Search

Many studies have examined obese/insulin-resistant mice to conclude that macrophages dominate pathogenic inflammation in metabolic disease, but few studies have ranked chronic sources of peripheral inflammation in obese people across a natural spectrum of metabolic health. We compared the relative contributions of myeloid and T cells to peripheral inflammation in samples from metabolically healthy and unhealthy obese people to address disappointing outcomes from clinical trials of anti-inflammatories in obesity-associated type 2 diabetes (T2D). We stimulated PBMCs from obese subjects with euglycemia, prediabetes, or T2D with T cell-targeting CD3/CD28 or myeloid-targeting LPS, and measured supernatant cytokines over time. Cytokines increased with time following CD3/CD28, but LPS responses were time-independent. A series of surface marker and intracellular staining assays taking into account these temporal differences in cytokine production unexpectedly showed that T cells contribute more TNFα to peripheral inflammation than do myeloid cells from all subject groups. Bioinformatic modeling combining cytokine outcomes from all subjects, stimuli, and time points, indicated IFNγ generated by CD3/CD28 stimulation was most important for differentiating peripheral inflammation in T2D compared to non-T2D samples. This result was consistent with the previously identified Th1/Th17 profile in T2D. We conclude that human T cells dominate peripheral inflammation in obesity-associated T2D, and thus targeting T cells may be an effective approach for prevention/management of obesity-associated metabolic diseases. Supported by NIH Training Grants T32 DK007778 and TL1 UL1TR001998, R01DK108056, the Shared Resource Facility of the University of Kentucky Markey Cancer Center P30 CA177558, University of Kentucky College of Medicine, Barnstable Brown Diabetes and Obesity Center, The Center for Clinical and Translational Research (UL1TR000117), and NIH National Center for Advancing Translational Sciences UL1TR001998.

Obesity prevalence within the 60+ years-old (yrs) population is drastically increasing. This alarmingly high rate of obesity compels comparative studies to identify common players involved in aging-derived and obesity-associated inflammation. Blood CD4+ T cells from lean older humans show an upregulated Th17 signature accompanied by a defective redox system. Treatment of these cells with the anti-diabetic drug metformin (100μM) promoted a more young-like anti-inflammatory/antioxidant phenotype. Here, we investigate whether the protective effects of metformin observed in CD4+ T cells from lean aging adults persist in obesity. Blood CD4+ T cells from obese older (60+ yrs) donors secrete twice the amount of the Th17 cytokines IL-17F and IL-21 when compared to their younger counterparts (25–35 yrs). Glutamate production, which supports Th17 cytokines secretion, is 1.5-times higher in cells from older compared to younger subjects. Mitochondrial superoxide production in these cells was 25% higher than in younger cells, with no changes in total peroxide. Contrary to findings from cells of lean subjects, metformin failed to reduce Th17 cytokines in cells from obese older subjects, perhaps due to metformin’s inability to reduce glutamate production. However, metformin reduced hydrogen peroxide production 1.75-fold, with no effect on superoxide, in cells from obese older subjects. These results collectively indicate that 1) obesity-associated upregulation of Th17 cytokines is mechanistically linked to glutamine metabolism and mitochondrial superoxide production and 2) metformin’s action targets peroxide production rather than superoxide, which is insufficient for reducing age-associated inflammation in obesity. The Biological mechanisms of Metformin Effects on Aging-Associated Inflammation 1R56AG069685-01

Diabetes interferes with fracture repair; therefore, we investigated mechanisms of impaired fracture healing in a model of multiple low-dose streptozotocin-induced diabetes. Microarray and gene set enrichment analysis revealed an up-regulation of gene sets related to inflammation, including tumor necrosis factor (TNF) signaling in the diabetic group, when cartilage is being replaced by bone on day 16, but not on days 12 or 22. This change coincided with elevated osteoclast numbers and accelerated removal of cartilage in the diabetic group (P < 0.05), which was reflected by smaller callus size. When diabetic mice were treated with the TNF-specific inhibitor, pegsunercept, the number of osteoclasts, cartilage loss, and number of TNF-alpha and receptor activator for nuclear factor kB ligand positive chondrocytes were significantly reduced (P < 0.05). The transcription factor forkhead box 01 (FOXO1) was tested for mediating TNF stimulation of osteoclastogenic and inflammatory factors in bone morphogenetic protein 2 pretreated ATDC5 and C3H10T1/2 chondrogenic cells. FOXO1 knockdown by small-interfering RNA significantly reduced TNF-alpha, receptor activator for nuclear factor kB ligand, macrophage colony-stimulating factor, interleukin-1alpha, and interleukin-6 mRNA compared with scrambled small-interfering RNA. An association between FOXO1 and the TNF-alpha promoter was demonstrated by chromatin immunoprecipitation assay. Moreover, diabetes increased FOXO1 nuclear translocation in chondrocytes in vivo and increased FOXO1 DNA binding activity in diabetic fracture calluses (P < 0.05). These results suggest that diabetes-enhanced TNF-alpha increases the expression of resorptive factors in chondrocytes through a process that involves activation of FOXO1 and that TNF-alpha dysregulation leads to enhanced osteoclast formation and accelerated loss of cartilage.

T cell inflammation plays pivotal roles in obesity-associated type 2 diabetes (T2D). Multiple T cell subsets, including Th1, Th2, Th17 and regulatory T cells are critical for the development of insulin resistance in animal models of T2D. However, the relative importance of T cell subsets in human disease pathogenesis remains a critical gap in knowledge with important implications for defining the most potent inflammatory mediators of T2D. To identify T cell subsets that disproportionately contribute to T2D-associated inflammation, we broadly quantified T cell cytokine production by circulating or adipose-associated immune cells from obese subjects clinically defined as non-T2D, pre-T2D or T2D. Cytokines traditionally associated with specific T cell subsets, including IL-2 (Th1), IL-4/10/13 (Th2) and IL-17A/F (Th17), were produced at highest amounts by T2D samples. We combined cytokine outcomes with multivariate mathematical approaches to show that T cell cytokine profiles predicted T2D status with >85% accuracy. Furthermore, Th17 signature cytokines were more powerful than either Th1 or Th2 cytokines for differentiation of non-T2D from T2D samples. We conclude that Th17 cells are an underappreciated source of T cell cytokines that support inflammation and predict human T2D. Study outcomes unify contradictory claims of the importance of single cytokines and justify including Th17 cells as druggable sources of inflammation in human T2D.

T cell inflammation plays critical roles in the development of obesity-associated type 2 diabetes (T2D). B cells support T cell-mediated inflammation in T2D, but the mechanisms underlying lymphocyte cross-talk and the relative importance of T cell inflammation in human T2D remain untested. Using peripheral blood mononuclear cells (PBMC) and/or purified cells from T2D and non-diabetic (ND) subjects, we show that B cell contact upregulates a pro-inflammatory CD4+ Th17 T cell program in T2D. Neutralization of either B cell CD80/86 or the downstream production of T cell IL-17A/F significantly decreased production of classical diabetogenic cytokines (TNFα, IL-6) in samples from T2D subjects. Although monocytes, B cells and T cells produced TNFα in response to treatment of PBMCs with αCD3/CD28, monocyte TNFα production was independent of IL-17A/F, while IL-17F was critical for maximal TNFα production by both T and B cells. IL-17A/F neutralization also reduced IL-17A and IL-17F production by CD4+ T cells, indicating Th17 function is auto-regulated. We conclude that human B cell co-stimulation broadly supports Th17 cells, which in turn stimulate lymphocytes (but not monocytes) to produce the classical diabetogenic cytokine TNFα.

Immune cells are dominant sources of the obesity-associated inflammation that predisposes type 2 diabetes (T2D), but triggers of immune cell hyper-responsiveness in obesity/T2D are poorly understood. T cells are key sources of cytokines that promote immune cell differentiation/activation, although T cells generally require contact with other immune cells to produce obesity-associated amounts of cytokines. To test the possibility that obesity associates with cytokine changes that impact immune cell differentiation/activation, we stimulated T cells from lean, obese non-diabetic (ND), or obese pre-diabetic (PD; HbA1c 5.6-6.5) subjects in the context of PBMCs and quantitated cytokines with the Milliplex Human High Sensitivity T Cell Panel. Samples from ND and PD subjects produced more T cell/Th1-supportive IL-2, IL-7 and IL-12p70, myeloid-supportive GM-CSF, and B cell-supportive IL-21 compared to samples from leans. In contrast, anti-inflammatory IL-10 was highest in samples from leans. Based on the anti-inflammatory actions of the T2D drug metformin, we also measured cytokine production by samples from PD subjects that were taking metformin. Metformin partially restored IL-10 production in samples from PD subjects, but had no impact on immune cell-activating cytokines. We conclude that obesity associates with inflammation due to over-expression of cytokines that support immune cell differentiation/activation, and that anti-inflammatory action of in vivo metformin is limited.

Chronic inflammation likely underlies the predisposition of type 2 diabetes (T2D) patients to periodontitis (PD), and the exacerbation of each disease when both are present. Several lines of evidence indicate that pro-inflammatory B cells promote T2D and PD individually, but the possibility that B cells are a key link between T2D to PD is untested. We compared outcomes from oral P. gingivalis challenge of lean WT or B cell-null mice to outcomes from mice that were obese and insulin resistance prior to oral challenge. Obese WT mice responded to oral P. gingivalis challenge with significant periodontal bone loss, while obese B cell-null mice were completely protected from PD. By contrast, lean WT and B cell-null mice suffer similar periodontal bone loss in response to oral pathogen. B cells from obese/insulin resistant hosts also support oral osteoclastogenesis, and both oral and systemic production of inflammatory cytokines, including the PD-associated cytokines TNF-α and MIP-2. B cells furthermore impact adipose tissue inflammation in obese P. gingivalis-infected hosts. Taken together, these data show that fundamentally different mechanisms regulate PD in lean and obese hosts, with B cells promoting PD only if the hosts are “primed” by obesity. These results justify more intense analysis of obesity-associated changes in B cells that predispose PD in human T2D, and may indicate the use of B cell depletion to alleviate PD and T2D simultaneously.

Lymphocytes play key roles in the chronic inflammation critical for T2D pathogenesis. We have shown T2D patients have an elevated ratio of pro- to anti-inflammatory T cells, and B cells that produce a pro-inflammatory cytokine profile. Thus lymphocytes promote T2D-associated inflammation. Numerous studies implicate the pro-inflammatory CD4+ T cell balance in T2D pathogenesis, but mechanisms that underlie elevated CD4+ T cell inflammation are poorly understood. We explored the possibility that the T2D-associated changes we identified in B cell function regulate T cell inflammation. We show that B cells control the T2D-associated increase in Th17-mediated inflammation in T2D patients and in obese/insulin resistant mice. Surprisingly, the disease-associated ability of B cells to regulate T cell function is contact-dependent, despite evidence that B cell cytokines hyper-secreted in T2D patients activate T cells. In contrast, elevated activation of Th1 cytokines is B cell-independent. We conclude that both T cell-intrinsic and T cell-extrinsic (B cell-dependent) changes regulate T cell inflammation in T2D. These data indicate that B cell depletion may partially curb T2D-associated T cell inflammation, and thus disease pathogenesis; however, combinatorial treatments aimed at multiple inflammatory axes may be required for favorable clinical outcomes.

Lymphocytes play key roles in the chronic inflammation critical for T2D pathogenesis. T2D patients have an elevated ratio of pro- to anti-inflammatory T cells, and B cells that fail to produce anti-inflammatory IL-10. New data show that B cells from the diet-induced obesity (DIO) mouse model of T2D secrete a pro-inflammatory balance of cytokines, including relatively low IL-10 production, which mirrors our findings in T2D patients. DIO B cells do not secrete auto-antibodies, as evidenced by anti-nuclear antigen staining. Complementary metabolic studies show that B cell-null μMT mice resist the development of glucose intolerance (but not obesity) in response to DIO. Taken together, these data support the conclusion that a pro-inflammatory B cell cytokine balance, rather than antibodies, promotes T2D. To further define roles for B cells in T2D, we immunophenotyped DIO μMT splenocytes. Regulatory T cells (Tregs) expand in DIO μMT, while Tregs fail to expand in WT mice. These data are consistent with new human studies showing B cells from T2D (but not healthy) subjects support pro-inflammatory T cells, and indicate clinical relevance of published studies showing decreased Th1 function in DIO μMT vs. WT mice. We conclude B cells are critical regulators of inflammation in T2D due to 1. Secretion of a pro-inflammatory cytokine balance; and 2. An ability to promote pro-inflammatory T cell function. Thus B cell depletion may represent a valuable tool in the T2D clinic.

Type 2 diabetes (T2D) is an inflammatory disease promoted by changes in immune cell function. Monocytes and T cells play roles in T2D inflammation as evidenced, in part, by our demonstration of pro-inflammatory T cell subset skewing in T2D patients. However, the role of cross-talk amongst immune system cells in T2D is poorly unstudied. PBMC depletion studies showed that T2D patient T cells, unlike non-diabetic (ND) donor cells, required monocytes to maintain IL-17 hyper-secretion. In contrast, B cell depletion did not affect T2D Th17 function, but surprisingly, B cell depletion from non-diabetic (ND) mixed cell cultures significantly elevated Th17 function. Based on the role B cell IL-10 plays in inflammation resolution, we tested the hypothesis that IL-10 regulates immune cell cross-talk to block Th17 function. IL-10 addition to B cell-depleted PBMCs decreased IL-17 to “healthy” levels in both ND and T2D samples, indicating that both cell types are physiologically capable of interpreting the anti-inflammatory signals from B cells despite the presence of pro-inflammatory monocytes in T2D samples. Critically, unlike ND B cells, T2D B cells fail to secrete IL-10, thus lack of B cell IL-10 in T2D samples compounds elevated pro-Th17 cytokine production by monocytes. We conclude that IL-10 can block monocyte-mediated increases in Th17 function, and that lack of B cell IL-10 in T2D associates with IL-17-induced inflammation thus metabolic imbalance.

Type 2 Diabetes (T2D) has recently been identified as a chronic inflammatory disease with changes in immune cell function. Th17 cells and their major product, IL-17, have been implicated in multiple chronic inflammatory diseases. However, the relationship between Th17s and T2D has not been reported. Pro-inflammatory cytokines hyper-secreted by T2D patient monocytes promote Th17 activation. These data suggest a functional link between Th17s and monocytes in T2D. To identify such a link, we first showed that fresh ex vivo T cells from T2D patients have molecular signatures of Th17 cells. T2D T cells stimulated in the context of peripheral blood mononuclear cells (PBMCs) activated Th17 signature genes and elevated IL-17 secretion. In contrast, LPS stimulation of PBMCs from T2D patients failed to elevate IL-17 secretion, indicating that increased monocyte cytokine production, characteristic of T2D, does not further stimulate Th17 cells. Unexpectedly, continuous co-culture of T cells with the non-T cell fraction was required to maintain elevated Th17 function. These data indicate a crucial role for non-T cells in maintaining the Th17 phenotype and elevating IL-17 production in T2D. We conclude the interaction between T cells and non-T cells, most likely monocytes, promotes inflammation in T2D through previously unappreciated mechanisms.