Single Cell RNAseq Analysis of Cytokine-Treated Human Islets: Association of Cellular Stress with Impaired Cytokine Responsiveness

Abstract Pancreatic β-cells are essential for survival, being the only cell type capable of insulin secretion. While they are believed to be vulnerable to damage by inflammatory cytokines such as interleukin-1 beta (IL-1β) and interferon-gamma, we have recently identified physiological roles for cytokine signaling in rodent β-cells that include the stimulation of antiviral and antimicrobial gene expression and the inhibition of viral replication. In this study, we examine cytokine-stimulated changes in gene expression in human islets using single-cell RNA sequencing. Surprisingly, the global responses of human islets to cytokine exposure were remarkably blunted compared to our previous observations in the mouse. The small population of human islet cells that were cytokine responsive exhibited increased expression of IL-1β-stimulated antiviral guanylate-binding proteins, just like in the mouse. Most human islet cells were not responsive to cytokines, and this lack of responsiveness was associated with high expression of genes encoding ribosomal proteins. We further correlated the expression levels of RPL5 with stress response genes, and when expressed at high levels, RPL5 is predictive of failure to respond to cytokines in all endocrine cells. We postulate that donor causes of death and isolation methodologies may contribute to stress of the islet preparation. Our findings indicate that activation of stress responses in human islets limits cytokine-stimulated gene expression, and we urge caution in the evaluation of studies that have examined cytokine-stimulated gene expression in human islets without evaluation of stress-related gene expression.


Introduction
Pancreatic β-cells, responsible for synthesis and secretion of insulin in response to a glucose c hallenge , reside in the islets of Langerhans and are essential for survival of the organism as the only cell type capable of producing this hormone.In the absence of pancreatic β-cells, which occurs during autoimmune-mediated destruction, type 1 diabetes ensues.While the killing of these cells is primarily mediated by Tcell-de pendent mechanisms, [1][2][3] inflammator y cytokines like interleukin-1 beta (IL-1 β) and interferon-gamma (IFN-γ ) produced by macr opha ges and T-l ymphocytes 4 , 5 ar e believ ed to contribute to β-cell damage and disease development.Islets ar e highl y v ascularized and r ecei v e a dispr oportionatel y large amount of blood flow, a feature that is crucial for proper blood glucose control but also facilitates exposure of β-cells to circulating cytokines produced during a viral or bacterial infection. 6 , 7It has long been known that these cytokines stimulate β-cells to express inducible nitric oxide synthase (iNOS), and the resulting production of micromolar levels of nitric oxide inhibit mitochondrial oxidation (aconitase activity and electron transport) and insulin secretion.[10][11][12][13] Although these effects of cytokines have been viewed as damaging to β-cells, our recent studies suggest that there are physiological roles for cytokine signaling in the endocrine islet that are aimed at protecting the β-cells from environmental threats.Interleukin-1 beta stimulates expression of a subset of antiviral and antimicrobial genes in β-cells and other islet endocrine cell types in a nitric oxide-independent manner. 14 , 15itric oxide, by inhibiting mitochondrial oxidation, attenuates viral r e plication in a β-cell-selecti v e manner, and nitric oxide is a potent inhibitor of insulinoma cell apoptosis. 16 , 17Importantly the inhibition of mitochondrial oxidation and insulin secretion ar e completel y r ev ersib le, and β-cells hav e efficient mechanisms to r e pair dama ged DNA; it is onl y pr olonged incubations with IL-1 β (greater than 36 h in vitro) that lead to irr ev ersib le damag e. [18][19][20][21] Tog ether, these pr evious studies performed in r odent and human islets support a physiological model in which IL-1 β signals to islet endocrine cells to increase the expression of protecti v e, anti-pathogen factors.
4][25] For example, it has been suggested that, while cytokines stimulate nitric oxide production in human islets, the inhibition of insulin secretion by cytokines is independent of nitric oxide. 23 , 257][28] Observ ed differ ences between r odent and human islets ar e not limited to cytokine signaling. 29Of note, it has also been r e ported that human islets express higher levels of heat shock proteins than rodent islets. 30 , 31Because of these r e ported differ ences, it is critical to determine if IL-1 β promotes protective responses in human β-cells.
In this study, we aimed to determine, by single-cell RNA sequencing (scRNA-seq), the effects of cytokines on gene expression in human islets.Specifically, we focused on identifying the earl y r esponses following a short 6-h exposure and nitric oxide-de pendent r esponses following a longer 18-h exposur e.This approach also allowed us to characterize the heterogeneity of human islet cell types with respect to their cytokine r esponse.Ov er 45,000 cells wer e anal yzed fr om 3 inde pendent, non-diabetic human islet preparations exposed to IL-1 β and IFN-γ with or without the nitric oxide synthase inhibitor N G -monomethyl-L -arginine (NMMA) for 6 or 18 h.In comparison to our previous analyses of mouse islet cells exposed to similar cytokine treatments, 14 , 15 human β-cells demonstrated markedl y b lunted r esponses to cytokine stim ulation, both in terms of the percentage of "responsive" cells and in terms of the percentage of differentially expressed genes.This blunted r esponse w as also observ ed in human α-, δ-, and PP-cells.We did identify a small subset of NOS2 + β-cells with similar cytokine-stim ulated gene expr ession changes to those pr eviousl y observ ed in the mouse, suggesting that the same signaling ev ents likel y occur in both species.The NOS2 − β-cells had higher expression of genes encoding several ribosomal proteins and heat shock proteins compared to the NOS2 + cells.One of these ribosomal protein-encoding genes, RPL5 , was positively correlated with indicators of cellular stress and was negatively correlated with cytokine-stimulated genes.Importantly, high expression of RPL5 pr edicted failur e to r espond to cytokines.This negati v e association between cytokine r esponsi v eness and ribosomal pr otein expr ession w as observ ed not onl y in β-cells but also in endocrine non-β-cells and non-endocrine cells.3][34][35] Taken together, our results suggest that there are no major species differences in the response of islet cells to cytokines and that the major differences are in the induction of a stress response .Therefore , caution should be used when interpreting results of studies using isolated human islets that do not assess str ess-r elated genes, as str ess is a welldefined r e pr essor of cytokine signaling and may impact multiple metabolic and signaling events.

Islet Isolation, Culture, and Treatment
Pancr eatic islets wer e cultur ed for 2-5 d after r eceipt at 37 • C and 5% CO 2 in CMRL supplemented with 10% heat-inacti v ated FBS and containing 5.5 mM glucose as pr eviousl y described. 36ntact islets were left untreated or were treated with following cytokines: IL-1 β, IFN-γ , or IL-1 β + IFN-γ for 6 or 18 h.We also examined the effects of the NOS inhibitor NMMA on islets treated for 18 h with or without IL-1 β + IFN-γ .Human recombinant IL-1 β was used at a concentration of 50 U/mL, human recombinant IFN-γ at a concentration of 500 U/mL, and NMMA at a concentration of 2 mM.Independent scRNAseq experiments were performed using human islets from 3 different donors.

Single-Cell RNA-Sequencing of Human Islets
Following treatment with cytokines, islets were incubated in 0.48 mM EDTA in phosphate-buffer ed saline and then a gitated in 1 mg/mL trypsin in Ca 2 + /Mg 2 + -free HBSS to disperse into single cells.Cells wer e filter ed and resuspended in CMRL media before being loaded into the Chromium Controller (10x Genomics).A wetting failure occurred during the generation of gel beads in emulsion (GEMs) of sample 5 (18 h IL-1 β treatment) from the first experimental replicate.For this reason, this sample was excluded from further analyses.scRNA-seq libr aries w er e pr e par ed using the Chr omium Single Cell 3 v3 Reagent Kit (10x Genomics) for replicate 1 and the Chromium Next GEM Single Cell 3 v3.1 Reagent Kit (10x Genomics) for r e plicates 2 and 3 according to the man ufactur er's pr otocol.Libr aries w ere sequenced using the NextSeq 500/550 High Output Kit v2.5 flow cell (150 cycles, Illumina) according to the 10x Genomics protocol.Samples were sequenced to a depth that r eturned appr oximatel y 30,000 r eads per cell on av era ge.Cell-Ranger (10x Genomics) functions "mkfastq" and "count" were used to demultiplex the sequencing data and generate genebarcode matrices (10x Genomics).Reads were aligned to human r efer ence genome assemb l y hg38.All scRNA-seq anal ysis w as performed in R (version 4.3.1)using the package Seurat (version 4.0.0). 37Number of genes detected per cell and percent of mitoc hondrial genes w ere plotted, and outlier cells were removed [number of genes fewer than 200 or greater than 3500 (r e plicate 1) or 5500 (r e plicates 2 and 3) or percent mitochondrial genes ov er 10% (r e plicate 1) or 15% (r e plicates 2 and 3)] to filter out doublets and cells with low read quality, leaving 47,121 of the original 70,236 cells.Cell cycle genes were re gressed.Seur at function "SCTransform" was used to integrate samples from all 3 experimental r e plicates into 1 dataset to reduce batch effects. 38rincipal component analysis was performed, and the top 50 principal components were used for Uniform Manifold Approximation and Pr ojection (UMAP) anal ysis, with clustering performed using the Louvain algorithm.All samples were normalized using Seurat's default normalization settings.

Mouse Islet Scrna-Seq Datasets
Our pr eviousl y pub lished single-cell RNA-sequencing datasets using mouse islets exposed to cytokines for 6 or 18 h were used for comparison to human islets.Data can be found under GEO accession numbers GSE156175 and GSE183010. 14 , 15

Functional Annotation Clustering Analysis
Lists of differ entiall y expr essed genes were inputted into the web interface of Database for Annotation, Visualization, and Inte gr ated Discov er y 39 and wer e anal yzed for enriched gene categories using the default settings.Similar annotations are grouped into "functional annotation clusters" and given an enrichment score defined as the geometric mean in −log scale of the P -values of the annotations in the functional annotation cluster.A higher enrichment score reflects lower P -values in the group and is more likely to be biologically meaningful.

Sta tistical Anal ysis
For differential expression analyses, P -values were calculated using the Wilcoxon test, and Bonferroni correction was used to av oid false positi v es.An av era g e log 2 (fold chang e) of 0.25 and adjusted P -value of 0.05 were the threshold used to declare significance .F isher's exact test w as used to compar e percenta ges of g enes chang ed by cytokine treatment in human islet cells to mouse islet cells.Linear r egr ession anal yses wer e performed using the ordinary least squares method in R using only cells with positi v e expr ession v alues of the genes being compar ed.

Single-Cell RNA-Sequencing of Human Islets Following Cytokine Exposure
To understand the heterogenous effects of inflammatory cytokines on gene expression in the individual cell types found in human islets, we performed single-cell RNA-sequencing (scRNA-seq) using islets isolated from 3 non-diabetic cadaveric donors ( Figure 1 A) following treatment with inflammatory cytokines for either 6 or 18 h.Islets were untreated; treated for 6 h with IL-1 β, IFN-γ , or IL-1 β + IFN-γ ; or treated for 18 h with IL-1 β, IL-1 β + IFN-γ , IL-1 β + IFN-γ + NMMA, or NMMA alone ( Figure 1 B).Islets from each donor wer e tr eated with all conditions for a total of 3 independent scRNA-seq replicates.The combination of IL-1 β + IFN-γ was chosen because this is the minimal cytokine combination necessary to stimulate iNOS expression and nitric oxide production in human islets. 12 , 40Cells from all samples and all 3 donors were combined into 1 dataset and were visualized using UMAP.After quality control, w e w ere left with 47,121 cells that were grouped unbiasedly into 19 clusters based on similarity of gene expression ( Figure 1 C and Table S1 ).Cells from each donor contributed to every cluster, and cells from each treatment contributed to every cluster ( Figure S1 ).We assigned endocrine cell identities ( β-, α-, δ-, and PP-cells) based on enrichment of genes encoding the primary islet hormones (insulin, glucagon, somatostatin, and pancreatic polypeptide, r especti v el y) ( Figur e 1 D).α-cells ( GCG ) comprised 50% (23,354

Human β-Cells Have a Blunted Cytokine Response Compared to Mouse β-Cells
To determine ho w c ytokine exposure alters human β-cell gene expr ession, we computationall y isolated Clusters 1, 6, 7, and 12 from the total dataset ( Figure 1 C).Because stimulation of iNOS mRNA ( NOS2 ) is a well-characterized response of β-cells to IL-1 β + IFN-γ exposure, 12 we used expression of this gene as a metric to assess the r esponsi v eness of the human β-cell population to cytokines.Surprisingly, only 1.6% (168/10,616) of the total human β-cell population expr essed detecta b le lev els of NOS2 ( Figure 2 A).This percentage is much lower than we observed in our previous single-cell RNA-sequencing studies using mouse islets. 14 , 15In those studies, 29% (2,948/10,265) of the whole β-cell population expressed Nos2 ( Figure 2 B) despite similar cytokine exposure conditions in vitro .This b lunted r esponse of the human β-cells was not limited to NOS2 .Visualization of the expression level and the percentage of cells expressing other selected cytokine-stimulated genes shows a markedly blunted response in human β-cells compared to mouse β-cells ( Figure 2 C and D).
As an unbiased approach to determine genes significantly changed in response to each cytokine tr eatment (compar ed to the untreated sample), we performed differential expression analysis of the human β-cell population ( Ta b le S2 ).Relati v e to untr eated contr ols, following a 6-h treatment, a total of 132 genes (0.36% of the detected genes) were significantly changed in response to IL-1 β, 143 (0.39%) in response to IFN-γ , and 339 (0.93%) in response IL-1 β + IFN-γ ( Figure 2 E).Relati v e to untr eated contr ols, following an 18-h tr eatment, a total of 302 (0.83%) genes were changed in response to IL-1 β and 627 (1.71%) in response to IL-1 β + IFN-γ ( Figure 2 E).By performing a similar differ ential expr ession anal ysis, but this time comparing genes changed in response to 18-h IL-1 β + IFN-γ to those changed in response to 18-h IL-1 β + IFN-γ + NMMA, we determined that the expression of only 24 genes (0.07% of the detected genes) w as significantl y c hanged by nitric oxide ( F igur e 2 E).These n umbers are significantly lower than we might expect based on the number of genes changed by each cytokine treatment in mouse β-cells ( Figure 2 E).Together, these observations suggest that human β-cells have a blunted gene expression response to cytokines compared to mouse β-cells.

Cytokine Responsi v eness is Also Blunted in Human Islet Endocrine Non-β-Cell Types
We demonstrated that, in the mouse, endocrine non-β-cells ( α-, δ-, and PP-cells) respond to cytokine stimulation in a manner that is nearly identical to β-cells. 14 , 15To test the hypothesis that human endocrine non-β-cells also respond to cytokines, we first determined the percentage of this population that expresses NOS2 mRNA as a metric for "cytokine r esponsi v eness."To do this, we first isolated clusters 0, 2, 4, 5, 10, 11, and 13.Similar to our human β-cell population, only 1% (220/26,651) of the entire endocrine non-β-cell population expressed NOS2 ( Figure 3 A).This is in stark contrast to our mouse studies, in which 20% (619/2,487) of the endocrine non-β-cells expressed Nos2 ( Figure 3 B).Also like the human β-cells, the blunted response of the human endocrine non-β-cells extended beyond NOS2 expression, with other expected cytokine-stimulated genes summarizing the percent of detected genes that were significantly changed by each cytokine treatment or genes that wer e significantl y changed in a nitric oxide-dependent manner in human and mouse β-cells.P -value determined by Fisher's exact test.Mouse data are from our previously published studies. 14 , 15ing blunted when compared to expression in mouse non-β endocrine cells ( Figure 3 C and D).We again performed differential expr ession anal ysis to determine the number of genes significantl y differ ent in each cytokine-tr eated population of endocrine non-β-cells compared to the untreated population ( Ta b le S3 ).In total, 88 genes (0.24% of the detected genes) were significantly changed after a 6-h treatment with IL-1 β, 57 (0.16%) with IFN-γ , and 156 (0.43%) with IL-1 β + IFN-γ .Following an 18-h treatment, 158 (0.43%) were changed in response to IL-1 β and 279 (0.76%) in response to IL-1 β + IFN-γ ( Figure 3 E).Six genes (0.02%) were changed in a nitric oxide-dependent manner ( Figure 3 E).These percenta ges ar e significantl y lower than the expected v alues fr om mouse endocrine non-β-cells ( Figure 3 E), suggesting that all human islet endocrine cells, not just β-cells, have a reduced cytokine response compared to mouse islet endocrine cells.

NOS2 + Islet Endocrine Cells Are Enriched for Other Cytokine-Stimulated Genes
To test our original hypothesis that cytokines stimulate protecti v e genes in human islet endocrine cells, we computationally isolated the β-cells expressing NOS2 at any level (ie, > 0 reads of NOS2 sequenced) from those not expressing NOS2 or the endocrine non-β-cells expressing NOS2 from those not expressing NOS2 and performed differential expression analyses.Cells from all samples were used for this comparison.A total of 1,307 genes (1,219 enriched in the NOS2 + cells, and 88 enriched in the NOS2 − cells) were significantly different between the 2 β-cell populations, and 1,822 genes (1,620 enriched in the NOS2 + cells, and 202 enriched in the NOS2 − cells) wer e significantl y differ ent between the 2 endocrine non-β-cell populations ( Ta b les S4 and S5 ).We found that the IL-1 β-regulated genes SOD2 and ICAM1  summarizing the percentage of detected genes that were significantly c hanged by eac h cytokine treatment or genes that wer e significantl y changed in a nitric oxide-dependent manner in human and mouse endocrine non-β-cells.P -value determined by Fisher's exact test.Mouse data are from our previously published studies. 14 , 15d the IFN-stimulated genes IRF1 and CXCL10 had significantl y higher expr ession lev els in endocrine cells expressing NOS2 compared to those not expressing NOS2 ( Figure 4 A and B).Importantly, genes encoding antiviral guanylate-binding proteins GBP2 and GBP5 , which are IL-1 β-stimulated in the mouse 14 , were also expressed at higher levels in the NOS2 + population ( Figure 4 A and B).Further, the identity genes MAFA and MAFB , which are known to be r e pr essed by IL-1 β in mouse islet endocrine cells, 14 were significantly lower in human islet endocrine cells expressing NOS2 compared to those not expressing NOS2 ( Figure 4 A and B).Functional annotation clustering analysis demonstrated that genes falling into categories associated with cytokine signaling, including "Innate Immunity," "Epstein-Barr virus infection," "NF-κB signaling," "guanylate-binding protein," and "Chemokine signaling" w ere enric hed in the NOS2 + human islet endocrine cells compared to the NOS2 − cells ( Figure 4 C and D).Together, these results demonstrate that although cytokine responses ar e generall y b lunted in human islet endocrine cells, cells that are responsive to cytokines ( NOS2 + cells) have similar g ene expression chang es to those pr eviousl y observ ed in the mouse, including stimulation of protective antiviral genes and r e pr ession of identity genes.

Genes Encoding Ribosomal Proteins Are Enriched in NOS2 − β-Cells
As shown in Figure 2 A, less than 2% of the total β-cell population expr esses detecta b le lev els of NOS2 mRNA, leading us to hypothesize that the remaining β-cells may have a common c har acteristic pr ev enting them fr om r esponding to cytokine stimulation in the expected manner.To test this hypothesis, we again performed functional annotation clustering analysis, this time focusing on genes increased in β-cells not expressing NOS2 as compared to β-cells expressing NOS2 .Strikingly, the most enriched genes were those that encode ribosomal proteins ( Figure 5 A).In fact, of the 88 genes significantly higher in NOS2 − β-cells compared to NOS2 + β-cells, 58% encode ribosomal proteins and another 16% encode proteins that play other roles in protein biosynthesis or protein folding ( Figure 5 B).The ribosomal protein genes that are most enriched in the NOS2 − β-cell population are shown in Figure 5 C, with ribosomal protein L5 ( RPL5 ) being the gene in this category that is the most different compared to NOS2 + β-cells.RPL5 is not increased by cytokine exposure, suggesting that it is basally high in NOS2 − β-cells ( Figure S2 and Table S2 ).Linear regression analysis of the β-cells demonstrated a significant negati v e corr elation between expression of RPL5 and the IL-1 β-stimulated gene SOD2 (super oxide dism utase 2) ( Figur e 5 D).Importantl y, further analyses demonstrate that expression of RPL5 is positively correlated with genes associated with cellular stress: HSPA1A (encodes the alpha subunit of heat shock protein 70) and DDIT3 (encodes DNA damage inducible transcript 3, also known as CHOP) ( Figure 5 E and F).The negati v e association between RPL5 and/or HSPA1A expression and cytokine responsiveness is mirr or ed by our original clustering anal ysis ( Figur e 1 C) in that βcells in clusters 7 and 12, c har acterized by enric hment for ribosomal proteins and heat shock proteins, respectively, have lower expression of cytokine-stimulated genes SOD2 , ICAM1 , and IRF1 ( Figure S3 and Table S1 ).These data together suggest that high expression of ribosomal proteins is an indicator of cellular stress and is negatively correlated with cytokine response in β-cells.

High Expression of RPL5 Predicts Failure to Respond to Cytokines in Human β-Cells
Since comparison of NOS2 -expressing β-cells to NOS2 -nonexpressing β-cells revealed a correlation to RPL5 expression ( F igure 5 ), w e hypothesized that β-cells with high expression of RPL5 would be less likely to respond to cytokines than those with low expression of RPL5 .To test this hypothesis, we computationall y se parated the β-cells fr om all samples into " RPL5 hi " and " RPL5 low " populations, using an expression level of 2.5 as our cutoff ( Figure 6 A).Over two-thirds of the β-cells fell into the " RPL5 hi " category, with the other third falling into the " RPL5 low " cate gory ( F igure 6 B).We performed differential expression analysis and functional annotation clustering analysis to determine which categories of genes ar e differ ent between the 2 βcell populations.A total of 2,840 genes were significantly different between the RPL5 hi and RPL5 low β-cells ( Ta b le S6 ).As expected, gene categories of "ribosomal protein," "small ribosomal subunit," and "chaperone" were enriched in the RPL5 hi β-cells ( Figure 6 C).Interestingly, categories of "innate immunity," "Epstein-Barr virus infection," and "guanylate binding protein" were enriched in the RPL5 low β-cells ( Figure 6 D).Consistent with this, IL-1 β-stimulated genes SOD2 and ICAM1 and IFNstimulated genes IRF1 and CXCL10 wer e significantl y higher in the RPL5 low β-cells ( Figure 6 E).Importantly, antiviral guanylatebinding proteins GBP1 , GBP2 , and GBP4 were also increased in the RPL5 low β-cells, while identity gene MAFA w as incr eased in the RPL5 hi β-cells ( Figure 6 E).These results suggest that low expression of RPL5 predicts cytokine responsiveness, and β-cells with low expression of this ribosomal protein are more likely to stimulate protective gene expression in response to cytokine exposure.

High Expression of RPL5 Predicts Failure to Respond to Cytokines in Human Endocrine Non-β-Cells
Like in the β-cells, genes that are enriched in endocrine nonβ-cells that are not expressing NOS2 largely fall into the category of ribosomal proteins and other categories related to protein biosynthesis ( Figure 7 A).Since RPL5 is the gene of this category that is most different between the NOS2 -expressing and non-expressing populations ( Figure 7 B), we hypothesized that its expression may be able to predict cytokine responsiveness in the endocrine non-β-cells like it did in the β-cells.To test this hypothesis, we computationally separated the endocrine nonβ-cells from all samples into " RPL5 hi " and " RPL5 low " populations, using an expression level of 2.5 as our cutoff ( Figure 7 C).In contrast to the β-cells, the endocrine non-β-cells were split evenly into the " RPL5 hi " and " RPL5 low " categories ( Figure 7 D).A total of 8,786 genes wer e significantl y differ ent between the RPL5 hi and RPL5 low endocrine non-β-cells ( Ta b le S7 ).Among those that were higher in the RPL5 low population were IL-1 β-stimulated genes SOD2 and ICAM1 , IFN-stimulated genes CXCL1 , CXCL8 , and STAT1 , and antiviral guanylate-binding proteins GBP1 , GBP2 , and GBP4 ( Figure 7 E).This observation indicates that high expression of ribosomal proteins is negatively correlated with cytokine r esponsi v eness not onl y in human β-cells but also in endocrine non-β-cells and that low expression of RPL5 predicts a higher likelihood of responding to cytokine stimulation in all islet endocrine cell types.

High Expression of RPL5 Predicts Failure to Respond to Cytokines in Human Non-Endocrine Cells
Appr oximatel y 20% of our dataset was made up of nonendocrine cells ( Figure 1 C).Of these, almost 4% (360 out of 9,824) expressed NOS2 (data not shown).When we computationall y se parated the non-endocrine cells fr om all samples into NOS2 + and NOS2 − populations and performed differential expr ession anal ysis, 3,227 genes wer e found to be significantl y differ ent between the 2 groups ( Table S8 ).Functional annotation clustering anal ysis r ev ealed that, like in the endocrine cell populations, categories of "innate immunity," "Epstein-Barr virus infection," "viral entry," and "interferon signaling" wer e incr eased in the NOS2 + non-endocrine cells ( Figure 8 A).Among the genes increased in this population were IL-1 β-stimulated genes SOD2 and ICAM1 , IFNstimulated genes IRF1 , CXCL1 , and CXCL10 , and antiviral guanylate-binding proteins GBP2 and GBP5 ( Figure 8 B).Conv ersel y, categories of "ribosomal protein" and "small ribosomal subunit" wer e incr eased in the NOS2 non-endocrine cells ( Figure 8 C), also consistent with our observations in the endocrine cells.While RPL5 was not the most differ entiall y expr essed ribosomal pr otein between the 2 non-endocrine cell populations, it was still among those of this category that were different ( Figure 8 D).To test the hypothesis that this gene might predict cytokine r esponsi v eness in the non-endocrine cells of the islet, we divided this population into " RPL5 hi " and " RPL5 low " subsets, using an expression level of 2.5 as our cutoff ( Figure 8 E).Nearly 60% of the non-endocrine cells fell into the " RPL5 hi " category, while the remaining 40% fell into the " RPL5 low " category ( Figure 8 F).Differential expression analysis identified 5,391 genes that were significantly different between the 2 populations ( Ta b le S9 ).Among those that were higher in the RPL5 low population wer e IL-1 β-stim ulated genes SOD2 and ICAM1 , IFN-stimulated genes IRF1 and CXCL10 , and antiviral guanylate-binding proteins GBP1 , GBP2 , GBP4 , and GBP5 ( Figure 8 B).Together, our analysis of the non-endocrine cells of our dataset demonstrates that high expression of genes encoding ribosomal proteins is negatively associated with cytokine r esponsi v eness in all islet cell types.

Donor Cellular Stress and Cytokine Responsi v eness
To determine potential differences in cytokine responsiveness among our 3 donors, we first determined markers that were enric hed in eac h of our 3 samples using all cells captured ( Ta b le S10 ).Genes encoding ribosomal proteins, including RPL5 , RPS10 , RPS18 , RPL8 , and RPS8 , as well as genes encoding heat shock proteins, like HSPA1A , HSPA1B , HSP90AA1 , HSP90AB1 , and HSPB1 , were significantly higher in cells from Donor 1 compared to cells from the other 2 donors ( Figure 9 A).Importantly, cells from Donor 1 were less responsive to cytokines compared to the cells from the other 2 donors.This was e videnced b y reduced expr ession of IL-1 β-stim ulated genes SOD2 and ICAM1 , antiviral guanylate binding protein GBP2 , and IFN-stimulated genes IRF1 and CXCL10 ( Figure 9 B-F).Interestingly, Donor 1 died from head trauma while the other 2 donors died from either a stroke or an anoxic event ( Figure 1 A).While our sample size is too small to make robust conclusions, these observations suggest that differences in donor c har acteristics, suc h as cause of death, may underl y differ ences in cellular stress and islet cytokine responsi v eness.

Discussion
5][16][17] Because these studies were performed using mouse islets and because differences in the responses of mouse and human islets to IL-1 β have been reported, 23-25 , 30 , 31 here, we aimed to test the hypothesis that IL-1 β stimulates protective genes in human pancreatic islet endocrine cells.
We found that gene expression changes in both β-cells and endocrine non-β-cells following cytokine exposure were markedl y b lunted in the human islets compared to our previous observations in mouse islets ( Figures 2 and 3 ). 14 , 15This was evident not only by a smaller percentage of cells expressing NOS2 but also by a smaller percentage of differentially expressed genes.However, when we computationally isolated the β-cells and the endocrine non-β-cells with detecta b le NOS2 expression and compared them to cells without detectable NOS2 expression, we observed significantly higher expression of known cytokine-stimulated genes, including IL-1 β-stimulated antiviral guanylate-binding proteins GBP2 and GBP5 ( Figure 4 ).This critical observation suggests that, although the global response to cytokine stimulation is blunted in the human islet endocrine cells assay ed here , a small per centa ge of them r espond to IL-1 β by incr easing pr otecti v e gene expr ession.It also suggests that there may be a common feature of the NOS2 − cells prev enting them fr om r esponding to cytokines.The identification of these 2 populations of cells ( NOS2 -expressing and NOS2 non-expr essing) w as onl y possib le because of our single-cell approach.It is also worth noting that our use of the minimum cytokine concentrations necessary to stimulate nitric oxide production in human islets may have led us to underestimate the size of the NOS2 + population. 12 , 40In other words, "cytokine r esponsi v eness" ma y ha v e incr eased if we had used higher concentrations of the c ytokines.Ho wever, the concentrations used her e ar e still gr eater than those used in our previous mouse islet studies, indicating that, even at higher cytokine concentrations, human islets are still less responsive to cytokines than mouse islets. 14 , 15 comparing NOS2 + and NOS2 − populations of β-cells ( Figure 5 ), endocrine non-β-cells ( Figure 7 ), and non-endocrine cells ( Figure 8 ), we identified a common association among all NOS2 − populations: higher expression of genes encoding ribosomal proteins than the respective NOS2 + population.This differ ence w as particularl y striking in β-cells wher e nearl y 60% of the genes that were significantly enriched in the NOS2 − population encoded ribosomal pr oteins ( Figur e 5 B).We further demonstrated that expression of ribosomal protein L5 ( RPL5 ) is negati v el y corr elated with expr ession of an IL-1 β-r egulated gene, SOD2 , and is positi v el y corr elated with expr ession of "stress" genes heat shock protein 70 ( HSPA1A ) and CHOP ( DDIT3 ) ( Figure 5 D-F).Most importantly, high expression of RPL5 predicted failure to respond to cytokine stimulation in β-cells ( Figure 6 ), endocrine non-β-cells ( Figure 7 ), and non-endocrine cells ( Figure 8 ), suggesting that RPL5 may provide a novel marker of "cellular stress" in human islets.This association between cellular stress and blunted cytokine signaling is consistent with pr evious observ ations by us and others that induction of heat shock stress or ER stress pr ev ents iNOS expr ession follo wing c ytokine stimulation in rodent and human islets. 30 , 33-35In our previous scRNA-seq studies using mouse islets, we observed populations of β-cells c har acterized by high expression of heat shock and ribosomal proteins that failed to respond to IL-1 β and IFN-γ exposure by increasing Nos2 mRNA, further emphasizing this negati v e relationship between cellular stress and cytokine signaling. 14 , 15t is critical to emphasize that cells in the current study with high expression of RPL5 had lower expression not only of IL-1 β-stimulated genes (like SOD2 and ICAM1 ) but also had b lunted expr ession of IFN-stim ulated genes (like IRF1 , CXCL10 , and STAT1 ).This observation demonstrates that the negati v e effects of cellular stress on cytokine signaling are not limited to IL-1 β but apply to cytokine responses more broadly, consistent with previous studies. 34 , 35e are not the first to r e port evidence of high levels of cellular stress in human islet preparations. 30 , 31 , 41-43However, the cause of this stress remains unclear.][45] Others have suggested that islets exhibit cellular stress even before the islet isolation process. 46Here, we observed differences in cytokine r esponsi v eness among our 3 islet samples that were correlated with differences in expression levels of genes encoding ribosomal proteins and heat shock proteins ( Figure 9 A).Interestingly, islets from the donor who died from head trauma (Donor 1) had higher levels of "stress-associated" genes and had a blunted cytokine response compared to islets from the donors who died from a stroke or an anoxic event ( Figure 9 B-F).This observation is consistent with the documented induction of a "cytokine storm" following traumatic brain injury in patients. 47 , 48This systemic cytokine release likely leads to local nitric oxide production in the islet microenvironment, which is known not only to stimulate expression of heat shock proteins, but also to blunt subsequent cytokine signaling in the β-cell. 33 , 34While our sample size here is too small to make any definiti v e conclusions, we would like to suggest that donor cause of death may contribute to cellular stress levels and cytokine responsiveness of human islet preparations.How ever, w e cannot exclude the possibility that factors other than donor cause of death contributed to differences in stress lev els observ ed among our 3 islet samples.Additional factors include different genetic and phenotypic makeup, disease pr ofiles, usa ge of pharmaceuticals of donors, as well potential differences in variables associated with islet isolation, from cold storage time of donor pancreas to days in culture .F inally, it is possible that human islets intrinsically express higher levels of str ess-r esponse genes than r odent islets.Indeed, Welsh et al. observed that human islets express higher levels of HSP70 than rat islets even following 4 wk of transplantation. 314][25] The observ ation pr esented her e that β-cells with lower expression of a stress-associated gene ( RPL5 ) respond to cytokines in the same manner as rodent β-cells (by increasing expression of antiviral guanylate-binding proteins) strongly suggests that cellular stress c har acteristic of many human islet pr e parations has pr ev ented a complete understanding of the cytokine responses of human β-cells.As human islets are increasingly being used in cytokine studies, our findings indicate that it is imperati v e to assess expr ession of "str ess" genes, like RPL5 and HSPA1A , before making conclusions regarding the effects, or lack of effects, of cytokines on human β-cell viability and function.

Figure 1 .
Figure 1.scRNA-sequencing of human islet following cytokine exposure.(A) Characteristics of human islet donors.(B) Schematic of experimental design.(C) Uniform manifold approximation and projection plot showing clusters of cells from all 3 scRNA-seq experimental r e plicates.Cell identity is shown to the right of the plot and was assigned based on enrichment for the genes indicated.(D) Dot plot indicating expression levels of and percentage of cells expressing marker genes in each of the 19 clusters.cells) of our dataset, β-cells ( INS ) 23% (10,616 cells), PP-cells ( PPY ) 4% (1,681 cells), and δ-cells ( SST ) 3% (1,616 cells).Using c har acteristic gene expression, we also identified the cell types of the non-endocrine clusters, which made up the remaining 20% of our dataset: fibr ob lasts ( LUM ), ductal cells ( KRT19 ), acinar cells ( CPA1 ), endothelial cells ( VWF ), macr opha ges ( AIF1 ), pericytes ( RGS5 ), T-cells ( CD3D ), and mast cells ( TPSB2 ) ( Figure 1 C and D).

Figure 2 .
Figure 2. Human β-cells have a blunted cytokine response compared to mouse β-cells.(A and B) Pie chart showing the percentage of human (A) or mouse (B) β-cells from all samples that express iNOS mRNA.(C and D) Dot plots depicting the expression levels of and percentage of human (C) or mouse (D) β-cells expressing selected genes in response to each of the cytokine tr eatments.(E) Ta b le summarizing the percent of detected genes that were significantly changed by each cytokine treatment or genes that wer e significantl y changed in a nitric oxide-dependent manner in human and mouse β-cells.P -value determined by Fisher's exact test.Mouse data are from our previously published studies.14 , 15

Figure 3 .
Figure 3. Cytokine r esponsi v eness is also b lunted in islet endocrine non-β-cells.(A and B) Pie chart showing the percentage of human (A) or mouse (B) endocrine non-β-cells from all samples that express iNOS mRNA.(C and D) Dot plots depicting the expr ession lev els of and percentage of human (C) or mouse (D) endocrine non-β-cells expressing selected genes in response to each of the cytokine treatments.(E) Tablesummarizingthe percentage of detected genes that were significantly c hanged by eac h cytokine treatment or genes that wer e significantl y changed in a nitric oxide-dependent manner in human and mouse endocrine non-β-cells.P -value determined by Fisher's exact test.Mouse data are from our previously published studies.14 , 15

Figure 4 .
Figure 4. NOS2 + islet endocrine cells are enriched for other cytokine-stimulated genes.(A and B) Violin plots showing the expression level of selected genes in human β-cells (A) or endocrine non-β-cells (B) expressing NOS2 compared to those not expressing NOS2 .All genes shown hav e P -v alues < 1 × 10 −8 .(C and D) Enriched categories of genes increased in human β-cells (C) or endocrine non-β-cells (D) expressing NOS2 compared to those not expressing NOS2 .

Figure 6 .
Figure 6.High expression of RPL5 pr edicts failur e to respond to cytokines in human β-cells.(A) Violin plot showing expr ession lev el of RPL5 in human β-cells with "high" RPL5 expression ( RPL5 hi ) compared to those with "low" RPL5 expression ( RPL5 low ).(B) Pie chart showing the percentage of RPL5 hi and RPL5 low β-cells.(C and D) Enric hed cate gories of genes incr eased in RPL5 hi β-cells compar ed to RPL5 low β-cells (C) and vice v ersa (D).(E) Violin plots showing the expr ession lev el of selected genes in RPL5 hi β-cells compared to RPL5 low β-cells.All genes shown have P -values < 1 × 10 −36 .

Figure 7 .
Figure 7. High expression of RPL5 predicts failure to respond to cytokines in endocrine non-β-cells.(A) Enriched categories of genes increased in human endocrine non-β-cells not expressing NOS2 compared to those that are expressing NOS2 .(B) Violin plots showing the expression level of selected ribosomal proteins in human endocrine non-β-cells expressing NOS2 compared to those not expressing NOS2 .All genes shown hav e P -v alues < 1 × 10 −8 .(C) Violin plot showing expression level of RPL5 in human endocrine non-β-cells with "high" RPL5 expression ( RPL5 hi ) compared to those with "low" RPL5 expression ( RPL5 low ).All genes shown have P -values < 1 × 10 −86 .(D) Pie chart showing the percentage of RPL5 hi and RPL5 low endocrine non-β-cells.(E) Violin plots showing the expr ession lev el of selected genes in RPL5 hi endocrine non-β-cells compared to RPL5 low endocrine non-β-cells.

Figure 8 .
Figure 8. High expression of RPL5 pr edicts failur e to r espond to cytokines in non-endocrine cells.(A and C) Enric hed cate gories of genes increased in human nonendocrine cells expressing NOS2 compared to those non expressing NOS2 (A) or vice versa (C).(B and D) Violin plots showing the expr ession lev el of selected genes in human non-endocrine cells expressing NOS2 compared to those not expressing NOS2 .(E) Violin plot showing expression level of RPL5 in human non-endocrine cells with "high" RPL5 expression ( RPL5 hi ) compared to those with "low" RPL5 expression ( RPL5 low ).(F) Pie chart showing the percentage of RPL5 hi and RPL5 low non-endocrine cells.(G) Violin plots showing the expression level of selected genes in RPL5 hi non-endocrine cells compared to RPL5 low non-endocrine cells.

Figure 9 .
Figure 9. Donor cellular stress levels and cytokine responsiveness.(A) Dot plot showing the expression levels of and percentage of cells from each of the 3 donors expressing selected ribosomal proteins and heat shock proteins.(B-F) Split violin plots showing the expression of SOD2 (B), ICAM1 (C), GBP2 (D), IRF1 (E), and CXCL10 (F) in response to cytokine stimulation in human islets from each of the 3 donors.All genes shown have P -values < 1 × 10 −15 .