Glucuronoxylomannan intranasal challenge prior to Cryptococcus neoformans pulmonary infection enhances cerebral cryptococcosis in rodents

The encapsulated fungus Cryptococcus neoformans is the most common cause of fungal meningitis, with the highest rate of disease in patients with AIDS or immunosuppression. This microbe enters the human body via inhalation of infectious particles. C. neoformans capsular polysaccharide, in which the major component is glucuronoxylomannan (GXM), extensively accumulates in tissues and compromises host immune responses. C. neoformans travels from the lungs to the bloodstream and crosses to the brain via transcytosis, paracytosis, or inside of phagocytes using a “Trojan horse” mechanism. The fungus causes life-threatening meningoencephalitis with high mortality rates. Hence, we investigated the impact of intranasal exogenous GXM administration on C. neoformans infection in C57BL/6 mice. GXM enhances cryptococcal pulmonary infection and facilitates fungal systemic dissemination and brain invasion. Pre-challenge of GXM results in detection of the polysaccharide in lungs, serum, and surprisingly brain, the latter likely reached through the nasal cavity. GXM significantly alters endothelial cell tight junction protein expression in vivo, suggesting significant implications for the C. neoformans mechanisms of brain invasion. Using a microtiter transwell system, we showed that GXM disrupts the trans-endothelial electrical resistance, weakening the human brain endothelial cell monolayers co-cultured with pericytes, supportive cells of blood vessels/capillaries found in the blood-brain barrier (BBB), and promotes C. neoformans BBB penetration. Our findings should be considered in the development of therapeutics to combat the devastating complications of cryptococcosis that results in an estimated ∼200,000 deaths worldwide each year. AUTHOR SUMMARY Cryptococcus neoformans infection of the central nervous system (CNS) typically begins by inhalation of fungal spores and results in devastating mortality rates worldwide. Over 200,000 deaths have been reported annually, with cryptococcal meningoencephalitis being the most severe form of the disease. This study investigates the ability of the fungus to invade, colonize, and cause damage to the host through properties of the fungal polysaccharide capsule, which allows the microbe a variety of both protective and offensive abilities. This capsule, made primarily of the polysaccharide glucuronoxylomannan (GXM), has been implicated in the progression and severity of cryptococcal infection in the CNS. We determined that GXM increases the fungal burden in the lungs of mice and enhances fungal migration to the brain. Interaction of GXM with the blood-brain barrier, which is a protective structure that regulates movement of particles into the CNS, demonstrated that GXM can disrupt the integrity of this barrier, compromising the delicate balances of fluids, immune cells, and other factors vital to the maintenance of the CNS. The findings of this study reveal the substantial role of GXM in establishing C. neoformans infection in the brain and necessitate future studies to further understand these interactions.

6 124 intranasal (i.n.) challenge prior to C. neoformans pulmonary infection enhances brain invasion and 125 colonization in rodents via disruption of the TJs on endothelial cells of the BBB. 126 To investigate the disruption of BBB integrity by GXM, we measured the activation of 127 RhoA, a member of the Ras-related small GTPase Rho family involved in the signaling pathway 128 for cytoskeletal regulation [18]. RhoA functions as a regulatory switch that confers increased 129 endothelial cell permeability [19] and BBB disruption [20] when activated. We assessed the 130 involvement of GXM in mediating RhoA activation in endothelial cells and found that RhoA 131 activation by GXM is significant and time-dependent, suggesting that disruption of cytoskeletal 132 regulation in endothelial cells may be a mechanism of virulence for C. neoformans to cross the 133 BBB and establish infection in the brain. These findings complement our in vitro observations that 134 GXM alters BBB permeability and contributes to increased fungal transmigration into the brain.

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GXM concentrations in murine tissues 24 h post-administration. 138 Given that we explored the impact of exogenous GXM on C. neoformans H99 strain infection, we 139 first determined whether GXM disseminated systemically from the airways and could be detected 140 in serum and brain tissue (Table 1). Therefore, C57BL/6 mice (n = 5) were challenged i.n. with a 141 single GXM dose of 125 µg/mL, and the CPS levels were quantified by enzyme-linked 142 immunosorbent assay (ELISA) 24 h later. Lung tissue GXM levels were 2.17 ± 0.09 µg/g. GXM 143 concentrations in serum and brain tissue were 0.34 ± 0.05 µg/mL and 0.21 ± 0.13 µg/g, 144 respectively. These measurements validated that GXM delivered into the nasal cavity can be 145 detected in tissues other than the lungs. Pre-conditioning with GXM exacerbates murine cryptococcosis and mortality. 148 We investigated the impact of GXM prior to C57BL/6 infection with C. neoformans H99 strain 149 cells (Fig. 1). Mice were sensitized i.n. with 125 µg/mL of GXM 24 h pre-inoculation with 10 5 C. 150 neoformans yeast cells (Fig. 1A). Cryptococcosis progression was compared between these mice 151 and rodents infected with the fungus only (untreated). i.n. administration of GXM significantly 152 accelerated the death of C. neoformans-infected mice relative to control mice (n = 10 mice per Exogenous GXM enhances pulmonary and systemic fungal load. 159 We quantified C. neoformans pulmonary burden in untreated and GXM-treated mice 3-and 7-dpi. 160 Although we did not observe cryptococcal load differences between untreated [4.1 x 10 4 colony 161 forming units (CFU)] and GXM (4.59 x 10 4 CFU)-challenged rodents 3-dpi ( Fig. 2A), we 162 demonstrated that the difference in pulmonary fungal burden was evident 7-dpi, with GXM-treated 163 animals (4.66 x 10 4 CFU) having a significantly higher burden than untreated mice (2.35 x 10 2 164 CFU; P<0.0001; Fig. 2A). Interestingly, untreated mice infected with cryptococci showed a 165 considerable reduction in fungal load 7-dpi (4.1 x 10 4 CFU) relative to 3-dpi (2.35 x 10 2 CFU).   181 We investigated the impact of exogenous GXM administration on C. neoformans dissemination to 182 the brain (Fig. 3). We demonstrated that brain fungal load in GXM-treated animals (3-dpi, 2.45 x 183 10 2 CFU; 7-dpi, 5.03 x 10 5 CFU) were significantly higher than in untreated (3-dpi, 1.67 x 10 1 184 CFU; 7-dpi, 1.94 x 10 1 CFU) mice (3-dpi, P < 0.05; 7-dpi, P < 0.0001; Fig. 3A). IF staining of C. 185 neoformans-infected brain tissue showed cryptococci of different size (yellow arrowheads) 186 embedded in neuronal tissue (Fig. 3B). A tissue section of the dentate gyrus in the hippocampus 187 of a GXM-challenged mouse exhibited a large cryptococcoma (red arrows) filled with yeasts cells 188 (yellow arrowhead) and substantial amounts of capsular GXM released (white arrowheads) (Fig.   189 3C). Extensive accumulation of GXM (white arrowheads) was also observed in cortical tissue (Fig. 190 3D) and a large blood vessel (Fig. 3E) in the brain parenchyma of GXM-treated and C. 191 neoformans-infected mice. IF staining showed a significantly higher number of lesions in the 192 brains of GXM-treated mice (average, 7.86 ± 0.91) than those of untreated animals (2.43 ± 0.43) 9 193 (P < 0.001) (Fig. 4A-B). The average areas of brain lesions of GXM-treated infected mice reached 194 241 μm 2 ± 27.79, whereas lesions of control mice averaged 166 μm 2 ± 75 (P < 0.05) (Fig. 4C). 195 These studies demonstrate that exogenous GXM administration increases the permeability of the 196 CNS to C. neoformans invasion and colonization.  To validate the results obtained in the murine model and to determine the impact of GXM on TJs, 217 we analyzed the distribution of TJs (claudin-5 and occludin) in HBECs after exposure to the 218 capsular component for 4 h using IF microscopy (Fig. 6). The distribution of claudin and occludin 219 on HBECs is considerably reduced after incubation with 10 μg/mL of GXM (Fig. 6A).

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Ethylenediaminetetraacetic acid (EDTA; 10 μg/mL) was used as a positive control [24] and, 221 similarly to GXM, substantially decreased the distribution of TJs on the surface of HBECs.

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Quantification of claudin-5 intensity on HBECs using the NIH ImageJ software demonstrated that 223 GXM (P < 0.01) and EDTA (P < 0.0001) significantly reduced this TJ intensity relative to 224 untreated control HBECs (Fig. 6B). There were no differences in the distribution of claudin-5 in 225 HBECs incubated with EDTA or GXM. Occludin intensity was also significantly decreased in 226 HBECs cultured with GXM (P < 0.05) or EDTA (P < 0.0001) compared to untreated cells (

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A previous study demonstrated that C. neoformans activates RhoGTPases to promote 233 transmigration across a HBEC monolayer in vitro, which is the critical step for cryptococcal brain 234 infection and development of cryptococcal meningoencephalitis (CME) [25]. To confirm that TJ 235 integrity is disrupted by GXM, levels of both RhoA (pan) and RhoA-GTP (activated form) were 236 measured in lysates of HBECs by WB analysis (Fig. 7). HBECs (n = 3) were plated in 24-well 237 plates and grown to confluency prior to stimulation with 10 µg/mL of GXM over time (0, 15, 30, 238 60, 120, and 180 min; Fig. 7A). RhoA activation was induced immediately upon stimulation by 11 239 GXM, reaching a peak 6-fold increase after 30 min (Fig. 7B). Interestingly, these levels dropped 240 to near-basal levels after 60 min before decreasing below basal levels at 180 min. RhoA activation 241 is associated with increased cell permeability and cytoskeletal fiber formation [19], and its 242 involvement in modulating TJ integrity is of interest during CME. Actin was used as a control,  with 50 (P < 0.05) and 100 (P < 0.01) μg/mL of GXM. Also, BBBs cultured with 100 μg/mL of 12 262 GXM exhibited lower TEER percentage than BBBs exposed to 10 μg/mL of the fungal 263 polysaccharide (P < 0.01). BBBs treated with 10 μg/mL of GXM maintained ~ 60% TEER   be retained in organ tissues for weeks, especially those of the liver and spleen [29]. Surprisingly, 312 we also detected low levels of GXM in brain tissue, validating that this delivery was appropriate 313 to study the impact of the CPS on the development of cryptococcosis.  Notably, the cortex and hippocampus of GXM-challenged mice were the most affected regions of 361 the brain, suggesting that these animals may also show behavioral and cognitive impairment. In 362 fact, altered mental status in HIV + patients with CME is associated with high mortality rates [45,  In conclusion, we demonstrated that exogenous GXM administration exacerbates lung        Slides were examined by confocal microscopy as described above and analyzed using NIH ImageJ    water. SMA that achieved relaxation to ACh were considered to have a preserved endothelium.