Role of IL-17 in Morphogenesis and Dissemination of Cryptococcus neoformans during Murine Infection

Cryptococcus neoformans is a pathogenic yeast that can form Titan cells in the lungs, which are fungal cells of abnormally large size. The factors that regulate Titan cell formation in vivo are still unknown, although an increased proportion of these fungal cells of infected mice correlates with induction of Th2-type responses. Here, we focused on the role played by the cytokine IL-17 in the formation of cryptococcal Titan cells using Il17a-/- knockout mice. We found that after 9 days of infection, there was a lower proportion of Titan cells in Il17a-/- mice compared to the fungal cells found in wild-type animals. Dissemination to the brain occurred earlier in Il17a-/- mice, which correlated with the lower proportion of Titan cells in the lungs. Furthermore, knockout-infected mice increased brain size more than WT mice. We also determined the profile of cytokines accumulated in the brain, and we found significant differences between both mouse strains. We found that in Il17a-/-, there was a modest increase in the concentrations of the Th1 cytokine TNF-α. To validate if the increase in this cytokine had any role in cryptococcal morphogenesis, we injected wild-type mice with TNF-α t and observed that fungal cell size was significantly reduced in mice treated with this cytokine. Our results suggest a compensatory production of cytokines in Il17a-/- mice that influences both cryptococcal morphology and dissemination.This work was supported by Grant SAF2017-86912-R and PID 2020-114546RB-100 from the Spanish Ministry for Science and Innovation. Roselletti E. is funded by an international collaboration with the company Lesaffre International Sarl. Garcia-Rodas R. is funded by a “Juan de la Cierva” contract from the Spanish Ministry for Economics, Industry, and Competitivity (Reference: IJCI-2015-25683). Trevijano-Contador N. is funded by an “Ayudas de Atracción de Talento Investigador” contract of the Community of Madrid (Reference: 2019-T2/BMD-14926).S

Secretory Aspartyl Proteinases Cause Vaginitis and Can Mediate Vaginitis Caused by Candida albicans in Mice

Vaginal inflammation (vaginitis) is the most common disease caused by the human-pathogenic fungus Candida albicans. Secretory aspartyl proteinases (Sap) are major virulence traits of C. albicans that have been suggested to play a role in vaginitis. To dissect the mechanisms by which Sap play this role, Sap2, a dominantly expressed member of the Sap family and a putative constituent of an anti-Candida vaccine, was used. Injection of full-length Sap2 into the mouse vagina caused local neutrophil influx and accumulation of the inflammasome-dependent interleukin-1β (IL-1β) but not of inflammasome-independent tumor necrosis factor alpha. Sap2 could be replaced by other Sap, while no inflammation was induced by the vaccine antigen, the N-terminal-truncated, enzymatically inactive tSap2. Anti-Sap2 antibodies, in particular Fab from a human combinatorial antibody library, inhibited or abolished the inflammatory response, provided the antibodies were able, like the Sap inhibitor Pepstatin A, to inhibit Sap enzyme activity. The same antibodies and Pepstatin A also inhibited neutrophil influx and cytokine production stimulated by C. albicans intravaginal injection, and a mutant strain lacking SAP1, SAP2, and SAP3 was unable to cause vaginal inflammation. Sap2 induced expression of activated caspase-1 in murine and human vaginal epithelial cells. Caspase-1 inhibition downregulated IL-1β and IL-18 production by vaginal epithelial cells, and blockade of the IL-1β receptor strongly reduced neutrophil influx. Overall, the data suggest that some Sap, particularly Sap2, are proinflammatory proteins in vivo and can mediate the inflammasome-dependent, acute inflammatory response of vaginal epithelial cells to C. albicans. These findings support the notion that vaccine-induced or passively administered anti-Sap antibodies could contribute to control vaginitis

Therapeutic activity of a Saccharomyces cerevisiae-based probiotic and inactivated whole yeast on vaginal candidiasis

Vulvovaginal candidiasis is the most prevalent vaginal infection worldwide and Candida albicans is its major agent. Vulvovaginal candidiasis is characterized by disruption of the vaginal microbiota composition, as happens following large spectrum antibiotic usage. Recent studies support the effectiveness of oral and local probiotic treatment for prevention of recurrent vulvovaginal candidiasis. Saccharomyces cerevisiae is a safe yeast used as, or for, the production of ingredients for human nutrition and health. Here, we demonstrate that vaginal administration of probiotic Saccharomyces cerevisiae live yeast (GI) and, in part, inactivated whole yeast Saccharomyces cerevisiae (IY), used as post-challenge therapeutics, was able to positively influence the course of vaginal candidiasis by accelerating the clearance of the fungus. This effect was likely due to multiple interactions of Saccharomyces cerevisiae with Candida albicans. Both live and inactivated yeasts induced coaggregation of Candida and consequently inhibited its adherence to epithelial cells. However, only the probiotic yeast was able to suppress some major virulence factors of Candida albicans such as the ability to switch from yeast to mycelial form and the capacity to express several aspartyl proteases. The effectiveness of live yeast was higher than that of inactivated whole yeast suggesting that the synergy between mechanical effects and biological effects were dominant over purely mechanical effects. The protection of epithelial cells to Candida-induced damage was also observed. Overall, our data show for the first time that Saccharomyces cerevisiae-based ingredients, particularly the living cells, can exert beneficial therapeutic effects on a widespread vaginal mucosal infection

Perinuclear Anti-Neutrophil Cytoplasmic Antibodies (pANCA) Impair Neutrophil Candidacidal Activity and Are Increased in the Cellular Fraction of Vaginal Samples from Women with Vulvovaginal Candidiasis

Vulvovaginal candidiasis (VVC) is primarily caused by Candida albicans and affects 75% of childbearing age women. Although C. albicans can colonize asymptomatically, disease is associated with an increased Candida burden, a loss of epithelial tolerance and a breakdown in vaginal microbiota homeostasis. VVC symptoms have been ascribed to a powerful inflammatory response associated with the infiltration of non-protective neutrophils (PMN). Here, we compared the immunological characteristics of vaginal fluids and cellular protein extracts obtained from 28 VVC women and from 23 healthy women colonized by Candida spp. We measured the levels of antibodies against fungal antigens and human autoantigens (anti-Saccharomyces cerevisiae antibodies (ASCA), C. albicans germ tube antibodies (CAGTAs) and perinuclear anti-neutrophil cytoplasmic antibodies (pANCA)), in addition to other immunological markers. Our results show that the pANCA levels detected in the cellular protein extracts from the vaginal fluids of symptomatic women were significantly higher than those obtained from healthy colonized women. Consistent with a potential physiologically relevant role for this pANCA, we found that specific anti-myeloperoxidase antibodies could completely neutralize the ex vivo killing capacity of polymorphonuclear cells. Collectively, this preliminary study suggests for the first time that pANCA are found in the pathogenic vaginal environment and can promptly impair neutrophil function against Candida, potentially preventing a protective response

Prevention and treatment of autoimmune diseases with plant virus nanoparticles

Plant viruses are natural, self-assembling nanostructures with versatile and genetically programmable shells, making them useful in diverse applications ranging from the development of new materials to diagnostics and therapeutics. Here, we describe the design and synthesis of plant virus nanoparticles displaying peptides associated with two different autoimmune diseases. Using animal models, we show that the recombinant nanoparticles can prevent autoimmune diabetes and ameliorate rheumatoid arthritis. In both cases, this effect is based on a strictly peptide-related mechanism in which the virus nanoparticle acts both as a peptide scaffold and as an adjuvant, showing an overlapping mechanism of action. This successful preclinical testing could pave the way for the development of plant viruses for the clinical treatment of human autoimmune diseases

NLRP3 inflammasome is a key player in human vulvovaginal disease caused by Candida albicans

The expression of host inflammatory and Candida albicans putative virulence factors was studied in women with vulvovaginal candidiasis (VVC; twenty) or colonized by the fungus but asymptomatic (carriers; fifteen) or non-colonized asymptomatic (ten subjects). Overexpression of genes encoding NLRP3 and caspase-1 inflammasome components sharply differentiated VVC patients from asymptomatic colonized or non-colonized women. Inflammasome expression was coupled with neutrophils recruitment in the vagina of VVC women and IL-1\u3b2 and IL-8 production. Both cytokines were present, though to a lower concentration, also in the vaginal fluid of colonized and non-colonized women. Secretory aspartyl proteinases (SAPs) and hyphae associated genes HWP1 and ECE1 were upregulated in VVC but with some differences among infected women. The most overexpressed SAP gene was SAP2, that correlated with neutrophils accumulation. Our data provide clinical evidence that the intracytoplasmic activation of NLRP3 inflammasome complex plays a critical, pathogenesis relevant role in human VVC

Inflammatory response during human vaginal infection with Candida albicans

Introduction. The microbiological, pathological and clinical factors determining vaginal candidiasis and recurrent vaginal candidiasis have long been studied, particularly using rodent models. The validity of which for understanding the pathogenesis of disease in women has been questioned. The most prevalent agent is critically determined by activation of microbial and host factors leading to persistent vaginal inflammation coupled to the inability of the inflammatory cells to resolve the fungal infection. Here we studied the activation of inflammasome complex neutrophil-recruiting and activating cytokines in the vaginal secretion of women with clinically established vaginal candidiasis. Materials and methods. In human vaginal samples positive for C. albicans with vaginal candidiasis (n=20) and carriage (n=15), infiltration of neutrophils, inflammatory mediators such as IL-8 and IL-1β, activation of inflammasome complex and expression of aspartyl proteases (SAPs) were examined. Results. In vaginal swabs of patients with vaginal candidiasis we found: i) consistent recruitment of neutrophils; ii) appreciable level of IL-8 and IL-1β; iii) activation of inflammasome complex; iv) consistent expression of SAP2, SAP5 and SAP6. Conclusions. These results show that immunopathogenesis of vaginal candidiasis is mediated by local recruitment of neutrophils, inflammatory cytokines secretion and inflammasome activation that mirror the upregulation of SAP2, SAP5 and SAP6 gene expression

Chronic Vaginal Candidiasis Is Achievable in Outbred CD-1 Mice

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Secretory aspartyl-proteinases of Candida albicans contribute to severe inflammation observed in the course of vaginal candidiasis

Candidiasis remains a notable health problem in subjects with underlying disease and several risk factors (diabetes, deep surgery, HIV infection, genetic predisposition and others) because of elevated mortality (candidemia and deep-seated infections), chronicity and recurrences in high prevalence clinical settings (oral and vaginal candidiasis), perceived increased risk of drug-resistance and lack of specific preventive measures such as vaccines. Within this framework, the high capacity of the major agent of candidiasis, Candida albicans (C. albicans), to express virulence factors causing inflammation and deceiving host immune system is remarkable and should constitute an important new concept/target to fight candidiasis by new tools providing an alternative or integration to the existing drugs. Secretory aspartyl-proteinases of C. albicans (SAP) are dominant virulence attributes of C. albicans and are considered to play an important role in the pathogenicity of this fungus. Using purified, enzymatically active, recombinant SAP, we demonstrated that SAP, including SAP2 and SAP6, are strong activators of NLRP3 inflammasome both in monocytes and epithelial cells. This occurs via NLRP3 and caspase-1 activation which cleaves pro-IL1\uf062 into secreted bioactive IL-1\uf062. The demonstration that proinflammatory cytokine production is induced by SAP through inflammasome activation implies new role for these virulence factors related to their structural features. Particularly, inflammasome activation may contribute to severe inflammation and recruitment of neutrophils, a scenario characteristic in the pathology of vaginal candidiasis, during SAP production or exposure in the vaginal environment. Our data strongly suggest that SAP2 production may contribute to the excessive inflammatory response observed during C. albicans infections, which may be the hallmark of at least some Candida pathologies

A role for secretory aspartyl proteinases of Candida albicans in mouse vaginal inflammation and vaginal candidiasis

We sought for a role of secretory aspartyl proteinase 2, (Sap2), a major virulence trait of Candida albicans, in a murine experimental model of vaginal candidiasis. Injection of enzymatically active, full-length Sap2 into the mouse vagina caused local PMN (neutrophil) influx and accumulation of the inflammasome-dependent interleukin (IL)-1beta but not of inflammasome-independent TNFalpha. No inflammatory response was detected following treatment with a N-terminus-truncated, enzymatically-inactive tSap2. Anti-Sap2 antibodies, in particular Fab from a human combinatorial antibody library inhibited or abolished the inflammatory response, provided they were able, like Pepstatin A, a known Sap inhibitor, to inhibit Sap2 enzyme activity. The same antibodies and Pepstatin A also caused strong inhibition of neutrophil influx and cytokine production early during experimental vaginal infection by Candida albicans. Sap2 induced expression of activated caspase-1 in murine vaginal epithelial cells as well as in a human vaginal epithelial cell line and caspase-1 inhibition down-regulated IL-1beta and IL-18 production by the vaginal epithelial cells. Overall, the data demonstrate that Sap2 is a critical determinant of the acute inflammatory response by the epithelial cells in vaginal candidiasis, and support the notion that vaccine-induced or passively administered anti-Sap antibodies could control vaginal disease