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The Journal of Immunology, 2004, 173: 4058-4065.
Copyright © 2004 by The American Association of Immunologists

Salmonella Escape from Antigen Presentation Can Be Overcome by Targeting Bacteria to Fc{gamma} Receptors on Dendritic Cells1

Jaime A. Tobar, Pablo A. González and Alexis M. Kalergis2

Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DCs) are professional APCs with the unique ability to activate naive T cells, which is required for initiation of the adaptive immune response against pathogens. Therefore, interfering with DC function would be advantageous for pathogen survival and dissemination. In this study we provide evidence suggesting that Salmonella enterica serovar typhimurium, the causative agent of typhoid disease in the mouse, interferes with DC function. Our results indicate that by avoiding lysosomal degradation, S. typhimurium impairs the ability of DCs to present bacterial Ags on MHC class I and II molecules to T cells. This process could correspond to a novel mechanism developed by this pathogen to evade adaptive immunity. In contrast, when S. typhimurium is targeted to Fc{gamma}Rs on DCs by coating bacteria with Salmonella-specific IgG, bacterial Ags are efficiently processed and presented on MHC class I and class II molecules. This enhanced Ag presentation leads to a robust activation of bacteria-specific T cells. Laser confocal microscopy experiments show that virulent S. typhimurium is rerouted to the lysosomal degradation pathway of DCs when internalized through Fc{gamma}R. These observations are supported by electron microscopy studies demonstrating that internalized S. typhimurium shows degradation signs only when coated with IgG and captured by Fc{gamma}Rs on DCs. Therefore, our data support a potential role for bacteria-specific IgG on the augmentation of Ag processing and presentation by DCs to T cells during the immune response against intracellular bacteria.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DCs)3 are professional APCs that have the unique ability to capture Ags and trigger the immune response by activating naive CD4+ and CD8+ T cells (1, 2, 3, 4). Therefore, they play an important role for the initiation of the adaptive immune response against infectious agents, such as bacteria. By recognizing pathogen-associated molecular patterns through specialized receptors, DCs detect the presence of bacteria in the host tissues, capturing them and their products, and migrate to lymph nodes to present bacterial Ags on MHC molecules to T cells. In response to pathogen-associated molecular patterns, DCs mature undergoing physiological changes that involve increased surface density of MHC and costimulatory molecules, which enhance them to activate Ag-specific naive T cells (1, 3). Because the immune response imposes a selective pressure on the pathogen, interference with DC function would be advantageous for microbial survival. Thus, pathogens have developed molecular mechanisms to interfere with the DC ability to activate T cells (5, 6, 7, 8). Characterizing these mechanisms is important for the design of efficient vaccines directed to confer protective antimicrobial immunity.

The efficiency of Ag uptake, processing, and presentation on MHC molecules can be significantly enhanced by targeting Ags to specific receptors on the surface of DCs (4, 9, 10, 11, 12, 13). One of such approaches consists of delivering Ags as immune complexes (ICs) to FcRs on DCs (10, 13, 14). Although it has been reported that these cells express most FcRs (Fc{gamma}RI, Fc{gamma}RIIb, Fc{gamma}RIII, Fc{epsilon}RI, Fc{epsilon}RII, and Fc{alpha}R) (15, 16, 17, 18), uptake of IgG-ICs by Fc{gamma}Rs is particularly efficient at enhancing Ag processing and presentation for both MHC class I and class II molecules by DCs (10, 13, 14). DCs express both activating (Fc{gamma}RIII) and inhibitory (Fc{gamma}RIIb) low affinity Fc{gamma}Rs (10, 14); however, the roles of Fc{gamma}Rs in bacterial Ag capture, processing, and presentation by DCs to T cells have not been determined.

In this study, we evaluated the capacity of DCs to present intracellular bacteria-derived Ags to T cells. The importance of T cell immunity for the clearance of intracellular bacterial infections has been demonstrated in several systems in which a deficiency of T cell function increases susceptibility to infection. Due to its ability to evade the mouse immune response, causing a typhoid-like disease, Salmonella enterica serovar typhimurium (S. typhimurium) provides a unique opportunity to test the ability of these intracellular bacteria to interfere with activation of T cells by DCs. Our data indicate that despite inducing DC maturation, virulent S. typhimurium blocks presentation of bacteria-derived Ags on both class I and class II MHC molecules to T cells. Consistent with this observation is the absence of colocalization between virulent S. typhimurium and lysosomal glycoproteins, suggesting that virulent S. typhimurium prevents Ag processing on DCs by avoiding the lysosomal degradation pathway.

However, this pathogenic mechanism of evasion fails to operate when bacteria are coated with S. typhimurium-specific IgG and captured by DCs through Fc{gamma}Rs. In this case, bacterial Ags are efficiently internalized and processed by DCs, leading to robust T cell activation. We observe that IgG-coated virulent S. typhimurium is rerouted to the lysosomal degradation pathway on DCs by Fc{gamma}Rs. These observations are supported by electron microscopy studies demonstrating that internalized S. typhimurium shows signs of bacterial degradation only when coated with IgG.

Our data show that by preventing Ag processing and presentation of bacterial Ags, virulent S. typhimurium can impair T cell activation by DCs. We provide a new model for bacterial Ag delivery to DCs that enhances T cell activation and could improve the design of vaccines aimed to confer protective immunity against intracellular bacteria.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6 mice were purchased from and maintained at the animal facilities of Pontificia Universidad Católica de Chile (Santiago, Chile). Animal work was performed according to institutional guidelines.

Bacterial strains

Pathogenic Salmonella enterica serovar typhimurium (ATCC 14028s; American Type Culture Collection, Manassas, VA) was provided by G. Mora (Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile). For Ag presentation assays, S. typhimurium-pOVA was generated by transforming 14028s with the pOVA plasmid. pOVA is a pUC-derivative encoding the full-length sequence for chicken egg OVA under the lac promoter and was provided by M. J. Wick (19). OVA-expressing the PhoP constitutive (PhoPc) S. typhimurium strain was a gift from J.-C. Sirard (University of Lausanne, Lausanne, Switzerland) and was described previously (20, 21, 22). This is an attenuated strain derived from 14028s by introducing a point mutation on the phoQ gene (21, 22). This point mutation leads to either constitutive expression or constitutive repression of PhoP-regulated virulence genes involved in invasion and intracellular survival. Genes encoded by the Salmonella pathogenicity island-1, such as those coding for the type three secretion system (invJICBAEG, prgKJIH, and spaSRQPO) and effector molecules (sopE, sopB, and sopD) are repressed by the mutation. The PhoPc mutation renders bacteria impaired for invasion of mammalian cells and survival in macrophages, which has pleiotropic effects on S. typhimurium pathogenesis, leading to attenuated virulence in mice (23).

OVA expression by S. typhimurium was confirmed by Western blot analysis (data not shown). For flow cytometry and confocal microscopy experiments, S. typhimurium-pGFP was generated by transforming 14028s with pGFP plasmid (BD Clontech, Palo Alto, CA). Bacteria were grown on Luria Bertani (LB) broth, and recombinant bacteria were selected on carbenicillin (50 µg/ml; Sigma-Aldrich, St. Louis, MO). Overnight cultures were subcultured starting a 1/100 dilution until reaching exponential phase (OD600 nm = 0.6). To evaluate infectivity for each S. typhimurium strain, DCs were infected at a multiplicity of infection (MOI) of 50 for 4 h, treated with 50 µg/ml gentamicin for 30 min to kill extracellular bacteria, permeabilized for 30 min with 0.1% Triton X-100 in PBS, and plated on LB-agar. For generation of bacteria-immune complexes, rabbit anti-Salmonella IgG (0.5 mg/ml; Denka-Seiken, Tokyo, Japan) was added and incubated for 6–8 h at 4°C. MOI and viability of bacteria-ICs were confirmed by serial dilutions on agar plates.

Ag presentation assays

Bone marrow-derived DCs were prepared as previously described (10, 24). Briefly, DCs were grown from bone marrow progenitors in RPMI 1640 containing 5% FCS supplemented at 3% (v/v) with supernatant from J558L cells transduced with murine GM-CSF. Day 6 DCs were pulsed for 4 h with either free or IgG-coated S. typhimurium- or PhoPc-pOVA, at an MOI of 50. DC cultures were routinely analyzed by flow cytometry for the expression of surface markers CD11c, I-Ab, H-2Kb, CD80, CD86, and CD40, revealing an immature phenotype (Fig. 1A and data not shown). After the pulse, DCs were washed and treated with gentamicin (50 µg/ml; Sigma-Aldrich) to eliminate extracellular bacteria, as previously described (25, 26, 27). After an additional 12-h culture, DC viability was determined by trypan blue exclusion, and live DCs were cocultured at different amounts with either 1 x 105 B3Z or 1 x 105 OT4H.2D5 (OT4H) T cell hybridomas. B3Z and OT4H.2D5 are specific for H-2Kb/OVA257–264 and I-Ab/OVA265–280, respectively, and secrete IL-2 upon TCR stimulation (28, 29, 30). After 20 h of DC-T cell coculture, IL-2 release was measured by cytokine ELISA as previously described (31, 32, 33). DC viability was evaluated 4 and 16 h after the Salmonella pulse. For low affinity Fc{gamma}R (CD16/CD32) or Fc{gamma}RIIB (CD32) blockade, before the Salmonella pulse, DCs were incubated for 30 min with 10 ng/ml 2.4G2 (Fc Block; BD Pharmingen, San Diego, CA) or Ly17.2 (supernatant from K9.361 hybridoma) mAbs, respectively.



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  		FIGURE 1. Virulent S. typhimurium prevents T cell activation by dendritic cells. A, DCs efficiently internalize S. typhimurium. Bone marrow-derived DCs were evaluated for the expression of surface markers CD11c, CD40, CD86, and I-Ab (left panel). These cells were incubated either with GFP-expressing S. typhimurium (right panel, thin line) or with control S. typhimurium (right panel, shaded), and green fluorescence was determined for CD11c+ DCs. B, Only virulent S. typhimurium prevented T cell activation by DCs. DCs were incubated with virulent S. typhimurium-pOVA ({circ}) or with attenuated S. typhimurium PhoPc-pOVA (•), washed, and cocultured with T cell hybridomas specific for either H-2Kb/OVA257–264 (B3Z; left panel) or I-Ab/OVA265–280 (OT4H; right panel). C, Virulent S. typhimurium does not prevent T cell activation by DCs pulsed with exogenous Ag. DCs were incubated with S. typhimurium-pOVA plus OVA257–264 or OVA265–280 peptides (•) or with whole OVA protein ({circ}), washed, and cocultured with T cell hybridomas specific for either H-2Kb/OVA257–264 (B3Z; left panel) or I-Ab/OVA265–280 (OT4H; right panel). D, Virulent and attenuated S. typhimurium strains show similar infectivity on DCs and do not significantly alter DC viability. Left panel, DCs were infected with virulent or attenuated S. typhimurium for 4 h, treated with gentamicin, and plated on LB-agar. Right panel, DCs were infected with virulent ({cjs2108}) or attenuated ({blacksquare}) S. typhimurium and evaluated for viability using trypan blue exclusion 4 and 16 h after Salmonella pulse. Data shown are means from five independent experiments, and error bars represent SDs.

 
Flow cytometry

All analyses were performed on a FACScan flow cytometer (BD Biosciences, Mountain View, CA). For Salmonella internalization experiments, DCs were pulsed with free or IgG-coated S. typhimurium-pGFP for 4 h. At the end of this period, DCs were treated with gentamicin as described above and were washed at 4°C to eliminate extracellular bacteria. This treatment has been shown to significantly reduce surface-bound green fluorescence (25, 26, 27) (data not shown). Then, DCs were stained with anti-CD11c-PE (clone HL3; BD Pharmingen), fixed on paraformaldehyde (2%, PBS), and analyzed by FACS. To evaluate MHC class I expression on DCs, control or S. typhimurium-pulsed DCs were double-stained with anti-CD11c-PE plus anti-H-2Kb-FITC (AF6-88.5; BD Pharmingen). To determine the density of H-2Kb/SIINFEKL complexes on the surface of DCs, cells pulsed with either free or IgG-coated S. typhimurium were stained with anti-CD11c-PE and supernatant from 25-D1.16 hybridoma ({kappa}-IgG1 mAb, specific for H-2Kb/SIINFEKL complex; provided by Dr. R. N. Germain, National Institute of Allergy and Infectious Diseases/National Institutes of Health, Bethesda, MD) (34). After washing, cells were stained with goat anti-mouse IgG-FITC (BD Pharmingen) and analyzed by FACS. Flow cytometry data were analyzed using WinMDI software (http://facs.scripps.edu).

Laser confocal microscopy

DCs pulsed with free or IgG-coated S. typhimurium-pGFP were stained with anti-CD11c-PE (clone HL3; BD Pharmingen). DCs were washed and fixed as described above. To detect lysosomes, fixed DCs were permeabilized for 20 min with Triton X-100 (0.5% Triton in 5% FCS-PBS) and incubated for 30 min on ice with purified rat anti-mouse lysosome-associated membrane protein 1 (LAMP-1) mAb (clone 1D4B; BD Pharmingen). After washing, cells were stained with Alexa Fluor 568-anti-rat IgG2a{kappa}, (clone R35-95; BD Pharmingen) for 30 min on ice and washed. Stained DCs were examined on an LSM 5 Pascal confocal microscope (Zeiss, Thornwood, NY). Fluorescence extension was plotted using LSM 5 Image examiner software. Semiquantitative analysis was performed by counting the number of DCs showing Salmonella-LAMP-1 colocalization on several fields that were selected randomly.

Electron microscopy

DCs pulsed with free or IgG-coated S. typhimurium were fixed overnight in PLP (4% paraformaldehyde, 0.01 M periodate, and 0.2 M L-lysine on 0.1 M phosphate buffer, pH 7.4). The samples were rinsed in distilled water and postfixed for 30 min at 4°C on 1% osmium tetroxide, dehydrated in ethanol and acetone, and embedded in Epon. Thin sections were cut with an OmU2 ultramicrotome (Reichert, Vienna, Austria) and were observed under a Tecnai 21 electron microscope (Phillips, Eindhoven, The Netherlands).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Virulent S. typhimurium interferes with presentation of bacterial Ags by DCs to T cells

Although they were readily infected by virulent Salmonella (Fig. 1A), DCs were unable to process and present peptides derived from OVA protein expressed by the bacteria to OVA-specific T cells. Thus, neither the H-2Kb/OVA-specific CD8+ B3Z cell line, nor the I-Ab/OVA-specific CD4+ OT4H cell line was activated by DCs infected with virulent S. typhimurium-pOVA (Fig. 1B). However, infection with virulent S. typhimurium did not interfere with the ability to activate T cells by DCs when pulsed with exogenously added MHC class I- or MHC class II-restricted peptides (Fig. 1C). In addition, S. typhimurium was only able to prevent OVA presentation to T cells when the protein was expressed by the infecting bacteria and not when the protein accompanied infection as bystander-soluble OVA (Fig. 1C). This observation suggests that only vacuoles containing virulent S. typhimurium were resistant to Ag processing and also that there is no inhibition of general DC function by Salmonella. In support of this idea, the ability to block processing and presentation of bacterial OVA was not observed for the attenuated PhoPc strain (Fig. 1B), which is deficient for the expression of the genes required for virulence that are contained in the Salmonella pathogenicity island-1. Consistent with previous observations (20), incubation of DCs with OVA-expressing PhoPc led to the activation of OVA-specific T cells (Fig. 1B). No significant changes in DC viability were observed after infection with virulent or attenuated Salmonella, and both strains showed equivalent ability to infect DCs (Fig. 1D). These results imply that inhibition of Ag processing is restricted to virulent strains of S. typhimurium, suggesting that virulence can in part be influenced by the ability to interfere with Ag presentation by DCs.

Targeting S. typhimurium to Fc{gamma}Rs on DCs restores presentation of bacterial Ags to T cells

Due to the fact that the efficiency of Ag uptake and processing can be enhanced by targeting Ags to Fc{gamma}Rs on DCs (10, 13, 14), we evaluated whether the ability of S. typhimurium to evade Ag processing on DC could be affected by directing bacteria to Fc{gamma}Rs. Thus, immune complexes containing bacteria were generated by incubating S. typhimurium with anti-Salmonella rabbit IgG, which is known to bind mouse Fc{gamma}Rs (35). After 4 h of incubation, capture of ICs containing viable bacteria by DCs was confirmed both by flow cytometry (Fig. 2A) and by plating DC lysates after gentamicin protection assay (data not shown).



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  		FIGURE 2. Targeting virulent S. typhimurium to Fc{gamma}R on DCs restores activation of bacteria-specific T cells. A, DCs efficiently internalize IgG-coated S. typhimurium. DCs were incubated with S. typhimurium-pGFP coated with S. typhimurium-specific IgG for 4 h. After this period, cells were treated with 50 µg/ml gentamicin, stained with anti-CD11c-PE, and analyzed by FACS. The histograms show green fluorescence for CD11c+ DCs incubated with GFP-expressing S. typhimurium (thin line) or with control S. typhimurium (shaded). B, T cell activation by DCs incubated with IgG-coated virulent S. typhimurium. DCs were incubated with IgG-coated S. typhimurium-pOVA for 4 h. After washing, DCs were cocultured with T cell hybridomas specific for H-2Kb/OVA257–264 (left panel) or I-Ab/OVA265–280 (right panel; •). For simultaneous blockade of Fc{gamma}RIIb and Fc{gamma}RIII, DCs were pretreated with 2.4G2 mAb ({circ}). For blockade of Fc{gamma}RIIb alone, DCs were pretreated with Ly17.2 mAb ({square}). Activation was determined by measuring IL-2 release with a cytokine ELISA. Data shown are means from four independent experiments, and error bars represent SDs.

 
In contrast with the results obtained with free virulent S. typhimurium-pOVA, DCs pulsed with ICs containing virulent S. typhimurium-pOVA induced efficient activation of T cells. As shown in Fig. 2B, capture of IgG-coated S. typhimurium-pOVA by DCs led to activation of both H-2Kb/OVA- and I-Ab/OVA-specific T cells. These findings indicate that presentation of S. typhimurium-derived Ags on MHC class I and class II molecules is restored when DCs capture bacteria in the form of ICs. This process was mediated by Fc{gamma}R IC uptake, because treatment of DCs with 2.4G2 (an mAb that blocks Fc{gamma}RIIb and Fc{gamma}RIII) before the pulse with ICs abrogated T cell activation (Fig. 2B). In contrast, activation of T cells was not significantly affected when DCs were treated with Ly17.2 (an mAb that selectively binds to Fc{gamma}RIIb) (36), suggesting that internalization of ICs was mainly mediated by Fc{gamma}RIII (Fig. 2B).

Virulent S. typhimurium prevents generation of pMHC complexes loaded with bacterial peptides

To determine the basis for the absence of activation of OVA-specific T cells by DCs infected with virulent S. typhimurium-pOVA, we measured the density of H-2Kb/SIINFEKL complexes on the surface of bacteria-infected DCs with a mAb specific for this particular pMHC complex (34). As shown in Fig. 3, A and D, H-2Kb/SIINFEKL complexes could be detected on DCs infected with attenuated S. typhimurium (PhoPc-pOVA), indicating that bacteria-derived OVA can be cross-presented on MHC class I. On the contrary, H-2Kb/SIINFEKL complexes were not detectable on the surface of DCs infected with virulent S. typhimurium-pOVA (Fig. 3, B and D). Interference with generation of H-2Kb/SIINFEKL complexes was specific for bacteria-derived OVA, because such pMHC complexes were present at the surface of DCs infected with virulent S. typhimurium-pOVA and pulsed simultaneously with soluble OVA (Fig. 3D). In addition, the absence of H-2Kb/SIINFEKL complexes from the surface of DCs infected with virulent S. typhimurium was not due to a reduction of surface levels of H-2Kb molecules, which, on the contrary, increased or did not change after S. typhimurium infection (Fig. 3, A–C).



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  		FIGURE 3. MHC molecules loaded with bacterial peptides are present on the DC surface only when virulent S. typhimurium is internalized via Fc{gamma}Rs. DCs were incubated for 4 h with either OVA-expressing S. typhimurium PhoPc (A) or wild-type S. typhimurium free (B) or coated with anti-S. typhimurium IgG (C). After incubation, DCs were treated with 50 µg/ml gentamicin, washed, and analyzed by flow cytometry after 12 h. Histograms show green fluorescence from either anti-H-2Kb (thin line, left panels) or anti-H-2Kb/SIINFEKL complex (thin line, right panels), gating on CD11c+ cells. Control DCs without S. typhimurium infection and autofluorescence controls are shown as filled histograms and dotted lines, respectively (one representative result is shown of five independent experiments). D, Bar graph showing the percentage of CD11c+ cells positive for H-2Kb/SIINFEKL complexes incubated with S. typhimurium-pOVA ({blacksquare}), S. typhimurium-pOVA plus 100 µg/ml soluble OVA ({cjs2113}), S. typhimurium-pOVA-IgG ({cjs2108}), PhoPc-pOVA ({cjs2098}), or unpulsed DCs ({square}). Data shown are means of four independent experiments, and error bars represent SDs.

 
Fc{gamma}R-mediated bacteria internalization by DCs restores generation of pMHC complexes containing bacterial peptides

The ability of virulent S. typhimurium to prevent the generation of H-2Kb/SIINFEKL complexes was no longer operative when bacteria were coated with IgG and internalized by DCs through Fc{gamma}Rs. As shown in Fig. 3, C and D, H-2Kb/SIINFEKL complexes were observed at the surface of DCs incubated with IgG-coated S. typhimurium-pOVA. Consistent with previous observations made using ICs containing purified protein Ags (10, 14), these data indicate that targeting bacteria to Fc{gamma}Rs enhances the ability of DCs to cross-present peptides derived from bacterial proteins on MHC class I.

Evasion of lysosomal degradation as a mechanism to prevent processing of S. typhimurium Ags by DCs

On macrophages, S. typhimurium has been shown to prevent fusion of bacteria containing vacuole with lysosomes (19, 37, 38). This mechanism of pathogenicity is important to avoid destruction by the macrophage oxidative burst response and degradation of bacterial proteins by lysosomal proteolysis. Although it was recently reported that S. typhimurium-containing vacuoles are devoid of lysosomal glycoproteins in a DC-like cell line (39), the biological significance of this finding remained unknown. To test whether the inability of bone marrow-derived DCs to present bacterial Ags to T cells after S. typhimurium infection was due to avoidance of lysosome fusion with bacteria-containing vacuoles, we determined whether S. typhimurium colocalizes with the lysosomal marker LAMP-1. As shown in Fig. 4, A, C, E, and G, S. typhimurium-pGFP did not colocalize with LAMP-1, indicating that in DCs, vacuoles containing bacteria are protected from lysosomal degradation. Analysis of confocal imaging data indicated that only 11.2% of DCs showed Salmonella-LAMP-1 colocalization (Fig. 4G, upper panel, and Fig. 4H). This result is consistent with the observation that DCs are unable to process S. typhimurium-derived proteins for presentation on MHC molecules.



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  		FIGURE 4. Virulent S. typhimurium is targeted for lysosomal degradation only when bacteria is internalized via Fc{gamma}Rs on DCs. DCs were incubated with free (A, C, E, and G) or IgG-coated (B, D, F, and G) S. typhimurium-pGFP at an MOI equal to 50 for 4 h, treated with gentamicin, and incubated for 12 h. Next, DCs were permeabilized and stained with goat anti-mouse LAMP-1 for 30 min, washed, and stained with Alexa Fluor 568-conjugated anti-goat IgG. After fixation, cells were analyzed by laser confocal microscopy. S. typhimurium-pGFP (A and B) and LAMP-1 of a DC (C and D) are at the same focal plane (1-µm width; bars = 10 µm). Merged images are shown in E and F, and fluorescence extension analyses for E and F are shown in G (green and red peaks correspond to GFP and LAMP-1 signals, respectively). H, A semiquantitative analysis of DCs showing Salmonella pGFP-LAMP-1 colocalization. All analyses were performed using Zeiss LSM 5 Examiner software.

 
Fc{gamma}R targeting restores presentation of S. typhimurium Ags to T cells by rerouting bacteria to the lysosomal degradation pathway

To evaluate the mechanism responsible for restoration of the ability of DC to process and present bacterial Ags to T cells by Fc{gamma}R-mediated capture, we determined the intracellular localization of S. typhimurium internalized through Fc{gamma}R by DCs. For these experiments, DCs were pulsed with IgG-coated S. typhimurium-pGFP, and after incubation, cells were permeabilized and stained with an anti-LAMP-1 mAb for confocal microscopic analysis. As shown in Fig. 4, B, D, F, and G, S. typhimurium-pGFP colocalized with LAMP-1-containing vesicles when DCs were pulsed with IgG-coated S. typhimurium-pGFP. Analysis of confocal imaging data indicated that 80.1% of DCs showed colocalization (Fig. 4G, lower panel, and Fig. 4H). These results contrast with the absence of significant Salmonella-LAMP-1 colocalization observed when DCs were pulsed with free S. typhimurium-pGFP (Fig. 4, A, C, E, and G). The idea that Fc{gamma}R internalization reroutes virulent Salmonella for lysosomal degradation is consistent with these data.

S. typhimurium shows signs of bacterial degradation only when internalized by DC through Fc{gamma}Rs

Consistent with the idea that virulent S. typhimurium is able to evade lysosomal degradation, electron micrographs of S. typhimurium-infected DCs show bacteria residing inside large vacuoles with intact ultrastructure and no signs of degradation (Fig. 5, A and B). Large and spacious vacuoles containing S. typhimurium in macrophages have been associated with pathogenicity and bacterial ability to prevent lysosomal degradation (40). In this study, we provide evidence for similar structures in DCs, which could ensure the survival of virulent S. typhimurium inside DCs, preventing processing and presentation of bacterial Ags to T cells.



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  		FIGURE 5. Only Fc{gamma}R-internalized S. typhimurium shows degradation signs inside DC vacuoles. Transmission electron micrographs of DCs incubated with free (A and B) or IgG-coated (C and D) virulent S. typhimurium. DCs were incubated with wild-type S. typhimurium (14028s) at an MOI of 50 for 4 h, washed, and cultured with 50 µg/ml gentamicin for 12 h. DCs were processed for microscopy as described in Materials and Methods. Boxed areas from A and C (magnification, x6800; bars = 500 nm) are magnified to x30,000 (bars = 500 nm) in B and D, respectively. Arrowheads in D show electron-light vesicles inside intracellular bacteria.

 
In contrast, evidence for bacterial degradation was observed as electron-light vesicles inside S. typhimurium in DCs that had internalized S. typhimurium through Fc{gamma}Rs, even if bacteria still resided inside large vacuoles (Fig. 5, C and D). Such electron-light structures have been previously associated with bacterial lysis (20), suggesting that Fc{gamma}R-mediated bacterial internalization targets bacteria for lysosomal degradation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we provide data supporting the ability of virulent S. typhimurium to interfere with DC function as a mechanism to evade immune recognition. Our results show that S. typhimurium is able to prevent Ag processing and presentation to T cells, probably by impairing endosome-lysosome fusion. These observations suggest an explanation for the fact that infection with virulent S. typhimurium does not lead to a significant expansion of S. typhimurium-specific T cells in the mouse. Avoidance of T cell activation by DCs might be a pathogenicity determinant for virulent S. typhimurium, as suggested by the observation that CD4+ and CD8+ T cells are involved in the clearance of attenuated S. typhimurium (41, 42, 43, 44). Thus, because DCs are the only APCs able to prime naive T cells, interference with DC function could enhance S. typhimurium virulence by preventing activation of CD4+ and CD8+ T lymphocytes. Our findings suggest that the mechanism responsible for the inability of S. typhimurium-infected DCs to activate T cells is a defective targeting of bacteria-containing endosomes to the lysosomal degradation pathway. This observation is consistent with previous confocal microscopy data indicating that DC vacuoles containing S. typhimurium lack lysosomal glycoproteins (39). In addition to confirm that S. typhimurium does not colocalize with lysosomes by confocal microscopy, we show that a functional consequence of this phenomenon is the inability of infected DCs to produce pMHC complexes containing bacteria-derived peptides, which prevents T cell activation (Figs. 1 and 3). However, it seems possible that the outcome of the DC-S. typhimurium interaction might vary depending on specific factors associated with the genetic background of the DCs as well as the different experimental conditions used for the Ag presentation assays. In support of this idea, irradiated BALB/c-derived DCs infected with virulent S. typhimurium at a lower MOI than that used in this study are able to activate MHC class II-restricted T cells when tested at only one DC:T cell ratio (20). The variance between BALB/c-DCs (20) and C57BL/6-DCs (present study) correlates with the reduced susceptibility to S. typhimurium infection shown by BALB/c compared with C57BL/6 mice (45, 46). Thus, it is likely that susceptibility to S. typhimurium infection could be modulated in part by the differential capacity of DCs derived from each of these strains to process and present S. typhimurium-derived Ags. Nevertheless, a systematic experimental analysis would be required to test this concept.

It has been shown that DCs are important at capturing bacteria in vivo, and it is believed that this function is critical for initiating adaptive immunity against bacterial Ags (19, 26, 47, 48, 49). A role for DCs in anti-pathogen immunity is supported by the observation that deletion of DCs from mice renders them susceptible to infection by pathogens such as rodent malaria (50). In addition, DCs are able to internalize bacterial proteins and target them for degradation and presentation on MHC class I and class II (51). Therefore, the ability of virulent S. typhimurium to interfere with T cell activation by DCs could enhance bacterial survival, promoting dissemination and systemic disease. In contrast, Ags expressed by the attenuated strain of S. typhimurium PhoPc would be efficiently presented on MHC class II and cross-presented in MHC class I, because this strain seems unable to avoid lysosomal degradation (52). In addition, presentation on MHC class I and class II of Ags expressed by bacteria was observed when DCs purified from Flt3L-treated C57BL/6 mice were pulsed with the Salmonella strain 14028r (27). Although it has been reported that these types of DCs have increased maturation and Ag presentation capacity (26, 53, 54), it is likely that the attenuation caused by the defect on LPS synthesis shown by this strain of Salmonella (55) is responsible for these data. Thus, the idea that DCs are able to process and present Ags only from attenuated strains of S. typhimurium is consistent with these findings. This feature of the S. typhimurium-DC interaction suggests an explanation for the observation that although infection with attenuated S. typhimurium leads to activation and expansion of bacteria-specific CD4+ and CD8+ T cells, infection with virulent S. typhimurium causes mouse mortality without significant T cell activation (41, 42, 43, 44).

Recently, evidence for induction of apoptosis by S. typhimurium as a strategy for interfering with DC function has been provided (56). Consistent with previous reports (20, 57), under the experimental conditions applied during this study we did not observe significant DC death as result of infection with S. typhimurium. The difference could be explained by the superior aggressiveness shown by strain SR-11 {chi}3041 used previously (54) compared with that of strain 14028s used in this study (LD50 = 2.4 x 104 and 105, respectively, determined in the same mouse strain and under equivalent experimental conditions) (58, 59). However, it is also possible that interference with Ag presentation by virulent S. typhimurium could precede bacteria-induced DC apoptosis. If this is the case, the existence of these two mechanisms that interfere with DC function underscores the molecular sophistication developed by S. typhimurium to evade initiation of adaptive immunity.

The ability of virulent S. typhimurium to interfere with Ag presentation is prevented when bacteria are internalized through Fc{gamma}Rs by DCs (Fig. 2). This observation is consistent with previous data showing that the efficiency of Ag processing and presentation to T cells is enhanced by Fc{gamma}R internalization (10, 11, 60, 61, 62). The observation that 2.4G2 totally blocks T cell activation by DCs pulsed with opsonized bacteria, whereas Ly17.2 does so only partially, suggests that the low affinity Fc{gamma}RIII is mainly responsible for the enhancement of processing and presentation of bacterial Ags (Fig. 2B). Our data indicate that targeting bacteria to Fc{gamma}Rs on DCs increases the amount of MHC molecules loaded with bacterial peptides (Fig. 3) by rerouting virulent S. typhimurium to the lysosomal degradation pathway (Fig. 4). This idea is supported by electron microscopy experiments that showed signs of bacterial degradation only when DCs internalize bacteria through Fc{gamma}Rs (Fig. 5). Lysosomal degradation of bacteria would also be consistent with the apparently reduced GFP signal observed when S. typhimurium-pGFP is internalized through Fc{gamma}Rs (Fig. 1A).

Similar to the observations made with Fc{gamma}Rs, other receptors expressed by DCs can increase the efficiency of internalization, processing, and presentation of exogenous Ags. Thus, facilitation of Ag processing has been demonstrated for receptors such as DEC-205 (63, 64), DC-SIGN (65, 66, 67, 68), mannose receptor (69), asialo-glycoprotein receptor (70), and, recently, CD91/low density receptor-related protein (71, 72). Ag targeting to each of these receptors has improved Ag processing and presentation on MHC molecules to T cells. The likely mechanisms responsible for the enhancement involve increased Ag uptake, lysosomal degradation, or targeting to MHC-rich compartments. However, the roles that these receptors play in the capture, processing, and presentation of bacterial Ags to T cells remain undefined.

In this study we provide evidence for an active role of Fc{gamma}Rs as enhancers for presentation of bacterial Ags to T cells. Our findings are consistent with those of recent studies, supporting a role for Fc{gamma}Rs in protective immunity against pathogenic bacteria (73, 74). Accordingly, Fc{gamma}RIII-deficient mice show increased susceptibility to infection with Streptococcus pneumoniae and reduced responsiveness to vaccination against this pathogen (73). In addition, mice deficient in the common {gamma}-chain (which lack Fc{gamma}RI and Fc{gamma}RIII) show reduced frequency of pathogen-specific T cells in response to infection with Chlamydia (74).

With regard to S. typhimurium infection, our data suggest an explanation for the previously reported dual requirement of T cells and Abs for protection against virulent strains (75, 76, 77). Although virulent S. typhimurium would prevent T cell immunity by interfering with DC function, internalization via Fc{gamma}Rs restores the ability of DCs to process and present Salmonella-derived Ags to T cells. Thus, bacteria-specific IgG would promote recognition of bacterial Ags by T cells and reduce the chance of systemic bacterial dissemination. These observations support the idea that targeting bacterial Ags to Fc{gamma}Rs on DCs could be an efficient approach to enhance T cell immunity against intracellular bacteria.


   Acknowledgments
 
We are indebted to María Rosa Bono and Mario Rosemblatt (Universidad de Chile) for accessibility to the FACS, to Mary Jo Wick (Goteborg University) for providing the pOVA plasmid and B3Z cell line, to Judy Kapp (Emory University School of Medicine) for providing the OT4H cell line and OVA265–280 peptide, to Jean-Claude Sirard (University of Lausanne) for providing the PhoPc-pOVA strain, to Ronald Germain (National Institutes of Health) for providing the 25-D1.16 hybridoma, to C. S. Koenig and Monica Pérez for electron microscopy assistance, and to Virginia Garretón (Rockefeller University), Guido Mora, Bernardo González, and Susan Bueno (Pontificia Universidad Católica de Chile) for critical reading of this manuscript.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants FONDECYT 1030557, DIPUC 2002/11E, and FONDAP 13980001. A.M.K. is a Helen Hay Whitney Foundation fellow, and J.A.T. is a Programa de Mejoramiento de la Calidad la Equidad de la Educación Superior fellow. Back

2 Address correspondence and reprint requests to Dr. Alexis M. Kalergis, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340, Santiago, Chile. E-mail address: kalergis{at}bio.puc.cl Back

3 Abbreviations used in this paper: DC, dendritic cell; IC, immune complex; LB, Luria Bertani; MOI, multiplicity of infection; PhoPc, PhoP constitutive; LAMP, lysosome-associated membrane protein. Back

Received for publication February 23, 2004. Accepted for publication July 16, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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I. M. Olazabal, N. B. Martin-Cofreces, M. Mittelbrunn, G. Martinez del Hoyo, B. Alarcon, and F. Sanchez-Madrid
Activation Outcomes Induced in Naive CD8 T-Cells by Macrophages Primed via "Phagocytic" and Nonphagocytic Pathways
Mol. Biol. Cell, February 1, 2008; 19(2): 701 - 710.
[Abstract] [Full Text] [PDF]

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 Proc. Natl. Acad. Sci. USAHome page
A. A. Herrada, F. J. Contreras, J. A. Tobar, R. Pacheco, and A. M. Kalergis
Immune complex-induced enhancement of bacterial antigen presentation requires Fc{gamma} Receptor III expression on dendritic cells
PNAS, August 14, 2007; 104(33): 13402 - 13407.
[Abstract] [Full Text] [PDF]

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 Proc. Natl. Acad. Sci. USAHome page
R. Ravindran, L. Rusch, A. Itano, M. K. Jenkins, and S. J. McSorley
CCR6-dependent recruitment of blood phagocytes is necessary for rapid CD4 T cell responses to local bacterial infection
PNAS, July 17, 2007; 104(29): 12075 - 12080.
[Abstract] [Full Text] [PDF]

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 J. Immunol.Home page
Y. Liu, X. Gao, E. Masuda, P. B. Redecha, M. C. Blank, and L. Pricop
Regulated Expression of Fc{gamma}R in Human Dendritic Cells Controls Cross-Presentation of Antigen-Antibody Complexes
J. Immunol., December 15, 2006; 177(12): 8440 - 8447.
[Abstract] [Full Text] [PDF]