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Mitogen-Activated Kinase Kinase 3 Potentiates Chlamydia HSP60-Induced Inflammatory Response through Distinct Activation of NF-κB This information is current as of September 23, 2021. Yanhua Kang, Fang Wang, Zhe Lu, Hangjie Ying, Hang Zhang, Wen Ding, Cuili Wang and Liyun Shi J Immunol published online 31 May 2013 http://www.jimmunol.org/content/early/2013/05/31/jimmun ol.1300481 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 31, 2013, doi:10.4049/jimmunol.1300481 The Journal of Immunology

Mitogen-Activated Protein Kinase Kinase 3 Potentiates Chlamydia HSP60-Induced Inflammatory Response through Distinct Activation of NF-kB

Yanhua Kang,*,1 Fang Wang,*,1 Zhe Lu,* Hangjie Ying,*,† Hang Zhang,* Wen Ding,* Cuili Wang,*,† and Liyun Shi*

Chlamydia pneumonia (C. pneumonia) remains one of the leading causes of bacterial pneumonia and has been implicated in the pathogenesis of some inflammation-related , such as asthma, chronic obstructive pulmonary , and vascular dis- eases. 60 is one of the pathogenic components of C. pneumonia that is closely associated with the inflammatory disorders. However, the molecular basis for the immunopathologic property of chlamydial heat shock protein (cHSP60) has not

been elucidated. In this article, we report that MAPK kinase 3 (MKK3) is essential for cHSP60-induced lung inflammation, Downloaded from because MKK3-knockout mice displayed significantly reduced lung neutrophil accumulation and decreased production of proin- flammatory mediators, correlating with the alleviated inflammatory response in lung tissues. Mechanistically, p38 kinase was selectively activated by MKK3 in response to cHSP60 and activated NF-kB by stimulating the nuclear kinase, mitogen- and - activated protein kinase 1. The specific knockdown of mitogen- and stress-activated protein kinase 1 in resulted in a defective phosphorylation of NF-kB/RelA at Ser276 but had no apparent effect on RelA translocation. Furthermore, TGF-b–

activated kinase 1 was found to relay the signal to MKK3 from TLR4, the major that sensed cHSP60 in the initiation of http://www.jimmunol.org/ the inflammatory response. Thus, we establish a critical role for MKK3 signaling in cHSP60 pathology and suggest a novel mechanism underlying C. pneumonia–associated inflammatory disorders. The Journal of Immunology, 2013, 191: 000–000.

hlamydia pneumoniae is an obligate intracellular bacte- ognized as a hallmark of chlamydial , and understanding rium that infects the respiratory tract of the host. This the mechanisms underlying the Chlamydia-associated inflamma- C bacterium causes airway infection, including bronchitis, tory response is of critical importance. pneumonia, sinusitis, and pharyngitis (1, 2). Persistent chlamydial In agreement with the fact that the majority of chlamydial re- infection is also associated with the development and exacerbation spiratory are asymptomatic or only mildly symptomatic, by guest on September 23, 2021 of asthma, chronic obstructive pulmonary disease, and vascular these generally exist in the host cells in an inert or non- diseases (3, 4). Despite its various manifestations, the pathogen- invasive form (7). The engagement of the bacteria with host cells, esis of Chlamydia seems to be largely ascribed to the immuno- likely through pathogen recognition receptors (PRRs) on the host pathological reaction, particularly the profound inflammatory cells, is assumed to be the premise for the induction of the im- response that it triggers. Essentially, the recurrence of chlamydial mune and inflammatory responses. However, recent studies sug- infection causes abundant production of proinflammatory cyto- gested that, in the case of intracellular bacterial, the generation of kines and chemokines, which promotes the influx of inflammatory proinflammatory chemoattractants or primarily occurred cells, damage to the host epithelium, scarring, and, ultimately, fibrosis in noninfected cells, indicating that the released microbial agent and scarring (5, 6). Therefore, the inflammatory response is rec- (s), and not the bacteria, initiate the inflammatory reaction (8–10). This finding prompted us to reconsider the mechanism underlying Chlamydia infection. Therefore, we sought to identify the key *Department of Basic Medical Science, Key Laboratory of Immunology and Molec- ular Medicine, School of Medicine, Hangzhou Normal University, Hangzhou, Zhejiang component in the infection with the potential to promote inflam- 310036, China; and †Department of Life Science and Technology, Hangzhou Normal matory signaling. Among the known virulence factors, chlamydial University, Hangzhou, Zhejiang 310036, China heat shock protein 60 (cHSP60) is believed to be critical for the 1Y.K. and F.W. contributed equally to this work. inflammatory pathology. Received for publication February 19, 2013. Accepted for publication April 22, 2013. cHSP60 is a conserved chlamydial Ag with ∼50% This work was supported by the National Key Scientific Research Project identity with heat shock protein 60 (11). This Ag can be (2012CB911200), National Natural Scientific Funds (81270066 and 30872262), abundantly synthesized from invaded host cells during persistent and funds from the Ministry of Education (201070, NCET-08-0927) and the Provin- cial and Municipal Government (2011R10027, LY12H1007, and 20100633B11). chlamydial infection and is released into the site of infection or the Address correspondence and reprint requests to Prof. Liyun Shi, Department of Basic circulation (12). In the respiratory system, cHSP60 was found to Medical Science, Key Laboratory of Immunology and Molecular Medicine, School be related to inflammatory diseases, such as asthma, chronic bron- of Medicine, Hangzhou Normal University, Hangzhou, Zhejiang 310036, China. chitis, and chronic obstructive pulmonary disease. cHSP60 Abs E-mail address: [email protected] were proven to correlate with atherosclerosis with a high predic- Abbreviations used in this article: BALF, bronchoalveolar lavage fluid; cHSP60, chlamydial heat shock protein 60; i.t., intratracheal(ly); MKK3, MAPK kinase 3; tive value (13). These observations clearly implicate cHSP60 in MPO, myeloperoxidase; MSK, mitogen- and stress-activated protein kinase; NS, Chlamydia-associated inflammatory disorders, although the caus- normal saline; PRR, pattern recognition receptor; siRNA, small interfering RNA; ative relationship is unknown. Functional studies indicated that TAK1, TGF-b–activated kinase 1; WT, wild type. cHSP60 is able to elicit the production of proinflammatory cytokines Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 and adhesive molecules in human endothelial and macrophages

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300481 2 MKK3 POTENTIATES THE cHSP60-INDUCED INFLAMMATORY RESPONSE

(14, 15). In addition, cHSP60 stimulated dendritic cells to up- translocation. Moreover, TGF-b–activated kinase 1 (TAK1), which regulate the phenotypic molecules and T cell–stimulatory capa- is downstream of TLR4, was found to initiate and deliver the sig- bility. The fact that pulmonary inflammation can be induced by naling cascade. Thus, we not only defined a critical role for the purified cHSP60 in mice further supported its proinflammatory MKK3/p38 pathway in the cHSP60-induced response, we also property (16). Moreover, a recent investigation indicated that revealed, for the first time to our knowledge, a novel action module Mycobacterium with a cpn60 was unable to induce the of the pathogenic factor that is likely integral to chlamydial in- inflammatory response and reduce the granulomatous response in flammatory diseases. an animal model, reinforcing the pathogenic role of this agent in infectious diseases (17). Despite these findings, the molecular basis Materials and Methods for the inflammation-promoting effects of cHSP60 have not been Mice and reagents elucidated. Previous studies indicated that cHSP60 engages with TLR4 MKK3-knockout mice were generated as previously described (32) and a maintained under specific pathogen–free conditions. All of the animal (and likely TLR2) to produce cytokines, including TNF- and experiments were performed in accordance with the National Institutes of IL-12p40, and the chemokines KC and MIP-2 in macrophages (16, Health Guide for the Care and Use of Laboratory Animals and with the 18, 19). The subordinate adaptor MyD88 is activated by cHSP60 approval and monitoring of the Animal Care and Use Committee of the and, in turn, activates NF-kB–dependent transcriptional activity School of Medicine at Hangzhou Normal University. All of the Abs, unless (20). However, the optimized activation of NF-kB involves the otherwise indicated, were obtained from Cell Signaling. The Ab for b- was obtained from Sigma-Aldrich. The pRL-TK plasmids were integration of the various signaling pathways triggered by a given obtained from Promega. The MAPK inhibitors UO126 (ERK1/2) and stimuli and is controlled by multiple steps, such as the selection SB203580 (p38) were purchased from Calbiochem. Polymyxin B1 was Downloaded from of genomic targets, binding kinetics, and the phosphorylation obtained from Sigma. k of RelA/NF- B and its cytoplasmic shuttling; thus, details of Preparation of recombinant cHSP60 the activation module of cHSP60 have to be explored (21). Addi- tionally, some studies indicated that MAPKs, a fundamental mech- The cHSP60 (1467608) was cloned from the genomic DNA of C. pneumoniae (ATCC 53592) into the expression vector pET-28a (Novagen) anism governing the cellular response to extracellular stimuli, with a hexahistidine tag attached to the C terminus. The recombinant are also involved in cHSP60 activity. MAPKs are proline-directed protein was overexpressed in Escherichia coli BL21 cells and induced by http://www.jimmunol.org/ /-specific protein kinases and include at least four isopropyl 1-thio-b-D-galactopyranoside. The recombinant protein was distinct classes of kinases: ERKs, JNK, p38 MAPK, and ERK5 purified by affinity chromatography using Ni-NTA-agarose (QIAGEN) and (22). In addition to the mitogenic actions of ERK induced by ion exchange with HiTrap Q ion exchange (33). To circumvent the effects of any possible endotoxin contamination in the recombinant protein, cHSP60 in human vascular cells, p38 in particular seems to play an cHSP60 was pretreated with Polymyxin B (20 mg/ml) or Endotoxin Re- essential role in chlamydial pathogenesis (15). It was shown that moval Resin (Pierce). Endotoxin levels in the cHSP60 protein remained p38 is potentially activated by Chlamydia or cHSP60 in human ,5 EU/ml, as measured by a Limulus amebocyte lysate kit (33). airway epithelial cells. The activation of p38, but not of ERK, c-Jun Animal experiments kinase/JNK, or PI3K, was required for the activation of NF-kB and the release of GM-CSF (23). A recent report indicated that the The mice were anesthetized i.p. with ketamine hydrochloride (100 mg/kg) by guest on September 23, 2021 m inhibition of p38 MAPK led to a 70–90% inhibition in IFN-b ex- and xylazine (10 mg/kg). A total volume of 50 l normal saline (NS) or cHSP60 (2 mg/g body weight) was instilled intratracheally (i.t.) (16, 34). pression induced by Chlamydia muridarum. This finding suggests a prominent role for p38 in chlamydial infection and Cell culture and generation of peritoneal macrophages (24). However, the mechanism driving p38 signaling upon cHSP60 The RAW264.7 cell lines were obtained from the American Type Culture stimulation and the in vivo relevance of this signaling, particularly Collection and grown in RPMI 1640 medium (Life Technologies) con- in the lung, have not been defined. Because mice with a genetic taining 10% (v/v) heat-inactivated FCS. To prepare murine peritoneal deletion of p38 are not available as a result of the associated em- macrophages, 8–20-wk-old mice were injected i.p. with 3% thioglycolate bryonic lethality (19–22), we sought to determine whether MAPK broth. After 72 h, the peritoneal cells were harvested, and the macrophages were enriched by quick adhesion. kinase 3 (MKK3), which is a presumed upstream kinase of p38, might be involved in cHSP60-associated inflammatory disorders. RNA isolation and quantitative RT-PCR MKK3 is one of the MAPK kinases that are hypothesized to Total RNA was isolated using TRIzol reagent (Invitrogen), following the phosphorylate p38 at the Thr-Gly-Tyr site. Previous studies dem- manufacturer’s protocol. SYBR Green PCR Master Mix (Bio-Rad) was used onstrated that MKK3 is rapidlyactivatedbyproinflammatory to detect mRNA levels, and relative expression levels were determined by ap- cytokines, such as TNF-a, IL-1b, and type I IFN, and is responsible plying the DDCt method using b-actin as the endogenous control. The fol- a 9 for full activation of the p38 pathway (25). The activation of p38 lowing primers were used in the tests: TNF- ,forward5-AAGGCCGGGG- TGTCCTGGAG-39 and reverse 59-AGGCCAGGTGGGGACAGCTC-39 and its subordinate kinase and the production of IL-8, IL-6, and and IL-6, forward 59-CCACTTCACAAGTCGGAGGCTTA-39 and reverse matrix metalloproteinase 3 are significantly impaired in MKK3- 59-AGTGCATCATCGTTGTTCATAC-39. deficient cells. Therefore, the pathway is critically involved in in- flammatory diseases, such as inflammatory arthritis and diabetic Bronchoalveolar lavage, cell differentiation, and histological nephropathy (26, 27). More importantly, MKK3 has been impli- analysis cated in the lung inflammation and injury caused by a broad range Briefly, the trachea was exposed through a midline incision and cannulated of innocuous insults, such as endotoxin, ventilator, oxidant, and with a sterile 22-gauge needle. Bronchoalveolar lavage fluid (BALF) was infectious agents (28–31). obtained by flushing three times with 1 ml 0.5 mmol/l EDTA/PBS. After centrifugation, the supernatants were stored at 280˚C until use. Total cell In this study, we used MKK3-knockout mice to show that numbers in BALF were counted with a hemocytometer, and differential MKK3-mediated signaling is essential for the inflammatory re- cell counts were determined on cytospin preparations with Diff-Quick sponse induced by cHSP60 in vivo or in vitro. p38 kinase was staining (IMEB) (34). Alternatively, neutrophils and macrophages in the selectively activated and further stimulated mitogen- and stress- BALF were assessed by immunostaining and subsequent flow cytometry k (35). For the histological analysis, mouse lung samples were washed activated protein kinase (MSK)1 kinase to activate RelA/NF- B thoroughly in PBS, fixed in 4% (w/v) formalin, and embedded in par- in a distinct manner, which was dependent on the phosphorylation affin. Then, 5-mM sections were stained with H&E using standard of RelA/p65 at Ser276, but not at Ser536, and independent of RelA procedures. The Journal of Immunology 3

Plasmid constructs, transfection, and luciferase reporter assays Determination of cytokines and myeloperoxidase levels For the IL-6 reporter plasmids, DNA sequences (2300 to +1) were amplified The levels of TNF-a, IL-6, KC, and MIP-2 were measured in the culture from RAW264.7 genomic DNA, and the products were inserted into the supernatants or BALF by ELISA (R&D Systems). Lung myeloperoxidase KpnI–HindIII sites of the pGL3-Basic vector (Promega). in the (MPO) levels were determined using mouse MPO ELISA (Hycult Bio- putative AP-1 (261 to 255), C/EBP (2161 to 2147), or NF-kB(273 to 264) tech), following the manufacturer’s instructions. binding sites in the mouse IL-6 promoter were generated using the Quik- Change Site-Directed Mutagenesis kit (Stratagene) (36). All of the con- Western blotting structs were confirmed by DNA . To test NF-kB–driven IL-6 transcriptional activity, the mouse IL-6 reporter plasmid was transfected in Western blots were performed following standard protocols. Total cell macrophages using Jet-ENDO transfection reagents (Polyplus). Twenty- lysates were prepared, and the protein concentration was determined by four hours later, the cells were stimulated with cHSP60 (10 mg/ml), the a BCA protein assay (Thermo Fisher Scientific). Cell extracts were then vehicle, or heated cHSP60, as indicated, collected, and then lysed for the subjected to SDS-PAGE, transferred onto a nitrocellulose membrane, and luciferase test. The luciferase activity was measured through dual-luciferase blotted as previously described. Targeted were visualized using an assays (Promega). ECL Western blotting kit (ECL Amersham Biosciences). Statistical analysis RNA interference All of the data, unless otherwise indicated, are presented as the mean 6 SD Small interfering RNA (siRNA) targeting mouse TAK1 or MSK1, as well as of independent experiments. The statistical significance of the differences the scramble siRNA, were synthesized by GIMA (Shanghai, China) (37). between two groups was analyzed with the Student t test. Differences with siRNA duplexes were transfected into macrophages using INTERFERin-HTS, a p value #0.05 were considered statistically significant. All of the cal- according to the standard protocol (Polyplus). The knockdown efficacy culations were performed using the Prism software program for Windows was determined by RT-PCR or Western blotting. Downloaded from (GraphPad Software). Immunofluorescence staining and confocal microscopy Results Wild type (WT) and MKK32/2 peritoneal macrophages were seeded Disruption of MKK3 prevents cHSP60-induced pulmonary onto slides at 30% confluence and stimulated with cHSP60 or vehicle inflammation for the indicated time. The cells were then collected, fixed with 100% methanol, washed, and permeabilized in 0.2% saponin. After blocking The correlation between high cHSP60 deposition and Chlamydia with 5% bovine serum, the cells were stained with primary rabbit anti- inflammatory disorders suggests that cHSP60 has an inflamma- http://www.jimmunol.org/ p65 overnight at 4˚C and then stained with goat anti-rabbit IgG conjugated tion-promoting effect (13, 14, 16, 39). To determine whether to Texas Red (Invitrogen). Finally, after the nuclei were labeled with DAPI MKK3 signaling is involved in cHSP60 activity in vivo, pulmonary (Invitrogen), the cells were mounted in VECTASHIELD and analyzed 2/2 using fluorescence confocal microscopy (LSM confocal microscope; Carl inflammation was induced in WT and MKK3 mice through i.t. Zeiss) (38). inoculation of purified cHSP60. The results showed that MKK3 by guest on September 23, 2021

FIGURE 1. MKK3 deficiency leads to an alleviated pulmonary inflammatory response induced by cHSP60. Age- and sex-matched WT and MKK32/2 mice (n = 5) were administered 50 ml of cHSP60 (2 mg/g body weight) or NS i.t. Six hours later, the animals were euthanized for histological and other functional analysis. (A) Representative H&E staining of lung tissues collected from WT and MKK32/2 mice (original magnification 3200). (B) Total cell and neutrophil counts in BALF. (C) MPO activity in the lung tissues. (D) Levels of IL-6, TNF-a, MIP-2, and KC in BALF from WT and MKK32/2 mice. All results are mean 6 SD. *p , 0.05, **p , 0.01 versus WT controls. 4 MKK3 POTENTIATES THE cHSP60-INDUCED INFLAMMATORY RESPONSE deficiency led to alleviated pulmonary inflammatory responses, as Because NF-kB is one of the principal transcription factors that evidenced by histological analysis (Fig. 1A). The numbers of total regulates proinflammatory , we tested cHSP60- cells and neutrophils in BALF were remarkably decreased in driven NF-kB activity using a luciferase reporter assay. The as- MKK32/2 mice compared with those in their WT littermates (Fig. say revealed that NF-kB–dependent promoter activation was 1B). The level of MPO in lung tissues, an indicator of neutrophil greatly induced by cHSP60; however, it was repressed by the loss activity, was diminished by MKK3 deficiency in mice (Fig. 1C). of MKK3, indicating that this factor is required for optimal NF-kB In addition, deletion of MKK3 downregulated the production of activation upon cHSP60 stimulation (Fig. 2D). Strikingly, phos- inflammatory cytokines and chemokines, including IL-6, TNF-a, phorylation of p65 at Ser276 was markedly inhibited, whereas no and MIP-2. This finding might explain, at least in part, the di- effect or even a slight increase in phospho-Ser536 was observed in minished recruitment of proinflammatory cells upon cHSP60 MKK32/2 macrophages compared with WT controls (Fig. 2E). challenge in MKK32/2 mice (Fig. 1D). These results indicate that Given that the release of RelA/NF-kB from its IkBa restraint MKK3 plays an essential role in the cHSP60-initiated inflamma- and the translocation of RelA into the nuclear compartment con- tory response. stitute the determinant step for the initiation of gene transcription, we then examined the effect of MKK3 on RelA translocation MKK3 mediates cHSP60-induced NF-kB activation in induced by cHSP60. As shown in Fig. 2F, a robust translocation macrophages of RelA into the nucleus was induced by cHSP60, peaking at We then examined the mechanism used by MKK3 to drive cHSP60 30 min postexposure. Unexpectedly, no apparent difference in activity in the macrophages. We first examined the activation of the nuclear abundance of RelA was observed between WT and 2 2 MKK3 by cHSP60 in RAW cells and found that a profound MKK3 / cells during the course of the reaction, suggesting that Downloaded from phosphorylation of MKK3 was markedly induced and sustained MKK3 had no effect on the RelA nuclear translocation. Taken for .2 h (Fig. 2A). To explore the relevance of MKK3 in the together, these results indicate a pivotal role for MKK3 in the cHSP60-initiated reaction, peritoneal macrophages from WT and cHSP60-initiated inflammatory response that is largely dependent MKK3-deficient mice were subjected to cHSP60 stimulation, and on a distinct activation of the NF-kB transcriptional program. the production of proinflammatory cytokines in these cells was p38 MAPK is critically involved in the cHSP60-induced response

compared. As shown in Fig. 2B and 2C, cHSP60-mediated in- http://www.jimmunol.org/ duction of IL-6 and TNF-a production was significantly attenu- It is known that MAPKs, particularly p38, are regulated by MKK3 ated in MKK3-deficient macrophages, at both the mRNA and and linked to the activation of NF-kB (22, 40). Therefore, we eval- protein levels. uated the involvement of MAPKs in MKK3-mediated cHSP60 by guest on September 23, 2021

FIGURE 2. MKK32/2 macrophages display a defective response to cHSP60. (A) Western blot analysis of p-MKK3 and total MMK3 in RAW cells treated with cHSP60 (10 mg/ml) for the indicated lengths of time. The expression of IL-6 and TNF-a at the mRNA level (B) and protein level (C)by cHSP60 stimulation was assessed by quantitative real-time PCR and ELISA, respectively, in WT and MKK32/2 peritoneal macrophages. NF-kB–driven promoter activity (D) and phosphorylation of p65 (Ser276 or Ser536)(E) in response to cHSP60 (or heated cHSP60 or LPS, as indicated). (F) Confocal analysis of RelA nuclear translocation upon stimulation with cHSP60. p65 immunoreactivity is shown in red, and the nuclei are stained with DAPI (blue). Scale bar, 5 mm. Data are mean 6 SD of three independent experiments. Representative results from three independent experiments are depicted in (E) and (F). *p , 0.05, **p , 0.01 versus WT or vehicle-treated controls. The Journal of Immunology 5 Downloaded from

FIGURE 3. p38 is selectively activated by MKK3. (A) The phosphor- ylation of p38 and ERK1/2 was analyzed in WT and MKK32/2 primary macrophages in response to cHSP60 treatment. The immunoblots are representative of three independent experiments. IL-6 and TNF-a ex- B C pression ( ) and IL-6 promoter activity ( ) induced by cHSP60 were de- http://www.jimmunol.org/ termined in RAW cells pretreated with DMSO (DM), the MEK inhibitor U0126 (U0; 10 mM), or the p38 inhibitor SB203580 (SB; 20 mM). Data are mean 6 SD of at least three independent experiments. *p , 0.05, **p , 0.01 versus vehicle-treated controls. FIGURE 4. MSK1 mediates NF-kB–dependent transcriptional activity. (A) Phosphorylation of MSK1 by cHSP60 was analyzed in RAW cells activity in vitro. We first examined the activation of MAPKs by pretreated or not with the p38 inhibitor SB203580 (SB; 20 mM). (B) Time- cHSP60 and found that ERK1/2 and p38 were strongly phos- dependent activation of MSK1 by cHSP60 was analyzed in macrophages phorylated in the primary macrophages. Interestingly, deletion of from WT and MKK32/2 mice. (C) The MSK1 level was tested by immu- MKK3 led to a specific inhibition of p38 phosphorylation and had noblotting in RAW cells transfected with MSK1-siRNA (siM) or non- by guest on September 23, 2021 a marginal effect on phospho-ERK1/2 (Fig. 3A). Consistently,, specific siRNA (siN), and cHSP60-induced p65 phosphorylation (Ser276) cHSP60-induced production of IL-6 and TNF-a was significantly was analyzed in siRNA-treated cells. cHSP60-induced NF-kB promoter reduced by the inhibitor for p38 but not by the ERK inhibitor (Fig. activity (D) and IL-6 and TNF-a expression (E) were analyzed in RAW 3B). This finding suggests a specific requirement of MKK3/p38 cells transfected with siM or siN. Data are mean 6 SD of at least three in the cHSP60-induced inflammatory response. To elucidate the independent experiments. The immunoblots are representative of three , , molecular mechanism by which p38 regulated the gene expression independent experiments. *p 0.05, **p 0.01 versus siN-treated controls. in this context, we focused our attention on the expression of IL-6, a classic proinflammatory effector molecule. The result showed that inhibition of p38 kinase significantly repressed cHSP60-induced data demonstrate that MSK1 is a critical component in the MKK3/ IL-6 promoter activity (Fig. 3C). Thus, we identified a critical role p38-signaling cascade that drives the NF-kB–dependent cHSP60 for p38 kinase, which acts downstream of MKK3, in the cHSP60- activity. initiated response. Predominant role for NF-kB in the MKK3-mediated MSK1 is essential for NF-kB activation upon cHSP60 inflammatory response stimulation As we demonstrated above, knockdown of MSK1 significantly p38 MAPK can regulate gene expression via direct activation of decreased NF-kB activity, which caused the reduced generation of transcription factors or by stimulation of nuclear kinases and other inflammatory mediators in response to cHSP60. Nevertheless, be- transcriptome components (41, 42). MSK1 is one of the substrates cause MSK1 is known to activate other transcription factors, such of p38 and was shown to activate NF-kB in response to various as AP-1 and C/EBPs, in addition to NF-kB, we constructed IL-6 stimuli (43, 44). We next examined whether MSK1 participated promoter reporter plasmids mutated at NF-kB, AP-1, and C/EBP in the cHSP60-triggered signaling pathway; we found that it was sites to dissect their respective contributions to cHSP60 activity rapidly phosphorylated in RAW cells and that this effect was almost (Fig. 5A). As shown in Fig. 5B, a promoter with mutations at the completely abrogated upon p38 inhibition (Fig. 4A). In agreement kB site exhibited a more profound downregulation of luciferase with the activation of p38 by MKK3 shown above, downregulation activity than did the other mutated constructs. This result indicates of MSK1 activation was observed in MKK32/2 macrophages (Fig. a predominant role for the NF-kB element in the cHSP60 response. 4B). Moreover, the specific interference of MSK1 led to diminished Accordingly, the NF-kB inhibitor pyrrolidine dithiocarbamate phosphorylation of p65/RelA (Ser276) and significantly lower NF- (PDTC) generated a defective expression of proinflammatory cy- kB–driven reporter activity upon cHSP60 stimulation (Fig. 4C, 4D). tokines in macrophages (Fig. 5C). The macrophages from RelA- Functionally, the expression of IL-6 and TNF-a by cHSP60 was deficient mice exhibited a diminished response to cHSP60 profoundly affected by MSK1 knockdown (Fig. 4E). Together, these (Fig. 5D). Therefore, we concluded that the transcriptional activity 6 MKK3 POTENTIATES THE cHSP60-INDUCED INFLAMMATORY RESPONSE Downloaded from

FIGURE 5. RelA/NF-kB drives cHSP60 activity. (A) Map of the IL-6 promoter indicating the elements present. (B) Luciferase activity of full- length or mutant mouse IL-6 promoter in response to cHSP60. The result was normalized and is presented as a percentage of maximal promoter FIGURE 6. TLR4 initiates cHSP60 activity in the murine macrophages. activity. (C) IL-6 and TNF-a expression levels induced by cHSP60 were mRNA-level (A) and protein-level (B) expression of IL-6 and TNF-a in- determined in RAW cells pretreated with the NF-kB inhibitor pyrrolidine duced by cHSP60 (10 mg/ml) were analyzed in WT, TLR42/2, TLR22/2, http://www.jimmunol.org/ dithiocarbamate (PDTC) or vehicle. (D) cHSP60-induced IL-6 and TNF-a and TLR2/42/2 macrophages. Data are mean 6 SD of three independent 2 2 production were analyzed in WT and RelA / macrophages. Data are experiments. cHSP60-induced phosphorylation of MKK3 (C), p38 and mean 6 SD of three independent experiments. *p , 0.05, **p , 0.01 ERK (D), and p65 (Ser276)(E) was detected in WT and TLR42/2 mac- versus vehicle-treated or WT controls. rophages. The immunoblots are representative of three independent experi- ments. *p , 0.05, **p , 0.01 versus WT controls. of RelA/NF-kB is essential for the MKK3-mediated proinflam- matory activity of cHSP60. family member (MAP3K7), has the potential to stimulate the downstream MAPKs, particularly p38 kinase. In addition, TAK1 TLR4 participates in the MKK3-promoted cHSP60-signaling was shown to activate the cytokine-induced NF-kBpathwayin by guest on September 23, 2021 pathway an IkBkinasea/b-dependent manner (45, 46). Thus, we tested The data from our study support the importance of MKK3/p38 whether TAK1 is involved in the cHSP60-induced signaling path- signaling in cHSP60 activity. However, we are still unaware of way. As shown in Fig. 7A, cHSP60 stimulation caused a sig- the upstream signaling pathways activated and the initial events in nificant activation of TAK1 in RAW cells. However, this effect the response. Some previous studies indicated that TLR4 senses was almost abrogated in TLR42/2 cells. To directly examine the cHSP60 and initiates the response, whereas other investigators function of TAK1 in cHSP60 activity, we specifically inhibited showed that TLR2 is responsible for the reaction (19, 39). Thus, we TAK1 expression in macrophages using RNA interference (siRNA). sought to evaluate the roles of these receptors in cHSP60 activity The results showed that cHSP60-induced phosphorylation of the using primary cells from TLR22/2 and TLR42/2 MKK3 and p38 kinases was remarkably inhibited by silencing mice. As shown in Fig. 6A and 6B, cHSP60-induced IL-6 and TNF-a of the TAK1 gene (Fig. 7B). Luciferase assays revealed that NF- production was markedly repressed in TLR42/2 macrophages kB transcription activity was severely impaired in the absence compared with WT cells. In contrast, TLR2 deficiency exerted of TAK1 and that this impairment was associated with the de- much lower inhibitory effects on the expression of these cytokines. fective expression of IL-6 and TNF-a upon cHSP60 stimulation Interestingly, cells deficient in both TLR2 and TLR4 exhibited (Fig. 7C–E). Thus, the data suggest that TAK1 might serve as a a more profound reduction in cHSP60 response compared with mediator that relays the signaling from the PRR to the downstream TLR42/2 cells, which suggests that there is a synergism between MKK3-signaling pathway. these two receptors during the response. More importantly, a re- 2 2 Alleviated pulmonary inflammation is induced in TLR4 / mice markable inhibition of MKK3 phosphorylation was induced by TLR4 deletion, and this inhibition was followed by selective im- To further confirm the in vivo relevance of the signaling pathways pairment of p38 activation (Fig. 6C, 6D). Congruent with this re- identified in this study, the pulmonary inflammatory responses 2/2 sult, cHSP60-induced RelA phosphorylation at Ser276,theend in TLR4 and WT mice caused by the i.t. administration of point of this pathway, was markedly inhibited in TLR42/2 mac- cHSP60 were compared. Consistent with the observation made by rophages (Fig. 6E). Thus, the engagement of TLR4 with cHSP60 Bulut et al. (16), the symptoms of lung inflammation were pro- 2/2 appears to be an early event that mediates the MKK3/p38-signal- foundlyrelievedinTLR4 mice compared with their WT ing cascade. counterparts, as evidenced by downregulation of the generation of proinflammatory cytokines, reduced neutrophil infiltration and TAK1 is responsible for the initiation of MKK3 signaling decreased MPO levels in the lung tissues, and the compromised Despite the essential role for TLR4 in the initiation of the inflammatory reaction observed through histological analysis cHSP60 reaction, the intermediate kinase required for MKK3 acti- (Fig. 8). The MKK32/2 and TLR42/2 phenotypes share many key vation has not been discovered. TAK1, a MAP kinase kinase kinase features, suggesting that TLR4 and downstream MKK3 signaling The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 7. TAK1 is involved in the cHSP60-induced reaction. (A)Phos- phorylation of TAK1 induced by cHSP60 was detected in WT and TLR42/2 macrophages. (B) TAK1 level was tested by immunoblotting in RAW cells transfected with TAK1-siRNA (siT) or nonspecific siRNA (siN). cHSP60- induced MKK3 and p38 phosphorylation was analyzed in siRNA-treated cells. cHSP60-induced IL-6 promoter activity (C) and expression of IL-6 and TNF-a (D, E) were analyzed in RAW cells transfected with siT or siN. FIGURE 8. Attenuated pulmonary inflammation induced by TLR4 de- Data are mean 6 SD of at least three independent experiments. The ficiency following cHSP60 challenge. Age- and sex-matched WT and 2 2 immunoblots are representative of three independent experiments. *p , TLR4 / mice (n = 5) were administered cHSP60 (2 mg/g body weight) or by guest on September 23, 2021 0.05, **p , 0.01 versus siN-treated controls. NS i.t. The animals were euthanized 6 h postadministration and subjected to the analysis. Neutrophil counts in BALF (A), MPO activity in the lungs (B), and IL-6 and TNF-a levels in BALF (C) were analyzed. Data are 6 D constitute the essential mechanistic machinery responsible for mean SD. ( ) Representative H&E staining of lung tissues collected from WT and TLR42/2 mice 6 h after treatment with cHSP60 or vehicle cHSP60-initiated pathological activity. (original magnification 3200). *p , 0.05, **p , 0.01 versus WT controls. Discussion It was suggested that many Chlamydia-associated diseases, either taminated bacterial components, most likely from E. coli LPS. localized or systemic, share a common pathology because of the Indeed, the strategy of using recombinant cHSP60 or other path- deposition of cHSP60. cHSP60 is highly expressed at the site of ogenic proteins has been widely adopted in functional studies (33, infection and/or the circulation, and this expression is concurrent 47, 48). In this study, to eliminate the trace endotoxin from the with the onset and development of Chlamydia inflammatory dis- preparation, cHSP60 was pretreated with LPS eliminator, Polymyxin eases. Despite the close link between cHSP60 and Chlamydia-as- B, throughout the experiments. To confirm that the recombinant sociated disorders, the action module of cHSP60 and the related protein, and not the contaminated LPS, caused the effects reported, molecular mechanism have not been elucidated. In this study, we we found that the rapid induction of inflammatory cytokines was identified that MKK3/p38 is a critical signaling pathway that reg- completely abrogated when cHSP60 was inactivated through a ulates the cHSP60-induced reaction in vivo and in vitro. MKK3 preheating treatment. Moreover, it was found that the Mycobac- is responsible for the activation of p38 and the subsequent MSK1 terium devoid of the cpn60 homolog failed to induce an inflam- activation, which, in turn, leads to NF-kB transcriptional activation matory response in mice, further confirming the inflammation- and inflammatory gene expression. Accordingly, MKK3-deficient promoting effect of cHSP60. Interestingly, a report showed that mice were unable to mount a pulmonary inflammatory response LPS-stimulated p38 phosphorylation, NF-kB activation, and IL-6 upon cHSP60 challenge. Furthermore, TAK1 was identified to production remained intact in MKK3-deficient fibroblast-like function downstream of TLR4 and to activate the MKK3/p38- synoviocytes, suggesting that action of LPS is different from that signaling pathway. Therefore, we revealed a previously unidenti- of cHSP60, at least in terms of MKK3 signaling (26). fied mechanism that is important in the context of C. pneumonia CHSP60 is one of the chlamydial major Ags involved in a vari- infection, because such a module generated by a specific bacterial ety of biological activities, such as cell proliferation, reactive virulence appears to contribute to a wide spectrum of diseases. oxygen species production, mitochondria stress, and the inflam- Our studies are based on in vivo and in vitro observations using matory and immune responses. Multiple key mediators participate a recombinant cHSP60 produced from E. coli.Thisraisesthe in cHSP60-induced signaling transduction, which exhibits pref- concern that the effects that we observed are derived from con- erential activation modules in distinct contexts. In this study, we 8 MKK3 POTENTIATES THE cHSP60-INDUCED INFLAMMATORY RESPONSE established the importance of MKK3 and downstream p38 sig- pathology. Thus, inhibition of MKK3 signaling may have poten- naling in the cHSP60-induced inflammatory response. This finding tial benefits for the treatment of such disorders. is consistent with the previous observation that p38 signaling is Another important finding from our study is the identification essential in Chlamydia pathology (15, 23, 24). Using a combina- of TAK1 as an essential component in the MKK3/p38-signaling tion of genetic-deletion and pharmacologic-blocking approaches, cascade induced by cHSP60. Using the specific gene interference, we defined a specific requirement for p38 in the activation of the we demonstrated that TAK1 is essential for the activation of nuclear kinase MSK1 and, thereby, NF-kB transcriptional activity. MKK3 and the downstream signaling molecules p38 and MSK1. p38 kinase is a stress-activated protein kinase with multivalent Macrophages with the TAK1 gene silenced exhibited repressed functions. This kinase can directly phosphorylate transcription MKK3/p38 activation and decreased NF-kB activation, correlating factors or activate several downstream protein kinases, such as with the downregulation of IL-6 and TNF-a production follow- MAPK-interacting kinases, MAPK-activated kinases, and MSKs ing cHSP60 exposure. Importantly, the activation of TAK1 and (22, 41, 49). Among these, MSK1 was shown to be activated by downstream MKK3 was profoundly impaired in cells deficient in proinflammatory or infectious stimuli; thus, it serves as a mediator TLR4, the putative sensor for the cHSP60 signal. This finding that delivers the upstream signal to the transcription machinery. In indicates a critical role for TAK1 in the relay of signaling from the the current study, we revealed that MSK1 was rapidly activated by PRR to the downstream-signaling pathways. In addition, TAK1 p38 kinase upon cHSP60 stimulation. Knockdown of MSK1 in was proven to directly activate IkB kinase complex through Lys63- macrophages resulted in defective phosphorylation of RelA/p65 at linked polyubiquitin, which causes IkBa phosphorylation and Ser276, but not at Ser536, which was associated with a significant results in NF-kB activation (46). Therefore, TAK1 might also downregulation of NF-kB–driven promoter activity. This result, participate in the MKK3-independent pathway that we suggested Downloaded from consistent with what we initially observed in MKK32/2 macro- above and might serve as the converging point of the two pathways phages, reflects a unique regulatory module of NF-kB activation underlying cHSP60 activity. Thus, further dissection of TAK1 in response to cHSP60. It seems that the potential of MKK3 to function in Chlamydia pathology might be warranted. Interestingly, mediate cHSP60 activity is not dependent on the translocation of an increasing amount of data suggests that TAK1 itself can be RelA or on the phosphorylation of RelA at Ser536, the two critical targeted directly by several pathogenic molecules, such as Heli-

steps in the classical activation of NF-kB. cobacter pylori CagA, HIV gp41, and T-lymphotropic virus-1 protein http://www.jimmunol.org/ NF-kB is generally composed of a heterodimer of p65 and p50 TAX (54–56). Through a physical interaction with or modification subunits, which are sequestered in the by the inhibitor by the pathogenic proteins, TAK1 is exploited by the respective IkBa. Upon stimulation, IkΒs become phosphorylated and de- microorganism to enhance NF-kB activation and expression of graded. This degradation results in the release of NF-kB and facili- the effector mediators, which are critically involved in the devel- tates its entry into the nucleus. Simultaneously, RelA/p65 becomes opment of infection and inflammation. Therefore, whether cHSP60 phosphorylated in a stimulus-dependent manner at multiple sites, secreted from the intracellular bacteria can directly induce NF-kB including Ser276, Ser529, and Ser536 (50, 51). In the current study, activation by targeting TAK1 is worthy of investigation. we provide compelling data suggesting that profound phosphor- Taken together, our findings establish a critical role for MKK3/ ylation of p65 at Ser276 caused by MKK3 signaling is indispens- p38 signaling in the cHSP60-induced inflammatory response and by guest on September 23, 2021 able for optimized NF-kB transcriptional activity and full acquisi- unveil a previously unknown activation module of this pathogenic tionofthecHSP60effect.Thisfindingwasevidencedbythe agent. Thus, this study may open new opportunities to protect remarkable downregulation of NF-kB–driven promoter activity and against chlamydial inflammatory diseases. effector cytokine expression in MKK3-deficient macrophages 2/2 and by the reduced susceptibility of MKK3 mice to cHSP60 Acknowledgments challenge. To support this conclusion, it was shown that Ser276 We thank Dr. Deborah A. Quinn and Dr. Hang Zhao (Massachusetts General phosphorylation of RelA constitutes the transcriptional machinery Hospital, Harvard Medical School, Boston, MA) for MKK3- and TLR- k by enhancing the binding of RelA to the Belements,whichpro- deficient mice and Dr. Eugene Y. Chin (Department of Surgery, Brown motes the phospho-acetylation of histones and the recruitment of University, Providence, RI) for the pGL3–NF-kB plasmid. We also thank the coactivator p300/CBP and PolII (52). Hongping Ying (Central Laboratory of Medical Research, Hangzhou Nor- Nevertheless, it should be noted that cHSP60 also induced the mal University) for histological analysis of lung tissues. phosphorylation of RelA at Ser536 and the cytoplasmic shuttling of RelA, as indicated in our results. Therefore, we suggest an Disclosures action model of cHSP60 in macrophages that includes at least The authors have no financial conflicts of interest. two pathways. The canonical pathway is mediated by the nuclear translocation of p65 and the phosphorylation of p65 at Ser536 upon cHSP60 stimulation. In contrast, MKK3, by activating the sub- References ordinate kinase p38 and MSK1, induced a distinct model of NF- 1. Grayston, J. T., C. C. Kuo, S. P. Wang, and J. Altman. 1986. A new Chlamydia psittaci strain, TWAR, isolated in acute respiratory tract infections. N. Engl. J. kB activation. Although the contribution of these two pathways in Med. 315: 161–168. cHSP60 activity has yet to be addressed, it is likely that these two 2. Hatch, T. 1998. Chlamydia: old ideas crushed, new mysteries bared. Science 282: 638–639. pathways act in concert to give rise to the full action of cHSP60. 3. Patel, K. K., E. Anderson, P. S. Salva, and W. C. Webley. 2012. The prevalence In support of this hypothesis, a recent study indicated that the and identity of Chlamydia-specific IgE in children with asthma and other chronic constitutive intestinal level of NF-kB was insufficient to trigger respiratory symptoms. Respir. Res. 13: 32. 4. Campbell, L. A., and C. C. Kuo. 2004. Chlamydia pneumoniae—an infectious destructive inflammation in a mice model of enteritis. Additional risk factor for atherosclerosis? Nat. Rev. Microbiol. 2: 23–32. activation of MAPK, in combination with the already active 5. He, X., S. Mekasha, N. Mavrogiorgos, K. A. Fitzgerald, E. Lien, and NF-kB, was necessary to induce significantly enhanced TNF-a R. R. Ingalls. 2010. Inflammation and fibrosis during Chlamydia pneumoniae infection is regulated by IL-1 and the NLRP3/ASC inflammasome. J. Immunol. production and led to widespread inflammation and tissue damage 184: 5743–5754. (53). The novel mechanistic configuration underlying cHSP60 ac- 6. Chen, S., K. Shimada, W. Zhang, G. Huang, T. R. Crother, and M. Arditi. 2010. IL-17A is proatherogenic in high-fat diet-induced and Chlamydia tivity that we provide in this article seems to be of particular in- pneumoniae infection-accelerated atherosclerosis in mice. J. Immunol. 185: terest, given the prevalence of cHSP60-associated inflammatory 5619–5627. The Journal of Immunology 9

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