Role of Th1/Th17 balance regulated by T-bet in a mouse model of Mycobacterium avium complex disease.

Th1 immune responses are thought to be important in protection against intracellular pathogens. T-bet is a critical regulator for Th1 cell differentiation and Th1 cytokine production. The aim of this study was to determine the role of T-bet in host defense against Mycobacterium avium complex (MAC) infection. Wild-type mice, T-bet-deficient mice, and T-bet-overexpressing mice were infected with MAC via intratracheal inoculation. Macrophages and dendritic cells obtained from these mice were incubated with MAC. T-bet-deficient mice were highly susceptible to MAC, compared with wild-type mice and T-bet-overexpressing mice. Neutrophilic pulmonary inflammation was also enhanced in T-bet-deficient mice, but attenuated in T-bet-overexpressing mice, following MAC infection. Cytokine expression shifted toward Th1 in the lung and spleen of T-bet-overexpressing mice, but toward Th17 in T-bet-deficient mice. IFN-γ supplementation to T-bet-deficient mice reduced systemic MAC growth but did not reduce pulmonary inflammation. In contrast, neutralization of IL-17 in T-bet-deficient mice reduced pulmonary inflammation but did not affect mycobacterial growth in any organs tested. T-bet-deficient T cells tended to differentiate toward Th17 cells in vitro following exposure to MAC. Treatment with NO donor suppressed MAC-induced Th17 cell differentiation of T-bet-deficient T cells. This study identified that the fine balance between Th1 and Th17 responses is essential in defining the outcome of MAC disease. T-bet functions as a regulator for Th1/Th17 balance and is a critical determinant for host resistance to MAC infection by controlling cytokine and NO levels.

Transcription factors GATA-3 and RORγt are important for determining the phenotype of allergic airway inflammation in a murine model of asthma.

In refractory asthma, neutrophils, rather than eosinophils, often predominate in the airways. Neutrophilic airway inflammation appears to be resistant to steroids and may be related to the Th17, rather than the Th2, cytokine milieu. However, the role of GATA-3 and RORγt, transcription factors for Th2 and Th17 cell differentiation, respectively, in the pathogenesis of steroid-insensitive asthma remains unclear. To examine the effect of GATA-3- and RORγt-overexpression backgrounds on airway inflammation and steroid sensitivity, we generated two strains of transgenic mice overexpressing GATA-3 or RORγt. Mice were sensitized and challenged with OVA. Some OVA-sensitized/challenged mice were treated with dexamethasone, anti-IL-17 Ab, CXCR2 antagonist, or anti-IL-6R Ab to demonstrate their therapeutic effects on airway inflammation. Although Ag-specific airway inflammation and hyperresponsiveness were induced in each mouse, the phenotype of inflammation showed a distinct difference that was dependent upon the genotype. GATA-3-overexpressing mice exhibited steroid-sensitive eosinophilic inflammation with goblet cell hyperplasia and mucus hyperproduction under Th2-biased conditions, and RORγt-overexpressing mice developed steroid-insensitive neutrophilic inflammation under Th17-biased conditions. The levels of keratinocyte-derived chemokine, MIP-2, IL-6, and other neutrophil chemotaxis-related mediators were significantly elevated in OVA-exposed RORγt-overexpressing mice compared with wild-type mice. Interestingly, airway hyperresponsiveness accompanied by neutrophilic airway inflammation in RORγt-overexpressing mice was effectively suppressed by anti-IL-17 Ab, CXCR2 antagonist, or anti-IL-6R Ab administration. In conclusion, our results suggest that the expression levels of GATA-3 and RORγt may be important for determining the phenotype of asthmatic airway inflammation. Furthermore, blockade of the Th17-signaling pathway may be a treatment option for steroid-insensitive asthma.

Carbocisteine reduces virus-induced pulmonary inflammation in mice exposed to cigarette smoke.

Carbocisteine (S-CMC) inhibits viral infection and prevents acute exacerbation of chronic obstructive pulmonary disease. We recently demonstrated the protective effects of NF-E2-related factor (Nrf) 2 against influenza virus (FluV)-induced pulmonary inflammation in mice exposed to cigarette smoke (CS). In our current study, we investigated the effects of S-CMC on Nrf2 activation in cultured macrophages, and in mice infected with influenza after exposure to CS. Nuclear translocation of Nrf2 and the expression of Nrf2-targeted antioxidant genes, such as heavy and light subunits of γ glutamyl cysteine synthetase and heme oxigenase-1, were enhanced in a dose-dependent manner after treatment with S-CMC in peritoneal and alveolar macrophages of wild-type mice, but not in those of Nrf2-deficient mice. Nuclear translocation of Nrf2 in macrophages was inhibited by the phosphatidylinositol 3-kinase inhibitor, LY294002. Phosphorylated Akt, Nrf2, and heme oxigenase-1 were induced in the alveolar macrophages of the lungs in wild-type mice after S-CMC administration. The extent of oxidative stress, inflammatory cell infiltration, pulmonary edema, and goblet cell hyperplasia was suppressed by S-CMC administration in the lungs of wild-type mice after exposure to both CS and FluV. Our findings suggest that S-CMC reduces pulmonary inflammation and mucus overproduction in mice exposed to CS after infection with FluV via the activation of Nrf2.

Phosphodiesterase inhibitors. Part 6: design, synthesis, and structure-activity relationships of PDE4-inhibitory pyrazolo[1,5-a]pyridines with anti-inflammatory activity.

We previously identified KCA-1490 [(-)-6-(7-methoxy-2-trifluoromethyl-pyrazolo[1,5-a]pyridin-4-yl)-5-methyl-4,5-dihydro-3-(2H)-pyridazinone], a dual PDE3/4 inhibitor. In the present study, we found highly potent selective PDE4 inhibitors derived from the structure of KCA-1490. Among them, N-(3,5-dichloropyridin-4-yl)-7-methoxy-2-(trifluoromethyl)pyrazolo[1,5-a]pyridine-4-carboxamide (2a) had good anti-inflammatory effects in an animal model.

Phosphodiesterase inhibitors. Part 5: hybrid PDE3/4 inhibitors as dual bronchorelaxant/anti-inflammatory agents for inhaled administration.

(-)-6-(7-Methoxy-2-(trifluoromethyl)pyrazolo[1,5-a]pyridin-4-yl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (KCA-1490) exhibits moderate dual PDE3/4-inhibitory activity and promises as a combined bronchodilatory/anti-inflammatory agent. N-alkylation of the pyridazinone ring markedly enhances potency against PDE4 but suppresses PDE3 inhibition. Addition of a 6-aryl-4,5-dihydropyridazin-3(2H)-one extension to the N-alkyl group facilitates both enhancement of PDE4-inhibitory activity and restoration of potent PDE3 inhibition. Both dihydropyridazinone rings, in the core and extension, can be replaced by achiral 4,4-dimethylpyrazolone subunits and the core pyrazolopyridine by isosteric bicyclic heteroaromatics. In combination, these modifications afford potent dual PDE3/4 inhibitors that suppress histamine-induced bronchoconstriction in vivo and exhibit promising anti-inflammatory activity via intratracheal administration.

Role of Nrf2 in host defense against influenza virus in cigarette smoke-exposed mice.

Influenza virus is a common respiratory tract viral infection. Although influenza can be fatal in patients with chronic pulmonary diseases such as chronic obstructive pulmonary disease, its pathogenesis is not fully understood. The Nrf2-mediated antioxidant system is essential to protect the lungs from oxidative injury and inflammation. In the present study, we investigated the role of Nrf2 in protection against influenza virus-induced pulmonary inflammation after cigarette smoke exposure with both in vitro and in vivo approaches. For in vitro analyses, peritoneal macrophages isolated from wild-type and Nrf2-deficient mice were treated with poly(I:C) and/or cigarette smoke extract. For in vivo analysis, these mice were infected with influenza A virus with or without exposure to cigarette smoke. In Nrf2-deficient macrophages, NF-κB activation and the induction of its target inflammatory genes were enhanced after costimulation with cigarette smoke extract and poly(I:C) compared with wild-type macrophages. The induction of antioxidant genes was observed for the lungs of wild-type mice but not those of Nrf2-deficient mice after cigarette smoke exposure. Cigarette smoke-exposed Nrf2-deficient mice showed higher rates of mortality than did wild-type mice after influenza virus infection, with enhanced peribronchial inflammation, lung permeability damage, and mucus hypersecretion. Lung oxidant levels and NF-κB-mediated inflammatory gene expression in the lungs were also enhanced in Nrf2-deficient mice. Our data indicate that the antioxidant pathway controlled by Nrf2 is pivotal for protection against the development of influenza virus-induced pulmonary inflammation and injury under oxidative conditions.

Phosphodiesterase inhibitors. Part 4: design, synthesis and structure-activity relationships of dual PDE3/4-inhibitory fused bicyclic heteroaromatic-4,4-dimethylpyrazolones.

(-)-6-(7-Methoxy-2-trifluoromethylpyrazolo[1,5-a]pyridin-4-yl)-5-methyl-4,5-dihydro-3-(2H)-pyridazinone (KCA-1490) is a dual PDE3/4 inhibitor that exhibits potent combined bronchodilatory and anti-inflammatory activity. Here we show that a 4,4-dimethylpyrazolone subunit serves as an effective surrogate for the 5-methyl-4,5-dihydropyridazin-3(2H)-one ring of KCA-1490 whilst lacking a stereogenic centre. The 2- and 7-substituents in the pyrazolo[1,5-a]pyridine subunit markedly influence the PDE-inhibitory profile and can be adjusted to afford either potent PDE4-selective inhibitors or dual PDE3/4 inhibitors. A survey of bicyclic heteroaromatic replacements for the pyrazolo[1,5-a]pyridine allowed further refinement of the inhibitory profile and identified 3-(8-methoxy-2-(trifluoromethyl)imidazo[1,2-a]pyridin-5-yl)-4,4-dimethyl-1H-pyrazol-5(4H)-one as an orally active, achiral KCA-1490 analog with well-balanced dual PDE3/4-inhibitory activity.

Aggravation of bleomycin-induced pulmonary inflammation and fibrosis in mice lacking peroxiredoxin I.

Oxidative stress plays an important role in the pathogenesis of acute lung injury and pulmonary fibrosis. Peroxiredoxin (Prx) I is a cellular antioxidant enzyme induced under stress conditions. In the present study, the protective effects of Prx I on the development of bleomycin-induced acute pulmonary inflammation and pulmonary fibrosis were investigated using Prx I-deficient mice. Survival of Prx I-deficient mice after bleomycin administration was significantly lower than that of wild-type mice, corresponding with enhanced acute pulmonary inflammation and fibrosis. The level of inflammatory cytokines and chemokines, such as TNF-α, macrophage inflammatory protein-2, and monocyte chemotactic protein-1, was significantly elevated in the bronchoalveolar lavage fluid of Prx I-deficient mice after bleomycin administration. Furthermore, the level of 8-isoprostane, an oxidative stress marker, and the concentration and alveolar macrophage expression of macrophage migration inhibitory factor were elevated in the lungs of Prx I-deficient mice after bleomycin administration. The exacerbation of bleomycin-induced pulmonary inflammation and fibrosis in Prx I-deficient mice was inhibited by treatment with N-acetyl-L-cysteine, a radical scavenger, or with (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester, a tautomerase inhibitor of macrophage migration inhibitory factor. These findings suggest that mice lacking Prx I are highly susceptible to bleomycin-induced pulmonary inflammation and fibrosis because of increases in pulmonary oxidant levels and macrophage migration inhibitory factor activity in response to bleomycin.

Nrf2 protects against pulmonary fibrosis by regulating the lung oxidant level and Th1/Th2 balance.

BACKGROUND: Pulmonary fibrosis is a progressive and lethal disorder. Although the precise mechanisms of pulmonary fibrosis are not fully understood, oxidant/antioxidant and Th1/Th2 balances may play an important role in many of the processes of inflammation and fibrosis. The transcription factor Nrf2 acts as a critical regulator for various inflammatory and immune responses by controlling oxidative stress. We therefore investigated the protective role of Nrf2 against the development of pulmonary fibrosis. METHODS: To generate pulmonary fibrosis, both wild-type C57BL/6 mice and Nrf2-deficient mice of the same background were administered bleomycin intratracheally. RESULTS: The survival of Nrf2-deficient mice after bleomycin administration was significantly lower than that of wild-type mice. The degree of bleomycin-induced initial pulmonary inflammation and pulmonary fibrosis was much more severe in Nrf2-deficient mice than in wild-type mice. The expression of antioxidant enzymes and phase II detoxifying enzymes was significantly reduced in the lungs of Nrf2-deficient mice, concomitant with an elevation of lung 8-isoprostane level, compared with wild-type mice. The expression of Th2 cytokines, such as interleukin-4 and interleukin-13, was significantly elevated in the lungs of Nrf2-deficient mice with an increase in the number of Th2 cells that express GATA-binding protein 3. CONCLUSIONS: The results indicated that Nrf2 protects against the development of pulmonary fibrosis by regulating the cellular redox level and lung Th1/Th2 balance. Thus, Nrf2 might be an important genetic factor in the determination of susceptibility to pulmonary fibrosis.

Intradermal nociceptin elicits itch-associated responses through leukotriene B(4) in mice.

Nociceptin, the endogenous peptide ligand for opioid receptor like-1 (ORL1) receptor, has been implicated in the inflammation and pain in the skin. We examined whether nociceptin is a pruritogen in mice. Intradermal injections of nociceptin (1-100 nmol per site) concentration dependently increased scratching in ICR mice; the effect started within 1 min, peaked at 10-20 min, and almost subsided by 30 min. The nociceptin action was absent in ORL1 receptor-deficient (ORL1(-/-)) mice. Systemic, but not local, treatment with naloxone significantly inhibited scratching induced by nociceptin. The action of nociceptin was inhibited by the leukotriene B(4) receptor antagonist ONO-4057 and azelastine, which inhibits the action and production of leukotriene B(4) in the skin. Prepronociceptin and ORL1 receptor mRNAs were substantially expressed in the skin, whereas their expression levels were very low in the dorsal root ganglia. In the skin, nociceptin- and ORL1 receptor-like immunoreactivities were localized in the epidermis. Administration of nociceptin to primary cultures of keratinocytes from ICR and C57BL/6 (ORL1(+/+)) mice, but not ORL1(-/-) mice, produced leukotriene B(4). The results suggest that nociceptin acts on ORL1 receptor on the keratinocytes to produce leukotriene B(4), which induces itch-associated responses in mice.

Evidence for separate involvement of different mu-opioid receptor subtypes in itch and analgesia induced by supraspinal action of opioids.

The common adverse effect of centrally-injected mu-opioid receptor (mu-OR) agonists is pruritus. This study was conducted using mice to examine whether different subtypes of mu-OR would be responsible for pruritus and analgesia. Intracisternal injections of morphine and morphine-6beta-glucronide (M6G), but not M3G, produced an antinociceptive effect. Morphine, but neither M6G nor M3G, induced facial scratching, a pruritus-related response. Facial scratching following morphine was not affected by the mu(1)-OR antagonist naloxonazine at doses that inhibited the antinociceptive effects. The results suggest that different subtype and/or splice variants of mu-OR are separately involved in pruritus and antinociception of opioids.

Intracisternal, but not intrathecal, injection of naloxone inhibits cutaneous itch-related response in mice.

The present study was conducted to determine whether cutaneous itch involves mu-opioid receptors in either of the spinal cord or lower brainstem or in both regions in mice. An intraplantar injection of serotonin hydrochloride (100 nmol/site) induced biting, an itch-related behavior. The behavior was inhibited by subcutaneous (0.3-1 mg/kg) and intracisternal (1--10 nmol/site), but not intrathecal (1--10 nmol/site), injections of naloxone hydrochloride. An intradermal injection of serotonin (100 nmol/site) to the rostral back induced scratching, an itch-related behavior, which was inhibited by subcutaneous (1 mg/kg) and intracisternal (10 nmol/site) injections of naloxone. These results suggest that mu-opioid receptor in the lower brainstem, but not spinal cord, is a site of central pruritogenic action of opioids and is involved in the facilitatory regulation of itch signaling.

Effects of imidafenacin (KRP-197/ONO-8025), a new anti-cholinergic agent, on muscarinic acetylcholine receptors. High affinities for M3 and M1 receptor subtypes and selectivity for urinary bladder over salivary gland.

Imidafenacin (CAS 170105-16-5, KRP-197, ONO-8025) is an antagonist for the muscarinic acetylcholine (ACh) receptor currently under development for the treatment of overactive bladder. Affinities of imidafenacin and other drugs for muscarinic ACh receptor subtypes were investigated by examining inhibitory effects on ACh release in the rat urinary bladder and K+ efflux in the rat salivary gland in functional and binding assays. In the functional assay, imidafenacin had higher affnities for M3 and M1 receptors than for the M2 receptor. In contrast, metabolites of imidafenacin (M-2, M-4 and M-9) had low affinities for muscarinic ACh receptor subtypes. Darifenacin had selectivity for the M3 receptor, while propiverine, tolterodine and oxybutynin had no selectivity for muscarinic ACh receptors. In carbamylcholine (CCh)-induced contraction in the urinary bladder, imidafenacin, propiverine, tolterodine and oxybutynin had affinities similar to those for the M3 receptor in the ileum. In the binding assay for human muscarinic ACh receptor subtypes, imidafenacin had higher affinities for m3 and m1 receptors than for m2 receptor, but tolterodine had no selectivity for m1, m2 and m3 receptors. In ACh release in the urinary bladder, inhibitory effects of imidafenacin, tolterodine, oxybutynin and darifenacin seemed to be partially mediated by the M1 receptor. In ACh-induced and electrical stimulation-induced K+ efflux from the salivary gland, inhibitory effects (IC50) of imidafenacin, propiverine, tolterodine, oxybutynin and darifenacin might be closely related to those for the M3 receptor in the ileum. These results suggest that imidafenacin more strongly antagonizes cholinomimetics on M3 and M1 receptors than on the M2 receptor. Moreover, imidafenacin seems to inhibit the contraction of the bladder smooth muscle by mediating antagonism to the M3 receptor and to regulate ACh release by mediating prejunctional facilitatory M1 receptor. Imidafenacin also inhibited K+ efflux from the salivary gland mainly by mediating the M3 receptor. Therefore, imidafenacin will have higher affinities for M3 and M1 receptors and higher selectivity for the urinary bladder than for the salivary gland.

Pharmacological effects of imidafenacin (KRP-197/ONO-8025), a new bladder selective anti-cholinergic agent, in rats. Comparison of effects on urinary bladder capacity and contraction, salivary secretion and performance in the Morris water maze task.

Imidafenacin (CAS 170105-16-5, KRP-197, ONO-8025) has been developed for the treatment of overactive bladder as a new anti-cholinergic with high affinities for muscarinic acetylcholine M3 and M1 receptors. The pharmacological profiles of imidafenacin on the urinary bladder function by determining carbamylcholine (CCh)-induced decrease in bladder capacity and distention-induced rhythmic bladder contraction in conscious rats were investigated. In addition, effects of imidafenacin on CCh-induced salivary secretion and performance in the Morris water maze task in rats were investigated to evaluate side effects, such as dry mouth and cognitive dysfunction in the central nervous system (CNS). Imidafenacin prevented the CCh-induced decrease in bladder capacity dose-dependently with an ID50 of 0.055 mg/kg. On the distention-induced rhythmic bladder contraction, imidafenacin, propiverine, tolterodine, oxybutynin and darifenacin showed inhibitory effects with ID30's of 0.17, 15, 3.0, 3.2 and 0.85 mg/kg, respectively. The rank order of inhibitory potency was: imidafenacin > darifenacin > tolterodine > or = oxybutynin > propiverine. Imidafenacin, propiverine, tolterodine, oxybutynin and darifenacin showed inhibitory effects on the CCh-stimulated salivary secretion with ID50's of 1.5, 14, 15, 4.4 and 1.2 mg/kg, respectively. The rank order of inhibitory potency was: darifenacin > or = imidafenacin > oxybutynin > propiverine > or = tolterodine. Imidafenacin at the doses of 1 and 10 mg/ kg did not affect the escape latencies in the Morris water maze task compared with those in vehicle controls. Oxybutynin at the dose of 100 mg/kg induced a significant increase in the escape latencies, but propiverine at the dose of 100 mg/kg did not induce significant changes. These results suggest that imidafenacin inhibits urinary bladder contraction to a greater extent than the salivary secretion (compared with the M3 receptor selective antagonist, darifenacin, and the non-selective antagonists, propiverine, tolterodine and oxybutynin) or the CNS functions, such as performance in the Morris water maze task (compared with oxybutynin). In conclusion, imidafenacin has organ selectivity for the bladder over the salivary gland, without influence on the central nervous system such as spatial learning and memory.