{"paper_id":"b4685b3d-20c8-4383-aad9-1505c5cbef8f","body_text":"1 \n \nPlacental malaria is associated  with a TLR–Endothelin-3–oxidative damage response in 1 \nhuman placenta tissues 2 \nSamuel Chenge1,2, Melvin Mbalitsi1, Harrison Ngure1, Moses Obimbo3, Mercy Singoei3, Mourine 3 \nKangogo2, Bernard N. Kanoi1, Jesse Gitaka1,*,#, and Francis M. Kobia1,*,# 4 \n 5 \n1Centre for Malaria Elimination, Mount Kenya University, Thika – Kenya.   6 \n2Department of Medical Microbiology, Jomo Kenyatta University of Agriculture and Technology, 7 \nNairobi – Kenya 8 \n3Department of Human Anatomy, University of Nairobi, Nairobi – Kenya 9 \n*Correspondence: Francis M. Kobia (fkobia@associates.mku.ac.ke), Center for Malaria 10 \nElimination, Mount Kenya University, P.O. Box 342-01000, Thika – Kenya. Jesse Gitaka 11 \n(jgitaka@mku.ac.ke), Center for Malaria Elimination, Mount Kenya University, P.O. Box 342-12 \n01000, Thika – Kenya. 13 \n#co-senior authorship 14 \n 15 \n 16 \n 17 \n 18 \n 19 \n 20 \n 21 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint \n\n2 \n \nAbstract 22 \nPlacental malaria, which is mainly caused by the sequestration of Plasmodium falciparum-23 \ninfected erythrocytes in the placenta, is an important driver of poor pregnancy outcomes, 24 \nincluding fetal growth restriction, preterm birth, and stillbirth. However, the mechanisms 25 \nunderlying its adverse outcomes are unclear. Mouse models have previously shown that 26 \nplacental malaria (PM) triggers a proinflammatory response in the placenta, which is 27 \naccompanied by a fetal Toll-like receptor (TLR)4-mediated innate immune response associated 28 \nwith improved fetal outcomes. Here, we used hematoxylin and eosin staining to identify PM-29 \npositive and negative samples in our biobank of placentas donated by women living in a 30 \nmalaria-endemic region of Kenya and assessed the impact of PM on the expression of TLRs, 31 \nEndothelins, and oxidative damage. RT-qPCR analysis revealed that PM was associated with 32 \nan upregulation of TLR4, TLR7, and Endothelin-3. Moreover, immunohistochemistry showed 33 \nthat PM was associated with elevated expression levels of the oxidative DNA damage marker, 34 \n8-hydroxy-2’-deoxyguanosine, while RT-qPCR revealed that this was accompanied by an 35 \nupregulation of p21, an inhibitor of cell cycle progression and marker of cellular response to 36 \nDNA damage. These findings allude to a novel mechanism of PM pathogenesis driven by a 37 \nTLR–Endothelin-3–oxidative DNA damage signaling axis. 38 \n 39 \nKeywords: Placental malaria, malaria in pregnancy, poor pregnancy outcomes, innate 40 \nimmunity, Plasmodium falciparum 41 \n 42 \n 43 \n 44 \n 45 \n 46 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint \n\n3 \n \n1. Introduction 47 \nAccording to the World Health Organization, globally, there were about 249 million malaria 48 \ncases and 608,000 malaria-associated deaths in 2022, with sub-Saharan Africa accounting for 49 \nmost of the cases and deaths [1]. Pregnant women have a higher susceptibility to malaria 50 \ninfection [2] and it is estimated that in 2022, there were about 12.7 million cases of malaria in 51 \npregnancy (MiP) in sub-Saharan Africa [1]. MiP is associated with several adverse outcomes on 52 \nthe mother, the fetus, and neonate [3]. For the fetus, MiP severely worsens pregnancy 53 \noutcomes and frequently leads to fetal growth restriction (including low birthweight, small for 54 \ngestational age, and intrauterine growth restriction) and may result in preterm birth and stillbirth 55 \n[3–5].  56 \nThe adverse effects of MiP on the fetus are attributable to malaria infection of the placenta [1], 57 \nleading to placental malaria (PM). PM is characterized by the sequestration of Plasmodium-58 \ninfected erythrocytes in placental intervillo us spaces. This phenomenon is most frequently 59 \nassociated with Plasmodium  falciparum (P. falciparum), the species associated with the most 60 \nsevere form of malaria [6]. The sequestration of P.  falciparum-infected erythrocytes to the 61 \nplacenta is mediated by the interaction between variant surface chondroitin surface antigen 2, a 62 \nPlasmodium falciparum protein expressed on the surface of infected erythrocytes [7], and 63 \nchondroitin sulfate A on the surface of the syncytiotrophoblast [7], the placental epithelial cell 64 \nlayer that contacts maternal blood [8]. 65 \nThe adverse impacts of PM on fetal well-being most likely results from the negative effects of 66 \nPM on placental health and function since the vertical transmission of malaria to the fetus is rare 67 \n[9]. Indeed, PM is reported to induce placental inflammation [10,11] and placental histological 68 \nchanges [12], which may contribute to placental insufficiency and poor pregnancy outcomes. 69 \nHowever, the mechanisms underlying PM-driven placental pathobiology are not fully understood 70 \nat the cellular and cell signaling levels.  71 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint \n\n4 \n \nInnate immune factors, such as Toll-like receptor (TLR)4, 7, and 9, which respond to infection by 72 \nrecognizing and clearing invading pathogens are reported to respond to malaria infection [13], 73 \nalthough their role in PM is unclear. Although several studies have investigated maternal 74 \nresponses to PM, few have studied how the fetus responds to parasite sequestration in the 75 \nplacenta. Nonetheless, a mouse model of PM revealed that PM triggers a TLR4-mediated 76 \ninnate immune reaction that adversely affects fetal outcomes, which is countered by a fetal 77 \ninnate immune reaction that led to better pregnancy outcomes [14]. This suggests the presence 78 \nof TLR-mediated innate immune responses to PM, although this has not been reported in the 79 \ncontext of human PM. Here, considering that mouse data show that TLR4 modulates 80 \nendothelin-1 expression [15], malaria is inflammatory and oxidative [16], oxidative DNA damage 81 \nupregulates TLR4 [17], and TLR signaling is thought to promote DNA repair [18], we used 82 \nbiobank placenta samples donated by women living in a malaria-endemic region of Kenya to 83 \nexamine the hypothesis that human PM triggers a TLR–Endothelin–oxidative damage signaling 84 \nresponse.  85 \n2. Materials and methods 86 \n2.1 The biobank and study participants 87 \nThe study used biobank placenta samples donated by residents of Bungoma County, a malaria-88 \nendemic region of Western Kenya. The biobank was established by a previous prospective 89 \nparent study. All placenta donors were aged ≥ 18 years and gave written informed consent to 90 \nparticipate in the parent study. Based on questionnaire responses, participants with a known 91 \nrecord of sexually transmitted disease infection during pregnancy, those with pregnancy-92 \nassociated noncommunicable diseases (preeclampsia and gestational diabetes) during the 93 \ncurrent pregnancy, and those with twin pregnancies were excluded from our analyses. During 94 \nparticipant recruitment and sample collection, participants were recorded as having MiP if they 95 \nhad at least one episode of hospital-diagnosed malaria during pregnancy. Maternal malaria 96 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint \n\n5 \n \nstatus was diagnosed using a rapid diagnostic test (Malaria Ag P.f, SD Biosensor). All data 97 \nunderlying the databank were deidentified. The characteristics of the biobank’s placenta donors 98 \nand samples are summarized in Table 1. Equal numbers of male and female placentas were 99 \nanalyzed. Demographic data (Table 1), including maternal age, history of malaria during 100 \npregnancy, and gravidity were collected using questionnaires, whereas pregnancy-associated 101 \ndata, including birthweight and placental weight, were recorded after birth. All participants gave 102 \nwritten informed consent before joining the study and agreed to the collection and use of their 103 \nplacenta samples in the study. 104 \n2.2 Histological analysis 105 \nFormalin-fixed placenta tissues were embedded in paraffin blocks as previously described [19] 106 \nusing an automated tissue embedding system (MediMeas). H&E analysis was used to confirm 107 \nthe presence of PM, which is indicated by the presence of infected erythrocytes in the placenta. 108 \nBriefly, formalin-fixed paraffin-embedded samples were sectioned onto charged microscope 109 \nslides (Dako, Cat No. K8020) at a 5-µm thickness, dried at 37 °C overnight on a slide warmer, 110 \ndewaxed in xylene (Finar Chemicals, Cat No. 21940LC250), rehydrated by dipping across an 111 \nalcohol gradient of absolute, 95%, 70%, and 50% ethanol (Scharlau, Cat No. ET00052500), and 112 \nthen in distilled water. They were then submerged in hematoxylin (Loba Chemie, Cat No. 113 \n04023) for seven minutes, rinsed with running water, and then destained through 10 dips in acid 114 \nalcohol (1% hydrochloric acid in 70% ethanol). Next, they were submerged in eosin (Griffchem, 115 \nCat No. 45380) for 45 seconds followed by dehydration in 95% ethanol and absolute ethanol 116 \n(five minutes each) and then cleared in xylene baths (10 minutes each) before being cover-117 \nslipped using dibutylphthalate polystyrene xylene mountant (Finar Chemicals, Cat No. 118 \n10525LM250). The slides were then examined under a microscope (Richter Optica UX1, M2 119 \nScientifics), followed by imaging in ≥ 10 fields of view per slide at a 40X magnification using a 120 \nMoticam microscope camera (Motic Scientific). PM was then diagnosed as described before 121 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint \n\n6 \n \n(Odongo et al., 2016) based on the presence of infected erythrocytes. PM-associated 122 \nhistopathological features were assessed by counting the number of syncytial knots as 123 \ndescribed previously [20] and measuring the fibrin-occupied placental areas using imageJ. 124 \nThese analyses were done in at least 10 fields of view per sample. The levels of PM burden in 125 \nthe PM-positive samples were determined by counting the number of identified infected 126 \nerythrocytes in at least 10 fields of view per slide imaged at a magnification of 40X. 127 \n2.4 RNA extraction and reverse transcription quantitative PCR (RT-qPCR) 128 \nTotal RNA was extracted from placenta tissue using a HigherPurity™ Tissue Total RNA 129 \nPurification kit as per the manufacturer’s guidelines (Canvax, cat No. AN0152) and quantified 130 \nusing a NanoDrop Microvolume Spectrophotometer (ThermoFisher Scientific) following the 131 \nmanufacturer’s instructions. For each sample, 500 ng of RNA were retrotranscribed into cDNA 132 \nusing a LunaScript™ RT SuperMix cDNA Synthesis Kit (NEB, Cat. No. E3010L) using the 133 \nmanufacturer’s protocol. RT-qPCR analysis was done on a QuantStudio™ 5 Real-Time PCR 134 \nSystem in a final volume of 20 µl containing 10 µl of GoTaq qPCR Master Mix (Promega, Cat 135 \nNo. PRA6001), 2 µl of the forward plus reverse primers (final primer concentration: 500 nM), 3 136 \nµl of nuclease free water (Promega, Cat No. P119E), and 5 µl of cDNA using the following 137 \nprogram: 50 °C for two minutes, 95 °C for 10 minutes, followed by 40 cycles at 95 °C for 15 138 \nseconds and 60°C for 30 seconds. Relative gene expression was determined using the 2 -ΔΔ ct 139 \nmethod [21], using β -actin as the reference gene. Primers were purchased from Macrogen and 140 \nprimer sequences are provided in Table 3. 141 \n2.5 P. falciparum detection PCR 142 \nThe presence of P. falciparum in placenta samples was evaluated using One Taq® Quick-143 \nLoad® 2X Master Mix with Standard Buffer (NEB, Cat No. M0486L) and the following 144 \nthermocycler program: Initial denaturation at 95 °C for five minutes, followed by 35 cycles of 145 \ndenaturation at 95 °C for 30 seconds, annealing at 55 °C for 60 seconds, and extension at 72 146 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint \n\n7 \n \n°C for 75 seconds, and then a final extension at 72 °C for five minutes. Primer sequences are 147 \nshown in Table 3. The PCR product was subjected to agarose (Sigma–Aldrich, Cat No. A9539) 148 \ngel electrophoresis using 1X tris–borate–EDTA buffer alongside a 100 base pair ladder using 149 \nSafeView™ Classic (Applied Biological Materials, Cat No. G108) nucleic acid stain and Gel 150 \nLoading Dye, Purple (6X) (NEB, Cat No. B7024S). The bands were developed and imaged 151 \nusing a UVITEC Gel Documentation System (Cleaver Scientific). 152 \n2.6 Immunohistochemistry 153 \nThe sections were deparaffinized by warming at 55 °C for 15 minutes followed by dipping in 154 \nthree xylene baths, about 10 dips each. They were then rehydrated and subjected to heat-155 \ninduced epitope retrieval by boiling for 30 minutes in Citrate Buffer, pH 6.0 (Sigma–Aldrich, cat. 156 \nNo. C9999). They were then cooled to room temperature, rinsed with distilled water for five 157 \nminutes and then blocked with 0.3% Triton-X in 1X phosphate-buffered saline (PBST). Next, 158 \nthey were blocked with 10% normal donkey serum (Abcam, cat. No. ab7475) in PBST for two 159 \nhours followed by overnight incubation (4 °C) with anti-DNA/RNA damage antibody [15A3] 160 \n(Abcam, cat. No. ab62623) at 1:2500 in blocking solution. Sections were then washed thrice (10 161 \nminutes each) using PBST and then incubated at room temperature for two hours with 162 \nhorseradish peroxidase-c onjugated goat anti-mouse se condary antibody  (Jackson 163 \nImmunoResearch, cat. No. 115-035-003) at 1:5000 in blocking solution. The sections were then 164 \nwashed thrice (10 minutes each) using PBST followed by signal development using an 165 \nImmPACT® DAB Substrate Kit (Vector, cat. No. SK-4105) as per the manufacturer’s protocol. 166 \nThey were then dehydrated using 95%, 95%, 100%, and 100% ethanol (five minutes each), 167 \ncleared by dipping in three xylene baths and then cover-slipped using a xylene-based mountant 168 \nand allowed to dry. They were then examined under a light microscope and imaged at a 169 \nmagnification of 40X. 170 \n2.7 Data analyses 171 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint \n\n8 \n \nStatistical analyses were done using GraphPad Prism version 9. Data are presented as 172 \npercentages, raw values, or mean ± standard deviation. Differences between two groups were 173 \ncompared using a t-test. Correlation analyses were done using nonparametric Spearman 174 \ncorrelation analysis. For each placenta, the PM burden in placentas was indicated by the total 175 \nnumber of infected erythrocytes in the placenta section. The birthweight-to-placenta weight 176 \n(BW:PW) ratio was obtained by dividing the birthweight (grams) with the corresponding 177 \nplacenta’s weight (grams). The correlation between PM status and birthweight, placental weight, 178 \nand birthweight-to-placental weight ratio was assessed using GraphPad prism to examine the 179 \nimpact of PM on fetal outcomes. P < 0.05 indicates statistically significant differences. 180 \n3. Results  181 \n3.1 Main characteristics of the placenta donors and donated placentas 182 \nAll placenta donors were ≥ 18-years-old and had received intermittent presumptive treatment 183 \nwith sulfadoxine pyrimethamine. The mean age, gravidity, birthweight (BW), placental weight 184 \n(PW), and BW:PW ratio of the cohort of placenta donors was 24.7 years, 2.69, 3077.64 g, 185 \n470.04 g, and 6.51, respectively (Table 1). Grouping the placenta donors into those with a 186 \nknown history of MiP and those without (NoMiP), revealed that in the MiP vs NoMiP groups, 187 \nmaternal age and gravidity were not significantly different (mean age: 24.4 [range: 18–30] vs 25 188 \n[range: 18–40] years, P  = 0.45; mean gravidity: 2.5 [range: 1–7] vs 2.9 [range: 1–7), P = 0.12). 189 \nHowever, in the MiP vs NoMiP groups, BW (mean: 2870.5 [range: 1600–5000] vs 3272.3 190 \n[range: 2000–4500) g), PW (mean: 464.3 [range: 280.4–675) vs 492.9 [range: 342.3–715] g, 191 \nand BW:PW ratio (mean: 6.27 [range: 3.2–10.2] vs 6.73 [range: 4.18–10], were significantly 192 \nlower in the MiP group ( P < 0.0001, = 0.009, and = 0.03, respectively). H&E analysis revealed 193 \nthat 92 placentas (51.4%) were PM-positive (had infected erythrocytes), 58 (32.4%) were PM-194 \nnegative (no infected erythrocytes observed), 29 (16.2%) had past malaria infection (hemozoin 195 \npresent in the absence of infected erythrocytes [12]), 92 (51.4%) belonged to male fetuses, and 196 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint \n\n9 \n \neight (4.5%) were preterm (Table 2). All downstream analyses were done on placenta samples 197 \nthat were confirmed to be PM-positive or PM-negative using H&E staining. 198 \n3.2 PM correlates negatively with birthweight and birthweight-to-placenta ratio 199 \nRepresentative images of PM-negative and PM-positive samples are shown on Figure 1A–B, 200 \nand the presence of P. falciparum in PM-positive tissues was confirmed using PCR (Figure 1C). 201 \nAnalysis of the data underlying the placental biobank revealed that when compared with the 202 \nPM-negative group, BW was significantly lower in the PM-positive group, which had more low 203 \nBW cases (Figure 1A, P = 0.03, low birthweight: <2500 g), but PW did not differ between the 204 \ntwo groups (Figure 2B, P = 0.8). However, the BW:PW ratio was lower in the PM-positive group, 205 \nalthough the difference did not reach statistical significance (Figure 1F, P = 0.08). Next, we used 206 \nthe H&E images to determine the proportion of infected erythrocytes, IEs (%), in each placenta 207 \nsample, and then used the obtained values to assess the correlation between the PM burden 208 \nand BW, PW, and the BW:PW ratio, i.e., the fetal weight obtained per gram of the placenta, 209 \nwhich is an indicator of placental efficiency, with higher BW:PW ratios indicating greater 210 \nplacental efficiency [22]. This analysis revealed negative correlation between IEs (%) (the 211 \nproportion of infected erythrocytes), and BW (correlation coefficient [rs]: -0.22, P < 0.005, 95% 212 \nconfidence interval [CI]: -0.359 – -0.071), and IEs (%) and BW:PW ratio (rs: -0.20, P = 0.007, 213 \n95% CI: -0.338 – -0.048). Expectedly, PW had a positive correlation with birthweight (rs: 0.29, P 214 \n< 0.001, 95% CI: 0.141 – 0.419) and a negative correlation with BW:PW ratio (rs: -0.42, P < 215 \n0.001, 95% CI: -0.539 – -0.290). However, the PM burden did not exhibit correlation with 216 \nplacental weight (rs: 0.01, P = 0.94, 95% CI: -0.156 – 0.146). Taken together, these findings 217 \nindicate that PM impairs placenta function, leading to low birthweight via reduced placenta 218 \nefficiency as indicated by the negative correlation between PM burden and the BW:PW ratios of 219 \nPM-exposed neonates. 220 \n3.3 PM markedly alters placental histological features 221 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint \n\n10 \n \nNext, we assessed the impact of PM on syncytial knotting and fibrin deposition in our placenta 222 \nsamples. This analysis revealed that when compared with PM-negative samples (A), PM-223 \npositive samples had more syncytial knots (B, yellow arrowhead) and greater fibrin-occupied 224 \nplacental area (C, FD in the broken line-demarcated area). Quantification analyses revealed 225 \nthat when compared with PM-negative samples, the levels of these histological features were 226 \nsignificantly higher in the PM-positive samples (D, SK: syncytial knots, P = 0.047 and E, fibrin 227 \narea, P < 0.0005). These observations indicate that in our study cohort, PM may have adversely 228 \naffected fetal outcomes at least in part, by altering normal placental histological features.  229 \n3.4 PM is associated with an upregulation of TLR4, TLR7, and Endothelin 3 230 \nWe then sought to determine if PM altered the expression of TLRs. To this end, we focused on 231 \nTLR4, TLR7, and TLR9, which have been associated with response to malaria infection in mice 232 \n[23,24], and with mouse PM in the case of TLR4 [25], although this has not been reported in 233 \nhuman PM. To evaluate the effect of PM on these innate immune system receptors, we 234 \nassessed their expression levels using RT-qPCR. The analysis revealed that when compared 235 \nwith PM-negative controls, PM-positive samples expressed significantly higher levels of TLR4 236 \nand TLR7, but not TLR9 (Figure 3A–C, P  = 0.002, 0.03, and 0.59, respectively). This is 237 \nconsistent with mouse data showing that PM upregulates TLR4-mediated expression of 238 \nendothelin-1 [15]. We therefore wondered if human PM alters the expression of Endothelin 239 \ngenes. RT-qPCR analysis of Endothelin-1 and -3 gene expression revealed that only 240 \nEndothelin-3 was detectable in our placenta samples and that when compared with PM-241 \nnegative placentas, PM-positive samples had significantly higher levels of Endothelin-3 (Figure 242 \n3D, P = 0.004), indicating the presence of a TLR–Endothelin signaling axis in response to 243 \nhuman PM. 244 \n3.5 PM is associated with high oxidative DNA damage 245 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint \n\n11 \n \nSince TLR4 was upregulated in PM-positive placenta samples, we wondered whether PM-246 \ndriven activation of TLR4 is associated with a dysregulation of other signaling processes that 247 \nmay underlie or contribute to PM-mediated placental pathobiology. Because malaria is known to 248 \nbe strongly inflammatory and oxidative, which may drive host tissue damage [16], and because 249 \noxidative DNA damage is associated with TLR4 upregulation [17] and that TLR signaling is 250 \nthought to promote DNA repair [18], we wondered if the TLR4 upregulation in the PM-positive 251 \nsamples might be associated with placental oxidative DNA damage. To assess this possibility, 252 \nwe used immunohistochemistry to assess the levels of 8-hydroxy-2’-deoxyguanosine, a marker 253 \nof oxidative DNA stress [26]. This analysis revealed that when compared with PM-negative 254 \nsamples, PM-positive samples express markedly higher levels of 8-hydroxy-2’-deoxyguanosine 255 \n(Figure 4A). Staining the same samples with the secondary antibody only (without the primary 256 \nantibody) confirmed signal specificity (Figure 4B). To further assess the effect of PM on 257 \noxidative stress, we used RT-qPCR to examine the level of p21, a mediator of cell cycle arrest 258 \nand indicator of cellular response to DNA damage [27]. This revealed that when compared with 259 \nPM-negative samples, PM-positive samples had significantly higher levels of p21 (Figure 4C, P 260 \n= 0.02). Taken together, these data indicate that PM triggers markedly high levels of placental 261 \noxidative DNA stress, placenta tissue damage, and cellular response to DNA stress, which may 262 \ncontribute to the pathobiology of PM, and that in response, at least in part, TLR signaling may 263 \nbe upregulated to counter these adverse effects through promotion of DNA repair.  264 \n4. Discussion 265 \nMalaria in pregnancy (MiP) often results in placental malaria (PM), where erythrocytes that are 266 \ninfected with P. falciparum , the parasite that most frequently causes PM, sequestrate in 267 \nplacental intervillous spaces [7]. PM may cause various adverse fetal outcomes, stillbirth, 268 \npreterm birth, and fetal growth restriction [3–5] and because P. falciparum rarely undergoes 269 \nvertical transmission [9], these effects are likely caused by PM-driven pathobiological effects in 270 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint \n\n12 \n \nthe placenta, which may impair placental function. However, although studies have implicated 271 \neffects like inflammation and histological changes in PM pathogenesis, the mechanisms 272 \nunderlying the adverse effects of human PM on the placenta are unclear. Moreover, because 273 \nmany MiP cases in malaria-endemic regions are asymptomatic [28,29] and the placenta is 274 \ninaccessible during pregnancy, there are no ways of detecting and intervening against PM 275 \nduring pregnancy. Thus, there is an urgent need to better understand the placental pathobiology 276 \nof PM to guide the development of effective diagnostic and therapeutic tools.  277 \nMouse models indicate that PM triggers innate immune responses (mainly via TLR4) that are 278 \nassociated with poor fetal outcomes and that TLR4-mediated fetal responses to PM lead to 279 \nimproved outcomes [14]. However, the effect of human PM on TLR-mediated immunity in the 280 \nplacenta has not been examined. In this study, we leveraged our well-characterized biobank of 281 \nplacenta samples from a malaria endemic region of Kenya (Table 1) and found that in our study 282 \ncohort, PM burden had a significant negative correlation with birthweight and BW:PW ratio, that 283 \nit was associated with significantly higher placental histological lesions, higher levels of TLR4 284 \nand Endothelin-3 expression, and enhanced oxidative DNA damage when compared with 285 \nsamples without PM. 286 \nConsistent with previous findings implicating PM in fetal growth restriction [30], we observed 287 \nthat in our study cohort, relative to the PM-negative cases, PM was associated with low 288 \nbirthweight. Moreover, we observed that PM was associated with lower BW:PW ratios, an 289 \nindicator of placental efficiency in which higher ratios indicate higher nutrient transfer for every 290 \ngram of placenta and vice versa [22], an observation that to our knowledge, has not been 291 \npreviously reported in human PM, but not with lower placental weight (Figure 1D–F). 292 \nInterestingly, our analyses also indicate that the PM burden (percentage of infected erythrocytes 293 \nin a sample’s intervillous spaces) correlates negatively with birthweight but not with placental 294 \nweight. Taken together, these observations indicate that PM contributes to fetal growth 295 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint \n\n13 \n \nrestriction primarily by impairing placental function and not via placental growth inhibition, 296 \nalthough the precise mechanisms remain unclear. This possibility is crucial considering that 297 \nwomen in malaria-endemic regions may experience multiple malaria reinfections throughout 298 \npregnancy, but further studies, such as using in vitro and organoid systems, are needed to 299 \ncomprehensively investigate this possibility.  300 \nOur analyses revealed that, PM-positive samples had markedly higher levels of fibrin deposition 301 \nand syncytial knotting than PM-negative samples, which is in line with earlier findings [31]. 302 \nThese changes, which indicate placental injury and have been associated with placental 303 \nmalperfusion and poor fetal outcomes, including fetal growth restriction [32,33], may contribute 304 \nto the low birthweight observed in our PM cohort. However, studies are needed to establish the 305 \nmechanisms by which PM alters placental histological features, how these changes correlate 306 \nwith fetal outcomes and postnatal wellbeing, and whether they can predict fetal wellbeing in 307 \npostnatal life. 308 \nTLRs are key innate immunity factors that sense host invasion by pathogens and activate host 309 \nimmune defenses [34]. Mouse models of malaria indicate that TLR4, TLR7, and TLR9 are 310 \ninvolved in detecting and responding to malaria infection [23,24]. Moreover, mouse models 311 \nindicate that at the fetal–maternal interface, PM activates TLR4-mediated immune responses 312 \nthat drive poor fetal outcomes, and that fetal TLR4-mediated counterresponses improve 313 \npregnancy outcomes [14,25,35]. However, this observation has not been previously made in 314 \nhuman PM. Here, we observed that the expression levels of TLR4 and TLR7, but not TLR9, 315 \nwere significantly upregulated in PM-positive samples, indicating that placental infection triggers 316 \nan innate immune reaction and that it is mainly driven by TLR4 and TLR7, although the status of 317 \nother TLRs during PM warrants investigation. Considering that TLRs are important drivers of 318 \ninflammation [36], which is implicated in PM pathogenesis [37], taken together with the 319 \nobserved changes in placental histological features, our findings indicate for the first time, that 320 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint \n\n14 \n \nTLR-mediated responses to PM may contribute to local placental inflammation, which may 321 \nunderlie the observed PM-associated low birth and placental weights, although the precise 322 \nmechanisms remain unclear. Mouse data show that PM-driven TLR4 expression drives 323 \nplacental endothelin-1 expression [15], and for the first time, our findings show that PM is also 324 \nassociated with the upregulation of Endothelin-3. The Endothelin ligands 1, 2, and 3 are a family 325 \nof vasoactive factors that influence a range of cellular processes, such as vascular remodeling 326 \nand angiogenesis [38]. Moreover, Endothelin-3 has been reported to have anti-inflammatory 327 \neffects [39,40], suggesting that its upregulation in the context of PM-mediated TLR4 328 \nupregulation is a mechanism of countering TLR4-dependent placenta inflammation. Collectively, 329 \nthese observations indicate that human placenta malaria may activate a TLR4–Endothelin-3 330 \nsignaling axis, but further studies are needed to test this hypothesis and to determine its 331 \nimplications in PM pathobiology and fetal outcomes. 332 \nBased on reports that malaria is strongly oxidative [16], oxidative stress causes tissue damage 333 \n[41], oxidative DNA damage upregulates TLR4 [17], and that the TLR pathway might promote 334 \nDNA repair [18], we reasoned that our observation of TLR4 and TLR7 upregulation in PM-335 \npositive samples might be accompanied by placental oxidative DNA damage. This hypothesis 336 \nwas confirmed by our immunohistochemistry data, which showed that 8-hydroxy-2’-337 \ndeoxyguanosine, a marker of oxidative DNA stress (Valavanidis et al., 2009), was markedly 338 \nupregulated in PM samples. Moreover, gene expression analysis revealed that these events 339 \nwere accompanied by a significant upregulation of p21, a cell cycle inhibitor and marker of 340 \ncellular response to DNA damage [27]. These observations align with previous findings that in a 341 \nmouse model, PM is associated with placental oxidative damage [42]. Furthermore, p21 342 \nupregulation in the placenta may arrest the cell cycle to allow for oxidative damage resolution, 343 \nwhich may contribute to the low placental weight observed in our cohort, but this possibility 344 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint \n\n15 \n \nrequires further investigation. Together, our data suggest the presence of a previously unknown 345 \nTLR–Endothelin–oxidative damage axis in human PM. 346 \n5. Conclusion 347 \nDespite its heavy burden and adverse effects on maternal and fetal outcomes, the mechanisms 348 \nunderlying the placental pathobiology of PM are unclear. Considering that malaria is rarely 349 \ntransmitted to the fetus [9], the adverse fetal outcomes of MiP are mainly driven by events that 350 \ndisrupt placenta physiology and function. Importantly, because of the placenta’s inaccessibility, 351 \nPM can only be confirmed via postnatal placental histopathology. These challenges highlight the 352 \nurgent need to better understand the mechanisms underlying placental pathobiology of PM, 353 \nwhich may inform the development of sensitive tools for diagnosing PM during pregnancy as 354 \nwell as effective therapeutic interventions. Our findings that PM may drive TLR-mediated 355 \nresponses in the placenta, raise the possibility that modulating innate responses to PM may 356 \nimprove fetal outcomes, as we previously discussed [13]. Moreover, our identification of an axis 357 \ninvolving TLRs, Endothelins, and oxidative DNA damage during PM (Figure 5), highlights 358 \nprocesses with the potential for intervention against human PM. However, further studies, such 359 \nas using primary human trophoblasts, human placental organoids, or human placental ex vivo 360 \nsystems are needed to validate our observations. Such approaches can investigate the 361 \nmechanisms of PM pathobiology more rigorously than can be done using term placentas. 362 \nFunding statement  363 \nF.M.K. is supported by the EDCTP2 programme supported by the European Union and Novartis 364 \nGlobal Health, Basel – Switzerland, Grant Number TMA2019CDF-2736.  365 \nAcknowledgments 366 \nWe thank our placenta donors and the staff at Webuye County and Mary Help of The Sick 367 \n(Thika) Hospitals for their generous support. We thank the staff of the histopathology and 368 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint \n\n16 \n \nanatomy departments at the University of Nairobi and Mount Kenya University for their help with 369 \nhistopathology. We are grateful to Prof. Walter Jaoko, Prof. Omu Anzala, and Dr. Daniel Muema 370 \nof KAVI–ICR for kindly sharing laboratory space and resources. We are thankful to Prof. Roger 371 \nSmith and Dr. Kaushik Maiti, Mothers and Babies Research Centre, Hunter Medical Research 372 \nInstitute, Newcastle, NSW – Australia, for sharing antibodies and other resources. 373 \nConflict of interest 374 \nThe authors declare no conflicts of interest. 375 \nEthics statement 376 \nThis study was approved by Mount Kenya University’s ethics review committee (approval 377 \nnumber 1314). 378 \n 379 \n 380 \n 381 \n 382 \n 383 \n 384 \n 385 \n 386 \n 387 \n 388 \n 389 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint \n\n17 \n \n 390 \n 391 \n 392 \n 393 \n 394 \n 395 \n 396 \nFigures 397 \n 398 \nFigure 1.  (A–B) Representative hematoxylin and eosin images of placental malaria (PM)-399 \nnegative (A) and PM-positive samples (B). When compared with a PM-negative sample, the 400 \npositive sample has malaria-infected erythrocytes (black arrowheads) in the placental 401 \nintervillous space. (C) PCR confirmed the presence of P. falciparum in the PM-positive sample 402 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint \n\n18 \n \nin B. L: 100 base pair ladder, N: PM-negative sample, S: PM-positive sample in B (histology), P: 403 \npositive control ( P. falciparum strain 3D7 genomic DNA). Expected PCR band size: 205 base 404 \npairs. (D–F) PM was associated with lower birthweight (BW) (D, P = 0.03) and lower 405 \nbirthweight-to-placental weight (BW:PW) ratio, although the difference did not reach statistical 406 \nsignificance (F, P  = 0.08), but not with lower placental weight (PW) (E, P = 0.8). In D–F, 407 \nwhiskers are drawn from the 10 th to 90 th percentile. (G) A correlation matrix shows that the 408 \nproportion of infected erythrocytes, IEs (%), in the placenta correlated negatively with BW ( P < 409 \n0.005) and the BW:PW ratio ( P = 0.007), but it did not correlate with PW ( P = 0.94). PW 410 \ncorrelated positively with BW and negatively with the BW:PW ratio (both P < 0.001).  411 \n 412 \nFigure 2 . (A–C) In the samples from the biobank underlying our study, when compared with 413 \nplacental malaria (PM)-negative samples (A), PM was associated with significantly higher rates 414 \nof syncytial knots (B, yellow arrowhead) and placental fibrin deposits (C; FD, marked with 415 \nbroken line). Black arrowheads indicate infected erythrocytes. (D–E) Quantification revealed 416 \nthat the levels of syncytial knots (SK [D], n = 21 and 38 for PM-neg and PM-pos, respectively; P 417 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint \n\n19 \n \n= 0.047) and the area of placenta intervillous spac e containing fibrin (E, n = 26 and 38 for PM-418 \nneg and PM-pos, respectively, P < 0.0005) were significantly higher in the PM-positive (PM-pos) 419 \nsamples than in the PM-negative (PM-neg) samples. Whiskers are drawn from the 10 th to 90 th 420 \npercentile. 421 \n 422 \nFigure 3. Placental malaria (PM) is associated with the upregulation of TLR4, TLR7, and 423 \nEndothelin-3. (A–C) When compared with PM-negative (PM-neg) samples, PM-positive (PM-424 \npos) samples expressed significantly higher levels of TLR4 and TLR7, but not TLR9 ( P = 0.002, 425 \n0.03, and 0.59, respectively). (D) PM-positive samples also expressed higher levels of 426 \nEndothelin-3 (P = 0.004). 427 \n 428 \n 429 \n 430 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint \n\n20 \n \n 431 \nFigure 4. Analysis of oxidative DNA damage in placental malaria (PM)-negative vs PM-positive 432 \nsamples. (A–B) Immunohistochemistry revealed that when compared with PM-negative 433 \nsamples, PM-positive tissues had markedly higher levels of 8-hydroxy-2’-deoxyguanosine (8-434 \nOHdG), an indicator of oxidative damage. Staining the same samples with the secondary 435 \nantibody only (B) confirmed signal specificity for this marker. (C) RT-qPCR showed that PM-436 \npositive samples express significantly higher levels of p21 (P = 0.02). 437 \n 438 \n 439 \n 440 \n 441 \n 442 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint \n\n21 \n \n 443 \nFigure 5. Schematic summary of the hypothesized TLR–Endothelin-3–oxidative stress axis in 444 \nhuman placental malaria. Further investigation is needed to validate this axis and determine its 445 \npotential for intervention against placental malaria. 446 \n 447 \n 448 \n 449 \n 450 \n 451 \n 452 \n 453 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint \n\n22 \n \nTables  454 \nTable 1. Summary of placenta donors’ demographics 455 \nGroup \nAge \n(range) \nGravidity  \n(range) \nBW (g) \n(range) \nPW (g) \n(range) \nBW:PW \n(range) \nAll donors \n24.7 years \n(18–40) \n2.69  \n(1–7) \n3077.64 \n(1600–5000) \n479.04 \n(280.37–715) \n6.51 \n(3.2–10.2) \nMiP \n24.4 years \n(18–30) \n2.5  \n(1–7) \n2870.5 \n(1600–5000) **** \n464.3 \n(280.4–675) ** \n6.27 \n(3.2–10.2) * \nNoMiP \n25 years \n(18–40) \n2.9  \n(1–7) \n3272.3  \n(2000–4500) \n492.9  \n(342.3–715) \n6.73 \n(4.18–10) \nAnalysis of the placenta donors’ data revealed that maternal age and gravidity were not 456 \nsignificantly different in the MiP (group with known history of malaria in pregnancy) vs the 457 \nNoMiP (group without known MiP history) groups ( P = 0.45 and 0.12, respectively), whereas 458 \nbirthweight (BW), placental weight (PW), and BW:PW ratios were significantly lower in the MiP 459 \ngroup when compared with the No MiP group ( P = < 0.0001, 0.009, and 0.03, respectively). *, 460 \n**, and **** indicate P < 0.05, 0.005, and 0.0005, respectively.  461 \nTable 2. Main characteristics of the donated placentas 462 \nNo. of placenta samples 179 \nPlacental malaria-positive placenta samples (infected erythrocytes present) 92 (51.4%) \nPlacental malaria-negative placenta samples 58 (32.4%) \nPlacentas with past placental malaria infection (hemozoin present) 29 (16.2%) \nPlacenta samples from male fetuses 92 (51.4%) \nPlacenta samples from female fetuses 87 (48.6 %) \nPlacenta samples from pre-term deliveries 8 (4.5%) \nThe general characteristics of the placentas used in this study are summarized. Placental 463 \nmalaria status was determined using hematoxylin and eosin (H&E) analysis. 464 \n 465 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.17.589949doi: bioRxiv preprint \n\n23 \n \nTable 3. List of primers used in the study 466 \n 467 \n 468 \n 469 \n 470 \n 471 \n 472 \n 473 \n 474 \n 475 \n 476 \n 477 \n 478 \n 479 \n 480 \nTarget Forward primer Reverse primer \nTLR4 5’-AGACCTGTCCCTGAACCCTAT-3’ 5’-CGATGGACTTCTAAACCAGCCA-3’ \nTLR9 5’-CTGCCTTCCTACCCTGTGAG-3’  5’-GGATGCGGTTGGAGGACAA-3’ \nTLR7 5’-TCCTTGGGGCTAGATGGTTTC-3’ 5’-TCCACGATCACATGGTTCTTTG-3’ \nβ-actin 5’-CATGTACGTTGCTATCCAGGC-3’ 5’-CTCCTTAATGTCACGCACGAT-3’ \np21 5’-TGTCCGTCAGAACCCATGC-3’ 5’-AAAGTCGAAGTTCCATCGCTC-3’ \nEndothelin-3 5’-GGGACTGTGAAGAGACTGTGG-3’ 5’-AGACACACTCCTTGTCCTTGTA-3‘ \nP. falciparum  5′-TTAAACTGGTTTGGGAAACCAAATATATT-3′ 5 ′-ACACAATGAACTCAATCATGACTACCCGTC-3′  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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