Cn is an opportunistic pathogen, which causes fatal meningoencephalitis, especially in AIDS patients. In order to cause meningoencephalitis, Cn must cross the BBB. A great deal of evidence supports the existence of the Trojan horse model of BBB transmigration of Cn. (1) Cn can survive in phagocytic cells via active phagosomal extrusion and spread to the phagocytes [59, 60]; (2) The incidence rate of fungemia and meningoencephalitis is higher in HIV-1-infected patients than that in HIV-1-negative patients because HIV-1 can cause severe monocyte dysfunction in host [61–63]; (3) Cn was carried and transported by circulating phagocytes in the murine model of cryptococcosis in a previous study by Chrétien F. et al. [64]. (4) Cn is a facultative intracellular pathogen and has been shown to survive and multiply inside phagocytes in vitro [65]. Previous research had shown that HIV-1 infection is able to increase the monocyte capacity to migrate across the BBB [36]. In present study, we have suggested that Cn and/or HIV-1 gp41-I90 is able to enhance the transmigration activities of monocytes across BBB by using the in vitro and in vivo BBB models [66]. Importantly, we found that HIV-1 gp41-I90 was able to synergistically enhance the transmigration activity of monocytes in HBMEC infected with Cn and in mice with Cn-caused meningoencephalitis. Thus, we have firstly demonstrated the relationship between HIV-1, Cn and monocytes, which point out a new potential mechanism of invasion for this pathogenic fungus into the brain tissues of HIV-1-infected patients.
Initially, we demonstrated that the transmigration of monocytes across the BBB in vitro could besynergistically enhanced by HIV-1 gp41 protein and Cn. The specificity of the synergistic effect is further confirmed by transmigration assays. Two experiments were designed. In the first experiment, we used H-Cn to examine whether H-Cn and HIV-1 gp41 could synergistically enhance the transmigrate ability of monocytes. Our results have shown that there is no synergistic effect on the transmigration of monocytes with a combination of H-Cn and gp41. Interestingly, we found that H-Cn could also increase monocyte transmigration ability. In the second experiment, HIV Tat and p24 proteins were used. HIV Tat is a regulatory protein that enhances viral transcription and replication, which plays a multifaceted role in pathogenesis of HIV infection, including favouring viral infection, contributing to inflammatory responses and inducing monocyte invasion into the brain [67–70]. Notwithstanding, we found there is no synergistic effect on enhancement of monocyte transmigration upon treatment by a combination of Cn and HIV-1 Tat protein. Similarly, HIV p24, which is a component of the HIV particle capsid, also has no synergistic effect on Cn-mediated enhancement of monocyte transmigration. Taken together, these results suggest that the synergistic enhancement by the HIV-1 gp41 protein on monocyte transmigration across the Cn-infected BBB is viral factor-dependent. This is most likely due to the fact that both HIV-1 gp41 and Cn may elicit a similar signal, such as up-regulating CD44 and ICAM-1 expression (Fig. 6), activating membrane lipid rafts (Fig. 8) and NF-?B [44], to facilitate the transmigration of monocytes. Thus, we speculate that the ectodomain of HIV-1 gp41 may play a role as a trans-predilection factor for cryptococcal CNS invasion, suggesting that the HIV-1 fusion inhibitors targeting gp41, such as T20 and C34, may be helpful in the prevention and treatment of cryptococcal meningitis in HIV/AIDS patients.
CD44 is a well-known type I transmembrane glycoprotein and functions as the major hyaluronan receptor, which is widely distributed in a variety of endothelial cells, mesenchymal cells, hematopoietic stem cells and mesodermal cells and tissues. Although, alternative splicing can produce a large number of different isoforms, they all retain the hyaluronan-binding link-homology region and a common transmembrane and cytoplasmic domain [19]. Recent studies have demonstrated that, the gene that encodes capsule hyaluronic acid synthase is a key virulence gene of Cn. The transmigration process of Cn across the BBB rely on HA binding to the BMEC receptor CD44, which activates the host signal pathway to induce cytoskeleton rearrangement required for Cn invasion [71, 72]. In present study, we used the CRISPR-Cas9 system and CD44 inhibitor to examine whether the enhancement of Cn and HIV-1 gp41-I90 in transmigration of monocytes across the BBB is related to CD44. Indeed, our results revealed that CD44 was involved in the enhancement of monocyte transmigration across the BBB by Cn and HIV-1 gp41.
Beside the effect of inducing monocyte transmigration across the BBB in vitro, in present study, we also found that Cn and/or HIV-1 gp41 could enhance CD44 redistribution to the membrane lipid rafts and up-regulate the expression level of ICAM-1 and CD44, which are two major endothelial adhesion molecules long known for its importance in facilitating leukocyte transmigration. These findings indicate that Cn and HIV-1 gp41-induced migration of monocytes across BMEC in a coordinate manner with up-regulation of ICAM-1 and CD44. Hence, we derived the conclusion that, HBMEC co-exposed with Cn and HIV-1 gp41 exhibited re-distribution of CD44 and over-expression of CD44 and ICAM-1, which lead to enhancement of the adhesion and transmigration rates of monocytes and facilitate cerebral invasion of Cn.
During the process of studying the effect of HIV-1 gp41-I90 on the transmigration of monocytes across the BBB, we found the facilitation of HIV-1 gp41-I90 induced transmigration of monocytes is dose-dependent. When the concentration of HIV-1 gp41 was raised to a certain level, the facilitation get subdued, which remind us that, there is a threshold in the over-expression of CD44 induced by HIV-1 gp41-I90. In order to test the above assumption, different doses of HIV-1 gp41 (2–25 ?M) was added to the HBMEC monolayers to observe the transmigration activities of monocyte. These results showed that the facilitation induced by HIV-1 gp41-I90 was significantly saturated with the higher concentrations of the recombinant protein (Fig. 7a). Furthermore, we performed BA-ELISAs to examine whether the over-expression of CD44 induced by HIV-1 gp41-190 is also dose-dependent. As we expected, the expression level of CD44 on HBMEC could became saturated when the concentration of HIV-1 gp41-I90 was increased from 20–25 ?M (Fig. 7b). These results have profound clinical significance in antiretroviral therapies for HIV-associated Cryptococoal meningoencephalitis, as it suggests that adherence to antiretroviral therapies may minimize the risk of Cryptococoal neurologic disease.