Successful management of infection and macrophage activation syndrome patient using low-dose etoposide:A case report

BACKGROUND Macrophage activation syndrome(MAS),a sub-type of hemophagocytic lymphohistiocytosis(HLH)secondary to autoimmune rheumatic diseases,is a critical and potentially fatal condition characterized by an excessive inflammatory response.Despite the established efficacy of the HLH-2004 guideline in diagnosing and treating HLH over the years,ongoing discussion persists regarding its application,especially for HLH secondary to complicated conditions,such as autoimmune rheumatic diseases combined with severe infection.Etoposide(VP-16),a topoisomerase II inhibitor that effectively induces DNA damage and subsequent apoptosis in hyperactivated immune cells,has been widely used for the treatment of HLH.However,its suppressive effect on immune system may also cause potential exacerbation of infection in autoimmune rheumatic disease-induced HLH patients complicated with severe infection.Therefore,the use of VP-16 in such cases was inconclusive.CASE SUMMARY In this case study,we propose a potentially effective strategy for managing a patient diagnosed with secondary HLH complicated with systemic lupus erythematosus(SLE)and chronic coronavirus disease 2019 infection.Our approach involves early administration of low-dose VP-16(100 mg twice a week,300 mg in total),combined with methylprednisolone,cyclophosphamide,and cyclosporine A.The administration of etoposide effectively led to improvements in various indices of HLH.CONCLUSION Low dose etoposide proves to be an effective approach in alleviating HLH while mitigating the risk of infection.

Macrophage Activation Syndrome as the Primary Presentation of Pediatric Systemic Lupus Erythematosus: A Case Report and Review of the Literature

Macrophage activation syndrome (MAS), in its secondary form, often complicates rheumatic diseases but rarely constitutes a mode of revelation. Systemic lupus erythematosus (SLE) is a systemic autoimmune disease of unknown etiology that primarily affects women in adulthood. MAS is a serious condition that may be the first presentation of SLE. Here, we report the case of a 4-year-old female with MAS as the primary manifestation of Systemic Lupus Erythematosus (SLE). In this case, we outline the characteristics of a complex case of SLE that was initially accompanied with MAS, and also review the literature to discuss the clinical, biological, and therapeutic aspects of this condition.

Toxoplasma ROP16Ⅰ/Ⅲ ameliorated inflammatory bowel diseases via inducing M2 phenotype of macrophages

BACKGROUND Inflammatory bowel disease（IBD） is characterized by chronic and non-specific inflammation of the intestinal mucosa and mainly includes ulcerative colitis and Crohn’s disease.AIM To explore the beneficial effect of Toxo ROP16Ⅰ/Ⅲ-induced M2 phynotype macrophages in homeostasis of IBDs through downregulation of M1 inflammatory cells.METHODS RAW264.7 macrophages stimulated by lipopolysaccharide（LPS）（M1 cells） were co-cultured with Caco-2 cells as an inflammatory model of IBD in vitro.The expression of Toxo ROP16Ⅰ/Ⅲ was observed in RAW264.7 macrophages that were transfected with p EGFP-rop16Ⅰ/Ⅲ.The phenotypes of M2 and M1 macrophage cells were assessed by quantitative real-time reverse transcriptase polymerase chain reaction and the expression of tumor necrosis factor（TNF）-α,interleukin（IL）-1β,IL-6,transforming growth factor（TGF）-β1,IL-10,inducible nitric oxide synthase（i NOS）,and arginase-1（Arg-1） was detected.The expression of i NOS,Arg-1,signal transducer and activator of transcription 3（Stat3）,p-Stat3,Stat6,pStat6,programmed death ligand-2（PD-L2）,caspase-3,-8,and-9 was analyzed by Western blotting,and Griess assays were performed to detect nitric oxide（NO）.TNF-α,IL-1β,IL-6,TGF-β1,and IL-10 expression in the supernatants was detected by enzyme-linked immunosorbent assay,and Caco-2 cell apoptosis was determined by flow cytometry after mixing M1 cells with M2 cells in a Caco-2 cell co-culture system.RESULTS M1 cells exhibited significantly increased production of i NOS,NO,TNF-α,IL-1β,and IL-6,while Toxo ROP16Ⅰ/Ⅲ induced macrophage bias to M2 cells in vitro,showing increased expression of Arg-1,IL-10 and TGF-β1 and elevated production of p-Stat3 and p-Stat6.The mixed M1 and M2 cell culture induced by Toxo ROP16 Ⅰ/Ⅲ exhibited decreased production of NO and i NOS and upregulated expression of Arg-1 and PD-L2.Accordingly,Caco-2 cells became apoptotic,and apoptosis-associated proteins such as caspase-3,-8 and-9 were dampened during co-culture of M1 and M2 cells.Flow cytometry analysis showed that co-culture of M1 cells with Caco-2 cells facilitated the apoptosis of Caco-2 cells,but co-culture of M1 and M2 cells alleviated Caco-2 cell apoptosis.CONCLUSION Toxo ROP16 Ⅰ/Ⅲ-induced M2 macrophages inhibited apoptosis of Caco-2 cells caused by M1 macrophages.This finding may help gain a better understanding of the underlying mechanism and represent a promising therapeutic strategy for IBDs.

Multimodal Identification by Transcriptomics and Multiscale Bioassays of Active Components in Xuanfeibaidu Formula to Suppress Macrophage-Mediated Immune Response

Xuanfeibaidu Formula (XFBD) is a Chinese medicine used in the clinical treatment of coronavirus disease 2019 (COVID-19) patients. Although XFBD has exhibited significant therapeutic efficacy in clinical practice, its underlying pharmacological mechanism remains unclear. Here, we combine a comprehensive research approach that includes network pharmacology, transcriptomics, and bioassays in multiple model systems to investigate the pharmacological mechanism of XFBD and its bioactive substances. High-resolution mass spectrometry was combined with molecular networking to profile the major active substances in XFBD. A total of 104 compounds were identified or tentatively characterized, including flavonoids, terpenes, carboxylic acids, and other types of constituents. Based on the chemical composition of XFBD, a network pharmacology-based analysis identified inflammation-related pathways as primary targets. Thus, we examined the anti-inflammation activity of XFBD in a lipopolysaccharide-induced acute inflammation mice model. XFBD significantly alleviated pulmonary inflammation and decreased the level of serum proinflammatory cytokines. Transcriptomic profiling suggested that genes related to macrophage function were differently expressed after XFBD treatment. Consequently, the effects of XFBD on macrophage activation and mobilization were investigated in a macrophage cell line and a zebrafish wounding model. XFBD exerts strong inhibitory effects on both macrophage activation and migration. Moreover, through multimodal screening, we further identified the major components and compounds from the different herbs of XFBD that mediate its anti-inflammation function. Active components from XFBD, including Polygoni cuspidati Rhizoma, Phragmitis Rhizoma, and Citri grandis Exocarpium rubrum, were then found to strongly downregulate macrophage activation, and polydatin, isoliquiritin, and acteoside were identified as active compounds. Components of Artemisiae annuae Herba and Ephedrae Herba were found to substantially inhibit endogenous macrophage migration, while the presence of ephedrine, atractylenolide I, and kaempferol was attributed to these effects. In summary, our study explores the pharmacological mechanism and effective components of XFBD in inflammation regulation via multimodal approaches, and thereby provides a biological illustration of the clinical efficacy of XFBD.

Macrophage Activation Syndrome in a Context of Pre-B Type Lymphoblastic Acute Leucemia: A Case Report

Macrophage activation syndrome (MAS) is linked to inappropriate stimulation of macrophage cells in the bone marrow and lymphoid system, resulting in abnormal phagocytosis of figurative blood elements and the release of pro-inflammatory cytokines. It is a rare and serious hyper-inflammatory condition of diagnostic and therapeutic emergency. MAS is characterized by non-specific clinical and laboratory signs associated with images of hemophagocytosis. MAS is either “primary” (familial or pediatric forms), or “secondary/reactive” to infection, neoplasia, or autoimmune disease. Hemopathies dominate MAS secondary to neoplasia. B-type acute lymphoblastic leukemia (ALL) is a hematological malignancy characterized by the proliferation and accumulation of B lymphoid progenitors, blocked at an early stage of differentiation, leading to suppression of polyclonal hematopoiesis and subsequent development of signs associated with bone marrow failure. In this context, we report the observation of a macrophage activation syndrome (MAS) associated with ALL, diagnosed at Hôpital Principal de Dakar/Senegal, in a 69-year-old patient with a well-controlled type 2 diabetes under oral antidiabetic therapy (OAD) and good general condition.

FXR agonist GW4064 alleviates endotoxin-induced hepatic inflammation by repressing macrophage activation

AIM: To examine the effect of farnesoid X receptor (FXR) activation by GW4064 on endotoxin-induced hepatic inflammation in nonalcoholic fatty liver disease (NAFLD) and the underlying mechanism.

Endothelial progenitor cell-conditioned medium promotes angiogenesis and is neuroprotective after spinal cord injury

Endothelial progenitor cells secrete a variety of growth factors that inhibit inflammation, promote angiogenesis and exert neuroprotective effects. Therefore, in this study, we investigated whether endothelial progenitor cell-conditioned medium might have therapeutic effectiveness for the treatment of spinal cord injury using both in vitro and in vivo experiments. After primary culture of bone marrow-derived macrophages, lipopolysaccharide stimulation was used to classically activate macrophages to their proinflammatory phenotype. These cells were then treated with endothelial progenitor cell-conditioned medium or control medium. Polymerase chain reaction was used to determine mR NA expression levels of related inflammatory factors. Afterwards, primary cultures of rat spinal cord neuronal cells were prepared and treated with H2O2and either endothelial progenitor cell-conditioned medium or control medium. Hoechst 33258 and propidium iodide staining were used to calculate the proportion of neurons undergoing apoptosis. Aortic ring assay was performed to assess the effect of endothelial progenitor cell-conditioned medium on angiogenesis. Compared with control medium, endothelial progenitor cell-conditioned medium mitigated the macrophage inflammatory response at the spinal cord injury site, suppressed apoptosis, and promoted angiogenesis. Next, we used a rat model of spinal cord injury to examine the effects of the endothelial progenitor cell-conditioned medium in vivo. The rats were randomly administered intraperitoneal injection of PBS, control medium or endothelial progenitor cell-conditioned medium, once a day, for 6 consecutive weeks. Immunohistochemistry was used to observe neuronal morphology. Terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling assay was performed to detect the proportion of apoptotic neurons in the gray matter. The Basso, Beattie and Bresnahan Locomotor Rating Scale was used to evaluate the recovery of motor function of the bilateral hind limbs after spinal cord injury. Compared with the other two groups, the number of axons was increased, cavities in the spinal cord were decreased, the proportion of apoptotic neurons in the gray matter was reduced, and the Basso, Beattie and Bresnahan score was higher in the endothelial progenitor cell-conditioned medium group. Taken together, the in vivo and in vitro results suggest that endothelial progenitor cell-conditioned medium suppresses inflammation, promotes angiogenesis, provides neuroprotection, and promotes functional recovery after spinal cord injury.

Adult-onset Still's disease evolving with multiple organ failure and death:A case report and review of the literature

BACKGROUND Adult-onset Still’s disease(AOSD)is a rare systemic inflammatory disease,which is characterized by daily fever and arthritis,with an evanescent rash and neutrophilic leukocytosis.To date,there has been no definite laboratory or imaging test available for diagnosing AOSD;the diagnosis is one of exclusion,which can be very challenging.In particular,AOSD patients may experience different complications affecting their clinical picture,management,and prognosis.The treatment of AOSD remains largely empirical and involves therapeutic agents.CASE SUMMARY We report the case of a 36-year-old woman who presented with fever,red rash,arthralgia,and sore throat.Her serum ferritin level and white blood cell count were markedly elevated,and the first diagnosis 22 years prior was"juvenile rheumatoid arthritis of systemic type".The patient was treated with prednisone,sulfasalazine,methotrexate,and leflunomide.After remission of her symptoms,the patient stopped taking the medications,and the disease recurred.Ultimately,the patient was diagnosed with adult-onset Still's disease.Relapse occurred several times due to self-medication withdrawal,and an interleukin-6 antagonist(tocilizumab/Actemra)was administered to control the disease.Recently,she was hospitalized because an incision did not heal,and the patient suddenly developed high fever and diarrhea during hospitalization.The patient's disease progressed violently and quickly developed into macrophage activation syndrome,disseminated intravascular coagulation,shock,and multiple organ failure.The patient had sudden cardiac arrest,and she died despite emergency rescue efforts.CONCLUSION AOSD patients need regular follow-up in the long-term treatment process,and must press formulary standard medication,and do not voluntarily withdraw or reduce the dose.Otherwise it may cause disease back-and-forth or serious lifethreatening complications.Meanwhile,strict management of trauma,infections,tumors,and other diseases may contribute to improved outcomes in patients with complications.

Cytokine nanosponges suppressing overactive macrophages and dampening systematic cytokine storm for the treatment of hemophagocytic lymphohistiocytosis

Hemophagocytic lymphohistiocytosis(HLH)is a highly fatal condition with the positive feedback loop between continued immune cell activation and cytokine storm as the core mechanism to mediate multiple organ dysfunction.Inspired by macrophage membranes harbor the receptors with special high affinity for proin-flammation cytokines,lipopolysaccharide(LPS)-stimulated macrophage membrane-coated nanoparticles(LMNP)were developed to show strong sponge ability to both IFN-γand IL-6 and suppressed overactivation of macrophages by inhibiting JAK/STAT signaling pathway both in vitro and in vivo.Besides,LMNP also efficiently alleviated HLH-related symptoms including cytopenia,hepatosplenomegaly and hepatorenal dysfunction and save the life of mouse models.Furthermore,its sponge effect also worked well for five human HLH samples in vitro.Altogether,it’s firstly demonstrated that biocompatible LMNP could dampen HLH with high potential for clinical transformation,which also provided alternative insights for the treatment of other cytokine storm-mediated pathologic conditions such as COVID-19 infection and cytokine releasing syndrome during CAR-T therapy.

Effect of short- and long-term immunization of recombinant disorganized muscle protein-1(rDIM-1) against human filarial parasite Brugia malayi in rodents

Objective: To evaluate the effect of short-term and long-term immunization of recombinant disorganized muscle protein-1(r DIM-1) in rodents against human filarial parasite Brugia malayi.Methods: Recombinant Brugia malayi DIM-1(rDIM-1 bm) protein was cloned, expressed and purified using a Ni-NTA affinity column. Mastomys coucha were immunized with rDIM-1 bm in three immunization schedules: short-term(3-dose of rDIM-1 bm), and long-term(booster doses till 3-and 6-week) and subsequently challenged with infective third-stage larvae of filarial parasite Brugia malayi(L3). Microfilaraemia was monitored in L3 exposed groups on day 90 post larval inoculation(p.l.i.) and continued till day 205 p.l.i. On day 205 p.l.i. all the infected animals were killed and total worm burden was estimated. Cellular proliferative response, macrophage activity, nitric oxide(NO) release, specific IgG and its subtypes, IgE, IgA and Th1(IFN-γ, TNF-ααand IL-2) and Th2(IL-4, IL-5, IL-6, IL-10 and IL-13) cytokine release were determined. Results: Of the 3 different immunization schedules, shortterm immunization(3-dose schedule) showed better reduction in microfilarial burden(36%-63%) in the peripheral circulation, adult worm load(52%), whereas long-term immunization(3-and 6-week schedule) exerted less effect on peripheral microfilariae count(9%-58%), and adult worm burden(9%-12.5%). Short-term immunization resulted in upregulation of cellular proliferation, macrophages activity, NO release, specific IgG, IgG1, IgG2 a, Ig G2 b, IgE and IgA levels and both Th1(IFN-γ, TNF-α and IL-2) and Th2(IL-4, IL-5, IL-6, IL-10 and IL-13) cytokine release whereas long-term immunization(3-and 6-week schedule) exerted less effect on parasite burden and showed mixed immunological responses. None of the rDIM-1 bm administration schedules induced any pathology in lymphoid tissues, or alteration in mast cell number and granularity. Conclusions: The short-term immunization with rDIM-1 bm(3-dose schedule) induces robust immune responses and protects the host from filarial parasite infection.

Macrophage activation syndrome as an initial presentation of systemic lupus erythematosus

In a recent article on World J Clin Cases 2019;7:3859-3865,Sun et al reported a case of 36-year-old female with macrophage activity syndrome as an onset of systemic lupus erythematosus.Although this is a very interesting case,some concerns still need to be addressed.First,the patient had an extremely elevated serum ferritin but a normal C-reactive protein level,which was unparallel with the inflammatory condition before she received any treatments.Second,the diagnosis of systemic lupus erythematosus seemed to be insufficient according to the patient’s medical information presented,most of which were not specific to lupus but could be explained by macrophage activity syndrome.Hence,more medical information on the patient should be provided,and a profound discussion needs to be addressed.

Macrophage activation syndrome as a complication of dermatomyositis: A case report

BACKGROUND Macrophage activation syndrome(MAS)can be a fatal complication of rheumatic disorders,which occurs most commonly in patients with systemic juvenile idiopathic arthritis or systemic lupus erythematosus.It has rarely been reported in patients with dermatomyositis.Here,we describe a fatal case of MAS that developed in an adult patient with dermatomyositis.CASE SUMMARY A 44-year-old woman was admitted to our hospital with fever,generalized rash and muscle weakness.Fifteen days later,the fever persisted after the use of antibiotics,and repeat blood culture was negative.The patient then exhibited a typical Gottron sign and diffuse erythema on the face and neck,which were consistent with a diagnosis of dermatomyositis.The patient exhibited limb muscle strength of 2,and electromyography was suggestive of muscle-derived damage,which also supported a diagnosis of dermatomyositis.In addition,the patient exhibited high serum ferritin level,cytopenia,liver dysfunction,coagulopathy,enlarged spleen and hypertriglyceridemia,all of which are typical manifestations of MAS.The patient was diagnosed with dermatomyositis complicated by MAS.Although a high dose of methylprednisolone was administered for 15 d,the patient’s condition continued to deteriorate and central nervous system symptoms developed.Eventually,treatment was discontinued,and the patient died.CONCLUSION MAS is an important,potentially fatal,complication of dermatomyositis.Although MAS is rare in dermatomyositis,it should be considered in the differential diagnosis of an unexplained change of hemoglobin,platelet,fibrinogen,ferritin and triglyceride,which may complicate dermatomyositis.

Single-cell RNA sequencing in juvenile idiopathic arthritis

Juvenile idiopathic arthritis (JIA) is one of the most common chronic inflammatory rheumatic diseases in children,with onset before age 16 and lasting for more than 6 weeks.JIA is a highly heterogeneous condition with various consequences for health and quality of life.For some JIA patients,early detection and intervention remain challenging.As a result,further investigation of the complex and unknown mechanisms underlying JIA is required.Advances in technology now allow us to describe the biological heterogeneity and function of individual cell populations in JIA.Through this review,we hope to provide novel ideas and potential targets for the diagnosis and treatment of JIA by summarizing the current findings of single-cell RNA sequencing studies and understanding how the major cell subsets drive JIA pathogenesis.

Glycyrrhizic acid activates chicken macrophages and enhances their Salmonella-killing capacity in vitro

Objective： Salmonella enterica remains a major cause of food-borne disease in humans, and Salmonella Typhimurium （ST） contamination of poultry products is a worldwide problem. Since macrophages play an essential role in controlling Salmonella infection, the aim of this study was to evaluate the effect of glycyrrhizic acid （GA） on immune function of chicken HD11 macrophages. Methods： Chicken HD11 macrophages were treated with GA （0, 12.5 25, 50, 100, 200, 400, or 800 pg/ml） and lipopolysaccharide （LPS, 500 ng/ml） for 3, 6, 12, 24, or 48 h. Evaluated responses included phagocytosis, bacteria-killing, gene expression of cell surface molecules （cluster of differentiation 40 （CD40）, CD80, CD83, and CD197） and antimicrobial effectors （inducible nitric oxide synthase （iNOS）, NADPH oxidase-1 （NOX-1）, interferon-γ （IFN-γ）, LPS-induced tumor necrosis factor （TNF）-α factor （LITAF）, interleukin-6 （IL-6） and IL-IO）, and production of nitric oxide （NO） and hydrogen peroxide （H202）. Results： GA increased the internalization of both fiuorescein isothiocyanate （FITC）-dextran and ST by HD11 cells and markedly decreased the intracellular survival of ST. We found that the messenger RNA （mRNA） expression of cell surface molecules （CD40, CDSO, CD83, and CD197） and cytokines （IFN-γ, IL-6, and IL-10） of HD11 cells was up-regulated following GA exposure. The expression of iNOS and NOX-1 was induced by GA and thereby the productions of NO and H202 in HD11 cells were enhanced. Notably, it was verified that nuclear factor-κB （NF-κB） and c-Jun N-terminal kinase （JNK） pathways were responsible for GA-induced synthesis of NO and IFN-γ gene expression. Conclusions： Taken together, these results suggested that GA exhibits a potent immune regulatory effect to activate chicken macrophages and enhances Salmonella-killing capacity.

A polysaccharide purified from Radix Adenophorae promotes cell activation and pro-inflammatory cytokine production in murine RAW264.7 macrophages

Radix Adenophorae, a traditional Chinese medicine, has been reported to have a variety of biological functions. In the present study, a polysaccharide component, Radix Adenophorae Polysaccharide （RAPS）, was purified from Radix Adenophorae by decoloring with ADS-7 macroporous adsorption resin, DEAE-52 cellulose ion-exchange chromatography, and Sephacryl S-300HR gel chromatography, with the purity of 98.3% and a molecular weight of 1.8 × 104 Da. The cell viability assay and microscopic examination revealed that RAPS promoted the proliferation and activation of macrophages. At 400 μg·mL-1, RAPS stimulated RAW264.7 cell proliferation by 1.91-fold compared with the control. Meanwhile, RAPS significantly increased the secretion of pro-inflammatory cytokines （TNF-a and IL-6） in a dose-dependent manner in the supernatant of RAW264.7 cell culture as determined by ELISA. At 400 μg·mL-1, the production of TNF-a was 20.8-fold higher than that of the control. Simultaneously, the production of nitric oxide （NO） and the expression of inducible nitric oxide synthase （iNOS） were increased in RAW264.7 cells incubated with RAPS, as measured by Griess assay and Western blot analysis. The NO production of cells treated with RAPS （400 μg·mL-1） reached 15.8 μmol·L-L, which was 30.4-fold higher than that of the control （0.53 μmol·L-1）. These data suggested that RAPS may enhance the immune function and protect against exogenous pathogens by activating macrophages.

Proteomic Analysis of Macrophages:A Potential Way to Identify Novel Proteins Associated with Activation of Macrophages for Tumor Cell Killing

One major mechanism through which macrophages effectively kill tumor cells requires cell to cell contact, indicating that certain molecules expressed on cell surface of activated macrophages may mediate the tumoricidal capability. Tumor necrosis factor （TNF） and nitric oxide （NO） are the two classical mediators of tumor cell death. However, evidence of discrepancy is accumulating indicating these known mediators do not appear to account for the broad and potent tumoricidal activity of macrophages. To obtain a full repertoire of tumoricidal activation-associated membrane proteins, we combined one-dimensional SDS-PAGE with capillary liquid chromatographytandem mass spectrometry （LC-MS/MS）. Using this technique, we identified 454 activated macrophage specifically expressed proteins with extremely high confidence, including most known activation markers of macrophages, such as NO synthase （iNOS）, Ym1, cyclooxygenase, etc. Membrane bound TNF-α was also identified on activated macrophages. However, it was also detected on thioglycolate elicited macrophages, indicating this molecule may not play a key role in conjugation-dependent tumor cell killing. In contrast, although NO has not been assigned as an effector molecule of conjugation-dependent tumoricidal pathway, iNOS was identified from membrane fraction of activated macrophages, suggesting NO may be involved in conjugation-dependent tumoricidal mechanism, because iNOS association with plasma membrane is ideally suited to deliver NO directly into the contacted tumor cells. This research provides not only new insights into macrophage conjugation-dependent tumoricidal mechanisms, but also a valuable data set of macrophage activation associated membrane proteins, thus providing better understanding of the functional mechanisms of macrophages in anti-tumor and other biological processes.

nduction of M2-1ike macrophages in recipient NOD-scid mice by allogeneic donor CD4＋CD25＋ regulatory T cells

CD4＋CD25＋ regulatory T cells （Tregs） play an important role in maintaining host immune tolerance via regulation of the phenotype and function of the innate and adaptive immune cells. Whether allogeneic CD4＋CD25＋ Tregs can regulate recipient mouse macrophages is unknown. The effect of allogeneic donor CD4＋CD25＋ Tregs on recipient mouse resident F4/80＋macrophages was investigated using a mouse model in which allogeneic donor CD4＋CD25＋ Tregs were adoptively transferred into the peritoneal cavity of host NOD-scid mice. The phenotype and function of the recipient macrophages were then assayed. The peritoneal F4/80＋ macrophages in the recipient mice that received the allogeneic CD4＋CD25＋ Tregs expressed significantly higher levels of CD23 and programmed cell death-ligand I（PD-L1） and lower levels of CD80, CD86, CD40 and MHC II molecules compared to the mice that received either allogeneic CD4＋CD25- T cells （Teffs） or no cells. The resident F4/80＋ macrophages of the recipient mice injected with the allogeneic donor CD4＋CD25＋ Tregs displayed significantly increased phagocytosis of chicken red blood cells （cRBCs） and arginase activity together with increased IL-IO production, whereas these macrophages also showed decreased immunogenicity and nitric oxide （NO） production. Blocking arginase partially but significantly reversed the effects of CD4＋CD25＋ Tregs with regard to the induction of the M2 macrophages in vivo. Therefore, the allogeneic donor CD4＋CD25＋ Tregs can induce the M2 macrophages in recipient mice at least in part via an arginase pathway. We have provided in vivo evidence to support the unknown pathways by which allogeneic donor CD4＋CD25＋ Tregs regulate innate immunity in recipient mice by promoting the differentiation of M2 macrophages.

Kv1.3 channel blockade enhances the phagocytic function of RAW264.7 macrophages

This study aimed to comprehend the largely unknown role of voltage-gated potassium channel 1.3 （Kvl.3） in the phagocytic function of macrophages. We found that blocking of the Kv 1.3 channel with 100 pmol L 1 Stichodactyla helianthus neurotoxin （ShK） enhanced the phagocytic capacities of both resting and lipopolysaccharide （LPS）-stimulated RAW264.7 macrophages in the chicken erythrocyte system. In the fluorescein isothiocyanate （FITC）-labeled Escherichia coli k-12 system, ShK increased the phagocytic capacities of resting RAW264.7 cells, but not of the LPS-stimulated cells, as LPS alone stimulated almost satu- rated phagocytosis of the macrophages. ShK increased the nitric oxide （NO） production in LPS-activated cells, but not in rest- ing RAW264.7 cells. There was no effect of ShK alone on the cytokine secretions in resting RAW264.7 cells, but it suppressed IL-113 secretion in LPS-stimulated RAW264.7 cells. At a concentration of 100 pmol L 1, ShK did not affect the viability of the tested cells. Kv 1.3 was expressed in RAW264.7 cells; this expression was downregulated by LPS, but significantly upregulat- ed by disrupting caveolin-dependent endocytosis with filipin III. In addition, cytochalasin D, an inhibitor of actin polymeriza- tion, did not affect the Kvl.3 expression. Thus, blocking of the Kvl.3 channel enhances the phagocytic capacity and NO pro- duction of this cell line. Our results suggest that Kv 1.3 channel serves as a negative regulator of phagocytosis in macrophages and can therefore be a potential target in the treatment of macrophage dysfunction.

Engineering nano-structures with controllable dimensional features on micro-topographical titanium surfaces to modulate the activation degree of M1 macrophages and their osteogenic potential

Modulating the activation state and degree of macrophages still remains as a challenge for the topographical design of Ti-based implants.In this work,micro/nano-structured coatings were prepared on Ti substrates by micro-arc oxidation(MAO)and subsequent hydrothermal(HT)treatment.By varying the HT conditions,plate-like nano-structures with an average length of 80,440 or 780 nm were obtained on MAO-prepared micro-topographical surfaces.Depending on the dimensional features of nano-plates,the specimens were noted as Micro,Micro/Nano-180,Micro/Nano-440 and Micro/Nano-780,respectively.The in vitro results showed that the activation state and degree of macrophages could be effectively modulated by the micro/nano-structured surfaces with various dimensional features.Compared to the Micro surface,the Micro/Nano-180 surface activated both M1 and M2 phenotype in macrophages,while the Micro/Nano-440 and Micro/Nano-780 surfaces polarized macrophages to their M1 phenotype.The activation degree of M1 macrophages followed the trend:Micro<Micro/Nano-180<Micro/Nano-440<Micro/Nano-780.However,the osteogenic potential of the activated macrophages in response to various surfaces were in the order:Micro≈Micro/Nano-780<Micro/Nano-180<Micro/Nano-440.Together,the findings presented in this work indicate that engineering nano-structures with controllable dimensional features is a promising strategy to modulate macrophage activation state and degree.In addition,it is essential to determine the appropriate activation degree of M1 macrophages for enhanced osteogenesis.

Optical-resolution photo acoustic microscopy continually monitors macrophages activities of acute inflammation in vivo

Photoacoustic imaging has been developed to image the immune study at the macro scale.Macrophages play diverse roles in the acute response to infection and tissue repair.However,macrophages activities in acute inflammation at the microscopic level still remain challenging.In this work,we proposed optical-resolution photoacoustic microscopy to promptly monitor the labeled macrophages activities in normal and inflammatory groups.The result showed that many labeled macrophages emerged around the vessels firstly,then exuded into tissues,and finally disappeared in the inflammatory group injected with labeled macrophages.In summary,our method allows us to exactly image and track the immune cells of inflammatory diseases.