LPS-stimulated Dendritic Cells Research Articles

Berry Extracts and Their Bioactive Compounds Mitigate LPS and DNFB-Mediated Dendritic Cell Activation and Induction of Antigen Specific T-Cell Effector Responses.

Berries have gained widespread recognition for their abundant natural antioxidant, anti-inflammatory, and immunomodulatory properties. However, there has been limited research conducted thus far to investigate the role of the active constituents of berries in alleviating contact hypersensitivity (CHS), the most prevalent occupational dermatological disease. Our study involved an ex vivo investigation aimed at evaluating the impact of black raspberry extract (BRB-E) and various natural compounds found in berries, such as protocatechuic acid (PCA), proanthocyanidins (PANT), ellagic acid (EA), and kaempferol (KMP), on mitigating the pathogenicity of CHS. We examined the efficacy of these natural compounds on the activation of dendritic cells (DCs) triggered by 2,4-dinitrofluorobenzene (DNFB) and lipopolysaccharide (LPS). Specifically, we measured the expression of activation markers CD40, CD80, CD83, and CD86 and the production of proinflammatory cytokines, including Interleukin (IL)-12, IL-6, TNF-α, and IL-10, to gain further insights. Potential mechanisms through which these phytochemicals could alleviate CHS were also investigated by investigating the role of phospho-ERK. Subsequently, DCs were co-cultured with T-cells specific to the OVA323-339 peptide to examine the specific T-cell effector responses resulting from these interactions. Our findings demonstrated that BRB-E, PCA, PANT, and EA, but not KMP, inhibited phosphorylation of ERK in LPS-activated DCs. At higher doses, EA significantly reduced expression of all the activation markers studied in DNFB- and LPS-stimulated DCs. All compounds tested reduced the level of IL-6 in DNFB-stimulated DCs in Flt3L as well as in GM-CSF-derived DCs. However, levels of IL-12 were reduced by all the tested compounds in LPS-stimulated Flt3L-derived BMDCs. PCA, PANT, EA, and KMP inhibited the activated DC-mediated Interferon (IFN)-γ and IL-17 production by T-cells. Interestingly, PANT, EA, and KMP significantly reduced T-cell proliferation and the associated IL-2 production. Our study provides evidence for differential effects of berry extracts and natural compounds on DNFB and LPS-activated DCs revealing potential novel approaches for mitigating CHS.

Nitric Oxide (NO) regulation of Dendritic cells (DC) metabolism and immune effector functions

Abstract Dendritic cell (DC) activation is characterized by a sustained commitment to glycolysis, a requirement for survival in DC subsets expressing inducible nitric oxide synthase (iNOS, encoded by Nos2 gene). Nitric oxide (NO) can mediate both host-protective and host-cytotoxic effects by interfering with pathogen replication and/or by inhibiting the host mitochondrial respiration, resulting in metabolic rewiring in DCs. These phenomena primarily have been studied in DCs from the classic laboratory inbred mouse strain C57BL/6J (B6), where DCs experience a loss of mitochondrial function due to NO accumulation. To assess the conservation of NO-driven metabolic regulation in DCs, we compared B6 mice to the wild-derived genetically divergent PWD/PhJ (PWD) strain. We show preserved mitochondrial respiration and enhanced post-activation survival in LPS-stimulated PWD DCs due to attenuated NO production. To genetically map PWD’s NO phenotype, we used a congenic mouse strain (B6.PWD-Chr11.2; 11.2) that carries a PWD-derived portion of chromosome 11, including Nos2, on a B6 background. The 11.2 DCs show comparable iNOS expression with lower NO production levels than B6 DCs. We demonstrate that activated 11.2 DCs maintain mitochondrial respiration and TCA cycle carbon flux, compared with B6 DCs. However, reduced NO production by the PWD Nos2 allele results in impaired cellular control of Listeria monocytogenes replication. These studies establish a natural genetic model for restrained endogenous NO production and suggest a useful model to investigate the contribution/regulation of NO in DC metabolism and immune function. This work was supported by the National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (P30GM118228, 1R21AI135385-01A, T32AI055402-15, and R21 AI145306), the NIH, National Institute of Neurological Disorders and Stroke (R01 NS097596), and the National Multiple Sclerosis Society (RG-1901-33309).

Extracellular vesicles from Trypanosoma cruzi-dendritic cell interaction show modulatory properties and confer resistance to lethal infection as a cell-free based therapy strategy.

Extracellular vesicles (EVs) include a heterogeneous group of particles. Microvesicles, apoptotic bodies and exosomes are the most characterized vesicles. They can be distinguished by their size, morphology, origin and molecular composition. To date, increasing studies demonstrate that EVs mediate intercellular communication. EVs reach considerable interest in the scientific community due to their role in diverse processes including antigen-presentation, stimulation of anti-tumoral immune responses, tolerogenic or inflammatory effects. In pathogens, EV shedding is well described in fungi, bacteria, protozoan and helminths parasites. For Trypanosoma cruzi EV liberation and protein composition was previously described. Dendritic cells (DCs), among other cells, are key players promoting the immune response against pathogens and also maintaining self-tolerance. In previous reports we have demonstrate that T. cruzi downregulates DCs immunogenicity in vitro and in vivo. Here we analyze EVs from the in vitro interaction between blood circulating trypomastigotes (Tp) and bone-marrow-derived DCs. We found that Tp incremented the number and the size of EVs in cultures with DCs. EVs displayed some exosome markers and intracellular RNA. Protein analysis demonstrated that the parasite changes the DC protein-EV profile. We observed that EVs from the interaction of Tp-DCs were easily captured by unstimulated-DCs in comparison with EVs from DCs cultured without the parasite, and also modified the activation status of LPS-stimulated DCs. Noteworthy, we found protection in animals treated with EVs-DCs+Tp and challenged with T. cruzi lethal infection. Our goal is to go deep into the molecular characterization of EVs from the DCs-Tp interaction, in order to identify mediators for therapeutic purposes.

Human Monocyte-Derived Dendritic Cells Are the Pharmacological Target of the Immunosuppressant Flavonoid Silibinin.

Silibinin, a natural polyphenolic flavonoid, is known to possess anti-inflammatory, anticancer, antioxidant, and immunomodulatory properties. However, the effects of Silibinin on the maturation and immunostimulatory functions of human dendritic cells (DC) remain to be elucidated. In this study, we have attempted to ascertain whether Silibinin influences the maturation, cytokine production, and antigen-presenting capacity of human monocyte-derived DC. We show that Silibinin significantly suppresses the upregulation of costimulatory and MHC molecules in LPS-stimulated mature DC and inhibits lipopolysaccharide (LPS)-induced interleukin (IL)-12, IL-23, and TNF-α production. Furthermore, Silibinin impairs the proliferation response of the allogenic memory CD4 T lymphocytes elicited by LPS-matured DC and their Th1/Th17 profile. These findings demonstrate that Silibinin displays immunosuppressive activity by inhibiting the maturation and activation of human DC and support its potential application of adjuvant therapy in the treatment of autoimmune diseases.

Inhibition of Notch Signaling Stimulates Osteoclastogenesis From the Common Trilineage Progenitor Under Inflammatory Conditions.

Osteoclasts, macrophages and dendritic cells (DCs) can be derived from a common trilineage myeloid progenitor of hematopoietic origin. Progenitor commitment is susceptible to regulation through Notch signaling. Our aim was to determine the effects of Notch modulation on trilineage progenitor commitment and functional properties of differentiated cells under inflammatory conditions. We used the conditional inducible CX3CR1CreERT2 mouse strain to achieve overexpression of the Notch 1 intracellular domain (NICD1) or to inhibit Notch signaling via deletion of the transcription factor RBP-J in a bone marrow population, used as a source of the trilineage progenitor (CD45+Ly6G−CD3−B220−NK1.1−CD11b–/loCD115+). Cre-recombinase, under the control of the CX3CR1 promoter, expressed in the monocyte/macrophage lineage, was induced in vitro by 4-hydroxytamoxifen. Differentiation of osteoclasts was induced by M-CSF/RANKL; macrophages by M-CSF; DCs by IL-4/GM-CSF, and inflammation by LPS. Functionally, DCs were tested for the ability to process and present antigen, macrophages to phagocytose E. coli particles, and osteoclasts to resorb bone and express tartrate-resistant acid phosphatase (TRAP). We found that Notch 1 signal activation suppressed osteoclast formation, whereas disruption of the Notch canonical pathway enhanced osteoclastogenesis, resulting in a higher number and size of osteoclasts. RANK protein and Ctsk gene expression were upregulated in osteoclastogenic cultures from RBP-J+ mice, with the opposing results in NICD1+ mice. Notch modulation did not affect the number of in vitro differentiated macrophages and DCs. However, RBP-J deletion stimulated Il12b and Cd86 expression in macrophages and DCs, respectively. Functional assays under inflammatory conditions confirmed that Notch silencing amplifies TRAP expression by osteoclasts, whereas the enhanced phagocytosis by macrophages was observed in both NICD1+ and RBP-J+ strains. Finally, antigen presentation by LPS-stimulated DCs was significantly downregulated with NICD1 overexpression. This experimental setting allowed us to define a cell-autonomous response to Notch signaling at the trilineage progenitor stage. Although Notch signaling modulation affected the activity of all three lineages, the major effect was observed in osteoclasts, resulting in enhanced differentiation and function with inhibition of canonical Notch signaling. Our results indicate that Notch signaling participates as the negative regulator of osteoclast activity during inflammation, which may be relevant in immune and bone diseases.

Reduced CXCL1 production by endogenous IL-37 expressing dendritic cells does not affect T cell activation.

The dendritic cell (DC)-derived cytokine profile contributes to naive T cell differentiation, thereby directing the immune response. IL-37 is a cytokine with anti-inflammatory characteristics that has been demonstrated to induce tolerogenic properties in DC. In this study we aimed to evaluate the influence of IL-37 on DC-T cell interaction, with a special focus on the role of the chemokine CXCL1. DC were cultured from bone marrow of human IL-37 transgenic (hIL-37Tg) or WT mice. The phenotype of unstimulated and LPS-stimulated DC was analyzed (co-stimulatory molecules and MHCII by flow cytometry, cytokine profile by RT-PCR and ELISA), and T cell stimulatory capacity was assessed in mixed lymphocyte reaction. The role of CXCL1 in T cell activation was analyzed in T cell stimulation assays with anti-CD3 or allogeneic DC. The expression of the co-stimulatory molecules CD40, CD80 and CD86, and of MHCII in LPS-stimulated DC was not affected by endogenous expression of IL-37, whereas LPS-stimulated hIL-37Tg DC produced less CXCL1 compared to LPS-stimulated WT DC. T cell stimulatory capacity of LPS-matured hIL-37Tg DC was comparable to that of WT DC. Recombinant mouse CXCL1 did not increase T cell proliferation either alone or in combination with anti-CD3 or allogeneic DC, nor did CXCL1 affect the T cell production of interferon-γ and IL-17. Endogenous IL-37 expression does not affect mouse DC phenotype or subsequent T cell stimulatory capacity, despite a reduced CXCL1 production. In addition, we did not observe an effect of CXCL1 in T cell proliferation or differentiation.

Mast cell–derived IL-13 downregulates IL-12 production by skin dendritic cells to inhibit the TH1 cell response to cutaneous antigen exposure

Atopic dermatitis (AD) is characterized by a skin barrier defect aggravated by mechanical injury inflicted by scratching, a TH2 cell-dominated immune response, and susceptibility to viral skin infections that are normally restrained by a TH1 cell response. The signals leading to a TH2 cell-dominated immune response in AD are not completely understood. Our aim was to determine the role of IL-13 in initiation of the TH cell response to cutaneously encountered antigens. Wild-type, Il13-/-, Il1rl1-/-, and Il4ra-/- mice, as well as mice with selective deficiency of IL-13 in mast cells (MCs) were studied; in addition, dendritic cells (DCs) purified from the draining lymph nodes of tape-stripped and ovalbumin (OVA)-sensitized skin were examined for their ability to polarize naive OVA-TCR transgenic CD4+ T cells. Cytokine expression was examined by reverse-transcriptase quantitative PCR, intracellular flow cytometry, and ELISA. Contact hypersensitivity to dinitrofluorobenzene was examined. Tape stripping caused IL-33-driven upregulation of Il13 expression by skin MCs. MC-derived IL-13 acted on DCs from draining lymph nodes of OVA-sensitized skin to selectively suppress their ability to polarize naive OVA-TCR transgenic CD4+ T cells into IFN-γ-secreting cells. MC-derived IL-13 inhibited the TH1 cell response in contact hypersensitivity to dinitrofluorobenzene. IL-13 suppressed IL-12 production by mouse skin-derived DCs invitro and invivo. Scratching upregulated IL13 expression in human skin, and IL-13 suppressed the capacity of LPS-stimulated human skin DCs to express IL-12 and promote IFN-γ secretion by CD4+ T cells. Release of IL-13 by cutaneous MCs in response to mechanical skin injury inhibits the TH1 cell response to cutaneous antigen exposure in AD.

Evaluation of immunomodulatory effects of Boswellia sacra essential oil on T-cells and dendritic cells

BackgroundBoswellia sacra resin has been commonly used as analgesic, antimicrobial, and anti-inflammatory properties, which reflect its immunomodulatory activity. Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) and sentinel cells that regulate the immune response. This study aims at investigating whether crude essential oil extracted from Boswellia sacra resin (BSEO), has a potential effect on the phenotype and functions of human monocyte-derived DCs.MethodsOil extract from the resin of Boswellia sacra was prepared by hydrodistillation using a custom made hydrodistiller. BSEO-mediated cell viability has been initially studied on human skin dermis cells (HSD) and DC precursors using quantitative and qualitative assays before applying on DCs. Human DCs were generated from differentiated peripheral blood monocytes cultured in media containing both GM-CSF and IL-4. DCs were exposed to 5 μg/mL or 10 μg/mL of BSEO in vitro. Morphological, phonotypical, and functional properties studied with microscopy, flow cytometry, and ELISA.ResultsCrude BSEO was found to interfere with the maturation and differentiation of DCs from precursor cells in the presence or absence of lipopolysaccharide (LPS). BSEO-treated DCs, cultured in the presence of LPS, reduced the ability of allogeneic T cells to proliferate compared to that co-cultured with LPS-stimulated DCs only. In addition, the endocytic capacity and secretion of IL-10 by DCs treated with BSEO was enhanced in comparison to LPS treated cells. Analysis of the chemical composition of BESO using GC-MS (Clarus 500 GC/MS, PerkinElmer, Shelton, CT) revealed the presence of compounds with several biological activities including antibacterial, antioxidant, and anti-inflammatory properties.ConclusionResults indicated that BSEO deviates the differentiation of monocytes into immature DCs. Furthermore, stimulation of immature DCs with BSEO was unable to generate full DC maturation. However, these findings may potentially be employed to generate DCs with tolerogenic properties that are able to induce tolerance in diseases with hypersensitivity, autoimmunity as well as transplantation.

Alleviation of Collagen-Induced Arthritis by Crotonoside through Modulation of Dendritic Cell Differentiation and Activation.

Crotonoside, a guanosine analog originally isolated from Croton tiglium, is reported to be a potent tyrosine kinase inhibitor with immunosuppressive effects on immune cells. Due to its potential immunotherapeutic effects, we aimed to evaluate the anti-arthritic activity of crotonoside and explore its immunomodulatory properties in alleviating the severity of arthritic symptoms. To this end, we implemented the treatment of crotonoside on collagen-induced arthritic (CIA) DBA/1 mice and investigated its underlying mechanisms towards pathogenic dendritic cells (DCs). Our results suggest that crotonoside treatment remarkably improved clinical arthritic symptoms in this CIA mouse model as indicated by decreased pro-inflammatory cytokine production in the serum and suppressed expression of co-stimulatory molecules, CD40, CD80, and MHC class II, on CD11c+ DCs from the CIA mouse spleens. Additionally, crotonoside treatment significantly reduced the infiltration of CD11c+ DCs into the synovial tissues. Our in vitro study further demonstrated that bone marrow-derived DCs (BMDCs) exhibited lower yield in numbers and expressed lower levels of CD40, CD80, and MHC-II when incubated with crotonoside. Furthermore, LPS-stimulated mature DCs exhibited limited capability to prime antigen-specific CD4+ and T-cell proliferation, cytokine secretions, and co-stimulatory molecule expressions when treated with crotonoside. Our pioneer study highlights the immunotherapeutic role of crotonoside in the alleviation of the CIA via modulation of pathogenic DCs, thus creating possible applications of crotonoside as an immunosuppressive agent that could be utilized and further explored in treating autoimmune disorders in the future.

Magnoliae Flos Essential Oil as an Immunosuppressant in Dendritic Cell Activation and Contact Hypersensitivity Responses

Magnoliae Flos is a commonly used traditional medicinal material in Asia. It is used to treat sinusitis, nasal congestion, and hypersensitive skin. Because Magonlia Flos was described as an aromatic material in ancient Chinese texts, we hypothesized that its essential oil may be used to treat immune disorders. Dendritic cells (DCs), regarded as a major target of immunomodulators to control immune responses, play a critical role in the adaptive immune response. In this study, Magnoliae Flos essential oil (MFEO) decreased the production of the cytokines TNF-, IL-6, and IL-12p70 in lipopolysaccharide (LPS)-stimulated DCs. It also suppressed the surface markers MHC II, CD80, and CD86 in LPS-stimulated DCs. Animal models demonstrated that the 2,4-Dinitro-1-fluorobenzene (DNFB) inducing a contact hypersensitivity response was inhibited following treatment with MFEO. In addition, MFEO inhibited the infiltration of T cells in the ears of DNFB-induced mice. To explore its bioactive compounds, the components of MFEO were analyzed using gas chromatography (GC) and GC-mass spectrometry. The results revealed that the major compounds in MFEO are camphor and 1,8-cineole. Additional DC bioassays confirmed that these compounds substantially suppressed cytokine production in LPS-induced DCs. Therefore, we demonstrated that MFEO exhibits an immunosuppressive effect both in vivo and in vitro, and camphor and 1,8-cineole may be the major components responsible for its immunosuppressive ability. The findings indicate that MFEO has the potential to be developed as a new immunosuppressant for excessive diseases.

Anti-inflammatory mediators ST2 and SIGIRR are induced by diphenyldifluoroketone EF24 in lipopolysaccharide-stimulated dendritic cells.

The objective of this study was to investigate the effect of EF24, an NF-κB-inhibitor, on the expression of negative regulators in IL-1R pathway, namely ST2 and SIGIRR. Murine JAWS II dendritic cells (DC) were challenged with lipopolysaccharide (LPS, 100 ng/ml) for 4 h, followed by treatment with 10 μM EF24 for 1 h. ST2 and SIGIRR expression was monitored by qRT-PCR and immunoblotting. ST2L and MyD88 interaction was studied by co-immunoprecipitation, and IL-33, a ST2L ligand, was assayed by ELISA. Activation of transcription factor SP1 was examined by confocal microscopy, immunoblotting, and EMSA. The effect of EF24 on accumulation of ubiquitinated proteins in DCs and proteolysis of fluorogenic peptides by purified proteasome was studied. We found that EF24 upregulated the expression of ST2 and SIGIRR and decreased the interaction of the membrane-bound ST2 (ST2L) with MyD88, and significantly reduced IL-33 levels in LPS-stimulated DCs. Simultaneously it increased the activation of transcription factor SP1and restored the basal level of ubiquitinated proteins in LPS-stimulated DCs. Moreover, EF24 inhibited trypsin- and chymotrypsin-like activity of proteasome by directly interacting with 26S proteasome. The results suggest that EF24 activates endogenous anti-inflammatory arm of IL-1R signaling, most likely by stabilizing SP1 against proteasomal degradation.

Age and sex determine CD4+ T cell stimulatory and polarizing capacity of rat splenic dendritic cells.

The study investigated influence of sex and age on splenic myeloid dendritic cells (DCs) from Dark Agouti rats. Freshly isolated DCs from young males exhibited less mature phenotype and greater endocytic capacity compared with those from age-matched females. Upon LPS stimulation in vitro they were less potent in stimulating allogeneic CD4+ cells in mixed leukocyte reaction (MLR), due to lower expression of MHC II, and greater NO and IL-10 production. In accordance with higher TGF-β production, young male rat DCs were less potent in stimulating IL-17 production in MLR than those from young females. Irrespective of sex, endocytic capacity and responsiveness of DCs to LPS stimulation in culture, judging by their allostimulatory capacity in MLR decreased with age, reflecting decline in MHC II surface density followed by their greater NO production; the effects more prominent in females. Additionally, compared with LPS-stimulated DCs from young rats, those from sex-matched aged rats were more potent in stimulating IL-10 production in MLR, whereas capacity of DCs from aged female and male rats to stimulate IL-17 production remained unaltered and decreased, respectively. This reflected age-related shift in IL-6/TGF-β production level ratio in LPS-stimulated DC cultures towards TGF-β, and sex-specific age-related remodeling CD4+ cell cytokine pathways. Additionally, compared with LPS-stimulated DCs from young rats, those cells from sex-matched aged rats were less potent in stimulating IFN-γ production in MLR, the effect particularly prominent in MLRs encompassing male rat DCs. The study showed that stimulatory and polarizing capacity of DCs depends on rat sex and age.

Quercetin‐Induced miR‐369‐3p Suppresses Chronic Inflammatory Response Targeting C/EBP‐β

Dendritic cells (DCs) are the most potent antigen-presenting cells that play an important role in the crosstalk between the innate and the adaptive immune response. Quercetin exposure is identified as an effective strategy to suppress the inflammatory response induced by LPS. In this study, using a next-generation sequencing analysis, the effect of quercetin on microRNAs (miRNAs) expression in DCs is examined. A signature of 113 miRNAs that are differentially regulated in LPS-stimulated DCs after quercetin exposure is defined. It is demonstrated that the loss of function of miR-369-3p in LPS-stimulated DCs during quercetin exposure led to an increase of CCAAT/enhancer binding protein β (C/EBP-β) mRNA and protein and its downstream targets tumor necrosis factor-α (TNF-α) and interleukin 6 (IL6). Conversely, it is shown that the ectopic induction of miR-369-3p without quercetin suppresses the inflammatory response of LPS reducing C/EBP-β, TNF-α, and IL6 production. In vivo, oral administration of quercetin in dextran-sulfate-sodium-induced colitis induces miR-369-3p expression. These findings indicate that quercetin-induced miR-369-3p regulates the inflammatory cascade in chronic inflammatory response and present promising therapeutic implications.

Pdlim4 is essential for CCR7-JNK-mediated dendritic cell migration and F-actin-related dendrite formation.

Dendritic cells (DCs) are the most potent professional antigen (Ag)-presenting cells and inducers of T cell-mediated immunity. A previous microarray analysis identified PDZ and LIM domain protein 4 (Pdlim4) as a candidate marker for DC maturation. The aim of this study was to investigate whether Pdlim4 influences DC migration and maturation. Mouse bone marrow-derived DCs were transduced lentivirally with Pdlim4 short hairpin RNA and examined by confocal microscopy, flow cytometry, ELISA, and Western blotting. Pdlim4 was highly induced in LPS-stimulated mature DCs (mDCs). Pdlim4-knockdown mDCs showed reduced expression of molecules associated with Ag presentation and T-cell costimulation, reduced cytokine production, and functional defects in their ability to activate T cells. Moreover, Pdlim4 was necessary for mDC migration via C-C chemokine receptor type 7 (CCR7)-JNK in in vitro Transwell assays. The importance of Pdlim4 in DC migration was confirmed with an in vivo migration model in which C57BL/6 mice were injected with fluorescently labeled DCs in the footpad and migration to the popliteal lymph nodes was assessed by flow cytometry. Moreover, dendrite formation in mDCs was remarkably attenuated under Pdlim4 knockdown. Taken together, these results demonstrate that Pdlim4 is necessary for DC migration via CCR7-JNK, dendrite formation, and subsequent development of functional T-cell responses.-Yoo, J.-Y., Jung, N.-C., Lee, J.-H., Choi, S.-Y., Choi, H.-J., Park, S.-Y., Jang, J.-S., Byun, S.-H., Hwang, S.-U., Noh, K.-E., Park, Y., Lee, J., Song, J.-Y., Seo, H. G., Lee, H. S., Lim, D.-S. Pdlim4 is essential for CCR7-JNK-mediated dendritic cell migration and F-actin-related dendrite formation.

Plant-Derived Polyphenols Modulate Human Dendritic Cell Metabolism and Immune Function via AMPK-Dependent Induction of Heme Oxygenase-1.

Polyphenols are important immunonutrients which have been investigated in the context of inflammatory and autoimmune disease due to their significant immunosuppressive properties. However, the mechanism of action of many polyphenols is unclear, particularly in human immune cells. The emerging field of immunometabolism has highlighted the significance of metabolic function in the regulation of immune cell activity, yet the effects of polyphenols on immune cell metabolic signaling and function has not been explored. We have investigated the effects of two plant-derived polyphenols, carnosol and curcumin, on the metabolism of primary human dendritic cells (DC). We report that human DC display an increase in glycolysis and spare respiratory capacity in response to LPS stimulation, which was attenuated by both carnosol and curcumin treatment. The regulation of DC metabolism by these polyphenols appeared to be mediated by their activation of the cellular energy sensor, AMP-activated Protein Kinase (AMPK), which resulted in the inhibition of mTOR signaling in LPS-stimulated DC. Previously we have reported that both carnosol and curcumin can regulate the maturation and function of human DC through upregulation of the immunomodulatory enzyme, Heme Oxygenase-1 (HO-1). Here we also demonstrate that the induction of HO-1 by polyphenols in human DC is dependent on their activation of AMPK. Moreover, pharmacological inhibition of AMPK was found to reverse the observed reduction of DC maturation by carnosol and curcumin. This study therefore describes a novel relationship between metabolic signaling via AMPK and HO-1 induction by carnosol and curcumin in human DC, and characterizes the effects of these polyphenols on DC immunometabolism for the first time. These results expand our understanding of the mechanism of action of carnosol and curcumin in human immune cells, and suggest that polyphenol supplementation may be useful to regulate the metabolism and function of immune cells in inflammatory and metabolic disease.

Distinct Role of IL-27 in Immature and LPS-Induced Mature Dendritic Cell-Mediated Development of CD4+ CD127+3G11+ Regulatory T Cell Subset.

Interleukin-27 (IL-27) plays an important role in regulation of anti-inflammatory responses and autoimmunity; however, the molecular mechanisms of IL-27 in modulation of immune tolerance and autoimmunity have not been fully elucidated. Dendritic cells (DCs) play a central role in regulating immune responses mediated by innate and adaptive immune systems, but regulatory mechanisms of DCs in CD4+ T cell-mediated immune responses have not yet been elucidated. Here we show that IL-27 treated mature DCs induced by LPS inhibit immune tolerance mediated by LPS-stimulated DCs. IL-27 treatment facilitates development of the CD4+ CD127+3G11+ regulatory T cell subset in vitro and in vivo. By contrast, IL-27 treated immature DCs fail to modulate development of the CD4+CD127+3G11+ regulatory T cell sub-population in vitro and in vivo. Our results suggest that IL-27 may break immune tolerance induced by LPS-stimulated mature DCs through modulating development of a specific CD4+ regulatory T cell subset mediated by 3G11 and CD127. Our data reveal a new cellular regulatory mechanism of IL-27 that targets DC-mediated immune responses in autoimmune diseases such as multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE).

Extracellular vimentin modulates human dendritic cell activation

Vimentin is an intermediate filament protein traditionally considered to be an intracellular protein with a structural role. However, recent evidence suggests that vimentin can also be found outside the cell in disease conditions such as cancer, traumatic tissue injury, and inflammation. Extracellular vimentin was previously found to stimulate innate immunity by increasing monocyte and macrophage ability to kill bacteria. However, vimentin has also been previously found to decrease neutrophil infiltration into inflamed tissue. How extracellular vimentin affects the initiation of adaptive immune responses is unknown. Initiation of adaptive immunity involves priming of naïve T cells by antigen-presenting cells, the most effective of which are dendritic cells (DCs). In this study, we demonstrate how extracellular vimentin modulates lipopolysaccharide (LPS) – induced activation of human DCs. Using cytometric bead arrays, we show that extracellular vimentin decreases LPS-activated DC secretion of pro-inflammatory cytokines IL-6 and IL-12 while increasing secretion of the anti-inflammatory cytokine IL-10. Using flow cytometry, we show that extracellular vimentin does not significantly affect LPS-induced DC surface expression of MHC I (HLA-ABC) or MHC II (HLA-DR) presentation molecules, costimulatory factors (CD80, CD86), or the DC maturation marker (CD83). Further, LPS-stimulated DCs co-cultured with allogeneic naïve CD4 + T cells (Th0) induced less secretion of the pro-inflammatory Th1 effector cytokine IFN-γ in the presence of vimentin than in the presence of LPS alone. This result suggests that vimentin reduces Th1 differentiation. Taken together, our data suggest that extracellular vimentin may inhibit pro-inflammatory adaptive immune responses, by blocking DC secretion of pro-inflammatory cytokines. Thus, extracellular vimentin may play an important role in cancer or trauma-complications by inducing suppression of the adaptive immune response. In a positive sense, the presence of extracellular vimentin may prevent tissue-damage from contributing to the development of autoimmunity. Consequently, extracellular vimentin may become a novel drug target for treatment of a variety of pro- and anti-inflammatory disease conditions.

IL-12/23p40 overproduction by dendritic cells leads to an increased Th1 and Th17 polarization in a model of Yersinia enterocolitica-induced reactive arthritis in TNFRp55-/- mice.

Dendritic cells (DCs) play critical functions in the initiation of immune responses. Understanding their role in reactive arthritis (ReA) will help delineate the pathogenesis of this arthropathy. In early studies, we detected IL-12/23p40 deregulation in Yersinia entercolitica (Ye)-induced ReA in TNFRp55-deficient (TNFRp55-/-) mice. In this study, we assessed the contribution of DCs in this overproduction. First, greater levels of IL-12/23p40, IFN-γand IL-17A were confirmed in supernatants of lipopolysaccharide (LPS)-stimulated TNFRp55-/-splenocytes obtained on arthritis onset (day 14 after Ye infection). Later, DCs were identified as a precise source of IL-12/23p40 since increased frequency of splenic IL-12/23p40+DCs was detected in TNFRp55-/- mice. After robust in vivo amplification of DCs by injection of Fms-like tyrosine kinase 3-Ligand (Flt3L)-transfected BL16 melanoma, DCs were purified. These cells recapitulated the higher production of IL-12/23p40 under TNFRp55deficiency. In agreement with these results, TNFRp55-/- DCs promoted Th1 and Th17 programs by co-culture with WT CD4+lymphocytes. A mechanistic study demonstrated that JNK and p38 MAPK pathways are involved in IL-12/23p40 overproduction in purified TNFRp55-/- DCs as well as in the JAWS II cell line. This deregulation was once again attributed to TNFRp55 deficiency since CAY10500, a specific inhibitor of this pathway, compromised TNF-mediated IL-12/23p40 control in LPS-stimulated WT DCs. Simultaneously, this inhibition reduced IL-10 production, suggesting its role mediating IL-12/23p40 regulation by TNFRp55 pathway. These results provide experimental data on the existence of a TNFRp55-mediated anti-inflammatory circuit in DCs. Moreover, these cells may be considered as a novel target in the treatment of ReA.

Guanabenz Prevents d-Galactosamine/Lipopolysaccharide-Induced Liver Damage and Mortality.

Multi-organ failure in response to uncontrolled microbial infection is characterized by low blood pressure accompanied by a systemic over-inflammation state, caused by massive pro-inflammatory cytokines release and liver damage. Recently, the integrated stress response (ISR), characterized by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, was involved with controlling apoptosis in stressed hepatocytes and associated with poor survival to endotoxin challenge. Lipopolysaccharide (LPS) alone is able to induce the ISR in hepatocytes and can trigger massive liver damage along with tumor necrosis factor-alpha (TNF-α) expression. Consequently, drugs interfering with eIF2α phosphorylation may represent potential candidates for the treatment of such pathologies. We, therefore, used Guanabenz (GBZ), a small compound with enhancing eIF2α phosphorylation activity to evaluate its effect on bacterial LPS sensing and endotoxemia. GBZ is confirmed here to have an anti-inflammatory activity by increasing in vitro interleukin-10 (IL-10) production by LPS-stimulated dendritic cells. We further show that in the d-galactosamine (d-galN)/LPS-dependent lethality model, intraperitoneal injection of GBZ promoted mice survival, prevented liver damage, increased IL-10 levels, and inhibited TNF-α production. GBZ and its derivatives could therefore represent an interesting pharmacological solution to control systemic inflammation and associated acute liver failure.

Glucose represses dendritic cell-induced T cell responses

Glucose and glycolysis are important for the proinflammatory functions of many immune cells, and depletion of glucose in pathological microenvironments is associated with defective immune responses. Here we show a contrasting function for glucose in dendritic cells (DCs), as glucose represses the proinflammatory output of LPS-stimulated DCs and inhibits DC-induced T-cell responses. A glucose-sensitive signal transduction circuit involving the mTOR complex 1 (mTORC1), HIF1α and inducible nitric oxide synthase (iNOS) coordinates DC metabolism and function to limit DC-stimulated T-cell responses. When multiple T cells interact with a DC, they compete for nutrients, which can limit glucose availability to the DCs. In such DCs, glucose-dependent signalling is inhibited, altering DC outputs and enhancing T-cell responses. These data reveal a mechanism by which T cells regulate the DC microenvironment to control DC-induced T-cell responses and indicate that glucose is an important signal for shaping immune responses.