Prmt2 Regulates the Lipopolysaccharide-Induced Responses in Lungs and Macrophages

This information is current as Emilie Dalloneau, Patricia Lopes Pereira, Véronique Brault, of September 30, 2021. Elizabeth G. Nabel and Yann Hérault J Immunol 2011; 187:4826-4834; Prepublished online 28 September 2011; doi: 10.4049/jimmunol.1101087

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Prmt2 Regulates the Lipopolysaccharide-Induced Responses in Lungs and Macrophages

Emilie Dalloneau,* Patricia Lopes Pereira,* Ve´ronique Brault,* Elizabeth G. Nabel,† and Yann He´rault*,‡

Precise control of the LPS stimulation in the lung modulates inflammation and airway hyperresponsiveness involving the well- known TLR4/NF-kB pathway. As a consequence, the expression and secretion of proinflammatory cytokines is tightly regulated with the recruitment of neutrophils. Changes in the LPS-induced responses have been observed in the Prmt2-Col6a1 monosomic model, suggesting the presence of dosage-sensitive controlling LPS pathway in the mouse. In this article, we report that the Prmt2 regulates the LPS-induced lung responses in lungs and macrophages. We demonstrate that Prmt2 dosage influences the lung airway hyperresponsiveness, the recruitment of neutrophils, and the expression of proinflammatory cytokines, such as IL-6 and TNF-a. In addition, Prmt2 loss of function also altered the nuclear accumulation of NF-kB in stimulated macrophages. Prmt2 Downloaded from should be considered as a new member of the NF-kB pathway controlling LPS-induced inflammatory and lung responses in a dosage-dependent manner, certainly through regulating nuclear accumulation of NF-kB as shown already in fibroblasts. The Journal of Immunology, 2011, 187: 4826–4834.

he LPS after intranasal instillation induces a complex LPS-induced inflammation is the consequence of the activation

response in the lungs that is tightly regulated (1). The LPS of the reticuloendothelial system leading to the synthesis of various http://www.jimmunol.org/ T binds adaptors and activates the TLR4, leading to the mediators that have a local but also a general action. The principal activation of different signaling pathways that will elicit the lung proinflammatory cytokines are the IL-1, the IL-6, and the TNF-a. and the inflammatory responses. Part of the control is due to Expressions of IL-6 and TNF-a are directly regulated by NF-kB specific adaptor proteins that contribute to the integrated response. (14, 15), and these molecules are responsible for the amplification In the classical TLR4/MyD88 pathway, NF-kB is normally se- of the inflammatory response and part of the systemic responses (16). questered in the cytoplasm by an IkB complex (2–6), and the In the course of our study to isolate mouse dosage-sensitive phosphorylation of IkB by inducible kinases, such as IkB kinase 1 genes homologous to human 21 (Hsa21) genes, we (IkK1) and IkK2, leads to subsequent ubiquitination and degra- developed a new mouse model of monosomy for the Prmt2-Col6a1 dation of these proteins. The released NF-kB then translocates to (noted Ms1Yah) genetic interval found on the mouse chromosome by guest on September 30, 2021 the nucleus, where it stimulates the transcription of genes involved 10 (17). The Ms1Yah model is deleted for 14 genes corresponding in immune and inflammatory responses (7). NF-kB DNA binding to the telomeric end of Hsa21, which was shown to display several and NF-kB–dependent transcription are attenuated by newly syn- copy number variants, in particular some that affect the Prmt2, thesized IkB-a in the nucleus, which associates with NF-kB/RelA S100B, and the genes. The only phenotype of the Ms1Yah mice is complexes. As IkB-a accumulates in the nucleus, there is a pro- an impaired airway response and an increased inflammatory re- gressive reduction of both NF-kB DNA binding and NF-kB– sponse after LPS stimulation. Further investigations showed mac- dependent transcription (8), presumably by export of NF-kB/IkB-a rophages (MF) as an important cellular compartment for the in- complexes from the nucleus (9–11). An additional pathway, in- creased production of proinflammatory cytokines. Several genes dependent of MyD88/MyD88-adaptor like and NF-kB, is regu- of the Pmrt2-Col6a1 region were found to be dosage sensitive in lated by TRAM and moderately by Toll/IL-1R domain containing lungs and MF, and are probably involved in phenotypes observed adaptor inducing IFN-b (TRIF), leading to the late activation of in Ms1Yah mice (17). NF-kB (12), and activates type I IFN synthesis (13). To further explain the increased production of IL-6 and TNF-a in Ms1Yah mice in response to LPS, we focused our attention on the Prmt2, which modifies arginine residues during posttranslational modification of proteins (18). PRMT2 is known to act as a nega- *Centre National de la Recherche Scientifique, Institut de Ge´ne´tique Biologie tive regulator of the NF-kB pathway in a dose-dependent manner. Mole´culaire et Cellulaire, INSERM, Universite´ de Strasbourg, Unite´ Mixte de Recherche 7104, Unite´ Mixte de Recherche 964, 67404 Illkirch, France; †Na- In fibroblasts, Prmt2 exerts its effect by causing nuclear accu- tional Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, ‡ mulation of IkB-a, which concomitantly decreases nuclear NF-kB MD 20892; and Institut Clinique de la Souris, 67404 Illkirch, France DNA binding (19). Thus, we hypothesized that the decrease in the Received for publication April 15, 2011. Accepted for publication August 22, 2011. copy number of this gene is at the origin of the increase of the This work was supported by the National Centre for Scientific Research and the inflammatory response observed in the Ms1Yah model. Indeed, European commission with the AnEUploidy project (LSHG-CT-2006-037627). the expression of proinflammatory cytokines IL-6 and TNF-a is Address correspondence and reprint requests to Dr. Yann He´rault, Institut de Ge´ne´t- ique Biologie Mole´culaire et Cellulaire, CNRS, INSERM, Universite´ de Strasbourg, NF-kB dependent and increases in Ms1Yah mice after stimula- UMR7104, UMR964, 1 rue Laurent Fries, 67404 Illkirch, France. E-mail address: tion with LPS. In this study, we demonstrated the involvement of [email protected] Prmt2 in the inflammatory response and determined how Prmt2 Abbreviations used in this article: ADMA, asymmetric dimethylarginine; AHR, air- heterozygotes could recapitulate the defects observed in Ms1Yah way hyperresponse; BALF, bronchoalveolar lavage fluid; IkK1, IkB kinase 1; MF, macrophage; MPO, myeloperoxidase; PenH, parameter of enhanced pause; TRIF, mice. The conclusion of this study is that Prmt2 is a dosage- Toll/IL-1R domain containing adaptor inducing IFN-b; wt, wild-type. sensitive gene contributing to the control of the LPS-induced www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101087 The Journal of Immunology 4827 inflammatory response by regulating nuclear level of NF-kB. RNA integrity number $6 and with a ratio [28S/18S] $1.5 (24). The More surprisingly, we found an inverse pulmonary function in synthesis of cDNA was done with the Absolute 2-step QRT-PCR SYBR a Prmt2 heterozygous mice compared with that of Ms1Yah mice, Green (Abgen). To determine the relative expression of IL-6 and TNF- , we used primers pairs from Qiagen. Each primer pair was tested suc- suggesting that Prmt2 should interact with other dosage-sensitive cessfully with the efficiency ranging from 90 to 110%. The quantitative genes, located in the Col6a1-Prmt2 region to recapitulate the PCR was performed with 15 ng cDNA and 200 nM of each primer in complete Ms1Yah phenotypes. a15ml final reaction in a Stratagene Mx4000 with a standard amplifica- tion procedure. In parallel, similar experiments were carried out for eight housekeeping genes, Actb, b2m, Gapdh, Pgk1, Rpl13a, Tbp, Tubb4, and Materials and Methods 18SRNA, used for normalization through the Genorm procedure (25). All Mouse strains the tested samples were performed in triplicate, and the results were reported as the mean 6 SEM. The Ms1Yah monosomic mice, Del(10Col6a1-Prmt2)Yah, were generated by chromosomal engineering and correspond to a deletion of 14 genes Bone marrow-derived MF culture and stimulation by LPS located in the Col6a1-Prmt2 interval. Ms1Yah mice were genotyped by +/2 2/2 Southern blot analysis as described previously (1). The Prmt2tm1Enbl allele Ten mice of each genotypes (Ms1Yah, Prmt2 , Prmt2 , and B6) were carries a G119X mutation and a neocassette replacing exons 4 and 5 and euthanized, and bone marrow cells were isolated from femurs and differ- entiated into MF (17). For the experiment, cells were plated in 96-well part of exon 6 (18, 20). The presence of the mutation is identified by PCR 5 in standard conditions with three primers: Prmt2,Nab.primer A: 59- plates at 10 cells/well. Cells from five mice of each genotype were CTGAGGTATTACCAGCAGACA-39; Prmt2,Nab.primer B: 59-CTC- stimulated with NaCl or LPS (serotype O111B4 at 100 ng/ml; Sigma- , . Aldrich) and IFN-g. Supernatants were harvested after 24 h and stored TCTGATGCAGGTCTAC-39; Prmt2 Nab primer C: 59-CCGGTGGAT- 2 GTGGAATGTGT-39. The primers A and B identified the wild-type (wt) at 20˚C for cytokine quantification. allele that corresponds to a 190-bp fragment, whereas the primers B and C NF-kB detection in nucleus of bone-marrow derived MF Downloaded from enable the amplification of the mutant allele with a 280-bp amplicon. All the lines used in the following experiments were maintained on C57BL/6J After differentiation, MF were transferred on permanox four-well slides, at (B6) with .10 generations of advanced backcross level. Y.H., as the 105 cells/well. Cells from two mice of each genotype were stimulated with principal investigator in this study, was granted the accreditation 67-369 to NaCl or LPS (serotype O111B4 at 30 ng/ml; Sigma-Aldrich) during 4 h. perform the reported experiments. The cells were washed twice with PBS for 5 min and fixed in para- formaldehyde 4% overnight. Cells were further washed on PBS twice and Airways resistance and bronchoalveolar lavage fluids twice in PBS plus glycine 0.1 M before 15 min of permeabilization in http://www.jimmunol.org/ Animals (n = 10) were treated by intranasal instillation with either isotonic Triton 0.5%. After two washes in PBS with BSA 1%, cells were incubated saline solution or 10 mg LPS (Escherichia coli, serotype O55B5, 10 mg; with NF-kB p65 Ab (SC-109, dilution 1/50 in PBS plus BSA 1%; Santa Sigma-Aldrich, St. Louis, MO) in deep anesthesia. Airway response was Cruz) for 90 min. Cells were washed three times in PBS plus BSA 1% and investigated over a period of 6 h after treatment using whole-body pleth- incubated with the second Ab (Alexa 488, 711-486-156 dilution 1/100 in ysmography and measurements of the parameter of enhanced pause PBS, BSA 1%; Jackson Immunoresearch) for 1 h. Cells were washed three (Penh), a dimensionless parameter that accounts for the respiratory pro- times in PBS, BSA 1% and mounted with Mowiol mounting medium file, taken in consideration the period of expiration and the variations of containing DAPI (diluted 1/100). To determine the effect of Prmt2 on the nuclear accumulation of NF-kB, the presence of the transcription factor pressure measured in a closed chamber during the respiratory cycle 2/2 (EMKA Technologies, Paris, France). PenH can be conceptualized as the was evaluated in wt and Prmt2 cells. Confocal microscopy was per- phase shift of the thoracic flow and the nasal flow curves. Increased phase formed using a Leica confocal microscope. Quantification of nuclear NF- shift correlates with increased respiratory system resistance. PenH is cal- kB in individual cells from several fields was done as follows. The nucleus by guest on September 30, 2021 culated by the formula PenH = (Te/RT 2 1) 3 PEF/PIF, where Te is was identified by DAPI staining, and the outline of cell nuclei was drawn expiratory time, RT is relaxation time, PEF is peak expiratory flow, and in a field with ImageJ. NF-kB pixel intensity from the nucleus of each PIF is peak inspiratory flow (21). Data are analyzed using Datanalyst individual cell in the field was measured as pixel intensity per square software (EMKA Technologies) and expressed as mean 6 SEM. Twenty- micrometer using ImageJ. For each condition, the data from 10 fields were four hours after stimulation, mice were euthanized and bronchoalveolar compiled (with at least 4–6 cells/field). lavages fluids (BALFs) were analyzed for cell composition and cytokine Endotoxic shock induced by administration of LPS quantification as described previously (22, 23). An aliquot was stained with trypan blue solution and analyzed to determine cellular content. After Mice of each genotype (Ms1Yah, Prmt2+/2, Prmt22/2, and control) re- centrifugation on microscopic slides, air-dried preparations were fixed and ceived an i.p. injection of 100 mg LPS (serotype O55B5; n = 10) or saline stained with Diff-Quick (Merz & Dade) using a May–Gru¨nwald–Giemsa control solution (n = 6) to evaluate the systemic inflammatory response coloration. Two hundred cells were counted twice for the determination of in vivo (17). After 90 min, mice were euthanized, the blood was collected the differential counts of each cell type in the BALF. Part of the lung was through the femoral vein and centrifuged at 2000 rpm for 15 min, and stored at 280˚C for the myeloperoxidase (MPO) assay. serum was collected and stored at 220˚C for cytokines assays. Measurement of cytokines and MPO assay Statistical analysis IL-6 and TNF-a concentration were estimated by ELISA test, in standard Statistical analysis was performed using either the parametric Fisher condition with the protocol of the supplier (R&D Systems). The sera were Student t test when applicable or the nonparametric Wilcoxon–Mann– diluted at one fourth and the supernatant of MF at half in PBS/BSA 1%. Whitney U test via the Statgraphics software (Centurion XV, Sigma plus, The 96-well plate was read by the reader EL 800 (BioTek Instruments). Levallois Perret). Values are presented as mean 6 SEM, and the significant For the MPO measurement, the right-heart ventricle was perfused with threshold was p , 0.05 or otherwise indicated. saline to flush the vascular content, and lungs were frozen at 280˚C until use. Lung was homogenized by polytron and centrifuged, and the super- natant was discarded. The pellets were resuspended in 1 ml PBS con- Results taining 0.5% hexadecyltrimethyl ammonium bromide and 5 mM EDTA. LPS-induced challenge of the respiratory function is altered in 2 2 2 After centrifugation, 50 ml supernatants were placed in test tubes with 200 Prmt2+/ and Prmt2 / mice ml PBS-hexadecyltrimethyl ammonium bromide EDTA, 2 ml HBSS, 100 ml o-dianisidine dihydrochloride (1.25 mg/ml), and 100 ml H2O2 0.05%. Using whole-body plethysmography and the PenH that reflects the After 15 min of incubation at 37˚C in an agitator, the reaction was stopped respiratory pattern, we previously demonstrated that Ms1Yah mice with 100 ml NaN3 1%. The MPO activity was determined as absorbance at carrying a single copy of Prmt2 have a reduced respiratory re- 460 nm against medium. sponse after LPS instillation (17). To investigate the precise role RNA extraction and quantitative RT-PCR analysis of Prmt2 in this phenotype, we used the loss-of-function allele of Prmt2 engineered previously (18), and we compared control Total RNA was extracted after LPS stimulation from the MF and from the +/2 2/2 lung of mice using the RNAeasyR mini-kit (Qiagen). The concentration of (B6), heterozygote (Prmt2 ), and homozygote (Prmt2 ) indi- RNA was measured by Nanodrop, and the quality was evaluated by the iduals instilled with LPS (10 mg) or saline control solution, and Agilent Bioanalyzer 2000 (Agilent). We kept all the samples that have an the experiment was carried out twice independently. As done 4828 Prmt2 DEPENDENCE OF LPS RESPONSES previously in the study of the Ms1Yah model (17), the airway To go further in the investigation of the inflammatory response response was monitored by whole-body plethysmography. The in the lung of Prmt2+/2 and Prmt22/2 mice, we measured the instillation of saline solution did not affect the respiratory function concentration of TNF-a and IL-6 in the BALFs 24 h after in- as assessed by Penh in wt and mutant mice (Fig. 1), whereas an flammatory induction. LPS significantly increased the concentra- intranasal administration of LPS induced a strong respiratory re- tion of the two proinflammatory cytokines in Prmt2+/2 and sponse characterized by an increase of the Penh values within 90– Prmt22/2 (Fig. 2C). Consequently, loss of function of one copy of 120 min, which lasted 3–4 h and decreased slowly, in the control Prmt2 is sufficient to deeply affect the respiratory and in- mice. Contrary to Ms1Yah mice, the Prmt2+/2 mice developed flammatory response toward LPS, highlighting a potential dual a delayed airway hyperresponse (AHR) compared with the control role for Prmt2. Prmt2 might certainly contribute to the early LPS- animal (from 80 to 145 min) with a higher level of the response induced responses but also mostly contributes later to inhibit the and no real restoration of the initial PenH level even after 6 h (Fig. TLR4/NF-kB pathway. 1A). This delayed response is comparable with the AHR observed Increased expression of TNF-a and IL-6 are correlated with in Prmt22/2 (Fig. 1B). The respiratory response induced by LPS a reduced number of copies of Prmt2 in lungs in the Prmt2+/2 mice showed that the absence of only one copy of Prmt2 is sufficient to modify the pulmonary function, and that the To better understand the role of Prmt2 in the NF-kB pathway, we LPS-induced response of the Ms1Yah mouse airways is controlled decided to study the impact of the partial and complete deletion by one or more additional gene(s) because these mice do not of Prmt2 on the expression of TNF-a and IL-6. present an AHR. Moreover, we demonstrated in this study a dual First, the expression of Prmt2 was evaluated by quantitative role of Prmt2 with a delayed response during the early phase and real-time PCR in total RNA isolated from lungs of B6, Ms1Yah, Downloaded from +/2 2/2 an increased amplitude and length of duration of the AHR. Prmt2 , and Prmt2 mice 90 min after LPS or NaCl ad- ministration. B6 mice stimulated with LPS were taken as the Partial and complete deletion of Prmt2 modifies the local reference condition, and data were normalized using the Genorm inflammatory response induced by LPS in lung method (25). Prmt2 is expressed with a basal level in control mice We previously determined that Ms1Yah mice instilled by LPS have and is further downregulated after LPS stimulation, as already

an enhanced recruitment of inflammatory cells and production of shown (17). In absence of LPS instillation, the expression of http://www.jimmunol.org/ TNF-a and IL-6 both in the lungs and in the bronchoalveolar space Prmt2 is reduced approximately by a factor 0.5 in Ms1Yah, in comparison with wt mice (17). Thus, we wanted to explore the strongly reduced in Prmt2+/2, and close to zero in Prmt22/2 (Fig. involvement of Prmt2 in the local inflammatory response. Twenty- 3A). Prmt2 appeared to be a dosage-sensitive gene, and the mutant four hours after the instillation of LPS or saline control solution, Prmt22/2, used in the study, completely abolished its expression. wt, Prmt2+/2, and Prmt22/2 mice were euthanized, and the in- Interestingly, the expression of Prmt2 is strongly decreased in flammation induced by LPS was followed by neutrophil re- Ms1Yah, Prmt2+/2, and Prmt22/2 individuals after LPS in- cruitment, MPO activity, and concentration of TNF-a and IL-6 in stillation. We conclude that the LPS pathway acts directly on BALFs. The mice instilled with the saline control solution did not Prmt2 transcription. Such an LPS-induced Prmt2 gene downreg- present any changes in the type of inflammatory cell recruitment. ulation is in agreement with the hypothesis that Prmt2 acts as an by guest on September 30, 2021 Similar to Ms1Yah mice (17), the Prmt2+/2 and Prmt22/2 mice inhibitor of the NF-kB pathway in the lung. showed an increase in the number of neutrophils when compared To check the impact of Prmt2 on the NF-kB pathway, we with control mice (Fig. 2A). Accordingly, the MPO activity, an studied the pattern of expression of TNF-a and IL-6, which are specifically expressed in neutrophil cells, is increased in direct targets of this pathway, in the lungs from B6, Prmt2+/2, and both mutant mice versus control (Fig. 2B). The data are in agree- Prmt22/2 mice. The saline control solution did not affect the ment with the increase in the number of neutrophils. expression of TNF-a or IL-6. The LPS induced an increase in the

FIGURE 1. LPS-induced respiration challenges in wt control, Prmt2+/2 (A), and Prmt22/2 mice (B). An intranasal administration of the control saline (NaCl) solution did not affect the Penh in wt (n = 2), Prmt2+/2 (n = 2), and Prmt22/2 mice (n = 2), whereas the re- sponse to LPS (10 mg) was delayed but strongly increased in Prmt2+/2 and Prmt22/2 animals (n =5/ genotype) compared with wt control (n = 5) treated in the same conditions. All results are expressed as mean + SEM. The Journal of Immunology 4829

FIGURE 2. Inflammatory responses in the lungs as measured by the recruitment of neutrophils (A), the MPO activity (B), and cytokine secretion in BALFs (C). NaCl did not affect any of the parameters. A, LPS-induced inflammation showed an increase in the recruitment of neutrophils in BALFs from Prmt2+/2 and Prmt22/2 (n = 5/genotype) in accordance with the increase of MPO (B), compared with control (wt). In response to LPS, Prmt2+/2 and Prmt22/2 mice showed an enhanced concentration of TNF-a and IL-6 compared with the one detected in control in- dividuals (C). All results are expressed as mean 6 SEM. Student t test, *p # 0.05, **p # 0.01. Downloaded from http://www.jimmunol.org/ expression of IL-6 and TNF-a in the lungs of Prmt2+/2 and Complete loss-of-function of Prmt2 is necessary for an Prmt22/2 mice in a manner that is inversely correlated to the enhanced inflammatory response in MF number of Prmt2 copies present in the mice (Fig. 3B). Altogether, TNF-a and IL-6 are primarily produced from the activated MF in these results support that Prmt2 acts on the expression of IL-6 and response to LPS. Therefore, we next asked whether the loss-of- a TNF- in a dose-dependent manner, certainly by interfering with function of Prmt2 in MF could influence directly the secretion of NF-kB activity. proinflammatory cytokines, such as TNF-a and IL-6, after LPS treatment. MF from Prmt2+/2, Prmt22/2, and control mice were

compared for their response toward LPS by measuring the pro- by guest on September 30, 2021 duction of TNF-a and IL-6 in vitro 24 h after stimulation by LPS or saline buffer (NaCl). We found that LPS-stimulated MF derived from Prmt22/2 mice produced significantly higher amounts of TNF-a and IL-6 than those derived from control animals (Fig. 4). The level of TNF-a and IL-6 secreted in the culture medium of LPS-stimulated Prmt22/2 MF was equivalent to that found in the medium of Ms1Yah-derived MF. Surprisingly, the amount of both cytokines in Prmt2+/2 MF was equivalent to that in B6-treated MF, sug- gesting that in Ms1Yah, additional genes should contribute to the Ms1Yah phenotypes.

The copy number of Prmt2 affects the expression level of IL-6 and TNF-a in MF We looked further at the expression of proinflammatory cyto- kines in MF 24 h after the LPS stimulation. As expected, control treatment (NaCl) did not induce expression of IL-6 and TNF-a genes in MF (Fig. 4). On the contrary, LPS induced a significant increase in the expression of the IL-6 and TNF-a genes in the Ms1Yah, Prmt2+/2, and Prmt22/2 mice, which is coherent with the increase in the concentration of the cytokines in the super- FIGURE 3. Comparison of Prmt2 (A), TNF-a, and IL-6 (B) expression natant of the MF. Nevertheless, clear differences are noted. In- +/2 2/2 in wt, Ms1Yah, Prmt2 , and Prmt2 animals. The analysis was carried deed, the expression of TNF-a is not affected when one copy of out by quantitative real-time PCR, without (NaCl) and with LPS induction. 2 Prmt2 is functional in heterozygotes, whereas similar level of Data are represented as mean (2 ddCt) 6 SEM and are representative of expression is achieved when there is a complete loss of function of n =5.Prmt2 is a dosage-sensitive gene, and its expression is decreased by LPS induction. This decrease showed that Prmt2 is involved in the in- Prmt2. On the contrary, the maximum level of expression of IL-6 flammation pathway because of LPS, and that the Prmt2 knockdown in LPS-induced MF is observed when only one copy of Prmt2 is is functional. The expressions of the cytokines were increased in both inactivated. The MF derived from Ms1Yah mice showed a stron- Prmt2+/2 and Prmt22/2 mice, suggesting a role of Prmt2 in the control of ger increase in the expression of IL6, similar to that observed in the expression of cytokines induced by LPS. Student t test, *p # 0.05. Prmt2 complete loss of function, confirming the hypothesis that 4830 Prmt2 DEPENDENCE OF LPS RESPONSES Downloaded from

FIGURE 4. Characterization of the inflammatory response induced by the LPS in the MF derived from B6, Ms1Yah, Prmt2+/2, and Prmt22/2 mice. A, The concentration of both cytokines in the culture supernatant is increased and similar in Ms1Yah and Prmt22/2 compared with B6, after stimulation with LPS, whereas the level of production of TNF-a and IL-6 in Prmt2+/2 are comparable with those in wt (n = 5 by group). B, Expression level of http://www.jimmunol.org/ TNF-a and IL-6 genes, in MF from B6, Ms1Yah Prmt2+/2,andPrmt22/2 mice after stimulation with LPS or NaCl (n = 5 by group). The LPS induces an increase in the expression of both cytokines in MF of Ms1Yah, Prmt2+/2, and Prmt22/2 mice. Nevertheless, the level of TNF-a in Prmt2 homozygote is similar to that of Ms1Yah, with little effect of the heterozygosity of Prmt2 FIGURE 5. PRMT2 affects nuclear accumulation of NF-kBinMF on TNF-a compared with B6. On the contrary, the removal of one functional treated with LPS. A, Overlay of NF-kB immunofluorescent detection 2/2 copy of Prmt2 in heterozygous mice is sufficient to obtain the maximum (green) and DAPI staining (blue) of wt and Prmt2 MF treated with level of expression of IL-6. Student t test, *p # 0.05, **p # 0.01. saline control (NaCl) solution or LPS for 4 h (original magnification 320). B, Quantification of the NF-kB signal (pixel intensity per mm2 from by guest on September 30, 2021 a the nucleus of labeled cells) showed that in control condition (NaCl), the another gene from the region deleted in Ms1Yah controls TNF- level of nuclear NF-kB is low and quite similar in both wt and Prmt22/2 expression, but not IL-6 (Fig. 4). MF. After LPS stimulation, NF-kB accumulated in the nucleus of control 2 2 MF and the inactivation of Prmt2 further increased the nuclear level of Accumulation of NF-kB in the nucleus of Prmt2 / MF after NF-kB. For each condition, ∼50 cells were measured and data are pre- LPS stimulation sented on a graph (wt in open bar and Prmt22/2 in black). Student t test, We found that the absence of Prmt2 induces an increase in ex- *p # 0.05. pression and production of TNF-a and IL-6. According to Ganesh et al. (20), we hypothesize that the loss of Prmt2 will lead to an increase of NF-kB accumulated into the nucleus of Prmt22/2 Ms1Yah mice (17). We supposed that the inhibitory role of Prmt2 cells, leading to a stronger and long-lasting NF-kB–dependent in the NF-kB signaling could explain this phenotype. To test transcription. To test this hypothesis, we stimulated MF from wt whether systemic LPS could lead to the same enhanced in- 2 2 and Prmt2 / mice with saline control solution or LPS for 4 h. flammatory response in Prmt2+/2 or in Prmt22/2 mice as in Immunofluorescence was performed on the cells, revealing an ac- Ms1Yah mice, we administered an i.p. injection of 100 mg LPS or 2 2 cumulation of NF-kB in nucleus of MF from both wt and Prmt2 / saline control solution in 10 mice of each genotype plus B6 mice mice (Fig. 5A). To determine the effect of Prmt2 on the nuclear as control. The proinflammatory response was assayed by mea- accumulation of NF-kB, the presence of the transcription factor was suring the concentration of cytokines and chemokines in the serum 2 2 evaluated in wt and Prmt2 / cells by fluorescence intensity 90 min after LPS stimulation. In particular, we selected the TNF-a (measured as pixel intensity per square micrometer). LPS induced and the IL-6, which are secreted during the inflammatory response 2 2 nuclear accumulation of NF-kB in wt and Prmt2 / MF, but the mediated through the innate immune system by the TLR4 and the quantity of fluorescent signal for the NF-kB transcription factor MyD88/NF-kB signaling pathway (22, 26). The Prmt22/2 mice 2 2 was more important in Prmt2 / cells (Fig. 5B).We thus confirmed had the same increase in the TNF-a concentration as Ms1Yah that Prmt2 inhibits nuclear export of NF-kBinMF as shown pre- mice, whereas sera of Prmt2+/2 mice presented the same con- viously in fibroblasts (20). The increase of NF-kB in the nucleus of centration of TNF-a as B6 treated mice (Fig. 6). The concentra- 2 2 Prmt2 / cells enlightens the increase of cytokine expression and tion of IL-6 is increased in Ms1Yah mice and in both Prmt2+/2 2 2 production in Prmt2 / lungs and MF compared with wt mice. and Prmt22/2 compared with control mice (Fig. 6). Haplo- insufficiency of Prmt2 is sufficient to induce a change in the Partial and complete deletion of Prmt2 modifies the systemic production of IL-6, whereas it has no impact on the release level inflammatory response induced by LPS of serum TNF-a, suggesting different levels of control mediated Systemic injection of LPS induced production of proinflamma- by Prmt2 for the regulation of the LPS response in vivo in dif- tory cytokines such as TNF-a and IL-6 as observed in the sera of ferent compartments. The Journal of Immunology 4831

Discussion (17). This observation led us to think that at least one gene of We report in this article a new role for the protein Prmt2 in the the Prmt2-Col6a1 region is responsible of the bronchoconstric- regulation of pulmonary inflammatory and airway distress syn- tion associated with the MyD88-dependent pathway. As opposed +/2 drome induced by LPS, but also in MF and after systemic stim- to mutants of the MyD88-dependent signaling, Prmt2 and 2/2 ulation. Prmt2 mice showed a delayed but enhanced AHR maintained in Aerogenic exposure to LPS induces pulmonary inflammation time compared with wt after LPS exposure. Prmt2 behaves as an in mice, characterized by recruitment and activation of MF and activator in the early phase of AHR, mainly as a consequence of its neutrophils in the airways, local TNF-a production, and direct own downregulation by LPS, and as an inhibitor during the regu- AHR (27). LPS first makes a complex with the LPS-binding lation of the response amplitude and length of the AHR. Recently, protein and the cell membrane CD14 coreceptor to activate the expression of the Prmt1-6 was studied in lungs and spleen from TLR4 (28). Then cell activation can occur via two pathways, both a rat model of asthma (41). Prmt1, Prmt2, Prmt3,andPrmt5 were leading to NF-kB activation (29–31). The first one is MyD88 overexpressed in lungs of all animals, whereas Prmt4 was decreased. dependent and need both the MyD88 and TIRAP adaptors. In the spleen, only Prmt2 and Prmt5 were overexpressed. This study MyD88 and TIRAP-deficient mice are both resistant to systemic suggests that PRMTs play an important role in the posttranslational LPS-induced shock (21, 32). Moreover, LPS-induced broncho- modification process of asthma-related genes that is reinforced by constriction, neutrophil recruitment, and TNF-a production in the our results demonstrating the involvement of Prmt2 in the AHR airways are abrogated in both lungs and MF of MyD88-and induced by LPS. Prmt2 is a new actor of the LPS/TLR4/MyD88- TIRAP-deficient mice (21, 33–35). However, LPS stimulation dependent induced bronchoconstriction. Nevertheless, at least an- still activates NF-kB in MyD88- and TIRAP-deficient cells, but other gene of the Prmt2-Col6a1 region is involved in the reduced Downloaded from with a delayed kinetics compared with wt cells (32), and preserve AHR observed in Ms1Yah mice. Finding such a gene is of interest their ability to induce IFN-inducible genes (36, 37). The MyD88- because it should also have the capacity of altering the increased independent pathway involves the TRIF and TRAM. TRIF- and AHR observed in Prmt2-deficient mice found in this report. TRAM-deficient MF were impaired in the LPS-induced inflam- Prmt2 has been shown to inhibit NF-kB–dependent transcrip- matory cytokine production but have normal NF-kB activation, tion by causing a nuclear accumulation of IkB-a that prevents suggesting that cooperation between both the MyD88-dependent DNA binding in a dose-dependent manner (20). Even if the exact http://www.jimmunol.org/ and -independent pathways is required for the TLR4-mediated mechanism is unclear, Prmt2 interacts with IkB-a by its ankyrin inflammatory cytokine production (38, 39). Whereas broncho- domain, which also mediates the interaction with NF-kB. The constriction is abrogated in MyD88-deficient mice, TRIF-deficient Prmt2/IkB-a complex still binds NF-kB molecules and prevents mice have normal bronchoconstriction in response to LPS expo- them from binding DNA (20). All these observations led us to sure. This observation indicates that bronchoconstriction induced hypothesize that Prmt2 loss of function can modify the in- by instillation of LPS is strictly dependent on the MyD88-depen- flammatory signaling mediated by NF-kB activation after LPS +/2 dent pathway (22, 40). Ms1Yah mice showed the same absence treatment. The analysis of the lung inflammation of the Prmt2 2/2 of bronchoconstriction as the MyD88- and TIRAP-deficient mice and Prmt2 mice after LPS stimulation showed an increase in the number of neutrophils, the MPO activity, and the concen- by guest on September 30, 2021 trations of both IL-6 and TNF-a. We then studied the expression of Prmt2, TNF-a, and IL-6 in lungs. In absence of LPS, the basal expression level of Prmt2 is more important in control lung and MF. Somehow, in absence of stimulus, the NF-kB factor is se- questered in the cytoplasm and the NF-kB–dependent transcrip- tion is “off”. Tam et al. (11) proposed a new model in which the maintenance of the p65/NF-kB subunit in the cytoplasm of unstimulated cells requires a continuous flow of nuclear protein. Accordingly, the NF-kB/IkB-a complexes are formed in the nu- cleus and then exported to the cytoplasm via the CRM1 chaperone protein. This leads to the accumulation of NF-kB/IkB-a in the cytoplasm, which cannot go back to the nucleus without degra- dation of IkB-a, which masks the nuclear localization signal of NF-kB. The expression level of Prmt2 in unstimulated conditions could facilitate the formation of NF-kB/IkB-a dimers. Indeed, Prmt2 promotes the nuclear accumulation of IkB-a (20). After LPS stimulation, expression of Prmt2 decreases, allowing the decrease of nuclear IkB-a, and thus the binding of NF-kBon DNA, leading to the expression of the target genes like IL-6 and TNF-a. This decrease is more important in Prmt2 mutants, almost to a null level, and can explain the increased expression level of TNF-a and IL-6 in lungs and MF. In parallel, we demonstrated that Prmt2-deficient MF showed an increased nuclear accumula- tion of NF-kB after LPS treatment. Overall, our results stressed the role of Prmt2 in controlling the NF-kB–dependent inflam- FIGURE 6. Characterization of the inflammatory response induced by systemic injection of LPS. The response was evaluated in the sera of wt, matory response and particularly the expression and secretion 2 a Ms1Yah, and Prmt2+/ mice (n = 10 for each group). The concentration of of both IL-6 and TNF- . Moreover, in lungs, there is a good TNF-a was significantly increased in Ms1Yah mice versus WT. The con- correlation between the number of copies of Prmt2 and expression centration of IL-6 is significantly increased in Ms1Yah but also in Prmt2+/2 of the two cytokines. Taken together, all these observations con- mice after LPS treatment. Student t test, **p # 0.01, ***p # 0.001. firm a role of Prmt2 as an inhibitor in the control of the NF-kB– 4832 Prmt2 DEPENDENCE OF LPS RESPONSES dependent inflammatory response in lungs, induced by the acti- can explain the enhanced AHR observed in Prmt2+/2 and Prmt22/2 vation of the TLR4 signaling. mice (56). The systemic response observed is different in Prmt2+/2 as In this report, we demonstrated that Prmt2 plays a direct role in expected. In fact, the loss of one copy of Prmt2 is sufficient to inflammation induced by LPS certainly through the control of the modify the systemic concentration of IL-6, but not TNF-a. This NF-kB pathway. Its sensitivity to gene dosage depends on the observation reinforces the involvement of Prmt2 in the inflam- compartments but is clearly observed in lungs and in MF. The mation signaling pathway and suggests the involvement of another staining of NF-kB in wt and Prmt22/2 MF (Fig. 5) clearly gene or a different mechanism to regulate TNF-a production. showed that Prmt2 allows a nuclear accumulation of NF-kB, After LPS stimulation, the activated MF is one of the major supporting the NF-kB–dependent transcription. Taking into ac- sources of TNF-a production. In MF, the loss of the two copies of count our data and those previously described (11, 20) led us to Prmt2 is necessary to find the same increase in TNF-a and IL-6 propose the following model for Prmt2 controlling NF-kB sig- expression and production as in Ms1Yah mice. In fact, expression naling. In unstimulated cells (Fig. 7), there is a constitutive pro- of the cytokines seems to be regulated by two different mecha- duction of IkB-a waiting for new synthesis of the NF-kB subunit nisms. One is dependent on Prmt2; indeed, in absence of the two p65. Prmt2 controls the nuclear accumulation of IkB-a.IkB- copies of Prmt2, we observed at the same time an increase of the a/p65 complexes are formed and are actively exported to the expression and the concentration of both cytokines, showing the nucleus through the CRM1 chaperon protein. After LPS treatment, same phenotype as the monosomic mice. In Prmt2+/2 MF, TNF-a Prmt2 is downregulated and the phosphorylated IkB-a is de- expression is not altered compared with WT, and no change in graded, which allows the translocation of NF-kB to the nucleus,

TNF-a concentration is observed in the supernatant, whereas where it binds the promoters of its target genes. This leads to the Downloaded from expression of Il-6 is upregulated in MF from Prmt2+/2 mice, inflammatory response. IkB-a is newly synthesized and goes to suggesting a different regulation between IL-6 and TNF-a ex- the nucleus to enter in competition with DNA to bind NF-kB. In pressions. Activation of TLR4 by LPS induces the expression of presence of LPS, we demonstrated that the expression of Prmt2 is IL-10 by MF to regulate excessive production of inflammatory decreased, but the produced quantity is sufficient to participate in cytokines (42). IL-10 has been shown to inhibit TNF-a at early (30 the regulation of the NF-kB transcription, allowing a return to

min) and late (120 min) time points, and IL-6 at 120 min (43), physiological conditions. We showed that in absence of Prmt2, the http://www.jimmunol.org/ which is consistent with the identification of these genes as pri- mary and secondary response genes (44). Because of their dif- ferent kinetic, IL-10 might act at different stages of their induction (42) and could explain the differences observed between TNF-a and IL-6 production by MF and during the systemic response. Although the expression of IL-6 is increased in Prmt2+/2 MF, the concentration of the IL-6 protein is lower than in WT MF, sug- gesting a posttranscriptional regulation. Prmt2 clearly interferes with different pathways. Indeed, it in- by guest on September 30, 2021 teracts with the retinoblastoma gene product, which is an impor- tant regulator of the transcription factor E2F (18, 45). Prmt2 can also directly regulate transcription factor activity like the estrogen receptor-a (45), Stat3 (46) and the androgen receptor (47). Moreover, Prmt2 directly modifies the structure of the chromatin, mediating an asymmetric methylation of histone H3(R8) after recruitment by b-catenin on its targeted genes, establishing a poised chromatin architecture necessary and sufficient to regulate expression of key organizer genes (48, 49). Thus, we could hy- pothesize an another mode of action of Prmt2, through the mod- ification of the chromatin structure, altering the long-term con- sequence of NF-kB–dependent transcription as suggested, but in opposition to the effect of the H3 lysine 4 methyltransferase, Seth 7/9, on NF-kB and inflammatory (50). LPS induces asymmetric dimethylarginine (ADMA) formation FIGURE 7. Model for Prmt2 regulating the NF-kB pathway. In unsti- in lung and MF (51–54). Increased ADMA level leads to both 2 mulated cells, NF-kB/IkB-a complexes are retained in the cytoplasm. decreased NO production and an increased O2 formation by Prmt2 participates in the accumulation of IkB-a in the nucleus. In absence uncoupling NO synthase (55, 56). Finally, high level of ADMA of stimulus, p65 complexes with NF-kB are actively exported to the cy- induced cell damage, AHR, and protein dysfunction. The type I toplasm by the CRM1 chaperon protein. After LPS stimulation leading to Prmts (1–4, 6) are directly involved in ADMA formation and the degradation of the IkB-a, Prmt2 is downregulated and the released p65 localize to bronchial epithelial cells, endothelial cells, and smooth translocates to the nucleus and binds to the promoters of target genes. The muscle cells. Prmt2 is a type I methyltransferase that participates NF-kB–dependent transcription induces expression of target genes such as in ADMA formation (51). Consequently, the loss of one or both TNF-a and IL-6, and the synthesis of IkB-a. Then IkB-a can go to the copies of Prmt2 should lead to a decrease in ADMA level, and nucleus to bind NF-kB. The formation of new NF-kB/IkB-a complexes modulates the NF-kB–dependent transcription and leads to basal physio- thus an increase in NO level. Accordingly, the concentration of logical conditions. In Prmt2 mutant, the inflammatory response is in- NO was increased in MF from Ms1Yah after LPS stimulation creased, supporting the fact that Prmt2 acts as an inhibitor of the NF-kB (17). We can reasonably suppose that the loss of functional copies transcription. Prmt2-dependent inhibition of NF-kB–dependent transcrip- of Prmt2 leads to a decrease in ADMA concentration in our tion is either due to a direct action of Prmt2 on the translocation of IkB-a +/2 2/2 Prmt2 and Prmt2 models versus control mice. This induces to the nucleus or to indirect action of Prmt2 on the chromatin by lysine an increase in NO with a reduced or no ADMA response, which modification. The Journal of Immunology 4833 inflammatory response is more important, supporting the fact that of human reveals haploinsufficient genes modulating the in- Prmt2 facilitates the formation of the IkB-a/NF-kB complex. In flammatory and airway responses. Hum. Mol. Genet. 16: 2040–2052. 18. Yoshimoto, T., M. Boehm, M. Olive, M. F. Crook, H. San, T. Langenickel, and lungs, Prmt2 exerts its effects in a dose-dependent manner. Prmt2 E. G. Nabel. 2006. The arginine methyltransferase PRMT2 binds RB and reg- is also involved in the systemic and MF responses, but there are ulates E2F function. Exp. Cell Res. 312: 2040–2053. other mechanisms such as posttranscriptional and/or translational 19. McBride, A. E., and P. A. Silver. 2001. State of the arg: protein methylation at arginine comes of age. Cell 106: 5–8. modifications. 20. Ganesh, L., T. Yoshimoto, N. C. Moorthy, W. Akahata, M. Boehm, E. G. Nabel, In conclusion, our results clearly demonstrate that Prmt2 is in- and G. J. Nabel. 2006. Protein methyltransferase 2 inhibits NF-kappaB function volved in the regulation of the inflammatory response induced and promotes apoptosis. Mol. Cell. Biol. 26: 3864–3874. 21. Yamamoto, M., K. Takeda, and S. Akira. 2004. TIR domain-containing adaptors by LPS, certainly via its inhibitory role of the NF-kB–dependent define the specificity of TLR signaling. Mol. Immunol. 40: 861–868. transcription. Given the importance of inflammation in a great 22. Noulin, N., V. F. Quesniaux, S. Schnyder-Candrian, B. Schnyder, I. Maillet, number of lung diseases, Prmt2 appeared as a new potential thera- T. Robert, B. B. Vargaftig, B. Ryffel, and I. Couillin. 2005. Both hemopoietic and resident cells are required for MyD88-dependent pulmonary inflammatory peutic target to modulate the inflammatory response. Furthermore, response to inhaled endotoxin. J. Immunol. 175: 6861–6869. it reinforces the role of the histone methyltransferase in contributing 23. Schnyder-Candrian, S., D. Togbe, I. Couillin, I. Mercier, F. Brombacher, to the regulation of NF-kB–dependent transcription (50). V. Quesniaux, F. Fossiez, B. Ryffel, and B. Schnyder. 2006. Interleukin-17 is a negative regulator of established allergic asthma. J. Exp. Med. 203: 2715–2725. 24. Fleige, S., and M. W. Pfaffl. 2006. RNA integrity and the effect on the real-time qRT-PCR performance. Mol. Aspects Med. 27: 126–139. Acknowledgments 25. Vandesompele, J., K. De Preter, F. Pattyn, B. Poppe, N. Van Roy, A. De Paepe, We thank members of the research group, the Institut de Ge´ne´tique Biologie and F. Speleman. 2002. Accurate normalization of real-time quantitative RT- Mole´culaire et Cellulaire laboratory, the Institut Clinique de la Souris, and PCR data by geometric averaging of multiple internal control genes. 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