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Divergent chemokine, cytokine and ß-defensin responses to gastric candidiasis in immunocompetent C57BL/6 and BALB/c mice

Department of Microbiology and Immunology¹ and Department of Stomatology², Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29403, USA

Correspondence David A. Schofield schofida{at}musc.edu

Received May 27, 2004
Accepted September 21, 2004

Previous studies of animal models of candidiasis have produced conflicting results concerning the cytokines and host defence mechanisms that are most relevant for protection against Candida infections. In this study, the host defence mechanisms evoked by two different immunocompetent murine strains following oral colonization with Candida albicans were assessed. ß-Defensin (mBD1, mBD3 and mBD4), chemokine (MIP-2 and KC) and cytokine (TNF-, IFN-, IL-4, IL-10, IL-12 and IL-15) gene expression in germ-free (gf) and C. albicans-infected (gastric) C57BL/6 and BALB/c mice was contrasted. Gf C57BL/6 and BALB/c mice expressed significantly different basal levels of mBD3, mBD4, TNF- and IL-12 in gastric tissues; however, gf C57BL/6 and BALB/c mice were equally susceptible to intestinal colonization with C. albicans and had similar fungal burdens in gastric tissues 4 weeks after oral challenge. C57BL/6 mice responded to colonization and gastric candidiasis with increased expression of mBD1, mBD3, mBD4, TNF-, MIP-2, KC and IL-12. Conversely, a much more specific and attenuated response was observed in Candida-infected gastric tissues from BALB/c mice. Therefore, different strains of mice that were equally susceptible to gastric candidiasis after oral challenge had divergent cytokine, chemokine and ß-defensin responses. This suggests that conflicting data as to the relevance of cytokines and other host factors in murine resistance to candidiasis may be explained, at least in part, by the strain of mouse studied.

	Introduction

TOP Introduction Methods Results and Discussion ACKNOWLEDGEMENTS References

 
Candida albicans can cause a variety of clinical diseases ranging from mild mucosal infections to life-threatening systemic disease in the immunocompromised host. Cell-mediated immunity at the site of infection is thought to be critical in the protection against both mucosal and systemic candidiasis. Cytokines and chemokines play important roles in stimulating the anti-Candida activity of phagocytes (Djeu, 1990) and in recruitment of neutrophils and macrophages to the site of infection (Balish et al., 1999). Antimicrobial peptides, such as ß-defensins, may also function directly in resistance to candidiasis by killing the fungus (Burd et al., 2002); however, animal models of infection using specific cytokine knockout mice, the administration of recombinant cytokines or the depletion of specific cytokines using anticytokine antibodies have generated conflicting data as to the cytokines that are most relevant to protection (Balish et al., 1998; Elahi et al., 2000; Kaposzta et al., 1998; Puccetti et al., 1994; Qian & Cutler, 1997) (and references therein). Such paradoxical findings may be explained by deriving data from different experimental models (guinea pigs, hamsters, rats or mice), different routes of challenge (oral, intranasal, intraperitoneal or intravenous challenges), different doses of C. albicans and even the choice of media used to grow the initial inocula (Odds et al., 2000). Susceptibility and survival of the animal host during disseminated candidiasis has also been shown to depend upon the strain of mouse used (Ashman, 1990; Ashman & Papadimitriou, 1987; Hector et al., 1982); however, some groups have reported that BALB/c mice are more susceptible to intravenous challenge with C. albicans than C57BL/6 mice (Ashman & Papadimitriou, 1987; Marquis et al., 1986), while others have shown no significant difference in their susceptibility (Hector et al., 1982).

To investigate the relationship between host immunity to candidiasis in different murine strains, we examined ß-defensin (mBD1, mBD3 and mBD4), chemokine (MIP-2 and KC) and cytokine (IFN-, TNF-, IL-4, IL-10, IL-12 and IL-15) gene expression in stomachs from germ-free (gf) immunocompetent BALB/c and C57BL/6 mice and C. albicans-monoassociated (Ca-ma, alimentary tract) BALB/c and C57BL/6 mice during gastric candidiasis. The two different strains of mice exhibited similar levels of C. albicans colonization and gastric infection, but differed significantly in the immune responses to colonization and gastric infection.

	Methods

TOP Introduction Methods Results and Discussion ACKNOWLEDGEMENTS References

 
Micro-organisms.

The clinical isolate C. albicans SC5314 was grown on Sabouraud Dextrose Agar or Broth (SDA or SDB; Difco) for 24 h at 37 °C and maintained at 4 °C.

Mice.

Immunocompetent inbred C57BL/6 and BALB/c mice were derived into the gf state by caesarian section. The mice were obtained from gf colonies maintained at the University of Wisconsin Gnotobiotic Research Laboratory.

Animal model of gastric candidiasis.

Gf male and female adult mice were orally inoculated with a pure culture of C. albicans (10⁶ viable cells) harvested from cultures grown in SDB. Fecal pellets from orally inoculated mice were serially diluted and plated onto SDA, which indicated that the gf mice became colonized (alimentary tract) within 24 h after inoculation. To limit variations between experiments, mice were inoculated and harvested at approximately the same time of day and a mixture of female and male adult mice were used for each experimental group. Stomachs, after the contents were removed, were rinsed three times with PBS prior to homogenization thereby ensuring that the number of c.f.u. represented the number of cells in the infected stomach tissue. The number of c.f.u. in the stomach and caecum were measured after disruption in PBS using the Stomacher II (Fisher). The homogenates were serially diluted and plated on SDA. The number of c.f.u. were counted after incubation at 37 °C for 24 h and are presented as the log₁₀ viable C. albicans c.f.u. [g (dry weight) tissue]^–1.

Histopathology.

Tissue samples were collected aseptically and stomach, liver, kidney and spleen were fixed in 10 % formaldehyde in PBS. The tissues were processed in graded (100, 95, 80 and 70 %) alcohol and xylene solutions and embedded in paraffin. Tissue sections (5 µm) were stained with the periodic acid–Schiff stain for fungi. Histopathology of Candida-colonized and control (gf) tissues was ranked by a clinical pathologist (Dr Thomas Warner, University of Wisconsin, Madison, WI, USA).

RNA extraction and relative RT-PCR.

RNA was prepared from tissues from gf control and C. albicans-infected mice as described previously (Schofield et al., 2004). The quantity and quality of RNA was measured spectrophotometrically at A₂₆₀ and A₂₈₀. RNA (2 µg) from infected and gf control tissues was reverse transcribed into cDNA using the retroscript kit (Ambion). PCRs were performed initially using primers (Table 1) designed against the housekeeping gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to ensure equal amounts of cDNA were used for each sample. If required, the amount of starting cDNA template was then adjusted accordingly. After an initial denaturation at 95 °C for 2 min, the samples were subjected to 30 (GAPDH) or 35 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C (mBD1, mBD3, mBD4, TNF-, MIP-2, IL-4, IL-10 and IL-15), 60 °C (IL-12) or 62 °C (KC) for 30 s, and extension at 72 °C for 30 s, with a final extension at 72 °C for 2 min. The PCR products were gel-purified and sequenced directly by the deoxy dye terminator method using an ABI 377 DNA sequencer to confirm their identity. PCRs lacking reverse transcriptase were subjected to PCR amplification to check for the presence of contaminating genomic DNA. PCR products were not obtained from these control reactions. In addition, the primers for mBD1, TNF- and KC amplification were designed to flank an intron thereby ensuring that these products were derived from cDNA as opposed to genomic DNA. Relative RT-PCR was used primarily when expression in one of the experimental groups was low to undetectable, and was therefore unsuitable for measurement by competitive RT-PCR.

View this table: [in this window] [in a new window]   Table 1. PCR primers used in this study The PCR primers for GAPDH, mBD1, mBD3 and mBD4 amplification and their corresponding competitor primers used to make the plasmid mimics have been described previously (Schofield et al., 2004), while the IFN-, IL-4, IL-10 and IL-12 (p40) primers were obtained from BioSource (Camarillo, CA, USA).

Competitive RT-PCR.

Competitor plasmid mimics containing the target DNA but missing an internal deletion (approx. 10 %) were constructed into pBluescript II SK^– (Stratagene) as described previously (Schofield et al., 2004). The primers (Table 1) used to construct the plasmid mimics contained XhoI and HindIII sites (bold) for cloning into the corresponding sites of pBluescript II SK^–. The sequence of the target drop out plasmids was confirmed by DNA sequencing.

PCRs containing cDNA and the plasmid mimic were prepared from master amplification mixes to minimize errors and the PCRs, after using the same cycling parameters as for the relative RT-PCR, were resolved using polyacrylamide (5 or 10 %) gel electrophoresis. The intensities of the bands were quantified using a FluorImager (Molecular Dynamics) and the ratio of target to competitor product was calculated for the target and for GAPDH. The ratio of target to competitor product was then normalized to the ratio of GAPDH internal control in each sample.

Statistical analysis.

The numbers presented are the means of three tissues from three mice plus standard deviations. The Student's t-test was used to determine statistical significance between the experimental groups. Differences were considered significant if the P value was less than 0.05.

	Results and Discussion

TOP Introduction Methods Results and Discussion ACKNOWLEDGEMENTS References

 
Basal gene expression in gf C57BL/6 and gf BALB/c mice

Competitive and relative RT-PCR was used to compare basal gene expression in stomach tissues from gf C57BL/6 and gf BALB/c mice (Fig. 1a, b). Expression of mBD1, MIP-2, KC, IL-10 and IL-15 was similar in stomach tissues from gf C57BL/6 and BALB/c; however, mBD4 expression was approximately threefold higher in gf C57BL/6. In contrast, TNF-, mBD3 and IL-12 were expressed at higher levels in gf BALB/c mice compared to gf C57BL/6 (Fig. 1a, b). Therefore, gf C57BL/6 and gf BALB/c mice expressed different basal levels of cytokine and ß-defensin, but not chemokine, mRNA in gastric tissues.

View larger version (37K): [in this window] [in a new window]   Fig. 1. Comparison of basal gene expression in stomach tissues harvested from gf immunocompetent C57BL/6 and BALB/c mice. (a) Competitive RT-PCR analysis of mBD1, mBD4, TNF-, IL-10, IL-12, MIP-2 and KC expression. *, P < 0.05 for BALB/c compared to the C57BL/6 mice. (b) Relative RT-PCR analysis of GAPDH (internal loading control), mBD3 and IL-15 expression in the latter gf tissues. The predicted sizes (bp) of RT-PCR were 217, 85 and 362, respectively. The analysis was performed using three tissues from three mice for each group. A PCR was also performed in the absence of cDNA template (lane 1). M, 100 bp DNA ladder.

Non-lethal mouse model of gastric candidiasis

Gf C57BL/6 and gf BALB/c mice, which were naturally susceptible to gastric candidiasis, were quickly colonized with C. albicans (1 day after oral inoculation) and remained chronically colonized (alimentary tract) for the duration of the study (4 weeks). Fecal pellets consistently yielded between 7 and 8 log₁₀(c.f.u. C. albicans g^–1) throughout the 4-week study period. Ca-ma C57BL/6 and BALB/c mice developed a low grade infection of the cardia-antrum section of the stomach. C. albicans colony counts from tissues indicated there was no significant difference in the degree of C. albicans colonization (caecum) or infection (gastric tissues) between the Ca-ma C57BL/6 and BALB/c mice (Fig. 2a). Histopathology of the infected tissues by a clinical pathologist (Dr Thomas Warner, University of Wisconsin) also indicated similar levels of gastric infection in both strains of mice at 4 weeks after oral challenge (data not shown). Microscopic examination of homogenized stomach tissues and of periodic acid–Schiff stained tissue sections cut from formalin-fixed paraffin-embedded stomach tissues for both Ca-ma C57BL/6 and BALB/c mice indicated that C. albicans existed as mostly hyphae/pseudohyphae in the infected gastric tissues (Fig. 2b). No dissemination to the internal organs (liver, kidney or spleen) was detected in these immunocompetent mice.

View larger version (53K): [in this window] [in a new window]   Fig. 2. (a) Alimentary tract colonization of Ca-ma immunocompetent C57BL/6 and BALB/c mice. Colony counts were from infected gastric tissues (stomach) and colonized tissue (caecum contents) isolated from chronically colonized mice harvested 4 weeks after oral inoculation. Numbers represent the mean of three individual stomachs or the contents of three caeca and the standard deviation. (b) Histopathology of C. albicans-infected stomachs harvested from C57BL/6 and BALB/c mice 4 weeks after oral association. The representative tissues shown were fixed in buffered formalin and stained using a standard periodic acid–Schiff reaction. Arrows denote the presence of infecting fungi. Magnification x400.

Divergent C57BL/6 and BALB/c immune responses to gastric candidiasis

Since previous studies using immunocompetent mice have shown that both C. albicans virulence factor expression and host immune responses were up-regulated at 4 weeks after Candida association (Schofield et al., 2003, 2004), we examined the specific cytokine, chemokine and ß-defensin response in Candida-infected stomach tissues from Ca-ma C57BL/6 and BALB/c mice 4 weeks after oral inoculation. The expression of mBD1 (2.5-fold), mBD4 (2.2-fold), TNF- (4.0-fold), IL-12 (2.9-fold), MIP-2 (13.0-fold), KC (3.7-fold) and mBD3 was increased in gastric tissues from Ca-ma C57BL/6 mice compared to gf C57BL/6 mice, while the expression of IL-10 and IL-15 was similar in the latter tissues (Fig. 3a, b). In contrast, the response of BALB/c mice to gastric candidiasis was attenuated and much more specific compared to that of C57BL/6 mice. For example, expression of mBD1, mBD4, TNF-, IL-10 and IL-12 was not significantly increased in tissues from Ca-ma BALB/c mice compared to gf BALB/c mice. The relative levels of IL-15 were also similar from the latter tissues (Fig. 4a, b). In addition, although expression of MIP-2 (1.7-fold) and KC (1.9-fold) was significantly increased in the response of BALB/c mice to infection, the increase in expression was much lower than that obtained for the Ca-ma C57BL/6 mice (13.0- and 3.7-fold, respectively). The exception to this was mBD3, which was increased in both Ca-ma BALB/c and C57BL/6 mice compared to gf mice. Stomach tissues from gf and Ca-ma mice were also tested for the presence of IFN- and IL-4; however, transcripts were low to undetectable from both strains of mice (data not shown).

View larger version (38K): [in this window] [in a new window]   Fig. 3. Gene expression in gf and Candida-infected gastric tissues from immunocompetent C57BL/6 mice. Stomachs were harvested from Candida-infected mice at 4 weeks after oral inoculation. (a) Competitive RT-PCR analysis was used to compare mBD1, mBD4, TNF-, IL-10, IL-12, MIP-2 and KC in stomach tissues. *, P < 0.05 for tissues from Ca-ma mice compared to the tissues from gf control mice. (b) Relative RT-PCR analysis of mBD3 and IL-15 expression in the latter tissues. The analysis was performed using three stomach tissues from three mice for each group. A PCR was also performed in the absence of cDNA template (lane 1). M, 100 bp DNA ladder.

View larger version (26K): [in this window] [in a new window]   Fig. 4. Gene expression in gf and Candida-infected gastric tissues from immunocompetent BALB/c mice. Stomachs were harvested from Candida-infected mice at 4 weeks after oral inoculation. (a) Competitive RT-PCR analysis was used to compare mBD1, mBD3, mBD4, TNF-, IL-10, IL-12, MIP-2 and KC in stomach tissues. *, P < 0.05 for tissues from Ca-ma mice compared to the tissues from gf control mice. (b) Relative RT-PCR analysis of IL-15 expression in the latter tissues. The analysis was performed using three tissues from three mice for each group. A PCR was also performed in the absence of cDNA template (lane 1). M, 100 bp DNA ladder.

Using two different strains of immunocompetent mice, we have shown that both strains exhibited a significantly different immune response to gastric candidiasis at the site of infection. Gene expression of mBD1, mBD3, mBD4, TNF-, IL-12, MIP-2 and KC was increased in C57BL/6 mice in response to the gastric infection. In contrast, only mBD3, and to a lesser extent MIP-2 and KC, were increased in the gastric tissues during infection in BALB/c mice. The different host response to gastric candidiasis between C57BL/6 and BALB/c mice was not due to differences in the level of colonization and infection since both strains of mice had similar C. albicans c.f.u. in the alimentary tract and infected stomach tissues. A single time point at 4 weeks after oral inoculation was chosen to examine the ß-defensin, chemokine and cytokine response since our previous results using different animal models of gastric candidiasis have indicated that these responses are ‘activated’ after 1–2 weeks and remain elevated for the duration of the infection (Schofield et al., 2004; D. A. Schofield & E. Balish, unpublished data); however, it is possible that the observed differences in expression between C57BL/6 and BALB/c mice might reflect differential temporal responses to infection and a more robust BALB/c response might have been evident if additional time points were analysed. The relative lack of stimulation of ß-defensin, chemokine and cytokine expression in Ca-ma BALB/c mice cannot be generally accounted for by a higher level of basal expression in gf BALB/c mice compared to in gf C57BL/6 mice. The fact that both murine strains were similar in their susceptibility to candidiasis therefore suggests that other immune functions may ‘compensate’ or be more/less important in resistance to candidiasis in relation to the strain studied. A similar suggestion was made by Kaposzta et al. (1998), who explained their paradoxical IL-4 results with previous reports by hypothesizing that ‘anti-IL-4 treatment can improve the resistance (to candidiasis) only in certain strains which exhibit a predominantly Th2-type immune response'. That some murine strains are more inherently susceptible to candidiasis than other strains has been described previously and partially explained by decreased IFN- expression and impaired T-cell responses (Ashman, 1990; Neta & Salvin, 1983); however, our data indicate that gf C57BL/6 and BALB/c mice were equally susceptible to gastric candidiasis using a natural route of infection (oral) yet mediate divergent immune responses that did not increase IFN- or IL-4 expression. The different responses in the two mouse strains also indicate that there was little direct correlation between resistance to candidiasis and basal or ‘stimulated’ ß-defensin, cytokine and chemokine mRNAs between the two strains. The only exception was mBD3, and to a lesser extent MIP-2 and KC, which were induced in both strains of mice during infection.

In summary, gf C57BL/6 and BALB/c were equally susceptible to colonization and gastric infection after oral inoculation with C. albicans; however, BALB/c mice exhibited a much more specific and attenuated immune response to alimentary tract colonization and gastric candidiasis compared to C57BL/6 mice. Therefore, conflicting data on the importance of host factors in resistance to candidiasis may be due, in part, to the different immune responses exhibited by the strain of mouse studied.

	ACKNOWLEDGEMENTS

TOP Introduction Methods Results and Discussion ACKNOWLEDGEMENTS References

 
Sequencing data were obtained by the Biotechnology Resource Laboratory of the Medical University of South Carolina. We thank Kimberly Brent, Peter J. Nicholas, Emily E. Paulling and Phillip A. Werner for technical assistance and Dr Thomas Warner for histopathology data. This work was supported by the National Institutes of Health (grant DE-13968).

	References

TOP Introduction Methods Results and Discussion ACKNOWLEDGEMENTS References