James V. Jackson

Inflammatory Signals shift from adipose to liver during high fat feeding and influence the development of steatohepatitis in mice

BackgroundObesity and inflammation are highly integrated processes in the pathogenesis of insulin resistance, diabetes, dyslipidemia, and non-alcoholic fatty liver disease. Molecular mechanisms underlying inflammatory events during high fat diet-induced obesity are poorly defined in mouse models of obesity. This work investigated gene activation signals integral to the temporal development of obesity.MethodsGene expression analysis in multiple organs from obese mice was done with Taqman Low Density Array (TLDA) using a panel of 92 genes representing cell markers, cytokines, chemokines, metabolic, and activation genes. Mice were monitored for systemic changes characteristic of the disease, including hyperinsulinemia, body weight, and liver enzymes. Liver steatosis and fibrosis as well as cellular infiltrates in liver and adipose tissues were analyzed by histology and immunohistochemistry.ResultsObese C57BL/6 mice were fed with high fat and cholesterol diet (HFC) for 6, 16 and 26 weeks. Here we report that the mRNA levels of macrophage and inflammation associated genes were strongly upregulated at different time points in adipose tissues (6-16 weeks) and liver (16-26 weeks), after the start of HFC feeding. CD11b+ and CD11c+ macrophages highly infiltrated HFC liver at 16 and 26 weeks. We found clear evidence that signals for IL-1β, IL1RN, TNF-α and TGFβ-1 are present in both adipose and liver tissues and that these are linked to the development of inflammation and insulin resistance in the HFC-fed mice.ConclusionsMacrophage infiltration accompanied by severe inflammation and metabolic changes occurred in both adipose and liver tissues with a temporal shift in these signals depending upon the duration of HFC feeding. The evidences of gene expression profile, elevated serum alanine aminotransferase, and histological data support a progression towards nonalcoholic fatty liver disease and steatohepatitis in these HFC-fed mice within the time frame of 26 weeks.

Small-molecule inhibitors of FABP4/5 ameliorate dyslipidemia but not insulin resistance in mice with diet-induced obesity.

Fatty acid binding protein-4 (FABP4) and FABP5 are two closely related FA binding proteins expressed primarily in adipose tissue and/or macrophages. The small-molecule FABP4 inhibitor BMS309403 was previously reported to improve insulin sensitivity in leptin-deficient Lepob/Lepob (ob/ob) mice. However, this compound was not extensively characterized in the more physiologically relevant animal model of mice with diet-induced obesity (DIO). Here, we report the discovery and characterization of a novel series of FABP4/5 dual inhibitors represented by Compounds 1–3. Compared with BMS309403, the compounds had significant in vitro potency toward both FABP4 and FABP5. In cell-based assays, Compounds 2 and 3 were more potent than BMS309403 to inhibit lipolysis in 3T3-L1 adipocytes and in primary human adipocytes. They also inhibited MCP-1 release from THP-1 macrophages as well as from primary human macrophages. When chronically administered to DIO mice, BMS309403 and Compound 3 reduced plasma triglyceride and free FA levels. Compound 3 reduced plasma free FAs at a lower dose level than BMS309403. However, no significant change was observed in insulin, glucose, or glucose tolerance. Our results indicate that the FABP4/5 inhibitors ameliorate dyslipidemia but not insulin resistance in DIO mice.

Regulatory effects of interleukin‐4 and interleukin‐10 on human neutrophil function ex vivo and on neutrophil influx in a rat model of arthritis

OBJECTIVE To assess the capacity of interleukin-4 (IL-4) and IL-10 to block polymorphonuclear neutrophil (PMN) activation in an ex vivo human model system, and to confirm their effect on neutrophil function in an animal model of arthritis. METHODS The ex vivo phagocytic capacity of cytokine-activated human PMNs was assessed by use of assays for measuring the ingestion of heat-killed yeast and by subsequent hexose-monophosphate shunt activation using nitroblue tetrazolium reduction. The in vivo activity of IL-4 and IL-10 was measured using a rat adjuvant arthritis model in which the mycobacterial antigen concentration was titrated to modify disease intensity. RESULTS IL-4 and IL-10 suppressed the ex vivo activation state of interferon-gamma- and tumor necrosis factor alpha-activated human neutrophils. In the rat adjuvant arthritis model, treatment with systemic murine IL-10 (mIL-10) effectively suppressed all disease parameters in rats that received the lower concentrations of mycobacteria, whereas systemic mIL-4 was effective against even the most severe disease. Both cytokines were effective in lowering the absolute PMN cell number recovered and the PMN activation state in the joint synovia. We also observed lower levels of the messenger RNA transcript for CINC protein (cytokine-induced neutrophil chemoattractant; a rat homolog for human IL-8) in the synovia. CONCLUSION IL-10 is an effective antiarthritic agent and has a major effect on the presence and function of PMNs in the joint synovia when disease intensity is not severe. IL-4 has an inhibitory profile that is similar to that of IL-10, but is effective in modifying even the most severe disease. Both cytokines reduced the phagocytic activation of human PMNs in response to proinflammatory cytokines. These data demonstrate that IL-4 and IL-10 can exert powerful regulatory effects on neutrophil function that translate into a therapeutic response in a disease model of arthritis. Treatment with these cytokines alone or in combination may therefore be very useful in the management of patients with rheumatoid arthritis.

Synthetic inhibitors of interleukin-6 II: 3,5-diaryl pyridines and meta-terphenyls

Abstract An efficient synthesis of the title compounds using the Ni (II) catalysed coupling of various aryl magnesium bromides with di-halo pyridines and di-halo benzenes is reported. Among the several compounds tested, 1 , 2 , 12 and 17 showed good inhibitory activity against interleukin-6.

Characterization of peripheral human cannabinoid receptor (hCB2) expression and pharmacology using a novel radioligand, [35S]Sch225336.

Studies to characterize the endogenous expression and pharmacology of peripheral human cannabinoid receptor (hCB2) have been hampered by the dearth of authentic anti-hCB2 antibodies and the lack of radioligands with CB2 selectivity. We recently described a novel CB2 inverse agonist, N-[1(S)-[4-[[4-methoxy-2-[(4methoxyphenyl)sulfonyl] phenyl]sulfonyl] phenyl]ethyl]methane-sulfonamide (Sch225336), that binds hCB2 with high affinity and excellent selectivity versus hCB1. The precursor primary amine of Sch225336 was prepared and reacted directly with [35S]mesyl chloride (synthesized from commercially obtained [35S]methane sulfonic acid) to generate [35S]Sch225336. [35S]Sch225336 has high specific activity (>1400 Ci/mmol) and affinity for hCB2 (65 pm). Using [35S]Sch225336, we assayed hemopoietic cells and cell lines to quantitate the expression and pharmacology of hCB2. Lastly, we used [35S]Sch225336 for detailed autoradiographic analysis of CB2 in lymphoid tissues. Based on these data, we conclude that [35S]Sch225336 represents a unique radioligand for the study of CB2 endogenously expressed in blood cells and tissues.

Impairment of leukocyte trafficking in a murine pleuritis model by IL-4 and IL-10.

We have characterized leukocyte migration to the pleural cavity in a methylated-BSA (mBSA)-induced model of murine delayed-type hypersensitivity and evaluated the ability of IL-4 and IL-10 to modulate this response. Neutrophils, macrophages, T cells, and dendritic cells migrated to the pleural cavity in a time-dependent fashion following direct intrapleural antigen challenge, with neutrophils comprising the majority of exudate leukocytes in the cavity within the first 24 h and the number of mononuclear cells increasing at later times. Real-time quantitative PCR analysis of infiltrating leukocytes revealed a marked elevation of steady-state mRNA levels of IL-1β and TNFα and the chemokines KC, MIP-2, CXCL9, CXCL10, CXCL11, CCL2, CCL3, and CCL4 at 6 h postchallenge, which diminished over time. In contrast, γIFN mRNA levels were maximal at 24 h and CCL5 expression was sustained throughout 72 h. ELISA analysis of pleural exudate fluid revealed significant elevations of KC and CCL2 protein levels at 6 h postantigen challenge and a peak increase in γIFN protein at 24 h, confirming our mRNA observations. Administration of recombinant murine IL-4 or IL-10 prior to challenge significantly blocked cell trafficking to the pleural cavity as well as peak levels of exudate γIFN, with IL-4 being more potent in impairing these responses. IL-4 administration also increased the proportion of naïve T cells in the pleural cavity, as judged by CD62L and CD45RB expression. These results indicate that this in vivo model demonstrates a pattern of events associated with Th1-mediated leukocyte trafficking and underscore the potential utility of this in vivo model for evaluating therapeutic inhibitors of leukocyte trafficking.

Cannabinoid CB2-Selective Inverse Agonist Protects Against Antigen-Induced Bone Loss

Work to improve the therapeutic properties of cannabinoid CB2 receptor-selective inverse agonists has led to the development of Sch.036, an aryl substituted triaryl bis-sulfone with improved oral pharmacokinetic parameters. In this report, we show that this compound blocks in vivo trafficking of various leukocyte populations, a property consistent with other members of this chemical series. This CB2-selective compound also shows efficacy in leukocyte recruitment models when added in concert with suboptimal doses of selected anti-inflammatory agents, consistent with its unique function and indicative of its potential therapeutic utility. Finally, studies with Sch.036 show that this cannabinoid CB2-specific inverse agonist can ameliorate bone damage in a rat model of relapsing-remitting arthritis. This result suggests that a cannabinoid CB2‐selective inverse agonist may help ameliorate a particularly harmful property of this inflammatory joint disease.

Potent and Selective Amidopyrazole Inhibitors of IRAK4 That Are Efficacious in a Rodent Model of Inflammation

IRAK4 is a critical upstream kinase in the IL-1R/TLR signaling pathway. Inhibition of IRAK4 is hypothesized to be beneficial in the treatment of autoimmune related disorders. A screening campaign identified a pyrazole class of IRAK4 inhibitors that were determined by X-ray crystallography to exhibit an unusual binding mode. SAR efforts focused on the identification of a potent and selective inhibitor with good aqueous solubility and rodent pharmacokinetics. Pyrazole C-3 piperidines were well tolerated, with N-sulfonyl analogues generally having good rodent oral exposure but poor solubility. N-Alkyl piperidines exhibited excellent solubility and reduced exposure. Pyrazoles possessing N-1 pyridine and fluorophenyl substituents were among the most active. Piperazine 32 was a potent enzyme inhibitor with good cellular activity. Compound 32 reduced the in vivo production of proinflammatory cytokines and was orally efficacious in a mouse antibody induced arthritis disease model of inflammation.

Oncostatin M stimulates proliferation in B9 hybridoma cells: Potential role of oncostatin M in plasmacytoma development

Oncostatin M was found to stimulate the IL-6-addicted hybridoma line B9. Leukaemia inhibitory factor did not stimulate proliferation of this line. Both of these factors bind to the gp130 of the IL-6 receptor. In another cell line that is stimulated by LIF (DA.1), neither IL-6 nor oncostatin M stimulated proliferation. Previously it had been thought that the gp130 alone is sufficient to bind ligand and transduce signal and that oncostatin M could bind to and activate the LIF receptor, but these data show this is not always the case. Mice primed with Pristane were found to have IL-6 in their sera and peritoneal fluid only at a few time points following Pristane treatment; this was determined by IL-6-specific ELISA. When the same samples were analysed on IL-6-addicted B9 cells, stimulatory activity was found at all time points. When the Pristane-primed samples were assayed for oncostatin M activity in the A375 melanoma assay, there was oncostatin M activity at various time points. IL-6 did not have activity on the A375 cells. These data indicate that oncostatin M play a role in the generation of plasmacytoma in vivo.

Evaluation of Chemokine- and Phlogistin-mediated Leukocyte Chemotaxis Using an In Vivo Sponge Model

We have directly compared the in vivo activity of a number of chemokines and phlogistins using a modified murine in vivo sponge model in which gelatin sponges are soaked with chemoattractant and implanted in the peritoneal cavity. Sponges soaked with murine JE/MCP-1 (monocyte chemoattractant protein-1) or zymosan promoted the chemotaxis of specific leukocyte populations in a time-dependent manner, as judged by multiparameter flow cytometry, with granulocytes predominating in zymosan-soaked sponges and granulocytes and macrophages present in JE/MCP-1-soaked sponges. Smaller numbers of B, T and dendritic cells were identified as well. Eotaxin selectively chemoattracted eosinophils in this model, while MIG induced significant T cell migration relative to other chemokines. Cell migration was inhibited by administration of methotrexate, piroxicam or dexamethasone, and JE/MCP-1-mediated trafficking was impaired by treatment with anti-JE antibody or with IL-10, suggesting a role for pro-inflammatory factors in amplifying the JE/MCP-1-induced response. This amplification phase involves the production of the chemokine KC, since anti-KC antibody significantly attenuated JE/MCP-1-induced chemotaxis. These results indicate that intraperitoneally implanted chemoattractant-soaked gelatin sponges are capable of inducing a pronounced inflammatory response characterized by the selective migration of leukocyte populations, and suggest that this model may be useful for delineating the activity of novel inhibitors of leukocyte chemotaxis.