Loss of the orphan nuclear receptor NR2F6 enhances CD8+ T-cell memory via IFN-γ.

Memory formation is a hallmark of T cell-mediated immunity, but how differentiation into either short-lived effector cells (SLECs, CD127-KLRG1+) or memory precursors cells (MPECs, CD127+KLRG1-) and subsequent regulation of long-term memory is adjusted is incompletely understood. Here, we show that loss of the nuclear orphan receptor NR2F6 in germ-line Nr2f6-deficient mice enhances antigen-specific CD8+ memory formation up to 70 days after bacterial infection with Listeria monocytogenes (LmOVA) and boosts inflammatory IFN-γ, TNFα, and IL-2 cytokine recall responses. Adoptive transfer experiments using Nr2f6-/- OT-I T-cells showed that the augmented memory formation is CD8+ T-cell intrinsic. Although the relative difference between the Nr2f6+/+ and Nr2f6-/- OT-I memory compartment declines over time, Nr2f6-deficient OT-I memory T cells mount significantly enhanced IFN-γ responses upon reinfection with increased clonal expansion and improved host antigen-specific CD8+ T-cell responses. Following a secondary adoptive transfer into naïve congenic mice, Nr2f6-deficient OT-I memory T cells are superior in clearing LmOVA infection. Finally, we show that the commitment to enhanced memory within Nr2f6-deficient OT-I T cells is established in the early phases of the antibacterial immune response and is IFN-γ mediated. IFN-γ blocking normalized MPEC formation of Nr2f6-deficient OT-I T cells. Thus, deletion or pharmacological inhibition of NR2F6 in antigen-specific CD8+ T cells may have therapeutic potential for enhancing early IFN-γ production and consequently the functionality of memory CD8+ T cells in vivo.

Effect of the melatonin nuclear receptor RORα on monochromatic light-induced T-lymphocyte proliferation in chicken thymus.

Present study clarified role of melatonin nuclear receptor RORα in monochromatic light-induced T-lymphocyte proliferation in chicks. Green light elevated plasma melatonin level and organ index, T-lymphocyte proliferation and IL-2 production in thymus, but decreased RORα, p-P65 and p-IκB expressions relative to red light. By contrast, pinealectomy decreased the melatonin content and reversed the stimulatory effect of green light, and resulted in that these thymus parameters were not significantly different among the light-treated groups. Exogenous melatonin supplementation enhanced T-lymphocyte proliferation and IL-2 production in cultured thymocytes. This stimulatory effect of melatonin was reversed by RORα agonist but was enhanced by RORα antagonist. In contrast to RORα antagonist, RORα agonist decreased cytoplasmic P65 level and increased nuclear P65 level. Supplementation with P65 antagonist increased T-lymphocyte proliferation. We conclude that RORα could negatively regulate green light-enhanced T-lymphocyte proliferation in chick thymus by upregulating IκB phosphorylation, which promotes P65 nuclear translocation and NF-κB activation.

The G protein-coupled receptors deorphanization landscape.

G protein-coupled receptors (GPCRs) are usually highlighted as being both the largest family of membrane proteins and the most productive source of drug targets. However, most of the GPCRs are understudied and hence cannot be used immediately for innovative therapeutic strategies. Besides, there are still around 100 orphan receptors, with no described endogenous ligand and no clearly defined function. The race to discover new ligands for these elusive receptors seems to be less intense than before. Here, we present an update of the various strategies employed to assign a function to these receptors and to discover new ligands. We focus on the recent advances in the identification of endogenous ligands with a detailed description of newly deorphanized receptors. Replication being a key parameter in these endeavors, we also discuss the latest controversies about problematic ligand-receptor pairings. In this context, we propose several recommendations in order to strengthen the reporting of new ligand-receptor pairs.

RORγt+ Innate Lymphoid Cells Promote Lymph Node Metastasis of Breast Cancers.

Cancer cells tend to metastasize first to tumor-draining lymph nodes, but the mechanisms mediating cancer cell invasion into the lymphatic vasculature remain little understood. Here, we show that in the human breast tumor microenvironment (TME), the presence of increased numbers of RORγt+ group 3 innate lymphoid cells (ILC3) correlates with an increased likelihood of lymph node metastasis. In a preclinical mouse model of breast cancer, CCL21-mediated recruitment of ILC3 to tumors stimulated the production of the CXCL13 by TME stromal cells, which in turn promoted ILC3-stromal interactions and production of the cancer cell motile factor RANKL. Depleting ILC3 or neutralizing CCL21, CXCL13, or RANKL was sufficient to decrease lymph node metastasis. Our findings establish a role for RORγt+ILC3 in promoting lymphatic metastasis by modulating the local chemokine milieu of cancer cells in the TME. Cancer Res; 77(5); 1083-96. ©2017 AACR.

AIM/CD5L: a key protein in the control of immune homeostasis and inflammatory disease.

CD5L, a soluble protein belonging to the SRCR superfamily, is expressed mostly by macrophages in lymphoid and inflamed tissues. The expression of this protein is transcriptionally controlled by LXRs, members of the nuclear receptor family that play major roles in lipid homeostasis. Research undertaken over the last decade has uncovered critical roles of CD5L as a PRR of bacterial and fungal components and in the control of key mechanisms in inflammatory responses, with involvement in processes, such as infection, atherosclerosis, and cancer. In this review, we summarize the current knowledge of CD5L, its roles at the intersection between lipid homeostasis and immune response, and its potential use as a diagnostic biomarker in a variety of diseases, such as TB and liver cirrhosis.

Macrophages engulfing apoptotic cells produce nonclassical retinoids to enhance their phagocytic capacity.

Previous work in our laboratory has shown that transglutaminase 2 (TG2) acting as a coreceptor for integrin ß3 is required for proper phagocytosis of apoptotic cells. In the absence of TG2, systemic lupus erythematosus-like autoimmunity develops in mice, similarly to other mice characterized by a deficiency in the clearance of apoptotic cells. In this study, we demonstrate that increasing TG2 expression alone in wild-type macrophages is not sufficient to enhance engulfment. However, during engulfment, the lipid content of the apoptotic cells triggers the lipid-sensing receptor liver X receptor (LXR), which in response upregulates the expression of the phagocytic receptor Mer tyrosine kinase and the phagocytosis-related ABCA1, and that of retinaldehyde dehydrogenases leading to the synthesis of a nonclassical retinoid. Based on our retinoid analysis, this compound might be a dihydro-retinoic acid derivative. The novel retinoid then contributes to the upregulation of further phagocytic receptors including TG2 by ligating retinoic acid receptors. Inhibition of retinoid synthesis prevents the enhanced phagocytic uptake induced by LXR ligation. Our data indicate that stimulation of LXR enhances the engulfment of apoptotic cells via regulating directly and indirectly the expression of a range of phagocytosis-related molecules, and its signaling pathway involves the synthesis of a nonclassical retinoid. We propose that retinoids could be used for enhancing the phagocytic capacity of macrophages in diseases such as systemic lupus erythematosus, where impaired phagocytosis of apoptotic cells plays a role in the pathogenesis of the disease.

The oxysterol receptor LXRß protects against DSS- and TNBS-induced colitis in mice.

We examined the function of the oxysterol receptors (LXRs) in inflammatory bowel disease (IBD) through studying dextran sodium sulfate (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice and by elucidating molecular mechanisms underlying their anti-inflammatory action. We observed that Lxr-deficient mice are more susceptible to colitis. Clinical indicators of colitis including weight loss, diarrhea and blood in feces appeared earlier and were more severe in Lxr-deficient mice and particularly LXRß protected against symptoms of colitis. Addition of an LXR agonist led to faster recovery and increased survival. In contrast, Lxr-deficient mice showed slower recovery and decreased survival. In Lxr-deficient mice, inflammatory cytokines and chemokines were increased together with increased infiltration of immune cells in the colon epithelium. Activation of LXRs strongly suppressed expression of inflammatory mediators including TNFα. While LXRα had anti-inflammatory effects in CD11b(+) immune cell populations, LXRß in addition had anti-inflammatory effects in colon epithelial cells. Lack of LXRß also induced CD4(+)/CD3(+) immune cell recruitment to the inflamed colon. Expression of both LXRA and LXRB was significantly suppressed in inflamed colon from subjects with IBD compared with non-inflamed colon. Taken together, our observations suggest that the LXRs could provide interesting targets to reduce the inflammatory responses in IBD.

Cyanidin-3-O-ß-glucoside ameliorates lipopolysaccharide-induced acute lung injury by reducing TLR4 recruitment into lipid rafts.

Cyanidin-3-O-ß-glucoside (C3G), a typical anthocyanin pigment that exists in the human diet, has been reported to have anti-inflammatory properties. The aim of this study was to detect the effect of C3G on LPS-induced acute lung injury and to investigate the molecular mechanisms. Acute lung injury was induced by intratracheal administration of LPS in mice. Alveolar macrophages from mice were stimulated with LPS and were treated with C3G. Our results showed that C3G attenuated lung histopathologic changes, myeloperoxidase (MPO) activity, TNF-α, IL-1ß and IL-6 production in LPS-induced acute lung injury model. In vitro, C3G dose-dependently inhibited TNF-α, IL-1ß, IL-6, IL-10 and IFN-ß production, as well as NF-κB and IRF3 activation in LPS-stimulated alveolar macrophages. Furthermore, C3G disrupted the formation of lipid rafts by depleting cholesterol and inhibited TLR4 translocation into lipid rafts. Moreover, C3G activated LXRα-ABCG1-dependent cholesterol efflux. Knockout of LXRα abrogated the anti-inflammatory effects of C3G. In conclusion, C3G has a protective effect on LPS-induced acute lung injury. The promising anti-inflammatory mechanisms of C3G is associated with up-regulation of the LXRα-ABCG1 pathway which result in disrupting lipid rafts by depleting cholesterol and reducing translocation of TLR4 to lipid rafts, thereby suppressing TLR4 mediated inflammatory response.

Liver X receptor regulates mouse GM-CSF-derived dendritic cell differentiation in vitro.

Liver X receptors (LXRs) are nuclear receptors that play an essential role in lipid and cholesterol metabolism. Emerging studies indicate a potential function for LXRs in regulating dendritic cell (DC)-dependent immune responses; however, the role of LXRs in DC differentiation is largely unknown. Here, we report that LXRα regulates the differentiation of mouse GM-CSF-derived DCs. Activation or overexpression of LXRα significantly enhanced myeloid DC differentiation from mouse bone marrow (BM) cells, while siRNA-mediated knockdown of LXRα suppressed DC differentiation. In addition, we demonstrated that LXR agonist-programmed DCs showed an increased capacity for stimulating T-cell proliferation. Mechanistic studies showed that activation of LXR could inhibit the phosphorylation of STAT3 and downregulate the expression of its target, S100A9, an important negative regulator of myeloid DC differentiation. We also found that Histone deacetylase (HDAC) inhibition interfered with the effect of LXR on STAT3 signaling via acetylation of STAT3. Chromatin immunoprecipitation assays confirmed that LXR activation and HDAC inhibition balanced the recruitment of STAT3 to the S100A9 promoter, which involved distinct post-translational modifications of STAT3. In conclusion, our observations demonstrated a novel role for LXRα in GM-CSF-derived DC differentiation and revealed the underlying mechanism.

The nuclear receptor LXRα controls the functional specialization of splenic macrophages.

Macrophages are professional phagocytic cells that orchestrate innate immune responses and have considerable phenotypic diversity at different anatomical locations. However, the mechanisms that control the heterogeneity of tissue macrophages are not well characterized. Here we found that the nuclear receptor LXRα was essential for the differentiation of macrophages in the marginal zone (MZ) of the spleen. LXR-deficient mice were defective in the generation of MZ and metallophilic macrophages, which resulted in abnormal responses to blood-borne antigens. Myeloid-specific expression of LXRα or adoptive transfer of wild-type monocytes restored the MZ microenvironment in LXRα-deficient mice. Our results demonstrate that signaling via LXRα in myeloid cells is crucial for the generation of splenic MZ macrophages and identify an unprecedented role for a nuclear receptor in the generation of specialized macrophage subsets.

Reciprocal negative cross-talk between liver X receptors (LXRs) and STAT1: effects on IFN-γ-induced inflammatory responses and LXR-dependent gene expression.

Liver X receptors (LXRs) exert key functions in lipid homeostasis and in control of inflammation. In this study we have explored the impact of LXR activation on the macrophage response to the endogenous inflammatory cytokine IFN-γ. Transcriptional profiling studies demonstrate that ∼38% of the IFN-γ-induced transcriptional response is repressed by LXR activation in macrophages. LXRs also mediated inhibitory effects on selected IFN-γ-induced genes in primary microglia and in a model of IFN-γ-induced neuroinflammation in vivo. LXR activation resulted in reduced STAT1 recruitment to the promoters tested in this study without affecting STAT1 phosphorylation. A closer look into the mechanism revealed that SUMOylation of LXRs, but not the presence of nuclear receptor corepressor 1, was required for repression of the NO synthase 2 promoter. We have also analyzed whether IFN-γ signaling exerts reciprocal effects on LXR targets. Treatment with IFN-γ inhibited, in a STAT1-dependent manner, the LXR-dependent upregulation of selective targets, including ATP-binding cassette A1 (ABCA1) and sterol response element binding protein 1c. Downregulation of ABCA1 expression correlated with decreased cholesterol efflux to apolipoprotein A1 in macrophages stimulated with IFN-γ. The inhibitory effects of IFN-γ on LXR signaling did not involve reduced binding of LXR/retinoid X receptor heterodimers to target gene promoters. However, overexpression of the coactivator CREB-binding protein/p300 reduced the inhibitory actions of IFN-γ on the Abca1 promoter, suggesting that competition for CREB-binding protein may contribute to STAT1-dependent downregulation of LXR targets. The results from this study suggest an important level of bidirectional negative cross-talk between IFN-γ/STAT1 and LXRs with implications both in the control of IFN-γ-mediated immune responses and in the regulation of lipid metabolism.

Myelin-derived lipids modulate macrophage activity by liver X receptor activation.

Multiple sclerosis is a chronic, inflammatory, demyelinating disease of the central nervous system in which macrophages and microglia play a central role. Foamy macrophages and microglia, containing degenerated myelin, are abundantly found in active multiple sclerosis lesions. Recent studies have described an altered macrophage phenotype after myelin internalization. However, it is unclear by which mechanisms myelin affects the phenotype of macrophages and how this phenotype can influence lesion progression. Here we demonstrate, by using genome wide gene expression analysis, that myelin-phagocytosing macrophages have an enhanced expression of genes involved in migration, phagocytosis and inflammation. Interestingly, myelin internalization also induced the expression of genes involved in liver-X-receptor signaling and cholesterol efflux. In vitro validation shows that myelin-phagocytosing macrophages indeed have an increased capacity to dispose intracellular cholesterol. In addition, myelin suppresses the secretion of the pro-inflammatory mediator IL-6 by macrophages, which was mediated by activation of liver-X-receptor ß. Our data show that myelin modulates the phenotype of macrophages by nuclear receptor activation, which may subsequently affect lesion progression in demyelinating diseases such as multiple sclerosis.

Converse regulation of CCR7-driven human dendritic cell migration by prostaglandin E2 and liver X receptor activation.

Migration and homing of DCs to lymphoid organs is pivotal for inducing adaptive immunity and tolerance. DC homing depends on the chemokine receptor CCR7. However, expression of CCR7 alone is not sufficient for effective DC migration. A second signal, mediated by prostaglandin E(2) (PGE(2)), is critical for the development of a migratory DC phenotype. PGE(2) is important for inducing efficient immune responses, but, if deregulated, contributes to chronic inflammation, autoimmune diseases through Th17-cell development and tumorigenesis. In contrast, activation of liver X receptor (LXR)α has recently been shown to interfere with CCR7 expression and migration of DCs resulting in a reduced immune response. Here, we demonstrate that PGE(2) downregulates LXRα expression in human monocyte derived as well as ex vivo DCs. Moreover, PGE(2) stimulation dampens LXR activation, auto-regulation and LXR-mediated gene transcription. Consequently, we show that PGE(2) enhances CCR7 expression and migration of LXR-activated DCs. Furthermore, we provide evidence that PGE(2) signaling and LXR activation specifically elicit converse effects on CCR7 expression and DC migration. In contrast, production of MMP9, CCL4, COX-2, and IL-23 is solely regulated by PGE(2) , but not by LXR activation, offering new perspectives for therapeutic interventions.

Control of the immune system by oxysterols and cancer development.

Oxysterols/oxysterol receptors have been shown to modulate several immune cell subsets, such as macrophages, T-cells and B-cells, neutrophils and dendritic cells (DCs). They participate in the control of several pathologic processes, that is, infectious diseases, atherosclerosis and autoimmunity. Moreover, some oxysterols have also been shown to favor tumor progression by dampening the antitumor immune response. The cellular responses generated by oxysterols depend on the engagement of Liver X Receptor (LXR) α and/or ß isoforms, which induce activation of target genes or trans-repression of pro-inflammatory gene transcription. Recently, some reports have described a different mechanism of action of oxysterols, mediated by the engagement of G-Protein Coupled Receptors. Here, we summarize LXR-dependent and LXR-independent responses of oxysterols on immune cells with possible effects on tumor development.

Lipid metabolism and neuroinflammation in Alzheimer's disease: a role for liver X receptors.

Liver X receptors (LXR) are nuclear receptors that have emerged as key regulators of lipid metabolism. In addition to their functions as cholesterol sensors, LXR have also been found to regulate inflammatory responses in macrophages. Alzheimer's disease (AD) is a neurodegenerative disease characterized by a progressive cognitive decline associated with inflammation. Evidence indicates that the initiation and progression of AD is linked to aberrant cholesterol metabolism and inflammation. Activation of LXR can regulate neuroinflammation and decrease amyloid-ß peptide accumulation. Here, we highlight the role of LXR in orchestrating lipid homeostasis and neuroinflammation in the brain. In addition, diabetes mellitus is also briefly discussed as a significant risk factor for AD because of the appearing beneficial effects of LXR on glucose homeostasis. The ability of LXR to attenuate AD pathology makes them potential therapeutic targets for this neurodegenerative disease.

Molecular evidence for the involvement of RORα and RORγ in immune response in teleost.

In mammals, retinoid-related orphan receptors (ROR) consist of three members as RORα, RORß and RORγ. It is well known that RORα plays a critical role in cerebellum development while RORγt directs T helper 17 (Th17) cell differentiation. So far, the knowledge on fish ROR family is limited as only zebrafish ROR family members have been characterized, showing that they play roles in embryonic and cerebellar development. In this study, we have cloned two paralogues for RORα (RORα1 and RORα2) and RORγ (RORγ1 and RORγ2) from grass carp (Ctenopharyngodon idellus). Phylogenetic analysis showed that grass carp RORα and RORγ were grouped in the clade of zebrafish RORα and RORγ, respectively. Real-time RT-PCR assay revealed that these four ROR transcripts exhibited similar expression patterns, in particular the high levels in pituitary, brain and some immune-related tissues, suggesting that all of them may play a role in endocrine and immune system of teleost. To explore the immune roles of grass carp RORα and RORγ, their expression was detected in periphery blood lymphocytes (PBLs) challenged by immune stimuli. Results showed that both RORα and RORγ mRNA levels were up-regulated by PHA but not LPS in PBLs, suggesting that their expression may be subject to different immune processes. In the same cell model, poly I:C stimulation induced RORγ1/2 but not RORα1/2 expression, pointing to the different roles of grass carp RORα and RORγ in immune response. Consistently, bacterial challenge significantly up-regulated the expression of these four ROR genes in spleen, headkidney and thymus. These results not only contribute to elucidate the roles of ROR in fish immunity but also facilitate to further clarify the existence of Th17-like cells in fish.

Biological roles of liver X receptors in immune cells.

Liver X receptors (LXRs) are members of the nuclear receptor superfamily that are activated by specific oxysterols. LXRs heterodimerize with retinoid X receptors to regulate positively the expression of a variety of target genes, many of which are involved in lipid and glucose metabolism. In the last few years, new targets of LXR activation have been identified with roles in the modulation of immune responses. Moreover, LXRs mediate repression of inflammatory pathways through mechanisms collectively known as transrepression. Here, we revise recent findings on the impact of LXR activation on immune responses, with an emphasis on advances in the understanding of the molecular mechanisms that mediate these effects.

Liver X receptors bridge hepatic lipid metabolism and inflammation.

Liver X receptors (LXRs) are members of the superfamily of metabolic nuclear receptors, which play central roles in the regulation of cholesterol absorption, efflux, transportation and excretion and many other processes correlating with lipid metabolism. LXRs can also regulate inflammation in vitro and in vivo. Accumulating evidence demonstrates that LXR are involved in the metabolism and inflammation in human diseases. Nonalcoholic fatty liver disease (NAFLD) is classically associated with lipid metabolic disorders and inflammatory responses, especially in the nonalcoholic steatohepatitis (NASH) phase. The effects of LXRs on cholesterol metabolism and inflammation make them attractive as a potential target for the treatment of NAFLD. Since the ability to synthesize triglycerides may be protective in obesity and fatty liver, the hepatic lipogenesis by LXRs should not rule out the possibility of the use of LXRs in NAFLD.

Metabolism, LXR/LXR ligands, and tumor immune escape.

The mechanisms of tumor immune evasion have gained increasing interest among the tumor immunologists, because of their ability to suppress spontaneous and immunotherapy-elicited antitumor responses. Recent studies clearly show that the deletion of cells/molecules involved in tumor evasion is capable of restoring antitumor immune responses, ultimately leading to tumor rejection in mouse tumor models. These studies further support and strengthen the idea to target not only the cancer cell-intrinsic defects but also those affecting cells of the microenvironment, such as immune cells. The alterations of cancer cell metabolism are also emerging as important regulators of immune cell function, with particular emphasis on immune-escape mechanisms. Indeed, intermediate or final products of cancer cell metabolism may interfere with the function of immune cells infiltrating the tumor microenvironment. This review will focus on the role of cholesterol metabolism, with particular emphasis on the axis LXR/LXR ligands. This axis has been shown to affect DC migration to lymphoid organs, thus dampening the induction of successful antitumor responses. Finally, we will discuss whether this pathway may interfere with other immune cells infiltrating tumors and how to improve spontaneous and immunotherapy-based antitumor responses by counteracting this immune-escape mechanism.

Decreased expression of liver X receptor-α in macrophages infected with Chlamydia pneumoniae in human atherosclerotic arteries in situ.

In in vitro experiments, Chlamydia pneumoniae has been shown to infect macrophages and to accelerate foam cell formation. It has been hypothesized that the C. pneumoniae infection affects foam cell formation by suppressing the expression of liver X receptors (LXR), but whether such an event occurs in human atherosclerosis is not known. In this study we examined carotid artery segments, obtained by endarterectomy, in which the presence of C. pneumoniae was confirmed by both polymerase chain reaction and immunohistochemistry. The expression of LXR-α in macrophages infected with C. pneumoniae and macrophages that were not infected was compared using a quantitative immunohistochemical analysis. The analysis revealed a 2.2-fold reduction in the expression of LXR-α in C. pneumoniae-infected cells around the lipid cores in atherosclerotic plaques. In the cytoplasm of laser-capture microdissected cells that were immunopositive for C. pneumoniae, electron microscopy demonstrated the presence of structures with the appearance of elementary, reticulate and aberrant bodies of C. pneumoniae. We conclude that LXR-α expression is reduced in C. pneumoniae-infected macrophages in human atherosclerotic lesions which supports the hypothesis that C. pneumoniae infection might suppress LXR expression in macrophages transforming into foam cells.