Multiple experimental designs to evaluate the role of T-cell-mediated immunity against experimental vaginal Candida albicans infection.

Studies to date suggest a limited protective role for Candida-specific Th1-type cell-mediated immunity (CMI) against Candida albicans vaginitis, despite protection against other mucosal Candida infections. Recent evidence suggests this may be due to immunoregulatory mechanisms that inhibit a more profound CMI response against C. albicans vaginal infections. The present study was designed to conduct an evaluation of the protective role of CMI against experimental C. albicans vaginitis using multiple approaches, including the use of T-cell-immunodeficient (SCID, Nude) and knockout (CD4) mice and several immunization designs in immunocompetent mice. Results showed, with few exceptions, that most T-cell-immunodeficient or knockout mice had a vaginal fungal burden similar to that of wild-type strains throughout the observation period. In addition, no correlation was observed between vaginal T-helper and proinflammatory cytokines and fungal burden, suggesting a generalized state of immunoregulation. Evaluation of the effects of various immunization designs that included different Candida antigens, routes of delivery and strains of mice yielded no protection against vaginal candidiasis. These studies provide further evidence of a lack of a protective role of T cells against C. albicans vaginitis, and continue to support the concept of immunoregulation against vaginal CMI responses.     

Rats clearing a vaginal infection by Candida albicans acquire specific, antibody-mediated resistance to vaginal reinfection.

Oophorectomized, estrogen-treated rats were susceptible to experimental vaginal infection by Candida albicans. After spontaneous clearing of the primary infection, the animals were highly resistant to a second vaginal challenge with the fungus. The vaginal fluid of Candida-resistant rats contained antibodies directed against mannan constituents and secretory aspartyl proteinase(s) of C. albicans and was capable of transferring a degree of anti-Candida protection to naive, nonimmunized rats. This passive protection was mediated by the immunoglobulin fraction of the vaginal fluid and was substantially abolished by preabsorption of the vaginal fluid with C. albicans, but not with Saccharomyces cerevisiae, cells. Vaginal anti-mannan antibodies were also produced by active immunization with heat-killed cells of C. albicans or with a mannan extract when administered via the vaginal route. The protection conferred was comparable to that resulting from clearing of the primary infection. In summary, the data suggest that acquired anticandidal protection in this vaginitis model is mediated at least in part by antibodies, among which those directed against the mannan antigen(s) might play a dominant role.     

Th17 cells confer long-term adaptive immunity to oral mucosal Candida albicans infections

Oropharyngeal candidiasis (OPC) is an opportunistic infection caused by Candida albicans. Despite its prevalence, little is known about C. albicans-specific immunity in the oral mucosa. Vaccines against Candida generate both T helper type 1 (Th1) and Th17 responses, and considerable evidence implicates interleukin (IL)-17 in immunity to OPC. However, IL-17 is also produced by innate immune cells that are remarkably similar to Th17 cells, expressing the same markers and localizing to similar mucosal sites. To date, the relative contribution(s) of Th1, Th17, and innate IL-17-producing cells in OPC have not been clearly defined. Here, we sought to determine the nature and function of adaptive T-cell responses to OPC, using a new recall infection model. Mice subjected to infection and re-challenge with Candida mounted a robust and stable antigen-specific IL-17 response in CD4+ but not CD8+ T cells. There was little evidence for Th1 or Th1/Th17 responses. The Th17 response promoted accelerated fungal clearance, and Th17 cells could confer protection in Rag1−/− mice upon adoptive transfer. Surprisingly, CD4 deficiency did not cause OPC but was instead associated with compensatory IL-17 production by Tc17 and CD3+CD4−CD8− cells. Therefore, classic CD4+Th17 cells protect from OPC but can be compensated by other IL-17-producing cells in CD4-deficient hosts.     

Chlamydia trachomatis infection does not enhance local cellular immunity against concurrent Candida vaginal infection

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.     

Protective immunity against murine candidiasis elicited by Candida albicans ribosomal fractions.

Candida albicans ribosomes were prepared from mechanically disrupted cells through differential centrifugation and purification in a sucrose-ammonium sulfate solution. The ribosomes were analyzed chemically and physically and exhibited characteristics of eucaryotic ribosomes (78S). ICR female mice were immunized with two subcutaneous inoculations, 2 weeks apart, of 100 microgram of ribosomes (expressed as ribosomal protein). Immunized mice were challenged either intraperitoneally or intravenously with a lethal dose of live C. albicans cells. The 31-day survival rate of immunized mice challenged intraperitoneally was 64% (mean value) versus 27% in controls; in intravenously challenged mice the survival rate of the immunized animals was about 60%, with no survivors among the controls. In intravenously challenged mice, incomplete Freund adjuvant enhanced the protection elicited by the ribosomes. Protection by ribosomal immunization was obtained against challenge doses causing chronic and acute infection.     

Adherence of Candida albicans and other Candida species to mucosal epithelial cells.

To study the possible involvement of candidal adherence in mucosal colonization, we examined the in vitro adherence capabilities of seven Candida species. Adherence was evaluated by direct microscopic examination and by a quantitative radiometric adherence test. The results indicate that C. albicans adheres to vaginal and buccal epithelial cells to a significantly greater degree (P less than 0.01) than the other species tested. C. tropicalis and C. stellatoidea demonstrated moderate adherence capabilities, while C. parapsilosis adhered only to a slight degree. Other species failed to interact with isolated mucosal cells. These findings suggest that there is a relationship between the adherence capabilities of the Candida species and their abilities to colonize mucosal surfaces, since those species which adhere are those which most frequently colonize mucosal surfaces. C. albicans was found to be adherent under a variety of environmental conditions. Stationary-phase blastospores of C. albicans were found to be more adherent than logarithmic-phase yeasts, and larger blastospore cell-to-epithelial cell ratios resulted in greater adherence values. The actual number of adherent yeasts varied considerably when epithelial cells were obtained from different donors.     

Effects of systemic cell-mediated immunity on vaginal candidiasis in mice resistant and susceptible to Candida albicans infections.

Studies to date with CBA/J mice suggest a limited role for systemic cell-mediated immunity (CMI) against vaginal Candida albicans infections. The results of the present study show that preinduced Candida-specific systemic CMI was equally nonprotective against C. albicans vaginal infections in mice with high (BALB/cJ), low (DBA/2), or intermediate (CBA/J) resistance to C. albicans infections. Similarly, the locally acquired partial protection against a second C. albicans vaginal infection was equally observed with BALB/cJ, DBA/2, and CBA/J mice. These results indicate that observations made previously with CBA/J mice were not murine strain specific and provide additional support for the hypothesis that systemic CMI does not represent a dominant host defense mechanism at the vaginal mucosa.     

Effect of progesterone on Candida albicans vaginal pathogenicity

Candida albicans is responsible for the majority of cases of vulvovaginal candidiasis (VVC), an infection which occurs mainly during the luteal phase of the menstrual cycle or during the pregnancy, when levels of progesterone are elevated. One of the most important candidal virulence factors is the ability to adhere to host surfaces and form biofilms.The aim of this study was to determine the influence of progesterone on C. albicans virulence, namely biofilm formation and colonisation/invasion of a reconstituted human vaginal epithelium (RHVE). Biofilm formation on the RHVE was evaluated by enumeration of culturable cells, total mass quantification and scanning electron microscopy. The capacity of C. albicans strains to invade and colonise the tissue was examined by fluorescence microscopy using species-specific peptide nucleic acid (PNA) probe hybridisation, and quantitatively evaluated by RT-PCR Candida quantification methodology. Furthermore, gene (BCR1 and HWP1) expression of biofilm and RHVE-colonising cells was evaluated by quantitative RT-PCR. Results confirmed that progesterone reduced the capacity of C. albicans strains to form biofilms and to colonise and invade RHVE. Additionally, it was demonstrated that progesterone decreased expression of BCR1 and HWP1, which are important virulence determinants of C. albicans. In conclusion, it was evident that progesterone can have a major influence on C. albicans pathogenicity on vaginal epithelial cells and may partly explain susceptibility of women to VVC at different stages of the menstrual cycle.     

Growth inhibition of Candida albicans by vaginal cells from na?ve mice.

Recurrent vulvovaginal candidiasis (RVVC) is a common idiopathic mucosal infection caused by Candida albicans. Current data suggests that local immunity is more important than that in the peripheral circulation for protection against infection. In the present study, anti-Candida innate resistance at the vaginal mucosa was investigated using a murine model. For this, splenic and vaginal cells were assessed for in vitro growth inhibition (GI) of C. albicans and cytotoxicity of natural killer (NK) cell-sensitive tumour targets (YAC-1). As expected, significant GI of C. albicans by splenic cells was mediated predominantly by polymorphonuclear leucocytes (PMNL) at effector to target (E:T) ratios of 100 and 50:1. From the vaginal mucosa, na?ve unfractionated, but not nylon wool non-adherent (NWN), cells extracted from whole vaginal tissue showed significant GI of C. albicans at E:T ratios as low as 1:1, but only modest killing of YAC-1 targets at all E:T ratios. Subsequent experiments showed significant GI of C. albicans by vaginal epithelioid-enriched cells and with several epithelial cell lines, but not in supernatants collected from the co-cultures. In contrast, lymphoid cell lines had no anti-Candida activity. These results suggest that anti-Candida activity is present at the vaginal mucosa, but unlike that from the spleen, the vaginal activity appears to be predominantly mediated by epithelial cells.     

Local anticandidal immune responses in a rat model of vaginal infection by and protection against Candida albicans

Humoral (antibody [Ab]) and cellular Candida-specific immune responses in the vaginas of pseudoestrus rats were investigated during three successive infections by Candida albicans. After the first, protective infection, Abs against mannan and aspartyl proteinase antigens were present in the vaginal fluid, and their titers clearly increased during the two subsequent, rapidly healing infections. In all animals, about 65 and 10% of vaginal lymphocytes (VL) were CD3(+) (T cells) and CD3(-) CD5(+) (B cells), respectively. Two-thirds of the CD3(+) T cells expressed the alpha/beta and one-third expressed the gamma/delta T-cell receptor (TCR). This proportion slightly fluctuated during the three rounds of C. albicans infection, but no significant differences between infected and noninfected rats were found. More relevant were the changes in the CD4(+)/CD8(+) T-cell ratio, particularly for cells bearing the CD25 (interleukin-2 receptor alpha) marker. In fact, a progressively increased number of both CD4(+) alpha/beta TCR and CD4(+) CD25(+) VL was observed after the second and third Candida challenges, reversing the high initial CD8(+) cell number of controls (estrogenized but uninfected rats). The CD3(-) CD5(+) cells also almost doubled from the first to the third infection. Analysis of the cytokines secreted in the vaginal fluid of Candida-infected rats showed high levels of interleukin 12 (IL-12) during the first infection, followed by progressively increasing amounts of IL-2 and gamma interferon during the subsequent infections. No IL-4 or IL-5 was ever detected. During the third infection, VL with in vitro proliferative activity in response to an immunodominant mannoprotein antigen of C. albicans were present in the vaginal tissue. No response to this antigen by mitogen-responsive blood, lymph node, and spleen cells was found. In summary, the presence of protective Ab and T helper type 1 cytokines in the vaginal fluids, the in vitro proliferation of vaginal lymphocytes in response to Candida antigenic stimulation, and the increased number of activated CD4(+) cells and some special B lymphocytes after C. albicans challenge constitute good evidence for induction of locally expressed Candida-specific Ab and cellular responses which are potentially involved in anticandidal protection at the vaginal level.     

Immunological Responses to Candida albicans II. Amyloidosis in Mice Induced by Candidiasis

Candida albicans infection in the mouse thigh rapidly induces amyloidosis in mice of the C57BL/Ks strain; amyloid is induced more slowly and to a lesser extent by viable candida in C3H and AKR mice, and by both viable Saccharomyces cerevisiae and heat-killed C. albicans in C57BL/Ks mice.     

Gastrointestinal Colonization by Candida albicans Mutant Strains in Antibiotic-Treated Mice

Antibiotic-treated mice orally inoculated with one of three Candida albicans strains (including two mutant strains) or indigenous Candida pelliculosa showed levels of candidal gastrointestinal colonization that were strain specific. However, regardless of strain, the numbers of viable candida were intermediate to high in the stomach, were consistently lowest in the upper small intestine, and increased progressively down the intestinal tract.     

High rate of vaginal infections caused by non-C. albicans Candida species among asymptomatic women.

A prospective observational study of patients attending a gynecological clinic and those referred to a clinic for genitourinary infections was undertaken with the purpose of evaluating the relative prevalence of non-C. albicans Candida species among Candida isolates from the vagina in different clinical settings in an area with high occurrence of vulvovaginal candidiasis. The rate of non-C. albicans Candida species was 44.5% among asymptomatic women, 19.4% among those with sporadic vaginitis and 21% among patients with chronic vaginal symptoms (p < 0.001 for asymptomatic vs. pooled symptomatic women). No increase in the rate of non-C. albicans Candida was observed during a period of 4 years (1995-1998) despite a 1.57-fold increase in the sales of azole antifungal agents. Unlike some previous reports we could not document an association of non-C. albicans Candida species with chronic vaginal symptoms or increased use of azole antifungal agents. The significantly higher rate of these yeasts in asymptomatic women is in accord with the known tendency of non-C. albicans Candida species to cause mild symptoms.     

Recognition and blocking of innate immunity cells by Candida albicans chitin

Chitin is a skeletal cell wall polysaccharide of the inner cell wall of fungal pathogens. As yet, little about its role during fungus-host immune cell interactions is known. We show here that ultrapurified chitin from Candida albicans cell walls did not stimulate cytokine production directly but blocked the recognition of C. albicans by human peripheral blood mononuclear cells (PBMCs) and murine macrophages, leading to significant reductions in cytokine production. Chitin did not affect the induction of cytokines stimulated by bacterial cells or lipopolysaccharide (LPS), indicating that blocking was not due to steric masking of specific receptors. Toll-like receptor 2 (TLR2), TLR4, and Mincle (the macrophage-inducible C-type lectin) were not required for interactions with chitin. Dectin-1 was required for immune blocking but did not bind chitin directly. Cytokine stimulation was significantly reduced upon stimulation of PBMCs with heat-killed chitin-deficient C. albicans cells but not with live cells. Therefore, chitin is normally not exposed to cells of the innate immune system but is capable of influencing immune recognition by blocking dectin-1-mediated engagement with fungal cell walls.     

Lymphokine-activated killer cell regulation of T-cell-mediated immunity to Candida albicans.

Monocytes are important accessory cells in the activation of T cells for specific antigen recognition yet little is known of their regulation. We demonstrated here that interleukin-2 (IL-2)-induced human lymphokine-activated killer (LAK) cells can inhibit monocyte antigen presentation, depending on the state of differentiation of the monocytes. Adherent monocytes cultured for 4 days in medium or granulocyte-macrophage colony-stimulating factor (GM-CSF) were found to equally process and present intact Candida albicans to autologous Percoll gradient-isolated T cells, as measured by [3H]thymidine uptake. However, only the GM-CSF-cultured monocytes were functionally inhibited by autologous 4-day IL-2-induced LAK cells. Even soluble candidal cell wall mannoprotein antigens could not be presented by these monocytes after exposure to LAK cells. Pretreatment of these monocytes with LAK cells for 1 h, followed by subsequent removal of the nonadherent LAK cells, was sufficient to cause significant inhibition, with maximal inhibition observed after 4 h. Northern (RNA) blot analysis indicated that mRNA expression for IL-1 alpha and IL-1 beta in response to C. albicans stimulation was also down-regulated in GM-CSF-cultured monocytes exposed to LAK cells. Interestingly, freshly isolated, Percoll gradient-purified large granular lymphocytes did not suppress antigen presentation in GM-CSF-treated monocytes. Another important finding was the inability of LAK cells to suppress the ability of freshly isolated or gamma interferon-cultured monocytes, which are resistant to LAK cell-mediated lysis, to present antigen to T cells. In contrast, IL-3 was similar to GM-CSF in inducing LAK cell susceptibility in monocytes. Taken together, these results indicated that IL-2 can induce LAK cells to down-regulate antigen presentation function in a select set of monocytes that have been activated by colony-stimulating factor (GM-CSF and IL-3) but not by gamma interferon. LAK cells may therefore play an important role in regulation of monocytes and their function, depending on their differentiation state.     

Suppression of Candida albicans by Human Oral Streptococci in Gnotobiotic Mice

Mixed human salivary bacteria and strains of Streptococcus salivarius and S. miteor suppressed colonization of Candida albicans in gnotobiotic mice. C. albicans attached in lower numbers to epithelial cells from conventional rats than from germ-free rats, and attachment inhibition by indigenous flora may explain in part the suppression of Candida colonization.     

Partial protection against experimental vaginal candidiasis after mucosal vaccination with heat-killed Candida albicans and the mucosal adjuvant LT(R192G).

The effectiveness of a mucosal vaccine composed of heat-killed Candida albicans (HK-CA) or C. albicans culture filtrate (CaCF) in conjunction with the mucosal adjuvant LT(R192G) against vulvovaginal candidiasis was examined in an estrogen-dependent murine model. Mice vaccinated intranasally with HK-CA + LT(R192G) exhibited a significant but short-lived protection accompanied by a vigorous delayed-type hypersensitivity response as well as high titers of circulating C. albicans-specific antibodies. Surprisingly, the levels of antigen-specific antibodies in the vaginal secretions of protected mice were negligible and no correlates of vaginal-associated Type 1 or Type 2 cytokines were observed. Vaginal priming with C. albicans before vaccination did not alter the protective outcome. Immunization with CaCF + LT(R192G) induced a discrete level of protection when administered intrarectally but not intranasally. These results suggest that mucosal vaccination can afford partial protection against vulvovaginal candidiasis, but the precise immune mechanisms responsible for protection are complex and as yet, not well understood.     

Mice lacking both G-CSF and IL-6 are more susceptible to Candida albicans infection: Critical role of neutrophils in defense against Candida albicans

Neutrophils play an important role in the host's defense against infection with various pathogenic organisms. Granulocyte colony stimulating factor (G-CSF) is regarded as a major regulator of neutrophil production and function. Mice lacking G-CSF or its receptor are neutropenic. IL-6 is another cytokine that has been shown to promote neutrophil production and modulate the function of many types of immune cells. We have analyzed G-CSF/IL-6 double deficient (G-CSF^{? / ?} /IL-6^{? / ?} ) mice to gain an insight into the possible contribution of IL-6 to the residual granulopoiesis in G-CSF-deficient (G-CSF^{? / ?} ) mice. Furthermore, we have evaluated the ability of G-CSF^{? / ?} /IL-6^{? / ?} mice to combat an experimental infection with Candida albicans. Our data shows that IL-6 plays a role in granulopoiesis during early post natal period but it is dispensable for steady-state granulopoiesis in adult mice. However, adult G-CSF^{? / ?} /IL-6^{? / ?} mice are more susceptible to Candida infection than similarly infected G-CSF^{? / ?} mice. Although, the candidacidal function of neutrophils of G-CSF^{? / ?} /IL-6^{? / ?} mice is deficient, the ability to produce IFN-γ and TNF-α in response to Candida infection is not compromised. Similarly, nitric oxide production by peritoneal macrophages from G-CSF^{? / ?} /IL-6^{? / ?} mice in response to Candida is comparable to G-CSF^{? / ?} mice.