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Malaria is a frequent parasitic infection becomes life threatening due to the disequilibrated immune responses of the host. Avid phagocytosis of malarial pigment hemozoin (HZ) and HZ-containing Plasmodium parasites incapacitates monocyte functions by bioactive lipoperoxidation products 4-hydroxynonenal (4-HNE) and hydroxyeicosatetraenoic acids (HETEs). CYP4F conjugation with 4-HNE is hypothesised to inhibit ω-hydroxylation of 15-HETE, leading to sustained monocyte dysfunction caused by 15-HETE accumulation. A combined immunochemical and mass-spectrometric approach identified 4-HNE-conjugated CYP4F11 in primary human HZ-laden and 4-HNE-treated monocytes. Six distinct 4-HNE-modified amino acid residues were revealed, of which C260 and H261 are localized in the substrate recognition site of CYP4F11. Functional consequences of enzyme modification were investigated on purified human CYP4F11. Palmitic acid, arachidonic acid, 12-HETE, and 15-HETE bound to unconjugated CYP4F11 with apparent dissociation constants of 52, 98, 38, and 73 µM, respectively, while in vitro conjugation with 4-HNE completely blocked substrate binding and enzymatic activity of CYP4F11. Gas chromatographic product profiles confirmed that unmodified CYP4F11 catalysed the ω-hydroxylation while 4-HNE-conjugated CYP4F11 did not. The 15-HETE dose dependently recapitulated the inhibition of the oxidative burst and dendritic cell differentiation by HZ. The inhibition of CYP4F11 by 4-HNE with consequent accumulation of 15-HETE is supposed to be a crucial step in immune suppression in monocytes and immune imbalance in malaria.

13 p.-4 fig., Common variable immunodeficiency disorders (CVID), the most common primary immune deficiency, includes heterogeneous syndromes characterized by hypogammaglobulinemia and impaired antibody responses. CVID patients frequently suffer from recurrent infections and inflammatory conditions. Currently, immunoglobulin replacement therapy (IgRT) is the first-line treatment to prevent infections and aminorate immune alterations in CVID patients. Intravenous Immunoglobulin (IVIg), a preparation of highly purified poly-specific IgG, is used for treatment of immunodeficiencies as well as for autoimmune and inflammatory disorders, as IVIg exerts immunoregulatory and anti-inflammatory actions on innate and adaptive immune cells. To determine the mechanism of action of IVIg in CVID in vivo, we determined the effect of IVIg infusion on the transcriptome of peripheral blood mononuclear cells from CVID patients, and found that peripheral blood monocytes are primary targets of IVIg in vivo, and that IVIg triggers the acquisition of an anti-inflammatory gene profile in human monocytes. Moreover, IVIg altered the relative proportions of peripheral blood monocyte subsets and enhanced the proportion of CD14+ cells with a transcriptional, phenotypic, and functional profile that resembles that of monocytic myeloid-derived suppressor cells (MDSC). Therefore, our results indicate that CD14 + MDSC-like cells might contribute to the immunoregulatory effects of IVIg in CVID and other inflammatory disorders., Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported by grants from Ministerio de Economía y Competitividad (SAF2017-83785-R)to MAV and ALC, Grant 201619.31 from Fundación La Marató/TV3 to ALC, and Red de Investigación en Enfermedades Reumáticas(RIER, RD16/0012/0007), and cofinanced by the European Regional Development Fund “A way to achieve Europe” (ERDF), to ALC, and grant 19/284-E from Instituto de Salud Carlos III (ISCIII) to SSR. MS-F was funded by a Formación de Personal Investigador predoctoral fellowship from MINECO (grant PRE2018-083396).

Made available in DSpace on 2021-06-25T11:14:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-03-04 OBJECTIVES: Propolis is a bee-made product used for centuries due to its diverse biological properties, including its immunomodulatory action. This work aimed at investigating whether propolis may affect monocyte functions challenged with retinoic acid (RA), B subunit of Escherichia coli heat-labile enterotoxin (EtxB), human melanoma-associated antigen-1 (MAGE-1) and lipopolysaccharide (LPS). METHODS: Monocytes from healthy donors were treated with the stimuli separately or in the presence of propolis. Cell viability was evaluated by MTT assay, cell marker expression was assessed by flow cytometry, cytokine production by ELISA, gene expression by RT-qPCR. KEY FINDINGS: Propolis alone maintained TLR-2, TLR-4, HLA-DR, CD40 and CD80 expression in the monocytes; however, its combination with either MAGE-1 or LPS decreased CD40 expression triggered by the stimuli. Propolis maintained RA action on cell marker expression. Propolis inhibited TNF-α (with either EtxB or MAGE-1) and IL-6 (with either RA or MAGE-1), and increased IL-10 (with MAGE-1) production. Propolis downmodulated LC3 expression induced by LPS. It also induced a lower NF-kB expression than control cells and its combination with RA induced a higher expression than the stimulus alone. CONCLUSIONS: Propolis potentially affected innate immunity by downmodulating the monocytes pro-inflammatory activity. Department of Chemical and Biological Sciences Institute of Biosciences São Paulo State University (UNESP) Botucatu Blood Center School of Medicine São Paulo State University (UNESP) Center for Neurosciences and Cellular Biology Faculty of Pharmacy University of Coimbra Department of Chemical and Biological Sciences Institute of Biosciences São Paulo State University (UNESP) Botucatu Blood Center School of Medicine São Paulo State University (UNESP)

Primary Sjogren’s syndrome (pSS) is a systemic autoimmune disease that is characterized by the infiltration of immune cells. Although the loss of salivary gland function is a major manifestation observed in pSS, the factors that could promote these changes in salivary gland tissue in pSS is not yet determined. Herein, we provide evidence that loss of alpha-1 antiproteinase antitrypsin could contribute to the induction of pSS. Alpha-1 antiproteinase antitrypsin belongs to the family of serpin proteins that function as protease inhibitors and protect secretory cells against proteases, especially to elastases that is secreted from lymphocytes. Importantly, expression of alpha-1 antiproteinase antitrypsin was decreased (more than 3-fold), along with an increase in elastase expression, in pSS samples when compared with age-matched non-SS-SICCA patients. Consistent with the human data, loss of alpha-1 antiproteinase antitrypsin, as well as an increase in immune infiltration, was observed in IL14α transgenic mice that exhibit SS like symptoms. Moreover, an age-dependent increase in elastase expression was observed in IL14α transgenic mice along with a decrease in total saliva secretion. Importantly, a 4-fold increase in microRNA132 expression, but not in other microRNAs, and increased DNA methylation in the promoter/noncoding region of serpina gene was observed in pSS, which could be responsible for the inhibition of alpha-1 antiproteinase antitrypsin expression in salivary gland cells of pSS patients. Together, these findings demonstrate that epigenetic regulations that include DNA methylation and microRNAs that could modulate the expression of alpha-1 antiproteinase antitrypsin in salivary glands and could be involved in the onset of pSS.

Decidual macrophages are in close contact with trophoblast cells during placenta development, and an appropriate crosstalk between these cellular compartments is crucial for the establishment and maintenance of a healthy pregnancy. During different phases of gestation, macrophages undergo dynamic changes to adjust to the different stages of fetal development. Trophoblast-secreted factors are considered the main modulators responsible for macrophage differentiation and function. However, the phenotype of these macrophages induced by trophoblast-secreted factors and the factors responsible for their polarization has not been elucidated. In this study, we characterized the phenotype and function of human trophoblast-induced macrophages. Using in vitro models, we found that human trophoblast educated macrophages (TEMs) were CD14(+)CD206(+)CD86(−) and presented an unusual transcriptional profile in response to TLR4/LPS activation characterized by the expression of type 1 Interferon beta (IFN-β) expression. IFN-β further enhance the constitutive production of soluble programmed cell death ligand 1 (PD-L1) from trophoblast cells. PD-1 blockage inhibited trophoblast induced macrophage differentiation. Soluble PD-L1 was detected in the blood of pregnant women and increased throughout the gestation. Collectively, our data suggest the existence of a regulatory circuit at the maternal fetal interface wherein IFN-β promotes sPD-L1 expression/secretion by trophoblast cells, which can then initiate a PD-L1/PD-1 mediated macrophage polarization towards an M2 phenotype, consequently decreasing inflammation. Macrophages then maintain the expression of sPD-L1 by the trophoblasts through IFN-β production induced through TLR4 ligation. Circulating PD-L1 might also function as a marker for normal trophoblast immune modulation during early pregnancy.

BackgroundRespiratory distress syndrome (RDS) is the most common respiratory disease in premature infants. Exogenous natural surfactant preparations are used in the treatment of RDS. In recent years, it has become increasingly evident that surfactant plays an immunoregulatory role. ObjectivesThe aim of this study was to evaluate cytokine and chemokine response following three different regimens of natural surfactant treatment in preterm newborns with RDS. MethodsPremature newborns below 32 weeks of gestation who were intubated for RDS and given early surfactant rescue therapy were included in the study. Newborns were randomly divided into three groups and Beractant 100 mg/kg (B-100), Poractant alfa 100 mg/kg (P alpha-100) and Poractant alfa 200 mg/kg (P alpha-200) were administered intratracheally. Blood samples and transtracheal aspirates (TA) were collected just before and 4-6 h after the surfactant treatment. Total eosinophil count, inducible T Cell alpha chemoattractant (ITaC), macrophage inflammatory protein 3 beta (MIP3b), interleukins (IL) 5, 8, 9, 10, 13, immunoglobulin E (IgE), interferon gamma (IFN-gamma), eotaxin and tumor necrosis factor beta-1 (TGF-beta 1) were measured from blood and tracheal aspirate samples. ResultsA total of 45 infants, 15 in each group, were included in the study. Mean gestational age, birth weight, antenatal, demographic and clinical characteristics of the study groups were similar. IFN gamma concentration and eosinophil counts in TA decreased after surfactant replacement in all groups, especially in the infants treated with P alpha-100 and P alpha-200. Eotaxin, TGF beta and IL-8 concentrations in TA increased significantly in the infants treated with P alpha-100 and P alpha-200. IL-9 levels in TA decreased in the B-100 group but increased in the P alpha-100 and P alpha-200 groups. Blood levels of cytokines and chemokines showed significantly decreased levels of ITaC and MIP3b only in the B-100 group, but no significant change was observed in the P alpha-100 and P alpha-200 groups. ConclusionIn our study, the different immunomodulatory effects of natural surfactant preparations on newborn lung is proven. We found that Poractant alpha, one of the natural surfactant preparations, shifted the lung immune system toward TH2., Ege University Scientific Research Projects Unit [MY-13-TIP-030], This study was carried out with the financial support of the Ege University Scientific Research Projects Unit (MY-13-TIP-030).

Das Immunsystem besitzt die herausragende F��higkeit anhand evolution��r konservierter, pathogen-assoziierter Muster zwischen k��rpereigenen und fremden Strukturen zu unterscheiden. Die Detektion von Mikroorganismen l��st eine skalierte Immunreaktion aus, die von einer Reihe von Faktoren abh��ngig ist. Im Falle einer Infektion mit Bakterien spielen dabei unter anderem die Virulenz, die Lokalisation der Infektion als auch die Lebendigkeit der Bakterien eine Rolle. Sander et al. konnten 2011 zeigen, dass murine Makrophagen die Vitalit��t von Bakterien anhand ihrer RNA erkennen und daraufhin das NLRP3-Inflammasom aktivieren. Dieses ist ein zytosolischer Proteinkomplex, welcher eine Reihe von endogenen und exogenen Stimuli erkennt und unter anderem die Vorstufe von IL-1�� in seine aktive Form ��berf��hrt. Die Mechanismen der Inflammasomaktivierung durch lebende Bakterien in humanen Monozyten sind jedoch unklar. Wir fanden eine NLRP3-abh��ngige IL-1�� Produktion, jedoch zeigte die NLRP3-Aktivierung durch lebende E. coli in humanen Monozyten keine Merkmale der kanonischen Inflammasomaktivierung, wie Abh��ngigkeit von der extrazellul��ren Kaliumkonzentration, Pyroptose und die Aggregation sogenannter Pyroptosome. Stattdessen ��hnelt der Prozess einer k��rzlich beschriebenen durch Toll-like Rezeptor 4 (TLR4) ausgel��sten alternativen Inflammasomaktivierung. Wir untersuchten daraufhin die zugrundeliegenden Mechanismen und die involvierten Rezeptor- und Adaptermolek��le. Dazu verwendeten wir neben prim��ren Monozyten die humane BlaER1-Zelllinie, die nach Transdifferenzierung einen monozyt��ren Ph��notyp annimmt und in welcher die Existenz des alternativen Inflammasoms zum ersten Mal beschrieben wurde. Wir konnten zeigen, dass die alternative Inflammasomaktivierung durch bakterielle RNA ausgel��st wird. Die Inflammasom-aktivierung und IL-1�� Freisetzung durch lebende E. coli wird vermittelt durch NLRP3, ASC und Caspase-1. W��hrend die Induktion von IL-1�� durch tote E. coli TLR4 ben��tigte und damit auf das LPS zur��ckzuf��hren ist, aktivierten lebende E. coli ��ber ihre RNA zus��tzlich einen Signalweg ��ber den RNA-Rezeptor TLR8 und das Adaptermolek��l MyD88. Diese Befunde stellen erneut die Rolle von TLR8 f��r die Erkennung von mikrobieller Vitalit��t heraus. Die Aktivierung von RNA-Rezeptoren wie TLR8 ist somit ein zentraler Mechanismus in der angeborenen Immunantwort gegen lebende Mikroorganismen., The immune system has the outstanding ability to distinguish between endogenous and foreign structures based on evolutionarily conserved pathogen-associated patterns. The detection of microorganisms triggers a scaled immune response that depends on a number of factors. In case of bacterial infection, virulence, localization of the infection as well as bacterial viability play a role among others. In 2011, Sander et al. showed that murine macrophages recognize the viability of bacteria by their RNA and subsequently activate the NLRP3 inflammasome. This is a cytosolic protein complex that recognizes a range of endogenous and exogenous stimuli and as a result converts the precursor of IL-1�� into its active form. However, the mechanisms of inflammasome activation by live bacteria in human monocytes remain unclear. We found NLRP3-dependent IL-1�� production, but NLRP3 activation by live E. coli in human monocytes did not show features of canonical inflammasome activation, such as dependence on extracellular potassium concentration, pyroptosis, and aggregation of so-called pyroptosomes. Instead, the process resembles a recently described alternative inflammasome activation via Toll-like receptor 4 (TLR4). Consequently, we investigated the underlying mechanisms and the receptor and adaptor molecules involved. For this purpose, in addition to primary monocytes, we used the human BlaER1 cell line, which assumes a monocytic phenotype after transdifferentiation and in which the existence of the alternative inflammasome was described for the first time. We demonstrated that alternative inflammasome activation is triggered by bacterial RNA. Inflammasome activation and IL-1�� release by live E. coli is mediated by NLRP3, ASC, and Caspase-1. Whereas the induction of IL-1�� by heat-killed E. coli required TLR4 and was thus due to the LPS, live E. coli additionally activated a signaling pathway via the RNA receptor TLR8 and the adaptor molecule MyD88. These findings again highlight the role of TLR8 in the recognition of microbial viability. The activation of RNA receptors such as TLR8 is a central mechanism in the innate immune response against living microorganisms.

Summary MV130 is an inactivated polybacterial mucosal vaccine that confers protection to patients against recurrent respiratory infections, including those of viral etiology. However, its mechanism of action remains poorly understood. Here, we find that intranasal prophylaxis with MV130 modulates the lung immune landscape and provides long-term heterologous protection against viral respiratory infections in mice. Intranasal administration of MV130 provides protection against systemic candidiasis in wild-type and Rag1-deficient mice lacking functional lymphocytes, indicative of innate immune-mediated protection. Moreover, pharmacological inhibition of trained immunity with metformin abrogates the protection conferred by MV130 against influenza A virus respiratory infection. MV130 induces reprogramming of both mouse bone marrow progenitor cells and in vitro human monocytes, promoting an enhanced cytokine production that relies on a metabolic shift. Our results unveil that the mucosal administration of a fully inactivated bacterial vaccine provides protection against viral infections by a mechanism associated with the induction of trained immunity., Graphical abstract, Highlights • MV130 reduces morbi-mortality in mouse models of viral respiratory infection • MV130 modulates mouse immunity complying with key features of trained immunity • MV130 promotes reprogramming of mouse bone marrow progenitors • MV130 induces trained immunity in human monocytes, Brandi et al. investigate the mechanism of action of an inactivated polybacterial mucosal immunomodulator, MV130, which confers protection in patients suffering from recurrent respiratory infections. They show that MV130 induces trained immunity both in mouse bone marrow progenitors, resulting in protection from viral infection, and in human monocytes in vitro.

Several studies have attributed the onset and progression of several diseases, including degenerative and cardiovascular diseases, cell aging and cancer, to the production of free radicals and inflammatory processes. Consequently, a large number of investigations have been carried out in order to find new, safer and potent antioxidant and anti-inflammatory drugs, and to elucidate their corresponding mechanisms of action. The evaluation of antioxidant vs antiradicalar and anti-inflammatory activity of phenolic compounds, either of natural or synthetic origin, is nowadays an important area of research in the field of medicinal chemistry. This work presents a brief introduction about oxidative stress and the origin of reactive oxygen and nitrogen species, and about inflammation and the cells involved in this process, specifically monocytes. The relationship between the inflammatory process and the oxidative burst produced by human monocytes is addressed. Polyphenols, secondary metabolites of several plants, are recognized as an important source of flavonoids, including 2-styrylchromones, which have demonstrated important biological activities. Thus, several examples of (E)-2- styrylchromones of natural and synthetic origin and the biological activities associated with them are presented herein, with emphasis on antioxidant and anti-inflammatory activities. The major goal of this work relies on the design and synthesis of a library of nine (E)-2-styrylchromones and their inhibitory effects on the oxidative burst produced by human monocytes and the establishment of structure-activity relationship studies (SARS). At last, the most used methods for the synthesis of (E)-2-styrylchromones containing relevant substituent groups to their antioxidant and anti-inflammatory activity, namely hydroxyl and halogen groups, as well as the procedures for the in chemico and in vitro biological assays, are described. O aparecimento e progressão de várias doenças, incluindo doenças neurodegenerativas, cardiovasculares, envelhecimento celular e cancro, têm sido associados à produção de radicais livres assim como a processos inflamatórios, de acordo com vários estudos realizados. Por conseguinte, têm sido realizadas diversas investigações com a finalidade de encontrar novos fármacos com potencial antioxidante e anti-inflamatório, mais eficazes e seguros, e de compreender os seus mecanismos de ação. Atualmente, a avaliação da atividade antioxidante e/ou antirradicalar e anti-inflamatória de compostos polifenólicos, classe de compostos onde se incluem as 2- estirilcromonas, quer de origem natural quer sintéticos, é uma área de estudo de grande interesse. Neste trabalho apresenta-se uma breve introdução sobre o stress oxidativo e a origem das espécies reativas de oxigénio e nitrogénio e sobre a inflamação e as células intervenientes neste processo, mais especificamente, acerca dos monócitos. Aborda-se a relação entre o processo inflamatório e o burst oxidativo produzido por monócitos humanos. Os polifenóis, metabolitos secundários de várias plantas, são reconhecidos como uma fonte importante de flavonóides, incluindo 2-estirilcromonas, que têm demonstrado importantes atividades biológicas. Seguidamente são apresentados vários exemplos de (E)-2-estirilcromonas de origem natural e sintética e as atividades biológicas que lhes estão associadas, destacando-se as atividades antioxidantes e anti-inflamatórias. O grande foco deste trabalho reside no desenho e síntese de um painel de nove (E)-2-estirilcromonas e o seu efeito inibitório no burst oxidativo produzido por monócitos humanos e ainda o estudo da relação estrutura-atividade (SARS). Por fim, são descritos os métodos mais utilizados para a síntese de (E)-2-estirilcromonas contendo grupos substituintes relevantes para a sua atividade antioxidante e antiinflamatória, nomeadamente grupos hidroxilo e halogéneos, assim como os métodos utilizados nos ensaios in chemico e in vitro. Mestrado em Bioquímica

At sites of inflammation, monocytes carry out specific immune functions while facing challenging metabolic restrictions. Here, we investigated the potential of human monocytes to adapt to conditions of gradually inhibited oxidative phosphorylation (OXPHOS) under glucose free conditions. We used myxothiazol, an inhibitor of mitochondrial respiration, to adjust two different levels of decreased mitochondrial ATP production. At these levels, and compared to uninhibited OXPHOS, we assessed phagocytosis, production of reactive oxygen species (ROS) through NADPH oxidase (NOX), expression of surface activation markers CD16, CD80, CD11b, HLA-DR, and production of the inflammatory cytokines IL-1β, IL-6 and TNF-α in human monocytes. We found phagocytosis and the production of IL-6 to be least sensitive to metabolic restrictions while surface expression of CD11b, HLA-DR, production of TNF-α, IL-1β and production of ROS through NOX were most compromised by inhibition of OXPHOS in the absence of glucose. Our data demonstrate a short-term hierarchy of immune functions in human monocytes, which represents novel knowledge potentially leading to the development of new therapeutics in monocyte-mediated inflammatory diseases.

At sites of inflammation, monocytes carry out specific immune functions while facing challenging metabolic restrictions. Here, we investigated the potential of human monocytes to adapt to conditions of gradually inhibited oxidative phosphorylation (OXPHOS) under glucose free conditions. We used myxothiazol, an inhibitor of mitochondrial respiration, to adjust two different levels of decreased mitochondrial ATP production. At these levels, and compared to uninhibited OXPHOS, we assessed phagocytosis, production of reactive oxygen species (ROS) through NADPH oxidase (NOX), expression of surface activation markers CD16, CD80, CD11b, HLA-DR, and production of the inflammatory cytokines IL-1 beta, IL-6 and TNF-alpha in human monocytes. We found phagocytosis and the production of IL-6 to be least sensitive to metabolic restrictions while surface expression of CD11b, HLA-DR, production of TNF-alpha, IL-1 beta and production of ROS through NOX were most compromised by inhibition of OXPHOS in the absence of glucose. Our data demonstrate a short-term hierarchy of immune functions in human monocytes, which represents novel knowledge potentially leading to the development of new therapeutics in monocyte-mediated inflammatory diseases.

The fusion process of mononuclear monocytes into multinuclear osteoclasts in vitro is an essential process for the study of osteoclastic resorption of biomaterials. Thereby biomaterials offer many influencing factors such as sample shape, material composition, and surface topography, which can have a decisive influence on the fusion and thus the entire investigation. For the specific investigation of resorption, it can therefore be advantageous to skip the fusion on samples and use mature, predifferentiated osteoclasts directly. However, most conventional detachment methods (cell scraper, accutase), lead to a poor survival rate of osteoclasts or to a loss of function of the cells after their reseeding. In the present study different conventional and novel methods of detachment in combination with different culture surfaces were investigated to obtain optimal osteoclast differentiation, yield, and vitality rates without loss of function. The innovative method&mdash, using thermoresponsive surfaces for cultivation and detachment&mdash, was found to be best suited. This is in particular due to its ability to maintain osteoclast activity, as proven by TRAP 5b-, CTSK-activity and resorption pits on dentin discs and decellularized osteoblast-derived matrix plates. In conclusion, it is shown, that osteoclasts can be predifferentiated on cell culture dishes and transferred to a reference biomaterial under preservation of osteoclastic resorption activity, providing biomaterial researchers with a novel tool for material characterization.

Background: At sites of inflammation, monocytes require energy and building blocks to fulfil immunological functions. They must cope with immunometabolic restrictions such as hypoxia and low glucose levels in the microenvironment. Objective: To investigate the adaptive potential of human monocytes, we modelled conditions of immunometabolic restriction by gradual inhibition of OXPHOS in the absence of glucose. Methods: Monocytes of healthy donors were cultivated under glucose free conditions, and mitochondrial ATP production was gradually decreased by Myxothiazol, an inhibitor of the respiration chain. For three different immunometabolic levels upon stimulation with LPS, we assessed phagocytosis, the expression of surface activation markers CD16, CD80, CD11b, HLA-DR and production of the inflammatory cytokines IL-1��, IL-6 and TNF-�� by flow cytometry. With a Clark type electrode, we assessed ATP production and the production of reactive oxygen species through NOX amperometrically. ATP content was determined by luminometric assessment. Results: Monocytes showed no sign of apoptosis following inhibition of mitochondrial respiration. In our model, the effect of immunometabolic restriction was most pronounced on the surface expression of CD11b, HLA-DR, production of TNF-��, IL-1�� and production of ROS through NOX. Phagocytosis and the production of IL-6 were less affected. Conclusion: Our data demonstrate a hierarchy of immune functions in LPSstimulated human monocytes. This represents novel knowledge, which could potentially lead to the development of new therapeutic targets in monocyte-mediated inflammatory diseases., Hintergrund: Monozyten ben��tigen zur Ausf��hrung spezifischer immunologischer Prozesse Energie und biosynthetische Substrate. Diese stehen in der Mikroumgebung einer Entz��ndung aufgrund von u.a. Hypoxie und Glukosemangel nur begrenzt zur Verf��gung. Fragestellung: Die Anpassung der Monozyten an metabolisch ung��nstige Bedingungen sollte hier mithilfe einer stufenweise gehemmten OXPHOS unter Glukoseentzug untersucht werden. Hierbei sollte herausgefunden werden, ob spezifische immunologische Prozesse unter diesen Bedingungen einer Hierarchie unterliegen. Methoden: Monozyten gesunder Spender wurden unter Glukoseentzug kultiviert, die mitochondriale ATP-Produktion wurde durch den Atmungskettenhemmstoff Myxothiazol stufenweise reduziert. F��r insgesamt 3 Belastungsstufen mit Myxothiazol untersuchten wir durchflusszytometrisch die Phagozytosekapazit��t, die Expression der Oberfl��chenmolek��le CD16, CD80, CD11b, HLA-DR sowie die Produktion der inflammatorischen Zytokine IL-1��, IL-6 und TNF-��. Mit einer Clark-Elektrode wurden die Produktion von ROS durch NOX und die mitochondriale ATP-Produktion amperometrisch untersucht, die ATP-Menge der Zellen wurde luminometrisch bestimmt. Ergebnisse: Die Monozyten zeigten keine Zeichen einer Apoptose unter Glukoseund/oder Energieentzug. Die metabolische Belastung beeintr��chtigte vor allem die Expression der Marker CD11b und HLA-DR, die Produktion von TNF-�� und IL-1�� sowie die ROS Produktion durch NOX. Sie zeigte einen geringeren Effekt auf die Phagozytose und die IL-6 Produktion. Schlussfolgerung: Unsere Ergebnisse sprechen klar f��r eine Hierarchie der Immunprozesse menschlicher, LPS-stimulierter Monozyten unter metabolischer Belastung. Dieses Wissen k��nnte zur Entwicklung zuk��nftiger Therapieans��tze f��r Monozyten-vermittelte entz��ndliche Erkrankungen f��hren.

The chlamydial plasmid, an essential virulence factor, encodes plasmid proteins that play important roles in chlamydial infection and the corresponding immune response. However, the virulence factors and the molecular mechanisms of Chlamydia psittaci are not well understood. In the present study, we investigated the roles and mechanisms of the plasmid-encoded protein CPSIT_P7 of C. psittaci in regulating the inflammatory response in THP-1 cells (human monocytic leukemia cell line). Based on cytokine arrays, CPSIT_P7 induces the expression of interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1) in THP-1 cells. Moreover, the expression levels of IL-6, IL-8, and MCP-1 stimulated by CPSIT_P7 declined after silencing of the Toll-like receptor 4 (TLR4) gene using small interfering RNA and transfection of a dominant negative plasmid encoding TLR4 (pZERO-hTLR4). We further demonstrated that transfection with the dominant negative plasmid encoding MyD88 (pDeNy-hMyD88) and the dominant negative plasmid encoding Mal (pDeNy-hMal) could also abrogate the expression of the corresponding proteins. Western blot and immunofluorescence assay results showed that CPSIT_P7 could activate nuclear factor κB (NF-κB) signaling pathways in THP-1 cells. Altogether, our results indicate that the CPSIT_P7 induces the TLR4/Mal/MyD88/NF-κB signaling axis and therefore contributes to the inflammatory cytokine response.

Cryptococcus neoformans is a human fungal pathogen that adapts its metabolism to cope with limited oxygen availability, nutrient deprivation and host phagocytes. To gain insight into cryptococcal metabolism, we optimized a protocol for the Seahorse Analyzer, which measures extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) as indications of glycolytic and respiratory activities. In doing so we achieved effective immobilization of encapsulated cryptococci, established Rotenone/Antimycin A and 2-deoxyglucose as effective inhibitors of mitochondrial respiration and glycolysis, respectively, and optimized a microscopy-based method of data normalization. We applied the protocol to monitor metabolic changes in the pathogen alone and in co-culture with human blood-derived monocytes. We also compared metabolic flux in wild-type C. neoformans, its isogenic 5-PP-IP5/IP7-deficient metabolic mutant kcs1∆, the sister species of C. neoformans, Cryptococcus deuterogattii/VGII, and two other yeasts, Saccharomyces cerevisiae and Candida albicans. Our findings show that in contrast to monocytes and C. albicans, glycolysis and respiration are tightly coupled in C. neoformans and C. deuterogattii, as no compensatory increase in glycolysis occurred following inhibition of respiration. We also demonstrate that kcs1∆ has reduced metabolic activity that correlates with reduced mitochondrial function. Metabolic inflexibility in C. neoformans is therefore consistent with its obligate aerobe status and coincides with phagocyte tolerance of ingested cryptococcal cells.

3-bromopyruvate (3-BP) is a small molecule with anticancer and antimicrobial activities. 3-BP is taken up selectively by cancer cells&rsquo, mono-carboxylate transporters (MCTs), which are highly overexpressed by many cancers. When 3-BP enters cancer cells it inactivates several glycolytic and mitochondrial enzymes, leading to ATP depletion and the generation of reactive oxygen species. While mechanisms of 3-BP uptake and its influence on cell metabolism are well understood, the impact of 3-BP at certain concentrations on DNA integrity has never been investigated in detail. Here we have collected several lines of evidence suggesting that 3-BP induces DNA damage probably as a result of ROS generation, in both yeast and human cancer cells, when its concentration is sufficiently low and most cells are still viable. We also demonstrate that in yeast 3-BP treatment leads to generation of DNA double-strand breaks only in S-phase of the cell cycle, possibly as a result of oxidative DNA damage. This leads to DNA damage, checkpoint activation and focal accumulation of the DNA response proteins. Interestingly, in human cancer cells exposure to 3-BP also induces DNA breaks that trigger H2A.X phosphorylation. Our current data shed new light on the mechanisms by which a sufficiently low concentration of 3-BP can induce cytotoxicity at the DNA level, a finding that might be important for the future design of anticancer therapies.

The obligate intracellular bacterium, Chlamydia pneumoniae, has been identified as a risk factor for several chronic inflammatory diseases in addition to respiratory tract infections. The dissemination of C. pneumoniae from respiratory tract to secondary sites of infection occurs via infected monocyte / macrophage line cells, in which C. pneumoniae can persist as an antibiotic-refractory phenotype. To allow more detailed studies on the epithelium-monocyte/macrophage transition of the infection, new in vitro bioassays are needed. To this end, a coculture system with human continuous cell lines was established. Respiratory epithelial HL cells were infected with C. pneumoniae and THP-1 monocytes were added into the cultures at 67 h post infection. After a 5 h coculture, THP-1 cells were collected with a biotinylated HLA antibody and streptavidin-coated magnetic beads and C. pneumoniae genome copy numbers in THP-1 determined by quantitative PCR. The assay was optimized for cell densities, incubation time, THP-1 separation technique and buffer composition, and its robustness was demonstrated by a Z' value of 0.6. The mitogen-activated protein kinase (MAPK) inhibitors: SP600125 (JNK inhibitor), SB203580 (p38 inhibitor) and FR180204 (ERK inhibitor) suppressed the transfer of C. pneumoniae from HL to THP-1 cells, making them suitable positive controls for the assay. Based on analysis of separate steps of the process, the MAPK inhibitors suppress the bacterial entry to THP-1 cells. The transfer of C. pneumoniae from epithelium to phagocytes represents a crucial step in the establishment of persistent infections by this pathogen, and the presented methods enables future studies to block this process by therapeutic means.

Human monocytes/macrophages play a central role in the immune response and defense of the host from influenza virus infection. They classically act as antigen-presenting cells for lymphocytes in the context of an immune cell cluster. In that setting, however, monocytes/macrophages exhibit additional, unexpected, roles. They are required for influenza virus infection of the lymphocytes in the cluster, and they are responsible for lymphocyte apoptosis via their synthesis and expression of the viral neuraminidase. Surprisingly, human alveolar macrophages, expected to be among the first cells to encounter the virus, are not susceptible to direct infection by a human influenza virus but can be infected when the virus is complexed with an antibody. Such monocyte/macrophage responses to influenza virus challenge should be considered part of a very complex but quite effective defense, since the common outcome is recovery of the host with development of immunity to the challenging strain of virus.

To date, no safe vaccine or antivirals for Zika virus (ZIKV) infection have been found. The pathogenesis of severe Zika, where host and viral factors participate, remains unclear. For the control of Zika, it is important to understand how ZIKV interacts with different host cells. Knowledge of the targeted cellular pathways which allow ZIKV to productively replicate and/or establish prolonged viral persistence contributes to novel vaccines and therapies. Monocytes and endothelial vascular cells are the main ZIKV targets. During the infection process, cells are capable of releasing extracellular vesicles (EVs). EVs are mediators of intercellular communication. We found that mosquito EVs released from ZIKV-infected (C6/36) cells carry viral RNA and ZIKV-E protein and are able to infect and activate na&iuml, ve mosquito and mammalian cells. ZIKV C6/36 EVs promote the differentiation of na&iuml, ve monocytes and induce a pro-inflammatory state with tumor necrosis factor-alpha (TNF-&alpha, ) mRNA expression. ZIKV C6/36 EVs participate in endothelial vascular cell damage by inducing coagulation (TF) and inflammation (PAR-1) receptors at the endothelial surface of the cell membranes and promote a pro-inflammatory state with increased endothelial permeability. These data suggest that ZIKV C6/36 EVs may contribute to the pathogenesis of ZIKV infection in human hosts.

Since induced pluripotent stem (iPS) cells have been established, in recent years, clinical transplantation of cells differentiated from iPS cells derived from human skin fibroblasts is been in progress. On the contrary, monocytes have complete genome information without damage and gene recombination, they are contained in the peripheral blood by ∼3%–8% and differentiate into dendritic cells that are the type of control tower for immune cells. However, generation of monocyte-derived iPS cells has only been successful when special persistent Sendai virus vectors have been used. Therefore, in this study, as a preculture method for monocytes, a culture method for maintaining activity without using any cytokine was established, and using a commercially available vector without genetic toxicity without damaging the chromosome of the cell, iPS cells derived from monocytes were successfully produced. This cell has the ability to differentiate into three germ layers, and when compared with commercially available iPS cells, there was no significant difference between self-renewal and gene expression in the three germ layers. In future, we will compare the differentiation induction of monocyte-derived iPS cells with dendritic cells and investigate the production of dendritic cells that can cope with various antigens.

Aims: H2O2 is produced by all eukaryotic cells under physiological and pathological conditions. Due to its enormous relevance for cell signaling at low concentrations and antipathogenic function at high concentrations, precise quantification of extracellular local hydrogen peroxide concentrations ([H2O2]) originating from single cells is required. Results: Using a scanning electrochemical microscope and bare platinum disk ultramicroelectrodes, we established sensitive long-term measurements of extracellular [H2O2] kinetics originating from single primary human monocytes (MCs) ex vivo. For the electrochemical techniques square wave voltammetry, cyclic and linear scan voltammetry, and chronoamperometry, detection limits for [H2O2] were determined to be 5, 50, and 500 nM, respectively. Following phorbol ester stimulation, local [H2O2] 5–8 μm above a single MC increased by 3.4 nM/s within the first 10 min before reaching a plateau. After extracellular addition of H2O2 to an unstimulated MC, the local [H2O2] decreased on average by 4.2 nM/s due to degradation processes of the cell. Using the scanning mode of the setup, we found that H2O2 is evenly distributed around the producing cell and can still be detected up to 30 μm away from the cell. The electrochemical single-cell measurements were validated in MC populations using electron spin resonance spectroscopy and the Amplex® UltraRed assay. Innovation and Conclusion: We demonstrate a highly sensitive, spatially, and temporally resolved electrochemical approach to monitor dynamics of production and degradation processes for H2O2 separately. Local extracellular [H2O2] kinetics originating from single cells is quantified in real time. Antioxid. Redox Signal. 29, 501–517.

Unbiased genomic screening analyses have highlighted novel immunomodulatory properties of the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D). However, clearer interpretation of the resulting gene expression data is limited by cell model specificity. The aim of the current study was to provide a broader perspective on common gene regulatory pathways associated with innate immune responses to 1,25(OH)2D, through systematic re-interrogation of existing gene expression databases from multiple related monocyte models (the THP-1 monocytic cell line (THP-1), monocyte-derived dendritic cells (DCs) and monocytes). Vitamin D receptor (VDR) expression is common to multiple immune cell types, and thus, pathway analysis of gene expression using data from multiple related models provides an inclusive perspective on the immunomodulatory impact of vitamin D. A bioinformatic workflow incorporating pathway analysis using PathVisio and WikiPathways was utilized to compare each set of gene expression data based on pathway-level context. Using this strategy, pathways related to the TCA cycle, oxidative phosphorylation and ATP synthesis and metabolism were shown to be significantly regulated by 1,25(OH)2D in each of the repository models (Z-scores 3.52–8.22). Common regulation by 1,25(OH)2D was also observed for pathways associated with apoptosis and the regulation of apoptosis (Z-scores 2.49–3.81). In contrast to the primary culture DC and monocyte models, the THP-1 myelomonocytic cell line showed strong regulation of pathways associated with cell proliferation and DNA replication (Z-scores 6.1–12.6). In short, data presented here support a fundamental role for active 1,25(OH)2D as a pivotal regulator of immunometabolism.

Two high molecular weight (MW larger than 788 kDa) mushroom polysaccharides extracted by hot water (PRW) and micronized sonication-assisted cold alkali (mPRSon), were isolated from the sclerotia of Polyporus rhinocerus. PRW was a polysaccharide-protein complex with a highly branched (degree of branching DB 0.60) β-D-mannoglucan while mPRSon was a hyper-branched β-glucan (DB 0.85). PRW could bind to complement receptor 3 (CR3) and significantly induced THP-1 differentiation evidenced by cell morphology and enhanced phagocytic activity. A significant increase in the expression of surface markers including CD14, CD11b and HLA-DR as well as enhanced production of cytokines including IL-8, RANTES, GRO, MCP-1, MCP-2 and IL-6 were observed on THP-1 cells treated with PRW. In contrast, mPRSon could not either activate the Dectin-1 signaling or induce the THP-1 differentiation. High MW and DB mushroom sclerotial polysaccharide which has a structure of heteroglycan and protein is more immunopotent than pure homoglycan.

Glutamine metabolism provides synergistic support for macrophage activation and elicitation of desirable immune responses; however, the underlying mechanisms regulated by glutamine metabolism to orchestrate macrophage activation remain unclear. Here we show that the production of α-ketoglutarate (αKG) via glutaminolysis is important for alternative (M2) activation of macrophages, including engagement of fatty acid oxidation (FAO) and Jmjd3-dependent epigenetic reprogramming of M2 genes. This M2-promoting mechanism is further modulated by a high αKG/succinate ratio, whereas a low ratio strengthens the proinflammatory phenotype in classically activated (M1) macrophages. As such, αKG contributes to endotoxin tolerance after M1 activation. This study reveals new mechanistic regulations by which glutamine metabolism tailors the immune responses of macrophages through metabolic and epigenetic reprogramming. ispartof: Nature Immunology vol:18 issue:9 pages:985-994 ispartof: location:United States status: published

QiHong Wu,1 RongRong Jin,2 Ting Feng,1 Li Liu,2 Li Yang,2 YuHong Tao,3 James M Anderson,4,5 Hua Ai,2,6 Hong Li1,3 1Key Laboratory of Obstetrics, Gynecology, Pediatric Disease, and Birth Defects, Ministry of Education, West China Second University Hospital, 2National Engineering Research Center for Biomaterials, 3Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu, China; 4Department of Biomedical Engineering, 5Department of Pathology, Case Western Reserve University, Cleveland, OH, US; 6Department of Radiology, West China Hospital, Sichuan University, Chengdu, China Abstract: Superparamagnetic iron oxide nanoparticles have been widely used in biomedical applications, but understanding of their interactions with the biological immune system is relatively limited. This work is focused on dextran-coated iron oxide nanoparticles and their induced autophagy in human monocytes. We found that these nanoparticles can be taken up by human monocytes, followed by localization within vesicles or free in cytoplasm. Autophagosome formation was observed with increased expression of LC3II protein, the specific marker of autophagy. The autophagy substrate p62 was degraded in a dose-dependent manner, and autophagy was blocked by autophagy (or lysosome) inhibitors alone or along with iron oxide nanoparticles, indicating that autophagosome accumulation was mainly due to autophagy induction, rather than blockade of autophagy flux. Interestingly, iron oxide nanoparticles increased the viability of human monocytes, but the mechanism was not clear. We further found that inhibition of autophagy mostly attenuated the survival of cells, with acceleration of the inflammation induced by these nanoparticles. Taken together, autophagic activation in human monocytes may play a protective role against the cytotoxicity of iron oxide nanoparticles. Keywords: autophagy, cytotoxicity, human monocytes, inflammation, iron oxide nanoparticles

QiHong Wu,1 RongRong Jin,2 Ting Feng,1 Li Liu,2 Li Yang,2 YuHong Tao,3 James M Anderson,4,5 Hua Ai,2,6 Hong Li1,3 1Key Laboratory of Obstetrics, Gynecology, Pediatric Disease, and Birth Defects, Ministry of Education, West China Second University Hospital, 2National Engineering Research Center for Biomaterials, 3Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu, China; 4Department of Biomedical Engineering, 5Department of Pathology, Case Western Reserve University, Cleveland, OH, US; 6Department of Radiology, West China Hospital, Sichuan University, Chengdu, China Abstract: Superparamagnetic iron oxide nanoparticles have been widely used in biomedical applications, but understanding of their interactions with the biological immune system is relatively limited. This work is focused on dextran-coated iron oxide nanoparticles and their induced autophagy in human monocytes. We found that these nanoparticles can be taken up by human monocytes, followed by localization within vesicles or free in cytoplasm. Autophagosome formation was observed with increased expression of LC3II protein, the specific marker of autophagy. The autophagy substrate p62 was degraded in a dose-dependent manner, and autophagy was blocked by autophagy (or lysosome) inhibitors alone or along with iron oxide nanoparticles, indicating that autophagosome accumulation was mainly due to autophagy induction, rather than blockade of autophagy flux. Interestingly, iron oxide nanoparticles increased the viability of human monocytes, but the mechanism was not clear. We further found that inhibition of autophagy mostly attenuated the survival of cells, with acceleration of the inflammation induced by these nanoparticles. Taken together, autophagic activation in human monocytes may play a protective role against the cytotoxicity of iron oxide nanoparticles. Keywords: autophagy, cytotoxicity, human monocytes, inflammation, iron oxide nanoparticles

The production of nitric oxide (NO) is a key defense mechanism against intracellular pathogens but it must be tightly controlled in order to avoid excessive detrimental oxidative stress. In this study we described a novel mechanism through which interleukin (IL)-6 mediates the regulation of NO release induced in response to Trypanosoma cruzi infection. Using a murine model of Chagas disease, we found that, in contrast to C57BL/6 wild type (WT) mice, IL-6-deficient (IL6KO) mice exhibited a dramatic increase in plasma NO levels concomitant with a significantly higher amount of circulating IL-1β and inflammatory monocytes. Studies on mouse macrophages and human monocytes, revealed that IL-6 decreased LPS-induced NO production but this effect was abrogated in the presence of anti-IL-1β and in macrophages deficient in the NLRP3 inflammasome. In accordance, while infected WT myocardium exhibited an early shift from microbicidal/M1 to anti-inflammatory/M2 macrophage phenotype, IL6KO cardiac tissue never displayed a dominant M2 macrophage profile that correlated with decreased expression of ATP metabolic machinery and a lower cardiac parasite burden. The deleterious effects of high NO production-induced oxidative stress were evidenced by enhanced cardiac malondialdehyde levels, myocardial cell death and mortality. The survival rate was improved by the treatment of IL-6-deficient mice with a NO production-specific inhibitor. Our data revealed that IL-6 regulates the excessive release of NO through IL-1β inhibition and determines the establishment of an M2 macrophage profile within infected heart tissue. Fil: Sanmarco, Liliana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Ponce, Nicolás Eric. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Visconti, Laura M.. Hospital Nuestra Señora de la Misericordia del Nuevo Siglo; Argentina Fil: Eberhardt, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Theumer, Martín Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Minguez, Ángel R.. Hospital Nuestra Señora de la Misericordia del Nuevo Siglo; Argentina Fil: Aoki, Maria del Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina

Objectives Extracellular vesicles (EVs) are important for intercellular signalling in cancer. Tumour-associated macrophages, expressing the haemoglobin-haptoglobin and mannose receptors CD163 and CD206, are crucial for cancer progression. We recently identified CD163 on EVs in the circulation as a fraction of total soluble CD163 (sCD163). Here, we investigated the presence of CD163 and CD206-positive EVs (EV-CD163, EV-CD206) in patients with multiple myeloma (MM).Methods We enrolled patients with MM (n = 32), monoclonal gammopathy of undetermined significance (MGUS) (n = 8) and healthy donors (n = 16). Plasma protein levels were determined by ELISA before and after vesicle precipitation. Monocytes were examined by flow cytometry, and leucocyte CD163 mRNA by qPCR.Results Fractions of EV-CD163 and EV-CD206 were significantly elevated in patients with newly diagnosed MM (median = 39.8%, 76.5%, respectively) compared to patients with relapse (15.6%, P = .02, 42.5%, P = .003), remission (16.9%, P Conclusions Our results indicate that macrophage-derived EVs may play a role in the late phase of malignant progression of MM, and encourage further EV investigations in functional experiments.

Background: CXCL13 is preferentially secreted by Follicular Helper T cells (T-FH) to attract B cells to germinal centers. Plasma levels of CXCL13 have been reported to be elevated during chronic HIV-infection, however there is limited data on such elevation during early phases of infection and on the effect of ART. Moreover, the contribution of CXCL13 to disease progression and systemic immune activation have been partially defined. Herein, we assessed the relationship between plasma levels of CXCL13 and systemic immune activation.Methods: Study samples were collected in 114 people living with HIV (PLWH) who were in early (EHI) or chronic (CHI) HIV infection and 35 elite controllers (EC) compared to 17 uninfected controls (UC). A subgroup of 11 EHI who initiated ART and 14 who did not were followed prospectively. Plasma levels of CXCL13 were correlated with CD4 T cell count, CD4/CD8 ratio, plasma viral load (VL), markers of microbial translocation [LPS, sCD14, and (1 -> 3)-beta-D-Glucan], markers of B cell activation (total IgG, IgM, IgA, and IgG1-4), and inflammatory/activation markers like IL-6, IL-8, IL-1 beta, TNF-alpha, IDO-1 activity, and frequency of CD38(+)HLA-DR+ T cells on CD4(+) and CD8(+) T cells.Results: Plasma levels of CXCL13 were elevated in EHI (127.9 +/- 64.9 pg/mL) and CHI (229.4 +/- 28.5 pg/mL) compared to EC (71.3 +/- 20.11 pg/mL), and UC (33.4 +/- 14.9 pg/mL). Longitudinal analysis demonstrated that CXCL13 remains significantly elevated after 14 months without ART (p 3)-beta-D-Glucan, total IgG, TNF-alpha, Kynurenine/Tryptophan ratio, and frequency of CD38+HLA-DR+ CD4 and CD8 T cells. In addition, CMV+ PLWH presented with higher levels of plasma CXCL13 than CMV- PLWH (p = 0.005).Conclusion: Plasma CXCL13 levels increased with HIV disease progression. Early initiation of ART reduces plasma CXCL13 and B cell activation without normalization. CXCL13 represents a novel marker of systemic immune activation during early and chronic HIV infection and may be used to predict the development of non-AIDS events.

Background Burkholderia mallei (Bm) is a facultative intracellular bacterial pathogen causing highly-fatal glanders in solipeds and humans. The ability of Bm to thrive intracellularly is thought to be related to exploitation of host immune response-related genes and pathways. Relatively little is known of the molecular strategies employed by this pathogen to modulate these pathways and evade intracellular killing. This manuscript seeks to fill gaps in the understanding of the interface between Bm and innate immunity by examining gene expression changes during infection of host monocytes. Methods The transcriptome of Bm-infected human Mono Mac-6 (MM6) monocytes was profiled on Affymetrix Human Transcriptome GeneChips 2.0. Gene expression changes in Bm-infected monocytes were compared to those of Burkholderia thailandensis (Bt)-infected monocytes and to uninfected monocytes. The resulting dataset was normalized using Robust Multichip Average and subjected to statistical analyses employing a univariate F test with a random variance model. Differentially expressed genes significant at p

Dissertation, Huntington’s Disease (HD) is an inherited, progressive, fatal, and late-onset neurodegenerative disorder. Its neuropathogenesis is characterized by selective loss of medium spiny neurons (MSNs) in the striatum and cortical neurons in the cortex. It is caused by an expansion of CAG repeats in the exon-1 of IT-15 gene, which results in a mutated polyglutamine domain in the huntingtin protein (HTT) (Collaborative, 1993). The mutation that causes HD was identified almost thirty years ago, yet the ultimate cause of neuron death is still uncertain. This dissertation is based on the hypothesis that monocytes infiltrate the brain of late stage HD patients and drive neuropathogenesis. To test this hypothesis, I developed a novel hybrid brain-slice experimental model in which human monocytes (MO) isolated from HD and non-disease control patients (CTRL) are engrafted into living postnatal striatal brain tissues prepared from neonatal rats.In the first set of experiments, the inflammatory response of stimulated HD and CTRL MO were tested. HD MO exhibited an increased inflammatory response demonstrated by increased production of cytokine IL-10. Furthermore, a late-stage HD patient exhibited an increased production of multiple cytokines, including IL-10. The second set of experiments utilized a brain-tissue based model to measure the impact of engrafting HD MO on MSN health. In brain slices engraftment with HD MO, there was a decrease in the number of healthy MSNs. A further experiment revealed that stimulating MO prior to their engraftment differentially influenced the survival of MSNs depending on whether the MO came from HD or CTRL patients. Measurement of cytokine production from these unstimulated/stimulated engraftment experiments revealed a trend for increased cytokine production in samples taken from a moderate stage HD patient. The third set of experiments assessed the ability of HD MO to influence the endogenous macrophages within the brain slice tissue, microglia. MO engraftment, independent of its genotype, altered microglia morphology within brain slices. Further results from these experiments demonstrated that the presence of HD MO increased microglial density and upregulated their phagocytosis of MSNs. While the mechanisms underlying these multicellular interactions remain to be determined, it is possible that such an increase in phagocytosis could lead to the observation of fewer healthy MSNs as quantified in the second set of experiments. Together, these three sets of experiments support the application of a novel model to study neuropathogenesis and highlight that the genotype of infiltrating MO has the potential to quantifiably alter intercellular interactions and neuronal health in the context of HD.

Human monocytes are divided in three major populations; classical (CD14+CD16−), non-classical (CD14dimCD16+), and intermediate (CD14+CD16+). Each of these subsets is distinguished from each other by the expression of distinct surface markers and by their functions in homeostasis and disease. In this review, we discuss the most up-to-date phenotypic classification of human monocytes that has been greatly aided by the application of novel single-cell transcriptomic and mass cytometry technologies. Furthermore, we shed light on the role of these plastic immune cells in already recognized and emerging human chronic diseases, such as obesity, atherosclerosis, chronic obstructive pulmonary disease, lung fibrosis, lung cancer, and Alzheimer's disease. Our aim is to provide an insight into the contribution of human monocytes to the progression of these diseases and highlight their candidacy as potential therapeutic cell targets.

Monocytes play a crucial role in immunity and tissue homeostasis. They constitute the first line of defense during the inflammatory process, playing a role in the pathogenesis and progression of diseases, making them an attractive therapeutic target. They are heterogeneous in morphology and surface marker expression, which suggest different molecular and physiological properties. Recent evidences have demonstrated their ability to enter the brain, and, as a consequence, their hypothetical role in different neurodegenerative diseases. In this review, we will discuss the current knowledge about the correlation between monocyte dysregulation in the brain and/or in the periphery and neurological diseases in humans. Here we will focus on the most common neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis and multiple sclerosis.

Human monocyte is an important cell type which is involved in various complex human diseases. To better understand the biology of human monocytes and facilitate further studies, we developed the first comprehensive proteome knowledge base specifically for human monocytes by integrating both in vivo and in vitro datasets. The top 2000 expressed genes from in vitro datasets and 779 genes from in vivo experiments were integrated into this study. Altogether, a total of 2237 unique monocyte-expressed genes were cataloged. Biological functions of these monocyte-expressed genes were annotated and classified via Gene Ontology (GO) analysis. Furthermore, by extracting the overlapped genes from in vivo and in vitro datasets, a core gene list including 541 unique genes was generated. Based on the core gene list, further gene-disease associations, pathway and network analyses were performed. Data analyses based on multiple bioinformatics tools produced a large body of biologically meaningful information, and revealed a number of genes such as SAMHD1, G6PD, GPD2 and ENO1, which have been reported to be related to immune response, blood biology, bone remodeling, and cancer respectively. As a unique resource, this study can serve as a reference map for future in-depth research on monocytes biology and monocyte-involved human diseases. Electronic supplementary material The online version of this article (doi:10.1007/s13238-016-0342-x) contains supplementary material, which is available to authorized users.

Monocytes-macrophages and lymphocytes are recruited to the respiratory tract in response to influenza virus challenge and are exposed to the virus during the establishment of immune defenses. The susceptibility of human lymphocytes to infection was assessed. The presence of monocytes-macrophages was required to attain infection of both resting and proliferating lymphocytes. Lymphocyte infection occurred in the context of immune cell clusters and was blocked by the addition of anti-intercellular adhesion molecule-1 (ICAM-1) antibody to prevent cell clustering. Both peripheral blood-derived and bronchoalveolar lymphocytes were susceptible to infection. Both CD4+ and CD8+ T lymphocytes were susceptible to influenza virus infection, and the infected CD4+ and CD8+ lymphocytes served as infectious foci for other nonpermissive or even virus-permissive cells. These data show that monocytes-macrophages and both CD4+ and CD8+ lymphocytes can become infected during the course of an immune response to influenza virus challenge. The described leukocyte interactions during infection may play an important role in the development of effective anti-influenza responses.

Bone remodeling can be mimicked in vitro by co-culture models. Based on bone cells, such co-cultures help to study synergistic morphological changes and the impact of materials and applied substances. Hence, we examined the formation of osteoclasts on bovine bone materials to prove the bone resorption functionality of the osteoclasts in three different co-culture set-ups using human monocytes (hMCs) and (I) human mesenchymal stem cells (hMSCs), (II) osteogenic differentiated hMSCs (hOBs), and (III) hOBs in addition of soluble monocyte-colony stimulating factor (M-CSF) and cytokine receptor activator of NFkB ligand (RANKL). We detected osteoclast-specific actin morphology, as well as the expression of cathepsin K and CD51/61 in single cells in set-up II and in numerous cells in set-up III. Resorption pits on bone material as characteristic proof of functional osteoclasts were not found in set-up I and II, but we detected such resorption pits in set-up III. We conclude in co-culture models without M-CSF and RANKL that monocytes can differentiate into osteoclasts that show the characteristic actin structures and protein expression. However, to receive functional bone resorbing osteoclasts in vitro, the addition of M-CSF and RANKL is needed. Moreover, we suggest the use of bone or bone-like materials for future studies evaluating osteoclastogenesis.

Anti-DNA autoantibodies are a hallmark of systemic lupus erythematosus (SLE). A subset of anti-DNA IgG autoantibodies is cell-internalizable; thus they can enter living cells in the form of free IgG. However, the contribution made by the Fc region of internalized free-form IgG to the cytokine response has not been studied, despite the recent discovery of tripartite motif-containing 21 (TRIM21), a cytosolic Fc receptor involved in immune signaling. This study used an internalizable IgG anti-DNA antibody (3D8) to examine the cytokine responses of human monocytes to the Fc region of cytosolic free IgG. Internalization of 3D8 IgG and a 3D8 single-chain variable fragment-Fc (scFv-Fc) induced production of IL-8 and TNF-α via activation of NF-κB. By contrast, a 3D8 scFv (comprising variable domains alone) did not. This suggests Fc-dependent cytokine signaling. A 3D8 IgG-N434D mutant that is not recognized by TRIM21 induced greater production of cytokines than 3D8 IgG. Moreover the amounts of cytokines induced by 3D8 IgG in TRIM21-knockdown THP-1 cells were higher than those in control cells, indicating that cytokine signaling is not mediated by TRIM21. The results suggest the existence of a novel Fc-dependent signaling pathway that is activated upon internalization of IgG antibodies by human monocytes.

Interleukin (IL)-30, the IL-27p28 subunit of the heterodimeric cytokine IL-27, acts as an antagonist of IL-27 and IL-6 signaling in murine cells via glycoprotein 130 (gp130) receptor and additional binding partners. Thus far, functions of IL-30 have not been fully elucidated in human cells. We demonstrate that like IL-27, IL-30 upregulated TLR4 expression to enhance lipopolysaccharide-induced TNF-α production in human monocytes; however, these IL-30-mediated activities did not reach the same levels of cytokine induction compared to IL-27. Interestingly, IL-30- and IL-27-mediated interferon-γ-induced protein 10 (IP-10) production required WSX-1 engagement and signal transducer and activator of transcription (STAT) 3 phosphorylation; furthermore, IL-30 induced STAT phosphorylation after 16 h, whereas IL-27 induced STAT phosphorylation within 30 min. This prompted us to examine if a secondary mediator was required for IL-30-induced pro-inflammatory functions, and hence we examined IL-6-related molecules. Combined with inhibition of soluble IL-6 receptor α (sIL-6Rα) and data showing that IL-6 inhibited IL-30/IL-27-induced IP-10 expression, we demonstrate a role for sIL-6Rα and gp130 in IL-30-mediated activity in human cells.

Interleukin-1β (IL-1β), a major pro-inflammatory cytokine, is a leaderless cytosolic protein whose secretion does not follow the classical endoplasmic reticulum-to-Golgi pathway, and for which the mechanism of secretion remains to be established. In murine macrophages, IL-1ß is known to be released through pyroptosis, a highly inflammatory cell death, mediated by gasdermin D (GSDMD) pores at the plasma membrane. Whether gasdermin D is dispensable for IL-1ß secretion in human cells was still unknown. Here we show that human blood monocytes stimulated with LPS alone secrete mature IL-1ß without cell death and without gasdermin D cleavage, while they undergo pyroptosis with gasdermin D cleavage when stimulated with LPS and an activator of the NLRP3 inflammasome. In addition, we show that LPS- stimulated human monocytic THP-1 cells secrete IL-1ß, even after invalidation of the gasdermin D- encoding gene, pointing out to the existence of a pathway of IL-1ß secretion that is cell death- and gasdermin D-independent. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2018

We report the detailed transcriptomic profiles of human innate myeloid cells using RNA sequencing. Monocytes migrate from blood into infected or wounded tissue to differentiate into macrophages, and control inflammation via phagocytosis or cytokine secretion. We differentiated culture primary monocytes with either GM- or M-CSF to obtain pro- or anti-inflammatory macrophages, and respectively activated them with either LPS/IFNγ or anti-inflammatory cytokines. We also treated the THP-1 monocytic cell line with PMA and similar cytokines to mimic differentiation and activation. We detected thousands of expression and alternative-splicing changes during monocyte-to-macrophage differentiation and activation, and a net increase in exon inclusion. MBNL1 knockdown phenocopies several alternative-splicing changes and strongly impairs PMA differentiation, suggesting functional defects in monocytes from Myotonic Dystrophy patients. This study provides general insights into alternative splicing in the monocyte–macrophage lineage, whose future characterization will elucidate their contribution to immune functions, which are altered in immunodeficiencies, autoimmunity, atherosclerosis and cancer. MOE (Min. of Education, S’pore) Published version

Aims/Introduction Resistin, secreted from adipocytes, causes insulin resistance in mice. In humans, the resistin gene is mainly expressed in monocytes and macrophages. Tunicamycin is known to induce endoplasmic reticulum (ER) stress, and reduce resistin gene expression in 3T3-L1 mouse adipocytes. The aim of the present study was to examine whether ER stress affects resistin gene expression in human monocytes. Materials and Methods The relationship between resistin messenger ribonucleic acid (mRNA) and ER stress markers mRNA was analyzed by reverse transcription polymerase chain reaction in isolated monocytes of 30 healthy volunteers. The effect of endotoxin/lipopolysaccharides or tunicamycin on resistin gene expression was analyzed in THP-1 human monocytes. Signaling pathways leading to resistin mRNA were assessed by the knockdown using small interfering RNA or overexpression of key molecules involved in unfolded protein response. Results Resistin mRNA was positively associated with immunoglobulin heavy chain-binding protein (BiP) or CAAT/enhancer binding protein-α homologous protein (CHOP) mRNA in human isolated monocytes. In THP-1 cells, lipopolysaccharides increased mRNA of BiP, pancreatic endoplasmic reticulum eukaryotic initiation factor 2α kinase (PERK) and CHOP, as well as resistin. Tunicamycin also increased resistin mRNA. This induction appeared to be dose- and time-dependent. Tunicamycin-induced resistin mRNA was inhibited by chemical chaperone, 4-phenylbutyric acid. The knockdown of either PERK, activating transcription factor 4 (ATF4) or CHOP reduced tunicamycin-induced resistin mRNA. Conversely, overexpression of ATF4 or CHOP increased resistin mRNA. Conclusions Endoplasmic reticulum stress induced by tunicamycin increased resistin mRNA through the PERK–ATF4–CHOP pathway in THP-1 human monocytes. ER stress could lead to insulin resistance through enhanced resistin gene expression in human monocytes.

Administration of prophylactic glucocorticoids has been suggested as a strategy to reduce postoperative AKI and other adverse events after cardiac surgery requiring cardiopulmonary bypass. In this post hoc analysis of a large placebo-controlled randomized trial of dexamethasone in 4465 adult patients undergoing cardiac surgery, we examined severe AKI, defined as use of RRT, as a primary outcome. Secondary outcomes were doubling of serum creatinine level or AKI-RRT, as well as AKI-RRT or in-hospital mortality (RRT/death). The primary outcome occurred in ten patients (0.4%) in the dexamethasone group and in 23 patients (1.0%) in the placebo group (relative risk, 0.44; 95% confidence interval, 0.19 to 0.96). In stratified analyses, the strongest signal for potential benefit of dexamethasone was in patients with an eGFR

We are increasingly exposed to low-frequency electromagnetic fields (LF EMFs) by electrical devices and power lines, but if and how these fields interact with living cells remains a matter of debate. This study aimed to investigate the potential effect of LF EMF exposure on calcium signalling in neutrophils. In neutrophilic granulocytes, activation of G-protein coupled receptors leads to efflux of calcium from calcium stores and influx of extracellular calcium via specialised calcium channels. The cytoplasmic rise of calcium induces cytoskeleton rearrangements, modified gene expression patterns, and cell migration. If LF EMF modulates intracellular calcium signalling, this will influence cellular behaviour and may eventually lead to health problems. We found that calcium mobilisation upon chemotactic stimulation was not altered after a short 30¿min or long-term LF EMF exposure in human neutrophil-like cell lines HL-60 or PLB-985. Neither of the two investigated wave forms (Immunent and 50¿Hz sine wave) at three magnetic flux densities (5¿µT, 300¿µT, and 500¿µT) altered calcium signalling in vitro. Gene-expression patterns of calcium-signalling related genes also did not show any significant changes after exposure. Furthermore, analysis of the phenotypical appearance of microvilli by scanning electron microscopy revealed no alterations induced by LF EMF exposure. The findings above indicate that exposure to 50¿Hz sinusoidal or Immunent LF EMF will not affect calcium signalling in neutrophils in vitro.

Background Leukocyte infiltration into the central nervous system is an important feature of multiple sclerosis (MS) pathology. Among the infiltrating cells, monocytes comprise the largest population and are considered to play a dual role in the course of the disease. The enzyme tissue transglutaminase (TG2), produced by monocytes, plays a central role in monocyte adhesion/migration in animal models of MS. In the present study, we questioned whether TG2 expression is altered in monocytes from MS patients compared to healthy control (HC) subjects. Moreover, we determined the inflammatory status of these TG2-expressing monocytes, what inflammatory factor regulates TG2 expression, and whether TG2 can functionally contribute to their adhesion/migration processes. Methods Primary human monocytes from MS patients and HC subjects were collected, RNA isolated and subjected to qPCR analysis. Human THP-1 monocytes were lentivirally transduced with TG2 siRNA or control and treated with various cytokines. Subsequently, mRNA levels of inflammatory factors, adhesion properties, and activity of RhoA were analyzed in interleukin (IL)-4-treated monocytes. Results TG2 mRNA levels are significantly increased in monocytes derived from MS patients compared to HC subjects. In addition, correlation analyses indicated that TG2-expressing cells display a more anti-inflammatory, migratory profile in MS patients. Using THP-1 monocytes, we observed that IL-4 is a major trigger of TG2 expression in these cells. Furthermore, knockdown of TG2 expression leads to a pro-inflammatory profile and reduced adhesion/migration properties of IL-4-treated monocytes. Conclusions TG2-expressing monocytes in MS patients have a more anti-inflammatory profile. Furthermore, TG2 mediates IL-4-induced anti-inflammatory status in THP-1 monocytes, adhesion, and cytoskeletal rearrangement in vitro. We thus propose that IL-4 upregulates TG2 expression in monocytes of MS patients, driving them into an anti-inflammatory status. Electronic supplementary material The online version of this article (10.1186/s12974-017-1035-y) contains supplementary material, which is available to authorized users.

Behcet’s disease (BD) and Vogt-Koyanagi-Harada disease (VKH) are systemic and recurrent autoimmune diseases associated with abnormal T cell immune response. Complement 3a receptor (C3aR) and complement 5a receptor (C5aR) have been reported to be involved in T cell mediated autoimmune disease. This study aimed to investigate the role of C3aR and C5aR in these two diseases. The C3aR expression in PBMCs was increased in patients with active BD (aBD) and active VKH (aVKH). No statistical difference was found concerning the expression of C5aR in PBMCs between patients with aBD or aVKH and normal controls. After the intraocular inflammation in BD and VKH patients was controlled, the C3aR expression returned back to normal levels. The serum from patients with aBD and aVKH significantly induced C3aR expression by PBMCs. C3a induced IL-6, IL-1β and TNF-α secretion, while inhibited the production of IL-10 by monocytes. Activation of C3aR in CD4+T cells could upregulate IL-17 production and inhibit IL-10 production, but had no detectable influence on IFN-γ production. Our data indicates that increased C3aR expression may lead to over activation of the Th17 cell response and may therefore contribute to the pathogenesis of BD and VKH disease.

Melaleuca alternifolia concentrate (MAC) is the refined essential oil of the Australian native plant Melaleuca alternifolia. MAC has been reported to suppress the production of pro-inflammatory cytokines in both murine RAW264.7 macrophages and human monocytes stimulated with lipopolysaccharide (LPS). However, the mechanisms involved in this effect remain unclear. This study aims to delineate the molecular mechanisms that drive the anti-inflammatory activity of MAC and its active component, terpinen-4-ol, in macrophages. The effects of MAC on RAW264.7 cells were studied using western blotting, real-time PCR, an electrophoretic mobility shift assay (EMSA), and NF-[Formula: see text]B luciferase reporter assays. Our results showed that MAC significantly increased both the mRNA and protein levels of heme oxygenase-1 (HO-1) via p38 and JNK MAPK activation. In addition, we showed that MAC significantly increased the activation and nuclear translocation of NF-E2-related factor 2 (Nrf2), a key transcription factor regulating HO-1 induction. MAC was also associated with significant inhibition of iNOS expression, NO production, and NF-[Formula: see text]B activation. HO-1 was required for these anti-inflammatory effects as tin protoporphyrin IX (SnPPIX), an HO-1 inhibitor, abolished the effects of MAC on LPS-induced iNOS, NO, and NF-[Formula: see text]B activation. Our results indicate that MAC induces HO-1 expression in murine macrophages via the p38 MAPK and JNK pathways and that this induction is required for its anti-inflammatory activity.

Background Concurrent diabetes mellitus and tuberculosis represent a significant health problem worldwide. Patients with diabetes mellitus have a high risk of tuberculosis, which may be mediated by an abnormal innate immune response due to hyperglycaemia or low vitamin D levels. Methods In the present study, we evaluated inactive vitamin D serum levels and the monocyte response to infection with M. tuberculosis, including phagocytosis of M. tuberculosis, antimycobacterial activity, LL-37, human β defensin-2 and IL-10 gene expression and nitric oxide production, between type 2 diabetes mellitus patients (n = 51) and healthy volunteers (n = 38). Results Twenty-seven type 2 diabetes mellitus patients had inadequate inactive vitamin D levels (

Lipid accumulation is a key characteristic of advancing atherosclerotic lesions. Herein, we analyzed the ultrastructure of the accumulated Lipids in endarterectomized human carotid atherosclerotic plaques using three-dimensional (3D) electron microscopy, a method never used in this context before. 3D electron microscopy revealed intracellular lipid droplets and extracellular Lipoprotein particles. Most of the particles were aggregated, and some connected to needle-shaped or sheet-like cholesterol crystals. Proteomic analysis of isolated extracellular Lipoprotein particles revealed that apolipoprotein B is their main protein component, indicating their origin from low-density lipoprotein, intermediate-density Lipoprotein, very-Low-density lipoprotein, lipoprotein (a), or chylomicron remnants. The particles also contained small exchangeable apolipoproteins, complement components, and immunoglobulins. Lipidomic analysis revealed differences between plasma lipoproteins and the particles, thereby indicating involvement of lipolytic enzymes in their generation. Incubation of human monocyte-derived macrophages with the isolated extracellular lipoprotein particles or with plasma lipoproteins that had been Lipolytically modified in vitro induced intracellular Lipid accumulation and triggered inflammasome activation in them. Taken together, extracellular Lipids accumulate in human carotid plaques as distinct 3D structures that include aggregated and fused lipoprotein particles and cholesterol crystals. The particles originate from plasma lipoproteins, show signs of lipolytic modifications, and associate with cholesterol crystals. By inducing intracellular cholesterol accumulation (ie, foam cell formation) and inflammasome activation, the extracellular lipoprotein particles may actively enhance atherogenesis.

The prevalence of diabetes around the world is increasing, and the complications of diabetes are becoming worse so that the global burden of diabetes-related complications is rising. The purpose of this study was to simulate the physiological condition of diabetes in the human body by culturing human microvascular endothelial cell 1 (HMEC-1) cells in Dulbecco’s modified Eagle’s medium (DMEM) containing 5.5mM glucose, 25mM glucose, and 50mM glucose. The cell viability of HMEC-1 cells at the indicated glucose concentrations was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The specific epithelial cell junction N-cadherin was measured by immunofluorescence. Furthermore, monocytic initiation of inflammatory reactions was studied by using Western blotting and enzyme-linked immunosorbent assay. Based on our results, hyperglycemia treatment influenced the distribution of N-cadherin-containing structures in HMEC-1 cells, whereas the change of the inflammatory profiles in HMEC-1 cells was affected after their coculture with the supernatant of diabetic THP-1 monocytes. In this study, we conclude that the removal of endothelial N-cadherin caused by hyperglycemia (at the 25mM glucose and 50mM glucose concentrations) may lead to endothelial dysfunction and subsequently endothelial cell death in the late stage of diabetes. Furthermore, the loss of intercellular adhesion molecule 1 and the upregulation of interleukin (IL)-1, IL-6, and tumor necrosis factor alpha mediated by diabetic monocytes showed altered interaction between the vascular endothelium and blood cells in the diabetic microenvironment.

Ob­jec­ti­ve: The vision of potential autologous cell therapy for the cure of diabetes encourages ongoing research. According to a previously published protocol for the generation of insulin-producing cells from human monocytes, we analyzed whether the addition of growth factors could increase insulin production. This protocol was then transferred to a non-human primate model by using either blood- or spleen-derived monocytes. Methods: Human monocytes were treated to dedifferentiate into programmable cells of monocytic origin (PCMO). In addition to the published protocol, PCMOs were then treated with either activin A, betacellulin, exendin 3 or 4. Cells were characterized by protein expression of insulin, Pdx-1, C-peptide and Glut-2. After identifying the optimal protocol, monocytes from baboon blood were isolated and the procedure was repeated. Spleen monocytes following splenectomy of a live baboon were differentiated and analyzed in the same manner and calculated in number and volume. Results: Insulin content of human cells was highest when cells were treated with activin A and their insulin content was 13 000 µU/1 million cells. Insulin-producing cells form primate monocytes could successfully be generated despite using human growth factors and serum. Expression of insulin, Pdx-1, C-peptide and Glut-2 was comparable to that of human neo-islets. Total insulin content of activin A-treated baboon monocytes was 16 000 µU/1 million cells. Conclusion: We were able to show that insulin-producing cells can be generated from baboon monocytes with human growth factors. The amount generated from one spleen could be enough to cure a baboon from experimentally induced diabetes in an autologous cell transplant setting.