Cutting Edge Communication: In Vitro Generation of Dendritic Cells from Human Blood Monocytes in Experimental Conditions Compatible for In Vivo Cell Therapy

Similar to IL-1α and IL-33, IL-1 family member IL-37b translocates to the nucleus and is associated with suppression of innate and adaptive immunity. Here we demonstrate an extracellular function of the IL-37 precursor and a processed form. Recombinant IL-37 precursor reduced LPS-induced IL-6 by 50% (P < 0.001) in highly inflammatory human blood-derived M1 differentiated macrophages derived from selective subjects but not M2 macrophages. In contrast, a neutralizing monoclonal anti–IL-37 increased LPS-induced IL-6, TNFα and IL-1β (P < 0.01). The suppression by IL-37 was consistently observed at low picomolar but not nanomolar concentrations. Whereas LPS induced a 12-fold increase in TNFα mRNA, IL-37 pretreatment decreased the expression to only 3-fold over background (P < 0.01). Mechanistically, LPS-induced p38 and pERK were reduced by IL-37. Recombinant IL-37 bound to the immobilized ligand binding α-chain of the IL-18 receptor as well as to the decoy receptor IL-1R8. In M1 macrophages, LPS increased the surface expression of IL-1R8. Compared with human blood monocytes, resting M1 cells express more surface IL-1R8 as well as total IL-1R8; there was a 16-fold increase in IL-1R8 mRNA levels when pretreated with IL-37. IL-37 reduced LPS-induced TNFα and IL-6 by 50–55% in mouse bone marrow-derived dendritic cells, but not in dendritic cells derived from IL-1R8–deficient mice. In mice subjected to systemic LPS-induced inflammation, pretreatment with IL-37 reduced circulating and organ cytokine levels. Thus, in addition to a nuclear function, IL-37 acts as an extracellular cytokine by binding to the IL-18 receptor but using the IL-1R8 for its anti-inflammatory properties.

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Expression of LAIR-1 (CD305) on Human Blood Monocytes as a Marker of Hepatic Cirrhosis Progression

Journal of Immunology Research ◽

10.1155/2019/2974753 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12 ◽

Cited By ~ 2

Author(s):

María Martínez-Esparza ◽

Antonio José Ruiz-Alcaraz ◽

Violeta Carmona-Martínez ◽

María Dolores Fernández-Fernández ◽

Gonzalo Antón ◽

...

Keyword(s):

Liver Cirrhosis ◽

Cell Surface ◽

Human Blood ◽

Total Content ◽

Cell Surface Expression ◽

Human Macrophage ◽

Blood Monocytes ◽

Cirrhotic Patients

Background and Aim. The presumed role of the inhibitory receptor LAIR-1 (CD305) in the inflammatory response suggests that it might contribute to the pathophysiology of chronic inflammatory diseases such as liver cirrhosis. We studied the LAIR-1 expression on liver macrophages and blood monocytes related to the progression of liver cirrhosis. Methods. The expression of LAIR-1 was analyzed by immunohistochemistry, flow cytometry, and Western blot. Results. We found a decreased number of macrophages expressing LAIR-1 in cirrhotic liver that could be due to a high presence of collagen, ligand of LAIR-1, in the fibrotic tissue which could downregulate its expression or interfere with the immunostaining. The expression of LAIR-1 decreased after cell differentiation, and the total content, but not the cell surface expression, increased after activation in the HL-60 human macrophage in vitro model. Blood monocytes exhibited higher LAIR-1 expression levels in cirrhotic patients, which were evident even in early clinical stages in all monocyte subsets, and greater in the “intermediate” inflammatory monocyte subpopulation. The in vitro activation of human blood monocytes did not increase its expression on the cell surface suggesting that the in vivo increase of LAIR-1 must be the result of a specific combination of stimuli present in cirrhotic patients. This represents an exclusive feature of liver cirrhosis, since blood monocytes from other chronic inflammatory pathologies showed similar or lower LAIR-1 levels compared with those of healthy controls. Conclusions. These results may indicate that monocyte LAIR-1 expression is a new biomarker to early detect liver damage caused by chronic inflammation in liver cirrhosis.

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In-VitroDifferentiation of Mature Dendritic Cells From Human Blood Monocytes

Developmental Immunology ◽

10.1155/1998/72054 ◽

1998 ◽

Vol 6 (1-2) ◽

pp. 25-39 ◽

Cited By ~ 19

Author(s):

Robert Gieseler ◽

Dirk Heise ◽

Afsaneh Soruri ◽

Peter Schwartz ◽

J. Hinrich Peters

Keyword(s):

Dendritic Cells ◽

Human Blood ◽

T Cell Response ◽

Blood Monocytes ◽

Gm Csf ◽

Hla Dr ◽

Hla Dq ◽

Specific Stimulation ◽

Antigen Presenting

Representing the most potent antigen-presenting cells, dendritic cells (DC) can now be generated from human blood monocytes. We recently presented a novel protocol employing GM-CSF, IL-4, and IFN-γto differentiate monocyte-derived DCin vitro. Here, such cells are characterized in detail. Cells in culture exhibited both dendritic and veiled morphologies, the former being adherent and the latter suspended. Phenotypically, they were CD1a-/dim, CD11a+, CD11b++, CD11c+, CD14dim/-, CD16a-/dim, CD18+, CD32dim/-, CD33+, CD40+, CD45R0+, CD50+, CD54+, CD64-/dim, CD68+, CD71+, CD80dim, CD86+/++, MHC class I++/+++HLA-DR++/+++HLA-DP+, and HLA-DQ+. The DC stimulated a strong allogeneic T-cell response, and further evidence for their autologous antigen-specific stimulation is discussed. Although resembling a mature CD 11c+CD45R0+blood DC subset identified earlier, their differentiation in the presence of the Thl and Th2 cytokines IFN-γand IL-4 indicates that these DC may conform to mature mucosal DC.

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Induction of nuclear lamins A/C in macrophages in in vitro cultures of rat bone marrow precursor cells and human blood monocytes, and in macrophages elicited in vivo by thioglycollate stimulation

Experimental Cell Research ◽

10.1016/0014-4827(90)90184-c ◽

1990 ◽

Vol 190 (2) ◽

pp. 185-194 ◽

Cited By ~ 30

Author(s):

Ruth-Ariane Röber ◽

Robert K.H. Gieseler ◽

J.Hinrich Peters ◽

Klaus Weber ◽

Mary Osborn

Keyword(s):

Bone Marrow ◽

Human Blood ◽

Precursor Cells ◽

In Vitro Cultures ◽

Blood Monocytes ◽

Nuclear Lamins ◽

Bone Marrow Precursor ◽

Rat Bone

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C-Reactive Protein Suppresses the Th17 Response Indirectly by Attenuating the Antigen Presentation Ability of Monocyte Derived Dendritic Cells in Experimental Autoimmune Encephalomyelitis

Frontiers in Immunology ◽

10.3389/fimmu.2021.589200 ◽

2021 ◽

Vol 12 ◽

Author(s):

Zhi-Yuan Shen ◽

Yi Zheng ◽

Maggie K. Pecsok ◽

Ke Wang ◽

Wei Li ◽

...

Keyword(s):

Dendritic Cells ◽

Blood Monocytes ◽

C Reactive Protein ◽

Autoimmune Encephalomyelitis ◽

Th17 Response ◽

Reactive Protein ◽

Antigen Presenting ◽

Experimental Autoimmune

Experimental autoimmune encephalomyelitis (EAE) is a classical murine model for Multiple Sclerosis (MS), a human autoimmune disease characterized by Th1 and Th17 responses. Numerous studies have reported that C-reactive protein (CRP) mitigates EAE severity, but studies on the relevant pathologic mechanisms are insufficient. Our previous study found that CRP suppresses Th1 response directly by receptor binding on naïve T cells; however, we did not observe the effect on Th17 response at that time; thus it remains unclear whether CRP could regulate Th17 response. In this study, we verified the downregulation of Th17 response by a single-dose CRP injection in MOG-immunized EAE mice in vivo while the direct and indirect effects of CRP on Th17 response were differentiated by comparing its actions on isolated CD4+ T cells and splenocytes in vitro, respectively. Moreover, the immune cell composition was examined in the blood and CNS (Central Nervous System), and a blood (monocytes) to CNS (dendritic cells) infiltration pathway is established in the course of EAE development. The infiltrated monocyte derived DCs (moDCs) were proved to be the only candidate antigen presenting cells to execute CRP’s function. Conversely, the decrease of Th17 responses caused by CRP disappeared in the above in vivo and in vitro studies with FcγR2B−/− mice, indicating that FcγR2B expressed on moDCs mediates CRP function. Furthermore, peripheral blood monocytes were isolated and induced to establish moDCs, which were used to demonstrate that the antigen presenting ability of moDCs was attenuated by CRP through FcγR2B, and then NF-κB and ERK signaling pathways were manifested to be involved in this regulation. Ultimately, we perfected and enriched the mechanism studies of CRP in EAE remission, so we are more convinced that CRP plays a key role in protecting against EAE development, which may be a potential therapeutic target for the treatment of MS in human.

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Monocyte Regulation By Soluble Uric Acid

10.26686/wgtn.17006731.v1 ◽

2021 ◽

Author(s):

◽

René Joel McLaughlin

Keyword(s):

Map Kinase ◽

Human Blood ◽

Nuclear Translocation ◽

Blood Monocytes ◽

Lps Challenge ◽

Inflammatory Stimuli ◽

Inflammatory Phenotype ◽

Inflammatory Signalling

<p>Hyperuricaemia is a chronic condition associated with diseases of the metabolic syndrome. However, the cause and effect relationship between increased serum uric acid (UA) levels and the pathophysiology of metabolic dysfunction is far from clear. From an immunological angle hyperuricaemia has been shown to modulate inflammatory signalling in both immune and nonimmune cell types. Blood monocytes are constantly exposed to soluble UA in the circulation but the direct effect of this exposure has not been examined. This research focuses on the how soluble UA alters blood monocyte responses to inflammatory stimuli using in vitro, in vivo and clinical manipulation of UA levels. The Harper group previously found that blood monocytes from hyperuricaemic individuals produced lower levels of inflammatory cytokines compared to monocytes from healthy controls when stimulated ex vivo with LPS. My research began by studying the direct effect of soluble UA on human blood monocytes in vitro. I found that soluble UA reduced monocyte production of pro-inflammatory cytokines and increased IL-10 in response to stimulation with LPS. I identified two inflammatory signalling pathways modulated by soluble UA that could be contributing to this suppressive monocyte phenotype: MAP kinase phosphorylation was reduced alongside increased expression of the regulatory protein DUSP10 and reduced ASC; there was a switch towards anti-inflammatory NFκB signalling illustrated by decreased p65 and increased p50 nuclear translocation. To study the modulation of soluble UA levels in a physiological context I raised serum UA levels in vivo with a model of acute hyperuricaemia and lowered serum UA using two clinically relevant medications: allopurinol and rasburicase. Consistent with in vitro UA treatment, raising serum UA levels in vivo suppressed pro-inflammatory cytokine responses to LPS, increased IL-10 and down-regulated monocyte MAP kinase and NFκB signalling pathways. Acute urate-lowering therapy (ULT) with allopurinol or rasburicase reversed this suppressive inflammatory cytokine and signalling pattern. The PLT2 mouse strain has had the purine metabolic pathway disrupted by random mutagenesis of the gene encoding 5-hiydroxyisourate hydrolase, the enzyme responsible for degradation of the molecule directly downstream of UA, 5-hydroxyisourate. I found that this mutation resulted in chronic hyperuricaemia with an average 2-fold increase in serum UA over C57 mice. LPS challenge resulted in increased IL-10 production in PLT2 mice compared to C57, however no differences in monocyte inflammatory signalling were observed between the two strains. Acute ULT with rasburicase reduced serum UA in the PLT2 strain and subsequent LPS challenge increased monocyte inflammatory signalling. Finally, I studied the effects of ULT on the inflammatory phenotype of human blood monocytes from patients with hyperuricaemia. ULT significantly reduced serum UA levels, which coincided with reduced blood monocyte percentages and adhesion molecule expression (CD11b and ICAM1). ULT increased the inflammatory potential of human blood monocytes: Monocytes stimulated with LPS produced less IL-10; MAP kinase phosphorylation increased alongside increased ASC expression; nuclear translocation of NFκB p65 was increased. ULT also increased expression of the NLRP3 inflammasome components procaspase1, pro-IL-1β and NLRP3. Taken together these results demonstrate a previously unidentified role for soluble UA in moderating monocyte immune responses to inflammatory stimuli. In vitro, in vivo and clinical experimentation all confirmed the immunosuppressive function of soluble UA. This potentially places UA in the centre of innate immune control through the dichotomy of its suppressive soluble effects, demonstrated herein, and the widely reported inflammatory crystalline effects. Importantly, this research illustrates that serum UA levels can be manipulated in a clinical setting to control the inflammatory phenotype of circulating immune cells.</p>

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Generation of Feline Dendritic Cells Derived from Peripheral Blood Monocytes for In Vivo Use

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.12.10.1202-1208.2005 ◽

2005 ◽

Vol 12 (10) ◽

pp. 1202-1208 ◽

Cited By ~ 13

Author(s):

Giulia Freer ◽

Donatella Matteucci ◽

Paola Mazzetti ◽

Leonia Bozzacco ◽

Mauro Bendinelli

Keyword(s):

Immune Response ◽

Dendritic Cells ◽

Peripheral Blood ◽

Poly I:C ◽

Interleukin 4 ◽

Blood Monocytes ◽

Autologous Plasma ◽

Peripheral Blood Monocytes

ABSTRACT Dendritic cells (DCs) are professional antigen-presenting cells that can prime T cells and polarize the cellular immune response. Because Th1-type immune responses have been connected to success in combating viral infection, a promising therapeutic application of DCs would be their differentiation in vitro and injection back into the host to boost an immune response in infected animals. This study was aimed both at developing a protocol to cultivate feline DCs in the absence of exogenous proteins for their use in vivo and at investigating what might be the most appropriate stimulus to induce their maturation in vitro and finding correlates of maturation. We generated DCs from peripheral blood monocytes in the presence of feline interleukin-4 and granulocyte-macrophage colony stimulating factor, and after 5 days their maturation was induced with either lipopolysaccharide, human recombinant tumor necrosis factor alpha, poly(I:C), or activated feline platelets. After 48 h, their CD14, CD1a, major histocompatibility complex class II, and B7.1 surface expression was analyzed in parallel with their ability to uptake antigen or prime a mixed leukocyte reaction. The results presented show that feline DCs cultured in autologous plasma differentiate and are able to mature in the presence of stimuli similar to the ones currently used for other species. The present work sets the grounds for future use of DCs obtained by the protocol described for in vivo vaccination and immunotherapy of feline immunodeficiency virus-infected cats.

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Monocyte Regulation By Soluble Uric Acid

10.26686/wgtn.17006731 ◽

2021 ◽

Author(s):

◽

René Joel McLaughlin

Keyword(s):

Map Kinase ◽

Human Blood ◽

Nuclear Translocation ◽

Blood Monocytes ◽

Lps Challenge ◽

Inflammatory Stimuli ◽

Inflammatory Phenotype ◽

Inflammatory Signalling

<p>Hyperuricaemia is a chronic condition associated with diseases of the metabolic syndrome. However, the cause and effect relationship between increased serum uric acid (UA) levels and the pathophysiology of metabolic dysfunction is far from clear. From an immunological angle hyperuricaemia has been shown to modulate inflammatory signalling in both immune and nonimmune cell types. Blood monocytes are constantly exposed to soluble UA in the circulation but the direct effect of this exposure has not been examined. This research focuses on the how soluble UA alters blood monocyte responses to inflammatory stimuli using in vitro, in vivo and clinical manipulation of UA levels. The Harper group previously found that blood monocytes from hyperuricaemic individuals produced lower levels of inflammatory cytokines compared to monocytes from healthy controls when stimulated ex vivo with LPS. My research began by studying the direct effect of soluble UA on human blood monocytes in vitro. I found that soluble UA reduced monocyte production of pro-inflammatory cytokines and increased IL-10 in response to stimulation with LPS. I identified two inflammatory signalling pathways modulated by soluble UA that could be contributing to this suppressive monocyte phenotype: MAP kinase phosphorylation was reduced alongside increased expression of the regulatory protein DUSP10 and reduced ASC; there was a switch towards anti-inflammatory NFκB signalling illustrated by decreased p65 and increased p50 nuclear translocation. To study the modulation of soluble UA levels in a physiological context I raised serum UA levels in vivo with a model of acute hyperuricaemia and lowered serum UA using two clinically relevant medications: allopurinol and rasburicase. Consistent with in vitro UA treatment, raising serum UA levels in vivo suppressed pro-inflammatory cytokine responses to LPS, increased IL-10 and down-regulated monocyte MAP kinase and NFκB signalling pathways. Acute urate-lowering therapy (ULT) with allopurinol or rasburicase reversed this suppressive inflammatory cytokine and signalling pattern. The PLT2 mouse strain has had the purine metabolic pathway disrupted by random mutagenesis of the gene encoding 5-hiydroxyisourate hydrolase, the enzyme responsible for degradation of the molecule directly downstream of UA, 5-hydroxyisourate. I found that this mutation resulted in chronic hyperuricaemia with an average 2-fold increase in serum UA over C57 mice. LPS challenge resulted in increased IL-10 production in PLT2 mice compared to C57, however no differences in monocyte inflammatory signalling were observed between the two strains. Acute ULT with rasburicase reduced serum UA in the PLT2 strain and subsequent LPS challenge increased monocyte inflammatory signalling. Finally, I studied the effects of ULT on the inflammatory phenotype of human blood monocytes from patients with hyperuricaemia. ULT significantly reduced serum UA levels, which coincided with reduced blood monocyte percentages and adhesion molecule expression (CD11b and ICAM1). ULT increased the inflammatory potential of human blood monocytes: Monocytes stimulated with LPS produced less IL-10; MAP kinase phosphorylation increased alongside increased ASC expression; nuclear translocation of NFκB p65 was increased. ULT also increased expression of the NLRP3 inflammasome components procaspase1, pro-IL-1β and NLRP3. Taken together these results demonstrate a previously unidentified role for soluble UA in moderating monocyte immune responses to inflammatory stimuli. In vitro, in vivo and clinical experimentation all confirmed the immunosuppressive function of soluble UA. This potentially places UA in the centre of innate immune control through the dichotomy of its suppressive soluble effects, demonstrated herein, and the widely reported inflammatory crystalline effects. Importantly, this research illustrates that serum UA levels can be manipulated in a clinical setting to control the inflammatory phenotype of circulating immune cells.</p>

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Functional Studies on the C-Type Lectin Receptor CD302 Present on Dendritic Cells and Macrophages

Blood ◽

10.1182/blood.v126.23.2198.2198 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2198-2198

Author(s):

Derek NJ Hart ◽

Pablo Silveira ◽

Tsun Ho Lo ◽

Nirupama Verma ◽

Ai Vu ◽

...

Keyword(s):

Flow Cytometry ◽

Dendritic Cells ◽

Immune Cell ◽

Blood Monocytes ◽

Lectin Receptors ◽

Functional Studies ◽

Cell Functions ◽

Equity Ownership

Abstract Introduction: C-type lectin receptors (CLR) play an important role in the immune system by recognising molecular patterns expressed by exogenous and endogenous threats. They have been shown to capture and internalise antigens and to mediate other important immune cell functions. DEC205 and CLEC9A are being actively investigated as targets for clinical therapeutic cancer vaccines. We discovered CD302 as a new CLR expressed on human dendritic cells (DC), monocytes and macrophages (J Immunol 2007;179:6052). Our initial studies suggested the molecule could play a role in cell adhesion or migration due to its co-localisation with migratory structures on macrophages. Our study set out to investigate the potential immunological function of CD302 using mouse models and to define its wider tissue expression in man. Methods: We generated CD302 knockout (KO) mice lacking exon 1 of its gene, abrogating transcription, for functional studies. We characterised the transcriptional expression of CD302 in mouse immune cells using real-time PCR. We developed monoclonal mAb to mCD302. Human studies utilized the anti-CD302 mAbs, MMRI-20 & 21 in flow cytometry and confocal microscopy studies of human immune cell populations. Results: CD302 was primarily expressed in mouse liver, lungs, lymph nodes (LN) and spleen. In spleen, macrophages, granulocytes and dendritic cells (DC) expressed CD302. Analysis of LN DC subsets revealed 2.5-fold higher CD302 mRNA expression in migratory compared to resident DC populations. Enumeration of various immune populations in lymphoid organs by flow cytometry uncovered a modest deficiency in migratory DC number and proportion within LN of CD302 KO mice compared to wild-type (WT) mice. In vitro studies showed CD302 KO and WT DC had an equivalent capacity to be activated by various stimuli, prime T cells and migrate towards the lymphoid-homing chemokines CCL19/CCL21. CD302 KO migratory DC exhibited a reduced in vivo migratory capacity to LN after FITC skin-painting. However, CD302 KO macrophages migrated similarly to WT macrophages in vivo in response to thioglycollate. In man, CD302 was present in high density in liver and peripheral blood monocytes and myeloid but not plasmacytoid DC. Current studies are aimed at clarifying its distribution on tissue DC and macrophage subsets. Anti-CD302 coated microbeads were taken up by human monocyte derived macrophages and anti-CD302 mAb was also internalized by DC. Confocal studies showed that CD302 co-localized with F-actin structures at the near basal surface such as filopodia and lamellipodia and podosomes of human macrophages and EGFP tagged CD302 expressed in COS-1 cells associated with F-actin. Conclusion: Our data suggests that CD302 may play a specialist role in DC and macrophage membrane functions. This appears to relate to its ability to associate with F-actin and may contribute to the membrane interactions required for DC to migrate towards the draining LN. Disclosures Hart: DendroCyte BioTech Pty Ltd: Equity Ownership. Clark:DendroCyte BioTech Pty Ltd: Equity Ownership.

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