ARID5B regulates fatty acid metabolism and proliferation at the Pre-B cell stage during B cell development.

During B cell development in bone marrow, large precursor B cells (large Pre-B cells) proliferate rapidly, exit the cell cycle, and differentiate into non-proliferative (quiescent) small Pre-B cells. Dysregulation of this process may result in the failure to produce functional B cells and pose a risk of leukemic transformation. Here, we report that AT rich interacting domain 5B (ARID5B), a B cell acute lymphoblastic leukemia (B-ALL) risk gene, regulates B cell development at the Pre-B stage. In both mice and humans, we observed a significant upregulation of ARID5B expression that initiates at the Pre-B stage and is maintained throughout later stages of B cell development. In mice, deletion of Arid5b in vivo and ex vivo exhibited a significant reduction in the proportion of immature B cells but an increase in large and small Pre-B cells. Arid5b inhibition ex vivo also led to an increase in proliferation of both Pre-B cell populations. Metabolic studies in mouse and human bone marrow revealed that fatty acid uptake peaked in proliferative B cells then decreased during non-proliferative stages. We showed that Arid5b ablation enhanced fatty acid uptake and oxidation in Pre-B cells. Furthermore, decreased ARID5B expression was observed in tumor cells from B-ALL patients when compared to B cells from non-leukemic individuals. In B-ALL patients, ARID5B expression below the median was associated with decreased survival particularly in subtypes originating from Pre-B cells. Collectively, our data indicated that Arid5b regulates fatty acid metabolism and proliferation of Pre-B cells in mice, and reduced expression of ARID5B in humans is a risk factor for B cell leukemia.

Lcn2 mediates adipocyte-muscle-tumor communication and hypothermia in pancreatic cancer cachexia.

OBJECTIVE: Adipose tissue is the largest endocrine organ. When activated by cancer cells, adipocytes secrete adipocytokines and release fatty acids, which are then transferred to cancer cells and used for structural and biochemical support. How this metabolic symbiosis between cancer cells and adipocytes affects skeletal muscle and thermogenesis during cancer cachexia is unknown. Cancer cachexia is a multiorgan syndrome and how the communication between tissues is established has yet to be determined. We investigated adipose tissue secretory factors and explored their role in crosstalk of adipocytes, muscle, and tumor during pancreatic cancer cachexia. METHODS: We used a pancreatic cancer cachexia mouse model generated by syngenic implantation of pancreatic ductal adenocarcinoma (PDAC) cells (KPC) intraperitoneally into C57BL/6 mice and Lcn2-knockout mice. For in vitro studies, adipocytes (3T3-L1 and primary adipocytes), cachectic cancer cells (Panc0203), non-cachectic cancer cells (Du145 cells), and skeletal muscle cells (C2C12 myoblasts) were used. RESULTS: To identify molecules involved in the crosstalk of adipose tissue with muscle and tumors, we treated 3T3-L1 adipocytes with conditioned medium (CM) from cancer cells. Upon screening the secretomes from PDAC-induced adipocytes, several adipocytokines were identified, including lipocalin 2 (Lcn2). We investigated Lcn2 as a potential mediator of cachexia induced by adipocytes in response to PDAC. During tumor progression, mice exhibited a decline in body weight gain, which was accompanied by loss of adipose and muscle tissues. Tumor-harboring mice developed drastic hypothermia because of a dramatic loss of fat in brown adipose tissue (BAT) and suppression of the thermogenesis pathway. We inhibited Lcn2 with an anti-Lcn2 antibody neutralization or genomic ablation in mice. Lcn2 deficiency significantly improved body temperature in tumor-bearing mice, which was supported by the increased expression of Ucp1 and ß3-adrenergic receptor in BAT. In addition, Lcn2 inhibition abrogated the loss of fat and muscle in tumor-bearing mice. In contrast to tumor-bearing WT mice, the corresponding Lcn2-knockout mice showed reduced ATGL expression in iWAT and decreased the expression of muscle atrophy molecular markers MuRF-1 and Fbx32. CONCLUSIONS: This study showed that Lcn2 is causally involved in the dysregulation of adipose tissue-muscle-tumor crosstalk during pancreatic cancer cachexia. Therapeutic targets that suppress Lcn2 may minimize the progression of cachexia.

Feedback regulation of Arid5a and Ppar-γ2 maintains adipose tissue homeostasis.

Immune cells infiltrate adipose tissues and provide a framework to regulate energy homeostasis. However, the precise underlying mechanisms and signaling by which the immune system regulates energy homeostasis in metabolic tissues remain poorly understood. Here, we show that the AT-rich interactive domain 5A (Arid5a), a cytokine-induced nucleic acid binding protein, is important for the maintenance of adipose tissue homeostasis. Long-term deficiency of Arid5a in mice results in adult-onset severe obesity. In contrast, transgenic mice overexpressing Arid5a are highly resistant to high-fat diet-induced obesity. Inhibition of Arid5a facilitates the in vitro differentiation of 3T3-L1 cells and fibroblasts to adipocytes, whereas its induction substantially inhibits their differentiation. Molecular studies reveal that Arid5a represses the transcription of peroxisome proliferator activated receptor gamma 2 (Ppar-γ2) due to which, in the absence of Arid5a, Ppar-γ2 is persistently expressed in fibroblasts. This phenomenon is accompanied by enhanced fatty acid uptake in Arid5a-deficient cells, which shifts metabolic homeostasis toward prolipid metabolism. Furthermore, we show that Arid5a and Ppar-γ2 are dynamically counterregulated by each other, hence maintaining adipogenic homeostasis. Thus, we show that Arid5a is an important negative regulator of energy metabolism and can be a potential target for metabolic disorders.

Regulatory T-Cells Mediate IFN-α-Induced Resistance against Antigen-Induced Arthritis.

Objective: CD4+FoxP3+CD25+ regulatory T-cells (Tregs) are important for preventing tissue destruction. Here, we investigate the role of Tregs for protection against experimental arthritis by IFN-α. Methods: Arthritis was triggered by intra-articular injection of methylated bovine serum albumin (mBSA) in wild-type mice, Foxp3DTReGFP+/- mice [allowing selective depletion of Tregs by diphtheria toxin (DT)] and CD4-Cre+/- IFNA1R flox/flox mice (devoid of IFNAR signaling in T-cells) earlier immunized with mBSA, with or without treatment with IFN-α or the indoleamine 2,3-dioxygenase (IDO)-metabolite kynurenine. Tregs were depleted in DT-treated Foxp3DTReGFP+/- mice and enumerated by FoxP3 staining. Suppressive capacity of FACS-sorted CD25+highCD4+ Tregs was tested in vivo by adoptive transfer and ex vivo in cocultures with antigen-stimulated CFSE-stained T-responder (CD25-CD4+) cells. IDO was inhibited by 1-methyl tryptophan. Results: Both control mice and mice devoid of IFNAR-signaling in T helper cells were protected from arthritis by IFN-α. Depletion of Tregs in the arthritis phase, but not at immunization, abolished the protective effect of IFN-α and kynurenine against arthritis. IFN-α increased the number of Tregs in ex vivo cultures upon antigen recall stimulation but not in naïve cells. IFN-α also increased the suppressive capacity of Tregs against mBSA-induced T-responder cell proliferation ex vivo and against arthritis when adoptively transferred. The increased suppressive activity against proliferation conferred by IFN-α was clearly reduced by in vivo inhibition of IDO at immunization, which also abolished the protective effect of IFN-α against arthritis. Conclusion: By activating IDO during antigen sensitization, IFN-α activates Tregs, which prevent arthritis triggered by antigen rechallenge. This is one way by which IFN-α suppresses inflammation.

Arid5a-deficient mice are highly resistant to bleomycin-induced lung injury.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are among the major causes of death worldwide due to acute inflammation in the lung. AT-rich interactive domain-containing 5a (Arid5a) is an RNA-binding protein involved in inflammatory autoimmune disease through post-transcriptional control of Il6, Stat3 and Tbx21 gene expression. We found that Arid5a-deficient mice were highly refractory to bleomycin (BLM)-induced lethality. Arid5a deficiency suppressed lung pathology, cytokine production (especially, IL-6), and clinical symptoms in BLM-treated mice. Production of reactive oxygen species (ROS) in response to BLM-induced cellular damage was inhibited in Arid5a-deficient mice, potentially affecting the level of oxidized 1-palmitoyl-2-arachidonoyl-phosphaticylcholine (OxPAPC) production. OxPAPC, which is supposed to be a TLR4/TLR2 ligand, stimulated expression of the Arid5a and Il6 genes. Thus, reduction of ROS production in Arid5a-deficient mice could mitigate OxPAPC production, which in turn decreases IL-6 production in vivo due to dysregulated post-transcriptional regulation by loss of Arid5a. Therefore, the control of Arid5a expression represents a potential therapeutic target for treatment of ALI and ARDS.

Arid5a exacerbates IFN-γ-mediated septic shock by stabilizing T-bet mRNA.

Adenine-thymine (AT)-rich interactive domain containing protein 5a (Arid5a) is an RNA-binding protein that has been shown to play an important immune regulatory function via the stabilization of IL-6 and STAT3 mRNA. However, the role of Arid5a in the overwhelming and uncontrolled immune response that leads to septic shock is unknown. Here, we report that Arid5a-deficient mice are highly resistant to lipopolysaccharide (LPS)-induced endotoxic shock and secrete lower levels of major proinflammatory cytokines, including IFN-γ, IL-6, and TNF-α, than WT mice in response to LPS. Arid5a deficiency resulted in decreased levels of IFN-γ under Th1 cell conditions, in which T-box expressed in T cells (T-bet) mRNA expression was inhibited. Arid5a bound to the conserved stem loop structure of the 3'UTR of T-bet and stabilized its mRNA. Arid5a-deficient mice were also resistant to Propionibacterium acnes-primed LPS injection, which is considered to be a T-cell-mediated IFN-γ dependent endotoxic shock mouse model. Thus, regulation of IFN-γ by Arid5a via the stabilization of T-bet mRNA in Th1 cells contributes to the development of septic shock in mice. In addition, our previous study suggests that Arid5a control the IL-6 level in vivo in response to LPS by stabilization of IL-6 mRNA. We also observed that neutralization of IFN-γ and IL-6 significantly recovered the mice from endotoxic shock. Taken together, we conclude that Arid5a regulates the augmentation of IL-6 and IFN-γ in response to LPS, which possibly works synergistically for amplification of various other cytokines that ultimately cause the development of septic shock in mice.

IDO1 and TGF-ß Mediate Protective Effects of IFN-α in Antigen-Induced Arthritis.

IFN-α prevents Ag-induced arthritis (AIA), and in this study we investigated the role of IDO1 and TGF-ß signaling for this anti-inflammatory property of IFN-α. Arthritis was induced by methylated BSA (mBSA) in mBSA-sensitized wild-type (WT), Ido1-/-, or Ifnar-/- mice, treated or not with IFN-α or the IDO1 product kynurenine (Kyn). Enzymatic IDO1 activity, TGF-ß, and plasmacytoid dendritic cells (pDC) were neutralized by 1-methyltryptophan and Abs against TGF-ß and pDC, respectively. IDO1 expression was determined by RT-PCR, Western blot, and FACS, and enzymatic activity by HPLC. Proliferation was measured by 3H-thymidine incorporation and TGF-ß by RT-PCR and ELISA. WT but not Ido1-/- mice were protected from AIA by IFN-α, and Kyn, the main IDO1 product, also prevented AIA, both in WT and Ifnar-/- mice. Protective treatment with IFN-α increased the expression of IDO1 in pDC during AIA, and Ab-mediated depletion of pDC, either during mBSA sensitization or after triggering of arthritis, completely abrogated the protective effect of IFN-α. IFN-α treatment also increased the enzymatic IDO1 activity (Kyn/tryptophan ratio), which in turn activated production of TGF-ß. Neutralization of enzymatic IDO1 activity or TGF-ß signaling blocked the protective effect of IFN-α against AIA, but only during sensitization and not after triggering of arthritis. Likewise, inhibition of the IDO1 enzymatic activity in the sensitization phase, but not after triggering of arthritis, subdued the IFN-α-induced inhibition of mBSA-induced proliferation. In conclusion, presence of IFN-α at Ag sensitization activates an IDO1/TGF-ß-dependent anti-inflammatory program that upon antigenic rechallenge prevents inflammation via pDC.

Local but Not Systemic Administration of Uridine Prevents Development of Antigen-Induced Arthritis.

OBJECTIVE: Uridine has earlier been show to down modulate inflammation in models of lung inflammation. The aim of this study was to evaluate the anti-inflammatory effect of uridine in arthritis. METHODS: Arthritis was induced by intra-articular injection of mBSA in the knee of NMRI mice pre-immunized with mBSA. Uridine was either administered locally by direct injection into the knee joint or systemically. Systemic treatment included repeated injections or implantation of a pellet continuously releasing uridine during the entire experimental procedure. Anti-mBSA specific immune responses were determined by ELISA and cell proliferation and serum cytokine levels were determined by Luminex. Immunohistochemistry was used to identify cells, study expression of cytokines and adhesion molecules in the joint. RESULTS: Local administration of 25-100 mg/kg uridine at the time of arthritis onset clearly prevented development of joint inflammation. In contrast, systemic administration of uridine (max 1.5 mg uridine per day) did not prevent development of arthritis. Protection against arthritis by local administration of uridine did not affect the anti-mBSA specific immune response and did not prevent the rise in serum levels of pro-inflammatory cytokines associated with the triggering of arthritis. In contrast, local uridine treatment efficiently inhibited synovial expression of ICAM-1 and CD18, local cytokine production and recruitment of leukocytes to the synovium. CONCLUSION: Local, but not systemic administration of uridine efficiently prevented development of antigen-induced arthritis. The protective effect did not involve alteration of systemic immunity to mBSA but clearly involved inhibition of synovial expression of adhesion molecules, decreased TNF and IL-6 production and prevention of leukocyte extravasation. Further, uridine is a small, inexpensive molecule and may thus be a new therapeutic option to treat joint inflammation in RA.

Dendritic cells activated by double-stranded RNA induce arthritis via autocrine type I IFN signaling.

Viral dsRNA can be found at the site of inflammation in RA patients, and intra-articular injection of dsRNA induces arthritis by activating type I IFN signaling in mice. Further, DCs, a major source of IFN-α, can be found in the synovium of RA patients. We therefore determined the occurrence of DCs in dsRNA-induced arthritis and their ability to induce arthritis. Here, we show, by immunohistochemistry, that cells expressing the pan-DC marker CD11c and the pDC marker 120G8 are present in the inflamed synovium in dsRNA-induced arthritis. Flt3L-generated and splenic DCs preactivated with dsRNA before intra-articular injection, but not mock-stimulated cells, clearly induced arthritis. Induction of arthritis was dependent on type I IFN signaling in the donor DCs, whereas IFNAR expression in the recipient was not required. Sorting of the Flt3L-DC population into cDCs (CD11c(+), PDCA-1(-)) and pDCs (CD11c(+), PDCA-1(+)) revealed that both subtypes were arthritogenic and produced type I IFN if treated with dsRNA. Taken together, these results demonstrate that viral nucleic acids can elicit arthritis by activating type I IFN signaling in DCs. Once triggered, autocrine type I IFN signaling in dsRNA-activated DCs is sufficient to propagate arthritis.

Type I IFN protects against antigen-induced arthritis.

Autoimmune diseases including rheumatoid arthritis (RA) involve immune reactions against specific antigens. The type I IFN system is suspected to promote autoimmunity in systemic lupus erythematosus, but may also dampen immune reactions in e.g. inflammatory bowel disease. This prompted us to investigate the role of type I IFN in antigen-induced arthritis (AIA). The importance of type I IFN in methylated (m) BSA-induced arthritis was studied by using mice deficient for the type I IFN receptor (IFNAR) and by administration of the IFN-α activator viral double-stranded (ds) RNA or recombinant IFN-α at antigen sensitization. In IFNAR knock-out mice, arthritis severity was significantly higher than in WT mice. Administration of dsRNA at antigen sensitization protected WT but not IFNAR KO mice from arthritis. Also, addition of recombinant IFN-α during the immunization, but not the induction phase of arthritis, almost abolished arthritis. Protection mediated by IFN-α was accompanied by delayed and decreased antigen-specific proliferative responses, including impaired lymph node recall responses after intra-articular antigenic challenge. In conclusion, we demonstrate that type I IFN can prevent joint inflammation by downregulating antigen-specific cellular immunity.

Antioxidant activity and polyphenol content in edible wild fruits from Nepal.

Fifteen fruits commonly used by the ethnic population in Nepal were studied for the antioxidant activity and total polyphenol content (TPC). Among them, Terminalia bellirica, Terminalia chebula, Phyllanthus emblica and Spondias pinnata were the most potent antioxidants as compared with vitamin C based on the 1,1-diphenyl-2-picryl hydrazyl radical assay. These fruits also contained high TPCs. Spondias pinnata, Pyrularia edulis, Melastoma malabathricum, Cipadema bacifera and Choerospondias axillaries fruits were evaluated for the first time. Moreover, Spondias pinnata was found to be more potent (16% radical scavenging activity at 5 microg/ml) than vitamin C (5% radical scavenging activity at 5 microg/ml). Antioxidant activity showed correlation to TPC with the correlation coefficients (R(2)) as 0.7189 and 0.7246 for the methanol and water extracts, respectively. This study suggests that a number of these fruits may have the potential to confer beneficial health effects due to their antioxidant activity and TPC.