Elena Gabrielli

Secreted aspartic proteases of Candida albicans activate the NLRP3 inflammasome

In a recent report, we demonstrated that distinct members of the secreted aspartic protease (Sap) family of Candida albicans are able to induce secretion of proinflammatory cytokines by human monocytes, independently of their proteolytic activity and specific pH optima. In particular, C. albicans Sap2 and Sap6 potently induced IL‐1β, TNF‐α, and IL‐6 production. Here, we demonstrate that Sap2 and Sap6 proteins trigger IL‐1β and IL‐18 production through inflammasome activation. This occurs via NLRP3 and caspase‐1 activation, which cleaves pro‐IL‐1β into secreted bioactive IL‐1β, a cytokine that was induced by Saps in monocytes, in monocyte‐derived macrophages and in dendritic cells. Downregulation of NLRP3 by RNA interference strongly reduced the secretion of bioactive IL‐1β. Inflammasome activation required Sap internalization via a clathrin‐dependent mechanism, intracellular induction of K+ efflux, and ROS production. Inflammasome activation of monocytes induced by Sap2 and Sap6 differed from that induced by LPS‐ATP in several aspects. Our data reveal novel immunoregulatory mechanisms of C. albicans and suggest that Saps contribute to the pathogenesis of candidiasis by fostering rather than evading host immunity.

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

ABSTRACT 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. IMPORTANCE Candidal vaginitis is an acute inflammatory disease that affects many women of fertile age, with no definitive cure and, in its recurrent forms, causing true devastation of quality of life. Unraveling the fungal factors causing inflammation is important to be able to devise novel tools to fight the disease. In an experimental murine model, we have discovered that aspartyl proteinases, particularly Sap2, may cause the same inflammatory signs of vaginitis caused by the fungus and that anti-Sap antibodies and the protease inhibitor Pepstatin A almost equally inhibit Sap- and C. albicans-induced inflammation. Sap-induced vaginitis is an early event during vaginal infection, is uncoupled from fungal growth, and requires Sap and caspase-1 enzymatic activities to occur, suggesting that Sap or products of Sap activity activate an inflammasome sensor of epithelial cells. Our data support the notion that anti-Sap antibodies could help control the essence of candidal vaginitis, i.e., the inflammatory response. Candidal vaginitis is an acute inflammatory disease that affects many women of fertile age, with no definitive cure and, in its recurrent forms, causing true devastation of quality of life. Unraveling the fungal factors causing inflammation is important to be able to devise novel tools to fight the disease. In an experimental murine model, we have discovered that aspartyl proteinases, particularly Sap2, may cause the same inflammatory signs of vaginitis caused by the fungus and that anti-Sap antibodies and the protease inhibitor Pepstatin A almost equally inhibit Sap- and C. albicans-induced inflammation. Sap-induced vaginitis is an early event during vaginal infection, is uncoupled from fungal growth, and requires Sap and caspase-1 enzymatic activities to occur, suggesting that Sap or products of Sap activity activate an inflammasome sensor of epithelial cells. Our data support the notion that anti-Sap antibodies could help control the essence of candidal vaginitis, i.e., the inflammatory response.

New Approaches in the Development of a Vaccine for Mucosal Candidiasis: Progress and Challenges

The commensal fungus Candida albicans causes mucosal candidiasis in the rapidly expanding number of immunocompromised patients. Mucosal candidiasis includes oropharyngeal, esophageal, gastrointestinal, and vaginal infections. Vulvovaginal candidiasis (VVC) and antimycotic-refractory recurrent VVC is a frequent problem in healthy childbearing women. Both these mucosal infections can affect the quality of life and finding new therapeutical and preventive approaches is a challenge. A vaccine against candidal infections would be a new important tool to prevent and/or cure mucosal candidiasis and would be of benefit to many patients. Several Candida antigens have been proposed as vaccine candidates including cell wall components and virulence factors. Here we discuss the recent progress and problems associated with vaccination against mucosal candidiasis.

Role of CD45 Signaling Pathway in Galactoxylomannan-Induced T Cell Damage

Previously, we reported that Galactoxylomannan (GalXM) activates the extrinsic and intrinsic apoptotic pathways through an interaction with the glycoreceptors on T cells. In this study we establish the role of the glycoreceptor CD45 in GalXM-induced T cell apoptosis, using CD45+/+ and CD45−/− cell lines, derived from BW5147 murine T cell lymphoma. Our results show that whereas CD45 expression is not required for GalXM association by the cells, it is essential for apoptosis induction. In CD45+/+ cells, CD45 triggering by GalXM reduces the activation of Lck, ZAP70 and Erk1/2. Conversely, in CD45−/− cells, Lck was hyperphosphorylated and did not show any modulation after GalXM stimulation. On the whole, our findings provide evidence that the negative regulation of Lck activation occurs via CD45 engagement. This appears to be related to the capacity of GalXM to antagonize T cell activation and induce T cell death. Overall this mechanism may be responsible for the immune paralysis that follows GalXM administration and could explain the powerful immunosuppression that accompanies cryptococcosis.

Peptides of the Constant Region of Antibodies Display Fungicidal Activity

Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA) of antibodies (Fc-peptides) exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia furfur, including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro. An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents.

Antibody Complementarity-Determining Regions (CDRs): A Bridge between Adaptive and Innate Immunity

Background It has been documented that, independently from the specificity of the native antibody (Ab) for a given antigen (Ag), complementarity determining regions (CDR)-related peptides may display differential antimicrobial, antiviral and antitumor activities. Methodology/Principal Findings In this study we demonstrate that a synthetic peptide with sequence identical to VHCDR3 of a mouse monoclonal Ab (mAb) specific for difucosyl human blood group A is easily taken up by macrophages with subsequent stimulation of: i) proinflammatory cytokine production; ii) PI3K-Akt pathway and iii) TLR-4 expression. Significantly, VHCDR3 exerts therapeutic effect against systemic candidiasis without possessing direct candidacidal properties. Conclusions/Significance These results open a new scenario about the possibility that, beyond the half life of immunoglobulins, Ab fragments may effectively influence the antiinfective cellular immune response in a way reminiscent of regulatory peptides of innate immunity.

A novel bioluminescence mouse model for monitoring oropharyngeal candidiasis in mice

Oropharyngeal Candida albicans (C. albicans) infection usually occurs in patients with altered cell-mediated immune response. Many animal models have been developed for studying the pathogenesis of disease. Here we describe a new model for real-time monitoring of oral candidiasis. Mice were rendered susceptible to oral candidiasis by injection with cortisone acetate. Oral infection was performed by placing a swab saturated with genetically engineered bioluminescent strain of C. albicans sublingually. An in vivo imaging technique, exploiting stably trasformed C. albicans that costitutively express luciferase, was adopted. This novel longitudinal study represents a powerful tool to: (1) test real-time progression of infection, (2) identify the target site of C. albicans in specific organs, (3) evaluate the efficacy of antifungal therapies and (4) explore the spread of C. albicans from the local to systemic compartment in a new way.

Induction of Caspase-11 by Aspartyl Proteinases of Candida albicans and Implication in Promoting Inflammatory Response

ABSTRACT We recently demonstrated that the secreted aspartyl proteinases (Saps), Sap2 and Sap6, of Candida albicans have the potential to induce the canonical activation of the NLRP3 inflammasome, leading to the secretion of interleukin-1β (IL-1β) and IL-18 via caspase-1 activation. We also observed that the activation of caspase-1 is partially independent from the NLRP3 activation pathway. In this study, we examined whether Sap2 and Sap6 are also able to activate the noncanonical inflammasome pathway in murine macrophages. Our data show that both Sap2 and Sap6 can activate caspase-11 through type I interferon (IFN) production. Caspase-11 cooperates to activate caspase-1, with a subsequent increase of IL-1β secretion. Endocytosis and internalization of Saps are required for the induction of type I IFN production, which is essential for induction of noncanonical inflammasome activation. Our study indicates a sophisticated interplay between caspase-1 and caspase-11 that connects the canonical and noncanonical pathways of inflammasome activation in response to C. albicans Saps.

A purified capsular polysaccharide markedly inhibits inflammatory response during endotoxic shock.

ABSTRACT Capsular material of the opportunistic fungus Cryptococcus neoformans is composed mainly of a polysaccharide named glucuronoxylomannan (GXM). In this study, the effects of GXM were analyzed in an in vivo experimental system of lipopolysaccharide (LPS)-induced shock. Endotoxic shock was induced in mice by a single intraperitoneal injection of LPS from Escherichia coli. GXM treatment reduced the mortality of mice at early stages. Mice treated with LPS alone showed markedly increased plasma levels of tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and IL-6, whereas mice that were also treated with GXM showed significantly lower plasma levels of these cytokines. This effect was related to a marked suppression of Akt and IκBα activation. Importantly, the inhibitory effect of GXM on proinflammatory cytokine secretion was reproduced by treatment with wortmannin, an inhibitor of the Akt transcription pathway. Our results indicate that GXM has a beneficial effect on endotoxic shock, resulting in a significant increase in the rate of survival by dampening the hyperinflammatory response.

Cryptococcus neoformans galactoxylomannan is a potent negative immunomodulator, inspiring new approaches in anti-inflammatory immunotherapy

Cryptococcus neoformans is an opportunistic fungal pathogen responsible for life-threatening infections in immunocompromised individuals and occasionally in those with no known immune impairment. The fungus is endowed with several virulence factors, including capsular polysaccharides that play a key role in virulence. The capsule is composed of 90-95% glucuronoxylomannan (GXM), 5-8% galactoxylomannan (GalXM) and <1% mannoproteins. Capsular polysaccharides are shed into tissue where they produce many deleterious effects. Since GalXM has a smaller molecular mass, the molar concentration of GalXM in polysaccharide that is shed could exceed that of GXM in C. neoformans exopolysaccharides. Moreover, GalXM exhibits a number of unusual biologic properties both in vitro and in vivo. Here, we summarize the principal immunomodulatory effects of GalXM described during the last 20 years, particularly the mechanisms leading to induction of apoptosis in T lymphocytes, B lymphocytes and macrophages. Since the capacity of GalXM to induce widespread immune suppression is believed to contribute to the virulence of C. neoformans, this property might be exploited therapeutically to dampen the aberrant activation of immune cells during autoimmune disorders.