Single-cell RNA sequencing of mouse islets exposed to proinflammatory cytokines

Abstract

Exposure to proinflammatory cytokines is believed to contribute to pancreatic β-cell damage during diabetes development. Although some cytokine-mediated changes in islet gene expression are known, the heterogeneity of the response is not well-understood. After 6-h treatment with IL-1β and IFN-γ alone or together, mouse islets were subjected to single-cell RNA sequencing. Treatment with both cytokines together led to expression of inducible nitric oxide synthase mRNA (Nos2) and antiviral and immune-associated genes in a subset of β-cells. Interestingly, IL-1β alone activated antiviral genes. Subsets of δ- and α-cells expressed Nos2 and exhibited similar gene expression changes as β-cells, including increased expression of antiviral genes and repression of identity genes. Finally, cytokine responsiveness was inversely correlated with expression of genes encoding heat shock proteins. Our findings show that all islet endocrine cell types respond to cytokines, IL-1β induces the expression of protective genes, and cellular stress gene expression is associated with inhibition of cytokine signaling.

Cytokine and Nitric Oxide-Dependent Gene Regulation in Islet Endocrine and Nonendocrine Cells

“…Briefly, reads in each cell for each gene were divided by the total number of reads within that cell, multiplied by a factor of 10,000, and transformed using the natural logarithm. Samples from this and our previous study of similar design 23 were generated in succession rather than in parallel.…”

“…While IL-1β is thought to solely damage β-cells, recent studies support an alternative primary role of IL-1β and nitric oxide in the protection of β-cells. The expression of a number of antiviral and other protective genes is increased in β-cells following acute (6 h) exposure to the combination of IL-1β and IFN-γ, and a subset of these protective genes is stimulated by IL-1β alone 23 . Furthermore, nitric oxide protects β-cells from apoptosis and viral infection in a manner that is associated with its ability to inhibit mitochondrial oxidation and deplete cellular energy 24–26 .…”

“…A single-cell approach accommodates the capture and sequencing of multiple cell types within the islet without the need for purification of individual subtypes that is challenging with rare cell populations. This approach allowed us to demonstrate that all islet endocrine cells respond to acute cytokine exposure by increasing Nos2 and other inflammatory genes, by increasing antiviral and other protective genes, and by decreasing expression of identity genes 23 . In this first report 23 , a short cytokine treatment (6 h) was used to examine nitric oxide-independent gene expression.…”

“…This approach allowed us to demonstrate that all islet endocrine cells respond to acute cytokine exposure by increasing Nos2 and other inflammatory genes, by increasing antiviral and other protective genes, and by decreasing expression of identity genes 23 . In this first report 23 , a short cytokine treatment (6 h) was used to examine nitric oxide-independent gene expression. In the current report, we have focused our efforts on nitric oxide-dependent gene expression that occurs in response to a cytokine treatment for a duration at which the actions of cytokines are reversible (18 h).…”

The Role of Thioredoxin/Peroxiredoxin in the β-Cell Defense Against Oxidative Damage

“…PRDX3, the mitochondrially-localized isoform, is also expressed in b-cells, and its expression is increased following 24-hr cytokine treatment (IL-1b, IFN-g, and TNF-a) in C57/BL6 mouse islets (68). However, acute exposure of C57/BL6 mouse islets to cytokines (IL-1b and IFN-g, 6 hr) does not stimulate a change the mRNA accumulation of any peroxiredoxin isoform but increases TXNRD1 expression (69).…”