PI3K Pathway Mediates Lung Caspase-1 Activation And HMGB1 Production In Toluene-diisocyanate Induced Murine Asthma Model

BACKGROUND AND AIM:Athma is characterized by chronic airway inflammation. Researches have showed that there are about 300 million people with asthma worldwide, while there are about 30 million asthma patients in China and the number is increasing.As a kind of bronchial asthma. Occupational asthma is caused by all kinds of exposure to the work environment, which accounts for 10% asthma in adult. With the rapid development of synthetic chemistry industry, the number of contact sensitization material increase, along with many new types of occupational allergens or half antigen. As a result, the incidence of Occupational asthma in recent years increases. The Toluene Diisocyanate (TDI) is reported as the most common cause of Occupational Asthma. The clinical pathological and manifestations changes of TDI-induced asthma is almost the same as allergic asthma. However, the pathogenesis involed in the disease is still largely unknowed. Our research team has successfully established a TDI-induced murine asthma model characterized by Th2-dominated airway inflammation along with eosinophil and neutrophil infiltrating in the airways.High mobility group box 1 (HMGB1) is a DNA-bindingprotein that belongs to the alarmins family. It acts as a key molecule of innate immunity, and an important downstream effector of persistent tissue injury, which orchestrates inflammation, stem cell recruitment/activation, eventually leading to tissue remodeling. These processes can occur during cell activation as well as cell death. So it is regarded as a critical regulator of innate immunity.Recently, higher level of HMGB1 has also been reported to be associated with impaired lung function and more severe disease in subjects with asthma.In a previous study, we have demonstrated a relationship between increased pulmonary expression of HMGB1 and allergic airway inflammation in the TDI-induced murine asthma model. Yet how HMGB1 is regulated in the lung remains largely uncertain.Inflammasome is currently emerging as a key mediator in inflammation and innate immunity, and there has been a growing body of evidence that the NLRP3 inflammasome is involved in airway inflammation of asthma. Inflammasome controls autoactivation of its effector caspase-1, which cleaves proIL-1β and proIL-18 to mature forms and promotes the release of IL-1β, IL-18followed by initiation and amplification of various inflammatory responses. Recently, HMGB1 was identified as another important downstream target of inflammasome and caspase-1. Also several researcheshave implied a role of caspase-1 in allergic diseases. Of note, more and more studiesindicated that inflammasome, caspase-1 and HMGB1 are associated with neutrophil inflammation.Phosphatidylinositol 3-kinases (PI3Ks) are a family of proteins that phosphorylate the 3-OH position of the inositol ring of phosphatidylinositols, which locate in the cellular membrane, resulting in the formation of phosphoinositide(3,4,5) triphosphate. PI3Ks play a prominent role in all eukaryotic cellular responses, including cell survival, proliferation, differentiation and cell migration as phosphoinositide(3,4,5) triphosphate activates various downstream signaling pathway. PI3Ks are thought to be involved in a number of pathological states such as cancer, inflammation and allergy. Study also showed that PI3K pathway play crucial role in the pathogenesis of asthma. In vitro, PI3K was required for activation of caspase-1 as well as secretion of IL-1β, IL-18 and HMGB1 nucleocytoplasmic translocation. However, whether it is also involved in the caspase-1 activation and downstream IL-1β and HMGB1 production in vivo asthma model remains to be investigated.Based on the above evidence, we hypothesized that PI3K signaling pathway mediates pulmonary HMGB1 production in TDI-induced asthma model and intended to explore the possible role of NLRP3 inflammasome and caspase-1 in this process.ContentsPart I:Establish a TDI-induced asthma model according to methods of our previuous studies. And then assess the role of PI3K inhibitor LY294002 in the process of allergic airway inflammation and HMGB1 production in the mice.Part II:Observe the effect of PI3K inhibitor LY204002 on the activation of NLRP3 inflammaome in the lungs of TDI-induced asthmatic mice.METHODSSix-week old specific-pathogen-free male BALB/c mice (20-22 g) were purchased from Southern Medical University. TDI, methacholine, acetone were obtained from Sigma-Aldrich. The vehicle (AOO) used to dissolve TDI consists of a mixture of 2 volumes of acetone and 3 volumes of olive oil for the dermal sensitization, and 1 volume of acetone and 4 volumes of olive oil for the challenge.1. Asthma modelThe mice were housed in a SPF house (temperature 23±2℃, humidity range40-70%, 12h light/dark cycle [lighting:7:00-19:00]). Foodand UV-sterilized tap water were provided ad libitum. Experimentalanimals used in this study were treated under a protocol approved by the committee of Southern Medical University on the use and care of animals. Standard guidelines for laboratory animal care followed the Guide for the Care and Use of Laboratory Animals. The TDI-induced asthma model was prepared by a modification of Hoet and Lee’s method26,27. All mice were randomized to the following 4 groups:(1) AOO-sensitized, AOO-challenged, and PBS-treated mice (AOO group); (2) TDI-sensitized, TDI-challenged and PBS treated mice (TDI group); (3) TDI-sensitized, TDI-challenged and LY294002-treated mice (TDI+LY294002 group) and (4) TDI-sensitized, TDI-challenged and DMSO-treated mice (TDI+DMSO group). Briefly, on days 1 and 8, mice were dermally treated with 0.3% TDI dropped on the dorsum of both ears (20μL/ear). On days 15,18, and 21 the mice were challenged for 3 h each time via the airways with 3% TDI by means of compressed air nebulization. Challenging was performed by placing mice in a Plexiglasbox, and annebulizerdriven bycompressed air was used to aerosolize TDI, with operating air flow rate of 3.5 L/min and Mass Median Aerodynamic Diameter (MMAD) of 3 um. As a control, mice was sensitized and challenged by the same procedures with the same amount of acetone and olive oil. LY294002 (1.5 mg/kg per mouse per time) was dissolved in DMSO and diluted with 0.9% NaCl. LY294002 in a volume of 50μL was given intratracheally 1 h before each challenge. By comparison, control mice received the same volume of vehicle (0.9% NaCl containing DMSO).2. Airway hyperresponsiveness (AHR)AHR to methacholine was assessed 24 h after the third challenge. Briefly, mice were placed in a barometric plethysmographic chamber and challenged with vehicle (sterile saline) followed by increasing concentrations of methacholine (1.25,2.5,5, and 10 mg/ml) by means of ultrasonic nebulization. Aerosols were generated with an matching ultrasonic nebulizer, and then nebulized into the main chamber through its inlet for 3 min. Pressure fluctuations caused by breathing of the mice were continuouslymonitored for 3 min after each nebulization, and subsequently these pressurefluctuations were quantified using the algorithm for enhanced pause (Penh), which represented an accurate indexof airway resistance 28.3. Animal euthanasia and necropsyThe mice were sacrificed with an overdose of sodium pentobarbitone (pentobarbital sodium,100 mg/kg body weight,administered intraperitoneally). Blood samples were taken from the retro-orbital plexus/sinus. Cervical lymph nodes were dissected and processed for each mouse separately (as described in the following section 2.5.). The chest cavity wasexposed, then the tracheawas carefully intubated and the catheter secured withligatures. Lungs were lavaged in situ, three times with 0.8 mL prewarmed sterile saline (0.9% NaCl,37℃), which was slowlyinfused into the lungs and withdrawn. The left lungs were infused with 4% neutral buffered formalin (0.2mL) then removed and immersed in 4% neutral buffered formalin to fixed for pathological section, while the right lungs were removed and stored at-80℃ for western blot.4. Preparing lymph node cells and dectecting the IL-4 in the supernatantsDissected cervical lymph nodes were kept on ice in RPMI-1640 medium and cell suspensions were obtained by pressing the lymph nodes through a cell strainer (40 μm) and rinsed with 10 mL tissue culture medium (RPMI-1640). Cells were counted using a Burker hemocytometer. Lymphocytes were then washed three times and suspended (107 cells/mL) in complete tissue culture medium (RPMI-1640 supplemented with 10% heat-inactivated FBS). Cells were seeded into 48-well culture plates at a density of 106 cells/mL and incubated in complete RPMI-1640 medium for 43 h with 5 mg/mL of concanavaline A, then centrifuged (1000 xg,4℃, 10 min) and the supernatants were stored at-80℃ for further measurement of IL-4 by ELISA.5. Total serum IgEBlood samples collected were rest for 1 h at room temperature, then centrifuged (3000 xg,20 min) and supernatants were harvested and stored at-80℃. Total serum IgE was measured by ELISA (BD Bioscience) according to the manufacturer’s instructions.6. Bronchoalveolar lavage (BAL)The recovered BAL fluid was pooled. Total cells in the BAL fluid were counted, and the BAL fluid was centrifuged (1000 xg,10 min). The supernatant was frozen (-80℃) for further analysis. For differential cell counts, a cytospin sample (Shandon Scientific, Runcorn, UK) was prepared and stained using hematoxylin and eosin (H&E). For each sample, a total of 200 cells were counted for the numbers of macrophages, eosinophils, neutrophils and lymphocytes.7. Lung histopathologyThe left lungs were embedded in paraffin. Lung sections (4μm) were cut with a Leica microtome 2030. Lung tissues were stained with hematoxylin and eosin to assess pathological changes. Lung inflammation was semiquantified as described in our previous study.8. Immunohistochemistry and western blotImmunohistochemistry and western blot for HMGB1, caspase-1, IL-1β and NLRP3 were performed as previously described8. Primary antibodies for HMGB1, caspase-1 and NLRP3 were obtained from Abcam (Hong Kong, China), and IL-1β from Santa Cruz (CA, USA).9. Statistical analysisStatistical analysis was performed using SPSS version 19.0. Data were expressed as mean±standard error (SE) and comparisons among groups were analyzed by one-way analysis of variance (ANOVA) accompanied by Bonferonni post hoc test for multiple comparisons. P<0.05 was considered statistically significant.RESULTS1.Effect of LY294002 on PI3K signaling in TDI-induced asthmatic miceTo confirm the effect of LY294002 on PI3K signal pathway, we detected p-Akt, the most important downstream of PI3K signal pathway. As shown in Fig.1A, TDI sensitization and challenge upregulated the expression of p-Akt compared with AOO group (p<0.05); while administration of LY294002 abolished this TDI induced elevated p-Akt expression (p<0.05). The vehicle DMSO alone did not affect p-Akt expression compared with TDI group (p>0.05).2. TDI-induced AHR and allergic airway inflammation are mediated by PI3KEffects of PI3K on TDI induced AHR are shown in Fig. 1B. Compared with AOO group, Penh values in TDI group were significantly increased when stimulated by methacholine (1.25,2.5,5, and 10 mg/mL) (p<0.05), all of which were partly recovered after LY294002 treatment (p<0.05). The release of total serum IgE was significantly increased in TDI group compared with AOO group (p<0.05), as well as IL-4 (p<0.05) in supernatant of cultured lymphocytes, and both were markedly decreased by LY294002 (p<0.05) (Fig.1C-D).Total cell counts, as well as numbers of macrophages, neutrophils, eosinophils and lymphocytes were assessed in BAL fluid (Fig.2A-B). In agreement with total cell counts, higher amounts of neutrophils and eosinophils were found after TDI stimulation (p<0.05). Pulmonary histological examination showed similar results. The TDI-exposed mice had heavier infiltration of inflammatory cells and notable epithelial proliferation than the AOO-exposed (Fig.2C-F). Changes in both BALF and lung tissue were partly reversed after LY294002 treatment (p<0.05).3. PI3K is required for HMGBl production and nucleocytoplasmic translocationIn accordance with AHR and allergic airway inflammation described above, the protein level of HMGB1 in TDI group was significantly increased compared with that in AOO group (Fig.3A). Similar results were obtained by immunohistochemistry. In AOO group, HMGB1 was detected almost only in the nuclei, however, after TDI challenge, we found that HMGB1 was translocated from the nuclei to the cytoplasm and scattered in bronchial epithelium, alveolar epithelium, airway fibroblasts and infiltrating inflammatory cells (Fig.3B-D). Notably, both the elevated protein expression and nucleocytoplasmic translocation of HMGB1 were largely alleviated after LY294002 adminstration (p<0.05) (Fig.3A-D).4. Effect of LY294002 on caspase-1 activationTo further explore how PI3K regulates HMGB1 production and release in TDI-induced airway response, we measured its well-established upstream caspase-1. Firstly, using immunohistochemistry, we found that after TDI challenge, the production of caspase-1 was remarkably increased with dense staining throughout the cytoplasm of bronchial and alveolar epithelium as well as infiltratinginflammatory cell, while inhibition of PI3K by LY294002 largely attenuate this TDI-induced caspase-1 upregulation (Figure 4A-C). Secondly, we measured the protein levels of procaspase-1 (45KD) and cleaved caspase-1 (20KD) in lung tissue by western blot. The protein level of cleaved caspase-1 rather than procaspase-1 was significantly elevated in TDI group compared with that in AOO group (p<0.05) (Figure 4D), indicating that the TDI-induced caspase-1 production observed in immunohistochemistry above mainly resulted from caspase-1 cleaving; we further found that this increased cleaved caspase-1 production was mostly reversed when administrating LY294002 before challenge, while the expression of procaspase-1 stayed nearly unchanged in all groups (P>0.05) (Figure 4D). These data demonstrated that the lung caspase-1 was activated in TDI induced asthma and PI3K activity was crucial for its maturation.5. Effect of LY294002 on IL-1β productionTo reconfirm the role of PI3K in caspase-1 activation and explore the downstream mechanism involved in TDI-induced asthma.We detected IL-1β, a proinflammatory factor well known as the downstream target of caspase-1. Immunohistochemistry (Figure 5A-B) showed faint expression of IL-1β in the lung in AOO group, while TDI treatment made more IL-1β thinly scattered in the airwayinfiltrating inflammatorycells, alveolar epithelium, bronchial epithelium andairway fibroblasts. Likewise, LY294002 administration remarkably reduced this TDI induced IL-1β expression in the cytoplasm. Similar result was obtained by western blot (Figure 4E-F). The TDI-induced elevated cleaved IL-1β(17KD) production (p<0.05) was nearly abolished when treated with LY294002 (p<0.05). We also observed increased expression of proIL-1β(31KD) after TDI stimulation (p<0.05) which was decreased after LY294002 treatment (p<0.05). But we failed to detect IL-1β in BALF by ELISA (data not shown) in all enrolled groups. In addition, we measure NLRP3, the sensor of NLRP3 inflammasome and the most well-established upstream of caspase-1. We found that although NLRP3 was fully expressed in the bronchial epithelium, alveolar epithelium as well as infiltrating Inflammatory cell (Figure 6A-B), the distribution (Figure 6A-B) and protein level (Figure 4E,G) of NLRP3remained unchanged either after TDI challenge or LY294002 treatment (p>0.05).CONCLUSIONS1. On the basis of a TDI-induced asthma model, we demonstrate that PI3K signal pathway is involved in TDI-induced allergic airway inflammation and HMGB1 production and nucleocytoplasmic translocation.2.PI3K is required for caspase-1 activation andIL-1β production in the lung of TDI induced asthmatic mice, which is a possible mechanism of TDI-induced HMGB1 production and nucleocytoplasmic translocation, as well as allergic airway inflammation.