Placental malaria is associated with a TLR–Endothelin-3–oxidative damage response in human placenta tissues

22 Placental malaria, which is mainly caused by the sequestration of Plasmodium falciparum-23 infected erythrocytes in the placenta, is an important driver of poor pregnancy outcomes, 24 including fetal growth restriction, preterm birth, and stillbirth. However, the mechanisms 25 underlying its adverse outcomes are unclear. Mouse models have previously shown that 26 placental malaria (PM) triggers a proinflammatory response in the placenta, which is 27 accompanied by a fetal Toll-like receptor (TLR)4-mediated innate immune response associated 28 with improved fetal outcomes. Here, we used hematoxylin and eosin staining to identify PM-29 positive and negative samples in our biobank of placentas donated by women living in a 30 malaria-endemic region of Kenya and assessed the impact of PM on the expression of TLRs, 31 Endothelins, and oxidative damage. RT-qPCR analysis revealed that PM was associated with 32 an upregulation of TLR4, TLR7, and Endothelin-3. Moreover, immunohistochemistry showed 33 that PM was associated with elevated expression levels of the oxidative DNA damage marker, 34 8-hydroxy-2’-deoxyguanosine, while RT-qPCR revealed that this was accompanied by an 35 upregulation of p21, an inhibitor of cell cycle progression and marker of cellular response to 36 DNA damage. These findings allude to a novel mechanism of PM pathogenesis driven by a 37 TLR–Endothelin-3–oxidative DNA damage signaling axis. 38 39

Placental malaria, malaria in pregnancy, poor pregnancy outcomes, innate 40 immunity, Plasmodium falciparum 41 42 43 44 45 46 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 3 1. Introduction 47 According to the World Health Organization, globally, there were about 249 million malaria 48 cases and 608,000 malaria-associated deaths in 2022, with sub-Saharan Africa accounting for 49 most of the cases and deaths [1]. Pregnant women have a higher susceptibility to malaria 50 infection [2] and it is estimated that in 2022, there were about 12.7 million cases of malaria in 51 pregnancy (MiP) in sub-Saharan Africa [1]. MiP is associated with several adverse outcomes on 52 the mother, the fetus, and neonate [3]. For the fetus, MiP severely worsens pregnancy 53 outcomes and frequently leads to fetal growth restriction (including low birthweight, small for 54 gestational age, and intrauterine growth restriction) and may result in preterm birth and stillbirth 55 [3–5]. 56 The adverse effects of MiP on the fetus are attributable to malaria infection of the placenta [1], 57 leading to placental malaria (PM). PM is characterized by the sequestration of Plasmodium-58 infected erythrocytes in placental intervillo us spaces. This phenomenon is most frequently 59 associated with Plasmodium falciparum (P. falciparum), the species associated with the most 60 severe form of malaria [6]. The sequestration of P. falciparum-infected erythrocytes to the 61 placenta is mediated by the interaction between variant surface chondroitin surface antigen 2, a 62 Plasmodium falciparum protein expressed on the surface of infected erythrocytes [7], and 63 chondroitin sulfate A on the surface of the syncytiotrophoblast [7], the placental epithelial cell 64 layer that contacts maternal blood [8]. 65 The adverse impacts of PM on fetal well-being most likely results from the negative effects of 66 PM on placental health and function since the vertical transmission of malaria to the fetus is rare 67 [9]. Indeed, PM is reported to induce placental inflammation [10,11] and placental histological 68 changes [12], which may contribute to placental insufficiency and poor pregnancy outcomes. 69 However, the mechanisms underlying PM-driven placental pathobiology are not fully understood 70 at the cellular and cell signaling levels. 71 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 4 Innate immune factors, such as Toll-like receptor (TLR)4, 7, and 9, which respond to infection by 72 recognizing and clearing invading pathogens are reported to respond to malaria infection [13], 73 although their role in PM is unclear. Although several studies have investigated maternal 74 responses to PM, few have studied how the fetus responds to parasite sequestration in the 75 placenta. Nonetheless, a mouse model of PM revealed that PM triggers a TLR4-mediated 76 innate immune reaction that adversely affects fetal outcomes, which is countered by a fetal 77 innate immune reaction that led to better pregnancy outcomes [14]. This suggests the presence 78 of TLR-mediated innate immune responses to PM, although this has not been reported in the 79 context of human PM. Here, considering that mouse data show that TLR4 modulates 80 endothelin-1 expression [15], malaria is inflammatory and oxidative [16], oxidative DNA damage 81 upregulates TLR4 [17], and TLR signaling is thought to promote DNA repair [18], we used 82 biobank placenta samples donated by women living in a malaria-endemic region of Kenya to 83 examine the hypothesis that human PM triggers a TLR–Endothelin–oxidative damage signaling 84 response. 85 2. Materials and methods 86 2.1 The biobank and study participants 87 The study used biobank placenta samples donated by residents of Bungoma County, a malaria-88 endemic region of Western Kenya. The biobank was established by a previous prospective 89 parent study. All placenta donors were aged ≥ 18 years and gave written informed consent to 90 participate in the parent study. Based on questionnaire responses, participants with a known 91 record of sexually transmitted disease infection during pregnancy, those with pregnancy-92 associated noncommunicable diseases (preeclampsia and gestational diabetes) during the 93 current pregnancy, and those with twin pregnancies were excluded from our analyses. During 94 participant recruitment and sample collection, participants were recorded as having MiP if they 95 had at least one episode of hospital-diagnosed malaria during pregnancy. Maternal malaria 96 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 5 status was diagnosed using a rapid diagnostic test (Malaria Ag P.f, SD Biosensor). All data 97 underlying the databank were deidentified. The characteristics of the biobank’s placenta donors 98 and samples are summarized in Table 1. Equal numbers of male and female placentas were 99 analyzed. Demographic data (Table 1), including maternal age, history of malaria during 100 pregnancy, and gravidity were collected using questionnaires, whereas pregnancy-associated 101 data, including birthweight and placental weight, were recorded after birth. All participants gave 102 written informed consent before joining the study and agreed to the collection and use of their 103 placenta samples in the study. 104 2.2 Histological analysis 105 Formalin-fixed placenta tissues were embedded in paraffin blocks as previously described [19] 106 using an automated tissue embedding system (MediMeas). H&E analysis was used to confirm 107 the presence of PM, which is indicated by the presence of infected erythrocytes in the placenta. 108 Briefly, formalin-fixed paraffin-embedded samples were sectioned onto charged microscope 109 slides (Dako, Cat No. K8020) at a 5-µm thickness, dried at 37 °C overnight on a slide warmer, 110 dewaxed in xylene (Finar Chemicals, Cat No. 21940LC250), rehydrated by dipping across an 111 alcohol gradient of absolute, 95%, 70%, and 50% ethanol (Scharlau, Cat No. ET00052500), and 112 then in distilled water. They were then submerged in hematoxylin (Loba Chemie, Cat No. 113 04023) for seven minutes, rinsed with running water, and then destained through 10 dips in acid 114 alcohol (1% hydrochloric acid in 70% ethanol). Next, they were submerged in eosin (Griffchem, 115 Cat No. 45380) for 45 seconds followed by dehydration in 95% ethanol and absolute ethanol 116 (five minutes each) and then cleared in xylene baths (10 minutes each) before being cover-117 slipped using dibutylphthalate polystyrene xylene mountant (Finar Chemicals, Cat No. 118 10525LM250). The slides were then examined under a microscope (Richter Optica UX1, M2 119 Scientifics), followed by imaging in ≥ 10 fields of view per slide at a 40X magnification using a 120 Moticam microscope camera (Motic Scientific). PM was then diagnosed as described before 121 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 6 (Odongo et al., 2016) based on the presence of infected erythrocytes. PM-associated 122 histopathological features were assessed by counting the number of syncytial knots as 123 described previously [20] and measuring the fibrin-occupied placental areas using imageJ. 124 These analyses were done in at least 10 fields of view per sample. The levels of PM burden in 125 the PM-positive samples were determined by counting the number of identified infected 126 erythrocytes in at least 10 fields of view per slide imaged at a magnification of 40X. 127 2.4 RNA extraction and reverse transcription quantitative PCR (RT-qPCR) 128 Total RNA was extracted from placenta tissue using a HigherPurity™ Tissue Total RNA 129 Purification kit as per the manufacturer’s guidelines (Canvax, cat No. AN0152) and quantified 130 using a NanoDrop Microvolume Spectrophotometer (ThermoFisher Scientific) following the 131 manufacturer’s instructions. For each sample, 500 ng of RNA were retrotranscribed into cDNA 132 using a LunaScript™ RT SuperMix cDNA Synthesis Kit (NEB, Cat. No. E3010L) using the 133 manufacturer’s protocol. RT-qPCR analysis was done on a QuantStudio™ 5 Real-Time PCR 134 System in a final volume of 20 µl containing 10 µl of GoTaq qPCR Master Mix (Promega, Cat 135 No. PRA6001), 2 µl of the forward plus reverse primers (final primer concentration: 500 nM), 3 136 µl of nuclease free water (Promega, Cat No. P119E), and 5 µl of cDNA using the following 137 program: 50 °C for two minutes, 95 °C for 10 minutes, followed by 40 cycles at 95 °C for 15 138 seconds and 60°C for 30 seconds. Relative gene expression was determined using the 2 -ΔΔ ct 139

[21], using β -actin as the reference gene. Primers were purchased from Macrogen and 140 primer sequences are provided in Table 3. 141 2.5 P. falciparum detection PCR 142 The presence of P. falciparum in placenta samples was evaluated using One Taq® Quick-143 Load® 2X Master Mix with Standard Buffer (NEB, Cat No. M0486L) and the following 144 thermocycler program: Initial denaturation at 95 °C for five minutes, followed by 35 cycles of 145 denaturation at 95 °C for 30 seconds, annealing at 55 °C for 60 seconds, and extension at 72 146 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 7 °C for 75 seconds, and then a final extension at 72 °C for five minutes. Primer sequences are 147 shown in Table 3. The PCR product was subjected to agarose (Sigma–Aldrich, Cat No. A9539) 148 gel electrophoresis using 1X tris–borate–EDTA buffer alongside a 100 base pair ladder using 149 SafeView™ Classic (Applied Biological Materials, Cat No. G108) nucleic acid stain and Gel 150 Loading Dye, Purple (6X) (NEB, Cat No. B7024S). The bands were developed and imaged 151 using a UVITEC Gel Documentation System (Cleaver Scientific). 152 2.6 Immunohistochemistry 153 The sections were deparaffinized by warming at 55 °C for 15 minutes followed by dipping in 154 three xylene baths, about 10 dips each. They were then rehydrated and subjected to heat-155 induced epitope retrieval by boiling for 30 minutes in Citrate Buffer, pH 6.0 (Sigma–Aldrich, cat. 156 No. C9999). They were then cooled to room temperature, rinsed with distilled water for five 157 minutes and then blocked with 0.3% Triton-X in 1X phosphate-buffered saline (PBST). Next, 158 they were blocked with 10% normal donkey serum (Abcam, cat. No. ab7475) in PBST for two 159 hours followed by overnight incubation (4 °C) with anti-DNA/RNA damage antibody [15A3] 160 (Abcam, cat. No. ab62623) at 1:2500 in blocking solution. Sections were then washed thrice (10 161 minutes each) using PBST and then incubated at room temperature for two hours with 162 horseradish peroxidase-c onjugated goat anti-mouse se condary antibody (Jackson 163 ImmunoResearch, cat. No. 115-035-003) at 1:5000 in blocking solution. The sections were then 164 washed thrice (10 minutes each) using PBST followed by signal development using an 165 ImmPACT® DAB Substrate Kit (Vector, cat. No. SK-4105) as per the manufacturer’s protocol. 166 They were then dehydrated using 95%, 95%, 100%, and 100% ethanol (five minutes each), 167 cleared by dipping in three xylene baths and then cover-slipped using a xylene-based mountant 168 and allowed to dry. They were then examined under a light microscope and imaged at a 169 magnification of 40X. 170 2.7 Data analyses 171 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 8 Statistical analyses were done using GraphPad Prism version 9. Data are presented as 172 percentages, raw values, or mean ± standard deviation. Differences between two groups were 173 compared using a t-test. Correlation analyses were done using nonparametric Spearman 174 correlation analysis. For each placenta, the PM burden in placentas was indicated by the total 175 number of infected erythrocytes in the placenta section. The birthweight-to-placenta weight 176 (BW:PW) ratio was obtained by dividing the birthweight (grams) with the corresponding 177 placenta’s weight (grams). The correlation between PM status and birthweight, placental weight, 178 and birthweight-to-placental weight ratio was assessed using GraphPad prism to examine the 179 impact of PM on fetal outcomes. P < 0.05 indicates statistically significant differences. 180 3. Results 181 3.1 Main characteristics of the placenta donors and donated placentas 182 All placenta donors were ≥ 18-years-old and had received intermittent presumptive treatment 183 with sulfadoxine pyrimethamine. The mean age, gravidity, birthweight (BW), placental weight 184 (PW), and BW:PW ratio of the cohort of placenta donors was 24.7 years, 2.69, 3077.64 g, 185 470.04 g, and 6.51, respectively (Table 1). Grouping the placenta donors into those with a 186 known history of MiP and those without (NoMiP), revealed that in the MiP vs NoMiP groups, 187 maternal age and gravidity were not significantly different (mean age: 24.4 [range: 18–30] vs 25 188 [range: 18–40] years, P = 0.45; mean gravidity: 2.5 [range: 1–7] vs 2.9 [range: 1–7), P = 0.12). 189 However, in the MiP vs NoMiP groups, BW (mean: 2870.5 [range: 1600–5000] vs 3272.3 190 [range: 2000–4500) g), PW (mean: 464.3 [range: 280.4–675) vs 492.9 [range: 342.3–715] g, 191 and BW:PW ratio (mean: 6.27 [range: 3.2–10.2] vs 6.73 [range: 4.18–10], were significantly 192 lower in the MiP group ( P < 0.0001, = 0.009, and = 0.03, respectively). H&E analysis revealed 193 that 92 placentas (51.4%) were PM-positive (had infected erythrocytes), 58 (32.4%) were PM-194 negative (no infected erythrocytes observed), 29 (16.2%) had past malaria infection (hemozoin 195 present in the absence of infected erythrocytes [12]), 92 (51.4%) belonged to male fetuses, and 196 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 9 eight (4.5%) were preterm (Table 2). All downstream analyses were done on placenta samples 197 that were confirmed to be PM-positive or PM-negative using H&E staining. 198 3.2 PM correlates negatively with birthweight and birthweight-to-placenta ratio 199 Representative images of PM-negative and PM-positive samples are shown on Figure 1A–B, 200 and the presence of P. falciparum in PM-positive tissues was confirmed using PCR (Figure 1C). 201 Analysis of the data underlying the placental biobank revealed that when compared with the 202 PM-negative group, BW was significantly lower in the PM-positive group, which had more low 203 BW cases (Figure 1A, P = 0.03, low birthweight: <2500 g), but PW did not differ between the 204 two groups (Figure 2B, P = 0.8). However, the BW:PW ratio was lower in the PM-positive group, 205 although the difference did not reach statistical significance (Figure 1F, P = 0.08). Next, we used 206 the H&E images to determine the proportion of infected erythrocytes, IEs (%), in each placenta 207 sample, and then used the obtained values to assess the correlation between the PM burden 208 and BW, PW, and the BW:PW ratio, i.e., the fetal weight obtained per gram of the placenta, 209 which is an indicator of placental efficiency, with higher BW:PW ratios indicating greater 210 placental efficiency [22]. This analysis revealed negative correlation between IEs (%) (the 211 proportion of infected erythrocytes), and BW (correlation coefficient [rs]: -0.22, P < 0.005, 95% 212 confidence interval [CI]: -0.359 – -0.071), and IEs (%) and BW:PW ratio (rs: -0.20, P = 0.007, 213 95% CI: -0.338 – -0.048). Expectedly, PW had a positive correlation with birthweight (rs: 0.29, P 214 < 0.001, 95% CI: 0.141 – 0.419) and a negative correlation with BW:PW ratio (rs: -0.42, P < 215 0.001, 95% CI: -0.539 – -0.290). However, the PM burden did not exhibit correlation with 216 placental weight (rs: 0.01, P = 0.94, 95% CI: -0.156 – 0.146). Taken together, these findings 217 indicate that PM impairs placenta function, leading to low birthweight via reduced placenta 218 efficiency as indicated by the negative correlation between PM burden and the BW:PW ratios of 219 PM-exposed neonates. 220 3.3 PM markedly alters placental histological features 221 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 10 Next, we assessed the impact of PM on syncytial knotting and fibrin deposition in our placenta 222 samples. This analysis revealed that when compared with PM-negative samples (A), PM-223 positive samples had more syncytial knots (B, yellow arrowhead) and greater fibrin-occupied 224 placental area (C, FD in the broken line-demarcated area). Quantification analyses revealed 225 that when compared with PM-negative samples, the levels of these histological features were 226 significantly higher in the PM-positive samples (D, SK: syncytial knots, P = 0.047 and E, fibrin 227 area, P < 0.0005). These observations indicate that in our study cohort, PM may have adversely 228 affected fetal outcomes at least in part, by altering normal placental histological features. 229 3.4 PM is associated with an upregulation of TLR4, TLR7, and Endothelin 3 230 We then sought to determine if PM altered the expression of TLRs. To this end, we focused on 231 TLR4, TLR7, and TLR9, which have been associated with response to malaria infection in mice 232 [23,24], and with mouse PM in the case of TLR4 [25], although this has not been reported in 233 human PM. To evaluate the effect of PM on these innate immune system receptors, we 234 assessed their expression levels using RT-qPCR. The analysis revealed that when compared 235 with PM-negative controls, PM-positive samples expressed significantly higher levels of TLR4 236 and TLR7, but not TLR9 (Figure 3A–C, P = 0.002, 0.03, and 0.59, respectively). This is 237 consistent with mouse data showing that PM upregulates TLR4-mediated expression of 238 endothelin-1 [15]. We therefore wondered if human PM alters the expression of Endothelin 239 genes. RT-qPCR analysis of Endothelin-1 and -3 gene expression revealed that only 240 Endothelin-3 was detectable in our placenta samples and that when compared with PM-241 negative placentas, PM-positive samples had significantly higher levels of Endothelin-3 (Figure 242 3D, P = 0.004), indicating the presence of a TLR–Endothelin signaling axis in response to 243 human PM. 244 3.5 PM is associated with high oxidative DNA damage 245 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 11 Since TLR4 was upregulated in PM-positive placenta samples, we wondered whether PM-246 driven activation of TLR4 is associated with a dysregulation of other signaling processes that 247 may underlie or contribute to PM-mediated placental pathobiology. Because malaria is known to 248 be strongly inflammatory and oxidative, which may drive host tissue damage [16], and because 249 oxidative DNA damage is associated with TLR4 upregulation [17] and that TLR signaling is 250 thought to promote DNA repair [18], we wondered if the TLR4 upregulation in the PM-positive 251 samples might be associated with placental oxidative DNA damage. To assess this possibility, 252 we used immunohistochemistry to assess the levels of 8-hydroxy-2’-deoxyguanosine, a marker 253 of oxidative DNA stress [26]. This analysis revealed that when compared with PM-negative 254 samples, PM-positive samples express markedly higher levels of 8-hydroxy-2’-deoxyguanosine 255 (Figure 4A). Staining the same samples with the secondary antibody only (without the primary 256 antibody) confirmed signal specificity (Figure 4B). To further assess the effect of PM on 257 oxidative stress, we used RT-qPCR to examine the level of p21, a mediator of cell cycle arrest 258 and indicator of cellular response to DNA damage [27]. This revealed that when compared with 259 PM-negative samples, PM-positive samples had significantly higher levels of p21 (Figure 4C, P 260 = 0.02). Taken together, these data indicate that PM triggers markedly high levels of placental 261 oxidative DNA stress, placenta tissue damage, and cellular response to DNA stress, which may 262 contribute to the pathobiology of PM, and that in response, at least in part, TLR signaling may 263 be upregulated to counter these adverse effects through promotion of DNA repair. 264 4. Discussion 265 Malaria in pregnancy (MiP) often results in placental malaria (PM), where erythrocytes that are 266 infected with P. falciparum , the parasite that most frequently causes PM, sequestrate in 267 placental intervillous spaces [7]. PM may cause various adverse fetal outcomes, stillbirth, 268 preterm birth, and fetal growth restriction [3–5] and because P. falciparum rarely undergoes 269 vertical transmission [9], these effects are likely caused by PM-driven pathobiological effects in 270 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 12 the placenta, which may impair placental function. However, although studies have implicated 271 effects like inflammation and histological changes in PM pathogenesis, the mechanisms 272 underlying the adverse effects of human PM on the placenta are unclear. Moreover, because 273 many MiP cases in malaria-endemic regions are asymptomatic [28,29] and the placenta is 274 inaccessible during pregnancy, there are no ways of detecting and intervening against PM 275 during pregnancy. Thus, there is an urgent need to better understand the placental pathobiology 276 of PM to guide the development of effective diagnostic and therapeutic tools. 277 Mouse models indicate that PM triggers innate immune responses (mainly via TLR4) that are 278 associated with poor fetal outcomes and that TLR4-mediated fetal responses to PM lead to 279 improved outcomes [14]. However, the effect of human PM on TLR-mediated immunity in the 280 placenta has not been examined. In this study, we leveraged our well-characterized biobank of 281 placenta samples from a malaria endemic region of Kenya (Table 1) and found that in our study 282 cohort, PM burden had a significant negative correlation with birthweight and BW:PW ratio, that 283 it was associated with significantly higher placental histological lesions, higher levels of TLR4 284 and Endothelin-3 expression, and enhanced oxidative DNA damage when compared with 285 samples without PM. 286 Consistent with previous findings implicating PM in fetal growth restriction [30], we observed 287 that in our study cohort, relative to the PM-negative cases, PM was associated with low 288 birthweight. Moreover, we observed that PM was associated with lower BW:PW ratios, an 289 indicator of placental efficiency in which higher ratios indicate higher nutrient transfer for every 290 gram of placenta and vice versa [22], an observation that to our knowledge, has not been 291 previously reported in human PM, but not with lower placental weight (Figure 1D–F). 292 Interestingly, our analyses also indicate that the PM burden (percentage of infected erythrocytes 293 in a sample’s intervillous spaces) correlates negatively with birthweight but not with placental 294 weight. Taken together, these observations indicate that PM contributes to fetal growth 295 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 13 restriction primarily by impairing placental function and not via placental growth inhibition, 296 although the precise mechanisms remain unclear. This possibility is crucial considering that 297 women in malaria-endemic regions may experience multiple malaria reinfections throughout 298 pregnancy, but further studies, such as using in vitro and organoid systems, are needed to 299 comprehensively investigate this possibility. 300 Our analyses revealed that, PM-positive samples had markedly higher levels of fibrin deposition 301 and syncytial knotting than PM-negative samples, which is in line with earlier findings [31]. 302 These changes, which indicate placental injury and have been associated with placental 303 malperfusion and poor fetal outcomes, including fetal growth restriction [32,33], may contribute 304 to the low birthweight observed in our PM cohort. However, studies are needed to establish the 305 mechanisms by which PM alters placental histological features, how these changes correlate 306 with fetal outcomes and postnatal wellbeing, and whether they can predict fetal wellbeing in 307 postnatal life. 308 TLRs are key innate immunity factors that sense host invasion by pathogens and activate host 309 immune defenses [34]. Mouse models of malaria indicate that TLR4, TLR7, and TLR9 are 310 involved in detecting and responding to malaria infection [23,24]. Moreover, mouse models 311 indicate that at the fetal–maternal interface, PM activates TLR4-mediated immune responses 312 that drive poor fetal outcomes, and that fetal TLR4-mediated counterresponses improve 313 pregnancy outcomes [14,25,35]. However, this observation has not been previously made in 314 human PM. Here, we observed that the expression levels of TLR4 and TLR7, but not TLR9, 315 were significantly upregulated in PM-positive samples, indicating that placental infection triggers 316 an innate immune reaction and that it is mainly driven by TLR4 and TLR7, although the status of 317 other TLRs during PM warrants investigation. Considering that TLRs are important drivers of 318 inflammation [36], which is implicated in PM pathogenesis [37], taken together with the 319 observed changes in placental histological features, our findings indicate for the first time, that 320 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 14 TLR-mediated responses to PM may contribute to local placental inflammation, which may 321 underlie the observed PM-associated low birth and placental weights, although the precise 322 mechanisms remain unclear. Mouse data show that PM-driven TLR4 expression drives 323 placental endothelin-1 expression [15], and for the first time, our findings show that PM is also 324 associated with the upregulation of Endothelin-3. The Endothelin ligands 1, 2, and 3 are a family 325 of vasoactive factors that influence a range of cellular processes, such as vascular remodeling 326 and angiogenesis [38]. Moreover, Endothelin-3 has been reported to have anti-inflammatory 327 effects [39,40], suggesting that its upregulation in the context of PM-mediated TLR4 328 upregulation is a mechanism of countering TLR4-dependent placenta inflammation. Collectively, 329 these observations indicate that human placenta malaria may activate a TLR4–Endothelin-3 330 signaling axis, but further studies are needed to test this hypothesis and to determine its 331 implications in PM pathobiology and fetal outcomes. 332 Based on reports that malaria is strongly oxidative [16], oxidative stress causes tissue damage 333 [41], oxidative DNA damage upregulates TLR4 [17], and that the TLR pathway might promote 334 DNA repair [18], we reasoned that our observation of TLR4 and TLR7 upregulation in PM-335 positive samples might be accompanied by placental oxidative DNA damage. This hypothesis 336 was confirmed by our immunohistochemistry data, which showed that 8-hydroxy-2’-337 deoxyguanosine, a marker of oxidative DNA stress (Valavanidis et al., 2009), was markedly 338 upregulated in PM samples. Moreover, gene expression analysis revealed that these events 339 were accompanied by a significant upregulation of p21, a cell cycle inhibitor and marker of 340 cellular response to DNA damage [27]. These observations align with previous findings that in a 341 mouse model, PM is associated with placental oxidative damage [42]. Furthermore, p21 342 upregulation in the placenta may arrest the cell cycle to allow for oxidative damage resolution, 343 which may contribute to the low placental weight observed in our cohort, but this possibility 344 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 15 requires further investigation. Together, our data suggest the presence of a previously unknown 345 TLR–Endothelin–oxidative damage axis in human PM. 346 5. Conclusion 347 Despite its heavy burden and adverse effects on maternal and fetal outcomes, the mechanisms 348 underlying the placental pathobiology of PM are unclear. Considering that malaria is rarely 349 transmitted to the fetus [9], the adverse fetal outcomes of MiP are mainly driven by events that 350 disrupt placenta physiology and function. Importantly, because of the placenta’s inaccessibility, 351 PM can only be confirmed via postnatal placental histopathology. These challenges highlight the 352 urgent need to better understand the mechanisms underlying placental pathobiology of PM, 353 which may inform the development of sensitive tools for diagnosing PM during pregnancy as 354 well as effective therapeutic interventions. Our findings that PM may drive TLR-mediated 355 responses in the placenta, raise the possibility that modulating innate responses to PM may 356 improve fetal outcomes, as we previously discussed [13]. Moreover, our identification of an axis 357 involving TLRs, Endothelins, and oxidative DNA damage during PM (Figure 5), highlights 358 processes with the potential for intervention against human PM. However, further studies, such 359 as using primary human trophoblasts, human placental organoids, or human placental ex vivo 360 systems are needed to validate our observations. Such approaches can investigate the 361 mechanisms of PM pathobiology more rigorously than can be done using term placentas. 362 Funding statement 363 F.M.K. is supported by the EDCTP2 programme supported by the European Union and Novartis 364 Global Health, Basel – Switzerland, Grant Number TMA2019CDF-2736. 365 Acknowledgments 366 We thank our placenta donors and the staff at Webuye County and Mary Help of The Sick 367 (Thika) Hospitals for their generous support. We thank the staff of the histopathology and 368 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 16 anatomy departments at the University of Nairobi and Mount Kenya University for their help with 369 histopathology. We are grateful to Prof. Walter Jaoko, Prof. Omu Anzala, and Dr. Daniel Muema 370 of KAVI–ICR for kindly sharing laboratory space and resources. We are thankful to Prof. Roger 371 Smith and Dr. Kaushik Maiti, Mothers and Babies Research Centre, Hunter Medical Research 372 Institute, Newcastle, NSW – Australia, for sharing antibodies and other resources. 373 Conflict of interest 374 The authors declare no conflicts of interest. 375 Ethics statement 376 This study was approved by Mount Kenya University’s ethics review committee (approval 377 number 1314). 378 379 380 381 382 383 384 385 386 387 388 389 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 17 390 391 392 393 394 395 396 Figures 397 398 Figure 1. (A–B) Representative hematoxylin and eosin images of placental malaria (PM)-399 negative (A) and PM-positive samples (B). When compared with a PM-negative sample, the 400 positive sample has malaria-infected erythrocytes (black arrowheads) in the placental 401 intervillous space. (C) PCR confirmed the presence of P. falciparum in the PM-positive sample 402 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 18 in B. L: 100 base pair ladder, N: PM-negative sample, S: PM-positive sample in B (histology), P: 403 positive control ( P. falciparum strain 3D7 genomic DNA). Expected PCR band size: 205 base 404 pairs. (D–F) PM was associated with lower birthweight (BW) (D, P = 0.03) and lower 405 birthweight-to-placental weight (BW:PW) ratio, although the difference did not reach statistical 406 significance (F, P = 0.08), but not with lower placental weight (PW) (E, P = 0.8). In D–F, 407 whiskers are drawn from the 10 th to 90 th percentile. (G) A correlation matrix shows that the 408 proportion of infected erythrocytes, IEs (%), in the placenta correlated negatively with BW ( P < 409 0.005) and the BW:PW ratio ( P = 0.007), but it did not correlate with PW ( P = 0.94). PW 410 correlated positively with BW and negatively with the BW:PW ratio (both P < 0.001). 411 412 Figure 2 . (A–C) In the samples from the biobank underlying our study, when compared with 413 placental malaria (PM)-negative samples (A), PM was associated with significantly higher rates 414 of syncytial knots (B, yellow arrowhead) and placental fibrin deposits (C; FD, marked with 415 broken line). Black arrowheads indicate infected erythrocytes. (D–E) Quantification revealed 416 that the levels of syncytial knots (SK [D], n = 21 and 38 for PM-neg and PM-pos, respectively; P 417 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 19 = 0.047) and the area of placenta intervillous spac e containing fibrin (E, n = 26 and 38 for PM-418 neg and PM-pos, respectively, P < 0.0005) were significantly higher in the PM-positive (PM-pos) 419 samples than in the PM-negative (PM-neg) samples. Whiskers are drawn from the 10 th to 90 th 420 percentile. 421 422 Figure 3. Placental malaria (PM) is associated with the upregulation of TLR4, TLR7, and 423 Endothelin-3. (A–C) When compared with PM-negative (PM-neg) samples, PM-positive (PM-424 pos) samples expressed significantly higher levels of TLR4 and TLR7, but not TLR9 ( P = 0.002, 425 0.03, and 0.59, respectively). (D) PM-positive samples also expressed higher levels of 426 Endothelin-3 (P = 0.004). 427 428 429 430 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 20 431 Figure 4. Analysis of oxidative DNA damage in placental malaria (PM)-negative vs PM-positive 432 samples. (A–B) Immunohistochemistry revealed that when compared with PM-negative 433 samples, PM-positive tissues had markedly higher levels of 8-hydroxy-2’-deoxyguanosine (8-434 OHdG), an indicator of oxidative damage. Staining the same samples with the secondary 435 antibody only (B) confirmed signal specificity for this marker. (C) RT-qPCR showed that PM-436 positive samples express significantly higher levels of p21 (P = 0.02). 437 438 439 440 441 442 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 21 443 Figure 5. Schematic summary of the hypothesized TLR–Endothelin-3–oxidative stress axis in 444 human placental malaria. Further investigation is needed to validate this axis and determine its 445 potential for intervention against placental malaria. 446 447 448 449 450 451 452 453 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 22 Tables 454 Table 1. Summary of placenta donors’ demographics 455 Group Age (range) Gravidity (range) BW (g) (range) PW (g) (range) BW:PW (range) All donors 24.7 years (18–40) 2.69 (1–7) 3077.64 (1600–5000) 479.04 (280.37–715) 6.51 (3.2–10.2) MiP 24.4 years (18–30) 2.5 (1–7) 2870.5 (1600–5000) **** 464.3 (280.4–675) ** 6.27 (3.2–10.2) * NoMiP 25 years (18–40) 2.9 (1–7) 3272.3 (2000–4500) 492.9 (342.3–715) 6.73 (4.18–10) Analysis of the placenta donors’ data revealed that maternal age and gravidity were not 456 significantly different in the MiP (group with known history of malaria in pregnancy) vs the 457 NoMiP (group without known MiP history) groups ( P = 0.45 and 0.12, respectively), whereas 458 birthweight (BW), placental weight (PW), and BW:PW ratios were significantly lower in the MiP 459 group when compared with the No MiP group ( P = < 0.0001, 0.009, and 0.03, respectively). *, 460 **, and **** indicate P < 0.05, 0.005, and 0.0005, respectively. 461 Table 2. Main characteristics of the donated placentas 462 No. of placenta samples 179 Placental malaria-positive placenta samples (infected erythrocytes present) 92 (51.4%) Placental malaria-negative placenta samples 58 (32.4%) Placentas with past placental malaria infection (hemozoin present) 29 (16.2%) Placenta samples from male fetuses 92 (51.4%) Placenta samples from female fetuses 87 (48.6 %) Placenta samples from pre-term deliveries 8 (4.5%) The general characteristics of the placentas used in this study are summarized. Placental 463 malaria status was determined using hematoxylin and eosin (H&E) analysis. 464 465 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 23 Table 3. List of primers used in the study 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 Target Forward primer Reverse primer TLR4 5’-AGACCTGTCCCTGAACCCTAT-3’ 5’-CGATGGACTTCTAAACCAGCCA-3’ TLR9 5’-CTGCCTTCCTACCCTGTGAG-3’ 5’-GGATGCGGTTGGAGGACAA-3’ TLR7 5’-TCCTTGGGGCTAGATGGTTTC-3’ 5’-TCCACGATCACATGGTTCTTTG-3’ β-actin 5’-CATGTACGTTGCTATCCAGGC-3’ 5’-CTCCTTAATGTCACGCACGAT-3’ p21 5’-TGTCCGTCAGAACCCATGC-3’ 5’-AAAGTCGAAGTTCCATCGCTC-3’ Endothelin-3 5’-GGGACTGTGAAGAGACTGTGG-3’ 5’-AGACACACTCCTTGTCCTTGTA-3‘ P. falciparum 5′-TTAAACTGGTTTGGGAAACCAAATATATT-3′ 5 ′-ACACAATGAACTCAATCATGACTACCCGTC-3′ .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 24

481 [1] World Health Organization. World malaria report 2023. 2023. 482 [2] Rogerson SJ, Mwapasa V, Meshnick SR. Malaria in pregnancy: Linking immunity and 483 pathogenesis to prevention. American Journal of Tropical Medicine and Hygiene 484 2007;77:14–22. 485 [3] Bauserman M, Conroy AL, North K, Patterson J, Bose C, Meshnick S. An Overview of 486 Malaria in Pregnancy. Semin Perinatol 2019;43:282. 487 https://doi.org/10.1053/J.SEMPERI.2019.03.018. 488 [4] Fried M, Kurtis JD, Swihart B, Pond-Tor S, Barry A, Sidibe Y , et al. Systemic Inflammatory 489 Response to Malaria During Pregnancy Is Associated With Pregnancy Loss and Preterm 490 Delivery. Clin Infect Dis 2017;65:1729. https://doi.org/10.1093/CID/CIX623. 491 [5] Schmiegelow C, Matondo S, Minja DTR, Resende M, Pehrson C, Nielsen BB, et al. 492 Plasmodium falciparum Infection Early in Pregnancy has Profound Consequences for 493 Fetal Growth. J Infect Dis 2017;216:1601–10. https://doi.org/10.1093/infdis/jix530. 494 [6] Trampuz A, Jereb M, Muzlovic I, Prabhu RM. Clinical review: Severe malaria. Crit Care 495 2003;7:315. https://doi.org/10.1186/CC2183. 496 [7] Ayres Pereira M, Mandel Clausen T, Pehrson C, Mao Y , Resende M, Daugaard M, et al. 497 Placental Sequestration of Plasmodium falciparum Malaria Parasites Is Mediated by the 498 Interaction Between VAR2CSA and Chondroitin Sulfate A on Syndecan-1. PLoS Pathog 499 2016;12:e1005831. https://doi.org/10.1371/journal.ppat.1005831. 500 [8] Frank HG. Placental Development. Fetal and Neonatal Physiology, 2-Volume Set 501 2017:101–13. https://doi.org/10.1016/B978-0-323-35214-7.00010-X. 502 [9] Harrington WE, Duffy PE. Congenital malaria: Rare but potentially fatal. Ped Health 503 2008;2:235–48. 504 https://doi.org/10.2217/17455111.2.2.235/ASSET/IMAGES/LARGE/GRAPHIC22.JPEG. 505 [10] Fried M, Duffy PE. Malaria during Pregnancy. Cold Spring Harb Perspect Med 506 2017;7:a025551. https://doi.org/10.1101/cshperspect.a025551. 507 [11] Rogerson SJ, Brown HC, Pollina E, Abrams ET, Tadesse E, Lema VM, et al. Placental 508 Tumor Necrosis Factor Alpha but Not Gamma Interferon Is Associated with Placental 509 Malaria and Low Birth Weight in Malawian Women. Infect Immun 2003;71:267. 510 https://doi.org/10.1128/IAI.71.1.267-270.2003. 511 [12] Zakama AK, Ozarslan N, Gaw SL. Placental Malaria. Curr Trop Med Rep 2020;7:162–71. 512 https://doi.org/10.1007/S40475-020-00213-2. 513 [13] Kobia FM, Maiti K, Obimbo MM, Smith R, Gitaka J. Potential pharmacologic interventions 514 targeting TLR signaling in placental malaria. Trends Parasitol 2022;38:513–24. 515 https://doi.org/10.1016/J.PT.2022.04.002. 516 [14] Rodrigues-Duarte L, Pandya Y , Neres R, Penha-Gonçalves C. Fetal and maternal innate 517 immunity receptors have opposing effects on the severity of experimental malaria in 518 pregnancy: Beneficial roles for fetus-derived Toll-like receptor 4 and type I interferon 519 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 25 receptor 1. Infect Immun 2018;86. https://doi.org/10.1128/IAI.00708-520 17/SUPPL_FILE/ZII999092383S1.PDF. 521 [15] Pandya Y , Marta A, Barateiro A, Bandeira CL, Dombrowski JG, Costa J, et al. TLR4-522 Endothelin Axis Controls Syncytiotrophoblast Motility and Confers Fetal Protection in 523 Placental Malaria. Infect Immun 2021;89. https://doi.org/10.1128/IAI.00809-524 20/ASSET/283CE60E-5A6D-46F3-9439-525 7A889F86217A/ASSETS/IMAGES/LARGE/IAI.00809-20-F001.JPG. 526 [16] Vasquez M, Zuniga M, Rodriguez A. Oxidative Stress and Pathogenesis in Malaria. Front 527 Cell Infect Microbiol 2021;11. https://doi.org/10.3389/FCIMB.2021.768182. 528 [17] Tawadros PS, Powers KA, Ailenberg M, Birch SE, Marshall JC, Szaszi K, et al. Oxidative 529 Stress Increases Surface Toll-Like Receptor 4 Expression in Murine Macrophages Via 530 Ceramide Generation. Shock 2015;44:157–65. 531 https://doi.org/10.1097/SHK.0000000000000392. 532 [18] Harberts E, Gaspari AA. TLR Signaling and DNA Repair: Are They Associated? J Invest 533 Dermatol 2013;133:296. https://doi.org/10.1038/JID.2012.288. 534 [19] Canene-Adams K. Preparation of Formalin-fixed Paraffin-embedded Tissue for 535 Immunohistochemistry. Methods Enzymol 2013;533:225–33. 536 https://doi.org/10.1016/B978-0-12-420067-8.00015-5. 537 [20] Senagore PK, Holzman CB, Parks WT, Catov JM. Working towards a Reproducible 538

for Quantifying Placental Syncytial Knots. Http://DxDoiOrg/102350/15-08-1701-539 OA1 2016;19:389–400. https://doi.org/10.2350/15-08-1701-OA.1. 540 [21] Livak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real-Time 541 Quantitative PCR and the 2−ΔΔ CT Method. Methods 2001;25:402–8. 542 https://doi.org/10.1006/METH.2001.1262. 543 [22] Hayward CE, Lean S, Sibley CP, Jones RL, Wareing M, Greenwood SL, et al. Placental 544 adaptation: What can we learn from Birthweight:placental weight ratio? Front Physiol 545 2016;7:177721. https://doi.org/10.3389/FPHYS.2016.00028/BIBTEX. 546 [23] Zhang Y , Zhu X, Feng Y , Pang W, Qi Z, Cui L, et al. TLR4 and TLR9 signals stimulate 547 protective immunity against blood-stage Plasmodium yoelii infection in mice. Exp 548 Parasitol 2016;170:73–81. https://doi.org/10.1016/J.EXPPARA.2016.09.003. 549 [24] Baccarella A, Fontana MF, Chen EC, Kim CC. Toll-Like Receptor 7 Mediates Early Innate 550 Immune Responses to Malaria. Infect Immun 2013;81:4431. 551 https://doi.org/10.1128/IAI.00923-13. 552 [25] Barboza R, Lima FA, Reis AS, Murillo OJ, Peixoto EPM, Bandeira CL, et al. TLR4-553 Mediated Placental Pathology and Pregnancy Outcome in Experimental Malaria. Sci Rep 554 2017;7. https://doi.org/10.1038/S41598-017-08299-X. 555 [26] Valavanidis A, Vlachogianni T, Fiotakis C. 8-hydroxy-2’ -deoxyguanosine (8-OHdG): A 556 critical biomarker of oxidative stress and carcinogenesis. J Environ Sci Health C Environ 557 Carcinog Ecotoxicol Rev 2009;27:120–39. https://doi.org/10.1080/10590500902885684. 558 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 26 [27] Villeneuve NF, Sun Z, Chen W, Zhang DD. Nrf2 and p21 regulate the fine balance 559 between life and death by controlling ROS levels. Cell Cycle 2009;8:3255. 560 https://doi.org/10.4161/CC.8.20.9565. 561 [28] Salgado C, Ayodo G, Macklin MD, Gould MP, Nallandhighal S, Odhiambo EO, et al. The 562 prevalence and density of asymptomatic Plasmodium falciparum infections among 563 children and adults in three communities of western Kenya. Malar J 2021;20. 564 https://doi.org/10.1186/S12936-021-03905-W. 565 [29] Idris ZM, Chan CW, Kongere J, Gitaka J, Logedi J, Omar A, et al. High and 566 Heterogeneous Prevalence of Asymptomatic and Sub-microscopic Malaria Infections on 567 Islands in Lake Victoria, Kenya. Scientific Reports 2016 6:1 2016;6:1–13. 568 https://doi.org/10.1038/srep36958. 569 [30] Menendez C, Ordi J, Ismail MR, Ventura PJ, Aponte JJ, Kahigwa E, et al. The Impact of 570 Placental Malaria on Gestational Age and Birth Weight. J Infect Dis 2000;181:1740–5. 571 https://doi.org/10.1086/315449. 572 [31] Crocker IP, Tanner OM, Myers JE, Bulmer JN, Walraven G, Baker PN. 573 Syncytiotrophoblast degradation and the pathophysiology of the malaria-infected 574 placenta. Placenta 2004;25:273–82. https://doi.org/10.1016/j.placenta.2003.09.010. 575 [32] Lampi K, Papadogiannakis N, Sirotkina M, Pettersson K, Ajne G. Massive perivillous 576 fibrin deposition of the placenta and pregnancy outcome: A retrospective observational 577 study. Placenta 2022;117:213–8. https://doi.org/10.1016/J.PLACENTA.2021.12.013. 578 [33] Brink LT, Roberts DJ, Wright CA, Nel DG, Schubert PT, Boyd TK, et al. Placental 579 pathology in spontaneous and iatrogenic preterm birth: Different entities with unique 580 pathologic features. Placenta 2022;126:54. 581 https://doi.org/10.1016/J.PLACENTA.2022.06.004. 582 [34] Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol 2003;21:335–76. 583 https://doi.org/10.1146/annurev.immunol.21.120601.141126. 584 [35] Pandya Y , Penha-Gonçalves C. Maternal-fetal conflict during infection: Lessons from a 585 mouse model of placental malaria. Front Microbiol 2019;10:1126. 586 https://doi.org/10.3389/FMICB.2019.01126/BIBTEX. 587 [36] Fukata M, Vamadevan AS, Abreu MT. Toll-like receptors (TLRs) and Nod-like receptors 588 (NLRs) in inflammatory disorders. Semin Immunol 2009;21:242–53. 589 https://doi.org/10.1016/J.SMIM.2009.06.005. 590 [37] Chua CLL, Khoo SKM, Ong JLE, Ramireddi GK, Yeo TW, Teo A. Malaria in Pregnancy: 591 From Placental Infection to Its Abnormal Development and Damage. Front Microbiol 592 2021;12:3452. https://doi.org/10.3389/FMICB.2021.777343/BIBTEX. 593 [38] Rodríguez-Pascual F, Busnadiego O, Lagares D, Lamas S. Role of endothelin in the 594 cardiovascular system. Pharmacol Res 2011;63:463–72. 595 https://doi.org/10.1016/J.PHRS.2011.01.014. 596 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint 27 [39] Sato A, Ebina K. Common mechanism in endothelin-3 and PAF receptor function for anti-597 inflammatory responses. Eur J Pharmacol 2013;718:30–3. 598 https://doi.org/10.1016/J.EJPHAR.2013.09.025. 599 [40] Sato A, Ebina K. Endothelin-3 at low concentrations attenuates inflammatory responses 600 via the endothelin B2 receptor. Inflamm Res 2013;62:417–24. 601 https://doi.org/10.1007/S00011-013-0594-3. 602 [41] Burton GJ, Jauniaux E. Oxidative stress. Best Pract Res Clin Obstet Gynaecol 603 2011;25:287. https://doi.org/10.1016/J.BPOBGYN.2010.10.016. 604 [42] Sarr D, Cooper CA, Bracken TC, Martinez-Uribe O, Nagy T, Moore JM. Oxidative Stress: 605 A Potential Therapeutic Target in Placental Malaria. Immunohorizons 2017;1:29. 606 https://doi.org/10.4049/IMMUNOHORIZONS.1700002. 607 608 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint