Murine Models of Cryptococcus Infection.

Cryptococcus is recognized as one of the emerging fungal pathogens that have major impact on diverse populations worldwide. Because of the high mortality rate and limited antifungal therapy options, there is an urgent need to understand the impact of dynamic processes between fungal pathogens and hosts that influence cryptococcal pathogenesis and disease outcomes. With known common limitations in human studies, experimental murine cryptococcosis models that can recapitulate human disease provide a valuable tool for studying fungal virulence and the host interaction, leading to development of better treatment strategies. Infection with Cryptococcus in mice via intranasal inhalation is mostly used because it is noninvasive and considered to be the most common mode of infection, strongly correlating with cryptococcal disease in humans. The protocols described in this article provide the procedures of establishing a murine model of Cryptococcus infection by intranasal inhalation and assessing the host immune response and disease progression during Cryptococcus infection. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Murine model of pulmonary cryptococcal infection via intranasal inhalation Basic Protocol 2: Assessment of the pulmonary immune response during Cryptococcus infection Support Protocol: Evaluation of pulmonary gene expression by real-time PCR Basic Protocol 3: Enumeration of survival rate and organ fungal burden.

Macrophages-derived exo-miR-4449 induced by Cryptococcus affects HUVEC permeability and promotes pyroptosis in BEAS-2B via the HIC1 pathway.

Macrophages have recently been discovered to assume a significant role in the progression of cryptococcosis. However, the potential involvement of macrophage-derived exosomes in the pathogenesis of cryptococcosis remains uncertain. In this study, we investigated the changes of microRNAs in macrophage exosomes (exo-miRNAs) in cryptococcal infections and the role of markedly altered exo-miRNAs in the modulation of Human Umbilical Vein Endothelial Cells (HUVEC) permeability and ROS accumulation and pyroptosis in Human Bronchial Epithelioid Cells (BEAS-2B). Techniques such as microarray analysis and real-time quantitative PCR were used to detect different exo-miRNAs and to screen for the most highly expressed exo-miRNAs. Then its mimics were transfected into HUVEC to study its effect on the monolayer permeability of HUVEC. Finally, the relationship between this exo-miRNAs and the ROS accumulation and pyroptosis was verified by bioinformatics analysis. The results showed that five exo-miRNAs were overexpressed and two exo-miRNAs were reduced, among which, exo-miR-4449 was expressed at the highest level. Exo-miR-4449 could be internalized by HUVEC and enhanced its monolayer permeability. Moreover, exo-miR-4449 was found to promote ROS accumulation and pyroptosis in BEAS-2B through HIC1 pathway. Thus, exo-miR-4449 plays an important role in the pathogenesis of cryptococcosis and holds promise as a significant biomarker for treatment.

Detrimental impact of the IL-33/ST2 axis in an animal infection model with Cryptococcus neoformans.

Cryptococcus neoformans and Cryptococcus gattii are pathogenic fungi that infect the human respiratory system and cause life-threatening pulmonary cryptococcosis. The immunopathology of cryptococcosis is completely different from that of other fungal allergies. In murine cryptococcal infection models, cryptococcal cells are usually injected via nasal or intratracheal routes. After the infection, the alveolar epithelial cells are impaired and release IL-33, an IL-1 family cytokine that functions as an alarmin. This cytokine detrimentally amplifies allergic responses, and also induces a protective immune response against parasitic infection. In the pulmonary cryptococcosis model, type-II alveolar epithelial cells are the major source of IL-33, and the alveolar epithelial cells, ILC2, and Th2 cells express the IL-33 receptor (ST2). In IL-33- or ST2-deficient mice, allergy-like immune responses are attenuated after the C. neoformans infection. The numbers of ILC2 and Th2 cells and the levels of type 2 cytokines, including IL-4, IL-5, and IL-13, are decreased in the mouse lungs in both models. In association with these changes, total blood IgE, bronchus mucus production, and the number of eosinophils are decreased. Conversely, lung neutrophils and M1-type macrophages are increased. These are protective immune subsets suppressing cryptococcal growth. As a result, the lung fungal burden of IL-33- and ST2-deficient mice is decreased post-infection, and both deficient mice show significantly improved mortality. This pathogenesis varies depending on the cryptococcal and murine strains used in the animal experiments. Here, we overview and discuss the itmmunopathology of the IL-33/ST2 axis in a murine lethal cryptococcal infection model.

Platelets and mast cells promote pathogenic eosinophil recruitment during invasive fungal infection via the 5-HIAA-GPR35 ligand-receptor system.

Cryptococcus neoformans is the leading cause of fungal meningitis and is characterized by pathogenic eosinophil accumulation in the context of type-2 inflammation. The chemoattractant receptor GPR35 is expressed by granulocytes and promotes their migration to the inflammatory mediator 5-hydroxyindoleacetic acid (5-HIAA), a serotonin metabolite. Given the inflammatory nature of cryptococcal infection, we examined the role of GPR35 in the circuitry underlying cell recruitment to the lung. GPR35 deficiency dampened eosinophil recruitment and fungal growth, whereas overexpression promoted eosinophil homing to airways and fungal replication. Activated platelets and mast cells were the sources of GPR35 ligand activity and pharmacological inhibition of serotonin conversion to 5-HIAA, or genetic deficiency in 5-HIAA production by platelets and mast cells resulted in more efficient clearance of Cryptococcus. Thus, the 5-HIAA-GPR35 axis is an eosinophil chemoattractant receptor system that modulates the clearance of a lethal fungal pathogen, with implications for the use of serotonin metabolism inhibitors in the treatment of fungal infections.

Yeast-amoeba interaction influences murine cryptococcosis.

The virulence of Cryptococcus spp. is modulated in the natural environment through interaction with abiotic and biotic factors, and this can occasionally have implications for the progression of cryptococcosis in mammals. Hence, we evaluated whether the prior interaction of highly virulent Cryptococcus gattii strain R265 with Acanthamoeba castellanii influenced the progression of cryptococcosis. The influence of the capsule on endocytosis was evaluated using amoeba and yeast morphometrics. Mice were intratracheally infected with yeast re-isolated from the amoeba (Interaction), yeast without prior contact with the amoeba (Non-Interaction), or sterile phosphate-buffered saline (SHAM). Morbidity signs and symptoms were monitored during the survival curve, while cytokine and fungal burden measurements and histopathological analysis were performed on the 10th day post infection. Morbidity and mortality parameters in experimental cryptococcosis were influenced by the prior interaction of yeast with amoeba, which led to phenotypic changes in the cryptococcal cells, polysaccharide secretion, and their tolerance to oxidative stress. Our results suggest that a prior yeast-amoeba interaction modulates yeast virulence, which is associated with a greater tolerance to oxidative stress related to the exo-polysaccharide content and influences the progression of cryptococcal infection.

Cryptococcal granulomas of basal ganglia due to Cryptococcus neoformans in a cat: a case report and literature review.

A 3-year-old spayed female domestic short-haired cat presented with a head turning to the left, circling to the right, seizures, and opisthotonos for approximately one month. Neurological examination revealed a deficit in the postural reaction of the left limbs and visual abnormalities. Forensic computed tomography revealed a hyperattenuating round mass of 1.3 cm diameter with a hypoattenuating center in the right hemisphere. Histopathology showed multifocal granuloma lesions with the major mass mostly affecting the right basal ganglia. Cryptococcus neoformans variety grubii molecular type VNI/ST31 was isolated from the cryptococcal granulomas. This report highlights the epidemiological distribution and differential diagnosis of a feline central nervous system cryptococcosis caused by C. neoformans that occurred in an Asian country.

Pulmonary cryptococcosis after immunomodulator treatment in patients with Crohn's disease: Three case reports.

BACKGROUND: Corticosteroids and anti-tumor necrosis factor α mAbs are widely used to treat Crohn's disease (CD). However, one disadvantage of this treatment is impairment of normal immune function, leading to an increased risk of infection. Cryptococcus infection is an opportunistic infection that occurs mainly in immunocompromised patients and poses a significant diagnostic challenge in patients with CD. CASE SUMMARY: Here, we report three cases of pulmonary cryptococcosis in patients with CD after receiving immunomodulatory treatment. The patients presented with no or mild respiratory symptoms. Chest computed tomography scans revealed pulmonary nodules in the unilateral or bilateral lobes. Diagnoses were made using pathological examination and metagenomic sequencing. The patients were treated with fluconazole 400 mg once daily for 1 to 6 mo, and symptoms were resolved. Literature searches were conducted in PubMed, Web of Science, and Embase to retrieve previously reported cases and summarize patient characteristics. CONCLUSION: The incidence of cryptococcus infection has increased along with immunomodulator use. Clinical vigilance is required for early identification and standardized treatment.

Cryptococcal lymphadenitis-First presentation in an HIV-positive patient.

Cryptococcal infection is a life-threatening, opportunistic infection in human immunodeficiency virus-infected individuals. The infection most commonly begins in the respiratory tract, with secondary involvement of the brain, skin, and lymph nodes. We report a rare case of isolated cervical cryptococcal lymphadenitis diagnosed on fine needle aspiration cytology, which was the initial presentation of secondary immunodeficiency in the patient. Periodic acid-Schiff stain, India ink preparation, and culture were done to confirm the diagnosis. He was diagnosed as HIV-positive on further investigation.

Cryptococcal granuloma of the the basal ganglia with fatal haemorrhage after stereotactic biopsy: case report.

This paper presents a male, immunocompetent case aged 62-year-old with cryptococcal granuloma in the basal ganglia. No cryptococcal infection occurred in other areas. The diagnosis was made by biopsy. Cryptococcal granuloma was tough and unsuitable for sterotactic biopsy. The patient died of postoperative bleeding and we suggest avoiding stereotactic biopsy of lesions suspected of being cryptococcal granulomas.

Primary laryngeal cryptococcosis mimicking laryngeal malignancy: a case report.

Primary laryngeal cryptococcosis is an extremely rare infection and presents with non-specific symptoms such as hoarseness or sore throat, resulting in delayed diagnosis. Here, we report the patient of a 56-year-old female patient with primary laryngeal cryptococcosis, who was being treated with oral and inhaled steroids for rheumatoid arthritis and bronchial asthma. The patient suffered from prolonged hoarseness and sore throat, and endoscopic biopsy was performed several times under local anesthesia, demonstrating only inflammatory cell infiltration. Considering the possibility of laryngeal malignancy, a third biopsy was performed by endoscopic laryngomicrosurgery under general anesthesia. Intraoperative frozen section revealed non-neoplastic laryngeal mucosa with erosion and severe inflammatory cell infiltration. However, we could not confirm the definite diagnosis of the lesion in the intraoperative consultation. Postoperative histopathological examination revealed a small number of yeast-type fungi and a definitive diagnosis was established by special stains including Alcian blue stain. Finally, the patient was diagnosed as primary laryngeal cryptococcosis. Daily oral administration of fluconazole (400 mg/day) was performed for 6 months according to the treatment protocol for pulmonary cryptococcosis. The symptoms gradually improved, and endoscopy revealed no recurrence 6 months post-treatment. Clinicians should consider the possibility of laryngeal cryptococcosis when severe inflammation is found in the larynx and discuss the disease history and pathological results with pathologists more closely.

Comparison of the clinical manifestations and chest CT findings of pulmonary cryptococcosis in immunocompetent and immunocompromised patients: a systematic review and meta-analysis.

OBJECTIVE: The purpose of our study was to perform a meta-analysis and systematic review to compare differences in clinical manifestations and chest computed tomography (CT) findings between immunocompetent and immunocompromised pulmonary cryptococcosis (PC) patients. METHODS: An extensive search for relevant studies was performed using the PubMed, EMBASE, Cochrane Library, and Web of Sciences databases from inception to September 30, 2021. We included studies that compared the clinical manifestations and chest CT findings between immunocompetent and immunocompromised PC patients. Study bias and quality assessment were performed using the Newcastle-Ottawa Scale (NOS). RESULTS: Nine studies involving 248 immunocompromised and 276 immunocompetent PC patients were included in our analysis. The NOS score of each eligible study was above 5, indicating moderate bias. The proportion of elderly patients (> = 60 years old) in the immunosuppressed group was significantly higher than that in the immunocompetent group (OR = 2.90, 95% CI (1.31-6.43), Z = 2.63, p = 0.01). Fever (OR = 7.10, 95% CI (3.84-13.12), Z = 6.25, p < 0.000) and headache (OR = 6.92, 95% CI (2.95-16.26), Z = 4.44, p < 0.000) were more common in immunosuppressed patients. According to thin-section CT findings, lesions were more frequently distributed in the upper lobe (OR = 1.90, 95% CI (1.07-3.37), Z = 2.2, p = 0.028) in immunocompromised individuals. The proportions of patients with cavity sign (OR = 5.11, 95% CI (2.96-8.83), Z = 5.86, p = 0.00), ground-glass attenuation (OR = 5.27, 95% CI (1.60-17.35), Z = 2.73, p = 0.01), and mediastinal lymph node enlargement (OR = 2.41, 95% CI (1.12-5.20), Z = 2.24, p = 0.03) were significantly higher in immunocompromised patients. CONCLUSION: No significant differences in nonspecific respiratory symptoms were found between immunocompromised and immunocompetent PC patients. Nevertheless, fever and headache were more common in immunocompromised patients. Among the CT findings, cavity, ground-glass attenuation, and mediastinal lymph node enlargement were more common in immunocompromised individuals.

[Pulmonary cryptococcosis coexisting with lung cancer confirmed by pathological examinations: report of 3 cases and literature review].

Objective: To describe the clinical characteristics of pulmonary cryptococcosis(PC)coexisting with lung cancer. Methods: We reported 3 cases of PC coexisting with lung cancer confirmed by pathology in Qingdao Municipal Hospital from January 2017 to December 2021.We reviewed the literature with"pulmonary cryptococcosis" and "lung cancer" as the keywords to search Wanfang database, China HowNet and PubMed database. Results: The patients consisted of 2 males and 1 female. Two patients were diagnosed with nodular type of PC and one with diffuse mixed type of PC. One patient had underlying cardiovascular diseases and the other two had no medical history. The clinical manifestations varied including fever, cough, sputum, and no specific symptoms. All the patients received surgery and postoperative medical therapy, and all 3 patients were pathologically confirmed with adenocarcinoma. A total of 18 cases were retrieved from related literatures. To our knowledge, one of our cases was the first one with diffuse mixed type of PC coexisting with lung cancer. Conclusions: Coexistence of pulmonary cryptococcosis and lung cancer is rare and the clinical symptoms are nonspecific. When PC coexists with lung cancer, it is extremely easy to be misdiagnosed. Therefore, PC should be considered in the differential diagnosis of pulmonary nodules and multiple imaging changes.

Secreted fungal virulence effector triggers allergic inflammation via TLR4.

Invasive fungal pathogens are major causes of human mortality and morbidity1,2. Although numerous secreted effector proteins that reprogram innate immunity to promote virulence have been identified in pathogenic bacteria, so far, there are no examples of analogous secreted effector proteins produced by human fungal pathogens. Cryptococcus neoformans, the most common cause of fungal meningitis and a major pathogen in AIDS, induces a pathogenic type 2 response characterized by pulmonary eosinophilia and alternatively activated macrophages3-8. Here, we identify CPL1 as an effector protein secreted by C. neoformans that drives alternative activation (also known as M2 polarization) of macrophages to enable pulmonary infection in mice. We observed that CPL1-enhanced macrophage polarization requires Toll-like receptor 4, which is best known as a receptor for bacterial endotoxin but is also a poorly understood mediator of allergen-induced type 2 responses9-12. We show that this effect is caused by CPL1 itself and not by contaminating lipopolysaccharide. CPL1 is essential for virulence, drives polarization of interstitial macrophages in vivo, and requires type 2 cytokine signalling for its effect on infectivity. Notably, C. neoformans associates selectively with polarized interstitial macrophages during infection, suggesting a mechanism by which C. neoformans generates its own intracellular replication niche within the host. This work identifies a circuit whereby a secreted effector protein produced by a human fungal pathogen reprograms innate immunity, revealing an unexpected role for Toll-like receptor 4 in promoting the pathogenesis of infectious disease.

Clinical Analysis of Non-AIDS Patients with Pulmonary Cryptococcosis and the Change in Their Clinical Features over 30 Years in a Tertiary Hospital in Beijing, China.

Over the past few decades, the clinical features of pulmonary cryptococcosis (PC) have progressed; however, there is a lack of data on the manifestations of PC over time. To investigate the differences in the clinical characteristics of PC across different time periods, we retrospectively reviewed 130 non-acquired immunodeficiency syndrome (AIDS) patients diagnosed with pathologically or microbiologically confirmed PC from 1990-2020. Among the 130 patients with PC, 24 (18.5%) exhibited immunosuppression, and 44 (33.8%) had underlying diseases. In radiology, 118 (90.8%) presented with subpleural lesions, and 68 (53.1%) presented with nodules with diameters ranging from 1-5 cm. Seventy-five (57.7%) patients underwent surgery alone. The clinical features of PC at different time periods showed that hospitalization days decreased (P = 0.009), and the number of patients with symptoms decreased over time. The number of patients exhibiting isolated lesions decreased (P = 0.022), and the number of patients exhibiting subpleural lesions increased (P = 0.020). In addition, the number of patients with lesions presenting 3-10 mm nodules increased (P = 0.028). In conclusion, an increasing number of patients have been diagnosed with PC over the last 30 years. The timing of PC diagnosis has shifted to the early stages of disease progression. Pulmonary lesions caused by cryptococcosis are easily misdiagnosed and may require unnecessary surgical treatment. Further research is needed to identify the lung lesions caused by cryptococcosis.

A novel alphaherpesvirus and concurrent respiratory cryptococcosis in a captive koala (Phascolarctos cinereus).

A novel alphaherpesvirus was detected in a captive adult, lactating, female koala (Phascolarctos cinereus) admitted to James Cook University Veterinary Emergency Teaching & Clinical Hospital in March 2019, showing signs of anorexia and severe respiratory disease. Postmortem examination revealed gross pathology indicative of pneumonia. Histopathology demonstrated a chronic interstitial pneumonia, multifocal necrotising adrenalitis and hepatitis. Intranuclear inclusion bodies were detected by light microscopy in the respiratory epithelium of the bronchi, bronchioles, alveoli, and hepatocytes, biliary epithelium and adrenal gland associated with foci of necrosis. Cryptococcus gattii was isolated from fresh lung on necropsy, positively identified by PCR, and detected histologically by light microscopy, only in the lung tissue. A universal viral family-level PCR indicated that the virus was a member of the Herpesviruses. Sequence analysis in comparison to other known and published herpesviruses, indicated the virus was a novel alphaherpesvirus, with 97% nucleotide identity to macropodid alphaherpesvirus 1. We provisionally name the novel virus phascolarctid alphaherpesvirus 3 (PhaHV-3). Further research is needed to determine the distribution of this novel alphaherpesvirus in koala populations and establish associations with disease in this host species.

Transcriptional Changes in Pulmonary Phagocyte Subsets Dictate the Outcome Following Interaction With The Fungal Pathogen Cryptococcus neoformans.

With over 220,000 cases and 180,000 deaths annually, Cryptococcus neoformans is the most common cause of fungal meningitis and a leading cause of death in HIV/AIDS patients in Sub-Saharan Africa. Either C. neoformans can be killed by innate airway phagocytes, or it can survive intracellularly. Pulmonary murine macrophage and dendritic cell (DC) subsets have been identified in the naïve lung, and we hypothesize that each subset has different interactions with C. neoformans. For these studies, we purified murine pulmonary macrophage and DC subsets from naïve mice - alveolar macrophages, Ly6c- and Ly6c+ monocyte-like macrophages, interstitial macrophages, CD11b+ and CD103+ DCs. With each subset, we examined cryptococcal association (binding/internalization), fungicidal activity, intracellular fungal morphology, cytokine secretion and transcriptional profiling in an ex vivo model using these pulmonary phagocyte subsets. Results showed that all subsets associate with C. neoformans, but only female Ly6c- monocyte-like macrophages significantly inhibited growth, while male CD11b+ DCs significantly enhanced fungal growth. In addition, cytokine analysis revealed that some subsets from female mice produced increased amounts of cytokines compared to their counterparts in male mice following exposure to C. neoformans. In addition, although cells were analyzed ex vivo without the influence of the lung microenviroment, we did not find evidence of phagocyte polarization following incubation with C. neoformans. Imaging flow cytometry showed differing ratios of cryptococcal morphologies, c-shaped or budding, depending on phagocyte subset. RNA sequencing analysis revealed the up- and down-regulation of many genes, from immunological pathways (including differential regulation of MHC class I in the antigen processing pathway and the cell adhesion pathway) and pathways relating to relating to metabolic activity (genes in the Cytochrome P450 family, genes related to actin binding, calcium voltage channels, serine proteases, and phospholipases). Future studies gaining a more in-depth understanding on the functionality of individual genes and pathways specific to permissive and non-permissive pulmonary phagocytes will allow identification of key targets when developing therapeutic strategies to prevent cryptococcal meningitis.

Anti-inflammatory actions of aspirin-triggered resolvin D1 (AT-RvD1) in bronchial epithelial cells infected with Cryptococcus neoformans.

BACKGROUND: The interaction of Cryptococcus neoformans with airway epithelial cells is crucial for the establishment of cryptococcosis. Aspirin-triggered-resolvin D1 (AT-RvD1) is a lipid mediator produced during the resolution of inflammation and demonstrates anti-inflammatory and pro-resolution effects in several inflammatory experimental models including in the airways. METHOD: Here, we evaluated the effects of AT-RvD1 (1, 10 or 100 nM) on human bronchial epithelial cells (BEAS-2B) stimulated with C. neoformans (1, 10 or 100 multiplicities of infection; MOI). RESULTS: After 24 h, C. neoformans (all MOI) demonstrated no cytotoxic effects and increased IL-8 production on BEAS-2B cells when compared to controls. In addition, C. neoformans (MOI 100) increased the concentration of IL-6, but not of IL-10. AT-RvD1 (100 nM) significantly reduced the concentration of IL-8 and IL-6 and increased IL-10 production in C. neoformans-stimulated BEAS-2B cells. C. neoformans increased the phosphorylation of NF-κB and ERK1/2, and ALX/FPR2 expression. AT-RvD1 reduced the activation of NF-kB without altering the ERK1/2 and ALX/FPR2 expression. The anti-inflammatory effects of AT-RvD1 were dependent on the ALX/FPR2, once its antagonist (BOC2) reversed its anti-inflammatory effects. No alteration on the fungal burden as well as interactions with BEAS-2B cells was observed by AT-RvD1. CONCLUSION: AT-RvD1 demonstrated significant anti-inflammatory effects in bronchial epithelial cells infected with C. neoformans without affecting the development of C. neoformans infection in the airways. TRIAL REGISTRATION: Not applicable.

Effects of cigarette smoke extract on bronchial epithelial cells stimulated with Cryptococcus neoformans.

In the airways, the adhesion of Cryptococcus neoformans with airway epithelial cells is crucial for the establishment of cryptococcosis. Tobacco smoke is considered a risk factor for cryptococcosis. Here, we evaluated the effects of cigarette smoke extract (CSE) on human bronchial epithelial cells (BEAS-2B) stimulated with C. neoformans. Multiplicities of infection (MOIs) of 1-100 of C. neoformans per cell led to increased IL-8 production and no cytotoxic effects when compared to those of controls. C. neoformans (MOI 100) also significantly increased the concentration of IL-6. In cells stimulated with CSE doses (1.0, 2.5 and 5.0%) from one or five cigarettes, increased IL-1ß production was observed only in doses from one (1.0%) and five (2.5%) cigarettes when compared to that of controls. However, only 1.0% CSE failed to show cytotoxic effects. In addition, CSE significantly increased the concentration of IL-8. Cells stimulated with both CSE and C. neoformans demonstrated a reduction in IL-6/STAT3 signalling compared to that in cells stimulated by C. neoformans. In addition, a significant increase in IL-10 production was also observed. No alterations in NF-kB or ICAM-1 expression were observed among the groups. The combination of CSE and C. neoformans favoured the increase of fungal numbers and extracellular adhering of C. neoformans on BEAS-2B cells. In addition, the internalization of C. neoformans on BEAS-2B cells was reduced after CSE stimulation. In conclusion, the association of CSE and C. neoformans induced an anti-inflammatory effect in bronchial epithelial cells, which might favour the development of C. neoformans infection in the airways.