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*Streptococcal Infections
The Journal of Immunology, 2005, 175: 3862-3872.
Copyright © 2005 by The American Association of Immunologists

The Role of the MHC on Resistance to Group A Streptococci in Mice1

Oliver Goldmann*, Andreas Lengeling{dagger}, Jens Böse{dagger}, Helmut Bloecker{ddagger}, Robert Geffers§, Gursharan S. Chhatwal and Eva Medina2,*

* Infection Immunology Group and {dagger} Infection Genetics Group, Department of Microbial Pathogenesis and Vaccine Research, {ddagger} Department of Genome Analysis, § Array Facility, and Microbial Pathogenesis Group, Department of Microbial Pathogenesis and Vaccine Research, Gesellschaft fur Biotechnologishe Forschung-German Research Center for Biotechnology, Braunschweig, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The severity of infection with Streptococcus pyogenes is strongly influenced by the host’s genetics. This observation extends to the murine model of streptococcal infection, where the background of the mouse strain determines the infection outcome (BALB/c are resistant, whereas C3H/HeN are susceptible). To determine the extent to which the MHC complex (H2) contributed to diseases susceptibility, the response to S. pyogenes of congenic BALB mice from a resistant background (BALB/c), but carrying the H2k region of susceptible C3H/HeN mice (BALB/k), was examined. BALB/k were as susceptible as the H2 donor strain (C3H/HeN). Linkage analysis performed in F2 backcross ([BALB/c x C3H/HeN] x BALB/c) mice confirmed the presence of a susceptibility locus within the H2 region on proximal chromosome 17. The possibility that modulation of T cell responses to streptococcal superantigens (GAS-SAgs) by different H2 haplotypes may influence disease severity was examined. BALB/k exhibited a significantly stronger response at the level of cell proliferation and cytokine production to GAS-SAgs than did BALB/c mice. However, the fact that T cell-deficient SCID-C3H/HeN mice also exhibited a susceptible phenotype suggests a more important contribution of innate effector cells to disease susceptibility. Lower transcriptional levels of certain inflammation-related regulatory genes located on chromosome 17 were detected in macrophages from susceptible than in those from resistant mice in response to infection. These results suggest that susceptibility to S. pyogenes may be associated with an altered transcription of specific genes that may compromise the endogenous regulatory processes controlling the inflammatory cascade and favor the progression to sepsis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Group A streptococci (GAS;3 Streptococcus pyogenes) are important human pathogens that cause infections of varying severity, ranging from uncomplicated self-limited infections such as pharyngitis and pyoderma to highly severe invasive diseases such as deep soft tissue infection (e.g., necrotizing fasciitis, colloquially referred to as flesh-eating bacteria), sepsis, and streptococcal toxic shock syndrome (1). Several studies have emphasized the importance of host genetic factors in determining the severity and outcome of GAS infections and, in particular, through the regulation of the inflammatory cytokine responses to streptococcal superantigens (GAS-SAgs) (2, 3, 4, 5). Thus, evidence has been provided that patients with a propensity to produce higher levels of inflammatory cytokines in response to GAS-SAgs developed significantly more severe systemic manifestations than patients with a propensity to produce lower levels of inflammatory cytokines to these superantigens (5).

S. pyogenes produces several superantigens that have been suggested to be major mediators of the systemic effects observed in severe invasive infections (6, 7, 8). The secreted streptococcal superantigens include SPE-A, -C, -F, -G, -H, and -J; streptococcal mitogenic exotoxins SmeZ and SmeZ2; and streptococcal superantigens SSA, among others (9, 10, 11, 12, 13). Cross-linking of T cells and APCs by superantigens results in potent stimulation of immune cells and subsequent massive cytokine production, which can be directly involved in the development of sepsis and septic shock (14, 15, 16). The role of GAS-SAgs in the severity of streptococcal infections has been supported by the presence of GAS-SAgs in the circulation and also at the local site of infection in patients with invasive GAS infection (17, 18).

Recent studies have suggested that allelic variations of the MHC class II (H-2) Ags may contribute to susceptibility to or protection from severe streptococcal diseases by their ability to modulate the magnitude of the inflammatory cytokine response elicited to GAS-SAgs (4, 19, 20). Thus, patients carrying the DRB1*14/DQB1*0503 or DRB1*07/DQB1*0201 haplotype have an increased risk of developing severe systemic streptococcal disease compared with those carrying the DRB1*1501/DQB1*0602 haplotype (4). However, significant association of a gene polymorphism with a certain disease susceptibility does not ensure that the gene polymorphism is a primary factor for the disease phenotype. There is a possibility that another polymorphism within closely linked genes may actually determine the disease susceptibility and that the observed association might be a secondary one resulting from linkage disequilibrium. Consequently, it is important to determine whether susceptibility to GAS infection is influenced directly by polymorphism of the H2 locus or by polymorphisms of other genes present in neighboring regions of this locus.

Mouse models of streptococcal infection have proved to be especially important in demonstrating the influence of genetic factors on the host response to S. pyogenes. We have previously shown that different inbred mouse strains show marked phenotypic differences in disease manifestations after infection with S. pyogenes (21, 22). Thus, although C3H/HeN mice are very susceptible, exhibiting high levels of bacterial growth that progress to systemic disease and death, BALB/c mice are very resistant and survive infection (21, 22). The objective of this study was to determine the extent to which polymorphism on the H2 complex, through modulation of T cell activation by streptococcal superantigens, influences the overall resistance or susceptibility of mice to infection with S. pyogenes. Using congenic BALB mice from a resistant background (BALB/c), but carrying the H2k haplotype region of the susceptible C3H/HeN strain (BALB/k mice), we have shown that although the H2 haplotype might influence the extent of the inflammatory response occurring during invasive streptococcal infection, non-H2-encoded genes present in this chromosomal region make a more critical contribution to the susceptibility of mice to S. pyogenes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Bacteria

S. pyogenes A20 strain is a human isolate obtained from the German Culture Collection (DSM 2071). Stock cultures were maintained at –70°C and were routinely cultured at 37°C in Todd-Hewitt broth (Oxoid) supplemented with 1% yeast extract for ~6 h. Bacteria was collected in midlog phase, washed twice with sterile PBS, and diluted to the required inoculum, and the number of viable bacteria was determined by counting CFU after diluting and plating in blood agar plates (Invitrogen Life Technologies) containing 5% sheep blood.

Detection of streptococcal exotoxin genes by PCR and Southern blot analysis

For PCR analysis, chromosomal DNA from S. pyogenes strain A20 was isolated and used as a template. Oligonucleotides used for analysis of the exotoxin genes and the expected amplification sizes are shown in Table I. PCR was performed using Taq polymerase (Qiagen) according to the manufacturer’s recommendations using the following protocol: denaturation at 94°C for 4 min, 30 cycles for 30 s at 94°C, annealing at 58°C for 30 s, and elongation for 70 s at 72°C, followed by 5 min at 72°C. The presence of toxin genes was confirmed by Southern blot analysis. Briefly, chromosomal DNA from S. pyogenes strain A20 was digested with BamHI, EcoRI, or HindIII (New England Biolabs), separated by agarose gel electrophoresis, and transferred to a positively charged nylon membrane. For detection, the membrane was hybridized at 42°C with toxin gene-specific digoxygenin-labeled DNA probes (Roche), prepared as recommended by the manufacturer.


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  		Table I. Exotoxin gene profile of S. pyogenes strain A20

 
Mice

Eight-week-old female congenic BALB mice of the H2c (BALB/c) and H2k (BALB/k) haplotypes, C3H/HeN, BALB/c mice, and immunodeficient BALB/c-SCID and C3H/HeN-SCID mice were purchased from Harlan-Winkelmann. Mice were maintained under specific pathogen-free conditions according to Federation of European Laboratory Animal Science Association guidelines and were used for experiments according to protocols approved by the local committee board.

Experimental infections

Mice were inoculated with 105 CFU of S. pyogenes in 0.2 ml of PBS via a lateral tail vein. For kinetic studies, infected mice were killed by CO2 asphyxiation, and bacteria were enumerated at progressive times in the systemic organs of infected mice by preparing homogenates of these organs in PBS and plating 10-fold serial dilutions on blood agar plates (Invitrogen Life Technologies). Colonies were counted after 24 h of incubation at 37°C. Viable bacterial counts were also determined in blood of infected mice by collecting blood samples from the tail vein at different times after inoculation and plating serial dilutions in blood agar.

Generation and phenotyping of the F2 backcross progeny

Resistant BALB/c females were crossed with susceptible C3H/HeN males to generate F1 hybrid mice. F1 males were backcrossed to BALB/c females to generate the F2 population. Females from this generation (n = 113) were used for further analysis. F2 and parental control mice were infected i.v. with 105 CFU of S. pyogenes and were monitored for survival and bacteremia on days 1 and 5 after inoculation.

Genotyping

Genomic DNA for PCR was prepared from tail clips using a nonorganic tail DNA extraction protocol (<www.jax.org/imr/tail_nonorg.html>). Each backcross mouse was genotyped using 66 microsatellite markers informative for crosses between BALB/c and C3H/HeN mice and selected at the Center for Inherited Disease Research database (<www.cidr.jhmi.edu/mouse/mouse_dif.html>). Primers for selected microsatellite markers were part of the Center for Inherited Disease Research set of highly informative mouse simple sequence length polymorphism markers (23) and were purchased from the ABI PRISM Mouse Mapping Primer version 1.0 set (Applied Biosystems). The forward primer of each marker pair was labeled on the 5' end with the fluorescent dyes FAM, VIC, or NED. PCR products labeled with different dye combinations were pooled and separated on an automated MegaBACE DNA Analysis System (Molecular Dynamics and Amersham Biosciences). Allele calling was performed using MegaBACE Genetic Profiler, version 1.1. Primer pairs for microsatellite markers on chromosome 17 were ordered from Research Genetics. PCR products were separated on 4% or 5% Metaphore agarose gels (Cambrex BioScience). PCRs for all microsatellite markers were conducted under the following conditions: initial denaturation for 12 min at 95°C, followed by 10 cycles with denaturation for 15 s at 94°C, annealing for 15 s at 55–60°C, extension for 30 s at 72°C and 20 cycles with denaturation for 15 s at 89°C, annealing for 15 s at 55–60°C, extension for 30 s at 72°C. A final extension step was performed for 10 min at 72°C. PCR assays were conducted in a 10-µl reaction volume using 25 mM MgCl2, 5 µM of each primer, 2.5 mM dNTPs, 50 ng of template DNA, and 0.5 U of AmpliTaq DNA polymerase (Applied Biosystems).

Statistical analysis

Genetic map construction and genome scan analyses of F2 backcross progenies were performed using Map Manager QTX17b (24). This program searched among the backcross progeny for significant associations between the markers genotypes (representing chromosomal regions) and quantitative phenotypes. Survival time after infection (TTD), bacteremia (CFU of S. pyogenes per milliliter of blood on days 1 and 5 after inoculation), and the ratio of CFU/TTD were used as quantitative traits to detect linkage. To calculate linkage based on bacteremia, values obtained on day 1 were used for animals that succumbed within the first 3 days of infection, and values obtained on day 5 were used for the rest of the animals. Map distances were calculated with the Kosambi map function. Quantitative trait linkage interval mapping analysis was performed using the implemented Map Manger functions.

Cytokine ELISAs

The determination of cytokines was performed by specific ELISA using matched Ab pairs and recombinant cytokines as standards. Briefly, 96-well microtiter plates were coated at 4°C with the corresponding purified rat anti-mouse capture rat mAbs (BD Pharmingen) at 2 µg/ml in sodium bicarbonate buffer overnight at 4°C. The wells were washed and then blocked with 1% BSA-PBS before serum samples and the appropriate standard were added to each well. Biotinylated rat mAbs (BD Pharmingen; 2 µg/ml) were added as the second Ab. Detection was conducted with streptavidin-peroxidase, and the plates were developed using ABTS. A standard curve was generated for each cytokine using recombinant murine proteins (BD Pharmingen).

Proliferation assay

Spleen cells from BALB/c or BALB/k congenic mice were dissociated into a single-cell suspension, adjusted to 2 x 106 cells/ml in complete medium (RPMI 1640 supplemented with 10% of FCS, 100 U/ml penicillin, 50 µg/ml streptomycin, 5 x 10–5 M 2-ME, and 1 mM L-glutamine), seeded at 100 µl/well in a flat-bottom, 96-well microtiter plate (Nunc), and incubated for 3 days with various concentrations of culture supernatant from S. pyogenes. During the final 18 h of culture 1 µCi of [3H]thymidine (Amersham Biosciences) was added per well. The cells were harvested on paper filters (Filtermat A; Wallac) using a cell harvester (Inotech), and the [3H]thymidine incorporated into the DNA of proliferating cells was determined in a beta scintillation counter (Wallac 1450, Micro-{beta} Trilux).

Depletion of Th1.2+ spleen cells

The Th1.2+ cell subset was depleted using MiniMACS magnetic microbeads according to the manufacturer’s instructions (Miltenyi Biotec). Depleted cell preparations contained <1% Th1.2+ cells.

FACScan analysis

Approximately 5 x 105 cells were incubated in staining buffer (PBS supplemented with 2% FCS and 0.1% sodium azide) with the desired Ab or combination of Abs for 30 min at 4°C. After washes, cells were analyzed on a FACScan (BD Biosciences). The mAbs used were PE-conjugated Thy-1.2 and FITC-conjugated Abs against the different V{beta} TCRs (BD Pharmingen).

Preparation of bacterial culture supernatants containing GAS-SAgs

S. pyogenes was cultured at 37°C in Todd-Hewitt broth (Oxoid), supplemented with 1% yeast extract to midlog phase. After centrifugation of bacterial cultures, supernatant was collected, filtered, and stored at –20°C.

In vivo infection of peritoneal macrophages

BALB/c and C3H/HeN mice were i.p. infected with 5 x 107 CFU of S. pyogenes and killed 1 h thereafter, and their peritoneums were lavaged with sterile PBS. Macrophages present in the lavage samples were labeled with anti-F4/80 Abs, further purified by positive selection using miniMACS magnetic microbeads according to the manufacturer’s instructions (Miltenyi Biotec), and used for cDNA microarray analysis.

Microarray analysis

Mice were injected i.p. with 5 x 107 CFU of S. pyogenes or PBS as a control, peritoneal washes were collected after 1 h, and pooled purified F4/80+ cells from 8 to 10 mice were used for the cDNA expression array. Total RNA isolated from infected or control macrophages was controlled by running all samples on a 2100 Bioanalyzer (Agilent Technologies). For biotin-labeled target synthesis starting from 3 µg of total RNA, reactions were performed using standard protocols supplied by the manufacturer (Affymetrix). Briefly, 5 µg of total RNA was converted to dsDNA using 100 pmol of a T7T23V primer (Eurogentec) containing a T7 promotor. The cDNA was then used directly in an in vitro transcription reaction in the presence of biotinylated nucleotides. The concentration of biotin-labeled cRNA was determined by UV absorbance. In all cases, 12.5 µg of each biotinylated cRNA preparation was fragmented and placed in a hybridization mixture containing four biotinylated hybridization controls (BioB, BioC, BioD, and Cre) as recommended by the manufacturer. Samples were hybridized to an identical lot of Affymetrix GeneChip MOE430A for 16 h. After hybridization, the GeneChips were washed, stained with streptavidin-PE, and read using an Affymetrix GeneChip fluidic station and scanner. Two replicate chips per group were used in two separate experiments performed with pooled macrophages harvested from 8 to 10 mice. Changes in the levels of some transcripts were verified by either PCR or bioassay.

Data analysis

Analysis of microarray data was performed using Affymetrix Microarray Suite 5.0, Affymetrix MicroDB 3.0, and Affymetrix Data Mining Tool 3.0. For normalization, all array experiments were scaled to a target intensity of 150, but otherwise used the default values of the Microarray Suite. Genes were considered as strongly regulated when their fold change was ≥2 or less than or equal to –2, the statistical parameter for a significant change was <0.001 (change in p value), and the difference between compared signal intensities of a certain gene was >200.

RT-PCR

Total RNA was isolated using peqGold TriFast (Peqlab) according to the manufacturer’s instructions. RNA was reverse transcribed with reverse transcriptase (Hoffmann-La Roche) and an Invitrogen Life Technologies RT-PCR kit was used for cDNA synthesis following the manufacturer’s protocols. The single-stranded cDNA was then subjected to PCR under standard reaction conditions. The following oligonucleotides were used: TNF-{alpha}, 5'-AGC CCA CGTC GTA GCA AAC CAC CA-3' and 5'-ACA CCC ATT CCC TTC ACA GAG CCA T-3'); and IL-6, 5'-CTG GTG ACA ACC ACG GCC GTC CCT A-3' and 5'-ATG CTT AGG CAT AAC GCA CTA GGT T-3'. The ribosomal housekeeping gene rsp9 (5'-CTG GAC GAG GGC AAG ATG AAG C-3' and 5'-TGA CGT TGG CGG ATG AGC ACA-3') was used as an internal standard. PCR was performed for 28 cycles at 94°C for 15 s, 58°C for 30 min, and 72°C for 15 s, followed by 72°C for 5 min. The resultant PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide, and photographed.

Statistical analysis

Comparisons between groups were performed by ANOVA. Comparison of survival curves was performed by Mann-Whitney U test. A value of p < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Susceptibility of BALB/c, C3H/HeN, and BALB/k mice to systemic infection with S. pyogenes

We have previously shown that different strains of mice infected with S. pyogenes exhibited marked differences in disease manifestations. Although C3H/HeN mice are very susceptible, exhibiting high levels of bacterial growth that progress to systemic disease and death, BALB/c mice are very resistant and survive infection (21, 22). To determine the extent to which the H2 haplotype affects resistance to S. pyogenes in these mice, the response to S. pyogenes of congenic mice from a resistant background (BALB/c), but carrying the H2k haplotype of the susceptible C3H/HeN mice, was evaluated. Congenic BALB mice of the H2c (BALB/c) and H2k (BALB/k) haplotypes as well as C3H/HeN mice were i.v. infected with 105 CFU of S. pyogenes, and mortality was recorded over a period of 10 days. The results in Fig. 1A show that BALB/k mice were as susceptible as C3H/HeN mice to S. pyogenes, with a mortality rate of 100% by days 5 and 3 after bacterial inoculation, respectively. In contrast, BALB/c mice were very resistant, and none of the mice succumbed to S. pyogenes infection during the observation period (Fig. 1A).



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  		FIGURE 1. A, Survival curves of BALB/c ({blacksquare}), C3H/HeN (•), and BALB/k ({blacktriangleup}) mice i.v. infected with 105 CFU of S. pyogenes (n = 10 mice/group). Animals were monitored daily, and mortality was recorded for a period of 10 days. One representative experiment of three is shown. Bacterial loads in the liver, B and spleen, C of BALB/c ({blacksquare}), C3H/HeN (•), and BALB/k ({blacktriangleup}) mice i.v. infected with 105 CFU of S. pyogenes. Each point represents the mean ± SD of five mice per group. One representative experiment of three is shown.

 
We next determined the growth kinetics of S. pyogenes in the in the livers and spleens of the above-mentioned mouse strains. For this purpose, BALB/c, C3H/HeN, and BALB/k mice were infected i.v. with 105 CFU of S. pyogenes, and bacterial burdens were assessed in the livers and spleens at different times after inoculation. The results obtained show that BALB/c mice had significantly lower bacterial burden in the liver (Fig. 1B) and spleen (Fig. 1C) than C3H/HeN and BALB/k mice at all time points examined. The failure of C3H/HeN and BALB/k mice to control bacterial growth strongly correlated with the high mortality observed in these mouse strains. These results suggest that the MHC region had a strong influence on severity of S. pyogenes infection in BALB congenic mice.

S. pyogenes susceptibility linkage analysis in ([BALB/c x C3H/HeN] x BALB/c) F2 mice

The presence of disease susceptibility loci in the H2 region on proximal mouse chromosome 17 was investigated by performing linkage analysis in 113 F2 ([BALB/c x C3H/HeN] x BALB/c) backcross mice. F2 female mice as well as BALB/c and C3H/HeN controls were i.v. infected with 105 CFU of S. pyogenes, and survival was determined over time. On days 1 and 5 after bacterial inoculation, a blood sample was taken for the determination of bacteremia. For the linkage analysis, we followed three traits: TTD, bacteremia (as measured by number of CFU in the blood of infected mice on days 1 and 5 after inoculation), and the ratio between CFU and TTD. The course of infection was followed over a period of 10 days; all animals surviving day 10 were considered to have survived the infection challenge. A linkage was found on proximal mouse chromosome 17 for bacteremia at D17Mit34 (likelihood ratio statistic = 6.0). Interval mapping for this trait on proximal chromosome 17 showed that the peak of linkage was between D17Mit101 and D17Mit10, a chromosomal region containing the H2 histocompatibility genes (Fig. 2A). In addition, a suggestive linkage was found on proximal mouse chromosome 7 (data not shown).



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  		FIGURE 2. A, Linkage analysis of susceptibility to infection with S. pyogenes in 113 F2 backcrosses of mice resulting from crossing F1 hybrids (BALB/c x C3H/HeN) with BALB/c mice. The logarithm of the odds score values along chromosome 17 by whole genome scan linkage analysis and interval mapping are shown. The map positions of microsatellite markers used are indicated, and chromosomal lengths are shown to scale. B, Genotyping with informative microsatellite markers on proximal chromosome 17. The origins of used DNAs are indicated on the top. Black arrows represent BALB/c alleles; gray arrows represent C3H/HeN alleles. C, Chromosomal interval of the H2K haplotype on chromosome 17. The positions of the analyzed microsatellite markers in centimorgans are shown on the right; the gray line on the chromosome represents the C3H/HeN-derived H2K interval; the black lines represent chromosomal regions derived from BALB/c.

 
To estimate the size of the congenic H2k haplotype interval in BALB/k mice, the allelic variants of several polymorphic microsatellite markers within the chromosomal region were examined. Genotyping of (BALB/k x BALB/c) F1 mice with the D17Mit101, D17Mit28, D17Mit51, and D17Mit68 microsatellite markers demonstrated that the H2k haplotype interval in BALB/k mice was flanked by D17Mit101 at the proximal site and by D17Mit68 on the distal site of the chromosome. Both markers displayed BALB/c alleles, whereas D17Mit28 and D17Mit51, which map between D17Mit101 and D17Mit68, showed C3H/HeN alleles (Fig. 2, B and C). This limits the H2k haplotype interval in BALB/k mice to a maximal chromosomal region of 8.1 cM (Fig. 2C).

Levels of IFN-{gamma} and IL-12 in sera of BALB/c and BALB/k mice 24 h after infection with S. pyogenes

It has been shown that patients with a propensity to produce higher levels of proinflammatory cytokines in response to streptococcal superantigens developed significantly more severe systemic manifestations than patients with a propensity to produce lower levels of inflammatory cytokines to the same superantigens (4, 5). Therefore, the levels of the inflammatory cytokines IFN-{gamma} and IL-12 were determined in sera from S. pyogenes-infected BALB/c and BALB/k mice 24 h after bacterial challenge. The levels of serum IFN-{gamma} (Fig. 3A) and IL-12 (Fig. 3B) were significantly higher in BALB/k mice than in BALB/c mice. These findings also confirm the previously reported association between a high level of inflammatory cytokines and the severity of streptococcal infection in mice (22).



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  		FIGURE 3. A, Level of IFN-{gamma} and B, IL-12 in sera of BALB/c and BALB/k mice 24 h after inoculation with 105 CFU of S. pyogenes ({square}). Mice inoculated with PBS were used as controls ({blacksquare}). Each bar represents the mean ± SD of data obtained from five mice per group. One experiment of three is shown. The level of statistical significance between samples from infected BALB/c and BALB/k mice is indicated (*, p < 0.05, by ANOVA).

 
In vitro responses of spleen cells isolated from BALB/c and BALB/k mice to GAS-SAgs

GAS-SAgs are believed to play an important role during severe infection (4, 5, 6, 7, 8, 9). Superantigens are potent immunostimulatory proteins that bind the H-2 and TCR molecules on the surface of APCs and T lymphocytes (25). Because proliferation of reactive T cells is a hallmark of SAg stimulation (26), we next determined whether the in vitro T cells responses to GAS-SAgs differed in BALB/c and BALB/k mice. For this purpose, spleen cells were isolated from BALB/c and BALB/k mice and stimulated in vitro with various concentrations of GAS-SAgs present in the culture supernatant from S. pyogenes. As shown in Fig. 4A, spleen cells from BALB/k mice responded more vigorously upon stimulation with GAS-SAgs than those from BALB/c mice. To verify that T cells were the responding population to GAS supernatant-induced proliferation, spleen cell suspensions from BALB/c and BALB/k mice were depleted of T cells before in vitro stimulation. The results presented in Fig. 4A show that the proliferative responses were almost completely abolished in both BALB/c and BALB/k mice after depletion of T cells, providing functional evidence that T cells were indeed the effector population. These results were also confirmed by the capacity of highly purified T cells isolated from both mouse strains to respond to stimulation with GAS-SAgs (data not shown). Differences in the capacity of GAS-SAgs to induce production of IFN-{gamma} by in vitro-stimulated spleen cells from BALB/c and BALB/k mice were also investigated. As shown in Fig. 4B and consistent with the proliferation data, GAS-SAgs induced higher levels of IFN-{gamma} production in spleen cells from BALB/k mice than in those from BALB/c mice.



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  		FIGURE 4. A, Proliferative responses of spleen cells from BALB/c and BALB/k mice after in vitro stimulation with culture supernatant from S. pyogenes. Total ({blacksquare} and {blacktriangleup}) or T cell-depleted ({square} and {triangleup}) spleen cells from either BALB/c ({blacksquare} and {square}) or BALB/k ({blacktriangleup} and {triangleup}) mice were stimulated with various concentrations of S. pyogenes supernatant for 72 h, and proliferation was determined by the incorporation of [3H]thymidine. Each point represents the mean ± SD of triplicate determinations. Spleen cells from BALB/c (•) or BALB/k ({diamondsuit}) stimulated with various concentration of medium without bacteria were used as controls. B, IFN-{gamma} production by spleen cells from BALB/c ({blacksquare}) and BALB/k ({blacktriangleup}) mice after 3 days of in vitro stimulation with 1% culture supernatant from S. pyogenes. Each point represents the mean ± SD of triplicate determinations. One representative experiment of three is shown.

 
Alterations in the TCR V{beta}-expressing T cell repertoire in congenic BALB/c and BALB/k mice during infection with S. pyogenes

Superantigens interact with the relatively invariable V{beta} region of the TCR and the H-2 molecules on APCs. Each superantigen exhibits specificity for V{beta} sequences, and although there may be overlap in specificities, each superantigen binds its own unique set of T cells (27, 28). Therefore, we next determined whether the differences in progression of disease between BALB/c and BALB/k mice were accompanied by changes in the relative abundance of splenic T cells expressing different V{beta}-chains. The phenotypes of splenic T cells expressing different V{beta}-chains were analyzed by flow cytometry in BALB/c and BALB/k mice 48 h after inoculation with S. pyogenes. The staining results for each of the V{beta}-specific TCR were calculated as a percentage of the total Thy 1.2+ cells. As shown in Fig. 5, the only significant expansion in infected BALB/c (Fig. 5A) and BALB/k (Fig. 5B) mice was observed in V{beta}13 TCR-bearing T cells. However, a significantly greater expansion of V{beta}13 T cells was detected in BALB/k mice than in BALB/c mice (11.2 ± 1.6 vs 6.3 ± 1.1, respectively; p < 0.05, by ANOVA test).



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  		FIGURE 5. V{beta} expression of murine T cells isolated from the spleen of either BALB/c (A) or BALB/k (B) mice 48 h after inoculation with 105 CFU of S. pyogenes ({blacksquare}). Spleen cells from uninfected mice were used as controls ({square}). Each bar represents the mean ± SD of four mice per group.

 
Phenotypic characterization of the in vitro-stimulated T cell repertoire from BALB/c and BALB/k mice

We next determined the TCR V{beta} repertoire on spleen cells from BALB/c and BALB/k mice after in vitro stimulation with GAS-SAgs. Spleen cells were isolated and stimulated in vitro with 1% bacterial supernatant for 72 h. Cells were then collected, washed, stained with Abs against the TCR V{beta}13, and analyzed by FACScan. The results presented in Fig. 6 show that the most significant expansion in response to in vitro stimulation with GAS-SAgs also occurred in V{beta}13 TCR-bearing T cells in both BALB/c (Fig. 6A) and BALB/k (Fig. 6B) mice. Again, a significantly increased number of V{beta}13+ T cells was observed in GAS-SAgs-stimulated cultures from BALB/k than in those from BALB/c mice (20.3 ± 2.5 vs 10.1 ± 1.02, respectively; p < 0.05, by ANOVA test). These results are compatible with the pattern of T cell changes observed during in vivo infection.



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  		FIGURE 6. V{beta} expression of murine T cells isolated from the spleen of either BALB/c (A) or BALB/k (B) mice after 3 days of in vitro stimulation with 1% culture supernatant from S. pyogenes. Each bar represents the mean ± SD of three independent determinations.

 
Capacity of superantigen-activated T cells to exacerbate S. pyogenes infection in resistant mice

A massive increase in the number of activated T cells is one of the biological factors that has been linked to diseases induced by superantigens (25, 26, 27, 28). To corroborate the hypothesis that highly activated T cells by stimulation with GAS-SAgs might contribute to infection severity, an experiment was performed to determine whether adoptively transferred, in vitro-activated T cells could influence the course of S. pyogenes infection. Spleen cells were isolated from BALB/c mice and stimulated in vitro with 1% GAS supernatant for 72 h. Nonstimulated spleen cells were used as controls. Different amounts (106 and 5 x 106) of either stimulated or nonstimulated cells were injected into S. pyogenes-infected SCID-BALB/c mice 24 h after infection, and bacterial loads were determined in blood, liver, and spleen 24 h thereafter. In some experiments, T cells were depleted before adoptive cell transfer. Bacterial clearance during infection with S. pyogenes is dependent on innate immune mechanisms, and we have previously shown that immunodeficient SCID mice from the BALB/c background are as resistant to streptococcal infection as the immunocompetent strain (21). All BALB/c-SCID mice reconstituted with GAS-SAg-activated spleen cells exhibited a significant increase in bacterial loads in blood and systemic organs compared with mice receiving nonstimulated cells (Fig. 7). The exacerbation of the infection was proportional to the amount of stimulated cells transferred into recipient mice (Fig. 7). Interestingly, depletion of T cells before cell transfer did not modify the course of infection, suggesting the contributory role of activated T cells to the severity of S. pyogenes infection (Fig. 7).



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  		FIGURE 7. Bacterial loads in systemic organs of S. pyogenes-infected BALB/c-SCID mice engrafted with in vitro-stimulated 106 total spleen cells ({cjs2110}), 5 x 106 total spleen cells ({square}), or 5 x 106 T cell-depleted spleen cells ({cjs2108}) from BALB/c mice. Mice engrafted with 5 x 106 nonstimulated spleen cells were used as a control ({blacksquare}). Spleen cells were isolated from BALB/c mice and stimulated in vitro with 1% culture supernatant from S. pyogenes for 72 h. Nonstimulated spleen cells were used as controls. Stimulated and nonstimulated cells were injected into S. pyogenes-infected SCID-BALB/c mice 24 h after infection, and bacterial loads were determined in blood, liver, and spleen 24 h after engrafting. Each bar represents the mean ± SD of five mice per group. *, p < 0.05, by ANOVA.

 
Susceptibility of C3H/HeN-SCID mice to infection with S. pyogenes

To determine to which degree GAS-SAg-activated T cells might be the major mediators of susceptibility to S. pyogenes infection in mice, the response to infection with S. pyogenes was examined in T cell-deficient mice (SCID) from either a susceptible (C3H/HeN) or a resistant (BALB/c) background strain. C3H/HeN-SCID, BALB/c-SCID, and immunocompetent C3H/HeN mice were challenged i.v. with 105 CFU of S. pyogenes, and mortality was determined over time. All animals from the C3H/HeN-SCID strain as well as immunocompetent C3H/HeN mice developed severe infection, resulting in a mortality rate of 100% by days 5 and 3 after infection, respectively (Fig. 8). In contrast, 90% of the BALB/c-SCID mice survived to infection with S. pyogenes (Fig. 8). These results indicate that the absence of T cells did not rescue the susceptible phenotype, and they also suggest a more important contribution of innate effector cells to disease susceptibility.



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  		FIGURE 8. Survival curves of T cell-deficient C3H/HeN-SCID ({diamondsuit}), BALB/c-SCID ({blacksquare}), and immunocompetent C3H/HeN ({blacktriangleup}) mice infected i.v. with 105 CFU of S. pyogenes (n = 10 mice/group). Animals were monitored daily, and mortality was recorded for a period of 14 days.

 
Transcriptional response of selected genes associated with the inflammatory response in macrophages from BALB/c and C3H/HeN mice after infection with S. pyogenes

Severe sepsis with organ failure may develop when bacteria and/or bacterial products lead to macrophage activation and production of macrophages and T cell-derived cytokines (29). Besides the highly deleterious exotoxins, S. pyogenes contains a number of immunogenic cell wall components, such as lipoteichoic acid and peptidoglycans, capable of stimulating enormous release of proinflammatory cytokines and other inflammatory mediators from monocytes/macrophages (30, 31, 32, 33, 34). Therefore, it can be hypothesized that differential induction of inflammation-related genes in macrophages from BALB/c and C3H/HeN mice by S. pyogenes may underlie the different levels of disease susceptibility expressed by these two mouse strains. Using microarray technology, the regulation of genes involved in the inflammatory response was analyzed in macrophages from both mouse strains 1 h after infection with S. pyogenes.

Upon infection with S. pyogenes, 26 inflammatory response-related genes were detected as differentially regulated in both mouse strains (Fig. 9A). Twenty-three genes were reproducibly up-regulated with a mean 2.0-fold increase (group I in Fig. 9A and Table II), and three genes were suppressed (group II in Fig. 9A) compared with the uninfected stated in both mouse strains. Genes coding for IL-6, IL-1{beta}, MCP-3, and MIP-1{alpha} as well as genes coding for CSFs, such as G-CSF and GM-CSF, were strongly up-regulated, with average increases of >100-fold (Table II). Induction of the gene coding for TNF-{alpha} was significantly stronger in BALB/c mice than in C3H/HeN mice (Table II). The transcription of anti-inflammatory genes such as Il1rn (IL-1R antagonist), Il4, Hspa1a (heat shock protein 1A), and Hspa1b (heat shock protein 1B) was also significantly enhanced in resistant BALB/c compared with that in susceptible C3H/HeN mice (Table II).



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  		FIGURE 9. A, Transcription of inflammatory response-related genes in macrophages from BALB/c and C3H/HeN mice upon infection with S. pyogenes. Genes induced by S. pyogenes are indicated in red (group I), and genes with reduced expression are indicated in green (group II). The degree of redness represents the level of induction, whereas the degree of greenness represents the level of repression. B, Level of inflammatory IL-6 in the serum of uninfected ({square}) and infected ({blacksquare}) BALB/c and C3H/HeN mice 48 h after i.v. inoculation with S. pyogenes. The level of circulating IL-6 was significantly higher in infected C3H/HeN than in infected BALB/c mice (*, p < 0.05, by ANOVA). Each bar represents the mean ± SD of four mice per group.

 

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  		Table II. Changes in the levels of selected transcripts associated with inflammatory response induced by S. pyogenes phagocytosis on macrophages from BALB/c and C3H/HeN mice

 
Focusing on the differentially regulated genes, the TNF-{alpha} gene (Tnf) and the gene coding for the 70-kDa heat-shock protein (Hspa1b) were located in the 8.1-cM interval of chromosome 17 harboring the susceptibility locus.

To further validate the gene array results, RT-PCR was performed on selected genes (Tnf and Il6). The housekeeping rps9 gene was used to ensure equal amounts of cDNA in each reaction and that PCR products were equally loaded onto the gel. The results obtained by RT-PCR and Western blot confirmed the gene array data (not shown).

Levels of TNF-{alpha} and IL-6 in susceptible C3H/HeN and resistant BALB/c mice during infection with S. pyogenes

TNF-{alpha} and IL-6 have been regarded as central mediators of the pathophysiological changes associated with bacteremia and sepsis syndrome (29, 35, 36). Although the gene is highly induced in both resistant and susceptible mice, we failed to detect TNF-{alpha} in the serum of infected mice. These data agree with our previous report regarding the production of this cytokines during S. pyogenes infection (22).

IL-6 is a widely recognized major mediator of Gram-positive sepsis (37, 38, 39, 40, 41). In particular, overproduction of IL-6 has been shown to correlate with poor outcome in patients with severe streptococcal sepsis and septic shock (42). To determine whether the development of S. pyogenes-induced septic shock in susceptible C3H/HeN mice was associated with high levels of circulating IL-6, the levels of this cytokine in the serum of both resistant BALB/c and susceptible C3H/HeN mice was measured 48 h after the animals were inoculated with 105 CFU of S. pyogenes. As shown in Fig. 9B, significantly higher levels of IL-6 were detected in the serum of infected susceptible C3H/HeN compared with infected resistant BALB/c mice (p < 0.05, by ANOVA test).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Haplotype variations of the H-2 Ags have been suggested to influence the severity of GAS infections (4, 19, 20). However, significant association of a gene polymorphism with a certain disease susceptibility does not ensure that the gene polymorphism is a primary factor for the disease phenotype, and the possibility exists that other polymorphisms within closely linked genes may also contribute. Therefore, it was important to investigate to what extent the HLA haplotype influences the overall resistance/susceptibility exhibited by the host to S. pyogenes.

Congenic strains of mice, which are different from the background strain only in the chromosomal region of interest, are one of the best ways to genetically dissect complex traits and to demonstrate the contribution of each locus to disease etiology. In this study, inbred mice of congenic BALB strains that differed in their H2 haplotype (BALB/c, H2c; BALB/k, H2k) were used to examine the effects of H2 genes on resistance/susceptibility to S. pyogenes. Notable differences were observed among these strains after i.v. infection with this pathogen. Although BALB/k were highly susceptible and succumbed to infection, BALB/c developed much less severe disease and survived infection with an effective bacterial clearance. The pattern of susceptibility observed in BALB/k mice resembles the susceptibility previously observed in C3H/HeN mice (21, 22). Because C3H/HeN is the donor strain of the H2k chromosome region of BALB/k mice, it can be suggested that the failure to control S. pyogenes infection in these mice might be associated with genes present within the MHC region. Indeed, linkage analysis using F2 backcross mice demonstrated that loci within the MHC gene region on mouse chromosome 17 contributed to host susceptibility. Furthermore, characterization of the H2k haplotype interval with polymorphic microsatellite markers in [BALB/c x C3H/HeN] F1 mice narrowed the critical gene interval size to 8.1 cM, defined by the flanking markers D17Mit101 and D17Mit68.

The molecular basis for the association between the human HLA haplotype and the severity of streptococcal infection has been related to a significant difference in the magnitude of the inflammatory cytokines responses of T cells elicited in response to GAS-SAgs when presented by different H-2 haplotypes (4). Therefore, we compared the induction of the inflammatory cytokines IFN-{gamma} and IL-12 in BALB/c and BALB/k mice during infection with S. pyogenes. Higher levels of these inflammatory cytokines were observed in BALB/k mice than in BALB/c mice, which might suggest a stronger response of T cells from the former mouse strain to SAgs produced by S. pyogenes. This possibility was confirmed by the greater proliferative response exhibited by spleen cells isolated from uninfected BALB/k mice after in vitro stimulation with culture supernatant from S. pyogenes, concomitant with a significantly higher production of IFN-{gamma}. Proliferation was mainly dependent on T cells, because depletion of this population completely abrogated the proliferative response. These results clearly suggested that genes within the MHC region are influencing the magnitude of cellular activation in response to GAS-SAgs in these mouse strains and, hence, the magnitude of the resulting pathology, as suggested for humans.

Each superantigen exhibits specificity for V{beta} sequences, and although there may be overlap in specificities, each superantigen binds its own unique set of T cells (26, 27). Therefore, we asked whether the S. pyogenes infection in this model was accompanied by visible changes in TCR V{beta} repertoire over the course of infection. TCR V{beta} analysis of spleen cells showed that S. pyogenes infection led to an increase in the percentage of T cells expressing V{beta}13 TCR in both BALB/c and BALB/k mice. However, a significantly higher expansion of V{beta}13 TCR-bearing T cells took place in mice that developed more severe infection (BALB/k) than in those that did not (BALB/c). These results were compatible with the pattern of V{beta} TCR changes observed after in vitro stimulation of spleen cells isolated from these mouse strains.

The hypothesis that a high level of strongly activated T cells by GAS-SAgs might contribute to infection severity was then corroborated by the capacity of high numbers of adoptively transferred, in vitro-activated T cells to enhance S. pyogenes infection. However, to what extent superantigen-activated T cells are responsible for the overall disease susceptibility was then determined in T cell-deficient SCID mice from a susceptible background strain. In these mice, the effect of H-2 on T cell activation by GAS-SAgs was absent. The results showed that although T cell-deficient mice from a susceptible background survive infection slightly longer than the corresponding immunocompetent mice, they remain fairly susceptible to S. pyogenes, suggesting that susceptibility might be mainly mediated by a cell population of the innate immune system. In this regard, it has been shown that the production of inflammatory mediators by monocytes/macrophages can significantly contribute to the pathogenesis of sepsis (43).

The possibility that S. pyogenes may induce a differential inflammatory transcriptional profile in macrophages from resistant and susceptible mice establishes a new hypothesis regarding the molecular mechanisms underlying the disease phenotype. In fact, genomic sequencing and annotation of the H2 chromosomal region containing the susceptibility loci have demonstrated the existence of a large number of genes putatively involved in inflammation and immune responses against infection (44).

In this study we have examined inflammation-related gene transcription of macrophages from BALB/c and C3H/HeN mice in response to S. pyogenes using a microarray approach. Based on our comparison of gene expression, it appears that S. pyogenes induced in both resistant and susceptible mice the expression of genes coding for proinflammatory cytokines (e.g., TNF-{alpha}, IL-6, IL-1{alpha}, and IL-1{beta}) as well as a rapid up-regulation of an arrangement of chemokine mRNAs, probably effective in mediating leukocyte recruitment to the infection foci.

Although Tnf was induced by S. pyogenes in macrophages from both C3H/He and BALB/c mice, higher levels of transcripts were found in the latter mouse strain. Intriguingly, the serum levels of TNF-{alpha} did not significantly increase during the course of infection in either resistant or in susceptible mice, confirming our previously reported data regarding this cytokine (22). The lack of TNF-{alpha} production in S. pyogenes-infected mice might be explained by the regulatory function attributed to IL-6. This cytokine has been shown to suppress TNF-{alpha} production either directly or indirectly through the stimulation of corticosterone production (45, 46, 47, 48). IL-6 is a cytokine produced by activated macrophages (49), and overproduction of IL-6 has been shown to correlate with poor outcome in patients with severe sepsis and septic shock (37, 38, 39, 40, 41, 42). In the studies we report, serum levels of IL-6 were significantly increased after infection with S. pyogenes in both mouse strains; however, these levels were greater in susceptible C3H/HeN than in resistant BALB/c mice. Because induction of Il6 was comparably induced after infection in macrophages from both mouse strains, differential regulation of IL-6 production may take place in BALB/c and C3H/HeN mice at the post-transcriptional level.

Adding an additional level of complexity to the inflammatory cascade, a recent study has shown that induction of the 70-kDa heat shock protein (HSP70) prevented high production of IL-6 and reduced tissue damage in septic mice (50). HSP70 is an endogenous cytoprotective molecule expressed in response to a variety of stressful stimuli, including microbial infections (51). Previous work has clearly and consistently shown that the induction of HSP70 gene transcription represses proinflammatory gene transcription and protects animals from sepsis-induced injury (50, 51, 52, 53, 54, 55). Interestingly, the gene coding for HSP70 (Hspa1b) is located in a region of chromosome 17 encompassing the locus associated with susceptibility to infection with S. pyogenes. Furthermore, this gene seems to be significantly more strongly up-regulated by S. pyogenes in macrophages from resistant BALB/c mice than in those from susceptible C3H/HeN mice. Therefore, the possibility that differences in the induction of Hspa1b between BALB/c and C3H/HeN mice may influence the inflammatory cascade and, in turn, the outcome of streptococcal infection should not be disregarded.

Interestingly, recent studies have shown that a particular functional polymorphism in the promoter region of HSPA1B affects HSP70 production and is a key determinant of individual susceptibility to a variety of infectious and inflammatory diseases (56). A significant association was also found between the HSPA1-B1267*A genotype and the risk of developing septic shock in a prospective cohort study of community-acquired pneumonia (57). Future studies should search for linkages between polymorphisms in the Hspa1b gene and resistance/susceptibility to infection with S. pyogenes.

The identification of susceptibility genes in the murine model of S. pyogenes infection followed by genomic analysis will be extremely useful for identifying their human homologues. Because the major histocompatibility gene region on human chromosome 6p21 is syntenic to the H2 gene region on proximal mouse chromosome 17 (58), identification of the specific loci may provide an entry point for elucidating pathways that are similar in humans and to search for polymorphisms associated with the variability of the response in humans. Elucidating the basis of host resistance/susceptibility to GAS will provide a rational basis for the development of new therapeutic and preventative strategies.


   Acknowledgments
 
We thank N. Janze and I. Wegener for excellent technical assistance, and also I. Sastalla and D. Zahner for characterization of the S. pyogenes strain.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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 in part by the Nationales Genomforschungsnetz I and II (Grants 01GS0404 and 01GR0439) and in part by Impuls und Vernetzungsfond, Helmholz Gemeinschaft Deutscher Forschungzentrum Präsidentenfonds. Back

2 Address correspondence and reprint requests to Dr. Eva Medina, Division of Microbiology, Department of Microbial Pathogenesis and Vaccine Research, Gesellschaft fur Biotechnologishe Forschung-German Research Center for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany. E-mail address: eme{at}gbf.de Back

3 Abbreviations used in this paper: GAS, group A streptococci; GAS-SAg, streptococcal superantigen; H-2, MHC class II; HSP70, 70-kDa heat shock protein; TTD, time to death (survival time after infection). Back

Received for publication February 3, 2005. Accepted for publication July 6, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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