Vulvovaginal candidiasis (VVC) is one of the most common infections caused by Candida albicans. VVC is characterized by an inadequate hyperinflammatory response and clinical symptoms associated with Candida colonization of the vaginal mucosa. Compared to other host niches in which C. albicans can cause infection, the vaginal environment is extremely rich in lactic acid that is produced by the vaginal microbiota. We examined how lactic acid abundance in the vaginal niche impacts the interaction between C. albicans and the human immune system using an in vitro culture in vaginal simulative medium (VSM). The presence of lactic acid in VSM (VSM+LA) increased C. albicans proliferation, hyphal length, and its ability to cause damage during subsequent infection of vaginal epithelial cells. The cell wall of C. albicans cells grown in VSM+LA displayed a robust mannan fibrillar structure, β-glucan exposure, and low chitin content. These cell wall changes were associated with altered immune responses and an increased ability of the fungus to induce trained immunity. Neutrophils were compromised in clearing C. albicans grown in VSM+LA conditions, despite mounting stronger oxidative responses. Collectively, we found that fungal adaptation to lactic acid in a vaginal simulative context increases its immunogenicity favouring a pro-inflammatory state. This potentially contributes to the immune response dysregulation and neutrophil recruitment observed during recurrent VVC.

Candida albicans is a fungus that colonizes the gut, oral, and vaginal mucosae of most humans without causing disease. However, under certain predisposing conditions this fungus can cause disease. Candida albicans has several factors and attributes that facilitate its commensal and pathogenic lifestyles including the transition from a yeast to a hyphal morphology, which is accompanied by the expression of virulence factors. These factors are central in candidiasis that can range from invasive to superficial. This review focuses on one example of a superficial disease, i.e. vulvovaginal candidiasis (VVC) that affects ~75% of women at least once with some experiencing four or more symptomatic infections per year (RVVC). During VVC, fungal factors trigger inflammation, which is maintained by a dysregulated innate immune response. This in turn leads to immunopathology and symptoms. Another unique characteristic of the vaginal niche, is its Lactobacillus-dominated microbiota with low species diversity that is believed to antagonize C. albicans pathogenicity. The importance of the interactions between C. albicans, the host, and vaginal microbiota during commensalism and (R)VVC is discussed in this review, which also addresses the application of this knowledge to identify novel treatment strategies and to study vaginal C. albicans infections.

Pathogenicity mechanisms of the yeast Candida albicans involve filamentous growth, adhesion, invasion, and toxin production. Interestingly, clinical isolates, and other Candida spp., can cause infection independent of filamentation or toxin production. These strains and species often are characterized as avirulent ex vivo, yet this does not correlate with their potential to cause infection. We hypothesized that specific host factors, which trigger pathogenicity in vivo, are absent in in vitro infection models and thereby clinical isolates can seem avirulent ex vivo. We investigated how albumin, the most abundant protein in humans, impacts infection and cytotoxic potential of C. albicans in vitro. The presence of albumin induces otherwise non-damaging and non-filamentous clinical isolates to cause host cell cytotoxicity. Moreover, avirulent deletion mutants deficient in filamentation, adhesion, or toxin production are restored in their cytotoxicity by albumin. This involves transcriptional and metabolic reprogramming of C. albicans, increasing biofilm formation and production of the oxylipin 13-hydroxyoctadecadienoic acid, driving host cell cytotoxicity. Collectively, our study uncoveres a pathogenicity mechanism by which C. albicans causes epithelial cytotoxicity independent of its conventional virulence mechanisms. This alternative pathogenicity strategy helps to explain the avirulence of clinical isolates ex vivo, when they are separated from the host environment.

Aspergillus fumigatus may cause infections in individuals with underlying lung damage, such as those with pulmonary tuberculosis (TB). Simultaneous exposure to A. fumigatus and Mycobacterium tuberculosis may worsen lung tissue damage, but the combined immune response to these pathogens has not yet been fully characterized. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were stimulated with A. fumigatus conidia, M. tuberculosis H37Rv lysate, or both combined. TNF and IL-1β were measured from culture supernatants. The role of different pattern recognition receptors (PRRs) important for the recognition of both pathogens were explored by using PRR inhibitors and PBMCs from donors deficient in certain pathways. A. fumigatus and M. tuberculosis synergistically induced TNF and IL-1β release by PBMCs, and this response was independent of TLR2 and dectin-1. We found that the synergy is regulated through distinct intracellular pathways. The activation of the intracellular receptor NOD2 by M. tuberculosis and NADPH oxidase complex-dependent ROS production triggered by A. fumigatus mediate TNF but not IL-1β synergy. Together, these findings indicate that A. fumigatus and M. tuberculosis jointly exacerbate proinflammatory responses. This may help to explain the persistent inflammation and immunopathology observed in patients with concurrent TB and aspergillosis.

BACKGROUND:Vulvovaginal candidiasis (VVC), mainly caused by Candida albicans, affects approximately 75% of women at least once during their lifetime. VVC is characterized by fungal colonization, which leads to inappropriate local hyperinflammation and symptoms. Although the trigger of C. albicans pathogenicity is often unknown, antibiotic use and vaginal dysbiosis are associated with increased susceptibility to VVC. A healthy vaginal microbiota is normally dominated by Lactobacillus species, which are believed to keep C. albicans infections at bay. Probiotic lactobacilli are, therefore, explored to treat symptomatic VVC episodes. However, the influence of probiotic lactobacilli on immune responses in the context of VVC remains underexplored. METHODS:We investigated how probiotic lactobacilli influence vaginal epithelial and downstream inflammatory responses during C. albicans infection, using in vitro vaginal epithelial infection models and stimulating primary human immune cells with supernatants from these vaginal epithelial cells. RESULTS:Our study shows that although most of the tested probiotic lactobacilli reduced C. albicans-induced vaginal epithelial cell damage, some species, particularly Limosilactobacillus fermentum and Lacticaseibacillus paracasei, elicited proinflammatory responses even in the absence of C. albicans. Probiotic lactobacilli also differentially modulated the C. albicans killing efficiency and production of reactive oxygen species by neutrophils. CONCLUSIONS:Overall, vaginal epithelial and downstream immune responses during co-cultivation with C. albicans and probiotic lactobacilli were mostly driven by specific bacterial species and their interactions with the vaginal epithelium. Therefore, the induction of "controlled" inflammation by probiotic lactobacilli may be beneficial to improve neutrophil function; however, whether this alleviates immunopathology warrants further investigation.

Pathogenic microorganisms can infect a variety of niches in the human body. During infection, these microbes can only persist if they adapt adequately to the dynamic host environment and the stresses imposed by the immune system. While viruses entirely rely on host cells to replicate, bacteria and fungi use their pathogenicity mechanisms for the acquisition of essential nutrients that lie under host restriction. An inappropriate deployment of pathogenicity mechanisms will alert host defence mechanisms that aim to eradicate the pathogen. Thus, these adaptations require tight regulation to guarantee nutritional access without eliciting strong immune activation. To work efficiently, the immune system relies on a complex signalling network, involving a myriad of immune mediators, some of which are quite directly associated with imminent danger for the pathogen. To manipulate the host immune system, viruses have evolved cytokine receptors and viral cytokines. However, among bacteria and fungi, selected pathogens have evolved the capacity to use these inflammatory response-specific signals to regulate their pathogenicity. In this review, we explore how bacterial and fungal pathogens can sense the immune system and use adaptive pathogenicity strategies to evade and escape host defence to ensure their persistence in the host.

BACKGROUND:Genetic predisposition plays a major role in the development of invasive pulmonary aspergillosis (IPA). The risk and course of IPA vary significantly among patients, yet continuously new genetic mechanisms that influence individual antifungal immune responses are being discovered. While genetic variability in interleukin (IL)-1 family cytokines is recognized as an important cause of disease susceptibility, it is unclear whether and how less-studied IL-1 family members, such as the IL-36 cytokine subfamily, are genetically regulated and influence the risk of infection. METHODS:We analyzed how genetic variants in the IL36 loci associate with the risk of IPA in 328 eligible recipients of allogeneic hematopoietic stem cell transplants and their corresponding donors. The functional consequences of relevant genetic variants were investigated using clinical samples and in vitro infection models. RESULTS:We report that recipient, but not donor, single-nucleotide polymorphisms (SNPs) rs895497 in IL36A and rs4849142 in IL36B increase the risk of IPA after transplantation. The strongest contribution of these SNPs to infection risk was observed in a combined analysis of transplant pairs. IL-36β was expressed in both human type II-like alveolar epithelial cells and macrophages following Aspergillus fumigatus infection. The risk genotype was associated with impaired production of IL-1β in bronchoalveolar lavage fluid samples from infected patients, as well as in fungal-stimulated macrophages. Moreover, macrophages harboring the risk genotype exhibited impaired fungicidal activity. CONCLUSIONS:Our findings suggest that genotype-specific mechanisms mediated by IL-36β act on the nonhematopoietic compartment, can impair antifungal immune responses in macrophages, and ultimately predispose to transplant recipient susceptibility to IPA.

Chitosan, the deacetylated product of chitin, is a significant component of the cell walls of nearly all fungi. In contrast with the high level of attention paid to plant immune recognition of chitin and chitosan of plant pathogenic fungi we know much less about the mammalian immune system immune recognition of chitosan during infections by human pathogenic fungal species. Here we show that the mammalian β-integrin CR3 complement scavenger receptor, that is expressed on monocytes and macrophages, recognises chitosan from a range of fungal sources and that this leads to the secretion of IL-6, IL-1β and TNF. The secretion of these pro-inflammatory cytokines was dependent on the phagocytosis of chitosan. The co-provision of chitosan along with a peptide (Aspf2 from Aspergillus fumigatus) presented by the MHCII complex potentiated a Th response leading to IL-22 production. Fungal cell wall chitosan therefore activates both the innate and adaptive arms of the human immune system.

The lung microbiome has recently gained attention for potentially affecting respiratory viral infections, including influenza A virus, respiratory syncytial virus (RSV) and SARS-CoV-2. We will discuss the complexities of the lung microenvironment in the context of viral infections and the use of organ-on-chip (OoC) models in replicating the respiratory tract milieu to aid in understanding the role of temporary microbial colonization. Leveraging the innovative capabilities of OoC, particularly through integrating gut and lung models, opens new avenues to understand the mechanisms linking inter-organ crosstalk and respiratory infections. We will discuss technical aspects of OoC lung models, ranging from the selection of cell substrates for extracellular matrix mimicry, mechanical strain, breathing mechanisms and air–liquid interface to the integration of immune cells and use of microscopy tools for algorithm-based image analysis and systems biology to study viral infection in vitro. OoC offers exciting new options to study viral infections across host species and to investigate human cellular physiology at a personalized level. This review bridges the gap between complex biological phenomena and the technical prowess of OoC models, providing a comprehensive roadmap for researchers in the field.

The formation of hyphae is one of the most crucial virulence traits the human pathogenic fungus Candida albicans possesses. The assessment of hyphal length in response to various stimuli, such as exposure to human serum, provides valuable insights into the adaptation strategies of C. albicans to the host environment. Despite the increasing high-throughput capacity live-cell imaging and data generation, the accurate analysis of hyphal growth has remained a laborious, error-prone, and subjective manual process. We developed an analysis pipeline utilizing the open-source visual programming language Java Image Processing Pipeline (JIPipe) to overcome the limitations associated with manual analysis of hyphal growth. By comparing our automated approach with manual analysis, we refined the strategies to achieve accurate differentiation between yeast cells and hyphae. The automated method enables length measurements of individual hyphae, facilitating a time-efficient, high-throughput, and user-friendly analysis. By utilizing this JIPipe analysis approach, we obtained insights into the filamentation behavior of two C. albicans strains when exposed to human serum albumin (HSA), the most abundant protein in human serum. Our findings indicate that despite the known role of HSA in stimulating fungal growth, it reduces filamentous growth. The implementation of our automated JIPipe analysis approach for hyphal growth represents a long-awaited and time-efficient solution to meet the demand of high-throughput data generation. This tool can benefit different research areas investigating the virulence aspects of C. albicans.

Inflammatory bowel disease (IBD) is an idiopathic gastrointestinal disease with drastically increasing incidence rates. Due to its multifactorial etiology, a precise investigation of the pathogenesis is extremely difficult. Although reductionist cell culture models and more complex disease models in animals have clarified the understanding of individual disease mechanisms and contributing factors of IBD in the past, it remains challenging to bridge research and clinical practice. Conventional 2D cell culture models cannot replicate complex host-microbiota interactions and stable long-term microbial culture. Further, extrapolating data from animal models to patients remains challenging due to genetic and environmental diversity leading to differences in immune responses. Human intestine organ-on-chip (OoC) models have emerged as an alternative in vitro model approach to investigate IBD. OoC models not only recapitulate the human intestinal microenvironment more accurately than 2D cultures yet may also be advantageous for the identification of important disease-driving factors and pharmacological interventions targets due to the possibility of emulating different complexities. The predispositions and biological hallmarks of IBD focusing on host-microbiota interactions at the intestinal mucosal barrier are elucidated here. Additionally, the potential of OoCs to explore microbiota-related therapies and personalized medicine for IBD treatment is discussed.

Candida albicans can cause mucosal infections in humans. This includes oropharyngeal candidiasis, which is commonly observed in human immunodeficiency virus infected patients, and vulvovaginal candidiasis (VVC), which is the most frequent manifestation of candidiasis. Epithelial cell invasion by C. albicans hyphae is accompanied by the secretion of candidalysin, a peptide toxin that causes epithelial cell cytotoxicity. During vaginal infections, candidalysin-driven tissue damage triggers epithelial signaling pathways, leading to hyperinflammatory responses and immunopathology, a hallmark of VVC. Therefore, we proposed blocking candidalysin activity using nanobodies to reduce epithelial damage and inflammation as a therapeutic strategy for VVC. Anti-candidalysin nanobodies were confirmed to localize around epithelial-invading C. albicans hyphae, even within the invasion pocket where candidalysin is secreted. The nanobodies reduced candidalysin-induced damage to epithelial cells and downstream proinflammatory responses. Accordingly, the nanobodies also decreased neutrophil activation and recruitment. In silico mathematical modeling enabled the quantification of epithelial damage caused by candidalysin under various nanobody dosing strategies. Thus, nanobody-mediated neutralization of candidalysin offers a novel therapeutic approach to block immunopathogenic events during VVC and alleviate symptoms.IMPORTANCEWorldwide, vaginal infections caused by Candida albicans (VVC) annually affect millions of women, with symptoms significantly impacting quality of life. Current treatments are based on anti-fungals and probiotics that target the fungus. However, in some cases, infections are recurrent, called recurrent VVC, which often fails to respond to treatment. Vaginal mucosal tissue damage caused by the C. albicans peptide toxin candidalysin is a key driver in the induction of hyperinflammatory responses that fail to clear the infection and contribute to immunopathology and disease severity. In this pre-clinical evaluation, we show that nanobody-mediated candidalysin neutralization reduces tissue damage and thereby limits inflammation. Implementation of candidalysin-neutralizing nanobodies may prove an attractive strategy to alleviate symptoms in complicated VVC cases.

Research on microbial pathogens has traditionally relied on animal and cell culture models to mimic infection processes in the host. Over recent years, developments in microfluidics and bioengineering have led to organ-on-chip (OoC) technologies. These microfluidic systems create conditions that are more physiologically relevant and can be considered humanized in vitro models. Here we review various OoC models and how they have been applied for infectious disease research. We outline the properties that make them valuable tools in microbiology, such as dynamic microenvironments, vascularization, near-physiological tissue constitutions and partial integration of functional immune cells, as well as their limitations. Finally, we discuss the prospects for OoCs and their potential role in future infectious disease research. This Review discusses how organ-on-chip models can applied for infectious disease research.

Candida albicans is a commensal yeast of the human intestinal microbiota that, under predisposing conditions, can become pathogenic and cause life-threatening systemic infections (candidiasis). Fungal-host interactions during candidiasis are commonly studied using conventional 2D in vitro models, which have provided critical insights into the pathogenicity. However, microphysiological models with a higher biological complexity may be more suitable to mimic in vivo-like infection processes and antifungal drug efficacy. Therefore, a 3D intestine-on-chip model was used to investigate fungal-host interactions during the onset of invasive candidiasis and evaluate antifungal treatment under clinically relevant conditions. By combining microbiological and image-based analyses we quantified infection processes such as invasiveness and fungal translocation across the epithelial barrier. Additionally, we obtained novel insights into fungal microcolony morphology and association with the tissue. Our results demonstrate that C. albicans microcolonies induce injury to the epithelial tissue by disrupting apical cell-cell contacts and causing inflammation. Caspofungin treatment effectively reduced the fungal biomass and induced substantial alterations in microcolony morphology during infection with a wild-type strain. However, caspofungin showed limited effects after infection with an echinocandin-resistant clinical isolate. Collectively, this organ-on-chip model can be leveraged for in-depth characterization of pathogen-host interactions and alterations due to antimicrobial treatment.

Our immune system possesses sophisticated mechanisms to cope with invading microorganisms, while pathogens evolve strategies to deal with threats imposed by host immunity. Human plasma protein α1-antitrypsin (AAT) exhibits pleiotropic immune-modulating properties by both preventing immunopathology and improving antimicrobial host defence. Genetic associations suggested a role for AAT in candidemia, the most frequent fungal blood stream infection in intensive care units, yet little is known about how AAT influences interactions between Candida albicans and the immune system. Here, we show that AAT differentially impacts fungal killing by innate phagocytes. We observed that AAT induces fungal transcriptional reprogramming, associated with cell wall remodelling and downregulation of filamentation repressors. At low concentrations, the cell-wall remodelling induced by AAT increased immunogenic β-glucan exposure and consequently improved fungal clearance by monocytes. Contrastingly, higher AAT concentrations led to excessive C. albicans filamentation and thus promoted fungal immune escape from monocytes and macrophages. This underscores that fungal adaptations to the host protein AAT can differentially define the outcome of encounters with innate immune cells, either contributing to improved immune recognition or fungal immune escape.

Protective immunity to opportunistic fungal infections consists of tightly regulated innate and adaptive immune responses that clear the infection. Immune responses to infections of the vaginal mucosa by Candida species are, however, an exception. In the case of vulvovaginal candidiasis (VVC), the inflammatory response is associated with symptomatic disease, rather than that it results in pathogen clearance. As such VVC can be considered an inflammatory disease, which is a significant public health problem due to its predominance as a female-specific fungal infection. Particularly, women with recurrent VVC (RVVC) suffer from a significant negative impact on their quality of life and mental health. Knowledge of the inflammatory pathogenesis of (R)VVC may guide more effective diagnostic and therapeutic options to improve the quality of life of women with (R)VVC. Here, we review the immunopathogenesis of (R)VVC describing several elements that induce an inflammatory arson, starting with the activation threshold established by vaginal epithelial cells that prevent unnecessary ignition of inflammatory responses, epithelial and inflammasome-dependent immune responses. These inflammatory responses will drive neutrophil recruitment and dysfunctional neutrophil-mediated inflammation. We also review the, sometimes controversial, findings on the involvement of adaptive and systemic responses. Finally, we provide future perspectives on the potential of some unexplored cytokine axes and discuss whether VVC needs to be subdivided into subgroups to improve diagnosis and treatment.

Candida albicans causes opportunistic infections ranging from mucosal mycoses to life-threatening systemic infections in immunocompromised patients. During C. albicans infection, leukotrienes and prostaglandins are formed from arachidonic acid by 5-lipoxygenase (5-LOX) and cyclooxygenases, respectively to amplify inflammatory conditions, but also to initiate macrophage infiltration to achieve tissue homeostasis. Since less is known about the cellular mechanisms triggering such lipid mediator biosynthesis, we investigated the eicosanoid formation in monocyte-derived M1 and M2 macrophages, neutrophils and HEK293 cells transfected with 5-LOX and 5-LOX-activating protein (FLAP) in response to C. albicans yeast or hyphae. Leukotriene biosynthesis was exclusively induced by hyphae in neutrophils and macrophages, whereas prostaglandin E2 was also formed in response to yeast cells by M1 macrophages. Eicosanoid biosynthesis was significantly higher in M1 compared to M2 macrophages. In HEK_5-LOX/FLAP cells only hyphae activated the essential 5-LOX translocation to the nuclear membrane. Using yeast-locked C. albicans mutants, we demonstrated that hyphal-associated protein expression is critical in eicosanoid formation. For neutrophils and HEK_5-LOX/FLAP cells, hyphal wall protein 1 was identified as the essential surface protein that stimulates leukotriene biosynthesis. In summary, our data suggest that hyphal-associated proteins of C. albicans are central triggers of eicosanoid biosynthesis in human phagocytes.

More than two million people worldwide are affected by life-threatening, invasive fungal infections annually. Candida species are the most common cause of nosocomial, invasive fungal infections and are associated with mortality rates above 40%. Despite the increasing incidence of drug-resistance, the development of novel antifungal formulations has been limited. Here we investigate the antifungal mode of action and therapeutic potential of positively charged, synthetic peptide mimics to combat Candida albicans infections. Our data indicates that these synthetic polymers cause endoplasmic reticulum stress and affect protein glycosylation, a mode of action distinct from currently approved antifungal drugs. The most promising polymer composition damaged the mannan layer of the cell wall, with additional membrane-disrupting activity. The synergistic combination of the polymer with caspofungin prevented infection of human epithelial cells in vitro, improved fungal clearance by human macrophages, and significantly increased host survival in a Galleria mellonella model of systemic candidiasis. Additionally, prolonged exposure of C. albicans to the synergistic combination of polymer and caspofungin did not lead to the evolution of tolerant strains in vitro. Together, this work highlights the enormous potential of these synthetic peptide mimics to be used as novel antifungal formulations as well as adjunctive antifungal therapy.