Use of Clinical Isolates to Establish Criteria for a Mouse Model of Latent Cryptococcus neoformans Infection.

Minna Ding,Kirsten Nielsen,Judith N Nielsen,Kyle D Smith,Katrina M Jackson,Darin L Wiesner

doi:10.3389/fcimb.2021.804059

Abstract

The mechanisms of latency in the context of C. neoformans infection remain poorly understood. Two reasons for this gap in knowledge are: 1) the lack of standardized criteria for defining latent cryptococcosis in animal models and 2) limited genetic and immunological tools available for studying host parameters against C. neoformans in non-murine models of persistent infection. In this study, we defined criteria required for latency in C. neoformans infection models and used these criteria to develop a murine model of persistent C. neoformans infection using clinical isolates. We analyzed infections with two clinical C. neoformans strains, UgCl223 and UgCl552, isolated from advanced HIV patients with cryptococcal meningitis. Our data show that the majority of C57BL/6 mice infected with the clinical C. neoformans isolates had persistent, stable infections with low fungal burden, survived beyond 90 days-post infection, exhibited weight gain, had no clinical signs of disease, and had yeast cells contained within pulmonary granulomas with no generalized alveolar inflammation. Infected mice exhibited stable relative frequencies of pulmonary immune cells during the course of the infection. Upon CD4+ T-cell depletion, the CD4DTR mice had significantly increased lung and brain fungal burden that resulted in lethal infection, indicating that CD4+ T-cells are important for control of the pulmonary infection and to prevent dissemination. Cells expressing the Tbet transcription factor were the predominant activated CD4 T-cell subset in the lungs during the latent infection. These Tbet-expressing T-cells had decreased IFNγ production, which may have implications in the capacity of the cells to orchestrate the pulmonary immune response. Altogether, these results indicate that clinical C. neoformans isolates can establish a persistent controlled infection that meets most criteria for latency; highlighting the utility of this new mouse model system for studies of host immune responses that control C. neoformans infections.

Highlights

Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening cryptococcal meningitis in immunocompromised individuals
We considered absence of extrapulmonary dissemination as a possible criterion for C. neoformans latency, but we did not include it for the following reasons: (1) Cases of extrapulmonary dissemination to cervical lymph nodes have been observed in immunocompetent patients (Baker, 1976; Bao et al, 2013; Gurung et al, 2014) and the prostate has been identified as a potential extrapulmonary site for latent C. neoformans infection (Larsen, 1989) (Table 1). (2) Extrapulmonary dissemination was observed in the brain, spleen, and kidney as early as 7 days postinfection in the rat model (Goldman et al, 1994) (Table 1). (3) there are documented clinical cases of extrapulmonary dissemination in latent Mycobacterium tuberculosis infections, which have similarity to latent cryptococcosis (Barrios-Payán et al, 2012; Pirofski and Casadevall, 2020)
There was no clinical or experimental basis to require the absence of systemic dissemination as a criterion for a latent C. neoformans infection animal model. Based on these data from human C. neoformans infections and existing animal models, we developed the following set of criteria to define latency in persistent C. neoformans infection models: 1) stable fungal counts in the lungs throughout the entirety of the infection, 2) generation of pulmonary granulomas and no alveolar inflammation in the surrounding lung parenchyma, 3) no clinical signs of disease throughout the entirety of infection, 4) no weight loss attributable to disease, 5) serum Cryptococcal Antigen (CrAg) (GXM) lateral flow assay (LFA) negative, and 6) no mortality associated with disease

Summary

Introduction

Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening cryptococcal meningitis in immunocompromised individuals. It is the second most common HIV/AIDS-associated infection worldwide, leading to 15% of all AIDS-related mortality annually (Rajasingham et al, 2017). The initial C. neoformans pulmonary infection is thought to establish a latent infection within the lungs that does not manifest any clinical symptoms (Pirofski and Casadevall, 2020). Immigrants presenting with HIV-associated cryptococcal meningitis that had lived in France for a median of 9 years were infected with C. neoformans isolates that are not commonly found in France, suggesting that these patients acquired their C. neoformans infection before immigrating to France (Garcia-Hermoso et al, 1999). There is sufficient evidence for latency in the context of C. neoformans infections

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Conclusion