Previous studies have shown that MyD88 and certain TLRs contribute to host protective responses in cryptococcal infection. MyD88 knockout mice had significantly reduced survival compared with WT C57BL/6 mice after pulmonary and intravenous challenge with C. neoformans
and TLR2 knockout mice died significantly sooner following pulmonary challenge but not intravenous challenge 
. TLR4 has been shown to recognize cryptococcal capsular polysaccharide 
. However, no strong phenotype for TLR4 has been observed in survival models using Cryptococcus 
. Furthermore, the impact of TLR9 has not been demonstrated in survival studies but has been shown to influence clearance of the organism from lungs 
Here we sought to establish the contributions of many of the receptors that utilize MyD88-dependent signaling pathways. We elected to intranasally inoculate mice with a highly virulent strain of Cryptococcus, H99, at a dose that kills WT C57BL/6 mice at an average time of 25–26 days. We confirmed that MyD88 knockout mice have reduced median survival time compared to WT mice and demonstrated that TLR9 knockout mice also have a significantly reduced median survival time compared to WT mice using this model. However, we found that neither TLR2 knockout nor TLR2/TLR4 double knockout mice exhibited differences in survival compared to WT mice using this infection model (unpublished data).
In our studies with IL-18R and IL-1R knockout mice, several observations were notable. First, IL-18R contributes to the protective response driven by MyD88, as demonstrated by the decreased median survival time in IL-18R knockout mice compared to WT mice. This corroborates with previously reported data demonstrating a protective role for IL-18 
. Secondly, IL-1R does not contribute to the overall survival in this infection model. Third, very high levels of IL-1β are induced in lungs of WT mice infected with H99 but IL-1β levels were markedly reduced in IL-1R, IL-18R, and MyD88 knockout mouse lungs, suggesting that expression of each of these molecules contributes substantially to IL-1β production. Nevertheless, diminished levels of pulmonary IL-1β do not necessarily correlate with survival outcomes, as evidenced by the similar survival curves for WT and IL-1R knockout mice. Also, while TLR9 knockout mice had a survival disadvantage, the levels of IL-1β were equal to those of WT mice.
Modestly diminished levels of IL-12p40 were observed in the lungs from IL-1R and IL-18R knockout mice at day 12 post-infection, suggesting that a reduced Th1 response contributed to the decreased survival of these knockouts during infection. The resolution of pulmonary C. neoformans
infection in experimental murine models has been associated with the induction of Th1-type cytokine responses 
. The cytokine IL-12p40, which is a subunit for both IL-12 and IL-23, plays a critical role in host defense through the induction of IFNγ and subsequent development of Th1 cells 
. In addition, IL-23 is important for the promotion of IL-17 production and driving Th17 responses, which participate in host immune responses following cryptococcal infection 
. A cooperative relationship between IL-18 and IL-12p40 has been described in Cryptococcus infection 
, so the diminished levels of IL-12p40 in IL-18R knockout mice are not surprising. Likewise, levels of the Th17 cytokine IL-17 were low but detectable in infected lungs at day 12. There was a trend, albeit not significant, towards diminished IL-17 levels for both IL-1R-deficient and IL-18R-deficient infected mice in comparison to wild-type infected mice (see ). IL-17 might peak in lungs at times other than day 12 post-infection, so the possibility exists that greater differences could be observed between wild-type and knockout mice.
We also found that IL-18R deficient mouse lungs have significantly elevated levels of IL-13 at day 12 following infection. IL-13 is cytokine produced by activated CD4+ lymphocytes, so the increase may reflect a shift towards a Th2 response, which has been associated with increased mortality 
. Other cytokines and chemokines associated with Th2 responses (e.g., IL-4, IL-5, and IL-10) did not differ between wild-type and knockout mouse lungs at day 12 in the experiments described here. However, the levels of these cytokines and chemokines were quite low and the possibility that they might peak in the lungs at a time other than day 12 post-infection cannot be excluded.
strain H99 is highly virulent in mouse models with central nervous system dissemination observed relatively early following pulmonary challenge. Perhaps because H99 is so virulent, significant differences in survival between mouse strains challenged with C. neoformans
have been generally difficult to demonstrate. Giles et al. investigated the role of surfactant protein A (SP-A), a putative mediator of host defense against Cryptococcus 
. While they were able to demonstrate that SP-A binds to Cryptococcus, SP-A-deficient and wild-type mice infected via intranasal inoculation with C. neoformans
H99 demonstrated no differences in lung CFU nor overall susceptibility to infection. In a similar infection model, McQuiston et al. assessed the role of sphingosine kinase 1 (SK1) in the host response to Cryptococcus infection 
. Following intranasal challenge of C57BL/6 wild-type mice and SK1 knockout mice with C. neoformans
H99, no significant differences in either survival or fungal burden in brain, liver, or spleen were observed between the two groups. Finally, Zhang et al. studied the impact of IL-4/IL-13 deletion on murine cryptococcosis 
. They compared wild-type Balb/C mice versus IL-4/IL-13 double knockout mice challenged intratracheally with C. neoformans
H99. While fungal burden was significantly diminished in the lungs of knockout mice, deletion of IL-4/IL-13 was insufficient to prevent CNS dissemination and the overall survival of the knockout mice was not statistically significant in comparison to wild-type mice. Given this context, the survival differences described in our study, while not dramatic, are nevertheless impressive.
The inflammatory response can be deleterious in cryptococcosis. Cryptococcosis is frequently associated with the immune reconstitution inflammatory syndrome (IRIS) in patients with AIDS on antiretroviral therapy 
. In the murine infection model used for our studies, considerable evidence of inflammation was observed through lung histopathology and cytokine levels, but enhanced or diminished inflammation did not appear to correlate with survival with any of the knockout strains of mice tested. The host inflammatory response may not directly correspond with a significant decrease in survival. Survival might instead correlate with dissemination outside the lung and the subsequent development of meningoencephalitis.
One observation was the paucity of BALT in lungs from MyD88 knockout mice infected with Cryptococcus. The presence of BALT is indicative of an immune response in mice but is not specific as it has been associated with either CD4 or CD8 cell recruitment in rodent models of infection 
. The significance of this isolated finding in our study is unclear. In our previous study, we did not observe any specific differences in inflammatory responses between WT and MyD88-deficient mice infected with C. neoformans
. However, in that study, we used a different strain (serotype A 145) and examined lungs at a different time point following infection 
In our infection model, no single cytokine specifically correlated with survival advantages or disadvantages. This emphasizes the complexity of signaling, cytokine and chemokine production, and host inflammatory cell recruitment and response during cryptococcal infection. The differential pattern of cytokine production in WT versus knockout mouse lungs following cryptococcal infection may be reflective of different types of cells that are recruited to the lungs.
Multiple factors impact survival following intranasal delivery of this dose of H99, including a combination of central nervous system disease and pulmonary disease. Our experimental findings suggest that IL-18R and TLR9 significantly contribute to MyD88-dependent host defense in cryptococcal infection. Further studies on the processing of the active form of IL-18 during cryptococcal activation are warranted.