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1.  Development of a Novel Severe Triple Allergen Asthma Model in Mice Which Is Resistant to Dexamethasone and Partially Resistant to TLR7 and TLR9 Agonist Treatment 
PLoS ONE  2014;9(3):e91223.
Severe asthma is characterised by persistent inflammation, hyperreactivity and remodeling of the airways. No efficient treatment is available, this is particularly the case for steroid resistant phenotypes. Our aim therefore was to develop a preclinical model showing characteristics of severe human asthma including steroid insensitivity. Mice were first sensitized with ovalbumin, extracts of cockroach or house dust mite followed by a challenge period of seven weeks. Further to this, an additional group of mice was sensitized with all three allergens and then challenged with allergen alternating weekly between allergens. All three allergens applied separately to the mice induced comparably strong Th2-type airway inflammation, airway hyperreactivity and airway remodeling, which was characterised by fibrosis and increased smooth muscle thickness. In contrast, application of all three allergens together resulted in a greater Th2 response and increased airway hyperreactivity and a stronger albeit not significant remodeling phenotype compared to using HDM or CRA. In this triple allergen model dexamethasone application, during the last 4 weeks of challenge, showed no suppressive effects on any of these parameters in this model. In contrast, both TLR7 agonist resiquimod and TLR9 agonist CpG-ODN reduced allergen-specific IgE, eosinophils, and collagen I in the lungs. The TLR9 agonist also reduced IL-4 and IL-5 whilst increasing IFN-γ and strongly IL-10 levels in the lungs, effects not seen with the TLR7 agonist. However, neither TLR agonist had any effect on airway hyperreactivity and airway smooth muscle mass. In conclusion we have developed a severe asthma model, which is steroid resistant and only partially sensitive to TLR7 and TLR9 agonist treatment. This model may be particular useful to test new potential therapeutics aiming at treating steroid resistant asthma in humans and investigating the underlying mechanisms responsible for steroid insensitivity.
doi:10.1371/journal.pone.0091223
PMCID: PMC3949744  PMID: 24618687
2.  The Absence of Mrp4 Has No Effect on the Recruitment of Neutrophils and Eosinophils into the Lung after LPS, Cigarette Smoke or Allergen Challenge 
PLoS ONE  2013;8(4):e61193.
The multidrug resistance protein 4 (Mrp4) is an ATP-binding cassette transporter that is capable of exporting the second messenger cAMP from cells, a process that might regulate cAMP-mediated anti-inflammatory processes. However, using LPS- or cigarette smoke (CS)-inflammation models, we found that neutrophil numbers in the bronchoalveolar lavage fluid (BALF) were similar in Mrp4−/− and Mrp4+/+ mice treated with LPS or CS. Similarly, neutrophil numbers were not reduced in the BALF of LPS-challenged wt mice after treatment with 10 or 30 mg/kg of the Mrp1/4 inhibitor MK571. The absence of Mrp4 also had no impact on the influx of eosinophils or IL-4 and IL-5 levels in the BALF after OVA airway challenge in mice sensitized with OVA/alum. LPS-induced cytokine release in whole blood ex vivo was also not affected by the absence of Mrp4. These data clearly suggest that Mrp4 deficiency alone is not sufficient to reduce inflammatory processes in vivo. We hypothesized that in combination with PDE4 inhibitors, used at suboptimal concentrations, the anti-inflammatory effect would be more pronounced. However, LPS-induced neutrophil recruitment into the lung was no different between Mrp4−/− and Mrp4+/+ mice treated with 3 mg/kg Roflumilast. Finally, the single and combined administration of 10 and 30 mg/kg MK571 and the specific breast cancer resistance protein (BCRP) inhibitor KO143 showed no reduction of LPS-induced TNFα release into the BALF compared to vehicle treated control animals. Similarly, LPS-induced TNFα release in murine whole blood of Mrp4+/+ or Mrp4−/− mice was not reduced by KO143 (1, 10 µM). Thus, BCRP seems not to be able to compensate for the absence or inhibition of Mrp4 in the used models. Taken together, our data suggest that Mrp4 is not essential for the recruitment of neutrophils into the lung after LPS or CS exposure or of eosinophils after allergen exposure.
doi:10.1371/journal.pone.0061193
PMCID: PMC3632556  PMID: 23613808
3.  Treatment of allergic asthma: Modulation of Th2 cells and their responses 
Respiratory Research  2011;12(1):114.
Atopic asthma is a chronic inflammatory pulmonary disease characterised by recurrent episodes of wheezy, laboured breathing with an underlying Th2 cell-mediated inflammatory response in the airways. It is currently treated and, more or less, controlled depending on severity, with bronchodilators e.g. long-acting beta agonists and long-acting muscarinic antagonists or anti-inflammatory drugs such as corticosteroids (inhaled or oral), leukotriene modifiers, theophyline and anti-IgE therapy. Unfortunately, none of these treatments are curative and some asthmatic patients do not respond to intense anti-inflammatory therapies. Additionally, the use of long-term oral steroids has many undesired side effects. For this reason, novel and more effective drugs are needed. In this review, we focus on the CD4+ Th2 cells and their products as targets for the development of new drugs to add to the current armamentarium as adjuncts or as potential stand-alone treatments for allergic asthma. We argue that in early disease, the reduction or elimination of allergen-specific Th2 cells will reduce the consequences of repeated allergic inflammatory responses such as lung remodelling without causing generalised immunosuppression.
doi:10.1186/1465-9921-12-114
PMCID: PMC3179723  PMID: 21867534
4.  Infection with Toxoplasma gondii Reduces Established and Developing Th2 Responses Induced by Nippostrongylus brasiliensis Infection  
Infection and Immunity  2004;72(7):3812-3822.
Oral infection of C57BL/6 mice with 100 cysts of the protozoan parasite Toxoplasma gondii results in the development of small intestinal Th1-type immunopathology. In contrast, infection with intestinal helminths results in the development of protective Th2-type responses. We investigated whether infection with the helminth Nippostrongylus brasiliensis influences the development of T. gondii-induced Th1 responses and immunopathology in C57BL/6 mice infected with T. gondii. Prior as well as simultaneous infection of mice with N. brasiliensis did not alter the course of infection with 100 cysts of T. gondii. Coinfected mice produced high levels of interleukin-12 (IL-12) and gamma interferon (IFN-γ), developed small intestinal immunopathology, and died at the same time as mice infected with T. gondii. Interestingly, local and systemic N. brasiliensis-induced Th2 responses, including IL-4 and IL-5 production by mesenteric lymph node and spleen cells and numbers of intestinal goblet cells and blood eosinophils, were markedly lower in coinfected than in N. brasiliensis-infected mice. Similar effects were seen when infection with 10 T. gondii cysts was administered following infection with N. brasiliensis. Infection of C57BL/6 mice with 10 T. gondii cysts prior to coinfection with N. brasiliensis inhibited the development of helminth-induced Th2 responses and was associated with higher and prolonged N. brasiliensis egg production. In contrast, oral administration of Toxoplasma lysate prior to N. brasiliensis infection had only a minor and short-lived effect on Th2 responses. Thus, N. brasiliensis-induced Th2 responses fail to alter T. gondii-induced Th1 responses and immunopathology, most likely because Th1 responses develop unchanged in C57BL/6 mice with a prior or simultaneous infection with N. brasiliensis. Our findings contribute to the understanding of immune regulation in coinfected animals and may assist in the design of immunotherapies for human Th1 and Th2 disorders.
doi:10.1128/IAI.72.7.3812-3822.2004
PMCID: PMC427426  PMID: 15213122
5.  Mice Deficient in Nuclear Factor of Activated T-Cell Transcription Factor c2 Mount Increased Th2 Responses after Infection with Nippostrongylus brasiliensis and Decreased Th1 Responses after Mycobacterial Infection  
Infection and Immunity  2003;71(11):6641-6647.
Infection of nuclear factor of activated T-cell transcription factor c2 (NFATc2)-deficient mice with the helminth Nippostrongylus brasiliensis led to a distinct increase in interleukin-4 (IL-4) and IL-5 protein synthesis by lymph node and spleen cells and to elevated serum immunoglobulin E (IgE) levels in comparison to those seen with infected control mice. While IL-4, IL-5, and IL-13 mRNA expression was also enhanced in lymph node cells from the lungs of infected NFATc2−/− mice, the number of T cells secreting Th2-type lymphokines remained the same in mice infected with N. brasiliensis. In contrast, lymphocytes from NFATc2-deficient mice infected with Mycobacterium bovis BCG secreted less gamma interferon than lymphocytes from infected control mice. These findings indicate that NFATc2 is an activator of Th1 responses and a suppressor of Th2 responses in vivo.
doi:10.1128/IAI.71.11.6641-6647.2003
PMCID: PMC219542  PMID: 14573689
6.  Infection with the Helminth Nippostrongylus brasiliensis Does Not Interfere with Efficient Elimination of Mycobacterium bovis BCG from the Lungs of Mice 
Infection with Mycobacterium tuberculosis continues to be one of the major global health threats. Strong mycobacterium-specific Th1 immune responses correlate with protection, and decreased Th1 responses correlate with disease progression. In contrast, the impact of Th2 responses on the development of protective immune responses to mycobacteria remains unclear. To analyze whether ongoing Th2 responses present in the lung influence the development of a protective Th1 immune response to mycobacteria, we coinfected mice with the helminth Nippostrongylus brasiliensis and Mycobacterium bovis BCG. We found that the T cells from the lymph nodes of coinfected mice secreted significantly less gamma interferon than did the T cells from mice infected with M. bovis BCG after in vitro stimulation with purified protein from M. tuberculosis when 108 CFU of M. bovis BCG were used for the infection. This result indicates that the helminth infection reduced the Th1 immune response to the mycobacteria in the lung. However, mycobacterial clearance was not delayed in the coinfected animals. Importantly, the infection with BCG after the helminth infection did not reduce the helminth-induced Th2 response in the lung, ruling out the possibility that the lack of a reduction in bacterial clearance in the coinfected mice was due to a downmodulation of the helminth-induced Th2 response. Taken together, our results suggest that ongoing Th2 responses in the lung do not necessarily lead to increased susceptibility to mycobacterial infection.
doi:10.1128/CDLI.9.3.727-730.2002
PMCID: PMC119979  PMID: 11986288
7.  Constitutive Expression of Interleukin (IL)-4 In Vivo Causes Autoimmune-type Disorders in Mice 
The transgenic (tg) expression of interleukin (IL)-4 under the control of a major histocompatibility complex (MHC) class I promoter leads to B cell hyperactivity in mice, characterized by increased B cell surface MHC class II and CD23 expression, elevated responsiveness of the B cells to polyclonal ex vivo stimulation, and increased immunoglobulin (Ig)G1 and IgE serum levels. Tg mice develop anemia, glomerulonephritis with complement and immune deposition in the glomeruli, and show increased production of autoantibodies. Treatment of IL-4 tg mice with anti-IL-4 neutralizing antibodies protected the mice from disease development, showing that IL-4 was responsible for the observed disorders. Deletion of superantigen responsive autoreactive T cells in the IL-4 tg mice was normal and treatment of mutant mice with deleting anti-CD4 antibodies failed to ablate the onset of autoimmune-like disease, suggesting that CD4+T cells were not the primary cause of the disorders. Furthermore, the deletion of B cells reacting against MHC class I molecules was also normal in the IL-4 tg mice. Therefore the most likely explanation for the increased production of autoantibodies and the autoimmunelike disorders is that IL-4 acts directly on autoreactive B cells by expanding them in a polyclonal manner. Taken together our results show that inappropriate multi-organ expression of IL-4 in vivo leads to autoimmune-type disease in mice.
PMCID: PMC2196114  PMID: 9016881

Results 1-7 (7)