Critical contribution of endothelial protein C receptor in experimental 2 malaria-associated acute respiratory distress syndrome 3 4

Plasmodium falciparum malaria severity is associated with parasite cytoadherence, but limited knowledge about its effect in malaria-associated acute respiratory distress syndrome (ARDS) is notorious. Our objective was to evaluate cytoadherence of infected red blood cells (iRBC) in a murine model of ARDS and appraise the role of endothelial protein C receptor (EPCR) in ARDS pathogenesis. Lungs from ARDS-developing mice showed evidences of iRBC accumulation in lungs besides increase of EPCR and TNF concentrations. Furthermore, TNF increased iRBC adherence in vitro. Dexamethasone-treated infected mice showed lower levels of TNF and EPCR mRNA expression and, finally, decreased the vascular permeability, protecting mice from ARDS. In addition, EPCR knockdown decreased the capacity of iRBC adherence in vitro. In conclusion, we identified that increased iRBC cytoadherence in lungs underlies malaria-associated ARDS in DBA/2 infected mice and that inflammation increased cytoadherence capacity through EPCR expression suggesting a potential target for drug development.

5 96 directly relevant to the complications associated with severe malaria [22]. Indeed, in 97 vitro studies with purified CIDRα1 domains and iRBCs with selected P. falciparum 98 laboratory strains confirm the loss of EPCR functionality upon CIDRα1 or iRBC 99 binding that includes loss of PC and APC binding to EPCR, inhibition of EPCR-100 mediated PC activation and obstruction of APC-mediated endothelial barrier protective 101 effects. Consequently, the binding of PfEMP1 to EPCR results in an acquired functional 102 PC system deficiency which supports new evidences that EPCR plays a central role in 103 the pathogenesis of severe malaria [22]. EPCR binding is mediated through the amino-104 terminal cysteine-rich interdomain region (CIDRa1) of DC8 and group PfEMP1 105 subfamilies, and that CIDRa1 interferes with protein C binding to EPCR [23]. 106 However, it has unexpectedly been shown that parasitized erythrocytes expressing the 107 DC13 HB3var03 or IT4var07 variants of PfEMP1 do not bind to the EPCR of brain 108 endothelial cells in vitro. On the other hand, it has been evidenced that the DC8 variant 109 IT4var19 may bind to the EPCR, but this interaction was inhibited when standard 110 human serum or plasma was added to the assays. Therefore, the discrepancy in the 111 binding activity of the EPCR and the recombinant PfEMP1 proteins indicates the need 112 for further studies to understand the current physiological significance resulting from 113 this PfEMP1-EPCR interaction [24]. 114 Although there are some suggestions that PfEMP1 can bind to EPCR with important 115 effects on the severe malaria, these data are controversial, and, in addition, there are any 116 evidences of P. berghei ANKA binding on EPCR and the effects of EPCR on ARDS 117 pathogenesis must be elucidated. Our study herein was developed in a murine model, In our previous study, it was shown that a proportion of DBA/2 mice infected with 128 PbA-iRBC developed singular characteristics of ARDS and died between the 7 th and 129 12 th dpi [12][13][14][15]. To investigate if adherence is essential in the evolution of this 130 phenotype, we observed that PbA-luciferase-infected erythrocytes were distributed in 131 the peripheral blood and tissues of DBA/2 mice. However, when they were perfused 132 with PBS 1x, the bioluminescence (luciferase/luciferin) signal remained concentrated 133 in spleen and lungs, especially in ARDS-developing mice (Fig 1 A-C), corroborating 134 with our published data [14]. We demonstrated that ARDS-developing mice showed 135 higher levels of 18s subunit PbA rRNA expression ( Fig 1D) and higher hemozoin 136 concentration in the lungs compared to HP-developing mice, on the 7 th dpi (Fig 1 E-G). 137 In addition, we analyzed histological lung section of ARDS-developing mice, finding 138 several iRBC in close contact with endothelial cells (Fig 1H). These results together 139 indicated that ARDS-developing mice accumulated a considerable amount of infected The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/348318 doi: bioRxiv preprint 7 144 ARDS-developing mice, on the 7 th dpi, displayed higher TNF levels in serum compared 145 to HP-developing mice (Fig 2A), suggesting that this inflammatory cytokine may be 146 critical to ARDS development. For this, we further investigated if iRBC could 147 contribute to TNF release by endothelial cells. First, we demonstrated that PMLEC 148 stimulated with iRBC can directly contribute to TNF production ( Fig 2B). Then, we 149 checked the influence of TNF in adhesion of iRBC in PMLEC through static and flow 150 conditions. It was observed that TNF-stimulated cells increased the capacity of iRBC 151 adherence into PMLEC after 24 and 72 hours in static assay (Fig 2 C-E). In the flow 152 adherence assay, which mimics the physiologic conditions, the iRBC had more 153 cytoadherence with TNF than without TNF stimulation (Fig 2 F-H). In addition, 154 PMLEC were seeded in transwell plates in indirect contact with peritoneal macrophages 155 (M), stimulated or not by red blood cells (RBC) or iRBC (Fig 2I). M stimulated by 156 iRBC produced more TNF than M without previous contact with iRBC ( Fig 2J). These 157 M also produced IFN- and IL-10 but with no difference between groups (data not 158 shown). Additionally, PMLEC that was submitted to indirect contact with M 159 stimulated with iRBC demonstrating more adherence of iRBC than PMLEC with no 160 previous contact with M ( Fig 2K). Finally, in order to evaluate the contribution of 161 TNF in the adhesion of iRBC in PMLEC, cells were blocked with TNF antibody and, as 162 a result, the blockage reduced the capacity of iRBC to adhere in PMLEC ( Fig 2L) Looking for adhesion molecules that could be involved in PbA-iRBC cytoadherence in 168 ARDS pathogenesis, we observed that ARDS-developing mice showed higher levels of 169 VCAM and ICAM-1 in lungs than HP-developing or non-infected mice (NI), analyzed 170 by immunohistochemistry (Fig S2 A-C). Also, recombinant TNF upregulated the 171 mRNA expression of ICAM-1 and VCAM in PMLEC (Fig S2 D-I). 172 However, the most interesting findings were the mRNA EPCR expression upregulated 173 in the lungs of ARDS-developing mice compared to NI mice (28.48-fold increase) and 174 HP-developing mice (13.16-fold increase) (Fig 3A). There was also an increase in   The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/348318 doi: bioRxiv preprint 9 192 4B) and sEPCR concentration in serum and bronchoalveolar lavage (BAL) (Fig 4C and 193 D). VEGF concentration, an essential factor to increase vascular permeability and 194 ARDS development, also decreased after dexamethasone treatment in the BAL of 195 infected mice, on the 7 th dpi ( Fig 4E). 196 Consequently, we analyzed the lung vascular permeability and dexamethasone-treated 197 infected mice showed to be protected compared to non-treated mice (Fig 4F-H). To   Additionally, our findings provide the first evidence that PbA-iRBC adhere more in 256 ARDS-developing mice than in those HP-developing. Moreover, we found that ARDS-257 developing mice have about 28.5 times more EPCR expression than those not infected, 258 implying that EPCR can be essential in ARDS progress. Finally, we demonstrated that 259 TNF-stimulated cells induced EPCR expression while iRBC did not.

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Previously, we had found that ARDS-developing mice showed more parasites load in 261 lungs, which can be important to induce the syndrome, than those HP-developing. On The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/348318 doi: bioRxiv preprint 12 266 lungs of DBA/2 mice, especially in those ARDS-developing, when the organs were 267 perfused we confirmed that the PbA-iRBC was adhered mainly in the lungs and spleen.

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On the other hand, in our experimental model, there was no adhesion in the brain as in 269 other models of ARDS [11,26].

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With the hypothesis that inflammation is essential to expose adherence molecules and 290 consequently ARDS pathogenesis, we treated DBA/2 mice with dexamethasone to . CC-BY 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/348318 doi: bioRxiv preprint 13 291 reduce inflammation, observing that these mice were protected from ARDS and had a 292 downregulation in EPCR expression, which indicates that EPCR may be important to 293 iRBC adherence and to the pathology progress of the disease. However, in a recent 294 study, the expression of EPCR decreased in the lungs of P. falciparum-infected patients 295 who developed ARDS when compared to those who did not develop, analyzed by

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The chamber was initially filled with DMEM medium, then an initial photo with the 506 parasites adhered to the CEPP-DBA/2 was taken, after, a medium continuous stream 507 was run through the chamber and maintained at 2 ml/hour with a pump syringe (Insight 508 Inc). Five images, every 3 minutes, of each well were captured, maintaining the same 509 field as the initial image, to determine the erythrocyte binding efficiency.      The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/348318 doi: bioRxiv preprint 25 588 membrane was then incubated with the primary anti-EPCR antibody (# 151403, abcam) 589 1: 1,000 stirring overnight at 4°C. After washing in TBS-T, the membrane was 590 incubated with the peroxidase-conjugated rabbit anti-rabbit IgG (HRP) secondary 591 antibody (# AP307P, Millipore) at 1: 6,000 concentration in TBS-T for 1 hour (RT).