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• W1972189052 abstract "Host responses during the later stages of Salmonella- macrophage interactions are critical to controlling infection but have not been well characterized. After 24 h of infection, nearly half of interferon-γ-primed murine RAW 264.7 macrophage-like cells infected by Salmonella enterica serovar Typhimurium contained filamentous bacteria. Bacterial filamentation indicates a defect in completing replication and has been previously observed in bacteria responding to a variety of stresses. To understand whether macrophage gene expression was responsible for this effect on Salmonella Typhimurium replication, we used gene arrays to profile interferon-γ-primed RAW 264.7 cell gene expression following infection. We observed an increase in MEK1 kinase mRNA at 8 h, an increase in MEK protein at 24 h, and measured phosphorylation of MEK's downstream target kinase, ERK1/2, throughout the 24-h infection period. Treatment of cells with MEK kinase inhibitors significantly reduced numbers of filamentous bacteria observed within macrophages after 24 h and increased the number of intracellular colony-forming units. Phagocyte NADPH oxidase inhibitors and antioxidants also significantly reduced bacterial filamentation. Either MEK kinase or phagocyte oxidase inhibitors could be added 4–8 h after infection and still significantly decrease bacterial filamentation. Oxidase activity appears to mediate bacterial filamentation in parallel to MEK kinase signaling, while inducible nitric-oxide synthase inhibitors had no significant effect on bacterial morphology. In summary, Salmonella Typhimurium infection of interferon-γ-primed macrophages triggers a MEK kinase cascade at later infection times, and both MEK kinase and phagocyte NADPH oxidase activity impair bacterial replication. These two signaling pathways mediate a host bacteriostatic pathway and may play an important role in innate host defense against intracellular pathogens. Host responses during the later stages of Salmonella- macrophage interactions are critical to controlling infection but have not been well characterized. After 24 h of infection, nearly half of interferon-γ-primed murine RAW 264.7 macrophage-like cells infected by Salmonella enterica serovar Typhimurium contained filamentous bacteria. Bacterial filamentation indicates a defect in completing replication and has been previously observed in bacteria responding to a variety of stresses. To understand whether macrophage gene expression was responsible for this effect on Salmonella Typhimurium replication, we used gene arrays to profile interferon-γ-primed RAW 264.7 cell gene expression following infection. We observed an increase in MEK1 kinase mRNA at 8 h, an increase in MEK protein at 24 h, and measured phosphorylation of MEK's downstream target kinase, ERK1/2, throughout the 24-h infection period. Treatment of cells with MEK kinase inhibitors significantly reduced numbers of filamentous bacteria observed within macrophages after 24 h and increased the number of intracellular colony-forming units. Phagocyte NADPH oxidase inhibitors and antioxidants also significantly reduced bacterial filamentation. Either MEK kinase or phagocyte oxidase inhibitors could be added 4–8 h after infection and still significantly decrease bacterial filamentation. Oxidase activity appears to mediate bacterial filamentation in parallel to MEK kinase signaling, while inducible nitric-oxide synthase inhibitors had no significant effect on bacterial morphology. In summary, Salmonella Typhimurium infection of interferon-γ-primed macrophages triggers a MEK kinase cascade at later infection times, and both MEK kinase and phagocyte NADPH oxidase activity impair bacterial replication. These two signaling pathways mediate a host bacteriostatic pathway and may play an important role in innate host defense against intracellular pathogens. Macrophages serve a central role in host defense against pathogenic microbes by nature of their ability to rapidly recognize bacterial components, phagocytose pathogens, and activate an arsenal of antimicrobial effectors to contain and eliminate the microbe. A macrophage's repertoire of antimicrobial effectors includes the phagocyte NADPH oxidase (phox), 1The abbreviations used are: phoxphagocyte NADPH oxidasecfucolony-forming unitd-NMMANG-d-monomethyl arginineDPIdiphenyleneiodoniumERKextracellular signal-regulated kinaseGFPgreen fluorescent proteinIFN-γinterferon-γiNOSinducible nitric-oxide synthasel-NMMANG-l-monomethylarginineLPSlipopolysaccharideROIreactive oxygen intermediateMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumPBSphosphate-buffered salineGAPDHglyceraldehyde-3-phosphate dehydrogenase inducible nitric-oxide synthase (iNOS), cationic antimicrobial peptides, and an endosomal system designed to restrict nutrients and traffic phagocytosed microbes to degradative lysosomes. Phox is a multisubunit complex that can be assembled on intracellular membranes, such as the phagosomal membrane and the plasma membrane. Phox activity produces superoxide that can lead to the generation of other toxic reactive oxygen intermediates (ROI), such as hydrogen peroxide, and combine with nitric oxide to generate peroxynitrite, all of which can directly cause oxidative damage to bacteria (1Vazquez-Torres A. Fang F.C. Trends Microbiol. 2001; 9: 29-33Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Macrophages activate many signaling pathways following recognition of bacterial components, although the relative contribution of each pathway to the induction of antibacterial effectors is not fully understood. For example, macrophages activate MEK/ERK kinase signaling in response to bacterial infection (2Procyk K.J. Kovarik P. von Gabain A. Baccarini M. Infect. Immun. 1999; 67: 1011-1017Crossref PubMed Google Scholar). MEK is a mitogen-activated protein kinase kinase that is activated by phosphorylation following Salmonella enterica serovar Typhimurium infection of macrophages in a Raf-dependent or -independent manner (3Jesenberger V. Procyk K.J. Ruth J. Schreiber M. Theussl H.C. Wagner E.F. Baccarini M. J. Exp. Med. 2001; 193: 353-364Crossref PubMed Scopus (54) Google Scholar). Upon activation, MEK phosphorylates the downstream kinase ERK (extracellular signal-regulated kinase), which then dimerizes and translocates to the nucleus where it activates transcription factors such as Elk-1 to modify gene expression (4Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1663) Google Scholar). MEK/ERK signaling is involved in the activation of oxidative and nitrosative bursts, endosomal trafficking, and increased macrophage differentiation and therefore is a strong candidate for being involved in the augmentation of macrophage defenses against intracellular pathogens (5Rao K.M. J. Leukoc. Biol. 2001; 69: 3-10PubMed Google Scholar). phagocyte NADPH oxidase colony-forming unit NG-d-monomethyl arginine diphenyleneiodonium extracellular signal-regulated kinase green fluorescent protein interferon-γ inducible nitric-oxide synthase NG-l-monomethylarginine lipopolysaccharide reactive oxygen intermediate mitogen-activated protein kinase/extracellular signal-regulated kinase kinase Dulbecco's modified Eagle's medium fetal bovine serum phosphate-buffered saline glyceraldehyde-3-phosphate dehydrogenase In the murine model of human typhoid fever, SalmonellaTyphimurium resides intracellularly within macrophages (6Richter-Dahlfors A. Buchan A.M. Finlay B.B. J. Exp. Med. 1997; 186: 569-580Crossref PubMed Scopus (421) Google Scholar) in a specialized vacuole, and macrophages appear to be a preferred site for bacterial replication (7Wijburg O.L. Simmons C.P. van Rooijen N. Strugnell R.A. Eur. J. Immunol. 2000; 30: 944-953Crossref PubMed Scopus (60) Google Scholar). As this intramacrophage niche helps to shield Salmonella from killing by components of the innate and humoral immune defenses, the responses of infected macrophages are thought to serve a central role in determining disease outcome (7Wijburg O.L. Simmons C.P. van Rooijen N. Strugnell R.A. Eur. J. Immunol. 2000; 30: 944-953Crossref PubMed Scopus (60) Google Scholar). The interplay between host resistance factors and bacterial virulence factors are critical to determining the outcome of infection. On the host side, macrophages serve to limit the course of infection by destroying intracellular Salmonella Typhimurium or restricting bacterial replication by modifying its intracellular environment. Macrophages limit the availability of cations and nutrients required by Salmonella within its intracellular vacuole (8Kingsley R.A. Baumler A.J. Oelschlaeger T.A. Hacker J. Bacterial Invasion into Eukaryotic Cells. 33. Plenum, New York2000: 321-342Google Scholar). Both phox and iNOS are required for effective host resistance against Salmonella Typhimurium in the murine typhoid model (9Vazquez-Torres A. Jones-Carson J. Mastroeni P. Ischiropoulos H. Fang F.C. J. Exp. Med. 2000; 192: 227-236Crossref PubMed Scopus (450) Google Scholar, 10Mastroeni P. Vazquez-Torres A. Fang F.C., Xu, Y. Khan S. Hormaeche C.E. Dougan G. J. Exp. Med. 2000; 192: 237-248Crossref PubMed Scopus (324) Google Scholar, 11Shiloh M.U. MacMicking J.D. Nicholson S. Brause J.E. Potter S. Marino M. Fang F. Dinauer M. Nathan C. Immunity. 1999; 10: 29-38Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar). Cytokines secreted during infection, including interferon (IFN)-γ (12Gulig P.A. Doyle T.J. Clare-Salzler M.J. Maiese R.L. Matsui H. Infect. Immun. 1997; 65: 5191-5197Crossref PubMed Google Scholar), are essential for host defense against Salmonella infection. IFN-γ-primed macrophages may be important in mediating bacterial clearance in immune mice (7Wijburg O.L. Simmons C.P. van Rooijen N. Strugnell R.A. Eur. J. Immunol. 2000; 30: 944-953Crossref PubMed Scopus (60) Google Scholar), and IFN-γ stimulation up-regulates the expression of many of these antimicrobial effectors and impairs replication of Salmonella Typhimurium within macrophages (12Gulig P.A. Doyle T.J. Clare-Salzler M.J. Maiese R.L. Matsui H. Infect. Immun. 1997; 65: 5191-5197Crossref PubMed Google Scholar). On the bacterial side, while Salmonella Typhimurium initiate a pro-inflammatory response by macrophages, some bacteria are able to secure an intracellular niche within a distinct endosomal compartment where replication occurs 4–8 h after infection. Bacterial virulence protein mutants that cannot replicate within macrophages are strongly attenuated for systemic disease within the murine typhoid model, reinforcing the importance of Salmonella-macrophage interactions (13Fields P.I. Swanson R.V. Haidaris C.G. Heffron F. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5189-5193Crossref PubMed Scopus (875) Google Scholar, 14Shea J.E. Beuzon C.R. Gleeson C. Mundy R. Holden D.W. Infect. Immun. 1999; 67: 213-219Crossref PubMed Google Scholar). Distinct antibacterial activities have been observed in macrophages at different times during infection (10Mastroeni P. Vazquez-Torres A. Fang F.C., Xu, Y. Khan S. Hormaeche C.E. Dougan G. J. Exp. Med. 2000; 192: 237-248Crossref PubMed Scopus (324) Google Scholar). The responses of macrophages to intracellular Salmonella Typhimurium at later times post-infection are likely critical in mediating the outcome of infection but have not been well characterized, with most of the work centered upon the first few hours of infection. We demonstrate here that IFN-γ-primed RAW 264.7 macrophage-like cells are capable of restricting the bacterial replication that is permitted by naı̈ve RAW 264.7 cells. To identify host factors mediating this control of bacterial replication, we have used gene arrays to examine the transcriptional responses of IFN-γ-primed RAW 264.7 cells to intracellular Salmonella Typhimurium at 8 h post-infection. We identified up-regulated MEK1 kinase mRNA levels, which were confirmed at the levels of RNA, protein, and kinase activity. MEK activity correlated with inhibition of bacterial replication and induction of bacterial filamentation, an indicator of bacterial stress. MEK kinase and phox activities can impact each other, and we observed that phox inhibitors mimicked the effect of MEK inhibitors in reducing bacterial filamentation. MEK kinase or oxidase inhibitors added later during infection could significantly decrease bacterial filamentation, suggesting that MEK and phox activities at later times are primarily responsible for mediating bacterial filamentation. While phox activity can positively regulate as well as be regulated itself by MEK kinase activity, our results suggest that MEK and phox activities function in parallel to mediate bacterial filamentation. In summary, we provide evidence that Salmonella Typhimurium infection of IFN-γ-primed macrophages triggers a MEK kinase cascade and ROI production at later infection times and that both MEK kinase and phox activities impair bacterial replication, which is reflected by filamentation. The Salmonella enterica serovar Typhimurium strain SL1344 was obtained from the American Type Culture Collection (ATCC; Manassas, VA) and grown in Luria-Bertani (LB) broth. The plasmid pAT113-GFP (kindly provided by Dr. J. L. Gaillard, Paris, France) was introduced into SL1344 by electroporation (kindly provided by Dr. L. Knodler, University of British Columbia, Vancouver, Canada). The Salmonella Typhimurium strain cs401 (14028S StrR) was kindly provided by Dr. S. Miller (University of Washington, Seattle, WA). For macrophage infections, 10 ml of LB in a 125-ml flask was inoculated from a frozen glycerol stock and cultured overnight with shaking at 37 °C to stationary phase. The murine macrophage cell line RAW 264.7 (TIB-72; ATCC) was maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Burlington, Ontario, Canada) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen) without antibiotics at 37 °C in 5% CO2. Cultures were used between passage numbers 6 and 20. For the bacterial colony-forming unit (cfu) enumeration experiments described in Fig. 1, RAW 264.7 cells were either unprimed or cultured with 20 units/ml (2 ng/ml) IFN-γ (R & D Systems, Minneapolis, MN) for 20–24 h prior to infection. For all subsequent experiments, cells were primed with IFN-γ prior to infection. For immunofluorescence and cfu experiments, IFN-γ-primed RAW 264.7 cells (1 × 105cells/well) were seeded in 24-well plates. Bacteria were diluted in culture medium to give a nominal multiplicity of infection of ∼100, bacteria were centrifuged onto the monolayer at 1000 rpm for 10 min to synchronize infection, and the infection was allowed to proceed for 20 min in a 37 °C, 5% CO2 incubator. Cells were washed three times with phosphate-buffered saline (PBS) to remove extracellular bacteria and then incubated in DMEM + 10% FBS containing 100 μg/ml gentamicin (Sigma, Oakville, Ontario, Canada) to kill any remaining extracellular bacteria and prevent re-infection. After 2 h, the gentamicin concentration was lowered to 10 μg/ml and maintained throughout the assay. Intracellular survival/replication of Salmonella Typhimurium SL1344 was determined using the gentamicin resistance assay, as described previously (15Steele-Mortimer O. Meresse S. Gorvel J.P. Toh B.H. Finlay B.B. Cell Microbiol. 1999; 1: 33-49Crossref PubMed Scopus (262) Google Scholar). Briefly, cells were washed twice with PBS to remove gentamicin, lysed with 1% Triton X-100/0.1% sodium dodecyl sulfate in PBS at various times post-infection, and numbers of intracellular bacteria enumerated from cfu counts on LB agar plates. Under these infection conditions, macrophages contained an average of 1 bacterium per cell after 2 h as assessed by standard plate counts, which permitted analysis of macrophages at 24 h post-infection. IFN-γ-primed RAW 264.7 cells (1 × 105 cells/well) were seeded on 12-mm diameter glass coverslips in 24-well plates. Following infection with Salmonella Typhimurium for 24 h, fixation was performed with 2.5% paraformaldehyde for 10 min at 37 °C. Fixed cells were washed three times with PBS and blocked in PBS containing 10% normal goat serum for 10 min. Extracellular bacteria were labeled by sequentially overlaying coverslips with a rabbit polyclonal primary antibody to Salmonella Typhimurium lipopolysaccharide (LPS; Difco, Detroit, MI) at 1:200 and an Alexa 568-conjugated mouse anti-rabbit secondary antibody (Molecular Probes, Eugene, OR) at 1:400 in PBS + 10% normal goat serum for 20 min. Coverslips were mounted onto 1-mm glass sides using Mowiol (Aldrich). To quantify cells containing filamentous bacteria, only intracellular Salmonella Typhimurium were counted (not labeled by the extracellularly applied LPS-specific antibody). Bacteria were scored as “filamentous” when they were >3× longer than a typical bacterium (approximately >5 μm). Three populations were scored: the number of infected cells containing predominantly filamentous bacteria, the number of infected cells where >50% of intracellular bacteria were of normal size, and the number of infected cells containing bacteria that were all of normal size. Significance was determined by calculating p values using an unpaired two-tailed t test. The level of terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive (Roche Molecular Biochemicals, Laval, Quebec, Canada) apoptotic cells was less than 10% for all conditions. At various times post-infection, IFN-γ-primed RAW 264.7 cells were washed once with PBS and scraped to detach the cells from the dish. RNA was then isolated using Trizol according to the manufacturer's directions (Invitrogen). RNA was extracted twice with phenol:chloroform:isoamyl alcohol (25:24:1) and once with chloroform. The RNA was then precipitated with 2.5 volumes of 100% ethanol and 0.10 volume sodium acetate, pH 5.2, resuspended in RNase-free water containing RNase inhibitor (Ambion, Austin, TX), and stored at −70 °C. RNA quality was assessed by gel electrophoresis and staining with ethidium bromide. Northern blots were prepared as described previously, using 5–10 μg of total RNA per lane (16Rosenberger C.M. Scott M.G. Gold M.R. Hancock R.E. Finlay B.B. J. Immunol. 2000; 164: 5894-5904Crossref PubMed Scopus (186) Google Scholar). To prepare templates for probe synthesis, cDNA was prepared from total RNA purified from RAW 264.7 cells using oligo(dT) and SuperScriptII reverse transcriptase (Invitrogen). The following primer pairs were designed to amplify portions of the indicated macrophage genes: MEK1, 5′-GTTGCTTTCAGGCCTCTCC-3′, 5′-AGTGATGGGCTCTGCTTAGG-3′; GAPDH, 5′-AGAACATCATCCCTGCATCC-3′, 5′-CTGGGATGGAAATTGTGAGG-3′. Antisense cDNA probes were prepared by PCR using 50 ng of the appropriate PCR product template, the reverse 3′-oligonucleotide, and modified nucleotides to facilitate repeated stripping of blots (Strip-EZ PCR, Ambion). These single-stranded PCR products were column-purified (Qiagen, Mississauga, Ontario, Canada) and labeled with biotin using psoralen-biotin (Ambion) and cross-linking with 365 nm ultraviolet light. Overnight hybridization at 42 °C was with labeled probe in UltraHyb (Ambion). The BrightStar non-isotopic detection kit (Ambion) was used for probe detection according to the manufacturer's protocols. Northern blots were quantified by densitometry using an AlphaImager system (Alpha Innotech Co., San Leandro, CA). AtlasTM Mouse cDNA Expression Arrays I (7741-1; CLONTECH, Palo Alto, CA) consist of a matched set of positively charged membranes containing duplicate spots of 588 murine partial cDNAs.32P-Radiolabeled first strand cDNA probes were prepared from 5 μg of total RNA from each cell population using Moloney murine leukemia virus transcriptase and pooled primers specific for the 588 genes. Hybridization conditions and data analysis have been described previously (16Rosenberger C.M. Scott M.G. Gold M.R. Hancock R.E. Finlay B.B. J. Immunol. 2000; 164: 5894-5904Crossref PubMed Scopus (186) Google Scholar). IFN-γ-primed RAW 264.7 cells (5 × 105/well) were seeded in six-well tissue culture plates and incubated overnight. At various times post-infection, cells were collected into 100 μl of boiling 5× SDS-PAGE loading buffer. Total protein lysates were resolved on a 12% acrylamide SDS-PAGE gel, electrotransferred to nitrocellulose membrane, and blocked with 5% skim milk in Tris-buffered saline-0.1% (v/v) Tween 20. Antibodies were used at the following concentrations: rabbit anti-MEK1, 1:1000 (New England Biolabs, Beverly, MA); rabbit anti-phosphorylated MEK1, 1:1000 (New England Biolabs; kindly provided by Dr. B. Ellis, University of British Columbia); rabbit anti-ERK, 1:2000 (New England Biolabs, Beverly, MA); monoclonal phosphospecific anti-p44/p42 (ERK1/2), 1:2000 (New England Biolabs); and monoclonal anti-actin 1:15,000 (ICN, Montreal, Quebec, Canada). Blots were incubated with primary antibodies overnight at 4 °C, followed by horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature and detected by enhanced chemiluminescence (Amersham Biosciences, Baie d'Urfé, Quebec, Canada). Western blots were quantified by densitometry using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). IFN-γ-primed RAW 264.7 cells were pretreated with inhibitors for 30 min prior to infection at the following concentrations: 50 μm PD98059 (Calbiochem), 50 μm U0126 (Promega, Madison, WI), 4 μmdiphenyleneiodonium (DPI; Sigma), 250 μm acetovanillone (apocynin; Aldrich), 1 mm ascorbic acid (Sigma), 30 mmN-acetylcysteine (Sigma), 2 mmNG-l-monomethylarginine (l-NMMA, Molecular Probes), or 2 mmNG-d-monomethylarginine (d-NMMA, Molecular Probes). Fresh inhibitors were added immediately after infection, at 2 h, and 6–8 h post-infection to ensure potency. Control cells were treated with equivalent volumes of dimethyl sulfoxide (Me2SO) per ml of media. To remove inhibitors from pretreated cells, monolayers were washed three times with PBS at 8 h post-infection and then cultured for 16 h in DMEM containing 10% FBS and 10 μg/ml gentamicin. Intracellular ROIs were quantified by a luminol-enhanced chemiluminescence assay as described previously (17Dahlgren C. Karlsson A. J. Immunol. Methods. 1999; 232: 3-14Crossref PubMed Scopus (656) Google Scholar, 18Karlsson A. Nixon J.B. McPhail L.C. J. Leukoc. Biol. 2000; 67: 396-404Crossref PubMed Scopus (170) Google Scholar). Briefly, 1 × 106 IFN-γ-primed RAW 264.7 cells were seeded per well in six-well tissue culture plates and primed with IFN-γ for 24 h. Cells were pretreated with inhibitors or Me2SO in media and infected as described above. After 6 or 24 h of infection, cells were washed once with PBS, scraped into 200 μl of substrate warmed to 37 °C (PBS containing 10% heat-inactivated FBS, 5 × 10−5m luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, Sigma) as an indicator of ROIs, and 50 units/ml superoxide dismutase (Sigma) and 2000 units/ml catalase (Sigma) to remove extracellular ROIs). Duplicate samples of 100 μl each were transferred to a clear-bottomed white 96-well plate, and chemiluminescence (light) units were quantified for 20 min using a TECAN spectrophotometer/luminometer (Männedorf, Switzerland), and the light units detected per minute over this time period were calculated. Nitrite concentration in extracellular medium of infected cells after 24 h was measured using a Griess reagent kit (Molecular Probes) according to the manufacturer's instructions. IFN-γ is essential for clearance of Salmonella Typhimurium within the murine typhoid model, and we have shown previously that IFN-γ has pleiotropic effects on macrophage transcriptional responses at early times to Salmonella Typhimurium infection (16Rosenberger C.M. Scott M.G. Gold M.R. Hancock R.E. Finlay B.B. J. Immunol. 2000; 164: 5894-5904Crossref PubMed Scopus (186) Google Scholar). To establish a model for investigating macrophage responses that are effective in restricting Salmonella Typhimurium replication, we assessed the effect of IFN-γ on the ability of RAW 264.7 macrophage-like cells to control intracellular numbers of Salmonella Typhimurium. As shown in Fig. 1A, the number of intracellular Salmonella Typhimurium increased 6-fold over a 22.5-h period in RAW 264.7 cells. These cells permit Salmonella Typhimurium replication after 4–8 h, although avoidance of macrophage-mediated killing could partially contribute to the increase. In contrast, intracellular bacterial numbers did not increase in RAW 264.7 cells primed with IFN-γ over this same period (Fig. 1A). We hypothesized that IFN-γ-primed RAW 264.7 cells provide a more relevant model for studying macrophage responses that are effective in limiting Salmonella Typhimurium infection, as host factors should be maximally expressed in IFN-γ-primed RAW 264.7 cells that restrict intracellular bacterial numbers. This choice of model was strengthened by the observation that both primary murine macrophages and IFN-γ-primed RAW 264.7 cells restrict the intracellular load of Salmonella Typhimurium (19Buchmeier N.A. Heffron F. Infect. Immun. 1989; 57: 1-7Crossref PubMed Google Scholar). IFN-γ priming of macrophages restricts intracellular Salmonella Typhimurium replication, but the precise mechanisms for this control are unclear (20Shtrichman R. Samuel C.E. Curr. Opin. Microbiol. 2001; 4: 251-259Crossref PubMed Scopus (258) Google Scholar). To better understand the interactions between macrophages and Salmonella Typhimurium, IFN-γ-primed RAW 264.7 cells were infected with Salmonella Typhimurium expressing green fluorescent protein (GFP) and examined by fluorescence microscopy. While intracellular bacteria exhibited normal morphology after 8 h of infection, 47 ± 12% of infected cells contained filamentous bacteria that were >3× the length of a typical bacterium after 24 h (Fig. 1B). Filamentous bacteria were observed using another Salmonella Typhimurium strain (14028s), indicating that filamentation is shared by more than one strain of Salmonella Typhimurium (data not shown). Filamentous bacteria had partial or absent septa, suggesting a defect in completion of cell division, an indicator of bacterial stress (21Weiss D.S. Chen J.C. Ghigo J.M. Boyd D. Beckwith J. J. Bacteriol. 1999; 181: 508-520Crossref PubMed Google Scholar, 22Hill T.M. Sharma B. Valjavec-Gratian M. Smith J. J. Bacteriol. 1997; 179: 1931-1939Crossref PubMed Google Scholar). To examine whether macrophage gene expression was responsible for this effect on bacterial replication at later infection times, we used gene array analysis to profile the transcriptional responses of IFN-γ-primed RAW 264.7 cells to intracellular SalmonellaTyphimurium after infection for 4, 8, and 24 h. Hybridization of cDNA arrays indicated that MEK1 kinase mRNA levels were elevated in IFN-γ-primed RAW 264.7 cells at 8 h post-infection but not at 4 h post-infection (data not shown). This observed modest increase in MEK1 mRNA after 8 h of Salmonella Typhimurium infection was confirmed by Northern blot analysis. As seen in Fig. 2A, MEK1 mRNA was transiently up-regulated at 8 h and 18 h post-infection, was not observed prior to 8 h, and reduced to the level in uninfected cells at 24 h (n = 3). The increase in MEK1 mRNA 8 h after infection ranged from 1.2- to 2.5-fold relative to uninfected cells in each of eight experiments (Fig. 2B). We observed similar kinetics in the increase in MEK1 mRNA abundance in cells stimulated with 1 μg/ml Salmonella Typhimurium lipopolysaccharide for 8 or 24 h (LPS; data not shown). This increase in MEK1 mRNA after infection for 8 h was abrogated when cells were treated with the MEK kinase inhibitor U0126 prior to infection, suggesting that transcriptional up-regulation of MEK1 is mediated by prior kinase activity (Fig. 2A and quantification in Fig. 2B). While activation of MEK/ERK kinase cascades have previously been shown to occur within 1 h of Salmonella Typhimurium infection or LPS stimulation (2Procyk K.J. Kovarik P. von Gabain A. Baccarini M. Infect. Immun. 1999; 67: 1011-1017Crossref PubMed Google Scholar), MEK kinase activity at much later times of infection or its transcriptional regulation following infection has not previously been reported. The observed elevation of MEK1 mRNA level in Salmonella Typhimurium-infected IFN-γ-primed RAW 264.7 cells relative to uninfected cells was followed by increased MEK1 protein abundance at 24 h, as determined by Western blot analysis (Fig. 2C). A modest increase in MEK1 protein of 1.5 ± 0.2-fold was detected at 24 h post-infection when normalized to actin protein and relative to uninfected cells (n = 4). This is of a comparable magnitude to the induction of MEK mRNA at 8 h post-infection. At the times when increases in MEK mRNA and protein abundance were measured, MEK protein was phosphorylated, an essential step in activation of MEK kinase. MEK phosphorylation was maximal at 1 h but remained sustained at a modest level in infected cells throughout 24 h, as seen in longer exposures of Western blots (Fig. 2C and data not shown). MEK activity could be detected throughout the infection period, as measured by Western blot analysis of phosphorylation of its downstream targets, the ERK1/2 kinases. As seen in Fig. 3,A–C, phosphorylation of ERK1/2 was maximal within 1 h following stimulation, but phosphorylation remained elevated throughout the 24-h period examined when compared with uninfected cells (quantification of ERK2 phosphorylation in Fig. 3D). MEK1 abundance and activity were similar in IFN-γ-primed RAW 264.7 cells infected by Salmonella Typhimurium or stimulated with 1 μg/ml purified Salmonella Typhimurium LPS over 24 h. Therefore, up-regulated MEK1 mRNA, protein, and activity in IFN-γ-primed RAW 264.7 cells during a 24-h infection by S almonella Typhimurium ca" @default.
• W1972189052 created "2016-06-24" @default.
• W1972189052 creator A5030919684 @default.
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• W1972189052 date "2002-05-01" @default.
• W1972189052 modified "2023-09-29" @default.
• W1972189052 title "Macrophages Inhibit Salmonella Typhimurium Replication through MEK/ERK Kinase and Phagocyte NADPH Oxidase Activities" @default.
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• W1972189052 doi "https://doi.org/10.1074/jbc.m110649200" @default.
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