The Roles of CD44 Membrane Ruffling and Signaling in Leukocyte Transmigration across Cryptococcus Neoformans-Coinfected Blood-Brain Barrier with HIV-1 gp41 in Vitro

Cryptococcus neoformans (Cn) is a critical human pathogen, mainly causes Cryptococcal meningitis with severe symptoms and high mortality. As an environmental yeast, Cn initiates infection after inhalation and spreads hematogenously to almost all organs. To cause Cryptococcal meningitis, Cn must invade the blood-brain barrier (BBB) that consists of brain microvascular endothelial cells (BMEC) and adhere to the host cell to resist the blood-flow. Previous studies have demonstrated that CD44, a cell adhesion molecule that mediates cell-cell and cell-matrix interactions, is involved in the invasion of Cn into human non-small cell lung cancer cells, providing a fundamental insight into the mechanism of Cn penetrating the blood-air barrier. Moreover, the binding between Cn and BMEC is enhanced by HIV-1 gp41 ectodomain (gp41-I90), which is related to its enhanced role in Cn-induced cytoskeletal changes. Both gp41-I90 and Cn are able to promote the CD44-mediated monocyte transmigration separately, and co-exposure to both would further accelerate the transmigration. The Cn CPS1 gene encodes a hyaluronic acid synthase, a product of which hyaluronic acid is the main receptor for CD44 in vivo. In addition, CD44 functions as the primary receptor during Cn infection. Although the mechanism how Cn crosses the BBB and causes CNS infection keeps mysterious, we suggest that CD44 plays a vital role in the interaction among Cn, leukocyte and BMEC.

To investigate the effect of CD44 in Cn infection into BMEC, we explored several cellular and molecular events during Cn invasion. Results showed that CD44 molecules from lipid rafts can interact with hyaluronic acid. The adherence and invasion of Cn were abrogated while CD44 was perturbed by Bikunin, a protein which can disturb the oligomerization of CD44 on membrane lipid rafts. The lipid raft marker, ganglioside GM1, co-localizes with CD44 on the plasma membrane, allowing Cn cells to adhere to host cells in GM1-enriched sites, illustrating that CD44 anchors on the lipid rafts and serves as the host receptor for Cn. Upon Cn engagement, GM1 internalized through vesicular structures to the nuclear membrane. This endocytic redistribution process is abolished by cytochalasin D, nocodazole, or anti-DYRK3 siRNA. The knockdown of DYRK3 significantly reduces Cn invasion across the BMEC. Cav1, a characteristic protein of caveolae/lipid rafts, colocalized with CD44 in certain regions on the plasma membrane (10~15%). The knockdown of Cav1 significantly reduced Cn invasion and the crossing of the BMEC. Upon Cn engagement, Cav1 was phosphorylated in a CD44-dependent manner, which was diminished by filipin, a disrupter of lipid raft structure. These findings imply that CD44 anchors on lipid rafts and plays a vital role in Cn infection and the perturbation of CD44, Cav-1 or DYRK3 would block Cn adherence and invasion to BMEC.

Furthermore, CD44 has an effect on leukocyte migration mediated by Cn. Cn infection into BMEC significantly enhanced the adhesion and migration of THP-1 cell (human leukemic monocytes) across the monolayers (P < 0.01), depending on the concentration and exposure time of Cn (P < 0.05). The addition of anti-CD44 and Bikunin to BMEC after Cn infection showed both adhesion and migration of THP-1 were significantly decreased (P < 0.01) in a dose dependent manner of the antibody (within 0-1 μg) and inhibitor (within 0-20 nmol/L). Both THP-1 adhesion and migration decreased in the BBB model infected with CPS1 gene-knockout Cn strain (P < 0.01, P < 0.05), but increased in complemented strain and wild-type strain. What's more, we also elaborated that gp41-I90 has an effect of enhancement in monocyte transmigration across Cn-infected BMEC, which may be induced by CD44. In conclusion, both CD44 and gp41-I90 play a vital role in THP-1 adhering to Cn-stimulated BMEC and migrating across BBB.

In summary, we indicate the CD44 molecule plays a crucial role in leukocyte transmigration. CD44 functions in conjunction with DYRK3, Cav1 and gp41-I90 on lipid rafts. Anti-CD44 and Bikunin, especially the latter, can significantly reduce leukocytes migration, and have certain application prospects in reducing inflammatory responses. The specific mechanism, however, is beneath the surface and studies is urgently appreciated. (Acknowledgements: Corresponding author: Hong Cao, gzhcao@smu.edu.cn; Grant from School of Public Health of Southern Medical University, China, No. GW202222 to H.C.)

No relevant conflicts of interest to declare.