Cryptococcosis, a systematic fungal infection caused by pathogenic Cryptococcus species, has emerged as a devastating cause of morbidity and mortality worldwide. It presents with a range of clinical outcomes that result from complex interactions between the pathogen and the mammalian host. During infection, Cryptococcus cells lodge in the alveoli of the lung where alveolar macrophages release cytokines in the response to the presence of antigens. These cytokines modulate the development and expression of T-cells stimulating a particular activation profile. A Th1 response is pro-inflammatory and leads to pathogen killing and clearance while a Th2 response is anti-inflammatory and leads to fungal growth.

In this study we are examining cytokine induction using two sets of Cryptococcus strains: a collection of 72 C. neoformans and C. gattii clinical isolates from two hospitals in Botswana, and a set of seven strains that have been derived from the virulent type strain C. neoformans H99 that vary in virulence despite being almost identical at the genotypic level. To assess host immune responses induced by different strains, J774.1 macrophages were infected with each isolate, the cells lysed, and purified RNA obtained. Cytokine expression was assessed by RNase Protection Assay (RPA) where radiolabelled anti-sense RNA probes are hybridised to purified macrophage RNA, non-hybridising RNA is degraded, and the protected RNA is electrophoresed, resulting in a profile of bands corresponding to the different cytokines.  

The results to date have revealed substantial variation in the amounts of TNF-α, IL-1β, and IL-1α induced by the different clinical isolates. However, statistical analysis found no significant association between cytokine profile and genotype or clinical outcome. We are currently optimising this analysis to ensure that it is thoroughly standardised and reproducible across all infection assays, and extending it to the H99 derivative strains.