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

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.

Importance of Clinical Isolates in Cryptococcus neoformans Research

“…C. neoformans is known to cause a latent, subclinical infection in 99% of the human population, although some researchers suggest that it could be considered to be a member of the lung microbiome [ 34 ]. Cryptococcosis is thought be caused by the reactivation of latent infection in the context of immune deficiency [ 35 , 36 ] However, H99 is highly pathogenic in animal models of cryptococcosis, and even as few as five cells can cause lethal disease [ 37 ], precluding its use for latent infection models. Instead, another reference strain, 52D, was initially used to develop latent models of cryptococcosis [ 38 , 39 , 40 ].…”

“…However, 52D is a C. deneoformans reference strain, and this species only causes 4% of infections in cryptococcal meningitis patients overall and only 1% in patients with HIV, as opposed to C. neoformans , which causes 95% and <99% of cryptococcosis cases, respectively [ 1 ]. To overcome this issue, we turned to clinical isolates to identify C. neoformans strains capable of establishing latent infections and used these strains to develop a latent murine model of cryptococcosis [ 35 ].…”

“…Similarly, mice infected with strains from patients who survived the infection tended to produce attenuated infections and longer survival in the murine model [ 51 ]. Importantly, this study also identified latent infections in mice and laid the foundation for developing a murine model for latent infection and reactivation following immune deficiency [ 35 ].…”

“…The murine inhalation model shows a strong association between human and murine mortality rates with C. neoformans clinical isolates, suggesting that this model is able to recapitulate human disease [ 51 ], and it was used to identify clinical isolates that produce latent infections in mice, broadening our ability to study latent infections and model the transition to lethal disease in immunocompromised hosts [ 35 ]. Importantly, the latent clinical isolates establish a persistent pulmonary infection that is controlled within granulomas in immunocompetent mice, mirroring many aspects of infection in healthy humans [ 35 ]. Upon immune depletion of these latently infected mice, the infection progresses to disseminated lethal disease similar to that observed in immunocompromised humans.…”

Murine Models of Cryptococcus Infection

“…Cryptococcus infection can be carried out in various ways, including intranasally, intratracheally, intracerebrally, intraperitoneally, and intravenously (Anderson & Sagha, 1988; Capilla et al., 2006; Coelho et al., 2019; Hamed et al., 2023; Kawakami et al., 1994; Lim et al., 1980; Mukaremera et al., 2019). Recent evidence indicated that disease progression from initial inhalation of Cryptococcus into the lungs to disseminated disease recapitulated human infection and disease outcomes (Coelho et al., 2019; Ding et al., 2021; Mukaremera et al., 2019; Normile et al., 2020). Compelling studies showed that humans and mice infected with the same C. neoformans strain exhibited similar outcomes for fungal burdens and mortality (Ding et al., 2021; Mukaremera et al., 2019; Normile et al., 2020).…”