It has been postulated that systemic cell-mediated immunity (CMI) is an important host defense factor against recurrent vaginal infections caused by Candida albicans. Using an estrogen-dependent murine model of vaginal candidiasis, we have previously shown that mice inoculated vaginally with C. albicans acquire a persistent vaginal infection and develop Candida-specific Th1-type systemic CMI. In the present study, experimental vaginitis was monitored in the presence of preinduced systemic Candida-specific CMI. Mice immunized systemically with C. albicans culture filtrate antigens (CaCF) in complete Freund's adjuvant (CFA) had Th1-type reactivity similar to that of vaginally infected mice. CaCF given to mice intravenously induced Candida-specific suppressor T (Ts) cells. Mice preimmunized with CaCF-CFA and given a vaginal inoculum of C. albicans had positive delayed-type hypersensitivity (DTH) reactivity from the time of vaginal inoculation through 4 weeks. Conversely, mice infected in the presence of Ts cells had significantly reduced DTH responses throughout the 4-week period in comparison with naive infected mice. However, the presence of Th1-type Candida-specific DTH cells or Ts cells, either induced in mice prior to vaginal inoculation or adoptively transferred at the time of inoculation, had no effect on the vaginal Candida burden through 4 weeks of infection. A similar lack of effects was obtained in animals with lower Candida population levels resulting from a reduction in or absence of exogenous estrogen. These results suggest that systemic Th1-type CMI demonstrable with CaCF is unrelated to protective events at the level of the vaginal mucosa.
Women with recurrent vulvovaginal candidiasis often demonstrate a down-regulation of cell-mediated immunity (CMI) to Candida albicans detected by a lack of cutaneous delayed-type hypersensitivity (DTH) to Candida antigens. However, the role of systemic CMI as a host defense mechanism against recurrent vulvovaginal candidiasis is not well understood, in part because of the lack of a well-defined murine model of vaginal candidiasis. The present study was undertaken to determine: (i) whether soluble Candida culture filtrate antigens (CaCF) could be used to induce and detect Candida-specific CMI in mice and (ii) whether these antigens would be useful in detecting systemic CMI in mice given an experimental Candida vaginal infection. To this end, mice were immunized subcutaneously with CaCF in complete Freund's adjuvant, and within 7 days they developed Candida-specific DTH reactivity detected by footpad swelling (increase in footpad thickness, 0.36 mm) 24 h after footpad challenge with CaCF. Adoptive transfer studies showed that the DTH responsiveness was elicited by CD4+ DTH T cells. In mice given a vaginal inoculum of C. albicans blastoconidia (5 x 10(5)), footpad challenge with CaCF resulted in positive DTH responses (0.24 mm) as early as 1 week, responses similar to immunization in 2 to 3 weeks (0.33 mm), and sustained low levels of DTH reactivity (0.15 mm) through 10 weeks of vaginal infection. Vaginal lavage cultures revealed that peak vaginal Candida burden occurred 1 week post-vaginal inoculation (10(5) CFU) and declined 16-fold by week 10. These results provide evidence that Candida-specific systemic CMI is generated and can be detected longitudinally in mice with Candida vaginitis by a multiantigen preparation of Candida organisms which both initiates and detects Candida-specific CMI.
The role of systemic cell-mediated immunity (CMI) as a host defense mechanism in the vagina is poorly understood. Using a murine pseudoestrus model of experimental vaginal candidiasis, we previously found that animals given a vaginal inoculum of viable Candida albicans blastoconidia acquired a persistent vaginal infection and developed Candida-specific delayed-type hypersensitivity (DTH) responses. The present study was designed to characterize the peripheral CMI reactivity generated from the vaginal infection in mice and to determine whether pseudoestrus is a prerequisite for the induction of peripheral CMI reactivity. Mice treated or not treated with estrogen and given a vaginal inoculum of C. albicans blastoconidia were examined for 4 weeks for their vaginal Candida burden and peripheral CMI reactivity, including DTH responsiveness and in vitro Th1 (interleukin-2 [IL-2], gamma interferon [IFN-gamma]/Th2 (IL-4, IL-10)-type lymphokine production in response to Candida antigens. Results showed that although mice not treated with estrogen before being given a vaginal inoculum of C. albicans blastoconidia developed only a short-lived vaginal infection and harbored significantly fewer Candida CFU in the vagina compared with those given estrogen and then infected; DTH reactivity was equivalent in both groups. In vitro measurement of CMI reactivity further showed that lymph node cells from both estrogen- and non-estrogen-treated infected mice produced elevated levels of IL-2 and IFN-gamma in response to Candida antigens during the 4 weeks after vaginal inoculation. In contrast, lymph node cells from the same vaginally infected mice showed no IL-10 production and only small elevations of IL-4 during week 4 of infection. These results suggest that mice with experimental vaginal candidiasis develop predominantly Th1-type Candida-specific peripheral CMI reactivity and that similar patterns of Th1-type reactivity occur in mice regardless of the persistence of infection and the estrogen status of the infected mice.
Recurrent vulvovaginal candidiasis, caused by Candida albicans, is a significant problem in women of childbearing age. Although cell-mediated immunity (CMI) due to T cells and cytokines is the predominant host defense mechanism against C. albicans at mucosal tissue sites, host defense mechanisms against C. albicans at the vaginal mucosa are poorly understood. Based on an estrogen-dependent murine model of vaginal candidiasis, our data suggest that systemic CMI is ineffective against C. albicans vaginal infections. Thus, we have postulated that local immune mechanisms are critical for protection against infection. In the present study, the kinetic production of chemokines normally associated with the chemotaxis of T cells, macrophages (RANTES, MIP-1α, MCP-1), and polymorphonuclear neutrophils (MIP-2) was examined following intravaginal inoculation of C. albicans in estrogen-treated or untreated mice. Results showed significant increases in MCP-1 protein and mRNA in vaginal tissue of infected mice as early as 2 and 4 days postinoculation, respectively, that continued through a 21-day observation period, irrespective of estrogen status. No significant changes were observed with RANTES, MIP-1α, or MIP-2, although relatively high constitutive levels of RANTES mRNA and MIP-2 protein were observed. Furthermore, intravaginal immunoneutralization of MCP-1 with anti-MCP-1 antibodies resulted in a significant increase in vaginal fungal burden early during infection, suggesting that MCP-1 plays some role in reducing the fungal burden during vaginal infection. However, the lack of changes in leukocyte profiles in vaginal lavage fluids collected from infected versus uninfected mice suggests that MCP-1 functions to control vaginal C. albicans titers in a manner independent of cellular chemotactic activity.
Protective host defense mechanisms against vaginal Candida albicans infections are poorly understood. Although cell-mediated immunity (CMI) is the predominant host defense mechanism against most mucosal Candida infections, the role of CMI against vaginal candidiasis is uncertain, both in humans and in an experimental mouse model. The role of humoral immunity is equally unclear. While clinical observations suggest a minimal role for antibodies against vaginal candidiasis, an experimental rat model has provided evidence for a protective role for Candida-specific immunoglobulin A (IgA) antibodies. Additionally, Candida vaccination-induced IgM and IgG3 antibodies are protective in a mouse model of vaginitis. In the present study, the role of infection-induced humoral immunity in protection against experimental vaginal candidiasis was evaluated through the quantification of Candida-specific IgA, IgG, and IgM antibodies in serum and vaginal lavage fluids of mice with primary and secondary (partially protected) infection. In naïve mice, total, but not Candida-specific, antibodies were detected in serum and lavage fluids, consistent with lack of yeast colonization in mice. In infected mice, Candida-specific IgA and IgG antibodies were induced in serum with anamnestic responses to secondary infection. In lavage fluid, while Candida-specific antibodies were detectable, concentrations were extremely low with no anamnestic responses in mice with secondary infection. The incorporation of alternative protocols—including infections in a different strain of mice, prolongation of primary infection prior to secondary challenge, use of different enzyme-linked immunosorbent assay capture antigens, and concentration of lavage fluid—did not enhance local Candida-specific antibody production or detection. Additionally, antibodies were not removed from lavage fluids by being bound to Candida during infection. Together, these data suggest that antibodies are not readily present in vaginal secretions of infected mice and thus have a limited natural protective role against infection.
It has been postulated that systemic cell-mediated immunity (CMI) is an important host defense mechanism against Candida infections of the vagina. However, in an estrogen-dependent murine model of experimental vaginal candidiasis, we recently showed that systemic Candida-specific Th1-type CMI induced by immunization with Candida culture filtrate antigen had no effect on vaginal Candida population levels during the course of a vaginal infection. In the present study, mice given a second vaginal inoculation in the presence of peripheral Candida-specific Th1-type CMI induced by prior vaginal infection had anamnestic-type increased delayed-type hypersensitivity (DTH) responses, concomitant with significantly fewer Candida organisms in the vagina than in primary-infected mice. In addition, organisms in secondary-infected mice were fragmented and superficial penetration into the epithelium was reduced. The systemic presence of Candida-specific T suppressor (Ts) cells that significantly suppressed the infection-derived anamnestic DTH reactivity did not abrogate the protective effect in the vagina. Additional experiments showed that vaginally immunized mice were not protected from gastrointestinal or systemic candidiasis and, in contrast to mice with a second vaginal infection, did not demonstrate anamnestic DTH reactivity. These results suggest that a moderate level of local protection against a Candida vaginal infection can be achieved by vaginal immunization but that the protective role of acquired peripheral Candida-specific Th1-type reactivity at the vaginal mucosa appears to be limited.
The etiology of recurrent vulvovaginal candidiasis in otherwise healthy women of child-bearing age remains an enigma. To date, results from both clinical studies and a murine model of vaginal candidiasis indicate that Candida vaginitis can occur in the presence of Candida-specific Th1-type cell-mediated immunity expressed in the peripheral circulation. The present study was designed to determine the role of circulating CD4 and CD8 cells in primary and secondary vaginal infections with Candida albicans. Vaginal fungal burden, Candida-specific delayed-type hypersensitivity (DTH), and lymph node cell Th1/Th2 cytokine production were monitored in CD4 and/or CD8 cell-depleted mice during persistent primary vaginal infections and secondary vaginal infections against which partial protection was observed. Treatment of mice with anti-CD4 or anti-CD8 antibodies resulted in 90% or greater depletion of the respective cell populations. Mice depleted of CD4 cells had significantly reduced Candida-specific DTH and lymph node cell Th1-type cytokine production during a primary vaginal infection, as well as reduced anamnestic DTH during a secondary vaginal infection. In contrast, mice depleted of CD8 cells showed only reduced gamma interferon production during a primary infection; no alterations in DTH were observed. Despite reductions in DTH and cytokine production, however, CD4 and/or CD8 cell depletion had no effect on vaginal C. albicans burden in mice after a primary or secondary vaginal inoculation. Taken together, these results suggest that while circulating CD4 and CD8 cells contribute to systemic Candida-specific cell-mediated immunity in vaginally infected mice, neither CD4 nor CD8 circulating T cells appear to provide significant host defenses against C. albicans at the vaginal mucosa.
Candida albicans is the causative agent of acute and recurrent vulvovaginal candidiasis (VVC), a common mucosal infection affecting significant numbers of women in their reproductive years. While any murine host protective role for cell-mediated immunity (CMI), humoral immunity, and innate resistance by neutrophils against the vaginal infection appear negligible, significant in vitro growth inhibition of Candida species by vaginal and oral epithelial cell-enriched cells has been observed. Both oral and vaginal epithelial cell anti-Candida activity has a strict requirement for cell contact to C. albicans with no role for soluble factors, and oral epithelial cells inhibit C. albicans through a cell surface carbohydrate moiety. The present study further evaluated the inhibitory mechanisms by murine vaginal epithelial cells and the fate of C. albicans by oral and vaginal epithelial cells. Similar to human oral cells, anti-Candida activity produced by murine vaginal epithelial cells is unaffected by enzymatic cleavage of cell surface proteins and lipids but sensitive to periodic acid cleavage of surface carbohydrates. Analysis of specific membrane carbohydrate moieties, however, showed no role for sulfated polysaccharides, sialic acid residues, or glucose and mannose-containing carbohydrates, also similar to oral cells. Staining for live and dead Candida in the coculture with fluorescein diacetate (FDA) and propidium iodide (PI), respectively, showed a clear predominance of live organisms, suggesting a static rather than cidal action. Together, the results suggest that oral and vaginal epithelial cells retard or arrest the growth rather than kill C. albicans through an as-yet-unidentified carbohydrate moiety in a noninflammatory manner.
The results from both clinical studies of women with recurrent vulvovaginal candidiasis and a murine model of experimental vaginitis indicate that systemic cell-mediated immunity may not represent a dominant host defense mechanism against vaginal infections by Candida albicans. Recent experimental evidence indicates the presence of local vaginal immune reactivity against C. albicans. The present study was designed to examine T-lymphocyte subpopulations in the vaginal mucosae of naive CBA/J mice. Vaginal lymphocytes (VL) were isolated by collagenase digestion of whole vaginal tissues. Cell populations were identified by flow cytometry, and the results were compared with those for both lymph node cells (LNC) and peripheral blood lymphocytes (PBL). The results of flow cytometry showed that 45% +/- 10% of lymphocytes in the vaginal mucosa are CD3+ compared with 75% +/- 5% in LNC and 50% +/- 5% in PBL. The majority (85%) of CD3+ VL are CD4+ and express the alpha/beta T-cell receptor (TCR), similar to the results for LNC and PBL. In contrast to LNC and PBL, VL contain a significantly higher percentage (15 to 20%) of gamma/delta TCR+ cells, 80% or more of which appear to express CD4. In addition, while CD4-CD8 cell ratios in LNC and PBL were 3:1 and 6:1, respectively, only 1% of VL expressed CD8, resulting in a CD4-CD8 cell ratio of > 100:1. Finally, while LNC and PBL recognized two epitope-distinct (GK 1.5 and 2B6) anti-CD4 antibodies, VL recognized only 2B6 anti-CD4 antibodies. Further analysis of VL showed that Thy-1 cells, but not CD4 cells, were reduced after intravaginal injection of complement-fixing anti-Thy-1.2 and GK 1.5 anti-CD4 antibodies, respectively. Taken together, these data suggest that T lymphocytes in the vaginal mucosae of mice are phenotypically distinct from those in the periphery and that CD4+ VL have an uncharacteristic or atypical expression of the CD4 receptor.
Recurrent vulvovaginal candidiasis (RVVC) is a prevalent opportunistic mucosal infection, caused predominantly by Candida albicans, which affects a significant number of otherwise healthy women of childbearing age. Since there are no known exogenous predisposing factors to explain the incidence of symptomatic vaginitis in most women with idiopathic RVVC, it has been postulated that these particular women suffer from an immunological abnormality that prediposes them to RVVC. Because of the increased incidence of mucosal candidiasis in individuals with depressed cell-mediated immunity (CMI), defects in CMI are viewed as a possible explanation for RVVC. In this review, we attempt to place into perspective the accumulated information regarding the immunopathogenesis of RVVC, as well as to provide new immunological perspectives and hypotheses regarding potential immunological deficiencies that may predispose to RVVC and potentially other mucosal infections by the same organism. The results of both clinical studies and studies in an animal model of experimental vaginitis suggest that systemic CMI may not be the predominant host defense mechanism against C. albicans vaginal infections. Rather, locally acquired mucosal immunity, distinct from that in the peripheral circulation, is now under consideration as an important host defense at the vaginal mucosa, as well as the notion that changes in local CMI mechanism(s) may predispose to RVVC.
Although Th1-type cell-mediated immunity (CMI) is the predominant host defense mechanism against mucosal Candida albicans infection, CMI against a vaginal C. albicans infection in mice is limited at the vaginal mucosa despite a strong Candida-specific Th1-type response in the draining lymph nodes. In contrast, Th1-type CMI is highly effective against an experimental Chlamydia trachomatis genital tract infection. This study demonstrated through two independent designs that a concurrent Candida and Chlamydia infection could not accelerate or modulate the anti-Candida CMI response. Together, these results suggest that host responses to these genital tract infections are independent and not influenced by the presence of the other.
Cell-mediated immunity by Th1-type CD4+ T cells is the predominant host defense mechanism against mucosal candidiasis. However, studies using an estrogen-dependent murine model of vaginal candidiasis have demonstrated little to no change in resident vaginal T cells during infection and no systemic T-cell infiltration despite the presence of Candida-specific systemic Th1-type responses in infected mice. The present study was designed to further investigate these observations by characterizing T-cell activation and cell adhesion molecule expression during primary and secondary C. albicans vaginal infections. While flow cytometry analysis of activation markers showed some evidence for activation of CD3+ draining lymph node and/or vaginal lymphocytes during both primary and secondary vaginal Candida infection, CD3+ cells expressing the homing receptors and integrins α4β7, αM290β7, and α4β1 in draining lymph nodes of mice with primary and secondary infections were reduced compared to results for uninfected mice. At the local level, few vaginal lymphocytes expressed integrins, with only minor changes observed during both primary and secondary infections. On the other hand, immunohistochemical analysis of vaginal cell adhesion molecule expression showed increases in mucosal addressin cell adhesion molecule 1 and vascular cell adhesion molecule 1 expression during both primary and secondary infections. Altogether, these data suggest that although the vaginal tissue is permissive to cellular infiltration during a vaginal Candida infection, the reduced numbers of systemic cells expressing the reciprocal cellular adhesion molecules may preempt cellular infiltration, thereby limiting Candida-specific T-cell responses against infection.
Mice from six genetically distinct strains were examined for their immune responses to Candida albicans in in vitro and in vivo assays, and naive mice and mice immunized with the fungus were challenged intravenously with three different doses of C. albicans to determine differences in susceptibility. Naive mice from the six groups showed substantial differences in resistance to challenge based on mortalities and quantitative cultures of kidneys, with mice from strains C57BL/6J and BALB/cByJ showing the most resistance; mice from strains A/J, C3H/HeJ, and CBA/J showing moderate susceptibility; and mice from strain DBA/2J showing the highest degree of susceptibility to challenge. Unimmunized mice from strains C57BL/6J and BALB/cByJ did not produce detectable levels of Candida-specific antibody by the end of the 28-day observation period when challenged intravenously, but the other strains did. Immunized mice showed a degree of protection to challenge, with all groups except mice from strain BALB/cByJ showing a reduction of two to three log units in the level of colonization in their kidneys and all strains producing significant levels of antibody. Additionally, the immunized mice of all strains developed substantial levels of delayed-type hypersensitivity and demonstrated nearly identical lymphocyte proliferative responses to Candida antigens. The results indicate that resistance to systemic candidiasis is dependent upon a combination of innate factors, predominately an intact complement system, and the acquisition of an immune response, most likely of a cell-mediated type. Additionally, the findings suggest that genetic control of acquired resistance to C. albicans may not be associated with the H-2 complex.
Studies with an estrogen-dependent murine model of vaginal candidiasis suggest that local cell-mediated immunity (CMI) is more important than systemic CMI for protection against vaginitis. The present study, however, showed that, compared to uninfected mice, little to no change in the percentage or types of vaginal T cells occurred during a primary vaginal infection or during a secondary vaginal infection where partial protection was observed. Furthermore, depletion of polymorphonuclear leukocytes (PMN) had no effect on infection in the presence or absence of pseudoestrus. These results indicate a lack of demonstrable effects by systemic CMI or PMN against vaginitis and suggest that if local T cells are important, they are functioning without showing significant increases in numbers within the vaginal mucosa during infection.
The immunologic effects of in vivo administration of recombinant murine gamma interferon (rMuIFN-gamma) were determined in a murine model of candidiasis. Naive mice were given graded doses of rMuIFN-gamma and then challenged intravenously with Candida albicans. Increased morbidity and mortality were noted in four different strains of mice, viz., BALB/c, A/J, Swiss Webster, and CBA/J, providing the mice had not been immunized with C. albicans before challenge. Quantitative culture of selected organs of Swiss Webster and CBA/J mice surviving treatment with rMuIFN-gamma revealed elevated numbers of C. albicans cells, particularly in the kidneys, but also in the liver, lungs, and spleen. The lungs, livers, and spleen of female CBA/J mice were more protected from increased multiplication of the fungus than were those of males of the same species or female Swiss Webster mice. On the basis of these initial findings, the effect of treatment with 5,000 U of rMuIFN-gamma on immune responses in a gastrointestinal model of candidiasis was determined. CBA/J mice that had been colonized with C. albicans as infants were boosted with a cutaneous inoculation of the fungus when 6 to 10 weeks old; development of delayed hypersensitivity (DH), antibodies, and protective responses was assayed at intervals thereafter. Daily treatment with rMuIFN-gamma (beginning 1 day before cutaneous inoculation) suppressed weak immune responses but had little effect on responses which were strong. For example, DH and anti-C. albicans antibody production were suppressed in animals colonized with C. albicans but not boosted by cutaneous inoculation, and DH was suppressed in uncolonized animals that had been inoculated once cutaneously with the fungus as well. There was no rMuIFN-gamma-induced suppressive effect of DH in mice which had been stimulated maximally with C. albicans, i.e., colonized animals that had been boosted cutaneously with the organisms. Collectively, these data indicate that naive mice or mice with minimal levels of anti-C. albicans sensitivity, females somewhat more so than males, were sensitive to suppressive effects of in vivo treatment with rMuIFN-gamma when challenged with C. albicans. In contrast, under conditions similar to those of humans, in whom underlying immunity to C. albicans is usually present, suppression of host responses to C. albicans was not observed in immunized mice in response to treatment with rMuIFN-gamma.
Vulvovaginal candidiasis, caused primarily by Candida albicans, presents significant health issues for women of childbearing age. As a polymorphic fungus, the ability of C. albicans to switch between yeast and hyphal morphologies is considered its central virulence attribute. Armed with new criteria for defining vaginitis immunopathology, the purpose of this study was to determine whether the yeast-to-hypha transition is required for the hallmark inflammatory responses previously characterized during murine vaginitis. Kinetic analyses of vaginal infection with C. albicans in C57BL/6 mice demonstrated that fungal burdens remained constant throughout the observation period, while polymorphonuclear leukocyte (PMN), S100A8, and interleukin-1β levels obtained from vaginal lavage fluid increased by day 3 onward. Lactate dehydrogenase activity was also positively correlated with increased effectors of innate immunity. Additionally, immunodepletion of neutrophils in infected mice confirmed a nonprotective role for PMNs during vaginitis. Determination of the importance of fungal morphogenesis during vaginitis was addressed with a two-pronged approach. Intravaginal inoculation of mice with C. albicans strains deleted for key transcriptional regulators (bcr1Δ/Δ, efg1Δ/Δ, cph1Δ/Δ, and efg1Δ/Δ cph1Δ/Δ) controlling the yeast-to-hypha switch revealed a crucial role for morphogenetic signaling through the Efg1 and, to a lesser extent, the Bcr1 pathways in contributing to vaginitis immunopathology. Furthermore, overexpression of transcription factors NRG1 and UME6, to maintain yeast and hyphal morphologies, respectively, confirmed the importance of morphogenesis in generating innate immune responses in vivo. These results highlight the yeast-to-hypha switch and the associated morphogenetic response as important virulence components for the immunopathogenesis of Candida vaginitis, with implications for transition from benign colonization to symptomatic infection.
Immune regulation in candidiasis is inferred from studies of both human and animal infection, with a suppressive role suggested for cell wall polysaccharide. To study the immunosuppressive potential of Candida albicans in a murine model, whole blastoconidia or purified cell wall components of C. albicans were tested for their effects on the development of acquired immune responses by superimposing a pretreatment regimen upon an established immunization protocol. CBA/J or BALB/cByJ mice were pretreated twice intravenously with 100 micrograms of mannan (MAN), 100 or 200 micrograms of glycoprotein (GP), or 5 X 10(7) heat-killed C. albicans blastoconidia, followed 1 week later by an immunization protocol of two cutaneous inoculations of viable C. albicans blastoconidia given 2 weeks apart. Delayed hypersensitivity (DTH) to GP or to a membrane-derived antigen, B-HEX, was tested 7 days after the second inoculation, and lymphocyte stimulation was tested with mitogens and Candida antigens after 12 days. To assess protection, mice were challenged intravenously with viable C. albicans blastoconidia 14 days after the second cutaneous inoculation and sacrificed 28 days later for quantitative culture of kidneys and brains. Sera were obtained for enzyme-linked immunosorbent assays at selected intervals. Pretreatment with GP resulted in specific in vivo suppression of DTH to GP but not to B-HEX antigen and specific in vitro suppression of lymphocyte stimulation to GP but not to other Candida antigens or mitogens. MAN and heat-killed C. albicans blastoconidia had no such effects. GP pretreatment also diminished the protective effect of immunization against challenge, demonstrable in the brain, while not altering significantly the production of antibody in response to infection. Contrary to clinical evidence, MAN was not immunosuppressive in this model, and in fact, the immunosuppressive potential of GP, which is composed largely of MAN, was found to be dependent upon the presence of its heat-labile protein moiety.
The 70-kDa recombinant Candida albicans heat shock protein (CaHsp70) and its 21-kDa C-terminal and 28-kDa N-terminal fragments (CaHsp70-Cter and CaHsp70-Nter, respectively) were studied for their immunogenicity, including proinflammatory cytokine induction in vitro and in vivo, and protection in a murine model of hematogenous candidiasis. The whole protein and its two fragments were strong inducers of both antibody (Ab; immunoglobulin G1 [IgG1] and IgG2b were the prevalent isotypes) and cell-mediated immunity (CMI) responses in mice. CaHsp70 preparations were also recognized as CMI targets by peripheral blood mononuclear cells of healthy human subjects. Inoculation of CaHsp70 preparations into immunized mice induced rapid production of interleukin-6 (IL-6) and tumor necrosis factor alpha, peaking at 2 to 5 h and declining within 24 h. CaHsp70 and CaHsp70-Cter also induced gamma interferon (IFN-γ), IL-12, and IL-10 but not IL-4 production by CD4+ lymphocytes cocultured with splenic accessory cells from nonimmunized mice. In particular, the production of IFN-γ was equal if not superior to that induced in the same cells by whole, heat-inactivated fungal cells or the mitogenic lectin concanavalin A. In immunized mice, however, IL-4 but not IL-12 was produced in addition to IFN-γ upon in vitro stimulation of CD4+ cells with CaHsp70 and CaHsp70-Cter. These animals showed a decreased median survival time compared to nonimmunized mice, and their mortality was strictly associated with organ invasion by fungal hyphae. Their enhanced susceptibility was attributable to the immunization state, as it did not occur in congenitally athymic nude mice, which were unable to raise either Ab or CMI responses to CaHsp70 preparations. Together, our data demonstrate the elevated immunogenicity of CaHsp70, with which, however, no protection against but rather some enhancement of Candida infection seemed to occur in the mouse model used.
Vulvovaginal candidiasis (VVC) caused by the commensal organism Candida albicans remains a significant problem among women of childbearing age, with protection against and susceptibility to infection still poorly understood. While cell-mediated immunity by CD4+ Th1-type cells is protective against most forms of mucosal candidiasis, no protective role for adaptive immunity has been identified against VVC. This is postulated to be due to immunoregulation that prohibits a more profound Candida-specific CD4+ T-cell response against infection. The purpose of this study was to examine the role of dendritic cells (DCs) in the induction phase of the immune response as a means to understand the initiation of the immunoregulatory events. Immunostaining of DCs in sectioned murine lymph nodes draining the vagina revealed a profound cellular reorganization with DCs becoming concentrated in the T-cell zone throughout the course of experimental vaginal Candida infection consistent with cell-mediated immune responsiveness. However, analysis of draining lymph node DC subsets revealed a predominance of immunoregulation-associated CD11c+ B220+ plasmacytoid DCs (pDCs) under both uninfected and infected conditions. Staining of vaginal DCs showed the presence of both DEC-205+ and pDCs, with extension of dendrites into the vaginal lumen of infected mice in close contact with Candida. Flow cytometric analysis of draining lymph node DC costimulatory molecules and activation markers from infected mice indicated a lack of upregulation of major histocompatibility complex class II, CD80, CD86, and CD40 during infection, consistent with a tolerizing condition. Together, the results suggest that DCs are involved in the immunoregulatory events manifested during a vaginal Candida infection and potentially through the action of pDCs.
Oropharyngeal and vaginal candidiases are the most common forms of mucosal fungal infections and are primarily caused by Candida albicans, a dimorphic fungal commensal organism of the gastrointestinal and lower female reproductive tracts. Clinical and experimental observations suggest that local immunity is important in host defense against candidiasis. Accordingly, cytokines and chemokines are present at the oral and vaginal mucosa during C. albicans infections. Since mucosal epithelial cells produce a variety of cytokines and chemokines in response to microorganisms and since C. albicans is closely associated with mucosal epithelial cells as a commensal, we sought to identify cytokines and/or chemokines produced by primary oral and vaginal epithelial cells and cell lines in response to C. albicans. The results showed that proinflammatory cytokines were produced by oral and/or vaginal epithelial cells at various levels constitutively with considerable interleukin-1α (IL-1α) and tumor necrosis factor alpha, but not IL-6, produced in response to C. albicans. In contrast, Th1-type (IL-12 and gamma interferon) and Th2-type-immunoregulatory (IL-10 and transforming growth factor β) cytokines and the chemokines monocyte chemoattractant protein 1 and IL-8 were produced in low to undetectable concentrations with little additional production in response to C. albicans. Taken together, these results indicate that cytokines and chemokines are variably produced by oral and vaginal epithelial cells constitutively, as well as in response to C. albicans, and are predominated by proinflammatory cytokines.
Male CBA/J mice were given a single dose of 200 mg of cyclophosphamide (CY) per kg 3 days before a first or second cutaneous inoculation with viable Candida albicans in an attempt to suppress antibody formation and determine the effects of such suppression on the development of acquired immunity. After cutaneous inoculation, mice not treated with CY developed acquired immunity to intravenous challenge, which was accompanied by the development of circulating antibodies, delayed hypersensitivity, and in vitro responsiveness of lymph node cells to Candida antigens. CY treatment resulted in an immediate depression of peripheral blood leukocytes, with polymorphonuclear leukocytes and monocytes rebounding quickly to normal or above normal levels while lymphocyte remained depressed throughout the 4-week observation period. In vitro stimulation of lymph node cells from CY-treated mice was depressed shortly after treatment; however, responses to phytohemagglutinin and three Candida antigens (a cell wall preparation, a membrane preparation, and soluble cytoplasmic substances) recovered, whereas the responses to lipopolysaccharide did not. CY effects on the cutaneous lesion were twofold; first, the number of viable Candida cells in the lesions was much higher in animals receiving CY 3 days before Candida inoculation, and second, the size of the dermal lesion was either greatly enhanced or reduced depending upon the time of CY treatment relative to the number of cutaneous Candida inoculations. CY-treated animals developed higher levels of delayed hypersensitivity to the membrane preparation when infected once cutaneously than did corresponding untreated animals. The number of mice responding with circulating antibodies to soluble cytoplasmic substances after cutaneous inoculation was greatly reduced in CY-treated groups, and this impaired ability to produce antibodies correlated with the poor survival of these mice after intravenous challenge. Our results suggest that the ability to produce antibody at the time of challenge is crucial to successful defense against systemic candidiasis in this murine model.
Reported targets of the specific immune responses to Candida albicans in human candidiasis include a 47-kDa breakdown product of a 90-kDa heat shock protein (HSP 90) (R. Matthews and J. Burnie, FEMS Microbiol. Lett. 60:25-30, 1989) and the 48-kDa enolase (K.M. Franklyn, J.R. Warmington, A.K. Ott, and R.B. Ashman, Immunol. Cell Biol. 68:173-178, 1990). These proteins are immunodominant antigens of C. albicans. Western blotting (immunoblotting) and immunoprecipitation were used to investigate the humoral response in a mouse model of systemic candidiasis. Resolution of systemic candidiasis in CBA/H mice is associated with a high level of antibody reactivity to C. albicans antigens. A significant antibody response against a non-HSP antigen of 96 kDa which was distinct from the C. albicans HSP 90 antigen was detected. Significant antibody reactivity against an HSP of 75 kDa was also detected. We concluded that resolution of C. albicans infections in CBA/H mice was associated with antibodies to an HSP and a non-HSP of 75 and 96 kDa, respectively.
Mannan (MAN) extracted from Candida albicans 20A was investigated for its potential as an antigen in the detection of cell-mediated immunity (CMI) in vivo and in vitro and for its ability to modulate CMI when administered intravenously (i.v.). CBA/J mice were either immunized as adults by the cutaneous inoculation of 10(6) viable blastoconidia or colonized as infants (primed) and then boosted cutaneously as adults. When immunized animals were footpad tested with MAN, highly significant delayed-type hypersensitivity (DH) responses were detected. The DH responses to MAN were of a greater magnitude than those noted with the same quantity of cell wall glycoprotein (GP), an ethylenediamine extract of the cell wall which contains both glucan and MAN. In contrast, GP was a better antigen for the detection of CMI responses in an in vitro lymphoproliferative assay with either spleen or lymph node cell suspensions. Mice treated with MAN i.v. prior to the initiation of immunization or between priming and secondary inoculations developed significantly suppressed DH reactions when tested with either MAN or GP. The lowest effective dose of MAN was 250 micrograms, maximum suppression occurred with 500 micrograms, and either dose given 1 week prior to immunization was suppressive. The suppression by MAN was specific for MAN or the MAN-containing GP. Responses to another unrelated candidal antigen, a membrane extract designated BEX, were relatively unaffected. MAN, therefore, was an effective antigen for the detection of CMI in vivo, and its administration i.v. created what appeared to be a MAN-specific suppression since it could be detected with both MAN and a MAN-containing extract from the cell wall. Caution must be exercised in the interpretation of these data, however, since the protein component of each of these extracts has not been characterized with respect to its potential role in the phenomena observed.
Oral candidiasis is a common fungal infection in patients infected with the human immunodeficiency virus (HIV). Although rare at the time of primary HIV infection, it is frequently found throughout the asymptomatic phase and is predictive of progressive immunodeficiency. However, the precise immune defect which results in outgrowth of commensal Candida albicans in HIV infection has not been identified. Mice infected with the Du5H(G6T2) mixture of mouse leukemia viruses develop a syndrome, designated murine AIDS (MAIDS), that has many of the immune abnormalities found in HIV infection. Retrovirus-infected C57BL/6 mice were examined for their ability to resist the development of oral candidiasis from the carrier state established after a self-limiting acute infection and to clear a subsequent secondary inoculum of oral C. albicans. Most of the mice orally colonized with C. albicans and then inoculated with the retrovirus mixture maintained a low-level oral carriage of C. albicans, while 30% of coinfected mice developed recurring 2- to 3-week episodes of acute Candida proliferation, separated by transient recoveries to the carrier state. The frequencies of CD4+ and CD8+ lymphocytes were, respectively, unchanged and significantly decreased (P < 0.05) in both cervical lymph nodes and spleens of coinfected mice compared to the corresponding frequencies in C. albicans-carrying, virus-free, age-matched control animals. Secretion of gamma interferon by concanavalin A (ConA)-stimulated spleen cells from Candida-carrying, retrovirus-infected mice was significantly decreased (P < 0.05) compared to that of C. albicans-carrying, retrovirus-free mice, in accordance with known abnormalities associated with MAIDS. However, production of this cytokine by ConA-stimulated or unstimulated cervical lymph node cells from coinfected mice was enhanced compared to that of virus-free animals colonized with C. albicans. Acquired resistance to reinfection with C. albicans was maintained in retrovirus-infected mice and was associated with a mucosal recruitment of CD8+ cells not observed in control mice. These results suggest that alterations in mucosal immunity which occur in MAIDS differ substantially from defects observed at other sites and that surrogate epithelial defense mechanisms may function locally to limit Candida proliferation.
Cytokine-mediated host defense against Candida glabrata infection was compared to that against C. albicans, using immunocompetent murine models of systemic candidiasis. The pathogenesis of infection was evaluated morphologically and by culture of target organs, while the kinetics of induction of cytokine mRNAs and corresponding proteins were determined in kidneys by real-time reverse transcription-PCR and cytokine-specific murine enzyme-linked immunosorbent assays, respectively. Systemic infection with C. glabrata resulted in a chronic, nonfatal infection with recovery of organisms from kidneys, while intravenous inoculation with C. albicans resulted in rapid mortality with logarithmic growth of organisms in kidneys and recovery of C. albicans from the spleen, liver, and lungs. Survival of C. glabrata-infected mice was associated with rapid induction of mRNAs and corresponding immunoreactive proteins for the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12), and gamma interferon (IFN-γ) and the lack of induction of protein for the anti-inflammatory cytokine IL-10. In contrast, mortality in C. albicans-infected mice was associated with induction of mRNA and corresponding protein for IL-10 but delayed (i.e., TNF-α) or absent (i.e., IL-12 and IFN-γ) induction of immunoreactive proinflammatory cytokines. Mice were subsequently treated with cytokine-specific neutralizing monoclonal antibodies (MAbs) to TNF-α, IL-12, or IFN-γ, and the effect on growth of C. glabrata in kidneys was assessed. Neutralization of endogenous TNF-α resulted in a significant increase in C. glabrata organisms compared to similarly infected mice administered an isotype-matched control MAb, while neutralization of endogenous IL-12 or IFN-γ had no significant effect on C. glabrata replication. These results demonstrate that in response to intravenous inoculation of C. glabrata, immunocompetent mice develop chronic nonfatal renal infections which are associated with rapid induction of the proinflammatory cytokines TNF-α, IL-12, and IFN-γ. Furthermore, TNF-α plays a key role in host defense against systemic candidiasis caused by either C. glabrata or C. albicans, as the absence of endogenous TNF-α activity was associated with enhanced tissue burden in both infection models.