Current antifungal treatment of cryptococcal meningitis consists of amphotericin B plus flucytosine followed by fluconazole for a long period of time [
24]. In clinical practice, AIDS patients show a reduced response to amphotericin B and flucytosine and often need lifelong prevention of relapses with fluconazole. For these immunocompromised individuals it would be highly beneficial to identify new therapeutic approaches helping the host to eliminate cryptococci from the CNS.
In vitro experiments have demonstrated that phagocytosis is dependent on the influence of opsonins [
25]. TLRs play a crucial role in recognition of PAMPs and the pathogen-triggered inflammatory response [
26]. We found that stimulation of microglial cells by different TLR agonists significantly increased the phagocytosis of fungi (present data) and bacteria [
20,
21]. This suggests that stimulation of microglial cells enhances the cellular innate immune response thereby increasing phagocytosis of invading bacteria or fungi and acts as an endogenous protective factor of the brain and myelon. The adaptor protein MyD88 is involved in the signaling cascade leading to the activation of nuclear factor-κB. All TLRs except TLR3 use the MyD88-dependent pathway. A mediator of the MyD88-independent pathway is the adaptor, TIR-domain-containing adapter-inducing interferon-β (TRIF), which relays signals of TLR3 and TLR4 [
15]. We have shown that the increase of phagocytosis of
C. neoformans by the different TLR agonists requires the functional MyD88 signaling cascade. After stimulation with Pam
3CSK
4, LPS and CpG, the phagocytic rate of MyD88-deficient microglial cells was strongly diminished compared to wild-type microglia. Conversely the difference of phagocytosed cryptococci was not significant different in poly (I:C)-stimulated wild-type and MyD88-deficient microglia.
Intracellular survival, expulsion of the yeast by phagocytic cells, intracellular replication and cell-to-cell spread of
C. neoformans play an important role in the pathogenesis of infection in immunosupressed patients [
27,
28]. Conversely, macrophages can phagocytose and kill
C. neoformans in immunocompetent individuals [
29]. When studying intracellular survival, intracellular replication of
C. neoformans is a very important issue that has to be considered. It has been observed that cryptococci replicate faster intracellularly than extracellularly [
30,
31]. In our intracellular survival experiment over 3 h, we found a decrease of intracellular cryptococci in microglial cells and a higher absolute number of
C. neoformans killed in Pam
3CSK
4-
, LPS- and CpG-stimulated microglial cells compared to unstimulated cells. Extracellular amphotericin B was necessary for the whole incubation time to avoid migration of viable cryptococci from the interior of the microglial cells to the medium, and subsequent extracellular growth. Microglia activated by TLR agonists produce proinflammatory cytokines (for example, TNF-α, IL-1 and IL-6) and nitric oxide, which can cause neuronal injury [
32‐
36]. For this reason, the approach described here will have to be tested
in vivo for efficacy and with respect to the possible induction of unintended neuronal injury. It may be necessary to design milder strategies for microglial activation in order to increase the infection resistance of the CNS without causing neuronal injury. In conclusion, our results suggest that the administration of TLR agonists is of potential therapeutic interest in the prevention and adjunctive treatment of
C. neoformans meningitis and meningoencephalitis in high-risk groups such as patients with AIDS, or organ transplant recipients.