Structural cells: from basic scaffolds to key players during immune responses

Immune functions are not unique to hematopoietic cells, as many other cell types display basic mechanisms of pathogen defense [1-3]. The epithelium, made by epithelial cells, constitute the structure of tissues and organs in the mammalian body, creating the internal and external surfaces and barriers; the endothelium, comprised of endothelial cells, form the ining of blood vessels; and the stroma, made of fibroblast, provide essential connective tissue. All of them are structural cells and have been shown to contribute in important ways to mammalian immunity [4-8]. 

A recent article published in Nature [9] has demonstrated the relationship between structural cells and immune system function by taking advantage of multi-omics profiling techniques. Multi-omics profiling allows the dissection of immune regulation in a systematic, genome-wide manner. The combination of multi-omics profiling with integrative bioinformatics allows for the establishment of a high-resolution atlas of both structural cells and non hematopoietic immune regulation in the mouse. They observed a widespread expression of immune regulators and cytokine signaling molecules in structural cells, organ-specific adaptation to the tissue environment, and unexpectedly diverse capabilities for interacting with hematopoietic cells. Moreover, cell-type-specific and organ-specific differences in immune gene activity were reflected by characteristic patterns of chromatin regulation. Structural cells showed an epigenetically encoded immune potential under homeostatic conditions, and those genes were preferentially upregulated in response to viral infection. 

Workflow for structural cell analysis. Krausgruber, T., et al., Structural cells are key regulators of organ-specific immune responses. Nature, 2020. 583(7815): p. 296-302

Authors performed a multi-omics profiling of endothelium, epithelium and fibroblasts from mouse brain, caecum, heart, kidney, large intestine, liver, lung, lymph node, skin, small intestine, spleen and thymus through three genome-wide assays: 1) RNA-seq; 2) chromatin accessibility by assay for transposase-accessible chromatin using sequencing (ATAC-seq); and 3) epigenome profiling by ChIPmentation with an antibody against the promoter and enhancer-linked histone 3 lysine 4 bimethylation (H3K4me2) mark. Structural cells were purified with the endothelium marker CD31, the epithelium marker EpCAM and the fibroblast marker GP38. The cellular identity of the sorted structural cells was further confirmed by RNA-seq data analysis, which showed the expected organ specific and cell type specific patterns of gene expression compared to published multi tissue expression profiles. They found that the expression profiles of structural cells within the same organ were globally more similar to each other than structural cells of the same type across organs, suggesting that the tissue and organ environment has a major effect on the transcriptomes of structural cells. Therefore, the context is highly relevant for the behavior and expression profile of structural cells. 

Analysis of cell cell interaction network between structural and immune cells predicted a frequent crosstalk under homeostasis conditions. Differences across cell types and organs were driven by the characteristic expression patterns of cell-surface proteins and secreted factors in structural cells. Structural cells also showed widespread activity of immune gene and regulatory modules, with highly cell type specific and organ specific patterns, which are expected to mediate cell type specific and organ specific interactions with the immune system cells. 

Network of potential cell-cell interactions between structural and immune cells. Krausgruber, T., et al., Structural cells are key regulators of organ-specific immune responses. Nature, 2020. 583(7815): p. 296-302.

Combining RNA-seq data with ATAC-seq profiles of chromatin accessibility and ChIPmentation maps for the promoter and enhancer linked H3K4me2, the gene regulatory networks across cell types and organs were compared, founding an extensive cell type specific and organ specific chromatin regulation at immune gene loci. A subset of crucial immune genes showed high chromatin accessibility in most samples, indicative of a shared core of immune regulation in structural cells. Although many key regulators of transcription showed cell type specific and organ specific activity, some groups of transcription factors were ubiquitously active in structural cells, which may constitute a shared regulatory basis of immune gene activity. Data suggest a model in which constitutively active regulators stablish a shared core of immune functions, while additional factors contribute to cell type specific and organ specific adaptions. 

Epigenome profile not only capture the current regulatory states of cells, but also reflect their future potential to respond to various stimuli. Structural cells may be epigenetically primed for immune gene activation, which would pre-program them for a rapid response to immunological challenges. Authors quantified the chromatin accessibility of each gene promoter and compared it to the expression level of the corresponding gene, thus identifying genes with low expression but high promoter accessibility, indicative of unrealized potential for increased expression. As expected, genes with unrealized epigenetic potential in structural cells were enriched for immune functions; furthermore, from the 1,665 genes with unrealized epigenetic potential, 335 were annotated with at least one immunological term. Liver, lymph node, spleen and thymus harbor a more pronounced epigenetic potential for gene activation in response to various stimuli, whereas brain, caecum, heart, kidney, large intestine and skin have less room for unrealized epigenetic potential. The epigenetic potential is expected to facilitate the rapid response to immunological challenges in a cell type specific and organ specific manner. 


Cell type specific and organ specific chromatin regulation at immune gene loci within structural cells of different tissues. Black boxes indicate the differences between tissues and organs. Krausgruber, T., et al., Structural cells are key regulators of organ-specific immune responses. Nature, 2020. 583(7815): p. 296-302.

Lymphocytic choriomeningitis virus (LCMV) infection in mice resulted in changes of structural cell composition in most organs, with a differential gene expression in a cell type specific and organ specific manner. The transcriptional response was globally more similar among structural cells of all three types within the same organ than between structural cells of a given type across organs, similar to homeostatic conditions. Genes with unrealized potential were overrepresented among the LCMV-induced genes, with the strongest association in liver, lung and spleen, although these organ-specific differences showed no clear correlation with differences in viral load, thus constituting intrinsic regulatory differences between cells types and organs. 

Upregulated genes in response to LCMV infection showed strong enrichment for immune functions, including positive regulation of those for immune responses, defense responses to virus, cellular response to IFNγ and antigen processing and presentation. Authors also found a widespread upregulation of interferon-induced and interferon-simulated genes, as well as of key transcriptional regulators in the interferon pathway, which indicates a strong interferon response to LCMV infection in structural cells. Furthermore, there was an increase in the strength and scope of predicted cell-cell interactions after infection as compared to homeostatic conditions, largely driven by upregulated gene expression levels for receptors and ligands in structural cells. Therefore, LCMV infection triggered widespread activation of immune genes that were lowly expressed but epigenetically poised under homeostatic conditions, thus supporting the model of epigenetically encoded potential for immune gene activation in structural cells in the context of viral infection. 

It seems that structural cells are epigenetically pre-programmed for a swift response to a variety of immunological challenges. It will be interesting to explore how the epigenetic potential of structural cells respond to other stimuli and whether it can be modulated for therapeutic purposes in the context of autoimmune diseases or the tumor microenvironment. 


References

1.     Schleimer, R.P., et al., Epithelium: at the interface of innate and adaptive immune responses. The Journal of allergy and clinical immunology, 2007. 120(6): p. 1279-1284.
2. Pober, J.S. and W.C. Sessa, Evolving functions of endothelial cells in inflammation. Nat Rev Immunol, 2007. 7(10): p. 803-15.
3. Buckley, C.D., et al., Stromal cells in chronic inflammation and tertiary lymphoid organ formation. Annu Rev Immunol, 2015. 33: p. 715-45.
4. Turley, S.J., V. Cremasco, and J.L. Astarita, Immunological hallmarks of stromal cells in the tumour microenvironment. Nat Rev Immunol, 2015. 15(11): p. 669-82.
5. Perez-Shibayama, C., C. Gil-Cruz, and B. Ludewig, Fibroblastic reticular cells at the nexus of innate and adaptive immune responses. Immunological reviews, 2019. 289(1): p. 31-41.
6. Nowarski, R., R. Jackson, and R.A. Flavell, The Stromal Intervention: Regulation of Immunity and Inflammation at the Epithelial-Mesenchymal Barrier. Cell, 2017. 168(3): p. 362-375.
7. Malhotra, D., A.L. Fletcher, and S.J. Turley, Stromal and hematopoietic cells in secondary lymphoid organs: partners in immunity. Immunol Rev, 2013. 251(1): p. 160-76.
8. Humbert, M., S. Hugues, and J. Dubrot, Shaping of Peripheral T Cell Responses by Lymphatic Endothelial Cells. Front Immunol, 2016. 7: p. 684.
9. Krausgruber, T., et al., Structural cells are key regulators of organ-specific immune responses. Nature, 2020. 583(7815): p. 296-302.





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