Sex-specific factors affecting intracellular infection of human primary macrophages by visceral Leishmania spp.

Abstract

Leishmaniasis is one of the most important neglected tropical diseases and is caused by protozoan parasites of the genus Leishmania. Depending on infecting parasite species and host immune status, the disease can manifest itself as self-healing skin ulcers, severe mucosal necrosis, or disseminated, often lethal, visceral infections of haematopoietic organs. To date, there are no licensed vaccines available and treatment options are limited and often accompanied by severe adverse effects. Many parasitic diseases occur with a sex bias in terms of incidence, morbidity and mortality towards male individuals. Leishmaniasis is such an example as it presents with higher numbers of clinical cases in male patients in various endemic areas. The reasons for this observed sex-specific difference are highly multifactorial and can include biological differences such as variances in chromosomes and hormones, as well as sociocultural disparities that can alter the susceptibility towards an infection and influence the outcome of the disease. Within the mammalian host, Leishmania parasites proliferate mainly in macrophages, while suppressing effective elimination by the host. In order to better understand the biological sex differences in leishmaniasis, the present project aimed to analyse the influence of donor sex on the infection of macrophages with Leishmania species at the cellular and transcriptional level. For the first time, using an in vitro infection model based on primary human macrophages with L. infantum and a high-throughput analysis, higher infection rates and parasite loads were detected in male macrophages than in female macrophages. These differences were most pronounced in later phases of infection (from 28 hours post infection). Due to their high plasticity in response to external stimuli, macrophages can polarise into pro-inflammatory M1 and anti-inflammatory M2 macrophages, which are distinguished by their different morphological and phenotypic characteristics. In this study, the polarisation during infection with L. infantum was investigated using microscopy- and flow cytometry-based methods. L. infantum-infected macrophages showed increased M1 polarisation and decreases in M2-like macrophages in both sexes, especially at early stages of infection, with a higher tendency towards M1 polarisation observed in female cells. Quantitative RNA sequencing of infected macrophages and corresponding controls of both sexes showed the most pronounced changes in mRNA levels at early time points of infection, but also more differentially expressed genes (DEGs) in female than in male macrophages. Over time (6, 28 and 52 hours after infection), the number of DEGs decreased in infected cells of both sexes. Genes that were similarly upregulated in both sexes were genes associated with important immunological signalling pathways, including TNF signalling, NF-𝜅B signalling, HIF-1𝛼 signalling, IL-17 signalling and FoxO signalling, mineral absorption, macrophage cytoskeletal remodelling and cytokine response. Specifically upregulated genes in female macrophages were associated with a more strongly induced interferon signalling pathway and proteasomal activities, while genes associated with interleukin response and Leishmania survival were preferentially upregulated in male macrophages. These results suggest that female macrophages exhibit increased transcriptomic activation and an enhanced anti-parasitic immune response, possibly leading to more effective elimination of the parasites in female-derived macrophages. The influence of sex hormones on L. infantum infection was investigated by pre-treating macrophages with androgens or estrogens prior to infection. While the administration of testosterone demonstrated no impact on infection rate or parasite burden, estradiol treatment correlated weakly with reduced parasite burden in infected female macrophages.

Taken together, this project identified several biological factors that may contribute to the divergent susceptibilities of sexes towards Leishmania infections. These factors include variances in macrophage polarisation, disparities in the expression of immunity-related genes, and the influence of sex hormones. These findings serve as a foundational framework for subsequent experiments which may identify therapeutic targets.