In summary, the 3D coculture system developed herein captures several key features of inflammatory OA and may prove useful in future screening of therapeutic agents and/or assessment of disease progression mechanisms.