We conducted the first study of viable neutrophil gene expression in the chronic inflammatory disease type 2 diabetes. We observed differential expression between T2D and healthy neutrophils of several inflammatory- or lipid-associated genes, indicating the potential for distinct dysbiotic transcriptomic profiles between these groups among a key and novel immune cell type. Further, as many of the significant differentially expressed genes had direct lipid- or glucogenesis-related roles, neutrophil transcriptomics may provide a window directly into impairment of the host response in diabetics and disease pathogenesis beyond their role in immune or inflammatory genes.
The top genes significantly down-regulated in T2D neutrophils compared to healthy neutrophils tended to be biologically relevant across inflammatory- and lipid-associated gene pathways and included SLC9A4, NECTIN2, and PLPP3 (Figure 1).
SLC9A4 is a plasma membrane solute carrier protein that acts in homeostatic pH regulation and proton transport, including to eliminate metabolism-generated acids and absorb sodium, with an important role in signal transduction (21,22). SLC9A4 is in a genomic region with genes involved in the IL-1 receptor (IL-1R) and IL-18 pathways (23,24). Ground-breaking studies have elucidated the function and structure of a network of proteins (termed the "inflammasome") responsible for processing and releasing key pro-inflammatory cytokines (e.g. IL-1β and IL-18) and controlling cell death and resolution of inflammation (25,26).
This SLC9A4/IL-1R/IL-18 locus has important roles in cytokine signaling and inflammatory response (23,24). The IL-18 pathway induces synthesis of the cytokine IFN- γ in T cells, which has been shown to act in mucosal inflammation in celiac disease (23,27). Celiac disease is a chronic inflammatory and autoimmune disease that shares several genetic risk loci with type 1 diabetes (23). A celiac disease GWAS identified associations with the SLC9A4/IL-1R/IL-18 locus, and a variant in this locus further showed an allelic dosage effect that reduced IL18RAP mRNA expression in celiac patients, indicating a direct link between this locus' genotype and biologically relevant gene expression in a chronic inflammatory disease (23). This reduced IL-18 cytokine expression in celiac disease is consistent with the overall reduced expression of SLC9A4 observed among diabetic neutrophils in our study. Another GWAS linked SLC9A4 with regulation of IL-33/ST2 (suppression of tumorigenicity 2, part of the IL-1R family) signaling, which has previously been implicated in both immune and inflammatory diseases, including cardiovascular disease (24). In addition, a variant in SLC9A4 was associated with eosinophil, though not neutrophil, cell counts in a GWAS of white blood cell subtypes utilizing subjects from the BioBank Japan Project, further indicating a direct role for SLC9A4 in immune cell activity (28).
NECTIN2 is a cholesterol responsive, adherens junction plasma membrane glycoprotein (29,30). NECTIN2 is also involved in extravasation and angiogenesis, and may act at vascular inflammation sites to regulate transendothelial leukocyte migration (29,30). This study is the first exploration of NECTIN2 expression in neutrophils. We observed downregulation of NECTIN2 among T2D subjects, which is consistent with a continuous cell migration phenotype. Further, we observed higher cholesterol levels among our T2D subjects, consistent with both the role of NECTIN2 and the known propensity for high cholesterol in diabetic patients. Previous evidence has shown that NECTIN2 also regulates immune system responses, including modulating T cell signaling and viral pathogenesis (31). Further, NECTIN2 has shown potential associations with several diseases, including chronic inflammatory diseases.
PLPP3 is a plasma membrane adherens junction phospholipid phosphatase that converts phosphatidic acid to diacylglycerol and has roles in lipid metabolism and Wnt signaling (21,32,33). The Wnt signaling pathway is involved in a menagerie of cellular functions and development, and its dysfunction has been linked to diseases including cancer, metabolic syndrome, diabetes, and diabetic neuropathy (34). It has also previously been demonstrated that silencing PLPP3 in an endothelial cell line enhanced inflammatory cytokine secretion, leukocyte adhesion, cell survival, and migration, while its overexpression reversed those effects and instead induced apoptosis (35). This is consistent with our observation of reduced PLPP3 expression among T2D neutrophils, as PLPP3 normally acts in a protective role as a negative regulator of inflammatory cytokines and leukocyte adhesion. In addition, GWAS have previously associated variants in PLPP3 with several chronic inflammatory diseases, including coronary artery disease (36–39) and eosinophilic esophagitis (40). Thus, the lipid regulatory and anti-inflammatory roles of PLPP3 in chronic inflammatory diseases lend further evidence for a metabolic-immune axis in diabetes (8).
It is worth note that our study of neutrophil transcriptomics found that most significant gene expression was downregulated in T2D neutrophils compared to healthy neutrophils. Out of several significant genes, the only upregulated FDR-corrected gene in T2D neutrophils was GTSCR1, a non-coding RNA with unknown biology. While non-coding RNA may act to alter gene expression, it is impossible to speculate on a role for GTSCR1 in T2D given the overall paucity of knowledge about its function. However, initial investigations into GTSCR1 showed it may have importance in cardiac inflammation (18). In Th17-promoting conditions, this was an important gene in regulating T-cell regulation and plasticity (18). Interestingly, in our study, promoter gene RN7SL2 demonstrated a dose-response to RvE1, showing that neutrophils were viable in cell culture and not terminally differentiated (41).
Overall, our findings on neutrophil transcriptomics provide a database to promote scientific survey of novel hypotheses of neutrophils studie It has traditionally been thought that gene expression in diabetic individuals would be increased, particularly among inflammatory and immune genes, and indeed gene expression studies of pancreatic islet cells showed increased gene expression in diabetes (42). However, as neutrophils experience severe dysregulation in individuals with chronic inflammatory disease (4,5,9), we do not believe it is surprising to see a downregulation of many key immune and inflammatory genes in neutrophils. Diabetic neutrophils are known to exhibit impaired immune responses and cell migration (4,5,9), and we observed a marked reduction in neutrophil chemotaxis in T2D compared to healthy neutrophils (data not shown), consistent with the downregulated gene expression observed in this study. Further, a recent study in adipose tissue showed that apolipoprotein M expression was decreased in T2D and obese individuals compared to lean individuals (43), further indicating a role for downregulation of genes in diabetes pathogenesis. A recent study investigating neutrophil gene expression in a small number of newly diagnosed diabetics compared to controls also observed neutrophil dysregulation, with >15% of neutrophil genes differentially expressed in new T2D, ~60% of which were upregulated (44). These vast differences in gene expression may be explained by neutrophil heterogeneity in a small number of samples, failure to fully exclude monocytes, or differences in neutrophil gene expression at onset of T2D compared to established disease.
When we further explored neutrophil gene expression by biological pathways, we observed strong links between lipid- and inflammatory-associated genes and pathways, further emphasizing the role of the metabolic-immune axis in diabetes (8). Plasma membrane cellular pathways were also over-represented, consistent with the roles of our top differentially expressed genes. Hierarchical clustering of over-represented and biologically relevant pathways by individual expression of significant differentially expressed genes tended to sort individuals by disease status (Figure 2). This is in agreement with our PCA clustering for disease status (Supplementary Figure S1), where type 2 diabetic and healthy individuals tended to cluster in different regions, consistent with hyperglycemia among diabetics. However, disease heterogeneity could be observed in the clustering, with some T2D individuals in a healthy cluster and vice versa, indicating the potential to identify subsets of disease severity through neutrophil transcriptomics.
To investigate the functional activity of T2D and healthy neutrophils and whether diabetic neutrophil dysregulation could be restored to a healthy, rather than diseased, phenotype, we treated cell cultured neutrophils from T2D and healthy individuals with the small lipid ligand resolution mediator RvE1. We previously investigated the impact of RvE1 perturbation in a murine model of T2D, where RvE1 treatment was able to rescue impaired neutrophil phagocytosis (45). In this study, we observed that RvE1 dose-dependently modified T2D neutrophil gene expression in cell culture, suggesting a tendency for RvE1 treatment to shift neutrophils toward a healthy phenotype, whereas healthy neutrophil gene expression required a minimum amount of the RvE1 ligand for perturbation, consistent with their lack of chronic inflammation for RvE1 to resolve and consistent with our previous receptor-ligand findings (12). Diabetic genes were mostly affected at 10 - 100 nM of RvE1, demonstrating that diabetic neutrophils are not permanently impacted by the chronic disease, and inflammation could be modulated in diabetes. While we showed that multiple genes were impacted by RvE1 treatment, after FDR correction only two genes were significantly differentially expressed between T2D and healthy neutrophils: LILRB5 and AKR1C1.
LILRB5 is an immune system gene that can bind major histocompatibility complex (MHC) class I molecules on antigen-presenting cells, including neutrophils, inhibiting stimulation of an immune response (46,47). The increased LILRB5 expression in T2D neutrophils may represent a functional neutrophil change, possibly reflecting the overall neutrophil dysregulation observed in T2D (47). AKR1C1 catalyzes the reaction of progesterone to its inactive form but may also regulate inflammatory cytokine signaling pathways (48), consistent with some altered cytokine concentrations observed in this study. As LILRB5 and AKR1C1 remained significantly differentially expressed regardless of RvE1 treatment dose, they may reflect molecular changes in neutrophil inflammatory profiles.
In general, the genes differentially expressed between untreated T2D and healthy neutrophils differed between the human serum and cell culture models. This may be due to in vivo vs. ex vivo neutrophil differences, inter-individual variation in neutrophil responses, as not all individuals whose neutrophils were cultured were also included in the baseline serum neutrophil analysis, or they may represent different genes targeting similar pathways. Indeed, when we investigated the top KEGG and GO pathways associated with differential gene expression between T2D and healthy cell cultured neutrophils with a low (1 nM) dose of RvE1, the top pathways were primarily immune related, though there was less of a lipid pathway role (data not shown).
Interestingly, three genes were differentially expressed in both neutrophil cell culture without RvE1 treatment and the baseline serum analysis, including NECTIN2, HTRA3, and ABCG1. Importantly, treatment of the T2D neutrophils in cell culture with a clinically relevant dose, 100 nM, of exogenous RvE1 was able to reduce both the overall number of genes differentially expressed relative to healthy neutrophils and the level of differential expression of NECTIN2 and ABCG1, suggesting a partial normalization of these neutrophils with RvE1 treatment. The top 50 significant genes from neutrophil gene expression in type 2 diabetic versus healthy subjects were listed (Supplementary Figure S2) .
We then used cytokine profiling to further interrogate the functional effect of RvE1 treatment on T2D and healthy neutrophils. We observed trends in differences in cytokine profiles between T2D and healthy neutrophils both with and without RvE1 treatment. In general, T2D neutrophils tended to have a stronger response to RvE1 treatment, particularly at higher, clinically relevant, RvE1 doses (Figure 3). Without any RvE1 treatment, we observed higher levels of pro-inflammatory cytokines P-Selectin and TNF-α among T2D compared to healthy neutrophils, consistent with recent evidence for enriched cytokine expression in T2D neutrophils (44), though levels of pro-inflammatory MIP-1β, IL-8, and sICAM-1 cytokines were decreased in T2D neutrophils (Figure 4). As cytokines have complex and often overlapping roles in pro- and anti-inflammatory pathways, the relative contribution of each cytokine to neutrophil dysfunction in T2D will require further investigation. We also observed differences in cytokine profiles by subject, consistent with known inter-individual variation (49). Our study aimed to understand unprimed and baseline levels of neutrophils in order to allow investigators to map exactly how neutrophils from diabetic subjects behave at baseline levels. We have previously shown that baseline levels of intracellular neutrophil signalling was detectable as soon as 15 mins, demonstrating the feasibility of our premise (12). We further aimed to investigate early stages of neutrophil cytokine production and detected limited amounts of cytokines in the current study; though due to limitations of sample size, a more detailed subgroup analysis was not feasible. However, even at this sample size, this novel cell type has shown distinct differences in transcriptomic and functional cytokine profiling between T2D and healthy individuals, including following RvE1 treatment, indicating that larger follow-up studies are warranted. While intracellular changes were mostly detected after RvE1 treatment, cytokine secretion was limited in the early stage settings. In the future, it will be possible to clarify the differences in the cytokine secretion between T2D and healthy volunteers in the presence of RvE1 by addition of stimulus or incubation for a longer time period. Importantly, our cytokine profiling experiments showed that both T2D and healthy neutrophils were viable and capable of both producing cytokines and responding to RvE1 treatment, demonstrating the importance of concentration-based therapeutics for exogenous lipid ligands. Notably, production of TNF-α was clearly increased in T2D neutrophils and modulated by RvE1 treatment. This cytokine trend was corroborated by gene regulation of CD177, demonstrating that that an intracellular regulator of TNF-α was also differentially expressed in diabetic neutrophils when compared to healthy controls (p < 0.05). Absolute values of cytokine expressions are listed in Supplementary Table S3 and S4.
Key strengths of our study include the novel exploration of a biologically relevant cell type (neutrophils) in chronic inflammatory diseases, investigations of both gene level transcriptomics and pathway analyses, and the further exploration of the impact of a key molecule in inflammation resolution (RvE1) on gene expression and functional cytokine levels. However, our study does have some limitations, including limited sample size, which made stratification on demographic and clinical characteristics unfeasible. While there were some differences in average demographics between T2D and healthy individuals in our cohorts that align with known risk factors for T2D, PCA of demographic and clinical characteristics (including age, BMI, and ethnicity) did not show any evidence that these differences explained the neutrophil gene expression results, though some residual confounding remains possible. Our T2D subjects were significantly older than our healthy subjects on average. While older individuals experience immune system changes that may include cell deterioration and DNA damage, the similar live neutrophil and monocyte cell counts for T2D and healthy individuals indicate we were able to capture healthy neutrophils from both groups despite any age-related effects on the immune system. A higher proportion of our diabetic subjects were African-American compared to our healthy subjects, who were primarily Caucasian, reflecting known differences in T2D risk across ethnicity. While racial and ethnic population stratification can bias DNA sequencing study results, this was an exploratory study of neutrophil gene expression and PCA did not show a significant impact of ethnicity, though some residual confounding remains possible.
Overall, we showed that neutrophils may act differently in individuals with chronic inflammatory diseases, specifically diabetes, compared to healthy individuals. Further validation of proteomic differences between diabetic and healthy individuals could elucidate important inflammation mechanisms and potential pathways related to resolvin treatment effects, including in subgroups of disease with clinically relevant diabetes comorbidities, such as those with cardiovascular disease or periodontitis. Unraveling these mechanisms of neutrophil dysregulation in chronic inflammatory diseases could ultimately elucidate inflammation and resolution targets for better diagnostic and therapeutic treatment options.