Exploring the molecular mechanisms that underpin M. tuberculosis and dendritic cell interactions

Abstract

Dendritic cells (DCs) are essential to the development of protective immunity through their potent ability to present antigens to T-cells. Once activated, T-cells promote the proliferation of antibody-producing cells and use antigen-targeted, cytotoxic mechanisms to neutralise infectious agents. In diseases such as Tuberculosis (TB), ineffective T-cell responses lead to uncontrolled dissemination of the pathogen, indicating that T-cells can be crucial to disease outcome. Phagocytic cells, in contrast, are believed to facilitate the survival of Mycobacterium tuberculosis (Mtb) in the host, by providing a protective, intracellular niche from other patrolling immune cells. As DCs are phagocytic and prime inducers of T-cell responses, they may play a central role in TB pathogenesis.
In this study, transcriptomic data from TB-infected patients and Mtb-infected DCs were analysed and compared to identify signatures of genes that may allude to a distinct response of DCs to Mtb. This began with a comparison between bulk RNA-seq and microarray datasets derived from DC’s infected with Mtb or challenged with other pathogens/pathogen-associated molecular patterns. A 211 gene signature was found to distinguish Mtb-infected DCs from other conditions, and contained within were genes related to a range of processes including antigen presentation and iron regulation. Principal component analysis further suggested the transcriptomic profile of DCs diverges from other pathogens in response to Mtb.
This signature was used to extrapolate a pattern of gene expression in bulk RNA-seq data derived from the lymph nodes of TB-infected patients. Using gene co-expression networks, 406 genes were found to be highly associated with the in vitro signature. Importantly, this showed that a DC-related pattern of gene expression could be detected in the lymph nodes, critical sites for antigen presentation. Similar to the in vitro signature, genes associated with iron regulation, production of reactive oxygen species and production of the antioxidant glutathione, were found.
In addition, two in vivo datasets, one derived from the lung tissue and the other derived from the blood of TB-exposed individuals were analysed to identify genes modulated in response to TB exposure. Across the two datasets, DC subpopulations: cDC1, cDC2, pDC and pre-DCs (AXL+SIGLEC6+) were identified, indicating that DCs were present in clinical samples. In addition, 896 genes were found to be differentially modulated across all DC subtypes, and included genes associated with iron regulation, superoxide metabolism and fatty acid synthesis.
Combining the DC signatures from each set of analyses indicated that SAT1, a gene involved in polyamine synthesis was upregulated in all three signatures. Combined with other genes found in at least two chapters, SAT1, TFRC, FTH1 and FTL enriched Ferroptosis, an iron-dependent cell death mechanism driven by an imbalance of fatty acid synthesis, iron-generated oxygen radicals and glutathione production, through gene ontology. Differential gene expression analysis showed consistent modulation of 34 genes in the pathway in Mtb-infected DCs and TB-infected lymph nodes. The modulation of some of these genes was found to be recapitulated in an in vitro cell culture model with monocyte-derived dendritic cells at the mRNA level using RT-qPCR and at the protein level for Haemoxygenase-1, Transferrin Receptor (CD71) and Prion Protein (CD230) using flow cytometry. Lipid peroxidation, a hallmark of Ferroptosis was shown to increase in infected DCs and correlated with cell death, suggesting Mtb-infection was driving the modulation of Ferroptosis genes, leading to increased lipid peroxidation and cell death. Finally, immunohistochemistry was used to show that Thioredoxin, a gene associated with Ferroptosis, was more prevalent in TB-infected lymph nodes compared to control tissue. Combined these findings provide compelling evidence that Ferroptosis, a mechanism known to increase cell death and suppress antigen presentation is driven by Mtb-infection. Ultimately, reduced DC function in clinical settings may result in a limited immune response and sustained infection.

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Identifiers

ORCID for Paul Elkington: orcid.org/0000-0003-0390-0613

ORCID for Andres Vallejo Pulido: orcid.org/0000-0002-4688-0598

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