Of Immune Responses Regulation Mannose Receptor Recognition To

The Journal of Immunology

Mycobacterium tuberculosis Activates Human Macrophage Peroxisome Proliferator-Activated Receptor g Linking Mannose Receptor Recognition to Regulation of Immune Responses

Murugesan V. S. Rajaram,* Michelle N. Brooks,*,† Jessica D. Morris,* Jordi B. Torrelles,*,‡ Abul K. Azad,*,‡ and Larry S. Schlesinger*,†,‡

Mycobacterium tuberculosis enhances its survival in macrophages by suppressing immune responses in part through its complex cell wall structures. Peroxisome proliferator-activated receptor g (PPARg), a nuclear receptor superfamily member, is a transcriptional factor that regulates inﬂammation and has high expression in alternatively activated alveolar macrophages and macrophage-derived foam cells, both cell types relevant to tuberculosis pathogenesis. In this study, we show that virulent M. tuberculosis and its cell wall mannose-capped lipoarabinomannan induce PPARg expression through a macrophage mannose receptor-dependent pathway. When activated, PPARg promotes IL-8 and cyclooxygenase 2 expression, a process modulated by

aPPARg agonist or antagonist. Upstream, MAPK-p38 mediates cytosolic phospholipase A2 activation, which is required for PPARg ligand production. The induced IL-8 response mediated by mannose-capped lipoarabinomannan and the mannose receptor is independent of TLR2 and NF-kB activation. In contrast, the attenuated Mycobacterium bovis bacillus Calmette-Gue´rin induces less PPARg and preferentially uses the NF-kB–mediated pathway to induce IL-8 production. Finally, PPARg knockdown in human macrophages enhances TNF production and controls the intracellular growth of M. tuberculosis. These data identify a new molecular pathway that links engagement of the mannose receptor, an important pattern recognition receptor for M. tuberculosis, with PPARg activation, which regulates the macrophage inﬂammatory response, thereby playing a role in tuberculosis pathogenesis. The Journal of Immunology, 2010, 185: 929–942.

uberculosis (TB) continues to be a worldwide public-health proinflammatory-mediated microbial killing (5). There is increasing threat with the emergence of multi- and extensively drug- evidence that these uniquely regulated resident macrophages play an by guest on October 1, 2021. Copyright 2010 Pageant Media Ltd. T resistant Mycobacterium tuberculosis strains (1). Inhaled important role in allowing for a host-adapted intracellular pathogen M. tuberculosis travels to the lung distal airways where it is phago- like M. tuberculosis to survive and replicate for an extended period cytosed by alveolar macrophages (AMs) via specific phagocytic prior to complete activation of protective innate and adaptive im- and pattern recognition receptors (PRRs) (2, 3). M. tuberculosis mune responses (6–9). We have recently termed this interval to infection proceeds with granuloma formation composed of multi- optimal macrophage activation the “switching time” within the al- ple cell types, including foamy macrophages with lipid bodies (4). veolar microenvironment (8). This concept raises questions as to Numerous TB animal studies have revealed an early phase of what the key molecular switches are in macrophages that regulate relatively unchecked growth of aerosolized M. tuberculosis within their immune response pathways (either pro- or anti-inflammatory) AMs. AMs are prototypic alternatively activated cells with in- to M. tuberculosis and pathogenic microbes in general. http://classic.jimmunol.org creased expression of a subset of PRRs that enhance microbial Peroxisome proliferator-activated receptors (PPARs), members clearance and a regulated inflammatory program with reduced of a lipid-activated nuclear receptor superfamily, have emerged as key regulators of cellular metabolism, proliferation, differentiation, and inflammation (10). However, their role in regulating *Center for Microbial Interface Biology, †Department of Microbiology, and ‡Division of Infectious Diseases, Department of Internal Medicine, The Ohio State University, Co- host immune responses to infectious agents is only now being lumbus, OH 43210 recognized. These receptors act as transcription factors, forming

Downloaded from Received for publication March 16, 2010. Accepted for publication May 12, 2010. heterodimers with the retinoid X receptor (RXR) and bind to This work was supported by the National Institutes of Health Grants AI059639 and specific PPAR response elements in the promoter region of the AI052458 (to L.S.S.). genes, which they regulate (11, 12). There are three types of Address correspondence and reprint requests to Dr. Larry S. Schlesinger, The Ohio human PPARs: PPARa,PPARg, and PPARb/d, and each type is State University, Biomedical Research Tower, 460 West 12th Avenue, Room 1004, Columbus, OH 43210. E-mail address: [email protected] the product of a different gene (13). Natural ligands include fatty 12,14 acids or fatty acid derivatives, whereas synthetic ligands include Abbreviations used in this paper: 15-d-PGJ2, 15-deoxy-D PGJ2; AA, arachidonic acid; AM, alveolar macrophage; BCG, bacillus Calmette-Gue´rin; cPLA2, cytosolic phospho- anti-diabetic thiazolidinediones, such as troglitazone (14, 15). lipase A2; EIA, enzyme immunoassay; LM, lipomannan; MAFP, methyl arachidonyl PPARg is highly expressed in adipose tissue and plays a crucial fluorophosphonate; ManLAM, mannose-capped lipoarabinomannan; MDM, monocyte- derived macrophage; MOI, multiplicity of infection; MR, mannose receptor; P–L, role in adipocyte differentiation and glucose metabolism, but it is phagosome–lysosome; PPAR, peroxisome proliferator-activated receptor; PRR, pattern also expressed in a variety of tissues and cell types including mac- recognition receptor; SIGN, specific intercellular adhesion molecule-3-grabbing noninte- rophages (16). PPARg serves as a negative regulator of macrophage grin; siRNA, small interfering RNA; TB, tuberculosis. activation; altering the expression of many inflammatory genes (17, Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 18), modulating macrophage differentiation and activation through

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000866 930 PPARg ALTERS THE MACROPHAGE RESPONSE TO M. tuberculosis

transrepression of the transcription factors NF-kB, AP-1, and STAT (Sigma-Aldrich), and glycerol (Fisher Scientific). Pam3Cys, SB 203580, and (19), and attenuating the respiratory burst (20). These attributes have UO126 were purchased from Calbiochem (Gibbstown, NJ). important implications for the control of M. tuberculosis infection. Abs Of additional relevance to M. tuberculosis pathogenesis, increased Abs specific for phospho-p38, total p38, phospho-cPLA2, total cPLA2, and PPARg expression is seen in IL-4/IL-13–mediated alternative PPARg were purchased from Cell Signaling Technology (Beverly, MA). activation of macrophages (5), in AMs (21), and in macrophage- b-Actin and anti-MR (CD 206-H300) Abs were purchased from Santa derived foam cells from atherosclerotic lesions (22). Cruz Biotechnology (Santa Cruz, CA). COX2 mAb was purchased from Two PRRs important for the recognition of M. tuberculosis by Cayman Chemical. Anti-CD206 mAb for confocal microscopy was pur- macrophages are TLRs and the mannose receptor (MR) (CD206) chased from BD Pharmingen (San Jose, CA). Accell PPARg small interfering RNA (siRNA) and scramble siRNA were purchased from Thermo (2, 3). TLRs are thought to protect against mycobacterial infection Scientific Dharmacon RNAi Technologies (Chicago, IL). The MR siRNA through induction of NF-kB and production of inflammatory and scramble siRNA were purchased from Qiagen (Valencia, CA). cytokines. The MR, in contrast, may promote infection because it Bacterial strains and culture is highly expressed on alternatively activated macrophages (5), and ligation of this receptor is associated with an anti-inflammatory Lyophilized M. tuberculosis H37Rv (ATCC 25618), M. bovis BCG (ATCC cytokine program and lack of activation of oxidative responses 35734), and M. smegmatis (ATCC 700084) were obtained from American Tissue Culture Collection (Manassas, VA), reconstituted, and used as de- (23, 24). In addition, mannose-capped lipoarabinomannan (Man- scribed previously (27). The concentration of bacteria (1–2 3 108 bacteria/ LAM) and higher-order phosphatidyl-myo-inositol mannosides, ml) and the degree of clumping (#10%) were determined by counting in major components of the M. tuberculosis cell wall, are MR ligands a Petroff-Hausser chamber. Bacteria prepared in this fashion are $90% during phagocytosis by human macrophages and MR-mediated viable by CFU assay. Killed M. tuberculosis was prepared by incubating bacteria with 4% paraformaldehyde for 30 min at room temperature, wash- entry leads to limited phagosome–lysosome (P–L) fusion (25, 26). ing extensively with PBS, and resuspending in RPMI 1640 plus 10 mM The expression and immune functions of PPARg in human mac- HEPES plus 0.4% human serum albumin for infection. rophages in response to M. tuberculosis infection and the link to Purification of ManLAM and lipomannan relevant PRRs have not been explored. In this study, we determined that infection with virulent M. tuberculosis or the addition of Man- ManLAM and lipomannan (LM) purification from M. tuberculosis H37Rv and LAM upregulates PPARg expression and concomitantly increases LM purification from Mycobacterium smegmatis were performed following our laboratory protocols as reported previously (28, 29). For quality control, IL-8 production, COX2 expression, and PGE2 production in macro- fractions containing pure H37Rv ManLAM, H37Rv LM, and M. smegmatis phages. Furthermore, we show that PPARg knockdown significantly LM were analyzed by electrophoresis using 15% SDS-polyacrylamine gel, reduces IL-8 release. We determined that MAPK-p38 and cytosolic followed by periodic acid-silver staining. In addition, sample purification was further confirmed by mass spectrometry (neutral sugar and fatty acid analy- phospholipase A2 (cPLA2), upstream mediators of PPARg, are in- 1 volved in IL-8 production. Surprisingly, we identified that MR liga- ses), H nuclear magnetic resonance, and endotoxin content determination (all samples tested had ,18 pg endotoxin/mg of sample). tion plays an important role in PPARg induction in macrophages. The M. tuberculosis- or ManLAM-induced PPARg-mediated IL-8 re- Isolation and culture of human monocyte-derived macrophages sponse was independent of NF-kB activation and TLR2 expression, Monocyte-derived macrophage (MDM) monolayers were prepared from suggesting that we have uncovered an MR-specific signaling path- healthy, purified protein derivative-negative human volunteers using an ap-

by guest on October 1, 2021. Copyright 2010 Pageant Media Ltd. way. In contrast, infection by the attenuated Mycobacterium bovis proved The Ohio State University Institutional Review Board protocol as bacillus Calmette-Gue´rin (BCG) induces less PPARg expression and described previously (30). Briefly, PBMCs were isolated from heparinized blood on a Ficoll cushion and then cultured in Teflon wells (Savillex, particularly PGE2 production, and IL-8 production in response to Minnetonka, MN) for 5 d in the presence of 20% autologous serum. The BCG was mediated by NF-kBratherthanbyPPARg. Finally, we wells were then placed on ice for 30 min, and the PBMCs were removed by determined that PPARg knockdown controls M. tuberculosis growth washing. MDMs in the cultured PBMCs were adhered to 12- or 24-well in macrophages with a concomitant increase in TNF production. tissue culture plates (Falcon; BD Biosciences Labware, Franklin Lakes, NJ) for 2–3 h at 37˚C/5% CO2 in 10% autologous serum. Lymphocytes were Thus, we have identified a role for PPARg as a key regulator of then washed away, and MDM monolayers were repleted with RPMI 1640 macrophage immune responses to M. tuberculosis and its major containing 10% autologous serum and incubated overnight. For ManLAM cell wall lipoglycan and have linked its activation to a signaling and LM stimulation experiments or infection with M. tuberculosis H37Rv, http://classic.jimmunol.org pathway initiated by ligation of the MR, an important PRR in the BCG or M. smegmatis, the MDM monolayers in tissue culture plates were phagocytosis and trafficking of M. tuberculosis. These studies sug- washed with warm RPMI 1640 and repleted with 1 ml RPMI 1640 plus 10 mM HEPES plus 0.4% human serum albumin, and 5 mg/ml ManLAM, 5 gest that PPARg functions as one important “molecular switch” in mg/ml LM, or mycobacteria at the indicated multiplicity of infection (MOI) regulating macrophage immune responses to M. tuberculosis and was subsequently added. The conditions for MDM transfection experiments potentially other microbial pathogens. In upregulating PPARg activ- and confocal microscopy studies are detailed below. ity, M. tuberculosis further drives the alternative activation state of Mycobacterial infection of macrophages, macrophage lysis, Downloaded from AMs to enhance its intracellular survival. and Western blotting Materials and Methods Following the addition of mycobacteria, MDM monolayers in tissue culture Reagents plates were placed on a platform shaker for 30 min at 37˚C/5% CO2 for effective dispersion of bacteria and then left stationary for an additional 90 2+ 2+ Dulbecco’s PBS with and without Ca and Mg ,RPMI1640withL- min (30). The cells were then washed three times with warm RPMI 1640 to glutamine, and HEPES buffer were purchased from Invitrogen Life Technol- remove extracellular bacteria and incubated in 2% autologous serum in ogies (Carlsbad, CA). Human serum albumin was purchased from CSL RPMI 1640 for different time periods. The infected and uninfected MDMs Behring (Kankakee, IL). BSAwas purchased from Sigma-Aldrich (St. Louis, as well as ManLAM-stimulated MDMs were rinsed with warm PBS and MO) and heat-inactivated Hyclone Standard FBS from Thermo Fisher lysed in TN1 buffer (50 mM Tris [pH 8.0], 10 mM EDTA, 10 mM Scientific (Waltham, MA). PPARg antagonist GW-9662, ligand 15-deoxy- Na4P2O7, 10 mM NaF, 1% Triton X-100, 125 mM NaCl, 10 mM Na3VO4, 12,14 D -PGJ2 (15-d-PGJ2), PGE2 enzyme immunoassay (EIA) kit– and 10 mg/ml each of aprotinin and leupeptin), incubated on ice for 10 monoclonal, and the cPLA2 inhibitor methyl arachidonyl fluorophos- min, and then centrifuged at 17,949 3 g at 4˚C to pellet the cell debris. phonate (MAFP) were purchased from Cayman Chemical (Ann Arbor, Protein concentrations of the cleared cell lysates were measured using the MI). Middlebrook 7H9 broth was purchased from BD Biosciences (Mountain Pierce BCA-protein assay kit (Thermo Scientific, Rockford, IL). Cell View, CA). 7H11 agar plates were prepared with Middlebrook 7H11 lysates were separated by SDS-PAGE under reduced and denaturing con- agar (BD Biosciences) to which was added oleic acid (Fisher Scientific, ditions and analyzed by Western blot by probing with the Ab of interest Pittsburgh, PA), BSA (Sigma-Aldrich), dextrose (Sigma-Aldrich), catalase and development using ECL (Amersham Biosciences, Pittsburg, PA). The Journal of Immunology 931

Band densitometry was measured using Scion image software (Scion, from each independent experiment varied among the donors; however, the Frederick, MD). To quantify the band in each sample, we first subtracted the pattern of experimental results was the same from donor to donor. To ac- background, normalized the signal to the amount of b-actin in the lysate, count for this variability, we normalized the data to an internal control in and plotted the values as band intensities. each experiment. A ratio of experimental results to control was obtained, and the mean ratio was then tested for a significant difference from one using Transfection of MDMs t statistics. Statistical significance was defined as p , 0.05. MDMs were transfected with scramble siRNA, PPARg siRNA (target sequence 59-GAUUGAAGCUUAUCUAUGA-39) (200 nM), MR siRNA Results (target sequence 59-GUGGUACGCAGAUUGCACGTT-39) (400 nM) and M. tuberculosis H37Rv infection or ManLAM stimulation TLR2 siRNA (sense sequence 59-CUGGUAGUUGUGGGUUGAAGCdTdT- upregulates PPARg expression in human macrophages 39, and antisense sequence 59-GCUUCAACCCACAACUACCAGdTdT-39) (200 nM) using the Amaxa Nucleofector (Amaxa Biosystems, Gaithersburg, Human AMs are reported to have high levels of PPARg expression 7 MD).Briefly, day 5 PBMCs(1 310 ) were resuspended in 100 ml nucleofector compared with other tissue macrophages (3, 19, 21). We began our solution, followed by the addition of scramble siRNA or target-specific siRNA, studies by determining the level of PPARg expression using an incubated at room temperature for 5 min, and nucleofected according to the manufacturer’s instructions. PBMCs were then seeded on 12-well tissue established human MDM model (30). As shown in Fig. 1A and 1B, culture plates (except for confocal microscopy studies below) with 1.0 ml the basal expression level of PPARg was low as measured in cell RPMI 1640 supplemented with 10% autologous serum and incubated for 2 h lysates by Western blot analysis. To assess whether M. tuberculosis at 37˚C/5% CO2. After 2 h, adhered transfected cells (MDMs) were washed and ManLAM induce PPARg expression, MDMs were infected and repleted with warm RPMI 1640 containing 20% autologous serum for 16 h (PPARg) or 48 h (MR and TLR2). Transfected cells were used for subsequent with live virulent M. tuberculosis H37Rv or stimulated with Man- experiments, such as CFU and cytokine assays, Western blots, and confocal LAM from M. tuberculosis H37Rv, and PPARg expression was microscopy. The transfection efficiency was analyzed by transfecting a GFP- assessed. We found that PPARg expression was significantly in- expressing plasmid pmaxGFP into day 5 PBMCs, and the number of GFP creased in M. tuberculosis-infected MDMs for up to 72 h (Fig. 1A). expressing cells was counted (200 consecutive cells in triplicatewells) by using A similar response was observed in ManLAM-stimulated MDMs inverted fluorescence microscopy. The transfection efficiency was .95%. (Fig. 1B), although the response followed a shorter time course. To Cytokine and PGE2 assays determine whether the viability of bacteria was required for PPARg MDMs (3 3 105) in monolayer culture were incubated with M. tubercu- expression, MDMs were incubated with killed M. tuberculosis for losis H37Rv or BCG or exposed to ManLAM for different time periods. different time points. Our results indicate that PPARg expression is Cytokines (TNF, IL-6, IL-12p40, IL-8, and IL-10) in cell supernatants enhanced under this condition; however, the time course is shorter were analyzed by ELISA (R&D Systems, Minneapolis, MN). For PGE2 than that seen with live bacteria, more closely resembling the time measurements, MDMs (3 3 105) in monolayer culture were incubated with course seen with ManLAM treatment (Fig. 1C). To examine the M. tuberculosis H37Rv or BCG for different time periods. The amount of PGE2 present in the cell supernatants was measured according to the specificity of the response, we next examined PPARg expression in manufacturer’s instruction by using a PGE2 EIA kit. MDMs incubated with the avirulent M. smegmatis at different Macrophage colocalization studies of PPARg and the nucleus MOIs (5:1 and 25:1) and observed that M. smegmatis did not upreg- by confocal microscopy ulate PPARg expression (Fig. 1D). We extended our studies on PPARg expression using LM, another major mycobacterial cell MDMs were transfected with scramble siRNA, PPARg siRNA, or MR wall component (Fig. 1E). LM from M. tuberculosis H R and siRNA by the Amaxa nucleofector system and plated onto Chromerge- 37 v by guest on October 1, 2021. Copyright 2010 Pageant Media Ltd. cleaned glass coverslips in 24-well tissue culture plates for 2 h at 37˚C (1 M. smegmatis failed to induce PPARg expression. Finally, we ver- 3 105 MDM/coverslip). The MDM monolayers were washed with warm ified that human AMs have a higher basal level of PPARg expres- RPMI 1640 and repleted with RPMI 1640 containing 20% autologous sion and that M. tuberculosis infection or ManLAM stimulation serum, incubated at 37˚C for an additional 16 h, and left untreated or further increased this level (Fig. 1F). Taken together, these results treated with ManLAM (5 mg/ml) for different time periods. MDM monolayers on coverslips were washed with PBS, fixed with 2% paraformalde- provide evidence that M. tuberculosis infection but not infection hyde, and permeabilized (cells were not permeabilized for MR surface with the avirulent M. smegmatis upregulates PPARg expression in expression experiments) with 100% methanol for 5 min at room temper- human macrophages and that ManLAM is one contributing cell ature (31). The cells were blocked overnight at 4˚C in blocking buffer wall component to this response. (Dulbecco’s PBS plus 5 mg/ml BSA plus 10% heat-inactivated FBS), in-

http://classic.jimmunol.org cubated with Abs against PPARg and CD206, followed by incubation with M. tuberculosis H37Rv infection or ManLAM stimulation the secondary Abs conjugated with rabbit Alexa Fluor 594 and Mouse enhances IL-8 production and COX2 expression in human Alexa Fluor 488, respectively. For isotype control, the permeablized or macrophages nonpermeablized MDMs were incubated with rabbit IgG or mouse IgG. MDM nuclei were labeled with 0.1 mg/ml of the DNA stain DAPI (Mo- We next investigated whether the M. tuberculosis-orManLAM- lecular Probes, Carlsbad, CA) in PBS for 5 min at room temperature. After mediated induction of PPARg leads to altered downstream immune extensive washing in blocking buffer, the coverslips were dried and functions in human macrophages. Activated PPARg heterodimerizes mounted on glass slides. PPARg and MR protein expression levels and

Downloaded from the colocalization of PPARg with the nucleus were examined by confocal with RXR and binds to PPAR response element in the promoter microscopy (LSM 510; Carl Zeiss MicroImaging, Thornwood, NY). region of IL-8 and COX2 genes to enhance their expression (33). MDMs were incubated with M. tuberculosis H R for different time M. tuberculosis intracellular growth assays in macrophages 37 v periods, and cell culture supernatants were analyzed for Intracellular growth assays using CFU were performed as described pre- IL-8 secretion by ELISA, and cell lysates were analyzed for COX2 viously (32). Briefly, scramble siRNA- or PPARg siRNA-transfected expression by Western blot. Our results demonstrate that M. tuber- MDM monolayers were incubated with M. tuberculosis H37Rv (MOI 1:1, triplicate samples in each test group) for 2 h (30 min on a platform shaker culosis infection induces IL-8 release (Fig. 2A) and COX2 expression and 90 min stationary as noted earlier). The MDM monolayers were then (Fig. 2C). Similarly, ManLAM-stimulated MDMs exhibited in- washed three times with warm RPMI 1640 and either lysed or repleted creased IL-8 production (Fig. 2B) and COX2 expression (Fig. 2D). with RPMI 1640 containing 2% human autologous serum and incubated Interestingly, MDMs and the monocytic cell line THP-1 stimulated for 24 h. The supernatants and MDM monolayers were then plated, the number of colonies counted after 4 wk, and CFUs were enumerated. with ManLAM did not induce other proinflammatory cytokines, such as TNF, IL-6, and IL-12p40 (data not shown). Thus, these results Statistics suggested that the observed PPARg upregulation is specifically in- For M. tuberculosis- or ManLAM-mediated cytokine responses and PPARg volved in IL-8 production and COX2 expression in M. tuberculosis- and COX2 expression analyses in MDMs, the magnitude of the response infected or ManLAM-stimulated macrophages. 932 PPARg ALTERS THE MACROPHAGE RESPONSE TO M. tuberculosis

FIGURE 1. M. tuberculosis H37Rv and its cell wall component ManLAM upregulate PPARg expression in human macrophages. MDM monolayers were incubated with M. tuberculosis H37Rv (MOI 5:1) (A) for 2 h; cells were washed and incubated in 2% autologous serum for different times (6, 24, 48, and 72 h) and ManLAM (5 mg/ml) (B) or killed M. tuberculosis H37Rv (MOI 5:1) (C) for different times (3, 6, 9, and 24 h) following 2 h and washed. MDM monolayers by guest on October 1, 2021. Copyright 2010 Pageant Media Ltd. were incubated with M. tuberculosis H37Rv (MOI 5:1) or M. smegmatis (MOI 5:1; 25:1) (D) or with M. tuberculosis LM (5 mg/ml) or M. smegmatis LM (5 mg/ ml) (E) for different time points (6 and 24 h). F, Human AMs were incubated with or without M. tuberculosis H37Rv (MOI 5:1) for 24 h or ManLAM (5 mg/ml) for 6 h. Cell lysates were analyzed for PPARg expression by Western blotting using PPARg Ab and for b-actin as a loading control. Shown are representative blots from three independent experiments (three donors), except C and F, which are representative blots from two independent experiments (two donors).

PPARg knockdown in human macrophages inhibits ManLAM- by its translocation and colocalization within the nucleus. In con- mediated IL-8 release trast, ManLAM-stimulated PPARg siRNA-transfected cells To confirm the direct involvement of PPARg in the ManLAM- showed a marked decrease in PPARg expression (Fig. 3C, lower http://classic.jimmunol.org mediated IL-8 response from macrophages, we used siRNA tar- panel). There was no staining of PPARg following incubation with geted to PPARg to knock down its expression in MDMs and sub- the isotype control Ab (data not shown). Taken together, these data sequently stimulated the cells with ManLAM. Cell culture demonstrate that ManLAM-induced PPARg expression is involved supernatants were used to measure IL-8 production. In control in the induction of IL-8 in human macrophages. scramble siRNA-transfected MDMs, IL-8 production in response to ManLAM was increased; in contrast, there was no increase in Modulation of PPARg activity alters IL-8 production in human macrophages Downloaded from IL-8 production from PPARg siRNA-transfected cells compared with unstimulated cells (Fig. 3A). This result provided strong evi- To further validate the involvement of PPARg in IL-8 production dence that PPARg is an important regulator of ManLAM-mediated in human macrophages, we treated MDMs with the PPARg an- IL-8 production in MDMs. In parallel, cell lysates were analyzed tagonist GW9662 (34) and subsequently stimulated them with for PPARg expression. Results in Fig. 3B show that PPARg siRNA ManLAM. Cell culture supernatants were analyzed for transfection nearly abolished PPARg expression. Simultaneously, IL-8 production. Our results show that treatment with the PPARg we assessed the expression and colocalization of PPARg within the antagonist significantly decreased IL-8 production (Fig. 3D). We nucleus by confocal microscopy. Scramble or PPARg siRNA- next tested whether PPARg activity was further enhanced by its transfected MDM monolayers were stimulated with or without natural ligand 15-d-PGJ2. MDMs were treated with exogenous ManLAM. The cells were fixed, permeabilized, and stained with 15-d-PGJ2 and then with ManLAM. PPARg ligand increased an isotype control or PPARg Ab and DAPI. Untreated control cells IL-8 production following ManLAM stimulation compared with showed very low levels of PPARg expression (Fig. 3C, upper ManLAM stimulation alone (Fig. 3E). MDMs treated with 15-d- panel). Consistent with our Western blot results, PPARg expression PGJ2 alone without ManLAM stimulation did not increase was dramatically increased by ManLAM stimulation following IL-8 production when compared with resting cells (data not scramble siRNA transfection (Fig. 3C, middle panel) as indicated shown). Thus, consistent with our previous PPARg knockdown The Journal of Immunology 933

FIGURE 2. M. tuberculosis H37Rv infection or ManLAM stimulation enhances IL-8 production and COX2 expression in human macrophages. The cell culture media and cell lysates of MDMs incubated with M. tuberculosis H37Rv or stimulated with ManLAM were analyzed for IL-8 production by ELISA and COX2 expression by Western blot, respectively. The time points were the same as in Fig. 1. The media of uninfected macrophages showed undetectable levels of IL-8. In contrast, the media of M. tuberculosis-infected cells (A) or ManLAM-stimulated cells (B) showed signiﬁcantly increased levels of IL-8. Shown are cumulative results of three independent experiments performed in triplicate in A and ﬁve independent experiments performed in triplicate in B

(mean 6 SEM). pp , 0.0001. Cell lysates from M. tuberculosis-infected cells showed increased COX2 expression relative to uninfected cells (C). MDMs stimulated with ManLAM showed increased expression of COX2; the expression peaked at 6 h and was sustained until 9 h (D). The Western blots shown are representative of three independent experiments. Immunostaining for b-actin was used as a loading control.

results, IL-8 production is regulated by PPARg levels in ManLAM- siRNA-transfected cells were stimulated with ManLAM for differ- stimulated human macrophages. ent time periods, and cell culture supernatants were analyzed for IL-8 production. MR knockdown in MDMs significantly reduced The macrophage MR regulates PPARg expression in response IL-8 production (Fig. 5A), providing evidence for the MR in regu- to ManLAM lating PPARg expression and downstream IL-8 production in mac- Engagement of the MR by ManLAM through its mannose caps rophages. In parallel, we examined whether MR knockdown by guest on October 1, 2021. Copyright 2010 Pageant Media Ltd. during phagocytosis directs M. tuberculosis to its initial phag- influences activity of another PRR that has been associated with osomal niche in human macrophages by limiting P–L fusion (25, the MR (i.e., TLR2) (36). MR siRNA-transfected macrophages 35). Because ManLAM preferentially binds to the MR, we hy- were stimulated with the TLR2 ligand Pam3Cys for 24 h, and the pothesized that the MR plays a role in increasing PPARg ex- cell culture supernatants were analyzed for TNF production. The pression in macrophages following ManLAM stimulation. To test results in Fig. 5B show that MR knockdown does not influence this hypothesis, MDMs were transfected with scramble siRNA or TLR2 function. MR siRNA and then stimulated with ManLAM. Cell lysates were Recent reports have demonstrated that coexpression of the MR analyzed for PPARg expression by Western blot analysis. The and TLR2 is required for Pnemocystis carinii-mediated IL-8 release results demonstrate that MR knockdown in MDMs significantly (36). Thus, we asked whether TLR2 is required for the ManLAM- http://classic.jimmunol.org reduces PPARg expression (Fig. 4A). In parallel, the same cell mediated IL-8 response. MDMs were transfected with scramble lysates were analyzed for MR protein expression to determine the siRNA or TLR2 siRNA and subsequently stimulated with Man- efficiency of siRNA-mediated knockdown. Transfection of MR LAM. We found no change in IL-8 production between control siRNA in MDMs decreased the MR protein level ∼75% when and TLR2 knockdown macrophages (Fig. 5C). In parallel, TLR2 compared with scramble siRNA-transfected cells (Fig. 4B). The knockdown was confirmed by Western blot analysis and by stimu- results were further confirmed by confocal microscopy. MR lating the transfected cells with Pam3Cys and measuring TNF pro- Downloaded from siRNA-transfected MDM monolayers were stimulated with Man- duction. The results show that TLR2 knockdown significantly LAM, fixed and immunostained for PPARg. The results in Fig. 4C reduces TNF production and TLR2 protein (Fig. 5D). Taken to- show that MR knockdown suppresses PPARg expression and gether, these results provide evidence that TLR2 is not involved colocalization with the nucleus. In addition, both surface and in- in IL-8 production in MDMs stimulated with ManLAM. tracellular levels of MR were markedly reduced in MR siRNA M. tuberculosis infection or ManLAM stimulation activates transfected macrophages (Fig. 4D). The confocal microscopy MAPK-p38 and cPLA in human macrophages results were consistent with Western blot results and provide ev- 2 idence that the MR is involved in ManLAM-mediated upregula- M. tuberculosis infection or stimulation with different cell wall tion of PPARg expression in human macrophages. components activates MAPKs, especially p38 (37). MAPK-p38 catalyzes the activation of cPLA2 by phosphorylating the serine ManLAM-mediated IL-8 response in human macrophages is 505 and 727 residues (38). Activation of cPLA2 then catalyzes the dependent on the MR but not on TLR2 release of arachidonic acids (AAs) from membrane phosholipids To further link involvement of the MR with increased PPARg ex- (33, 39), which are hydrolyzed and further processed by lipo- pression in response to ManLAM, we measured the IL-8 response oxygenase and cyclooxygenases (COX1 and COX2) to generate from MR knockdown macrophages. Scramble siRNA- or MR leukotrienes and PGH2, respectively (40). PG synthase converts 934 PPARg ALTERS THE MACROPHAGE RESPONSE TO M. tuberculosis

FIGURE 3. PPARg knockdown and modulation of activity alter ManLAM-mediated IL-8 release. MDMs were transfected with scramble siRNA (control) or PPARg siRNA by nucleofection. After 16 h, cells were stimulated with ManLAM (5 mg/ml) for 6 and 24 h. Supernatants and cell lysates

by guest on October 1, 2021. Copyright 2010 Pageant Media Ltd. were analyzed for IL-8 production by ELISA (A) and PPARg expression by Western blot (B). The results in A are the mean 6 SD from a representative experiment performed in triplicate (n = 3) and in B from the same experiment (representative of blots that were performed in each experiment with immunostaining for b-actin as a loading control). In parallel experiments, scramble siRNA- or PPARg siRNA-transfected MDMs on coverslips were stimulated with or without ManLAM (5 mg/ml) for 6 h. The MDMs were ﬁxed with paraformaldehyde, permeabilized, stained with anti-PPARg Ab (red) and DAPI (blue) for nuclear localization, and examined by confocal microscopy (magniﬁcation 3630) (C). The upper panel shows untreated cells, the middle panel shows scramble siRNA-transfected ManLAM-stimulated cells, and the lower panel shows PPARg siRNA-transfected ManLAM-stimulated cells (from two independent experiments). Scale (white line) represents 20 mm. MDMs were pretreated with the PPARg antagonist, GW9662 (100 nM) (D),

the PPARg ligand 15-d-PGJ2 (PGJ2)(2mM) (E), or DMSO for 30 min and subsequently stimulated with ManLAM (5 mg/ml). Supernatants were collected and analyzed for IL-8 production by ELISA. The results in D and E are the mean 6 SD from a representative experiment performed in triplicate (n = 3). pp , 0.0398; ppp , 0.0025; and pppp , 0.0001 for the control versus inhibitor group. http://classic.jimmunol.org

PGH2 to PGD2, which is further converted to15-d-PGJ2, which significantly reduces IL-8 production in response to ManLAM serves as a ligand for PPARg. Therefore, we examined whether (Fig. 7A). The Western blot results confirmed that SB-203580 M. tuberculosis infection or ManLAM stimulation of MDMs efficiently suppresses the phosphorylation of p38 in response to leads to activation of MAPK-p38 and cPLA2. Our results show ManLAM (Fig. 7B). Inhibition of p38 activation also reduced

Downloaded from that M. tuberculosis infection or ManLAM stimulation enhances activation of the downstream signaling molecule cPLA2 in re- the phosphorylation of MAPK-p38 and cPLA2 (Fig. 6). Thus, sponse to ManLAM (Fig. 7C). In contrast to the results with M. tuberculosis or ManLAM stimulation leads to the activation MAPK-p38, we found that ManLAM stimulation does not activate of MAPK-p38 and cPLA2 which is critical for AA cleavage. MAPK-Erk and also that inhibition of MAPK-Erk by the pharmacological inhibitor UO-126 (2.5 mM) does not affect the activation ManLAM activation of MAPK-p38 regulates the PPARg- of cPLA2 in response to ManLAM (data not shown).Thus, our mediated IL-8 response through the activation of cPLA2 in results indicate that MAPK-p38 is directly involved in the regu- human macrophages lation of cPLA2 activation in this pathway. We next hypothesized that MAPK-p38 activation and p38- To determine more directly whether cPLA2 is critical for the mediated cPLA2 phosphorylation were involved in the observed ManLAM-mediated IL-8 response, we examined the effect of PPARg-mediated IL-8 response. To test this, MDMs were pre- a cPLA2 inhibitor on IL-8 production. MDMs were pretreated treated with the MAPK-p38 inhibitor SB-203580 (5 mM) and with 2.5 nM MAFP or vehicle control and stimulated with Man- subsequently stimulated with ManLAM. IL-8 was measured in LAM, and culture supernatants were analyzed for IL-8 production. cell culture supernatants, and cell lysates were analyzed for Our results conﬁrmed that inhibition of cPLA2 signiﬁcantly re- MAPK-p38 activation. We found that inhibition of MAPK-p38 duced the production of IL-8 (Fig. 7D). Taken together, these The Journal of Immunology 935

FIGURE 4. The macrophage MR regulates PPARg expression in response to ManLAM. MDMs were transfected with MR siRNA or scramble siRNA by nucleofection and plated in RPMI 1640 containing 20% autologous serum. After 48 h, the cells were washed and stimulated with ManLAM (5 mg/ml) for 6, 9, and 24 h. Cell lysates were examined for PPARg and b-actin expression (A) by Western blot analysis. B, MR knockdown was confirmed by Western blot using a MR Ab. The Western blots shown are representative of greather than or equal to three independent experiments. In parallel, MR siRNA or scramble siRNA-transfected MDMs were treated with ManLAM for 6 h. The cells were fixed, permeabilized, stained with anti-PPARg Ab (red) and DAPI (blue) for nuclear localization, and then examined by confocal microscopy (C; n = 2) (magnification 3400). MR expression in MR siRNA-transfected cells was by guest on October 1, 2021. Copyright 2010 Pageant Media Ltd. analyzed by confocal microscopy (D; n = 2) (magnification 3400). Both permeabilized (I and III) and nonpermeabilized (II and IV) cells (scramble siRNA or MR siRNA) were stained with anti-MR Ab (green) and DAPI (blue) for the nucleus. Scale (white line) represents 20 mm.

results provide evidence that activation of MAPKp38 and cPLA2 downstream products of PG biosynthesis and an indicator of natural is required for the PPARg-mediated IL-8 response in macro- PPARg ligand biosynthesis from PGD2 in MDMs (42). The results phages. This effect is likely due to the cPLA2-mediated release show that M. tuberculosis H37Rv infection signiﬁcantly increases of AAs and generation of the PPARg ligand 15-d-PGJ2 during PGE2 production relative to BCG infection (Fig. 8C). In the experi- ManLAM stimulation (also see Fig. 3E). ments that directly compared M. tuberculosis- and BCG-induced http://classic.jimmunol.org production of IL-8 and PGE by MDMs, the levels of M. tubercu- M. bovis BCG infection of macrophages induces IL-8 2 losis and BCG cell association were equivalent as assessed by production despite low expression of PPARg ﬂuorescence microscopy (data not shown). Taken together, these On the basis of a recent report that the attenuated M. bovis BCG results provide evidence that although BCG infection leads to the induces PPARg expression in murine macrophages (41), we di- induction of PPARg to a more limited extent, it is not involved in rectly compared MDMs infected with virulent M. tuberculosis the production of IL-8. Thus, BCG may activate an alternative

Downloaded from H37Rv or the attenuated M. bovis BCG for PPARg expression by pathway for induced IL-8 production. Western blot analysis of cell lysates. The results indicate that BCG infection leads to a more limited expression proﬁle of PPARg than NF-kB mediates IL-8 production following BCG infection of M. tuberculosis (Fig. 8A). Although PPARg expression levels were human macrophages, whereas PPARg mediates IL-8 similar between M. tuberculosis and BCG at the 6-h time point, production following M. tuberculosis H37Rv infection or PPARg expression in M. tuberculosis-infected MDMs was greater ManLAM stimulation at 24, 48, and 72 h. We next determined whether the difference in Following exposure to pathogens and endotoxins, NF-kB activation PPARg expression observed between M. tuberculosis- and BCG- in macrophages induces proinﬂammatory cytokine production infected MDMs led to a difference in IL-8 production. Unexpect- including IL-8. To delineate the roles of NF-kB and PPARg in edly, there was no difference in IL-8 production between the two mediating IL-8 production following infection of macrophages with mycobacterial species (Fig. 8B). This result suggested that BCG M. tuberculosis or BCG, MDMs were pretreated with the NF-kB activates other signaling pathways for IL-8 production, which are inhibitor SN-50 (75 mg/ml) and subsequently infected with M. tu- not activated or may be inhibited by M. tuberculosis. To further berculosis H37Rv or BCG or stimulated with ManLAM. As a positive assess the role of PPARg during M. tuberculosis and BCG infec- control, MDMs were stimulated with LPS (100 ng/ml). Cell culture tion, we elected to measure the production of PGE2, one of the key supernatants were analyzed for IL-8 and TNF secretion. The results 936 PPARg ALTERS THE MACROPHAGE RESPONSE TO M. tuberculosis

FIGURE 5. The MR modulates the macrophage IL-8 response to ManLAM without involvement of TLR2. MDMs were transfected with scramble siRNA, MR siRNA, or TLR2 siRNA as described in Fig. 4. A, ManLAM-stimulated IL-8 production was analyzed in MDMs transfected with MR siRNA or

scramble siRNA by ELISA. B, MR siRNA-transfected MDMs were stimulated with the TLR2 ligand Pam3Cys (100 ng/ml) for 24 h, and culture supernatants were analyzed for TNF production by ELISA. C, TLR2 siRNA or scramble siRNA-transfected MDMs were stimulated with ManLAM for 24 h. The cell culture supernatants were analyzed for IL-8 production by ELISA. The dotted line represents the level of IL-8 production in resting cells. For

a positive control (D), the TLR2-mediated cytokine response was analyzed by stimulating transfected cells with Pam3Cys (100 ng/ml) and measuring the by guest on October 1, 2021. Copyright 2010 Pageant Media Ltd. amount of TNF produced by ELISA. TLR2 knockdown was conﬁrmed by Western blot using a TLR2 Ab. The Western blots shown are representative of more than or equal to three independent experiments. Results in A–D are representative experiments (mean 6 SD of triplicate samples in each test group) of three independent experiments for each condition. pp , 0.0003.

show that IL-8 secretion was significantly decreased (∼5-fold) in 9A). In contrast, inhibition of NF-kB significantly reduced TNF SN-50–treated BCG-infected cells at 6 h, whereas in M.tb-infected production in both M. tuberculosis- and BCG-infected MDMs cells, the NF-kB inhibitor did not influence IL-8 production (Fig. (Fig. 9B). Thus, these results indicate that PPARg rather than NF-kB http://classic.jimmunol.org

FIGURE 6. M. tuberculosis infection or ManLAM

stimulation activates MAPK-p38 and cPLA2 in human

Downloaded from macrophages. MDMs were infected with M. tuberculo-

sis H37Rv (MOI 5:1) for 2 h (A) or left uninfected. Cells were washed and incubated in 2% autologous serum for different times (6, 24, 48, and 72 h). B, MDMs were stimulated with ManLAM (5 mg/ml) for 3, 6, 9, and 24 h. Cell lysates from M. tuberculosis-infected or Man- LAM-stimulated cells were analyzed for the activation

of MAPK-p38 (A, B) or cPLA2 (C, D) by Western blot using anti–phospho-speciﬁc Abs and reprobed for total

p38 and total cPLA2, respectively. The Western blots shown are representative blots of three independent experiments. The Journal of Immunology 937

FIGURE 7. ManLAM activation of MAPK-p38 regulates the PPARg-mediated IL-8 response through activation of cPLA2 in macrophages. MDMs were pretreated with the p38 inhibitor SB-203580 (5 mM) or DMSO for 30 min and subsequently stimulated with ManLAM (5 mg/ml) or left unstimulated for 3, 6, 9, and 24 h. Cell culture supernatants were analyzed for IL-8 production by ELISA (A); a representative experiment is shown. Mean 6 SD of triplicate samples. n =

3. pp , 0.013; ppp , 0.0003. Cell lysates were used to analyze the activation of p38 (B) and cPLA2 (C) by Western blotting using phospho-speciﬁc Abs and

by guest on October 1, 2021. Copyright 2010 Pageant Media Ltd. reprobed for total p38 and cPLA2 or actin. The Western blots shown are representative of three independent experiments. D, MDMs were pretreated with the cPLA2 inhibitor MAFP (2.5 nM) for 30 min and subsequently stimulated with ManLAM (5 mg/ml) for the indicated times. Cell culture supernatants were analyzed for IL-8 production by ELISA; a representative experiment is shown. Mean 6 SD of triplicate samples. n =3.pp , 0.0024; ppp , 0.0003.

is the major transcription factor for inducing IL-8 production during (Fig. 10A, cumulative data showed a 35 6 4% decrease, mean 6 M. tuberculosis but not BCG infection, despite BCG being able to SEM, n =7;p , 0.0001). Cell culture supernatants from these induce low levels of PPARg. Instead, BCG uses NF-kBas survival experiments contained significantly increased amounts of a transcription factor for IL-8 production. In addition, the NF-kB TNF in infected PPARg siRNA-transfected MDMs (Fig. 10B). inhibitor did not affect the production of IL-8 during ManLAM Thus, these data provide evidence that M. tuberculosis-induced http://classic.jimmunol.org stimulation (Fig. 9C). ManLAM stimulation also did not induce PPARg is an important mediator in controlling M. tuberculosis the production of other NF-kB–mediated proinflammatory growth in human macrophages at least in part through transcrip- cytokines, such as TNF, IL-6, and IL 12-p40 (data not shown). tional regulation of inflammatory cytokines. PPARg knockdown in human macrophages controls Discussion M. tuberculosis H37Rv infection Modulation of the host immune response is an essential component of

Downloaded from PPARg has been shown to be a marker of alternative activation in mycobacterial pathogenesis. AMs play a critical role in clearing in- macrophages, and several reports indicate that PPARg activation haled particles and pathogens and function to inhibit the amplification efficiently transrepresses NF-kB activity within the nucleus that of signaling, which leads to a vigorous proinflammatory response (5). blocks the expression of many proinflammatory cytokines, such These prototypic alternatively activated cells are endowed with as TNF and IL-6, as well as oxidant generation. These inflamma- unique immune properties and are increasingly recognized as playing tory mediators play a crucial role in pathogen clearance. In addi- an important role in TB pathogenesis. We are interested in identi- tion, there is increasing recognition of the importance of fying the key molecular signaling pathways, intracellular “switches,” alternatively activated macrophages in the survival of M. tubercu- and inflammatory mediators that regulate the biology of macro- losis (6–9). Therefore, we sought to determine whether PPARg phages in general and in response to M. tuberculosis in particular. knockdown in MDMs alters the control of M. tuberculosis H37Rv PPARg is a prime candidate for an intracellular molecular switch intracellular survival along with the production of proinflammatory based on its central role in controlling macrophage inflammatory cytokines. MDMs were transfected with scramble or PPARg responses (43) and lipid metabolism, including foam cell generation siRNA and subsequently infected with M. tuberculosis. The results (44). AMs express high levels of PPARg (19), and elevated PPARg in show that M. tuberculosis intracellular growth is significantly de- human macrophages is one of the biological markers of IL-4/IL-13– creased in PPARg knockdown MDMs compared with control cells mediated alternative activation. Foamy appearing macrophages are 938 PPARg ALTERS THE MACROPHAGE RESPONSE TO M. tuberculosis

FIGURE 8. M. bovis BCG infection induces limited PPARg expression in macrophages. A,MDMs were infected with M. tuberculosis

H37Rv or BCG at an MOI of 5:1. After 2 h, the cells were washed and incubated in 2% autologous serum for 6, 24, 48, and 72 h. Cell lysates were examined for PPARg expression by Western blot analysis (upper panel). The band intensities (lower panel) were measured in each experiment by densitometry, and the bar graphs shown represent the mean 6 SEM from three independent experiments. pp , 0.0043; ppp , 0.0001. B,The cell culture supernatants were har- vested from M. tuberculosis and BCG-infected MDMs and analyzed for IL-8 production by ELISA;

PGE2 production was analyzed by an EIA kit (C). Shown is a representative experiment. Mean 6 SD of triplicate samples. n =3.pp = 0.0030; ppp , 0.0001.

being recognized as playing an important role in TB pathogenesis, necrotic changes in granuloma (55). A recent study demonstrated particularly within granulomas (4, 45). Finally, PPARg has been that IL-8 and COX2 expression was induced by cPLA2 in human implicated in the downregulation of proinﬂammatory responses. lung cells (33). Although previous studies had established that For these reasons, we explored the role of PPARg in regulating the COX2 expression is induced by MAPK-p38 and NF-kB– immune response of human macrophages to virulent M. tuberculosis dependent pathways in human lung epithelial cells for another and its major cell wall immune regulatory lipoglycan, ManLAM. respiratory pathogen (56), the M. tuberculosis- and/or ManLAM- Although PPARg has been extensively investigated for its role mediated biochemical signaling pathway(s) involved in macro- in other diseases (10), its immunoregulatory role(s) in infectious phages was not known. Our results provide evidence that COX2 diseases is just now being recognized (46–49). There are two re- expression in response to M. tuberculosis infection or ManLAM

by guest on October 1, 2021. Copyright 2010 Pageant Media Ltd. cent reports for altered PPARg levels during mycobacterial in- stimulation involves the upregulation and activation of PPARg in fection in humans, both involving the attenuated BCG. BCG MDMs. PPARg knockdown in MDMs blocked the release of infection has been found to induce PPARg expression in bladder IL-8 during ManLAM stimulation. This was further confirmed by cancer cells (50), whereas transcriptional profiling of PBMCs blocking or enhancing the transcriptional activity of PPARg in from BCG-vaccinated infants stimulated ex vivo has revealed ManLAM-stimulated macrophages. Last, the induction of PGE2 a downregulation of PPARg (51). The role of PPARg during in- by M. tuberculosis is also consistent with a role for PPARg in fection with virulent M. tuberculosis and human macrophages has mediating COX2 expression because it is a key intermediate in not been explored previously. PG biosynthesis. In this study, we identify PPARg as an important contributor to Previous reports using murine and human macrophages have http://classic.jimmunol.org regulation of the macrophage immune response to M. tuberculosis shown thatmycobacterial infection or ManLAM stimulation leads to infection. Expression and activity of PPARg in human macro- activation of the MAPK pathway and subsequent proinflammatory phages were enhanced by both M. tuberculosis and ManLAM. As cytokine production in infected cells (37, 57, 58). Consistent with reported, we found that AMs express a high basal level of PPARg, these earlier findings, our results demonstrate that virulent M. tu- which is further increased by M. tuberculosis and ManLAM. Be- berculosis infection or ManLAM stimulation activates MAPK-p38 cause PPARg knockdown in macrophages led to reduced in- in MDMs (59). There is also evidence that MAPK-p38 activation is 505 727 Downloaded from tracellular growth of M. tuberculosis,PPARg activation appears to necessary for the phosphorylation of Ser and Ser in cPLA2, correlate with a more susceptible host cell phenotype for this which leads to an increase in its enzymatic activity (38, 60). Our bacterium. Thus, our findings indicate that M. tuberculosis in- study shows that cPLA2 phosphorylation is induced by M. tuber- fection alters AM function further toward an alternative activation culosis and/or ManLAM in MDMs. Inhibition of MAPK-p38 acti- state, which is advantageous for this host-adapted intracellular vation by a pharmacological inhibitor significantly decreased pathogen within the lung microenvironment particularly during IL-8 production and also the phosphorylation of cPLA2 in MDMs. primary infection. In human lung epithelial cells, cPLA2 activation induces the pro- We observed an increase in IL-8 and COX2 following PPARg duction of IL-8 and COX2 gene expression through PPARg, and induction in MDMs by M. tuberculosis infection and/or ManLAM this induction was significantly reduced by cPLA2 inhibitor MAFP stimulation. IL-8 has been implicated as a chemoattractant in the (61). Our results demonstrate that suppression of cPLA2 activation lung in a variety of lung diseases (52, 53). A previous study in AMs also leads to reduced IL-8 release in response to ManLAM in showed that infection with M. tuberculosis or stimulation with its human macrophages. cell wall components significantly enhances IL-8 production, An important discovery in the current study is the identification of which serves to attract both neutrophils and T cells (54) to the site a pathway that links engagement of the MR with increased ex- of infection. IL-8 may also play a role in granuloma formation and pression and activation of PPARg. The MR plays an important role The Journal of Immunology 939

FIGURE 10. PPARg knockdown leads to decreased growth of M. tuberculosis in human macrophages and increased TNF production. A, MDMs were transfected with scrambled siRNA or PPARg siRNA by nucleofection.

After 24 h, the cells were incubated with M. tuberculosis H37Rv at an MOI of 1:1 for 2 h, washed, and either lysed or incubated in 2% autologous serum for another 24 h before lysing. CFUs were obtained from the cell lysates by serial dilution and plating on 7H11 agar in triplicate. The colonies were counted after 30 d. The graph shows the mean 6 SD of triplicate wells from a representative experiment (n =7;pp , 0.034). B, Culture supernatants were used to measure TNF production by ELISA. Shown is a representative experiment (mean 6 SD of triplicate samples; n =3; pp , 0.037).

by guest on October 1, 2021. Copyright 2010 Pageant Media Ltd. stimulated iNOS production in murine macrophages (65), and attenuates the respiratory burst (20). PPARg depletion in AMs FIGURE 9. NF-kB mediates IL-8 production following BCG but not leads to a Th1 pulmonary inﬂammatory response (66). Further M. tuberculosis infection of human macrophages. MDMs were pretreated supporting this concept, PPARg as well as PGE2 production have with the NF-kB inhibitor SN-50 (75 mg/ml) for 30 min and subsequently been shown to increase MR expression and activity on macrophages

infected with M. tuberculosis H37Rv or BCG at an MOI of 5:1 or stimu- (67, 68). lated with LPS (100 ng/ml). The cell culture supernatants were analyzed Specific biochemical signaling pathways triggered by MR liga- for IL-8 (A) and TNF-a (B) production by ELISA. A and B are tion have been elusive. It has a short cytoplasmic tail and lacks an representative experiments (mean 6 SD of triplicate samples; n =3;pp intracellular ITIM. The lack of discernable signaling motifs has led , pp , http://classic.jimmunol.org 0.0080; p 0.0006). C, In parallel, NF-kB inhibitor-treated cells some to question its role in phagocytosis and signaling (69). Despite were stimulated with ManLAM (5 mg/ml) for 6 and 24 h or with LPS this, recent studies continue to implicate the MR in signaling (100 ng/ml) for 24 h. Cell culture supernatants were used to measure IL-8 production by ELISA. Shown is a representative experiment (mean 6 SD cascades, most often in conjunction with other PRRs like TLR2 of triplicate samples; n =3;ppp , 0.0006). and CD14 (36, 63, 70, 71). Thus, another important finding in the current study is the identification of a new pathway activated by MR ligation that leads to enhanced expression and activity of

Downloaded from in the phagocytosis and trafficking of virulent M. tuberculosis by PPARg and excludes TLR2 and the NF-kB pathway. Our results human macrophages by binding ManLAM and higher-order show that the ManLAM/MR pathway-mediated IL-8 response is phosphatidyl-myo-inositol mannosides (30, 62) and regulating the independent of TLR2. This result together with earlier findings that intracellular trafficking of M. tuberculosis following phagocytosis ManLAM does not induce other proinflammatory cytokines in resulting in limited P–L fusion (25). MR ligation leads to an anti- macrophages identifies a key branch point in IL-8 production, inflammatory program (23), suppresses proinflammatory cytokine and potentially in the production of other inflammatory cytokines, release (63), and bypasses oxidative responses (24). The MR is that differentiates the known NF-kB–mediated pathway from the highly expressed on AMs as a marker of alternative activation (5). PPARg-mediated pathway. Importantly, activation of the latter For these reasons, its role in regulating the early host response to M. pathway can block NF-kB–mediated gene expression and there- tuberculosis infection in the lung is likely to be important. In this by augment an immunosuppressive program in the cells (18). Our context, it is important that the biological consequences of MR results identify the MR as a key PRR in initiating this pathway, and ligation are generally in concert with the activity of PPARg as these results place us in an excellent position to further explore a negative regulator of macrophage activation (64). PPARg down- upstream biochemical signaling mediators. regulates proinflammatory gene expression by antagonizing the ac- Our studies show differential PPARg expression following in- tivity of several transcription factors (19), downregulates IFN-g– fection with virulent M. tuberculosis and the attenuated BCG, but 940 PPARg ALTERS THE MACROPHAGE RESPONSE TO M. tuberculosis

this difference in expression did not lead to a difference in production is not clear at present. One possibility is that there are IL-8 production. In addition, infection with virulent M. tuberculosis differences between species in the involvement of the MR or in the led to a significantly greater release of PGE2 when compared with signaling response downstream of MR engagement. Another pos- BCG. Similarly, previous studies reported that heat-killed BCG did sibility is that there are differences in MR expression and/or re- not induce expression of COX2 and the production of PGE2 in ceptor partners for the MR between human and mouse macro- macrophages (72). Production of PGE2 is important in that it damp- phages. For example, most unstimulated human macrophages ens the Th1 response (73) and cell-mediated immunity in part (including the MDMs used in the current study) do not express through enhanced activity of regulatory T cells (74). We found that dendritic cell-specific intercellular adhesion molecule-3-grabbing inhibition of NF-kB translocation significantly reduced the nonintegrin (SIGN), whereas murine macrophages express various IL-8 response from BCG-infected MDMs; however, inhibition of related SIGN receptors (75). Because SIGN receptors can bind the NF-kB did not affect the IL-8 release from M. tuberculosis-infected same carbohydrate motifs as the MR, it is possible that signaling in MDMs. In contrast, NF-kB–mediated TNF production was murine macrophages involves coligation of the MR and SIGN inhibited in both M. tuberculosis- and BCG-infected macrophages, receptors or other lectin PRRs, such as Dectin-1 (69). and similar results were observed in ManLAM-stimulated cells. It On the basis of our results, we propose a model (Fig. 11) whereby has recently been reported that BCG induces PPARg expression in virulent M. tuberculosis or ManLAM upregulates PPARg expres- murine macrophages (41). However, in contrast to our findings, sion through an MR-mediated signaling pathway and simulta- PPARg expression and PGE2 production were dependent on neously activates the MAPK-p38 pathway, leading to activation TLR2 in that study. The potential difference(s) between human of downstream cPLA2 and release of AAs from membrane phos- and mouse macrophages in the PPARg pathway leading to PGE2 holipids. Hydrolyzed AAs are further processed by lipooxygenase by guest on October 1, 2021. Copyright 2010 Pageant Media Ltd. http://classic.jimmunol.org Downloaded from

FIGURE 11. Model of the PPARg signaling pathway in human macrophages in response to virulent M. tuberculosis or ManLAM. Shown is a schematic based on our results along with those in the literature of how virulent M. tuberculosis infection or ManLAM stimulation induces PPARg-mediated immunosuppression in macrophages. M. tuberculosis or ManLAM binds to the MR, which leads to upregulation of PPARg expression through an MR-

dependent signaling pathway and, simultaneously, to activation of MAPK-p38-mediated cPLA2, which leads to the release and hydrolysis of AA from the plasma membrane to generate the ligand for PPARg (15-d-PGJ2). Activated PPARg heterodimerizes with RXR, and they bind to PPREs in the IL-8 and COX2 promoters, thereby inducing their expression as well as expression of the MR. Activation of PPARg leads to transrepression of NF-kB activity but promotes the generation of Th2 cytokines through PGE2 production. The Journal of Immunology 941

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