Animal studies were performed according to the regulations of the institutional animal care and use committee. Approval was obtained from the University of Washington Institutional Animal Care and Use Committee (IACUC) under protocol 4317-01. The University of Washington IACUC adheres to the NIH Office of Laboratory Animal Welfare standards (OLAW welfare assurance # D16-00292).

Male and female BALB/cJ (4–6 wks old) were obtained from Jackson Laboratories (Barr Harbor, ME) and housed at the University of Washington in an IACUC-approved animal facility. All mice were used under an approved IACUC protocol (4317–01 to SCM). Primary euthanasia was conducted with CO₂ inhalation at 30-70% of chamber volume per minute, followed by a secondary dissection method.

Female Anopheles stephensi mosquitoes infected with wild-type Py (strain 17XNL) were reared at Seattle Children’s Research Institute (Seattle, WA) or UW Medicine (Seattle, WA). Fresh sporozoites (spz) were obtained by salivary gland dissection 14–18 days post-infection. All spz were diluted in Schneider’s insect media for administration (Gibco, Thermo Fisher). Figure legends specify the dose for each experiment. For Py wild-type (WT) spz administrations, 1x10⁵ freshly dissected Py-WT spz were injected intravenously (IV, retro-orbital) unless stated otherwise. Each independent replicate involved a distinct mosquito cage and independently administered parasite infection.

Following infection with Py or mock injection with loading media (Schneider’s), livers from mice were harvested at 12, 24, or 44 hours post infection (hpi) and left lateral lobe was fixed in 10% neutral buffered formalin solution (Millipore Sigma) for 24 hours then transferred to 70% ethanol. Tissues were then paraffin embedded, cut into 5 µm sections, and mounted on positively charged glass slides. Mounted liver slices were then used for digital spatial profiling or immunofluorescence staining.

Slide-mounted liver slices were washed twice in xylene (Fisher Scientific) for 3 minutes followed by washes in 100% (twice), 95%, 70% and 50% ethanol for 3 minutes each. Slides were then washed with DI water and heated to 90°C for 30 minutes in 1% citrate-based antigen unmasking solution (Vector Laboratories) using a Decloaking Chamber (Biocare Medical). After primary and secondary antibody staining, autofluorescence was quenched using Vector TrueView (Vector Laboratories), and Fluoromount G (Southern Biotech) was applied for mounting.

For Kupffer cell localization experiments and quantification of spatial localization and parasite size at 24hpi, slides were washed with Tris-buffered saline (TBS) + 1% Triton X-100 (Sigma Aldrich), blocked for 2 h in 1.5% Bovine Serum Albumin (BSA; GoldBio) + 15% goat serum (Sigma Aldrich), and incubated with PyHSP70 antibodies overnight at 4 °C. After washing, secondary antibodies and DAPI were applied for 1 h at room temperature. Because CLEC4F antibodies are mouse-derived, slides were re-blocked and incubated with conjugated CLEC4F-AF647 antibodies overnight. Antibody concentrations: PyHsp70 (gift from Kaushansky Lab) 1:500, CLEC4F-AF647 (BioLegend 156804) 1:100, anti-mouse AlexaFluor-488 (Thermo Fisher Scientific A-11001) 1:1000.

For quantification of spatial localization and parasite size at 44 hpi, blocking was performed in PBS containing 0.3% Triton X-100 (Sigma Aldrich), 1% BSA (Fisher Scientific), and 10% natural goat serum (Jackson ImmunoResearch) for 1 h. Slides were incubated for 1 h at room temperature with primary antibodies plus Hoechst (1:1000; Invitrogen) and DAPI (1:400; Invitrogen), followed by PBS washes and secondary antibody incubation (1 h, room temperature). Antibody concentrations: PfHsp70 (StressMarq SPC-186; cross-reacts with Py) 1:500, anti-ASS1-AF488 (Abcam ab208412) 1:250, anti-GS (Abcam ab64613) 1:500; secondary antibodies anti-rabbit AlexaFluor-594 (Invitrogen A32740) and anti-mouse AlexaFluor-647 (Invitrogen A32728), both at 1:500.

For Kupffer cell localization experiments, images (40X) were acquired using a high-resolution widefield microscope (Nikon) focused on infected hepatocytes and uninfected regions. For cell quantification within regions of interest (ROIs), 4x4 image panels were taken with a 60-pixel overlap and stitched together using the DAPI channel. Images were stitched and deconvolved using the NIS-Elements software (Nikon) and were visualized using ImageJ/FIJI software (37, 38). ImageJ was used to quantify fluorescence intensity within defined ROIs of 200μm radii. Distances from parasitized cells to Kupffer cells (KCs) were measured between nucleus centers. Only KCs with a visible, DAPI-stained nucleus and a clear surrounding CLEC4F signal were included in counts. Whole lobe images were also captured on the 24hpi samples in Figures 1c, d. In this case, whole lobe images were taken with a high-resolution widefield microscope (Nikon) at 10X with 60-pixel overlap and stitched together using the DAPI channel. Only parasites with visible, DAPI-stained nuclei were included in counts and measurements.

For quantification of spatial localization and parasite size at 44 hpi, images (20X) were acquired and stitched using an Automated Microscope (Keyence). For liver zonation, periportal, interzonal, and pericentral regions were identified by ASS1 and GS staining. Periportal regions were classified as ASS1^hiGS^lo, interzonal regions were classified as ASS1^intGS^lo, and pericentral regions were classified as within 3 nuclei of ASS1^loGS^hi. Images were visualized for analysis using ImageJ software. ImageJ was used to count the number of parasites in each liver region and define ROIs around parasites to measure parasite area. Only parasites with visible, DAPI-stained nuclei were included in counts and measurements.

To quantify liver burden by reverse transcription polymerase chain reaction (RT-PCR), mice were sacrificed, half of the liver was excised and pulverized by bead beating in NucliSENS lysis buffer (bioMérieux, Durham, NC, USA), and nucleic acid was extracted on an EasyMAG instrument (bioMérieux). RNA was subjected to RT-PCR using the SensiFAST™ Probe Lo-ROX Kit (Bioline, London, UK) using a predesigned HEX-labelled mouse GAPDH RT-PCR assay (IDT Inc, Coralville, IA) multiplexed with a Pan-Plasmodium 18S rRNA RT-PCR assay as described (39). RT-PCR conditions of 45 °C for 10 min, 95 °C for 2 min and 45 cycles of 95 °C for 5 s, 50 °C for 35 s were performed on a QuantStudio 5 real-time PCR machine (Thermo Fisher Scientific). Copy numbers per reaction were determined using custom lot of quantified Armored RNA encoding full-length Plasmodium 18S rRNA (Asuragen, Austin, TX).

Acyline (lot # RDZ001) was provided by John K. Amory, MD, MPH at the University of Washington. Lyophilized acyline vials were stored at -20 °C until resuspension. At the time of use, acyline was resuspend to a concentration of 2 mg/mL with sterile Molecular Biology Grade Water (Cytiva). Resuspended vials were stored for a maximum of 1 week at 4 °C. Mice were subcutaneously injected with 300µg of acyline on 5 and 4 days prior to infection to give time for the hormone environment to equilibrate (36). For testosterone treatment, testosterone propionate (Sigma Aldrich) was dissolved in sesame oil (Sigma Aldrich) at a concentration of 20 µg/µL. Mice were weighed to determine average weight per group, and then subcutaneously injected with 100 µg testosterone/g body weight at 2 timepoints (3 days and 1 day prior to infections).

Serum was collected via chin bleed day of harvest and stored in -20 °C until testing. To measure testosterone levels in serum, Testosterone ELISA kits (Crystal Chem) were used per manufacturer guidelines. Singlet absorbance values of each sample were measured with CLARIOstar Plus Microplate Reader (BMG Labtech) and analyzed using MARS data analysis interface. A calibration curve was generated using manufacturer provided standards (0,0.1, 0.4, 1.5, 6.0, and 25 ng/mL) and a four parametric logistic (4-PL) curve fit was used to calculate sample values.

Male mice were orchiectomized at least 30 days prior to treatment. Male mice were anesthetized by isoflurane inhalation (Isoflurane, USP, Dechra Veterinary Products), with doses of 4-5% for induction and, 2-3% for maintenance. A 5 mg/kg dose of meloxicam (Pivetal^® Alloxate™, Aspen Veterinary Resources^®, LTD) was administered subcutaneously (SC) to each mouse for systemic analgesia. Lubricant ophthalmic ointment (Pivetal^® Artificial Tears, Aspen Veterinary Resources^®, LTD) was applied to both eyes to prevent corneal desiccation. Thermal support was provided using a warm water circulating heating pad. The incision site was surgically prepped with three alternating scrubs of 10% povidone-iodine and 70% ethanol. A small, midline, scrotal incision was made with tissue scissors. Each testicle was identified singly, and the associated spermatic cord was clamped with hemostats for at least 10 seconds, and the testicle removed. The incision was closed using surgical tissue adhesive (3M™ Vetbond Tissue Adhesive 1469, 3M). Sham ORX surgeries were performed using the same surgical protocol without surgical removal of the testicles.

Spatial transcriptomics was conducted using the GeoMx Digital Spatial Profiler (Nanostring) with a previously described protocol (40). Tissues were cut onto positively charged slides at a 5μm thickness, dried overnight, and baked at 65 °C for 1 hour. All subsequent steps were performed in an RNAse-free environment with diethyl pyrocarbonate (DEPC) water. Slides were de-paraffinized with xylene, rehydrated using graded alcohols, and held in 1X PBS for 1 minute. Tissues were incubated in Tris/EDTA pH 9.0 antigen retrieval solution (Invitrogen #00-4956-58) pre-heated to 100 °C for 15 minutes, washed in 1X PBS for 5 minutes, and transferred to 37 °C 1.0 µg/ml proteinase K (Ambion #AM2546) for 15 minutes. Slides were washed in 1X PBS for 5 minutes, then post-fixed with a 5-minute incubation in 10% neutral-buffered formalin (Epredia #5701) followed by two 5-minute incubations in Tris-glycine fixation stop buffer (Sigma #77-86-1, #G7126) and one 5-minute wash in 1X PBS. Slides were hybridized overnight at 37 °C with the Mouse Whole Transcriptome Atlas probe set (Nanostring #121400313) diluted in Buffer R (Nanostring #121300313) and DEPC water. Unbound probes were removed using two 25-minute incubations in stringency wash buffer comprised of 50% deionized formamide (Sigma #4650) and 50% 4X Saline Sodium Citrate (SSC) buffer (Sigma #S6639). Slides were washed twice with 2X SSC buffer for 2 minutes, then incubated in Buffer W (Nanostring #121300313) for 30 minutes. Slides were stained overnight in morphology marker PfHSP70 antibody (Rabbit polyclonal, 1:42 dilution, StressMarq #SPC-186D), washed twice with 2X SSC for 5 minutes, and detected with a 1 hour incubation in secondary antibody conjugated to AlexaFluor 647 (Goat polyclonal, 1:200 dilution, Jackson ImmunoResearch #111-605-144). This was followed by two 5 minute 2X SSC washes and a 1-hour incubation of labeled antibodies ASS1-AlexaFluor 488 (Rabbit clone EPR12398, 1:250 dilution, Abcam #ab208412) and GS-AlexaFluor 594 (Mouse clone 3B6, 1:60 dilution, Abcam #ab64613) cocktailed with nuclear counterstain Syto83 (2 uM, 1:10, Invitrogen #S11364). Slides were held overnight in 2X SSC buffer, then re-stained for 10 minutes with Syto83 immediately prior to loading the instrument.

Following whole-slide fluorescent imaging, circular ROIs were placed on tissue areas. Syto83 immunofluorescence was utilized for autofocus of GeoMx imaging. Immunofluorescence for ASS1 was used to identify periportal (PP), pericentral (PC), and interzonal (IZ) regions and PfHSP70, which cross reacts with Py, was used to identify the location of parasites. ROIs were generated using the circle tool and measured to a diameter of 200 µm, 100 µm, 75 µm, and 50 µm. Each ROI was selectively exposed to UV light, cleaving the barcode sequences and collecting them in a unique well in a 96-well collection plate (three plates in total). Libraries were prepared with Nanostring SeqCode construction kits according to manufacturer instructions, sequenced using NovaSeq 6000 SP flowcell, and processed using the GeoMX NGS Pipeline.

Differential expression of gene targets was determined with DESeq2 package (43). The regression model implemented was as follows:

Where Group is a designation for sex/hormone status of mouse (eg. male (M), female (F), and orchiectomized male (ORX)); Type is a designation for the ROI’s infection type (e.g. mock, bystander, and parasite); and Liver zone is a designation for liver zone (e.g. periportal (PP), interzonal (IZ), pericentral (PC)).

In order to quantify the joint effect of Group and Type using GSEA, we concatenated the Group and Type variables leading to the simplified version of the previous regression formula, omitting the bystander effects to focus the effects between mock and parasite stratified by group and controlling for liver zone:

Where Group_Type is a combined designation for sex/hormone status and infection type (e.g. F mock, F parasite, M mock, M parasite, ORX mock, ORX parasite); and Liver zone is a designation for liver zone (e.g. periportal (PP), interzonal (IZ), pericentral (PC)).

In order to provide an exhaustive set of differential expressed genes between all comparison, a fully stratified set of comparison for each independent variable (Group, Type, Zone) individually is included in the supplements (Supplementary Figures S9–S12).

Differentially expressed genes (DEGs) were defined as genes with an adjusted p-values (false-discovery rate) < 0.05 and |Log2 Fold Change| > 1.0.

Hypergeometic enrichment tests against Broad MSigDBHallmark (H) and KEGG (K) pathway gene sets (44, 45) were performed to determine pathways with significant overrepresentation. Gene set enrichment analysis (GSEA) was performed again H and KEGG gene sets with contrast fold changes using fast GSEA (46).

We used BayesPrism’s (47) (via InstaPrism (48)) cell-type deconvolution algorithm to determine the cell-type proportions in our spatial transcriptomics data. We determined cell-type proportions by deconvoluting our data with single-nucleus data from Hildebrandt et al. (23), which was conducted on C57BL/6 mice during Pb infection and included 14 cell types. The gene expression profiles for deconvolution were computed via genes averaged across each cell types within the snRNASeq dataset. To determine statistical significance of difference in cell type proportions, we compared groups with a Wilcoxon test with Benjamini–Hochberg adjusted p-values.

Details of the statistical tests applied to datasets shown in figures can be found in the above methods and in corresponding figure legends. All data points and n values reflect biological replicates. Data analyzed included outliers unless these could be explained by technical error. GeoMx data collection and gene expression analysis were not performed blind to the conditions of the experiments. Fluorescent microscopy with CLEC4F antibody and liver zonation were counted blinded to experimental conditions. Fluorescent microscopy and Plasmodium 18S rRNA RT-PCR data were assumed to be normal thus parametric tests were employed. Where relevant, statistical tests were two-sided. Individual p-values are provided in the figures. Statistical analyses were performed in R (version 4.3.2) or GraphPad Prism 9.1.2 Software. Plots used in this manuscript were generated with GraphPad Prism and the following R packages: ggplot2 (49), ggpubr (50), ComplexHeatmap (51), and UpSet (52).

To test if biological sex modify susceptibility to infection or subsequent liver stage development, we infected adult male and female BALB/cJ mice with 1 x10⁵Py spz intravenously (IV). Livers were isolated at 12, 24, and 44 hours post infection (hpi) for quantification of Plasmodium parasite RNA by RT-PCR (Figure 1a). At 12 and 24 hpi, no significant sex difference was observed. However, at 44 hpi, males had an approximately 3-fold higher liver burden compared to female age-matched mice (Figure 1b), indicating a sex difference in parasite growth or survival.

Given the established role of androgens in modulating the pathogenesis of hepatotropic infections (9) and recent evidence of androgen-mediated effects on immune responses to malaria liver stage vaccination (36, 53), we hypothesized that androgens play a role in regulating increased liver stage parasite burden during Plasmodium infection. To test this, we orchiectomized (ORX) adult male mice at least 14 days prior to spz injection and quantified liver burden as described above (Figure 1a). ORX is a common method for removing testicular hormones, and is frequently applied to reduce androgen levels in murine systems (54). ORX males exhibited reduced liver burden at 44 hpi, but not at 12 or 24 hpi (Figure 1b) when compared to intact male mice. The apparent bimodality observed at 24 hpi reflects differences between two independent experimental batches rather than biological heterogeneity. To further assess the role of androgens in regulating parasite survival, we employed an acute and reversible model to suppress testosterone production in male mice. Acyline is a gonadotropin releasing hormone (GnRH) antagonist that suppresses downstream production of testosterone in a dose-dependent manner and has previously been applied in mice (55), and humans (56, 57). We depleted testosterone with acyline prior to Py infection and measured liver burden at 44 hpi (Supplementary Figures S1a, b). We found reducing testosterone was sufficient to decrease liver burden outcomes in male mice. Testosterone repletion before infection restored Plasmodium 18S rRNA levels relative to acyline-only (testosterone-depleted) group (Supplementary Figure S1c), confirming its association with liver burden. Together, these findings lead us to conclude that androgens significantly influence parasite burden during the liver stage.

At least two factors could influence the quantity of Plasmodium rRNA at 44 hours: 1) the number of infected hepatocytes or 2) the rate of parasite replication within hepatocytes. To evaluate if sex influenced these factors, the number of infected hepatocytes was counted and parasite size was measured by fluorescence microscopy. There were more parasites in males compared to females and ORX males at 44 hpi, but not at 24 hpi (Figure 1c). Parasite size was not significantly different between groups at 44 hpi (Figure 1d), even when stratifying by liver zone (Supplementary Figure S2). Interestingly, parasite size was modestly, but significantly greater in intact males at the 24-hour timepoint, indicating that initial rate or initiation of replication might differ. Taken together, these data suggest that the higher liver burden at 44 hpi in males compared to females and ORX males reflects parasite survival rather than initial invasion or parasite replication rate.

To interrogate the impact of sex and sex hormones on the host response to Plasmodium infection, we performed GeoMx spatial genome-wide transcriptomic profiling on Py-infected BALB/cJ mouse liver tissue. Adult intact male, female, and ORX male mice were infected IV with 3 x10⁵Py spz or mock injected. The comparison with mock-infected mice enabled us to evaluate the baseline contributions of biological sex in this model. At 44 hpi, liver sections from the left lateral lobe were collected and prepared for digital spatial profiling. Regions of interest (ROIs) were selected based on staining with three markers: one for Plasmodium parasites (HSP70, a cytosolic marker), one for liver zonation (argininosuccinate synthetase 1, ASS1, which marks the periportal region of the liver), and one nuclear stain (Syto83). ROIs were selected in tissue from infected and mock infected mice. In infected mice, ROIs were selected to capture both local and distal immune responses—one encircling the infected hepatocyte and its immediate neighboring cells and another bystander region positioned away from any detectable parasites (Figure 2a). As zonal location (Periportal (PP), Interzonal (IZ), and Pericentral (PC)) is known to influence the host response to infection (23, 25), we selected around 3 ROIs per mouse per zone per infection type (Supplementary Table S1) and confirmed expected zonal gene expression phenotypes (Supplementary Figure S3).

First, we performed unsupervised clustering to identify overall trends in the data. We identified 5 clusters (0–4) (Figure 2b). Many of these clusters represented the hormone status of the mouse, with intact males (Cluster 0) differentiating from females (Clusters 1, 3, 4) and ORX males (Clusters 1, 2, 4) regardless of infection status (Figure 2c). Clusters associated with females and ORX males primarily differentiated on infection status (Cluster 4 – uninfected; Cluster 1, 2, and 3 – infected). In contrast, infected and uninfected intact males primarily clustered together (Cluster 0). We found genes enriched in female and ORX clusters were connected to the cytochrome P450 superfamily, fatty acid metabolism, and hormone biosynthesis (Figure 2d). Cluster 3 captured liver zone-specific expression with enrichment more in the PC and IZ regions (Figure 2c). With these clusters defined, we next conducted a supervised analysis to further investigate the impact of biological sex, liver zone, and infection type on gene expression outcomes.

To evaluate how sex and sex hormones impact the liver independent of liver zonation and infection type, we overlaid phenotypic information of all ROIs collected. Globally, male mice differentiate from female and ORX male mice with high expression of Cyp7b1, a cytochrome p450 family gene that is known to be regulated by biological sex in the murine liver (58). Female and ORX male mice also differentiate from intact male mice with higher expression of Cyp2b13, a gene connected to lipid and fatty acid metabolism (59) (Figures 3a, b). We were able to capture known differences in gene expression by biological sex and hormone status (18, 58, 60), and identified that clustering is principally determined by androgen presence, rather than biological sex.

To identify differentially expressed genes at steady state, we performed pairwise comparisons between all intact males and females, and intact males and ORX males, while accounting for covariates of infection and liver zone (Equation 1, β₁). We found most genes differentially expressed between females and intact males were also differentially expressed between ORX and intact males (Figure 3c; Supplementary Figure S4). Upregulated genes in females and ORX males were enriched for genes associated with fatty acid metabolism (e.g. Acot3, Hao2) and xenobiotic metabolism (e.g. Fmo3). Upregulated genes in males were linked to bile acid metabolism (e.g. Cyp7b1, Scp2) and complement/coagulation cascades (e.g. C4a, C9) (Figure 3d; Supplementary Figure S4), corroborating previously identified biases (61).

We next explored if biological sex and hormone status modified gene expression patterns by liver zone. Previous studies have identified sex-bias gene expression patterns by liver zones (18, 62). We identified three significantly differentially expressed genes, using an interaction term between sex and liver zone on all mice, that were modified by biological sex and hormone status and downregulated between PC and PP region: Cyp2c29, Csad, and Cyp2a4 (Supplementary Figure S5). While previous single-cell studies have shown that biological sex can influence liver zone-specific gene expression, the fact that GeoMX DSP ROIs represent multiple types of cells within the liver may have diluted this signal.

To estimate cell type proportions for each ROI in the spatial gene expression data we leveraged a single-nucleus RNAseq data set from livers of C57BL/6 mice infected with Pb published by Hildebrandt et al. (23). This dataset defined liver cell populations through cluster-based gene expression profiles, which we used for deconvolution (47, 48). We focused on mock-infected groups to define cellular composition by sex in the liver at baseline (Figure 3e). Comparing the proportions of 14 annotated cell types, we found that within our selected ROIs that hepatocytes comprised 20 – 40% and remaining cells type comprised 60 – 80% of the RNA measured in the tissue (Supplementary Figure S6). We note this was different than Hildebrandt et al., likely due to differences between in situ- and droplet-based sequencing platforms, divergent mouse strains, and snRNAseq method versus targeted probe-based sequencing. Four immune cell categories were captured with this method: KCs, monocytes and dendritic cells (DCs), T and NK cells, and B cells. Analysis of cell proportions revealed that ORX males had the highest levels of KCs, monocytes, DCs, and T and NK cells, followed by females, with intact males exhibiting the lowest levels (Figure 3f). Thus, this analysis recapitulates similar sex-specific differences in baseline immune cell proportions in tissue as previously identified in rodent models (63, 64).

To confirm the finding that females and ORX males have a higher density of KCs compared to males in mock-infected tissues, we used fluorescent microscopy and quantified KCs with a CLEC4F marker (Figure 3g). We found that females and ORX males have higher baseline levels of CLEC4F⁺ cells compared to the males (Figure 3h). Overall, this suggests that differences in baseline gene expression and immune cell composition between intact male and female mice are strongly influenced by androgen status, establishing a distinct metabolic and immune homeostatic state.

After establishing that biological sex and androgens alter innate immune cells and gene expression patterns at baseline, we aimed to evaluate whether these effects influence the host response to Py infection. First, we identified that Py infection induces a similar inflammatory state in both the ROIs around the parasite and bystander ROIs further from infected cells (Supplementary Figure S7). The ROIs around parasite-infected hepatocytes are also capturing uninfected cells, potentially masking a stronger differential signal to uninfected, bystander regions. To focus on the response closest to the infected-hepatocyte, we conducted pairwise comparisons between parasite and mock ROIs for female, male, and male ORX mice (Figure 4a; Equation 2). Differential gene expression analysis found that regardless of zonal location, IFNα responses were upregulated in infected female, males, and ORX males. Gene enrichment analysis conducted separately for females, males and ORX males confirmed enrichment of pathways previously identified as induced in response to Pb in female C57BL/6 mice (25). For example, all groups demonstrated an upregulation of response to type I interferons (IFNs) and antigen processing and both females and males showed downregulation of metabolism of fatty acids and bile acid in response to parasite infection (Figure 4b).

Further investigation in infected mice of the specific genes differentially induced between groups revealed a restricted inflammatory response within intact male mice. Overall, fewer genes were differentially induced in males, compared to female and ORX males (Figure 4c). While all groups demonstrated an upregulation of genes associated with cellular stress response (Saa1, Saa2, Orm2), the interferon response was strongest in females, intermediate in ORX males, and lowest in intact males. (Figure 4d). Upregulated genes at 44 hpi shared between female and ORX male mice, but not intact male mice, were linked to cellular stress responses and inflammation (e.g. Saa3, Cxcl9) and type I IFN response (e.g. Irf7, Tgtp1, Gbp6, Ly6e) (Figure 4e; Supplementary Table S2). At 44 hpi, females exhibited the greatest breadth of IFN response, while ORX males showed an intermediate phenotype between females and intact males (Figure 4d). Many differentially expressed genes in females were interferon-stimulated genes (ISGs), including transcription of Cxcl10, Isg15, Bst2, and Ifitm3 and ISGs connected to autophagy, including Igtp, Gbp2, Irmg1, Irmg2, and Ifi47 (Figure 4e; Supplementary Table S2). Thus, both female and ORX mice mount a more extensive inflammatory responses compared to intact male counterparts.

Further investigation into which immune responses are differentially regulated by biological sex revealed pathways associated with antigen processing and presentation that were more enriched within female and ORX male mice. We identified a coordinated set of expression of genes involved in antigen presentation via MHC class II (Cd74, H2-Eb1, H2-Ea), MHC class I (H2-D1, Tap1) (Figure 4f), and Mpeg1, a macrophage-expressed gene1 that plays a central role in antigen cross-presentation in DCs (65, 66). Taken together, this may indicate either reduced frequency or function of antigen-presenting cells (APCs) and monocyte-derived APCs involved in T cell priming of CD8⁺ T cells (67, 68) in male mice.

Genes associated with iron homeostasis were also differentially induced in response to Py infection between sexes. Plasmodium parasites are known to shift iron flux during hepatocyte invasion (69). Lsn2 was specifically increased within female and ORX male mice, which is constituently expressed primarily by neutrophils and is responsible for iron-trafficking from the cell. In contrast, Hamp2 encoding hepcidin-2, a regulator of iron metabolism, was specifically downregulated in female mice, which could be connected to low iron levels. Interestingly, Scd1, a gene involved in fatty acid metabolism, was reduced within male mice, but not female and ORX male mice (Supplementary Table S2). Together, this data demonstrates during Py infection, biological sex and sex hormones influence metabolic processes related to liver stage parasite development.

While we did not identify major gene expression differences based on zonal location, we had yet to evaluate potential sex difference in zonal patterns in infection rates. We quantified the location of parasites in the liver in a separate set of validation mice that were infected with 1 x10⁵Py IV and harvested 44 hpi (Supplementary Figure S8a). Here, zonation was determined with the markers HSP70, ASS1 (periportal marker), and GS (pericentral marker) (Supplementary Figure S8b). Across all samples, regardless of biological sex and hormone status, there were more periportally located parasites than pericentral (Supplementary Figure S8c). Parasite numbers were slightly higher in the PP zone and lower in the IZ region of female mice compared to males and ORX males, with no apparent difference in the PC region. Overall, liver zonation contributes minimally to sex- and hormone-based differences, though parasites in females may be modestly skewed toward the PP zone over the IZ region.

To identify the immune cell source driving sex-specific inflammatory response, we sought to evaluate the impact of biological sex on immune cell proportions around parasites in infected tissues. We again used the single-nucleus RNAseq data set by Hildebrandt et al. (23) to deconvolve the spatial gene expression data to estimate cell type proportion across the tissue. For this analysis, we compared immune cells between female, male and ORX male mice in the 200 µm diameter around a parasite. We found female and ORX male mice were enriched for KCs and monocytes and DCs compared to intact male mice (Figure 5a).

To identify if females and ORX males have higher density of KCs compared to intact males in infected tissues, we used fluorescent microscopy and quantified KCs with a CLEC4F marker in the set of mice from Figure 1 that were infected with 1x10⁵Py sporozoites and harvested at 44hpi (Figure 5b). We further found that following infection, females and ORX males retain this increased density, while all groups increase overall density above mock-infected (Figure 5c). To assess differences in recruited KCs while accounting for baseline variations, we calculated the fold difference relative to mock-infected samples based on sex and hormone status. Notably, although we observed a significant decrease in KC distance from the parasite between infected and mock-infected samples, there was no significant change in the fold difference by biological sex. (Figures 5c, d). This data overall indicates that sex difference in KC recruitment at 44 hpi is potentially related to innate difference in baseline cell densities rather than an intrinsic activity difference between recruitment of KC toward site of infection.