Upon infecting its vertebrate host, the malaria parasite initially invades the liver where it undergoes massive replication, whilst remaining clinically silent. The coordination of host responses across the complex liver tissue during malaria infection remains unexplored. Here, we perform spatial transcriptomics in combination with single-nuclei RNA sequencing over multiple time points to delineate host-pathogen interactions across Plasmodium berghei-infected liver tissues. Our data reveals significant changes in spatial gene expression in the malaria-infected tissues. These include changes related to lipid metabolism in the proximity to sites of Plasmodium infection, distinct inflammation programs between lobular zones, and regions with enrichment of different inflammatory cells, which we term ‘inflammatory hotspots’. We also observe significant upregulation of genes involved in inflammation in the control liver tissues of mice injected with mosquito salivary gland components. However, this response is considerably delayed compared to that observed in P. berghei-infected mice. Our study establishes a benchmark for investigating transcriptome changes during host-parasite interactions in tissues, it provides informative insights regarding in vivo study design linked to infection and offers a useful tool for the discovery and validation of de novo intervention strategies aimed at malaria liver stage infection.

The liver is a major entry point and gatekeeper for invasive pathogens. However, high-resolution, spatiotemporal transcriptomic analysis of host–pathogen interactions has remained challenging. Afriat et al. have deconvoluted Plasmodium berghei liver-stage maturation at an unprecedented scale and discovered molecular signatures of heterogeneity during pre-erythrocytic development of malarial parasites.

Unicellular parasites of the apicomplexan phylum have a considerable effect on global health and agriculture. Two prominent examples of this phylum include malaria causing parasites of the Plasmodium genus and the widely prevalent parasite Toxoplasma gondii. While sharing a common ancestor, these parasites occupy unique biological niches, follow distinct life cycles, and result in different courses and outcomes of disease. In response to the parasite, the mammalian host has developed efficient and effective defense strategies. However, both Plasmodium and Toxoplasma have evolved strategies to evade the host’s defense response. Plasmodium parasites infect distinct tissues and cell types whereas T. gondii parasites are highly promiscuous and infect all nucleated cells. The identification of key factors involved in the interaction between the host and parasite is crucial for disease intervention, prevention, and eventually eradication efforts.

Next-generation sequencing technologies have proven effective tools to investigate the response in a tissue or cell population of an infected organism. Novel genomics methods such as single-cell RNA-seq and spatial transcriptomics have enabled the investigation of heterogeneous transcriptional responses of individual cells in a population as well as heterogeneous expression profiles at spatially distinct tissue positions across entire tissue sections. This thesis pioneers the exploration of these methods in discerning the enormous complexity underlying host-parasite interplay.

In Paper I, we determine spatial components of naive mouse liver in its true tissue context. We define gene expression gradients of pericentral and periportal zones in the liver and predict vein types with ambiguous annotations, based on in situ transcriptional profiles. We further identify novel spatial structures with distinct transcriptional profiles, associated with tissue integrity and integrate cell type proportions across the tissue.

In Paper II we investigate host-pathogen interactions in P. berghei infected liver sections with spatiotemporal resolution. We establish spatial gene expression gradients from infection sites exhibiting upregulation of lipid metabolism associated genes 38 hours post-infection, suggesting a potential role of these pathways in immune evasion. We further show that local and systemic inflammation are delayed but not ablated in salivary gland lysate challenged control livers and propose that local inflammatory hotspots may represent an important spatial component for parasite development in the liver.

In Paper III we use dual scRNA-seq to investigate heterogeneous transcription of mouse bone marrow-derived dendritic cells (BMDCs) infected with two distinct genotypes of T. gondii parasites. We show differential responses towards the two T. gondii genotypes in two distinct subpopulations of BMDCs over multiple time points post infection. Moreover, we generate co-expression networks that define host and parasite genes, which are likely involved in the modulation of host immunity.

In summary, this thesis aims to characterize host-pathogen interactions of two major apicomplexan genera in two distinct cell niches of the murine host with spatiotemporal or single cell resolution. In detail, this encompasses the study of spatial structures of the host in the liver environment and the spatiotemporal consequences of an infection with P. berghei. Furthermore, the aims include deciphering heterogeneous interactions between two distinct T. gondii strains and infected BMDCs.

Dendritic cells and macrophages are integral parts of the innate immune system and gatekeepers against infection. The protozoan pathogen, Toxoplasma gondii, is known to hijack host immune cells and modulate their immune response, making it a compelling model to study host-pathogen interactions. Here we utilize single cell Dual RNA-seq to parse out heterogeneous transcription of mouse bone marrow-derived dendritic cells (BMDCs) infected with two distinct genotypes of T. gondii parasites, over multiple time points post infection. We show that the BMDCs elicit differential responses towards T. gondii infection and that the two parasite lineages distinctly manipulate subpopulations of infected BMDCs. Co-expression networks define host and parasite genes, with implications for modulation of host immunity. Integrative analysis validates previously established immune pathways and additionally, suggests novel candidate genes involved in host-pathogen interactions. Altogether, this study provides a comprehensive resource for characterizing host-pathogen interplay at high-resolution.

Background: Plasmodium vivax was endemic in northern Europe until the early twentieth century. Considering climate change and the recent emergence of other vector borne diseases in Europe, historical insight into the relationship between malaria and environmental factors in northern Europe is needed. This article describes malaria epidemiology in late-nineteenth century Denmark.

Methods: We described the seasonality and spatial patterns of malaria, and the relationship of the disease with environmental factors such as soil types, clay content and elevation for the period 1862–1914. We studied demographic and seasonal patterns and malaria mortality in the high-morbidity period of 1862–1880. Finally, we studied the relationship between malaria seasonality and temperature and precipitation using a Spearman correlation test.

Results: We found that the highest incidence occurred in eastern Denmark. Lolland-Falster medical region experienced the highest incidence (14.5 cases per 1000 pop.) and Bornholm medical region experienced the lowest incidence (0.57 cases per 1000 pop.). Areas with high malaria incidence also had high soil clay content, high agricultural production, and Lolland-Falster furthermore has a low elevation. Malaria incidence typically peaked in May and was associated with high temperatures in July and August of the previous year but not with precipitation. The case fatality rate was 0.17%, and the disease affected both sexes and all age groups except for infants. In 1873, a large epidemic occurred following flooding from a storm surge in November 1872.

Conclusions: Malaria gradually declined in Denmark during our study period and had essentially disappeared by 1900. The high adult and low child morbidity in 1862–1880 indicates that malaria was not highly endemic in this period, as malaria is most frequent among children in highly endemic areas today. The association of high malaria incidence in spring with warmer temperatures in the previous summer suggests that transmission took place in the previous summers. The close geographical connection between malaria and soil types, agricultural production and elevation suggests that these factors are detrimental to sustain endemic malaria. Our findings of a close connection between malaria and environmental factors such as climate and geography provides insights to address potential reintroduction of malaria in temperate climates.

The inflamed rheumatic joint is a highly heterogeneous and complex tissue with dynamic recruitment and expansion of multiple cell types that interact in multifaceted ways within a localized area. Rheumatoid arthritis synovium has primarily been studied either by immunostaining or by molecular profiling after tissue homogenization. Here, we use Spatial Transcriptomics, where tissue-resident RNA is spatially labeled in situ with barcodes in a transcriptome-wide fashion, to study local tissue interactions at the site of chronic synovial inflammation. We report comprehensive spatial RNA-Seq data coupled to cell type-specific localization patterns at and around organized structures of infiltrating leukocyte cells in the synovium. Combining morphological features and high-throughput spatially resolved transcriptomics may be able to provide higher statistical power and more insights into monitoring disease severity and treatment-specific responses in seropositive and seronegative rheumatoid arthritis.

Reconstruction of heterogeneity through single cell transcriptional profiling has greatly advanced our understanding of the spatial liver transcriptome in recent years. However, global transcriptional differences across lobular units remain elusive in physical space. Here, we apply Spatial Transcriptomics to perform transcriptomic analysis across sectioned liver tissue. We confirm that the heterogeneity in this complex tissue is predominantly determined by lobular zonation. By introducing novel computational approaches, we enable transcriptional gradient measurements between tissue structures, including several lobules in a variety of orientations. Further, our data suggests the presence of previously transcriptionally uncharacterized structures within liver tissue, contributing to the overall spatial heterogeneity of the organ. This study demonstrates how comprehensive spatial transcriptomic technologies can be used to delineate extensive spatial gene expression patterns in the liver, indicating its future impact for studies of liver function, development and regeneration as well as its potential in pre-clinical and clinical pathology.

Background: Understanding of the impacts of climatic variability on human health remains poor despite a possibly increasing burden of vector-borne diseases under global warming. Numerous socioeconomic variables make such studies challenging during the modern period while studies of climate-disease relationships in historical times are constrained by a lack of long datasets. Previous studies have identified the occurrence of malaria vectors, and their dependence on climate variables, during historical times in northern Europe. Yet, malaria in Sweden in relation to climate variables is understudied and relationships have never been rigorously statistically established. This study seeks to examine the relationship between malaria and climate fluctuations, and to characterise the spatio-temporal variations at parish level during severe malaria years in Sweden 1749-1859.

Methods: Symptom-based annual malaria case/death data were obtained from nationwide parish records and military hospital records in Stockholm. Pearson (r(p)) and Spearman's rank (r(s)) correlation analyses were conducted to evaluate inter-annual relationship between malaria data and long meteorological series. The climate response to larger malaria events was further explored by Superposed Epoch Analysis, and through Geographic Information Systems analysis to map spatial variations of malaria deaths.

Results: The number of malaria deaths showed the most significant positive relationship with warm-season temperature of the preceding year. The strongest correlation was found between malaria deaths and the mean temperature of the preceding June-August (r(s) = 0.57, p < 0.01) during the 1756-1820 period. Only non-linear patterns can be found in response to precipitation variations. Most malaria hot-spots, during severe malaria years, concentrated in areas around big inland lakes and southern-most Sweden.

Conclusions: Unusually warm and/or dry summers appear to have contributed to malaria epidemics due to both indoor winter transmission and the evidenced long incubation and relapse time of P. vivax, but the results also highlight the difficulties in modelling climate-malaria associations. The inter-annual spatial variation of malaria hot-spots further shows that malaria outbreaks were more pronounced in the southern-most region of Sweden in the first half of the nineteenth century compared to the second half of the eighteenth century.