Association of proinflammatory cytokines and islet resident leucocytes with islet dysfunction in type 2 diabetes
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Chronic inflammation in type 2 diabetes is proposed to affect islets as well as insulin target organs. However, the nature of islet inflammation and its effects on islet function in type 2 diabetes remain unclear. Moreover, the immune cell profiles of human islets in healthy and type 2 diabetic conditions are undefined. We aimed to investigate the correlation between proinflammatory cytokine expression, islet leucocyte composition and insulin secretion in type 2 diabetic human islets.
Human islets from organ donors with or without type 2 diabetes were studied. First and second phases of glucose-stimulated insulin secretion were determined by perifusion. The expression of inflammatory markers was obtained by quantitative PCR. Immune cells within human islets were analysed by FACS.
Type 2 diabetic islets, especially those without first-phase insulin secretion, displayed higher CCL2 and TNFa expression than healthy islets. CD45+ leucocytes were elevated in type 2 diabetic islets, to a greater extent in moderately functional type 2 diabetic islets compared with poorly functional ones, and corresponded with elevated ALOX12 but not with CCL2 or TNFa expression. T and B lymphocytes and CD11c+ cells were detectable within both non-diabetic and type 2 diabetic islet leucocytes. Importantly, the proportion of B cells was significantly elevated within type 2 diabetic islets.
Elevated total islet leucocyte content and proinflammatory mediators correlated with islet dysfunction, suggesting that heterogeneous insulitis occurs during the development of islet dysfunction in type 2 diabetes. In addition, the altered B cell content highlights a potential role for the adaptive immune response in islet dysfunction.
Keywords12 Lipoxygenase B cell CCL2 Dendritic cells Flow cytometry Insulin secretion Macrophages Perifusion T cell TNF-α
Chemokine (C-C motif) ligand
Chemokine (C-X-C motif) ligand
Glucose-stimulated insulin secretion
Integrated Islet Distribution Program
The pathogenesis of type 2 diabetes consists of insulin resistance and islet dysfunction . Metabolic stress from excessive nutrition contributes to insulin resistance by provoking chronic inflammation in insulin target organs [1, 2, 3]. It is plausible that islet dysfunction is also related to global chronic inflammation in type 2 diabetes. Indeed, some clinical studies targeting IL-1β- and nuclear factor κB-related inflammatory pathways improved aspects of type 2 diabetic beta cell function [4, 5, 6]. IL-1β and IL-12, as well as the chemokine (C-C motif) ligand 2 (CCL2) and CCL13, are increased in type 2 diabetic islets and circulation [2, 7, 8]. Similarly, some alterations in circulating leucocyte subsets have been reported in individuals with type 2 diabetes [3, 9]. Peripheral blood T helper (Th)17 and Th1 cells are increased, but T regulatory cells are diminished in obese individuals with type 2 diabetes compared with obese controls [10, 11]. Circulating T cells reactive to islets are detectable in type 2 diabetic patients . Similarly, peripheral blood circulating B cells from individuals with type 2 diabetes secrete more IL-8 but less IL-10, and support contact-dependent T cell activation . However, it is unclear whether B or T cells are present and participate in inducing islet dysfunction. Thus, the mechanisms that connect adipose and islet tissue inflammation and dysfunction in type 2 diabetes are unclear .
In this respect, there is limited information concerning the impact of local inflammation on the deterioration of human islet function or the profile of islet-associated leucocytes during the development of type 2 diabetes. While most knowledge about the potential role of leucocytes in islet dysfunction is derived from mouse models of type 1 diabetes , several recent studies have aimed to examine the immune cell content of human type 2 diabetic islets. CD68+ leucocytes, which are mostly macrophages, were increased in histological studies of humans with type 2 diabetes [16, 17], suggesting that islet macrophages may play a role in type 2 diabetes. However, a comprehensive analysis is required to determine whether other leucocyte subsets exist within human islets, where they might orchestrate inflammation during type 2 diabetes.
Immune cell accumulation has been reported in multiple rodent models of type 2 diabetes, including ob/ob mice, high-fat-fed mice, Goto–Kakizaki rats and Zucker diabetic fatty rats, supporting the notion that inflammation may contribute to islet dysfunction [16, 18, 19]. Although animal models offer valuable insight into islet biology, human islets are known to differ from rodent islets in morphology [20, 21] and functionality , highlighting the importance of studying human islets. The scarcity and difficulty of procuring human islets has been a major hurdle in understanding the pathogenesis of islet failure during type 2 diabetes. In the present study, we applied a flow cytometry-based approach to examine the distribution of leucocyte subsets in non-diabetic and type 2 diabetic human islets, in combination with assessments of islet function and proinflammatory marker expression, to determine the relationship between inflammation and islet function.
Human islet culture
Human islets were acquired from the Integrated Islet Distribution Program (IIDP; Duarte, CA, USA, for 40 donors, see electronic supplementary material [ESM] Methods) and Beta-Pro (Charlottesville, VA, USA, for three donors), with approval from the institutional review board at the Eastern Virginia Medical School. Islets were incubated overnight in CMRL-1066 containing 10% FBS and 1% penicillin–streptomycin at 37°C and 5% CO2 to recover from shipment. For cytokine treatments, a mixture of 0.57 mmol/l TNF-α, 5.9 mmol/l IFN-γ and 0.29 mmol/l IL-1β (all from BD Bioscience, San Jose, CA, USA) were added to the culture overnight.
Ex vivo perifusion assay
A total of 500 islet equivalents (IEQ) of human islets were perifused at 3 or 23 mmol/l glucose (between 45 and 65 min) . The samples were collected at 1 ml/min for human insulin measurement by ELISA (Mercodia, Winston Salem, NC, USA). The islet insulin content was measured by ELISA after extraction by acidified ethanol . Variables used to compare glucose-stimulated insulin secretion (GSIS) are detailed in ESM Methods.
Gene expression analyses
cDNA was prepared from 500 IEQ of human islets, as described in ESM Methods. Gene expression was analysed using the TaqMan gene-expression assay (Invitrogen, Carlsbad, CA, USA), normalised against β actin expression.
A total of 5,000–7,000 IEQ islets digested with 0.025% trypsin and dispersed into single-cell suspensions were used for flow cytometry experiments (detailed in ESM Methods).
Characteristics of human islets
Non-diabetes (n = 21)a
Type 2 diabetes (n = 18)a
Cytokine treatment (Fig. 4g, n = 4)
44.8 ± 2.9 (47)
51.2 ± 1.7 (54)
31.3 ± 4.2 (33.5)
28.5 ± 1.2 (29.1)
34.5 ± 1.9* (33.3)
24.0 ± 3.7 (22.8)
Cause of death
85.1 ± 1.6 (90)
86.3 ± 1.4 (90)
86.3 ± 3.2 (85)
Time in culture, days
3.3 ± 0.3 (3)
3.4 ± 0.3 (3)
3 ± 0 (3)
Impairment of GSIS in human type 2 diabetic islets
The correlation between GSIS and proinflammatory gene expression in type 2 diabetic islets
Increased number of leucocytes in human type 2 diabetic islets
We next estimated the overall leucocyte cellularity within human islets based on CD45-positive leucocytes normalised to 1,000 IEQ. After correcting for cell loss during sample preparation, the abundance of CD45+ leucocytes per 1,000 IEQ obtained by flow cytometry was similar to that previously reported in histological estimates of islet leucocyte cellularity [16, 33]. The number of CD45+ leucocytes was increased within type 2 diabetic islets (Fig. 3f). Since the degree of islet functionality varied widely within the type 2 diabetic islet cohort (Fig. 1), the number of islet leucocytes was compared between high and low insulin-secreting type 2 diabetic islets (Fig. 3g). In contrast to the negative relationship between proinflammatory CCL2 expression and insulin secretion, the number of CD45+ leucocytes was elevated within type 2 diabetic islets with preserved insulin secretion, in comparison with dysfunctional type 2 diabetic islets (Fig. 3g). A positive correlation was observed between GSIS (first/second-phase ratio) and the number of CD45+ leucocytes within type 2 diabetic islets (Fig. 3h); however, BMI and CCL2 and TNFa expression did not correlate with CD45+ leucocytes (ESM Fig. 3b–d). These data suggest that the accumulation of leucocytes within islets is associated with the pathology of type 2 diabetes, although the peak accumulation of leucocytes did not coincide with CCL2 expression. Altogether, other factors may affect the recruitment of immune cells, resulting in variable amounts of islet inflammation within both high- and low-insulin-secreting type 2 diabetic islets. Interestingly, the increase in CD45+ leucocytes was strongly associated with elevated expression of ALOX12 in type 2 diabetic islets (‘Hi’ vs ‘Lo’, Fig. 3i), suggesting that islet leucocytes may be an important source of 12LO production within type 2 diabetic islets. Alternatively, 12LO, which is also produced by beta cells , may support the recruitment of leucocytes to the islets.
Type 2 diabetic islets contain elevated levels of B cells
An important role for B cells in the development of systemic and adipose inflammation has been reported in a mouse model of insulin resistance . In parallel, a recent study with peripheral blood circulating B cells from type 2 diabetic individuals highlighted the unique phenotype and functions of these lymphocytes . To test whether B cells are present within islets, we performed staining for CD20, a marker that is expressed by the majority of B cells, including pre-, resting and memory B cells. A small but distinguishable resident population of CD20+ B cells was present within non-diabetic and type 2 diabetic islets (Fig. 4c). Overall, the percentage of B cells was increased up to 2.2-fold in type 2 diabetic islets compared with non-diabetic controls (Fig. 4f). To assess whether human islet T cells may be activated in response to cytokines, we assessed TNF-α-, IL-1β- and IFN-γ-treated non-diabetic islets for upregulation of the early T cell activation marker CD69 on intraislet memory T cells (CD3+CD44+CD45RA– T cells). In this ex vivo system, memory T cells significantly upregulated CD69 in comparison to vehicle-treated controls (Fig. 4g). These results suggest that islet T cells are functional and can be activated in response to proinflammatory cytokines, likely through islet antigen presenting myeloid or B cells.
In this study, we have established a strong correlation between the proinflammatory cytokine TNF-α and the chemokine CCL2 with GSIS in type 2 diabetic human islets. We found an increased content of CD45+ leucocytes within type 2 diabetic islets, specifically within moderately functional type 2 diabetic islets. Importantly, CD20+ B cells were increased in type 2 diabetic human islets in comparison with non-diabetic islets. This study is the first to attempt simultaneous examination of both physiological and immunological variables in human islets, in order to determine if intraislet immune cells are associated with islet dysfunction in human type 2 diabetes.
Low first-phase insulin secretion upon i.v. glucose injection is a pathognomonic feature of human type 2 diabetes [25, 26]. It is noteworthy that the perifusion of human islets from clinically labelled type 2 diabetic donors unanimously showed blunting or loss of first-phase insulin secretion, suggesting that islets from type 2 diabetic donors retain features of islet dysfunction ex vivo. In comparison with previous studies that used glucose-ramp or batch assays [40, 41], we determined both the first- and second-phase GSIS responses in a large cohort of type 2 diabetic islets. The wide spectrum of impairment in first- and second-phase insulin secretion that we observed may reflect a gradual decline in functional beta cell mass, which is considered to be responsible for the progression of type 2 diabetes in humans . The IIDP in the USA is an invaluable resource that facilitates access to a large number of islet donors. However, the medical history details available to researchers are currently limited (see ESM Methods). Future studies should determine whether the clinical history of a donor, including the duration and severity of type 2 diabetes, correlates with GSIS and islet leucocyte content ex vivo.
Several lines of evidence suggest that the levels of proinflammatory cytokines such as IL-1β, IL-8 and IL-12 and chemokines CCL2, CCL13 and chemokine (C-X-C motif) ligand 10 (CXCL10) are elevated in type 2 diabetic islets [7, 8, 14, 42]. However, it is unclear whether proinflammatory cytokines and chemokines are definitively associated with the development of islet dysfunction in type 2 diabetes. Our study provides compelling evidence for a negative correlation between the levels of CCL2 and TNF-α within type 2 diabetic islets and their functions, as determined by GSIS. CCL2 and TNF-α levels were significantly elevated in dysfunctional type 2 diabetic islets in comparison with moderately functional type 2 diabetic islets, indicating the existence of several subgroups with different levels of inflammation within type 2 diabetic individuals. Surprisingly, ALOX12, which is strongly associated with inflammation in adipose and other tissues , was upregulated within moderately functional but not dysfunctional type 2 diabetic islets, indicating that 12LO may play a role in islet dysfunction at early certain stages of the pathogenesis and that beta cell production of 12LO might participate in the early homing of leucocytes to islets. Alternatively, increased ALOX12 expression may originate from non-beta cells, considering the correlation between ALOX12 expression and the abundance of CD45+ leucocytes.
M1-like macrophages are recruited into islets in rodent type 2 diabetes models and, similarly, CD68+ macrophages are elevated within human T2D islets [16, 17, 43]. To further determine if the immune system is associated with the pathology of type 2 diabetes, we examined the overall abundance of leucocytes and leucocyte subsets within human islets from non-diabetic and type 2 diabetic individuals. Importantly, we observed evidence of increased accumulation of CD45+ leucocytes within type 2 diabetic islets, which strongly correlated with islet function. To our surprise, individuals with moderately functional islets possessed significantly more leucocytes than markedly dysfunctional type 2 diabetic islets. Based on these results, initial steps in the development of type 2 diabetes may be accompanied by an influx of CD45+ leucocytes, which likely supports active islet inflammation. In contrast, at an advanced stage of type 2 diabetes, significant beta cell damage and apoptosis due to oxidative stress, endoplasmic reticulum stress and mitochondrial dysfunction may provide less support for the recruitment of leucocytes to type 2 diabetic islets. Together, these results suggest that the accumulation of leucocytes within islets is associated with the pathology of type 2 diabetes, but the recruitment of leucocytes may occur in a temporal, stage-dependent manner. Considering the variable degrees of islet inflammation within high- and low-insulin-secreting type 2 diabetic islets, other factors such as the management of the individual’s diabetes and end-of-life treatment may modify the recruitment of leucocytes.
Chronic low-grade inflammation resulting from changes in lymphocyte and myeloid cell subsets promotes and exacerbates insulin resistance, which, together with islet dysfunction, defines type 2 diabetes. We detected an increased accumulation of B cells within type 2 diabetic islets. Recent reports suggest that circulating B cells are hyperactivated in type 2 diabetic patients and elicit T cell-derived proinflammatory TNF-α, IL-17A and IL-6 production in T cell/B cell cocultures . Thus, an elevated proportion of islet B lymphocytes may work together with costimulatory molecule-expressing myeloid cells (CD86+ cells) to affect islet T cell activation or serve as “accessory cells” to promote islet-specific T cell proliferation, as shown for islet B lymphocytes in the non-obese diabetic mouse model .
Interestingly, comparable levels of CD3+ T cells and CD11c+ myeloid cells were present in both non-diabetic and type 2 diabetic islets. Islets treated with TNF-α, IL-1β and IFN-γ demonstrated that islet memory T cells are responsive to cytokines and may become activated during inflammation. Importantly, subtle changes in the proportion of T and B cell subsets may exist in type 2 diabetic islets, in addition to changes in the overall abundance of T and B cells within the islet. Future studies are required to determine if the overall abundance and differentiation of T cell subsets are affected by the elevated number of islet B cells.
Recent studies have demonstrated that several DC subsets are present within rodent pancreatic islets, where they may play proinflammatory or tolerogenic roles in the context of type 1 diabetes [32, 45]. Two subsets of islet DCs with distinct functions are found within healthy murine islets. CD11b+CD103−CX3CR1+ DCs represent the majority of islet DCs with high phagocytic, but low antigen presentation activity . In contrast, a small subset of CD11blow CD103+CX3CR1− DCs is able to migrate to the draining lymph node and cross-present antigens. In addition, suppressive CD11b+CD11c+ tolerogenic DCs have been described in a non-obese diabetic mouse model . Multiple myeloid cell subsets may be similarly present within human islets, where they may participate in T cell activation and the development of islet dysfunction in type 2 diabetes. Indeed, sizeable populations of CD11c+ cells are present in non-diabetic and type 2 diabetic human islets, and further characterisation of CD11c+ cells will be required to determine their roles in type 2 diabetes.
Collectively, our results demonstrate that insulitis is associated with and may participate in the deterioration of islet function in human type 2 diabetes. CCL2 and TNFa expression were strongly correlated with severe GSIS impairment, while the abundance of ALOX12 and CD45+ leucocytes correlated with moderate impairment in GSIS, providing evidence for a complex interplay between islets and the immune system. Moreover, the current study highlights the potential importance of B lymphocytes in human islet biology. The detection of leucocyte populations in non-diabetic and type 2 diabetic human islets may not only provide insight into the physiologic role of immune cells associated with islets, but also highlight new directions for studying inflammation in type 2 diabetes. This could lead to new, more targeted therapies to prevent the decline of functional beta cell mass in patients with type 2 diabetes.
Human islets were provided to J. Nadler and Y. Imai by the IIDP. We thank J. Kaddis and B. Olack of the IIDP for assisting with documentation on the IIDP and donor data.
This work was supported by the Juvenile Diabetes Research Foundation (JN), grants from the National Institutes of Health to JN (R01-HL112605) and YI (R01-DK090490), and by the IIDP pilot programme (YI), a BD Pharmingen Research Grant (EVG), AstraZeneca (JN) and start-up funds from the Eastern Virginia Medical School (EVG).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
JN, EVG and YI contributed to the study conception and design. MJB, EVG (flow cytometry), DH, EG, YM (GSIS, quantitative PCR), SC (quantitative PCR) and YI (GSIS, quantitative PCR, flow cytometry) were responsible for the acquisition and analysis of the data. MJB, EVG and YI interpreted the data, drafted the manuscript and critically revised the manuscript for important intellectual content. DH, EG, YM, SC and JN also revised the manuscript. All authors approved the final version of the manuscript.
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