Elizabeth A. Lilly

Epithelial cell-derived S100 calcium-binding proteins as key mediators in the hallmark acute neutrophil response during Candida vaginitis

ABSTRACT Vulvovaginal candidiasis (VVC), caused by Candida species, is a significant problem in women of childbearing age. Similar to clinical observations, a robust vaginal polymorphonuclear neutrophil (PMN) migration occurs in a subset of mice without affecting vaginal fungal burden. We hypothesize that the vaginal PMN infiltrate and accompanying inflammation are not protective but instead are responsible for the symptoms of infection. The purpose of this study was to identify the signal(s) associated with the PMN response in the established mouse model. Vaginal lavage fluid from inoculated mice were categorized base on PMN counts, evaluated for PMN chemotactic activity and analyzed by SDS-PAGE and mass spectrometry (MS) for unique protein identification. The lavage fluid from inoculated mice with high, but not low, PMN levels showed increased chemotactic activity. Likewise, SDS-PAGE of lavage fluid with high PMN levels showed distinct protein patterns. MS revealed that bands at 6 and 14 kDa matched the PMN chemotactic calcium-binding proteins (CBPs), S100A8 and S100A9, respectively. The presence of the CBPs in lavage fluid was confirmed by Western blots and enzyme-linked immunosorbent assay. Vaginal tissues and epithelial cells from inoculated mice with high PMN levels stained more intensely and exhibited increased mRNA transcripts for both proteins compared to those in mice with low PMN levels. Subsequent antibody neutralization showed significant abrogation of the chemotactic activity when the lavage fluid was treated with anti-S100A8, but not anti-S100A9, antibodies. These results reveal that the PMN chemotactic CBP S100A8 and S100A9 are produced by vaginal epithelial cells following interaction with Candida and that S100A8 is a strong candidate responsible for the robust PMN migration during experimental VVC.

Immunohistochemical Evaluation of T Cells in Oral Lesions from Human Immunodeficiency Virus-Positive Persons with Oropharyngeal Candidiasis

ABSTRACT Oropharyngeal candidiasis (OPC), caused by Candida albicans, is the most frequent opportunistic fungal infection in human immunodeficiency virus (HIV)-positive persons. Although Th1-type CD4+ T cells are considered important for host defense against mucosal C. albicans infections, there is a paucity of information regarding the presence and/or role of T cells in OPC lesions. In pursuit of this, initial chromophore immunohistochemical studies showed a majority of CD8+ rather than CD4+ cells equally distributed throughout the buccal mucosa of OPC− persons (HIV− or HIV+), irrespective of blood CD4+ cell numbers. In contrast, CD8+ cells in lesions from HIV+ OPC+ persons were in significantly higher numbers and concentrated at the lamina propria-epithelium interface, a considerable distance from the Candida at the outer epithelium. Dual fluorescence and confocal microscopy confirmed that the majority of CD8+, but not CD4+, cells were T cells by the presence or absence, respectively, of CD3 on each cell type. These results suggest that CD8+ T cells may be important for oral host defense against OPC, especially when CD4 cell numbers are reduced, with a potential CD8 cell-specific dysfunction associated with susceptibility to OPC.

Chemical screening identifies filastatin, a small molecule inhibitor of Candida albicans adhesion, morphogenesis, and pathogenesis

Infection by pathogenic fungi, such as Candida albicans, begins with adhesion to host cells or implanted medical devices followed by biofilm formation. By high-throughput phenotypic screening of small molecules, we identified compounds that inhibit adhesion of C. albicans to polystyrene. Our lead candidate compound also inhibits binding of C. albicans to cultured human epithelial cells, the yeast-to-hyphal morphological transition, induction of the hyphal-specific HWP1 promoter, biofilm formation on silicone elastomers, and pathogenesis in a nematode infection model as well as alters fungal morphology in a mouse mucosal infection assay. We term this compound filastatin based on its strong inhibition of filamentation, and we use chemical genetic experiments to show that it acts downstream of multiple signaling pathways. These studies show that high-throughput functional assays targeting fungal adhesion can provide chemical probes for study of multiple aspects of fungal pathogenesis.

Tissue-Associated Cytokine Expression in HIVPositive Persons with Oropharyngeal Candidiasis

Oropharyngeal candidiasis (OPC), caused by Candida albicans, is the most common infection in human immunodeficiency virus (HIV)-positive persons. Although CD4(+) T cells are considered to be important for host defense against C. albicans at the oral mucosa, a recent immunohistochemical evaluation of T cells in OPC lesions of HIV-positive persons with reduced CD4(+) T cells showed high numbers of CD8(+) T cells. The present study investigated tissue-associated expression of cytokine and chemokine mRNA at the site of infection. Results showed some effects of HIV (primarily increased chemokine mRNA levels) but little effect of blood CD4(+) T cells. In contrast, mRNA for several proinflammatory, T helper, and CD8(+) T cell-associated cytokines and chemokines were increased in subjects with OPC versus those without. These results support the presence of CD8(+) T cells in OPC lesions and suggest evidence for a response against OPC, despite reduced levels of CD4(+) T cells.

Assessment of the association between HIV viral load and CD4 cell count on the occurrence of oropharyngeal candidiasis in HIV-infected patients.

Background: Oropharyngeal candidiasis (OPC) is the most frequently observed oral infection in HIV-infected individuals. Historically, lower CD4 counts have been associated with an increased prevalence of OPC in HIV-infected patients, but HIV viral load has also recently been recognized as a possible predictive factor. Objective: We examined the impact of viral load and blood CD4 cell count on the occurrence of OPC using modern exploratory statistical analyses. Methods: The exploratory and inferential methods of classification and regression trees (CARTs) and logistic regression were used to compare the impact of viral load and CD4 cell counts on OPC status in 161 HIV-infected individuals from an outpatient clinic population in New Orleans. Results: The use of stepwise logistic regression and CART to classify individual OPC status both identified viral load as the most important covariate, followed by CD4 cells counts. Age, sex, and highly active antiretroviral therapy use were also found to be associated with OPC status. Conclusions: These data strongly suggest that low viral load distinguishes those not at risk for OPC with high viral load, which also includes a heterogeneous set of predictors for OPC status, has the highest impact on OPC classification.

Annexin-A1 identified as the oral epithelial cell anti-Candida effector moiety

Innate and adaptive immunity are considered critical to protection against mucosal candidal infections. Among innate anti-Candida mechanisms, oral and vaginal epithelial cells have antifungal activity. The mechanism is fungistatic, acid-labile and includes a requirement for cell contact by intact, but not necessarily live, epithelial cells. The purpose of this study was to use the acid-labile property to further characterize the effector moiety. Surface material extracted from phosphate-buffered saline (PBS) -treated, but not acid-treated, epithelial cells significantly inhibited the growth of Candida blastoconidia in a dose-dependent manner which was abrogated by prior heat and protease treatment. Proteins extracted from PBS-treated cells bound blastoconidia and hyphae more intensely than those from acid-treated cells. Proteins from PBS-treated cells eluted from Candida revealed two unique bands of approximately 33 and 45 kDa compared with acid-treated cells. Mass spectrometry identified these proteins as Annexin-A1 and actin, respectively. Oral epithelial cells stained positive for Annexin-A1, but not actin. Western blots showed reduced Annexin-A1 in proteins from acid-treated epithelial cells compared with those from PBS-treated epithelial cells. Lastly, it was demonstrated that immunoprecipitation of Annexin-A1 from proteins extracted from PBS-treated oral epithelial cells resulted in abrogation of inhibitory activity. Taken together, these results indicate that Annexin-A1 is a strong candidate for the epithelial cell anti-Candida effector protein.

Candida-induced oral epithelial cell responses.

Objective: Oropharyngeal candidiasis (OPC), caused by Candida albicans, is the most common oral infection in HIV+ persons. Oral epithelial cells are considered important for innate host defense against OPC with production of cytokines in response to C. albicans and the ability to inhibit Candida growth in vitro. The purpose of this study was to determine if Candida similarly induces cytokines by oral epithelial cells from HIV+ persons, including those with OPC, as well as to determine if cytokines can influence the oral epithelial cell anti-Candida activity. Methods: Supernatants from oral epithelial cells from HIV+ persons with and without OPC cultured with Candida were evaluated for cytokines by ELISA, or cytokines were added to the standard growth inhibition assay using epithelial cells from HIV− persons. Results: Results showed low Candida-induced epithelial cell cytokine production from HIV+ persons, but with some elevated proinflammatory cytokines (TNF-α, IL-6) in those with OPC compared to those without OPC. The addition of specific proinflammatory or Th cytokines had no effect on oral epithelial cell anti-Candida activity in healthy HIV− persons. Conclusion: These results suggest that oral epithelial cells from HIV+ persons can contribute at some level to the oral cytokine milieu in response to Candida during OPC, but that cytokines do not appear to influence oral epithelial cell anti-Candida activity.

CD8 T cells and E-cadherin in host responses against oropharyngeal candidiasis.

BACKGROUND Oropharyngeal candidiasis (OPC) is the most common oral infection in HIV(+) persons. Previous studies suggest a role for CD8(+) T cells against OPC when CD4(+) T cells are lost, but enhanced susceptibility to infection occurs when CD8(+) T-cell migration is inhibited by reduced tissue E-cadherin. OBJECTIVE   To conduct a longitudinal study of tissue CD8(+) T-cells and E-cadherin expression before, during, and after the episodes of OPC. METHODS Oral fungal burden was monitored and tissue was evaluated for CD8(+) T cells and E-cadherin over a 1-year period in HIV(+) persons with a history of, or an acute episode of, OPC. RESULTS While longitudinal analyses precluded formal interpretations, point prevalence analyses of the data set revealed that when patients experiencing OPC were successfully treated, tissue E-cadherin expression was similar to that in patients who had not experienced OPC, and higher numbers of CD8(+) T cells were distributed throughout OPC(-) tissue under normal expression of E-cadherin. CONCLUSION These results suggest that (1) reduction in tissue E-cadherin expression in patients with OPC(+) is not permanent, and (2) high numbers of CD8(+) T cells can be distributed throughout OPC(-) tissue under normal E-cadherin expression. Together, these results extend our previous studies and continue to support a role for CD8(+) T cells in host defense against OPC.

Oral epithelial cell antifungal activity: approaches to evaluate a broad range of clinical conditions.

Anti-Candida activity by oral epithelial cells is considered one of several innate mucosal defense mechanisms against oropharyngeal candidiasis (OPC). OPC is the most common fungal infection in HIV disease. Previously we reported that oral epithelial cell anti-Candida activity is reduced in those with OPC, potentially representing a contributing factor to OPC. However, testing clinical epithelial cells possessing high levels of Candida has been limiting due to high background in the assay controls. HIV+ smokers often develop OPC sooner than non-smokers during progression to AIDS, suggesting additional immune aberrations. The purpose of this study was to design a means to reduce Candida associated with epithelial cells collected from saliva without affecting their in vitro growth inhibitory activity, and to employ that approach to evaluate antifungal activity in HIV+ smokers. To do so, oral epithelial cells with and without known levels of Candida were subjected to various treatments including azole, polyene, or echinocandin antifungal drugs or fixation followed by the standard growth inhibition (GI) assay. The results indicated that antifungal drugs, while effectively reducing cell-associated Candida, also affected epithelial cell function. In contrast, fixation with paraformaldehyde eliminated cell-associated Candida and had minimal effects on epithelial cell anti-Candida activity. Employing the fixation design that allowed a broad range of patients to be evaluated showed no difference in oral epithelial anti-Candida activity between HIV+ smokers and non-smokers. Therefore, oral epithelial cell antifungal activity does not appear compromised in those who smoke, reducing it as a contributing factor in susceptibility to premature OPC.

Immune Protection against Lethal Fungal-Bacterial Intra-Abdominal Infections

ABSTRACT Polymicrobial intra-abdominal infections (IAIs) are clinically prevalent and cause significant morbidity and mortality, especially those involving fungi. Our laboratory developed a mouse model of IAI and demonstrated that intraperitoneal inoculation with Candida albicans or other virulent non-albicans Candida (NAC) species plus Staphylococcus aureus resulted in 70 to 80% mortality in 48 to 72 h due to robust local and systemic inflammation (sepsis). Surprisingly, inoculation with Candida dubliniensis or Candida glabrata with S. aureus resulted in minimal mortality, and rechallenge of these mice with lethal C. albicans/S. aureus (i.e., coninfection) resulted in >90% protection. The purpose of this study was to define requirements for C. dubliniensis/S. aureus-mediated protection and interrogate the mechanism of the protective response. Protection was conferred by C. dubliniensis alone or by killed C. dubliniensis plus live S. aureus. S. aureus alone was not protective, and killed S. aureus compromised C. dubliniensis-induced protection. C. dubliniensis/S. aureus also protected against lethal challenge by NAC plus S. aureus and could protect for a long-term duration (60 days between primary challenge and C. albicans/S. aureus rechallenge). Unexpectedly, mice deficient in T and B cells (Rag-1 knockouts [KO]) survived both the initial C. dubliniensis/S. aureus challenge and the C. albicans/S. aureus rechallenge, indicating that adaptive immunity did not play a role. Similarly, mice depleted of macrophages prior to rechallenge were also protected. In contrast, protection was associated with high numbers of Gr-1hi polymorphonuclear leukocytes (PMNLs) in peritoneal lavage fluid within 4 h of rechallenge, and in vivo depletion of Gr-1+ cells prior to rechallenge abrogated protection. These results suggest that Candida species can induce protection against a lethal C. albicans/S. aureus IAI that is mediated by PMNLs and postulated to be a unique form of trained innate immunity. IMPORTANCE Polymicrobial intra-abdominal infections are clinically devastating infections with high mortality rates, particularly those involving fungal pathogens, including Candida species. Even in patients receiving aggressive antimicrobial therapy, mortality rates remain unacceptably high. There are no available vaccines against IAI, which is complicated by the polymicrobial nature of the infection. IAI leads to lethal systemic inflammation (sepsis), which is difficult to target pharmacologically, as components of the inflammatory response are also needed to control the infection. Our studies demonstrate that prior inoculation with low-virulence Candida species provides strong protection against subsequent lethal infection with C. albicans and S. aureus. Surprisingly, protection is long-lived but not mediated by adaptive (specific) immunity. Instead, protection is dependent on cells of the innate immune system (nonspecific immunity) and provides protection against other virulent Candida species. This discovery implies that a form of trained innate immunity may be clinically effective against polymicrobial IAI. IMPORTANCE Polymicrobial intra-abdominal infections are clinically devastating infections with high mortality rates, particularly those involving fungal pathogens, including Candida species. Even in patients receiving aggressive antimicrobial therapy, mortality rates remain unacceptably high. There are no available vaccines against IAI, which is complicated by the polymicrobial nature of the infection. IAI leads to lethal systemic inflammation (sepsis), which is difficult to target pharmacologically, as components of the inflammatory response are also needed to control the infection. Our studies demonstrate that prior inoculation with low-virulence Candida species provides strong protection against subsequent lethal infection with C. albicans and S. aureus. Surprisingly, protection is long-lived but not mediated by adaptive (specific) immunity. Instead, protection is dependent on cells of the innate immune system (nonspecific immunity) and provides protection against other virulent Candida species. This discovery implies that a form of trained innate immunity may be clinically effective against polymicrobial IAI.