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. 2006 Oct;146(1):93–100. doi: 10.1111/j.1365-2249.2006.03184.x

Mycobacterium tuberculosis-susceptible I/St mice develop severe disease following infection with taxonomically distant bacteria, Salmonella enterica and Chlamydia pneumoniae

L N Nesterenko *, D V Balunets *, A S Tomova *, J M Romanova *, J S Alyapkina *, N A Zigangirova *, M A Kapina †, E V Kondratieva †, A V Pichugin †, K B Majorov †, A S Apt †
PMCID: PMC1809730  PMID: 16968403

Abstract

Mice of I/St strain develop severe lung inflammation and die shortly following infection with virulent mycobacteria. To find out whether tuberculosis (TB)-susceptible I/St mice are susceptible to other intracellular bacteria, we investigated two different taxonomically distant pathogens, Chlamydia pneumoniae and Salmonella enterica serovar Typhimurium. Comparison of I/St and TB-resistant A/Sn mice (both Nramp1r) demonstrated that the former are more susceptible to both salmonella and chlamydia, displaying a significantly shortened survival time following challenge. Lung pathology develops more rapidly in I/St compared to A/Sn mice following infection with chlamydia, despite their similar ability to control bacterial multiplication. Following infection with salmonella, substantial (∼ 3 log) but very short (second day post-infection) interstrain differences in bacterial loads were observed, accompanied by higher levels of interleukin (IL)-6 and tumour necrosis factor (TNF)-α in the peritoneal cavities of I/St mice. I/St macrophages were more permissive for salmonella growth during the first 24 h following infection in vitro. Because the prominent differences in survival time did not correlate with permanent differences in bacterial multiplication, we suggest that both infections trigger fatal pathological processes whose dynamics depend strongly upon the host genetics.

Keywords: Chlamydia, mouse models, Salmonella, susceptibility

Introduction

The outcome of diseases caused by intracellular pathogens strongly depends upon host genetic factors [1]. Genetic variations in susceptibility to infections are under polygenic control and usually demonstrate complex patterns of inheritance [1,2]. This makes investigation of genetic mechanisms of infectious disease control very difficult, especially in heterogeneous human populations. Animal model systems have contributed to the discovery of some of the genetic pathways which determine the outcome of host–parasite interactions, e.g. the murine Nramp1 and Tlr4 (formerly Lps) genes [3–5]. Allelic variants of these genes determine, in part, the susceptibility to and severity of a number of intracellular and lipopolysaccharide (LPS)-producing Gram-negative bacterial infections in mice. Participation of their human homologues in the control of susceptibility to tuberculosis [6,7] and the airway responsiveness to LPS [8] has been confirmed recently. Development of new methodologies in mouse genetics for the analysis of complex genetic traits resulted in the mapping of several genetic loci involved in the control of intracellular infections [9–11]. However, researchers mainly use a particular combination of inbred mouse strains and/or a single set of recombinant inbred/congenic strains that prove to be informative in their own experimental system, usually in response to a single infectious agent. Combinations of genes and their alleles involved in the genetic control of any infection consist of an unknown number of players (probably, dozens). In each particular genetic cross, a unique combination of segregating and non-segregating alleles occurs. Very often the phenotype under study arises from epistatic interactions between several genes. For these reasons, the genetic mechanisms of resistance/susceptibility to intracellular infections remain largely unknown.

Infectious agents affect different organs and cause various pathological manifestations; accordingly, the host responses to infections display a high degree of diversity regarding thegenes, reactions and cells involved. Nevertheless, for some infections caused by taxonomically distant intracellular parasites there is both direct and indirect evidence for the existence of common genetic regulatory networks. Thus, quite expectedly, an increased susceptibility to various infections is caused by the lack-of-function mutations in genes encoding key cytokines and their receptors, e.g. interferon (IFN)-gamma and interleukin (IL)-12 [12–15]. The sensitivity to different infections may also depend upon allelic variations in a single gene whose function is less obviously linked to immune deficiencies. For example, the Nramp1 gene affects susceptibility of mice to Mycobacterium bovis bacille Calmette–Guérin (BCG), Leishmania donovani and Salmonella enterica serovar Thyphimurium [16], while the sst1 gene determines severity of M. tuberculosis and Listeria monocytogenes infections [17,18]. Less direct, but suggestive, evidence for the existence of genes that regulate host interactions with many different parasites comes from genome-wide scans for quantitative trait loci (QTL) involved in the control of different infections. These studies provided some intriguing matches for the chromosomal location of independently identified QTL [19–22], indicating that the existence of genetic networks that control general aspects of intracellular parasitism may be a reality.

Previously, we demonstrated that mice of the I/St inbred strain are exceptionally susceptible to tuberculosis (TB) infection and develop a severe pulmonary disease in various experimental models of acute, chronic and reactivation TB [23–25]. Susceptibility does not depend upon the Nramp1 gene, as I/St mice carry its resistant allele [19], but is controlled by a few interacting QTL mapped to chromosomes 3, 9, 17 and X [19,22]. The I/St mouse strain is not widely available and has never been studied in other intracellular infection models. We reasoned that the comparison between TB-susceptible I/St and TB-resistant mice regarding their susceptibility to non-mycobacterial intracellular parasites will provide useful information about universal versus specific mechanisms of infection control. Therefore, in the present study, we investigated manifestations of and immune responses to experimental infections caused by two different intracellular pathogens taxonomically distant from mycobacteria. TB-susceptible I/St and TB-resistant A/Sn mice were infected with the virulent strains of either Chlamydophila, formerly Chlamydia pneumoniae, or S. enterica serovar Typhimurium. The choice of these particular experimental infections was dictated by their localization: in mice C. pneumonia causes inflammatory lung disease, as does M. tuberculosis, whereas salmonella, sharing an intramacrophage location with mycobacteria, affects predominantly the intestine, spleen and liver. The experiments were designed to determine whether TB-susceptible mice are susceptible to other intracellular parasites despite their Nranp1r genotype, and how inflammatory and immune responses differ between mice of the two strains under study.

Materials and methods

Animals

Mice of inbred strains I/StSnEgYCit (I/St), A/JSnYCit (A/Sn), BALB/cJCit (BALB/c) and their F1 hybrids were maintained under conventional, non-specific pathogen free (SPF) conditions at the Animal Facilities of the Central Institute for Tuberculosis (CIT, Moscow, Russia) in accordance with guidelines from the Russian Ministry of Health no. 755, and the NIH Office of Laboratory Animal Welfare (OLAW) Assurance no. A5502-01. Water and food were provided ad libitum. Female mice of 20–22 g and 9–11 g body weight were used in salmonella and chlamydia infection models, respectively. All experimental procedures were approved by the CIT institutional animal care committee.

Microbes and infection models

C. pneumoniae strain K6 (a kind gift of Dr P. Saikku, Finland) was maintained by passage in the sensitive HL cell line [26]. Experimental infection was performed exactly as described [26,27]. In brief, chlamydia were isolated from the lysed HL cells and partially purified by one cycle each of low- and high-speed centrifugation, resuspended in sucrose-phosphate–glutamic acid buffer and stored at −70°C until used. Their content in the infecting inoculum was estimated by titration of a sample in HL cells using fluorescent microscopy to assess the numbers of inclusion-forming bodies (IFB) stained with the genus-specific fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (MoAb) CF-2. To initiate acute lethal infection, 1-month-old (∼10 g body weight) I/St and A/Sn mice were infected intranasally (i.n.) under light hexenal anaesthesia with 1·5 × 105 inclusion-forming units (IFU)/mouse in 30 µl. The rational for usage such young mice is the following. In a series of preliminary experiments we established that infection of young mice with Chlamydia results in a semi-acute lethal disease. This allows both modelling of the acute variant of human chlamydial pneumonia and the use of mortality curves as one of the readouts to estimate the severity of infection. Infection of adult I/St and A/Sn mice also results in markedly more severe early pathology in I/St compared to A/Sn lungs (data not shown); however, inflammation is eventually taken under control and mice of both strains cure the infection spontaneously. To estimate the degree of chlamydial multiplication in the lungs, we established a quantitative real-time polymerase chain reaction (PCR)-based method which allows enumeration of the parasite's genome equivalents in infected tissue. Whole lung tissue from individual mice was homogenized vigorously using the Ultra Turrax T8–S8N-8G tool (Heidolph, Germany), and DNA was extracted using the QIAmp DNA Mini Kit (Qiagen, Valencia, CA, USA). Specific primers and the TaqMan probe for the gene encoding chlamydial 16S rRNA were: primer CPN90: 5′-GGTCTCAACCCCATCCGTGTCGG-3′; primer CNP91: 5′-TGCGGAAAGCTGTATTTCTACAGTT-3′; probe: 5′-CAAGTCCAGGTAAGGTCCTTCGCGTTGC-3′.

PCR was performed under the following regimen: 95°C 10 min, 50 cycles 94°C−1 min, 64°C−1·5 min. Ten-fold serial dilutions of a known concentration of chlamydial DNA were used to create a calibration curve.

S. enterica serovar Typhimurium (var. C53), was grown in broth overnight, centrifuged, resuspended in sterile saline and serial dilutions were plated on blood agar for 1·5 h to estimate colony-forming unit (CFU) numbers, while the bulk sample was stored at 4°C. The sample was diluted in sterile saline to achieve a concentration of 100 CFU/ml, and 0·1 ml (10 CFU/mouse) were injected intraperitoneally (i.p.). Similar i.p. models of experimental salmonella infection were reported previously to be adequate for the study of genetic variations in susceptibility and bacterial killing [28]. Mortality was monitored daily. To estimate the salmonella loads, serial dilutions of peritoneal cavity lavages and whole spleen homogenates were plated onto SS-agar. Colonies were counted following 12 h incubation at 37°C.

Macrophage–salmonella interaction in vitro

The capacity of peritoneal macrophages to inhibit salmonella growth was assessed in vitro using a modification of the culture system developed earlier for mycobacterial infection [29]. Peritoneal macrophages were isolated exactly as described [29], with > 92% viability and 95–98% purity, as revealed by trypan blue exclusion and non-specific esterase staining, respectively. Macrophages (6 × 104 macrophages/well) were allowed to adhere for 1 h to 96-well flat-bottomed plates, and a salmonella suspension in supplemented RPMI-1640 without antibiotics was added at a multiplicity of infection (MOI) 10 : 1 and 100 : 1. Plates were centrifuged (400 g, 7 min) for salmonella sedimentation and incubated for 60 min to allow macrophages to ingest bacteria. Medium containing 200 µg/ml gentamycin was added for 15 min to kill extracellular salmonella, plates were washed twice and further incubated without antibiotics. At the indicated time-points (see Fig. 4, later) macrophages were lysed using sodium dodecyl sulphate (SDS)-containing medium, SDS was blocked with bovine serum albumin (BSA) and the well contents were plated onto SS-agar for CFU counting.

Fig. 4.

Fig. 4

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Peritoneal macrophages from A/Sn mice better inhibit salmonella growth following infection in vitro. Macrophages from A/Sn (squares) and I/St (triangles) mice were isolated and infected as described in Materials and methods, and the numbers of salmonella CFU per well were assessed in triplicate at indicated time points. Results of one out of three similar experiments are expressed as mean ± SD for triplicates. *P < 0·01; **P < 0·05, Student's t-test.

Histopathology

Six days post-infection with chlamydia, lung tissue was examined for pathology. Mice were euthanized by a thiopental overdose. Lung tissue (the middle right lobe) was frozen in the regimen of −60°C to −20°C temperature gradient in the electronic Cryotome® (ThermoShandon, Astmoor Runcorn, Cheshire, UK), and serial 8 mm-thick sections were made across the widest area of the lobe. Sections were stained with haematoxylin and eosin, and examined by an experienced pathologist without knowledge of the experimental group.

Immune responses

Local responses to chlamydia infection in the lung tissue and to salmonella infection in the peritoneal cavity were assessed. Lung cell suspensions were prepared following enzymatic disruption of lungs from individual mice exactly as described previously [24,29]. Peritoneal exudates cells were washed out from peritoneal cavities of individual mice with 1 ml of heparin-containing phosphate-buffered saline (PBS) 24 h after infection. After centrifugation and washing, cells were resuspended in PBS supplemented with 0·5% BSA and 0·01% NaN3 and incubated for 5 min at 4°C with CD16/CD32 MoAbs (clone 2·4G2, Pharmingen, San Diego, CA, USA) to block Fc-receptors. Cells were then stained with the following directly conjugated antibodies, according to the manufacturer's instructions: FITC-anti-CD4 (clone H129·19, Pharmingen), FITC-anti-CD8a (clone 53–6·7, Pharmingen) and FITC-anti-MAC-3 (clone M3/84, Pharmingen). Stained cells (104 cells per sample) were washed twice, fixed with 1% paraformaldehyde and analysed by flow cytometry, using a fluorescence activated cell sorter (FACS)Calibur cytometer (Becton Dickinson, San Diego, CA, USA) and BD-CellQuestPro (Becton Dickinson) and FlowJo 4·5.9 (Tree Star, Inc., San Carlos, CA, USA) software.

IL-6, IL-10, IL-12, tumour necrosis factor (TNF)-α and IFN-γ content in individual 1 ml cell-free whole-lung homogenates or peritoneal lavage samples was assessed using sandwich enzyme-linked immunosorbent assays (ELISA). The following ELISA kits were purchased from Pharmingen and used according to the manufacturer's instructions: OptEIA mouse IL-6 (sensitivity 15·6 pg/ml), OptEIA mouse IL-10 set (63 pg/ml), OptEIA mouse IL-12 set (31 pg/ml), OptEIA mouse TNF-α set (31 pg/ml), OptEIA mouse IFN-γ set (63 pg/ml).

Results and discussion

I/St mice are susceptible to chlamydia infection

To evaluate possible differences between I/St and A/Sn mice in susceptibility to or severity of infection caused by C. pneumoniae, young (∼ 10 g body weight) mice were infected intranasally with 1·5 × 105 chlamydial IFU/mouse. As shown in Fig. 1a, (I)/St and A/Sn mice differed profoundly in time to death following challenge [mean survival time (MST) = 9·2 ± 1·2 and 22·0 ± 2·0, respectively, P < 0·001]. Chlamydia are obligate intaracellular parasites, do not grow in artificial media, and their enumeration in infected lung tissue by titrating samples in HL cells often provides variable results. Thus, to find out whether or not mortality is linked to the parasite accumulation in the lungs, we estimated bacterial loads by a surrogate qrt-PCR method, which yields genome equivalents per organ. As shown in Fig. 1b, multiplication of chlamydia in the lungs was controlled efficiently after day 4 of infection, and the numbers of genome equivalents dropped slightly by day 8 both in I/St and A/Sn mice. Nevertheless, I/St mice started to die at this time-point, and A/Sn mice showed a sharp peak of mortality around day 20 post-infection despite the ∼ 1·5 log decrease in parasite loads in their lungs (Fig. 1b).

Fig. 1.

Fig. 1

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Mice of the I/St strain rapidly develop fatal disease (a), despite their ability to control multiplication of the parasite (b) following infection with C. pneumoniae. (a) I/St (triangles) and A/Sn (squares) mice in groups of 15 were infected i.n. with 1·5 × 105 chlamydial IFB/mouse, and mortality was monitored daily (P < 0·01 between mean survival time of the strains, Gohan's criterion for survival curves). (b) Whole lung tissue homogenates were obtained from individual I/St and A/Sn mice (3 animals of each strain per time point), and the numbers of genome equivalents per organ were estimated by qrt-PCR as described in the Materials and methods. Results are expressed as mean ± SEM. Results of one out of two similar experiments are displayed in (a) and (b).

Taking into account that the multiplication of chlamydia did not appear to be a direct cause of mortality in our model and that infection with C. pneumoniae may cause acute pneumonitis both in humans [30] and mice [27], we compared lung pathology in I/St and A/Sn animals following infection. Histological examination of the lung tissue sections at day 6 post-infection revealed prominent interstrain differences in the severity of lung disease. In I/St mice, lungs were severely infiltrated with a mixture of inflammatory cells, including neutrophils, macrophages and lymphocytes, which resulted in the development of condensed pneumonic foci and almost full obstruction of alveolar spaces (Fig. 2a). In contrast, only limited pathology, characterized by the increased thickness of alveolar septae and the presence of small infiltrative foci, was observed in the lungs of A/Sn mice at this time-point (Fig. 2b). However, by day 18 of infection, severe lung pathology also developed in A/Sn mice (not shown) and apparently caused death. Thus, the C. pneumoniae-induced disease in our model progressed as a triggered pathological reaction in the lung tissue, whose rate of development depended upon the host genotype rather than the degree of parasite multiplication.

Fig. 2.

Fig. 2

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Differences in the degree of lung pathology between mice of the two strains at day 6 post-infection with chlamydia. Severe lung pathology with alveolar obstruction and numerous condensed pneumonic foci (arrows) were seen in the lungs of I/St mice (a). In A/Sn mice (b), pathological changes in the lung tissue remained mild. Hematoxilin–eosin staining of the 8 micrometre-thick lung tissue cryoslides (magnification × 150).

To understand better the nature of inflammatory response, we also assessed whether I/St and A/Sn mice differ regarding the influx of distinct leucocyte populations and production of key effector and regulatory cytokines in the lung tissue following infection with C. pneumoniae. In our previous experiments it was shown that in the absence of infection there was no significant difference between the two mouse strains regarding cellular composition of the lung tissue [24]. As shown in Table 1, at day 6 post-infection the lung tissue of susceptible I/St mice was markedly infiltrated with macrophages (MAC-3-positive cells comprised ∼ 15% of the total cell content), which differed significantly from the lungs of resistant A/Sn mice. The latter showed no significant shifts in cellular composition of the lung tissue compared to naive animals. In agreement with higher macrophage content in the lungs, significantly more macrophage-derived proinflammatory cytokines TNF-α and IL-6 were detected in lung tissue homogenates obtained from I/St mice (Table 1). The content of effector T cell cytokines IFN-γ and IL-10 was very low and equal in the two mouse strains (data not shown), presumably reflecting a pre-T cell phase of inflammatory response at day 6 of infection.

Table 1.

Infiltration with lymphoid cells and cytokine production in the lungs of susceptible I/St and resistant A/Sn mice 6 days following chlamydia infection.

Lung cell content (% of live cells) Cytokine content (pg/ml)
Mouse strain
B (CD19) T (CD4) T (CD8) Mph (MAC-3) TNF-α IL-6
A/Sn 11·7 ± 4·4 15·5 ± 4·5 11·1 ± 2·1 5·7 ± 1·5 56·8 ± 20·1 110 ± 30·4
I/St 12·6 ± 1·7 22·3 ± 5·2 11·5 ± 3·2 15·6 ± 2·8 101·0 ± 13·3 240 ± 50·7
Significance (P) 0·81 0·15 0·83 0·01* < 0·05* < 0·05*
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Mice of the two strains in groups of four were infected intranasally with with 1·5 × 105 inclusion-forming units (IFU)/mouse in 30 µl. Six days post-challenge, individual left lungs were homogenized in 1 ml of phosphate-buffered saline and frozen for further estimation of cytokine content. Right lungs were individually digested with collagenase-DNAase mixture [24,29], single-cell suspensions prepared, stained with indicated labelled antibodies and propidium iodide (PI), and analysed using a fluorescence activated cell sorter, with gating for the PI-negative population in each probe. Results of one of two similar experiments are expressed as mean ± s.d.

*

Statistically significant differences.

Previous assessments of interstrain differences in the severity of chlamydial disease provided a highly variable picture of mortality/survival within individual mouse strains; moreover, the severity of the disease was not compared adequately between strains [27]. Our results suggest that the comparison of I/St and A/Sn mouse strains may be a useful tool for the study of natural genetic variations in susceptibility and innate immunity to C. pneumoniae. So far, mouse models based upon gene disruption have been the main source of information about the role of distinct populations of lymphoid cells and cytokines produced by these cells in protection against chlamydia [31–33]. Gene targeting, which results in a complete abrogation of key functions of the immune system, leads to extreme defects in protection against infections. Given that such defects are normally eliminated rapidly from a population by natural selection, they could hardly account for the much more common, modestly susceptible, phenotypes. We suggest that the correct choice of strain combination and experimental challenge protocol allows a more rational study of natural variations in susceptibility to chlamydia in traditional murine models, i.e. by combining interstrain comparisons with segregation genetic analysis.

I/St mice are susceptible to salmonella infection

In the next series of experiments we evaluated whether TB-susceptible I/St and TB-resistant A/Sn mice differ in susceptibility to a non-mycobacterial, non-pulmonary intracellular pathogen. To this end, we infected the animals with different doses of virulent salmonella via the i.p. route. Because susceptibility to salmonella depends strongly upon the expression of resistant and susceptible alleles of Nramp1 gene [16], and A/Sn and I/St mice both carry the resistant Nramp1r allele [19], BALB/c-Nramp1s mice were included as the susceptible control. Infection with 10 CFU/mouse provided a clear-cut picture of interstrain differences. As shown in Fig. 3a, I/St mice were almost as susceptible as Nramp1s BALB/c mice (MST = 7·2 ± 0·9 days and 5·0 ± 0·8 days, respectively), whereas A/Sn mice displayed a significantly (P < 0·001) more resistant phenotype (MST = 16·3 ± 1·5 days; ∼ 20% of animals survived infection indefinitely). Similar, albeit somewhat less pronounced, differences were observed following the 102 CFU/mouse challenge (not shown). To demonstrate further that the genetic traits determining susceptibility to salmonella in I/St and BALB/c mice are different, we infected (I/St × BALB/c) F1 hybrids and obtained a clear picture of genetic complementation: F1 hybrids between two susceptible parental strains (one Nramp1s, the other Nramp1r) survived infection, displaying an even more resistant phenotype than the relatively resistant A/Sn animals (Fig. 3a). As expected (I/St × A/Sn) F1 hybrids were also resistant and survived infection indefinitely (data not shown). Inheritance of resistance to infection that follows the pattern of superdominance (genetic heterosis) was demonstrated earlier for TB infection in several F1 combinations that involved I/St and BALB/c parents [34], and is indicative of the polygenic character of genetic control of the trait.

Fig. 3.

Fig. 3

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Interstrain differences in susceptibility to salmonella infection. Mice of the A/Sn (squares), I/St (triangles), and BALB/ c (diamonds) strains and (I/St × BALB/c) F1 hybrids (circles) were infected i.p. with 10 CFU of S. enteridica var. Typhimurium. Mortality (a) was monitored daily in groups of 10 animals of each strain. To assess the dynamics of salmonella multiplication in peritoneal cavities (b) and spleens (c), mice in groups of 4 were sacrificed at indicated time points post infection, and serial dilutions of peritoneal lavages and spleen homogenates were plated individually on SS blood agar. Results of one out of two similar experiments are displayed as mean CFU ± SD (b, c: P < 0·0001 between I/St and A/Sn mice at the 24-h time point, Student's t-test).

In addition to survival curves, the differential susceptibility of I/St and A/Sn mice to salmonella was underlined by the differences in the speed of bacterial multiplication in the peritoneal cavity and spleen at the early phase of infection: 3 log more salmonella CFU were recovered from I/St compared to A/Sn mice 24 h post-challenge (Fig. 3b,c). Although this difference was only transient and disappeared by day 3 of infection, rapid multiplication of the parasite in susceptible mice immediately following challenge could have a significant impact on their survival time, given the acute nature of infection caused by LPS-producing salmonella and the paramount importance of rapid, innate, macrophage-mediated mechanisms for host protection [35]. To evaluate whether the difference in susceptibility to salmonella between I/St and A/Sn mice depended upon different capacities of macrophages which accumulated at the site of infection to inhibit bacterial growth, as was shown earlier for TB infection [29], we performed in vitro experiments with peritoneal macrophages. Macrophages were isolated, purified and infected as described in Materials and methods and, at selected time-points, the numbers of live salmonella were assessed by counting CFU. In full agreement with results obtained in vivo (Fig. 3b,c), inhibition of bacterial growth started earlier and occurred faster in A/Sn compared to I/St macrophages at MOI 100 : 1 (Fig. 4) and 10 : 1 (not shown).

Local immune response to salmonella infection

Because interstrain differences in the capacity to inhibit salmonella multiplication in vivo and in vitro became apparent at 24 h following infection, we assessed whether mice of the two strains differ regarding cell influx and the level of key cytokines in the peritoneal cavities at this time-point. The numbers of all types of cells in peritoneal cavities of control noninfected I/St and A/Sn mice was low and equal (data not shown). As shown in Table 2, 24 h post-infection macrophages represented about a half of the total cell content of peritoneal exudates, whereas T cells were present in very low numbers in both mouse strains. Interestingly, significantly more CD19+ B cells were recovered from A/Sn compared to I/St mice (Table 1). Due to technical difficulties (to obtain reasonably uniform samples peritoneal cavities were lavaged with relatively large volumes of liquid which diluted the exudates) we were unable to compare directly the level of early anti-salmonella IgM and IgA antibodies during the first 2 days of infection. In contrast to most other intracellular bacteria, antibodies are protective against salmonella [36,37]. Thus, one may speculate that a more vigorous B cell response played a role in protection of the relatively resistant A/Sn mice.

Table 2.

Infiltration of peritoneal cavities of susceptible I/St and resistant A/Sn mice with lymphoid cells 24 h following salmonella infection.

Cell content in peritoneal cavity (% of live cells)
Mouse strain B (CD19) T (CD4) T (CD8) Mph (MAC-3)
A/Sn 36·8 ± 3·8 1·7 ± 0·5 0·5 ± 0·1 45·8 ± 3·8
I/St 16·4 ± 4·8 0·9 ± 0·3 0·7 ± 0·1 48·4 ± 6·6
Significance (P) 0·02 0·28 0·45 0·86
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Mice of the two strains in groups of four were infected intraperitoneally with 10 colony-forming units/mouse; 24 h post-challenge peritoneal cavities were washed with 1 ml of warm phosphate-buffered saline containing heparin, cells from individual mice were collected, stained with indicated labelled antibodies and propidium iodide (PI), and analysed using a fluorescence activated cell sorter, with gating for the PI-negative population in each probe. Results of one of two similar experiments are expressed as mean ± s.d. Statistically significant difference in the B cell content is marked in bold type.

We also compared mice of the two strains regarding their capacity to produce key effector and inflammatory cytokines early at the site of infection. As shown in Fig. 5, significantly (P < 0·05) higher amounts of two proinflammatory cytokines, TNF-α and IL-6, were secreted into the peritoneal cavities of I/St compared to A/Sn mice. The latter effectively controlled salmonella multiplication during the first 24 h of infection, keeping bacterial loads very low (Fig. 3b,c). It is likely that antigenic and toxic stimulation of the immune system remained low in A/Sn mice during this period whereas, in I/St animals, the bacterial loads reached a level sufficient to induce the release of proinflammatory cytokines into the peritoneal cavity. No detectable amounts of IL-10, IL-12 and IFN-γ were found, indicating that these types of responses in peritoneal cavity either occurred later or were too weak to be detected by ELISA.

Fig. 5.

Fig. 5

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Higher amounts of proinflammatory cytokines tumour necrosis factor (TNF)-α and interleukin (IL)-6 are secreted into the peritoneal cavities of susceptible I/St compared to resistant A/Sn mice following infection with salmonella. Mice of the two strains in groups of four were infected intraperitoneally with 10 colony-forming units of salmonella. After 24 h, IL-6 and TNF-α content in individual 1 ml cell-free peritoneal lavage samples was assessed using sandwich enzyme-linked immunosorbent assays. Results (pg/ml) of one of two similar experiments are expressed as mean ± s.e.m. Interstrain differences were significant (P < 0·05, Student's t-test) for both cytokines.

In summary, this is the first report that Nramp1r I/St mice develop rapidly progressive, fatal diseases after infection, not only with M. tuberculosis but also with two other virulent intracellular bacteria, C. pneumoniae and S. enterica. The picture of a poorly controlled inflammatory response that develops in I/St mice following infection with all three agents indicates that an intrinsic defect(s) in the ability to maintain the balanced pattern of immune reactions following infection is probably a common and important host genetic factor operating in these experimental models. Segregation analysis is in progress to find out whether or not genetic traits that influence susceptibility to Salmonella and Chlamydia infections co-localize with QTL involved in the control of experimental TB.

Acknowledgments

This work was supported by R01 grant no. HL-68532 from NIH and by grant no. 05-04-49861 from the Russian Foundation for Basic Research.

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