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Previous Article | Next Article 
Infection and Immunity, August 2007, p. 4148-4157, Vol. 75, No. 8
0019-9567/07/$08.00+0 doi:10.1128/IAI.00181-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Infection Immunology Research Group, Department of Microbial Pathogenesis, Helmholtz Center for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany,1 Department of Microbial Pathogenesis, Helmholtz Center for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany,2 Mucosal Immunity, Helmholtz Center for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany3
Received 2 February 2007/ Returned for modification 13 April 2007/ Accepted 15 May 2007
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ABSTRACT |
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INTRODUCTION |
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Previous studies examining host responses to S. pyogenes in a mouse model of infection have shown the importance of resident macrophages for controlling infection (18, 19). Macrophages are capable of recognizing, phagocytosing, and destroying S. pyogenes in order to eliminate the invading pathogen, while also producing cytokines and chemokines that are crucial in controlling the recruitment and activation of inflammatory cells at the site of infection (18, 19). Although it is assumed that the activation of macrophages is directed toward the elimination of the invading pathogens, it is equally likely that the excessive and unregulated stimulation of macrophages can lead to a continuous release of inflammatory mediators that act synergistically and thus lead to sepsis and septic shock (12). Therefore, the functional activities of macrophages during S. pyogenes infection may greatly influence the character, course, and outcome of the pathogenic process.
To improve our understanding of the complex response of macrophages to S. pyogenes and to identify new targets for which therapeutic options might be possible, we have analyzed the global gene expression profile of murine resident peritoneal macrophages after in vivo infection with this pathogen by gene array technology. We have identified more than 400 genes differentially transcribed in macrophages following 1 h of infection with S. pyogenes. Infection-induced genes fell into several functional categories, including immune response and inflammation, transcription, signaling, apoptosis, cell cycle, electronic transport, and cell adhesion; other induced genes have unknown functions.
Macrophages are plastic cells that respond to microenvironmental signals with distinct activation programs (16, 20, 30). Classically activated macrophages (M1 phenotype) are induced by inflammatory molecules such as lipopolysaccharide (LPS) and gamma interferon (IFN-
). These M1 macrophages produce proinflammatory cytokines and chemokines, such as tumor necrosis factor alpha (TNF-
), interleukin 1ß (IL-1ß), IL-6, IL-12, and macrophage inflammatory protein 1 (MIP-1), and generate reactive nitrogen species, such as nitric oxide (NO), via expression of inducible nitric oxide synthase (iNOS) (16, 20, 30). Alternatively activated macrophages (M2 phenotype) are generated after exposure to certain stimuli, such as IL-4, IL-13, transforming growth factor beta (TGF-ß), or glucocorticoids (16, 20). The M2 macrophages express anti-inflammatory molecules, such as IL-10 and IL-1 decoy receptor (IL-1ra), and metabolize arginine through arginase rather than iNOS (16, 20). Arginase blocks iNOS activity by a variety of mechanisms, including competing for the arginine substrate that is required for NO production (5). Classically activated M1 macrophages are potent effector cells integrated into Th1 responses, which kill microorganisms and tumor cells and produce copious amounts of proinflammatory cytokines (29). In contrast, M2 macrophages tune the inflammatory responses, promoting angiogenesis and tissue remodeling and repair. However, the M1 and M2 phenotypes seem to represent the two extremes of a spectrum of possible forms of macrophage activation, and different versions of the M2 phenotype—M2a, M2b, and M2c—with different functional properties have been described. Specifically, M2a macrophages are induced by IL-4 or IL-13 and are involved in promotion of Th2 responses; M2b macrophages are induced by exposure to agonists of Toll-like receptors (TLRs) or IL-1 receptor and play a role in suppression and regulation of inflammation and immunity; and the M2c phenotype, induced by IL-10 and glucocorticoid hormones, participates in matrix deposition and tissue remodeling (1, 26, 27).
The phenotype of macrophages activated by S. pyogenes is currently unknown but may be important in understanding the contribution of these phagocytic cells to disease pathogenesis. In this regard, we have shown here that S. pyogenes induces an atypical activation phenotype in macrophages that includes markers characteristic of both M1 and some of the M2 activation pathways.
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MATERIALS AND METHODS |
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Mice. Inbred female C3H/HeN and BALB/c mice were purchased from Harlan-Winkelmann (Borchen, Germany). Mice with either a targeted disruption in the iNOS gene (B6.129P2-Nos2tm1Lau/J) (iNOS–/–) or a targeted deletion in the cytosolic p47phox gene [B6(Cg)-Ncf1m1J/J] (p47phox–/–), as well as wild-type control mice (C57BL/6J), were purchased from Jackson Laboratories (Bar Harbor, ME). Animals were housed in microisolator cages and given food and water ad libitum. All studies were approved by the local ethics board.
In vivo infection of peritoneal macrophages. Mice were intraperitoneally infected with 5 x 107 CFU of S. pyogenes and euthanized 1 h thereafter, and the peritoneum was lavaged with sterile PBS. Macrophages present in the lavage samples were labeled with anti-F4/80 antibodies, further purified by positive selection with miniMACS magnetic microbeads, according to the manufacture's instructions (Miltenyi Biotec Inc., Germany), and used for the cDNA microarray analysis or reverse transcriptase PCR (RT-PCR).
For macrophage killing assays, peritoneal macrophages isolated from infected mice (1 h postinoculation) were seeded into 48-well microtiter plates and cultured at 37°C, 5% CO2, in Dulbecco modified Eagle medium (GIBCO) containing 10 mM HEPES, 2 mM L-glutamine, and 100 µg/ml of gentamicin. At several time points, the macrophages were lysed with distilled water (dH2O) and surviving bacteria were enumerated by plating of serial dilutions in blood agar.
In some experiments, peritoneal macrophages were stimulated with 1 µg/ml of LPS from Salmonella enterica serovar Typhimurium (Sigma-Aldrich, Taufkirchen, Germany) plus 100 U/ml of recombinant murine IFN- (PeproTech, Rocky Hill, NJ).
Array analysis. Total RNA was isolated from highly purified F4/80+ cells obtained from the peritoneal cavity of BALB/c or C3H/HeN infected and uninfected control mice with peqGold TriFast (Peqlab), according to the manufacturer's instructions, and hybridized to an Affymetrix GeneChip MOE430A by standard Affymetrix protocols, as described elsewhere (36). Two replicate chips per group were used with pooled macrophages harvested from 8 to 10 mice. The data set used in this study is available in a MIAME-compliant format at the NCBI Gene Expression Omnibus (GEO) database under accession number GSE7769 (http://www.ncbi.nlm.nih.gov/geo/). Normalized gene expression intensities were compared, and genes were considered to be differentially expressed between infected and uninfected samples when their fold change was greater than or equal to 2 or less than or equal to –2. The statistical parameter used to define significant change (P) was less than 0.001, and the difference between compared signal intensities of a certain gene was more than 200.
RT-PCR.
To independently confirm the microarray results, RT-PCR was carried out on arbitrarily selected genes that were found to be up-regulated in infected macrophages in the microarray analysis (Il-1, Il-1ß, Il-6, Csf2, Tnf-, Mip-1, Mip-1ß) and two unaffected genes (Ifn-, Il-12p40). Total RNA was prepared as described above. RNA was reverse transcribed with RT (Hoffman La Roche), and cDNA synthesis was performed with a Gibco RT-PCR kit, according to the manufacturer's instructions. The single-stranded cDNA was then subjected to PCR under standard reaction conditions. The PCR primer sequences for these genes, as well as those of the housekeeping genes ß-actin and Rsp9, are provided in Table 1. The resultant PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide, and photographed.
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Measurement of NO. The Griess reaction was used to determine NO concentrations in supernatants of S. pyogenes-infected macrophages, as previously described (11). Briefly, supernatant from cultured uninfected or S. pyogenes-infected macrophages was mixed with an equal volume of Griess's reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, 2.5% H3PO4). Absorbance at 550 nm was recorded. Serial dilutions of sodium nitrite were used to construct a standard curve.
Determination of ROS radicals. For detection of reactive oxygen species (ROS) generated by S. pyogenes-infected macrophages, mice were intraperitoneally infected, as described above, with either unlabeled S. pyogenes or S. pyogenes labeled green with carboxyfluorescein (Molecular Probes, Göttingen, Germany). For labeling, a suspension of 5 x108 bacteria was centrifuged, resuspended in 1 ml of Hanks balanced salt solution containing 0.2 mg/ml of carboxyfluorescein, and incubated 30 min 4°C in the dark. After incubation, labeled bacteria were washed several times to remove unbound dye. Infected mice were euthanized 1 h after bacterial inoculation and subjected to peritoneal lavage. Mice intraperitoneally injected with PBS were used as controls. Lavage samples were added to wells containing glass coverslips and incubated for 1 h at 37°C, 5% CO2. After a wash to remove nonadherent cells, macrophages were incubated with 1 mg/ml of Nitro Blue Tetrazolium (NBT) dissolved in Krebs-Ringer phosphate glucose buffer (KRPG) (144 mM NaCl, 5 mM KCl, 8.5 mM Na2HPO4, 1.4 mM NaH2PO4, 1.3 mM MgSO4, 5 mM glucose, 10 mM HEPES [pH 7.4]) for 45 min at 37°C. After incubation, the cells were washed twice with KRPG buffer, fixed in 4% paraformaldehyde, and counterstained with Giemsa stain. Samples were examined by light and fluorescence microscopy for the presence of green-fluorescing bacteria and blue-black formazan precipitate.
Experimental infection of mice. A previously described murine model of S. pyogenes infection was used (17). In brief, mice were inoculated with 105 CFU of S. pyogenes in 0.2 ml of PBS via a lateral tail vein. Viable bacterial counts were determined in the blood of infected mice by collecting blood samples from the tail vein at 24 h postinoculation and plating serial dilutions in blood agar.
Statistical analysis. Statistical significance between samples was determined by analysis of variance (ANOVA) and the Mann-Whitney (Wilcoxon) W test, with a P value of <0.05 considered significant.
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RESULTS |
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, IL-1ß, TNF-, and IL-6), chemokines (monocyte chemoattractant protein 1 [MCP-1], MCP-3, MIP-1, MIP-1ß, and MIP-2), and growth factors (granulocyte-macrophage colony-stimulating factor [GM-CSF], G-CSF). Genes encoding anti-inflammatory molecules (Ilrn and Il10) were also induced in macrophages upon stimulation with S. pyogenes. Also, the gene encoding type II arginase (Arg2) and the gene encoding the p47phox component of the NADPH oxidase (Ncf1), were both up-regulated after infection.
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Other genes whose expression was greatly increased were associated with the cell cycle (Gadd45a, Cdkn1a, Fosb), transcription (Maff, Egr2, Relb, Nfkb1), or cell signaling (Rab20, Socs3 or Plaur). Elevated mRNA levels were also observed for apoptosis-regulating genes (Gadd45b, Bcl2a1a, Birc2, Cflar), as well as for apoptosis-associated genes (Pmaip1, Casp4, Bcl211).
Among the down-regulated genes were transcription factors such as Gata 6, which has been shown to regulate innate immune responses (39), and Tieg (TGFB-inducible early growth response) and signaling genes such as Il16.
To independently confirm the microarray results, RT-PCR was carried out on seven arbitrarily selected genes that were found to be up-regulated in infected macrophages in the microarray analysis (Il-1, Il-1ß, Il-6, Csf2, Tnf-, Mip-1, and Mip-1ß) and on two unaffected genes (Ifn- and Il-12p40). As shown in Fig. 2A, expression of mRNA was detected by RT-PCR for IL-1, IL-1ß, IL-6, TNF-, MIP-1, MIP-1ß, and GM-CSF in infected macrophages but not in uninfected cells. The levels of mRNA for IL-12 p40 and IFN- were undetectable in both S. pyogenes-infected and uninfected control macrophages in the microarray data and were also undetectable by RT-PCR (Fig. 2A). Similar results were obtained when the transcription of these genes was investigated in resident macrophages after exposure to a different strain of S. pyogenes (M-type 1 strain SF370) (data not shown).
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Activation phenotype of S. pyogenes-infected macrophages.
Some markers of classical activation (M1 phenotype) and some markers compatible with alternative activation (M2 phenotype) were induced in macrophages after exposure to S. pyogenes. Thus, the levels of transcripts encoding cytokines and chemokines, such as TNF-, IL-1, IL-6, IP-10, MIP-1, and MCP-1, implicated in the classical activation phenotype were significantly increased after infection (Table 2). On the other hand, several transcripts typical of the alternative activation phenotype (e.g., IL-1ra and IL-10) were also up-regulated in infected macrophages. As classically activated macrophages are developed in response to IFN-, along with exposure to a microbe or microbial product such as LPS (30), peritoneal macrophages stimulated with IFN- plus LPS were used for comparison. Treatment of murine macrophages with LPS plus IFN- also resulted in up-regulation of inflammatory genes, such as the TNF-, Mip-1, and Mip-1ß genes, typical of the classical activation pathway (Fig. 3A).
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and LPS resulted in induction of Nos2 within 1 h of exposure, Nos2 transcripts were undetectable in macrophages both at 1 h and at 4 h or 16 h after infection. For that reason, NO was undetectable in the supernatant of cultured S. pyogenes-infected macrophages at the selected time points (data not shown).
The up-regulation of Arg2 observed in the array data was then confirmed by RT-PCR. As shown in Fig. 3C, Arg2 was induced in infected macrophages as early as 1 h of infection, and gene transcription is maintained after 4 h and 16 h of infection (Fig. 3C). The Arg2 gene was not induced in macrophages stimulated with IFN- and LPS (Fig. 3C).
NADPH oxidase is involved in the S. pyogenes-killing activity of murine macrophages. Since NO produced by iNOS and ROS produced by phagocyte oxidase (phox) are the major antimicrobial mechanisms involved in host defense (4, 22, 34), we hypothesized that production of oxygen radicals may play a major role in the elimination of phagocytosed S. pyogenes by murine macrophages. To confirm this hypothesis, the production of ROS by S. pyogenes-infected macrophages was determined with the NBT reaction. Uninfected macrophages were used as a control (Fig. 4A). As shown in Fig. 4B, significantly high levels of NBT-reducing activity (dark blue precipitate) were detected in infected macrophages (red arrows). Similar experiments were performed with S. pyogenes labeled with fluorescein to determine whether production of ROS takes place in macrophages that are associated with bacteria. Figure 4C shows that the oxidative response (black precipitate) largely occurred in macrophages with associated S. pyogenes (green). Colocalization of ROS and S. pyogenes was also evident in infected macrophages (Fig. 4D, red arrows).
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DISCUSSION |
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Another important difference between M1 and M2 macrophages involves the balance between arginase and iNOS activities. Classically activated murine macrophages undergo a respiratory burst and express iNOS, whereas alternatively activated macrophages metabolize arginine through arginase rather than iNOS. Arginase and iNOS both utilize L-arginine as a substrate (46). However, while iNOS generates reactive NO species with microbicidal and proinflammatory effects important in immune responses, arginase competes with iNOS for arginine as a substrate and generates L-ornithine, an important precursor for proline that enhances collagen biosynthesis, promoting cell growth and tissue repair (21). The induction of either iNOS or arginase is usually associated with suppression of the opposing enzyme, indicating a competitive nature in these alternative states of macrophage metabolism. We report in this study that during experimental S. pyogenes infection, arginase II mRNA, but not iNOS mRNA, was up-regulated in macrophages. These results also suggest that the bactericidal activity of macrophages against S. pyogenes is mediated by phagocyte oxidase and not by iNOS. This assumption was further confirmed by the impaired capacity of macrophages with dysfunctional phagocyte oxidase (p47phox–/–) to kill S. pyogenes. In contrast, this capability was preserved in macrophages deficient in iNOS expression. Besides S. pyogenes, oxygen radical formation has been shown to participate in the killing of a diverse group of pathogens, including the promastigote form of Leishmania donovani (32), Toxoplasma gondii (33), Plasmodium falciparum (45), and Staphylococcus aureus (24). The critical role of the phagocyte oxidase for control of S. pyogenes infection in humans is reflected by the enhanced susceptibility of patients with chronic granulomatous disease, an inherited disease characterized by deficient functional activity of phagocyte oxidase complex, to recurrent pyogenic infections (15, 37).
Little is known regarding the interaction of S. pyogenes with human macrophages. Thulin et al. (42) recently reported that some S. pyogenes microorganisms are capable of surviving intracellularly in human macrophages during acute invasive infection, as well as in in vitro-infected human monocytes/macrophages. In contrast to this observation, murine macrophages infected in vivo with S. pyogenes were refractory to bacterial persistence. However, Thulin et al. (42) also showed that the percentage of human monocytes associated with viable microorganisms was reduced to almost 50% between 4 and 12 h after in vitro infection. This observation clearly indicates that, although some human monocytes allowed S. pyogenes persistence, other infected monocytes were also capable of efficiently eliminating ingested S. pyogenes. Whether the killing of S. pyogenes by human monocytes/macrophages is mediated by the phagocyte oxidase system or by iNOS remains to be elucidated.
The phagocyte oxidase system produces superoxide after bacterial phagocytosis, which is rapidly converted to other potent ROS, such as hydrogen peroxide, within forming phagosomes (3). In addition to participating in bacterial killing, ROS released in high levels by overstimulated immune cells have been implicated in inflammation and tissue injury (13). In blood, ROS can be neutralized by the antioxidant activity of red cell and plasma components (44). However, local generation of ROS can cause tissue injury, as has been shown in Pseudomonas aeruginosa pneumonia (41), pneumococcal meningitis (2), and Helicobacter pylori gastritis (35). Therefore, immune cells also require adequate levels of antioxidants in order to avoid the harmful effects of an excessive production of ROS. The excess of oxygen radicals can be neutralized by a wide array of antioxidant molecules, including superoxide dismutase (28). In this regard, our array data show that the genes encoding metallothioneins (Mt1 and Mt2), thioredoxin reductase (Txnrd1), and superoxidismutase 2 (Sod2), which are reactive oxygen scavengers and play an important role in the detoxification of free radicals (38, 43), were up-regulated in S. pyogenes-infected macrophages. The up-regulation of these genes may be critical for protection against the potential harmful effects for cells of high levels of oxygen radicals.
The gene encoding prostaglandin-endoperoxidase synthase 2 (Ptgs2) was also strongly induced following S. pyogenes infection, with an average increase of more than 1,000-fold. While the role of Ptgs2 expression in response to S. pyogenes is unknown, it may serve as a potent regulator of inflammation. Up-regulation of this gene is responsible for the increased production of inflammatory prostaglandins implicated in the pathogenesis of many inflammatory diseases, including sepsis (6).
Of particular interest was the up-regulation of Gadd45 family proteins, which have been implicated in DNA repair following stress (40), and the cytoprotective Tnfaip3 protein, which is antiapoptotic through inhibition of the caspase cascade at the level of the initiator caspase 8 (10). These cytoprotective molecules may be critical for maintenance of cellular homeostasis under the severe stress conditions associated with streptococcal infection. Interestingly, the gene encoding the suppressor of cytokine signaling 3 (Socs3) was also strongly up-regulated. Socs3 protein is a key negative regulator of cytokine signaling (8). Up-regulation of Socs3 may constitute a negative feedback mechanism for the maintenance of homeostasis.
Taken together, these data strongly suggest that resident macrophages activate an array of specific survival pathways after infection with S. pyogenes directed at maintaining cell integrity and ensuring survival. In contrast to this survival program activated in macrophages, exposure of human neutrophils to S. pyogenes results in the induction of apoptotic genes and acceleration of apoptosis (23). This divergence in response between these two phagocytic cells may most likely reflect fundamental differences either in the cellular receptors recognizing S. pyogenes or in their phagocytic pathways.
In summary, the results of our study indicate that S. pyogenes induces an uncommon activation profile in murine macrophages that does not strictly fit either the M1 or M2 activation phenotype. This profile can be explained by the high plasticity of the mononuclear phagocyte system. Thus, the activation response of macrophages to S. pyogenes infection may be the result of exposure to a multiplicity of polarizing signals emerging from the pathogen (e.g., cell wall components and exotoxins, etc.), yielding either a mixed M1/M2 activation phenotype or a mixed population of macrophages belonging to the M1 and M2 phenotypes. The characterization of the transcriptional profile adds a new dimension to the analysis of the macrophage response to S. pyogenes with the identification of potential fingerprints useful for defining states within the complex infection process.
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ACKNOWLEDGMENTS |
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We thank Tanja Toepfer and Sabine Lehne for excellent technical work.
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FOOTNOTES |
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Published ahead of print on 25 May 2007.
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