Using the Collaborative Cross to Dissect Immunological Mechanisms Governing Latent Infection, Granuloma Formation, and Neuroinvasive Disease Following Repeated Very Low-Dose Inhalation Exposure to Cryptococcus

BackgroundDecades of cryptococcal research have relied on a small number of hypervirulent laboratory strains, principally H99 and its derivatives, which cause rapid and lethal central nervous system infection and bypass the latent pulmonary phase that defines the natural history of human disease. As a result, the immune mechanisms that govern durable fungal containment in the lung, the reasons these mechanisms fail in genetically susceptible hosts, and whether the pulmonary immune response can engage the brain before any fungal cell reaches the central nervous system remain poorly understood. We address these gaps using a physiologically relevant repeated low-dose inhalation model in members of the Collaborative Cross and the BXD mouse panels. MethodsA repeated low-dose intranasal exposure model, consisting of four doses of 50 C. neoformans UgCl223 cells over 18 days, was compared with a single primary challenge in C57BL/6J mice. Six Collaborative Cross founder strains (A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, NZO/H1LtJ, CAST/EiJ) and one BXD founder (DBA/2J) were assessed for fungal burden, granuloma architecture, and CD4 T cell polarisation. The neuroimmune axis was interrogated by exposing BV2 microglia to serum from infected mice with confirmed lung-restricted infection or disseminated disease. ResultsRepeated low-dose exposure, in contrast to single-dose primary challenge, was linked with enhanced fungal containment, elevated frequency of CD44+ CD4 T cells, expansion of regulatory T cells, and organised granuloma formation. Across seven genetically diverse strains, host genetic background determined disease outcome, spanning four orders of magnitude in lung fungal burden, from near-sterilising control in NZO mice to overt central nervous system dissemination with brain lesions in CAST mice. Granuloma architecture varied markedly across backgrounds in a pattern that mirrors human granuloma heterogeneity. The Th17 and regulatory T cell axis, rather than Th1 or Th2 polarisation, distinguished progressors from controllers. Critically, serum from mice with confirmed lung-restricted infection induced morphological microglial activation in a strain-dependent pattern associated with the disease spectrum, providing initial evidence supporting the existence of a lung-brain immune axis in cryptococcal disease. ConclusionsThese data demonstrate that the immune dynamics of latent cryptococcal infection are fundamentally distinct from those characterised in primary high-dose models. Host genetic background influences not only pulmonary containment of Cryptococcus but may also impact the magnitude of distal brain immune responses, potentially through mechanisms that extend beyond direct fungal dissemination to the central nervous system. These findings suggest that early pulmonary immune events may shape subsequent neuroimmune outcomes and identify a previously underappreciated lung-brain axis in cryptococcosis that warrants in vivo validation.