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IL6 receptor-mediated lung Th2 cytokine networking in silica-induced pulmonary fibrosis

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Pulmonary silicosis is a deadly disease which kills thousands of people every year worldwide. The disease initially develops as an inflammatory response with recruitment of inflammatory cells into the lung controlled by multiple cytokines. The
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  Arch Toxicol (2010) 84:947–955DOI 10.1007/s00204-010-0559-z  1 3 ORGAN TOXICITY AND MECHANISMS IL-6 receptor-mediated lung Th2 cytokine networking in silica-induced pulmonary W brosis Shambhoo Sharan Tripathi · Vani Mishra · Mamta Shukla · Mukesh Verma · Bhushan Pradosh Chaudhury · Pradeep Kumar · Jasmeet Kaur Chhabra · Haushila Prasad Pandey · Bholanath Paul Received: 27 March 2010 / Accepted: 4 May 2010 / Published online: 20 May 2010 ©  Springer-Verlag 2010 Abstract Pulmonary silicosis is a deadly disease whichkills thousands of people every year worldwide. The dis-ease initially develops as an in X ammatory response withrecruitment of in X ammatory cells into the lung controlledby multiple cytokines. The question whether these cyto-kines exert biological functions through signal transducingpathway remains unanswered along with the potential roleof interleukin-6 receptor   (IL-6R  ) in regulating in X am-matory cytokines. We aimed to assess the status of signaltransducers and activator of transcription (Stat3), suppres-sor of cytokine signalling 3(Socs3) and in X ammatorycytokines in airways of silica-exposed mice, and theirrelationship with IL-6R  . Silica-exposed and silica-exposed IL-6R   gene knockdown Balb/c mice were used inthe study. Lung function was measured by plethysmogra-phy, mRNA expression of cytokines and signal moleculesby qRT 2 -PCR and lung architecture by histopathology; Thelper cell-type 2 (Th2) cytokines in broncho-alveolar lav-age X uids were evaluated by ELISA and hydroxyproline inlung by colorimetry. Elevated levels of collagen deposition,signs of lung W brosis, in W ltration of in X ammatory cells andpresence of exfoliated mucosa in the lung of silica-exposedmice with concurrent increase in methacholine-inducedspeci W c resistance of airways were observed on day 60post-exposure. In parallel, heightened expression of Th2cytokines (IL-4, IL-5, IL-6) and signal molecules (Stat3and Socs3) were observed in the airways of silica-exposedmice. Th1 (IL-1   and TNF-  ) cytokines are underexpressedin majority of the airways tissues of silica-exposed mice.Silencing IL-6R   in lung of silica-exposed mice down reg-ulated the hypermorphic mRNA pool of potential Th2 cyto-kines and signal molecules. Hypermorphic expression of Th2 cytokines and signal molecules in airways of silica-exposed mice are mediated through IL-6R  . Keywords Silica · Th2 cytokines · IL-6R   · Stat3 · Socs3 Introduction Although crystalline silica has been a known industrial haz-ard since the early 1900s, the onslaught of pulmonary dam-age and death caused by it continues till date (LegalResource Centre 2009). It kills thousands of people everyyear worldwide (Dunne 2009). Goldmine workers developsilicosis while exposed to a quartz concentration below therecommended occupational exposure limit (OEL) of 0.1mg/m 3 . This accord with a mounting body of evidencethat an OEL of 0.1mg/m 3  is not protective against silicosis(Churchyard etal. 2004). Occupational exposure to crystalline silica is associatedwith the development of pulmonary silicosis (Hnizdo andVallyathan 2003; Reiser and Last 1979) and an increased risk for lung cancer (McDonald and McDonald 1995) and S. Sharan Tripathi · V. Mishra · M. Shukla · M. Verma · J. K. Chhabra · B. Paul ( & )Immunobiology Division, Indian Institute of Toxicology Research, Mahatma Gandhi Marg, Lucknow 226001, Indiae-mail: bholanathpaul@yahoo.com; bnpaul@iitr.res.inB. P. ChaudhuryHistopathology Division, Indian Institute of Toxicology Research, Mahatma Gandhi Marg, Lucknow 226001, IndiaP. KumarAnimal Facility Centre, Indian Institute of Toxicology Research, Mahatma Gandhi Marg, Lucknow 226001, IndiaH. P. PandeyDepartment of Biochemistry, Banaras Hindu University, Varanasi, India  948Arch Toxicol (2010) 84:947–955  1 3 pulmonary tuberculosis (Corbett etal. 1999; Hnizdo and Murray 1998; Park etal. 2009; teWaternaude etal. 2006) as well as chronic obstructive pulmonary disease (Hnizdo andVallyathan 2003). Silica can cause direct DNA damage andmammalian cell transformation (Daniel etal. 1995; Shi etal. 1994) attributing to disease pathogenesis. However,the initial event is an in X ammatory response, including oxi-dant production and recruitment of in X ammatory cells intothe lung.Numerous in X ammatory mediators have been implicatedin silica-induced pathology including interleukin(IL)-1ß(Goodman etal. 1982), IL-6 (Gosset etal. 1991), IL-10 (Huaux etal. 1998), tumour necrosis factor-   (TNF-  )(Dubois etal. 1989) and transforming growth factor (Williams etal. 1993; Williams and Sa Y otti 1995);Chemokines, such as monocyte chemoattractant protein-1(Barrett etal. 1999); macrophage in X ammatory protein-2(Driscoll etal. 1998) and the non-protein in X ammatorymediator nitric oxide, generated mainly through induciblenitric oxide synthase (Castranova etal. 1998). Adhesion molecules, such as intercellular adhesion molecule-1 (Narioand Hubbard 1996), have been implicated in silica-inducedpathology. Silicosis is characterised by activation of resi-dent macrophages that engulf the silica particles, as well asby a rapid and persistent in X ux of polymorphonuclear cells.Monocyte and macrophages secrete IL-1, IL-6 and TNF-  that modulate T-lymphocyte recruitment and type switch-ing (Olszewski etal .  2000). IL-6 is a pleiotropic cytokine(Qiu etal. 2004) and exerts important biological e V  ects onin X ammation, immunity and stress (Kishimoto etal. 1995; Papanicolaou etal. 1998). Accumulating evidence reveals that IL-6 levels increase in blood (Yokoyama etal. 1995), bronchoalveolar lavage X uid (BALF) (Broide etal. 1992) and lung tissues (Marini etal. 1992) of patients with lung disease. It has also been implicated in the pathogenesisof pulmonary W brosis induced by asbestos and silica(Simeonova etal. 1997). IL-6 binds to the surface IL-6 receptor (IL6R), leading tothe dimerization of gp130/IL-6R   into tetra- or hexametricstructures, thereby forming the active IL-6R complex(Boulanger etal. 2003). IL-6 engages receptors that recruit Janus Kinases(Jak); and activated Jaks phosphorylate thecytoplasmic domain of the receptor, creating a docking sitefor Src homology 2-containing proteins such as Stat tran-scription factors (Ihle 2001). In the lung, Stat3 is activatedby phosphorylation at residues Tyr705 and Ser727 duringIgG-IC-induced acute lung injury (Gao etal. 2004). Phos- phorylated Stats bind to the promoter sequences andactivate the transcription of a suite of genes, includingthose encoding the suppressor of cytokine signalling-1(Socs1) and Socs3 proteins (Starr etal. 1997; Warmald andHilton 2003). Stat3 activates the transcription of Socs3mRNA byinteracting with the 5  promoter of the Socs3gene (Auernhammer etal. 1999). Socs3 protein can inhibit Jak phosphorylation of Stat, thus creating a negative feed-back loop that attenuates cytokine signal transduction.Socs3 inhibit signalling by binding to phosphorylated tyro-sine sites on the cytoplasmic domain of the receptor (Nich-olson etal. 1999).Although insight into the pathophysiology of silica-induced lung injury has increased substantially over theyears, a number of issues remain to be clari W ed. As multiplecytokines are responsible for silica-induced in X ammatoryresponse in the airways, it is not known whether these cyto-kines exert biological functions through Jak/Stat pathway.Elucidation of the regulatory mechanisms of the Jak-Stat3pathway is important in understanding the in X ammatoryprocess associated with lung injury. Therefore, we assessedthe status of Stat3 and Socs3 in relation to in X ammatorycytokines following Si-exposure to elucidate, whetherinduction of the in X ammatory cytokines is routed throughIL-6R  . In this study, we have explored the relationshipbetween the kinetics of Stat3 and Socs3 mRNA expressionfor silica-induced airways in X ammation and demonstratedmarked increase in Stat3 and Socs3 expression, along withheightened expression of Th2 cytokines, reduced lung func-tions and W brotic architecture of the lung. Interestingly, theheightened expressions of in X ammatory cytokines arerouted through IL-6R. This study elucidates the molecularmechanism of in X ammatory response leading to lung W bro-sis by silica and identi W es the functional receptor that regu-lates the in X ammatory response. Our study data alsosuggests that IL-6 can be a target for prevention and thera-peutic interventions in silica-induced airways injury. Materials and methods Characterisation of silica particlesSilica particles (SiO 2 ·xH 2 O; MW 60; HiMedia LaboratoriesP Ltd., Mumbai, India) were crushed in heavy duty ironmortar and pestle for 2h and thereafter grounded in REMIgrinder for 30min. Silica particles adhering on the roof of grinder cover were collected on glass petri plates, dryheated at 170°C for 24h, weighed and suspended in sterilesaline by vortexing before being administered on the nares.The mean particle count diameter of silica particles wasmeasured by Scanning electron microscopy. Silica particleswere suspended in distilled water and a drop of it was dis-pensed on glass plate and air dried under table lamp. Theplate was mounted on aluminium stub and silver paintapplied on the corners of the plate for conduction of elec-tron beam. The plate mounted stub was coated with goldpalladium with the help of a Polaron sputter coater andscanned under Leo 430 (England) conventional electron  Arch Toxicol (2010) 84:947–955949  1 3 microscope (Birbal Sahani Institute of Paleobotany,Lucknow). Photographs were taken at 7.86KX magni W ca-tion. Mean particle count diameter was 2.5   m, (Fig.1) andthus merit being of respirable size.Silica exposure and experimental designEight- to 10–week-old female Balb/c mice procured fromInstitutional Animal Facilities were used for the experi-ments with consent from the Committee for the Purpose of control and Supervision on Experiments on Animals(CPCSEA), India. Mice were anesthetized by injecting0.05mL ketamine hydrochloride, i.p. and 2.5mg silica in75   L phosphate-bu V  ered saline (PBS) was administeredon days 0, 14 and 21; the animals were killed on day 60.Mice administered 75   L PBS represent sham controlgroup. For administration of silica, mice were held perpen-dicular to the counter top and the mouths pressed shut.Silica droplets in PBS (0.84mg Silica in 25  L PBS) weredelivered per cycles at an interval of » 2min on the nares; 3cycles were administered with a micropipette. Mice inhaledthe droplets involuntarily. Noses were decontaminated with70% ethanol. Before killing, lung function was monitoredby plethysmography followed by collection of broncho-alveolar lavage X uid (BALF) from airways. Post-killinglungs and trachea were dissected out for RNA isolation andqRT 2 -PCR for the detection of Stat3, Socs3, IL-1  , IL-4,IL-5, IL-6, TNF-  , eotaxin and  -actin.Measurement of airways hyper-reactivity (AHR)AHR induced by di V  erent concentrations (0, 3.12, 12.5 and50mg/mice) of methacholine (Mch) was measured by adouble-chambered whole-body plethysmograph (Buxco;model No.PLY 4451). Nebulised Mch was administeredthrough inhalation route by dispensing various concentra-tions on the nebulizer head and passed to the nasal chamberat a X ow rate 0.5LPM for 3min for each concentration.Irregularities in the breathing pattern were often encoun-tered due to movement of the neck in most cases and to alesser extent, sigh-breath and cough. Data arising fromthese irregularities in breathing pattern were not consideredfor quantitative analysis. Before the start of the experi-ments, animals were conditioned to stay in the plethysmo-graph chamber for at least 3 or 4 sessions of 10-minduration each. All the groups of mice were conditioned in asimilar way to nullify the e V  ect of preconditioning of ani-mals on the measurement of speci W c resistance of airways(sRaw). sRaw was monitored with the Biosystem XA SFT3410 version 2.11(Buxco).Collection of BALF and evaluation of Th2 cytokinesFor collection of BALF, mice were anesthetized withketamin hydrochloride (0.05ml/mice), the tracheaexposed by mid-line incision in the neck region and 1mlof PBS was injected into lungs through the trachea andwithdrawn after 10s. The X uid recovered from eachmouse was centrifuged at 2,000rpm, for 5min at 25°C,and the supernatants used for Th2 cytokine analysis. IL-4,IL-5 and IL-6 protein levels in BALF were quanti W edwith commercial mouse ELISA kit (R&D system) accord-ing to manufacturer’s instructions. Cell pellets were usedfor RNA isolation.Hydroxyproline assayCollagen deposition was estimated by measuring thehydroxyproline content of the lung. Lungs were excised,homogenised in acetic acid (0.5M) and hydrolysed in 6NHCl overnight at 110°C. Hydroxyproline was assessed bycolorimetric analysis and data were expressed as micro-grams of hydroxyproline per lung.Isolation of RNA and qRT 2 -PCR of cytokines and signal moleculesTotal RNA was isolated from lung, trachea and BALF cellsusing RNAeasy mini kit as per the manufacturer’ instruc-tion (Qiagen, Hilden,Germany). RT–PCR was carried outusing a QuantiTect SYBR green RT–PCR kit (Qiagen,Germany). The RT–PCR conditions were 50°C for 30minfor reverse transcription; initial PCR activation at 95°C for15min followed by 40 cycles of denaturation at 94°C for15s, annealing at a 5°C below the T  m  of primers for 30sand extension of 30s at 72°C. Melting curve analysis (notshown) was performed to verify the speci W city and identityof the RT–PCR products. One microlitre of template RNAcontaining 50ng of total RNA was used in a 50-  L RT–PCR Fig.1 Scanning electron micrograph of silica particles  950Arch Toxicol (2010) 84:947–955  1 3 cocktail. All reactions were performed in triplicates. Relativequanti W cations were performed in LightCycler 480 calibra-tor normalised relative quanti W cation assay in which thetarget concentration is calculated relative to a non-regulatedreference. The results are expressed as the target/referenceratio of each sample normalised by the target/referenceratio of the calibrator using LightCycler 480 relative quan-ti W cation software release 1.2.0625. Primers (Operon Bio-technologis Gmbh, Germany) used for RT–PCR were:Stat3 forward 5  -GAAGACCAAGTTCATCATCATCTGTGTG-3   reverse 5  -GTAGCACACTCCGAG GTCAGAT-3  ;Socs3 forward 5  -TGAGCGTCAAGACCCAGTCG-3  reverse 5  -CACAGTCGAAGGCGGGGAACT-3  ; IL-6forward 5  -TTGCCTTCTTGGGACTGAT GCT-3   reverse5  -GTATCTCTCTGAAGGACTCTGG-3  ; IL-5 forward5  -AAGGATGCT TCTGCACTTGA-3   reverse 5  -TATCTCTCTGAAGGACTCTGG-3  , IL-4 forward 5  -GACAAAAATCACTTGAGAGAGA-3   reverse 5  -ACGAGTAATCCATTTGCATGAT-3  ; IL1   forward 5  -TGACGGACCCCAAAAGATGAAG-3   reverse 5  -CTGCTTGTGAGGTG °CTGATGTA-3  ; TNF-   forward 5  -CCAGACCCTCACACTCCAGAT-3   reverse 5  -AACACCCATTCGCTTCACAG-3  ; Eotaxin forward 5  -GGGCAGTAACTTCCATCTGTCTCC-3   reverse 5  -CACTTCTTCTTGGGGTCAGC-3  ;  -actin forward 5  -GACATGGAGAAGATCTGGCAC-3   reverse 5  -TCCAGACGCAGG ATGGCGTGA-3  .In vivo silencing of IL-6R   geneTo determine the optimum concentration of siRNA thatinhibited >80% gene activity, a pool of 3 target-speci W c20–25 nucleotide siRNA designed to knockdown mouseIL-6R   expression, varying concentrations of siRNA,40–80pmols (sc-40065; Santa Cruze Biotech, California,USA) complexed with 5% glucose solution was diluted inExGen 500 in vivo transfection reagent containing a cat-ionic polymer, polyethylenimine (Fermentas Life Sciences,Canada) and administered through nasal route in anesthe-tized silica-exposed mice according to the manufacturer’sinstructions on days 49 and 56, and killed on day 60.Administration of siRNA onto the nares was performed in asimilar way as done for silica. Optimum dose of siRNAwas monitored by measuring the mRNA expression pro W leof IL-6R   and  -actin using speci W c primers. One stepRT–PCR was performed in Thermal cycler (Esco, Japan)using 25ng RNA as template in a 25   L reaction volume.The RT–PCR products (344bp for IL-6R   and 307bp for  -actin) were analysed by using Agilent DNA 750 kit andanalysed in Bioanalyzer 7500 (Agilent). Sense strandsequence of the scrambled siRNA(ss) (Qiagen) is:UUUUCCGAACGUGUCACGUdTdT. The sequence of the primers used is: IL-6R   (F) 5  -AGGAGTGAAGCACGTGGTCCAGGT-3  , (R) 5  -TCTGACTTCCATTTCTGCTTGAGT;  -Actin (F) 5  -GACATGGAGAAGATCTGGCAC-3  , (R) 5  -TCCAGACGCAGGATGGCG TGA-3  .Statistical analysisAll results are expressed as mean § SD. Groups were com-pared using the Students t   test with P  value<0.05 taken asindicator of statistical signi W cance. Results Impaired lung function in silica-exposed micesRaw is a sensitive and reproducible measure of lung func-tion; an inverse correlation exists between sRaw and lungfunction (Paul etal. 2009; Ranganathan etal. 2008; Simpson etal. 2007). We monitored Mch-induced sRaw on day 1 and 60 post–silica exposure in mice and observed sig-ni W cant ( P <0.05) increase in sRaw on day 60 in silica-exposed mice in comparison with sham control (Fig.2a),indicating impairment of lung function by silica exposure.However, signi W cant changes in Mch-induced sRaw on day1 between silica exposed and sham group were notobserved which indicated that silica-driven impairment of lung physiology is a function of time and ruled out acutealteration in lung function. Silica does not act directly onlung function to evoke immediate response and seems toinvolve time drawn processes of inducing a pool of in X am-matory molecules that are responsible for W brogeniclung. Interestingly, signi W cant ( P <0.05) decrease inMch-induced sRaw was observed in IL-6R  -silenced sil-ica-exposed mice in comparison with SS control (Fig.2b).These data suggest that IL-6 receptors play an importantrole in silica-induced reduction of lung function.Histopathological alterations in lung architectureHistological studies on the lungs of silica-exposed micerevealed signs of lung W brosis, in W ltration of in X ammatorycells, disrupted bronchiolar epithelial lining and presence of exfoliated mucosa (Fig.3b) with concurrent increase inMch-induced sRaw of airways. These observations werenot seen in sham control (Fig.3a). Total hydroxyprolinelevels in the lung, an index of W brosis (Kehrer and Margolin1997), were measured on 60 days post–silica exposure inmice and observed to be signi W cantly ( P <0.05) higher(Fig.3f) in comparison with sham control (Fig.3c). Essen- tially, all the hydroxyproline in the tissues of vertebrates isfound in collagen and, except for the small amount of hydroxyproline in elastin, no other animal protein containssigni W cant amounts of this amino acid (Prockop andKivirikko 1967). The deposition of excess or abnormal  Arch Toxicol (2010) 84:947–955951  1 3 collagen is characteristic of pulmonary W brosis and candisrupt gas exchange resulting in severe respiratory impair-ment. High level of hydroxyproline in lungs, 60 days post-exposure of silica particles, correlates with the extensivelung W brosis in histological analysis. In IL-6R  -silencedsilica-exposed mice, marginal reductions in foci of W brosis,exfoliated mucosa and partially healthy bronchiolar liningepithelium were observed (Fig.3e) in comparison withSS control (Fig.3d). Hydroxyproline content in lung of IL-6R-silenced silica-exposed mice decreased marginallyin comparison with SS control, thus re X ecting of a possiblerole of IL-6 receptor in silica-induced pulmonary injury.Cytokine expression pro W le in silica-exposed miceLung W brosis and tissue W brosis in general is regulated by acomplex network of cytokines (Fenton 2003). We mea- sured the relative expression of both Th1 cell- and Th2cell-associated cytokines and signal molecules in silica-exposed mice. TNF-  , IL-1  , IL-1   and IFN-   are thesignature cytokines of Th1 cells; and IL-4, IL-5, IL-6 andIL-10, are secreted by Th2 cells (Mosmann and Co V  man1989; Mosmann and Sad 1996). Total RNA from lung, tra- chea and BALF cells were subjected to qRT 2 -PCR to studythe expression of Th1 and Th2 cytokines. Aberrant expres-sion of the Th1 cytokines, TNF-   and IL-1  , were seen indi V  erent tissues of airways; among the Th2 cytokines, IL-4,IL-5 and IL-6 were consistently overexpressed in BALFcells, trachea and lung on day 60 post-silica exposure(Fig.4). Protein expression pro W le of Th2 cytokines inBALF (Table1) was also in line with the mRNA expressionpro W le. Eotaxin—a chemokine preferentially expressed in Fig.2 Lung function in silica-exposed mice. a  E V  ect of varying con-centration of Mch-induced sRaw in silica-exposed mice (  W lled square )and sham control (  W lled diamond  ) on day 60 post-exposure or day 1(  W lled triangle ) post-exposure. b  E V  ect of varying concentration of Mch-induced sRaw in silica-exposed IL-6R   knockdown mice (  W lled rectangle ) and scrambled sequence control (  W lled diamond  ) on day 60.  N  =6 mice per point Fig.3 Silica-induced altera-tions of lung architecture in mice. Representative section showing histological changes in lung of silica-exposed ( a ); Sham control ( b ); IL-6R  -silenced silica-exposed ( d ) and SS-trans-fected silica-exposed ( e ) mice (), in W ltration of in X amma-tory cells; [], disrupted bronchiolar lining epithelium; [], oedema; [], foci of W brosis; [], exfoliated mu-cosa. Hydroxy proline pro W le in lung of silica-exposed and sham control mice ( c ) and IL6R   knockdown silica-exposed and SS control mice ( f  ). *  P >0.05;  N  =6 per group
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