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.

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

Inflammation and mucus production in the lung of the mice subjected to the different sensitization and challenge protocols.

<p>Sections of the lungs were stained with haematoxilin/eosin (H&E) (A) and periodic acid-schiff (PAS) (B). Shown are representative sections of 8–12 mice/group. Scale bar  =  100 µm. Wet weight of left lung is shown (C) and mucus production (D) was assessed by automated analysis of the main bronchus epithelium. Results represent mean ± SEM from 8–12 mice/group. **<i>P</i><0.01; ***<i>P</i><0.001 in comparison to the PBS control mice and ^§<i>P</i><0.05 in comparison to the triple allergen combination (TAC) group. Mucus plugging (E) was scored manually analyzing whole lung cross sections from 7–12 mice/group as described in methods.</p

Effects of dexamethasone, TLR7 or TLR9 agonists on cellular influx and airway hyperreactivity in mice subjected to the triple allergen challenge model.

<p>Therapeutic treatment with dexamethasone, R848, or CpG-ODNs was started in the 4^th week of allergen challenge. CpG-ODNs and R848 were given i.tr. (1 mg/kg) and dexamethasone was administered orally (1 m/kg) as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091223#pone-0091223-g001" target="_blank">Figure 1C</a>. Treatment was given twice weekly one hour prior to allergen challenge. 24 h after the last allergen challenge mice were sacrificed and absolute numbers of total cells, eosinophils, neutrophils and macrophages (A) were measured in whole lung lavage. Data are presented as mean ± SEM, n  =  8–12/group. Lung function was assessed 22 h after final challenge on day 70 via invasive measurement of resistance (B) and compliance (D). The area under the curve (AUC) is shown for resistance (C) and compliance (E). Results represent mean ± SEM for 8–12 mice/group. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001 in comparison to the PBS control mice and ^§§<i>P</i><0.01, ^§§§<i>P</i><0.001 in comparison to the triple allergen combination (TAC) group.</p

The absence of mrp4 has no effect on the recruitment of neutrophils and eosinophils into the lung after LPS, cigarette smoke or allergen challenge.

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

Levels of cytokines in the lungs of mice subjected to the different sensitization and challenge protocols.

<p>Values are expressed as mean ± SEM from 8–14 mice per group. *P <0.05, **P <0.01, ***P<0.001 in comparison to the PBS control and §< 0.05, §§< 0.01, and §§§<0.001 compared to the triple allergen combination (TAC) group.</p

Treatment protocols.

<p>Mice were sensitized on day 0, 14 and 21 with ovalbumin (OVA), house dust mite (HDM) or cockroach (CRA) together with alum and challenged intra-tracheally starting on day 26 with the respective allergens twice a week for 7 weeks (A). In the triple allergen combination model (TAC), mice were sensitized with the three extracts plus alum and challenged with one single extract twice a week alternating between the allergens (B). Therapeutic treatment with dexamethasone (per os), R848, or CpG-ODNs (both intratracheal) was started in the 4^th week of allergen challenge (C). 1 mg/kg of dexamethasone or TLR agonist was given twice weekly, 1 hour prior to allergen challenge.</p

Smooth muscle thickening and lung fibrosis is strongest in the mice treated with all three allergens.

<p>Smooth muscle increase was detected by α-smooth muscle actin staining (SMA) (A). Sirius red staining was used to detect sub epithelial collagen deposition (B). Shown are representative sections of 8–12 mice/group. Scale bar  =  100 µm. Smooth muscle area (C) and smooth muscle thickness (D) were assessed by automated analysis of α-smooth muscle actin stained lungs. Fibrosis was quantified by ELISA measurement of collagen I (E) and collagen III (F) in lung homogenates. Results represent mean ± SEM from 8–14 mice/group. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001 in comparison to the PBS control mice and ^§<i>P</i><0.05in comparison to the triple allergen combination (TAC) group.</p

IL-4, IL-5, IL-10, TGF- β 1, IFN-γ, IL-2, IL-12, and IL-1β cytokine levels in the lungs of mice treated with dexamethasone, TLR7 or TLR9 agonists in the triple allergen challenge model (TAC).

<p>Treatment was started in the 4^th week of allergen challenge with 1 mg/kg dexamethasone given orally, 1 mg/kg of R848, or 1 mg/kg CpG-ODNs, both given intratracheal. Values are expressed as mean ± SEM from 8–12 mice/group. *P <0.05, ***P<0.001 in comparison to the triple allergen combination (TAC) group.</p

Lung remodeling and allergen specific IgE levels in mice treated with dexamethasone, TLR7 or TLR9 agonists.

<p>Mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091223#pone-0091223-g001" target="_blank">Figure 1C</a> and in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091223#pone-0091223-g005" target="_blank">Figure 5</a>. Mucus production was assessed by automated analysis of the epithelium of PAS stained sections (A). Mucus plugging was scored manually analyzing whole lung cross sections as described in methods (B). Smooth muscle area (C) and smooth muscle thickness (D) were assessed by automated analysis of the smooth muscles surrounding the main bronchus in lungs stained for α-smooth muscle actin. Collagen I levels were determined in lung homogenate via ELISA (E). Levels of IgE specific for the allergens OVA (F), CRA (G), and HDM (H) were measured in the serum of animals and are expressed in arbitrary units. Results represent mean ± SEM for 8–12 mice/group and 5–9 mice/group for mucus plugging. #<i>P</i><0.05; ##<i>P</i><0.01; ###<i>P</i><0.001 in comparison to the triple allergen combination (TAC) group.</p

IL36 is a critical upstream amplifier of neutrophilic lung inflammation in mice

IL-36, which belongs to the IL-1 superfamily, is increasingly linked to neutrophilic inflammation. Here, we combined in vivo and in vitro approaches using primary mouse and human cells, as well as, acute and chronic mouse models of lung inflammation to provide mechanistic insight into the intercellular signaling pathways and mechanisms through which IL-36 promotes lung inflammation. IL-36 receptor deficient mice exposed to cigarette smoke or cigarette smoke and H1N1 influenza virus had attenuated lung inflammation compared with wild-type controls. We identified neutrophils as a source of IL-36 and show that IL-36 is a key upstream amplifier of lung inflammation by promoting activation of neutrophils, macrophages and fibroblasts through cooperation with GM-CSF and the viral mimic poly(I:C). Our data implicate IL-36, independent of other IL-1 family members, as a key upstream amplifier of neutrophilic lung inflammation, providing a rationale for targeting IL-36 to improve treatment of a variety of neutrophilic lung diseases.publishe