Edited by: Guochang Hu, University of Illinois at Chicago, United States
Reviewed by: Shulin Qin, University of Pittsburgh, United States; Hussein Al-Mossawi, University of Oxford, United Kingdom
This article was submitted to Inflammation, a section of the journal Frontiers in Immunology
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Type 2 diabetes (T2D) is characterized by systemic low-grade inflammation and activation of innate and adaptive immunity, which supports the novel medical theory that T2D is an autoimmune disease. The bone marrow (BM) is a primary immune organ. People with T2D have a fatty BM characterized by inflamed adipocytes that accumulate at the expense of hematopoietic cells and vascular cells.
Abatacept, an immunomodulatory drug that acts through the selective inhibition of T-cell co-stimulation, was approved for rheumatoid arthritis in 2007. The drug has shown favorable action in preserving beta cell function in patients with rheumatoid arthritis, possibly due to its anti-inflammatory and immunomodulatory properties.
Is adaptive immunity activated in the BM of patients with T2D?
Do mice with T2D model the condition seen in patients?
If so, can abatacept treatment rescue T-cell activation, thereby benefiting metabolic control and cardiac function?
Patients and mice with T2D show a shift in the prevalent subsets of BM T lymphocytes, with enrichment of activated cells.
Treatment of obese, T2D mice with abatacept rescued the altered adaptive immune profile and improved heart performance, without improving insulin sensitivity.
The BM requires clinical attention as a target and trigger of inflammatory responses involving cells of the adaptive immunity.
Multicenter trials are warranted to extend the implication of the study to the general population affected by T2D, to determine if those with an activation of adaptive immunity develop more cardiovascular complications.
Modern immunomodulatory drugs that disable just the initial stage of lymphocyte activation leaving the remaining immune army unscathed may represent a novel approach to prevent cardiovascular complications.
Chronic low-grade inflammation plays an important role in the progression of type 2 diabetes (T2D) mellitus and in the increased propensity of diabetic people to develop cardiovascular disease, infections, and cancer (
The BM not only functions as a primary B lymphocyte-producing organ but also represents a site where immune cells are formed, mature, and/or recirculate. Furthermore, the BM plays a key role as a secondary lymphoid organ, crucial for CD4 and CD8 cell-related responses, as well as a site of preferential homing and persistence for memory T cells (
Several studies have shown that people with T2D incur a remarkable remodeling of BM, consisting of an accumulation of inflamed adipocytes and depletion of hematopoietic, neuronal, and vascular cells (
Modern immunomodulatory drugs, which—at variance from classical immunosuppressive agents—disable just a particular type of lymphocyte or interfere with the initial state of lymphocyte activation, leaving the global immune response intact, are attracting much clinical attention. One of these compounds, abatacept, exerts anti-inflammatory activity
The present study was a proof-of-concept investigation assessing the frequencies and activation state of immune cell subsets in the BM of patients with T2D compared with non-diabetic (ND) individuals, with a focus on activated and memory T cells. After confirming human results in a murine model of T2D, we performed a therapeutic preclinical study in a murine model of T2D to determine the effect of abatacept on adaptive immunity, cytokine secretion, metabolic control, and cardiac function.
Patients undergoing hip replacement surgery were recruited under informed consent at the Avon Orthopedic Centre, Southmead Hospital, Bristol, UK. The study protocol complied with the Declaration of Helsinki was covered by institutional ethical approval (REC14/SW/1083 and REC14/WA/1005) and was registered as an observational clinical study in the National Institute for Health Research Clinical Research Network Portfolio, UK Clinical Trials Gateway. Demographic and clinical data of the 24 enrolled subjects are reported in
Characteristics of study subjects.
Age, years | 64.0 ± 3.5 | 70.8 ± 3.0 | N.S. |
Male, % | 50 | 66 | N.S. |
BMI, kg/m2 | 26.6 ± 1.6 | 33.4 ± 1.9 | 0.018 |
HbA1c, mmol/mol | N.D. | 53.3 ± 2.7 | |
Smoking, % | 42 | 50 | N.S. |
Hypertension, % | 66 | 75 | N.S. |
Coronary Artery Disease, % | 17 | 66 | 0.01 |
Insulin, % | 0 | 42 | 0.01 |
Oral anti-diabetic drugs, % | 0 | 66 | 0.01 |
Statins, % | 25 | 83 | 0.01 |
Anti-hypertensive drugs, % | 42 | 50 | N.S. |
Type 2 diabetes was diagnosed according to the American Diabetes Association guidelines. Specifically, it was defined as (1) patient/referring doctor reports a previous diagnosis of diabetes, and (2) HbA1c > 48 mmol/mol. We excluded subjects with acute disease/infection, inflammatory/immune diseases, current or past hematological disorders or malignancy, unstable angina, recent (within 6 months) myocardial infarction or stroke, heart failure, liver failure, dialysis, and pregnancy.
Bone marrow samples were obtained from scooped femur heads remaining from hip replacement surgery. Only material that would otherwise be discarded was collected for the study. During the replacement procedure, the femoral head was removed with a saw, and the proximal femoral canal was opened with reamers and rasps. The BM displaced into the wound was scooped into a sterile pot with a curette. The sample was decanted into a collection tube with 0.5 mol/L ethylenediaminetetraacetic acid (EDTA), pH 8 (Thermo Fisher Scientific, Gloucester, UK, #28348) labeled, and placed in a fridge for collection by the investigator within 1 h.
The diluted BM suspension was passed through a 100-μm filter to remove bone fragments and cell clumps. The sample was stratified on Ficoll Histopaque 1077 (Sigma-Aldrich, St. Louis, MO, USA, #10771) and centrifuged without acceleration or brake at 300 × g for 45 min at 24°C. Mononuclear cells sedimented at the interphase were then collected, washed twice with phosphate buffered saline (PBS), assessed for viability by trypan blue staining (Thermo Fisher Scientific, #15250061), and counted at microscopy. Plasma was separated from freshly collected human BM mononuclear cells (BMMCs) through centrifugation at 3,500 rpm for 10 min and frozen immediately at −80°C.
The following monoclonal antibody combinations were used to characterize the phenotype of different immune cell subsets: anti-CD45RO-BB515 (#564529), anti-CD3-PE-cy5.5 (#340949), anti-CD4-PE-cy5.5 (#35-0047-42), anti-CD8-PE-cy5 (#15-0088-42), anti-CD45RA-BV605 (#562886), anti-CD25-PE-cy7 (#557741), anti-CD69-APC (#310910), anti-CCR7-Aleza700 (#565867), anti-CD45 BV771 (#304050), anti-CD56PE-cy7 (#557747), anti-CD8-BV510 (#563256), anti-CD19-APC (#340722), anti-CD4BV421 (#565997), anti-CD3PE #340662), and anti-CD19BV785 (#363028). All antibodies were titrated for optimal staining performance.
Cell viability was determined using Zombie NIR™ Fixable Viability Kit (BioLegend, San Diego, CA, USA; #423106). Briefly, 70 × 106 BMMCs (at a concentration of 1 × 106 cells/ml) were stained with Zombie NIR™ Fixable Viability Kit for 30 min at room temperature (RT). Then, the cells were washed with Flow Cytometry Staining Buffer (FACS buffer) (eBioscience™ Flow Cytometry Staining Buffer, eBioscience, Inc., San Diego, CA, USA; #00-4222-57) at 300 × g for 10 min. The staining protocol included the use of an Fc blocking antibody (True-Stain Monocyte Blocker, BioLegend, #426102) to control for non-specific binding and background fluorescence. Each sample was added with 100 μl Fc blocking antibody (diluted in FACS buffer at 1:50 ratio) and incubated on ice for 20 min. The cells were then centrifuged at 500 × g for 5 min at 4°C. Subsequently, BMMCs were stained with a defined combination of antibodies, at the appropriate dilutions, in FACS buffer for 20 min at 4°C. Cells were washed twice in FACS buffer and stained with 1% PFA. A minimum of 2 × 106 cells/ml was assayed in the flow cytometry studies. Fluorescence minus one (FMO) controls were included in building multicolor flow cytometry panels. Moreover, BD Comp Beads (#560497) were used to optimize fluorescence compensation settings in multicolor flow cytometry studies. The analysis was performed on a BD LSR II Fortessa X20 (Becton Dickinson, San Jose, CA, USA) using FlowJo software (TreeStar, San Carlos, CA, USA).
Experiments were performed following the Guide for the Care and Use of Laboratory Animals (The Institute of Laboratory Animal Resources, 1996) and with the approval of the University of Bristol and the British Home Office (license 30/3373). As a model of T2D, we used a 10-week-old male obese leptin receptor homozygous mutant BKS.Cg-+Leprdb/+Leprdb/OlaHsd (db/db) mice (Envigo, Blackthorn, UK). Age- and sex-matched lean heterozygous + BKS.Cg-+Leprdb/+LeprWT/OlaHsd (db/WT) mice served as controls. Animals were maintained in the high standard animal facilities of the University of Bristol, under the surveillance of specialists trained in husbandry and welfare of the species. The animals fed standard chow (EURodent Diet 22%,
Abatacept (250 mg lyophilized powder per vial, Bristol-Myers Squibb, Princeton, NJ, USA) was purchased from the Bristol University Hospital Pharmacy. The drug was reconstituted at 2× of the concentration required in sterile distilled water (25 mg/ml). The vial was gently swirled until the complete dissolution of the compound. The reconstituted material was diluted with sterile pH-checked PBS for
A cohort of twelve 10-week-old male db/db mice was randomized for treatment with abatacept or vehicle. Mice were injected intraperitoneally with 300 μg abatacept in 100 μl PBS, three non-consecutive times a week, for 4 weeks, while controls received the vehicle. The dosage and control choice were adapted from a previous abatacept study in a murine model of cardiomyopathy (
At sacrifice, whole BM cells were obtained from the mouse tibiae and femora. Both ends of the bones were cut using sterile, sharpened scissors. BM cells were harvested by repeated flushing of the bone shaft in 1 ml PBS in a tube using a syringe provided with a 23-gauge needle. After removal of aggregates from the BM suspension by vigorous pipetting and filtration through a 70-μm mesh nylon strainer (#07-201-431, Thermo Fisher Scientific), the sample was centrifuged at 300 × g for 10 min. The pellets were resuspended to obtain a suspension of 2 × 106 cells/ml in fresh PBS for use in cytometry analysis. Supernatants were collected for cytokine assays and stored at −20°C.
Mononuclear spleen suspensions were prepared by gently pressing the spleen tissue with the flat end of a syringe in 5 ml of Roswell Park Memorial Institute Medium (RPMI) containing 1% pen/strep, on a 100-mm culture dish. Then, the cell suspension was passed through on a 70-μm cell strainer into a 50-ml tube. After washing twice with 5-ml RPMI, the cell suspension was centrifuged at 300 × g for 10 min, and cells were resuspended in eBioscience™ 1 × red blood cell (RBC) Lysis Buffer (#00-4333-57) on ice for 3 min. Finally, splenocytes were washed, resuspended in PBS at a concentration of 2 × 106 cells/ml, and counted.
At sacrifice, the blood was harvested with an EDTA-coated syringe from the left ventricle and placed into Eppendorf tubes (Eppendorf, Hamburg, Germany). Blood was centrifuged for 15 min at 3,000 rpm (1,500 × g) at 4°C. After centrifugation, the plasma was removed, and the pellet was incubated with 1× RBC Lysis Buffer on ice for 10 min. Mononuclear cells were then resuspended at 2 × 106 cells/ml in PBS. Plasma was stored at −80°C for later analysis.
Immunofluorescence surface staining was performed by adding a panel of directly conjugated mAb to freshly prepared BMMCs, splenocytes, and peripheral blood mononuclear cells. Before staining, cell viability was assessed using Zombie NIR™ Fixable Viability Kit (#423106) for 30 min at RT. For all experiments, cell suspensions were preincubated with anti-CD16/CD32 mAb (BioLegend) to block FcγRII/III receptors. Cell staining was performed in the dark for 20 min at 4°C in FACS staining buffer (BD, #554656). The following reagents were used: CD4-PE-cy5.5 (#35-0042-82), CD8-PE-cy5 (#15-0083-81), CD25-FiTC (#130-120-172), CD45-BV785 (#103149), CD69 PE-Vio®770 (#30-103-944), NK1.1-AF700 (#108729), CD19-BV510 (#115545), CD44-Super Bright (#63-0441-82), CD62l BV650 (#564108), F4/80-APC (#123115), CD206-PE (#141705), CD11b-PE-cy 7 (#101215), CD80-PEdazzle (#104737), CD11c-BV605 (#117333), and MHCII-AF700 (#107621). The proliferation of CD4+ and CD8+ cells was assessed using the KI67-BV510 marker (#563462). Cells were fixed and permeabilized using the BD Pharmingen™ Transcription Factor Buffer Set Kit (#562574; BD Pharmingen Inc., San Diego, CA, USA). A minimum of 2 × 106 cells/ml was assayed in the flow cytometry studies. BD Anti-Mouse Ig, κ/Negative Control (BSA) Compensation Plus (7.5 μm) Particles Set (#560497) were used to optimize fluorescence compensation settings in multicolor flow cytometry analysis. Cells were analyzed on LSR II Fortessa X20 Flow Cytometry instrument using the FlowJo software.
Cytokine content in plasma separated from BM was measured using the Proteome Profiler Human Cytokine Array Kit (Research & Diagnostic Systems, Inc, Minneapolis, MN, USA; #ARY005B). A dedicated mouse Cytokine Array Kit (#ARY006) was used to measure cytokines in the BM supernatants and the plasma isolated from the peripheral blood. The cytokines were detected by exposing the membrane to X-ray film, which was subsequently developed with ChemiDoc XRS+ System by Bio-Rad (Hercules, California, USA). The mean luminescence was normalized to reference spots from the same membrane following background correction. Pixel densities on developed X-ray film were analyzed using Fiji—ImageJ image analysis software (National Institutes of Health, Bethesda, MD, USA).
Plasma levels of insulin and glucose were measured using colorimetric assay kits (from Thermo Fisher Scientific, #EMINS, and Abcam, #ab65333, respectively). The quantitative insulin sensitivity check index (QUICKI) was calculated according to the formula QUICKI = 1/(logI0 + logG0), where I is insulin (μU/ml) and G is glucose (nmol/μl). In addition, the HOMA-IR formula was calculated as fasting insulin multiplied by fasting glucose divided by 22.5.
Values are presented as mean ± SEM. Two-tailed independent-samples
The two studied groups were similar regarding age and sex distribution (
The flow cytometry analysis of BM cells was performed on data of the CD45/SSC gating, where lymphocytes showed the highest CD45 fluorescence intensity and the lowest SSC signal (
Flow cytometry analysis of immune cells in human bone marrow shows type 2 diabetes (T2D) is associated with an increased abundance of CD4+ and CD8+ cells. Scooped bone marrow (BM) from femoral head leftovers of orthopedic surgery was obtained from patients previously diagnosed to have T2D and controls without T2D (ND).
T cells are activated through the presentation of antigens expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete cytokines that regulate and sustain the immune response. Previous studies have shown that the human BM contains T cells in the activated state as denoted by the expression of CD69 (
Increased relative abundance of activated T cells in the bone marrow (BM) of patients with T2D.
The CC-chemokine receptor 7 (CCR7) and its ligands play a key role in lymphocyte homing to lymphoid tissue. In the T2D group, both CD4+ and CD8+ lymphocytes expressed CCR7 with higher frequency than the ND group (2.27-fold,
Increased relative abundance of CCR7 expressing T lymphocytes in BM of patients with T2D.
A multivariable regression model including age, sex, and BMI as covariates confirmed the differences emerging from the univariate analysis illustrated in previous paragraphs.
We next examined the expression of a panel of cytokines, chemokines, and proteins in BM perfusates from three patients with T2D and three ND subjects. Of 104 measured factors, 65 were upregulated and 2 downregulated in T2D BM, considering a threshold fold change ≥2 (
Altered chemokine profile in BM of patients with T2D.
List of modulated factors in human bone marrow with value of the fold change vs. ND.
CD40LG | CD40 ligand; Cytokine that binds to CD40/TNFRSF5. Costimulates T-cell proliferation and cytokine production. Its cross-linking on T-cells generates a costimulatory signal which enhances the production of IL4 and IL10 in conjunction with the TCR/CD3 ligation and CD28 costimulation. Induces the activation of NF-kappa-B and kinases MAPK8 and PAK2 in T- cells. Induces tyrosine phosphorylation of isoform 3 of CD28. Mediates B-cell proliferation in the absence of co-stimulus as well as IgE production in the presence of IL4. Involved in immunoglobulin class switching (By similarity) | 0.48 | 0.41 |
LIF | Leukemia inhibitory factor; LIF has the capacity to induce terminal differentiation in leukemic cells. Its activities include the induction of hematopoietic differentiation in normal and myeloid leukemia cells, the induction of neuronal cell differentiation, and the stimulation of acute-phase protein synthesis in hepatocytes; Endogenous ligands | 0.48 | 0.85 |
BDNF | Brain-derived neurotrophic factor; During development, promotes the survival and differentiation of selected neuronal populations of the peripheral and central nervous systems. Participates in axonal growth, pathfinding and in the modulation of dendritic growth and morphology. Major regulator of synaptic transmission and plasticity at adult synapses in many regions of the CNS | 2.01 | 0.24 |
GGH | Gamma-glutamyl hydrolase; Hydrolyzes the polyglutamate sidechains of pteroylpolyglutamates. Progressively removes gamma-glutamyl residues from pteroylpoly-gamma-glutamate to yield pteroyl-alpha- glutamate (folic acid) and free glutamate. May play an important role in the bioavailability of dietary pteroylpolyglutamates and in the metabolism of pteroylpolyglutamates and antifolates; Belongs to the peptidase C26 family | 2.10 | 0.27 |
CXCL8 | Interleukin-8; IL-8 is a chemotactic factor that attracts neutrophils, basophils, and T-cells, but not monocytes. It is also involved in neutrophil activation. It is released from several cell types in response to an inflammatory stimulus. IL-8(6-77) has a 5-10-fold higher activity on neutrophil activation, IL-8(5-77) has increased activity on neutrophil activation and IL-8(7-77) has a higher affinity to receptors CXCR1 and CXCR2 as compared to IL-8(1-77), respectively; Chemokine ligands | 2.13 | 0.13 |
IL15 | Interleukin-15; Cytokine that stimulates the proliferation of T- lymphocytes. Stimulation by IL-15 requires interaction of IL-15 with components of IL-2R, including IL-2R beta and probably IL-2R gamma but not IL-2R alpha; Belongs to the IL-15/IL-21 family | 2.20 | 0.59 |
DKK1 | Dickkopf-related protein 1; Antagonizes canonical Wnt signaling by inhibiting LRP5/6 interaction with Wnt and by forming a ternary complex with the transmembrane protein KREMEN that promotes internalization of LRP5/6 | 2.23 | 0.58 |
LEP | Leptin; Key player in the regulation of energy balance and body weight control. Once released into the circulation, has central and peripheral effects by binding LEPR, found in many tissues, which results in the activation of several major signaling pathways. | 2.30 | 0.13 |
RETN | Resistin; Hormone that seems to suppress insulin ability to stimulate glucose uptake into adipose cells (By similarity). Potentially links obesity to diabetes (By similarity). Promotes chemotaxis in myeloid cells | 2.30 | 0.10 |
IL32 | Interleukin-32; Interleukin 32; Interleukins | 2.40 | 0.31 |
CXCL9 | C-X-C motif chemokine 9; Cytokine that affects the growth, movement, or activation state of cells that participate in immune and inflammatory response. Chemotactic for activated T-cells. Binds to CXCR3; Belongs to the intercrine alpha (chemokine CxC) family | 2.49 | 0.59 |
IGFBP2 | Insulin-like growth factor-binding protein 2; Inhibits IGF-mediated growth and developmental rates. IGF-binding proteins prolong the half-life of the IGFs and have been shown to either inhibit or stimulate the growth promoting effects of the IGFs on cell culture. They alter the interaction of IGFs with their cell surface receptors | 2.50 | 0.008 |
PDGFB | Platelet-derived growth factor subunit B; Growth factor that plays an essential role in the regulation of embryonic development, cell proliferation, cell migration, survival and chemotaxis. Potent mitogen for cells of mesenchymal origin. Required for normal proliferation and recruitment of pericytes and vascular smooth muscle cells in the central nervous system, skin, lung, heart and placenta. Required for normal blood vessel development, and for normal development of kidney glomeruli | 2.50 | 0.15 |
CSF2 | Granulocyte-macrophage colony-stimulating factor; Cytokine that stimulates the growth and differentiation of hematopoietic precursor cells from various lineages, including granulocytes, macrophages, eosinophils and erythrocytes; Belongs to the GM-CSF family | 2.52 | 0.55 |
CST3 | Cystatin-C; As an inhibitor of cysteine proteinases, this protein is thought to serve an important physiological role as a local regulator of this enzyme activity; Belongs to the cystatin family | 2.55 | 0.45 |
FGF2 | Fibroblast growth factor 2; Plays an important role in the regulation of cell survival, cell division, angiogenesis, cell differentiation and cell migration. Functions as potent mitogen |
2.55 | 0.32 |
IL19 | Interleukin-19; May play some important roles in inflammatory responses. Up-regulates IL-6 and TNF-alpha and induces apoptosis (By similarity); Interleukins | 2.60 | 0.36 |
TNFSF13B | Tumor necrosis factor ligand superfamily member 13B; Cytokine that binds to TNFRSF13B/TACI and TNFRSF17/BCMA. TNFSF13/APRIL binds to the same 2 receptors. Together, they form a 2 ligands −2 receptors pathway involved in the stimulation of B- and T-cell function and the regulation of humoral immunity. A third B-cell specific BAFF-receptor (BAFFR/BR3) promotes the survival of mature B-cells and the B-cell response; CD molecules | 2.71 | 0.31 |
C19orf10 | Myeloid-derived growth factor; Bone marrow-derived monocyte and paracrine-acting protein that promotes cardiac myocyte survival and adaptive angiogenesis for cardiac protection and/or repair after myocardial infarction (MI). Stimulates endothelial cell proliferation through a MAPK1/3-, STAT3-, and CCND1-mediated signaling pathway. Inhibits cardiac myocyte apoptosis in a PI3K/AKT-dependent signaling pathway (By similarity). Involved in endothelial cell proliferation and angiogenesis | 2.90 | 0.38 |
CXCL12 | Stromal cell-derived factor 1; Chemoattractant active on T-lymphocytes, monocytes, but not neutrophils. Activates the C-X-C chemokine receptor CXCR4 to induce a rapid and transient rise in the level of intracellular calcium ions and chemotaxis. Also binds to atypical chemokine receptor ACKR3, which activates the beta-arrestin pathway and acts as a scavenger receptor for SDF-1. SDF-1-beta(3-72) and SDF-1- alpha(3-67) show a reduced chemotactic activity | 2.90 | 0.27 |
IL1B | Interleukin-1 beta; Potent proinflammatory cytokine. Initially discovered as the major endogenous pyrogen, induces prostaglandin synthesis, neutrophil influx and activation, T-cell activation and cytokine production, B-cell activation and antibody production, and fibroblast proliferation and collagen production. Promotes Th17 differentiation of T-cells | 2.90 | 0.51 |
PDGFA | Platelet-derived growth factor subunit A; Growth factor that plays an essential role in the regulation of embryonic development, cell proliferation, cell migration, survival and chemotaxis. Potent mitogen for cells of mesenchymal origin. Required for normal lung alveolar septum formation during embryogenesis, normal development of the gastrointestinal tract, normal development of Leydig cells and spermatogenesis | 3.00 | 0.09 |
MMP9 | Matrix metalloproteinase-9; May play an essential role in local proteolysis of the extracellular matrix and in leukocyte migration. Could play a role in bone osteoclastic resorption. Cleaves KiSS1 at a Gly-|-Leu bond. Cleaves type IV and type V collagen into large C-terminal three quarter fragments and shorter N-terminal one quarter fragments. Degrades fibronectin but not laminin or Pz-peptide; M10 matrix metallopeptidases | 3.10 | 0.20 |
RLN1 | Prorelaxin H1; Relaxin is an ovarian hormone that acts with estrogen to produce dilatation of the birth canal in many mammals. May be involved in remodeling of connective tissues during pregnancy, promoting growth of pubic ligaments and ripening of the cervix; Belongs to the insulin family | 3.10 | 0.61 |
TGFA | Protransforming growth factor alpha; TGF alpha is a mitogenic polypeptide that is able to bind to the EGF receptor/EGFR and to act synergistically with TGF beta to promote anchorage-independent cell proliferation in soft agar | 3.20 | 0.69 |
MOK | MAPK/MAK/MRK overlapping kinase; Able to phosphorylate several exogenous substrates and to undergo autophosphorylation. Negatively regulates cilium length in a cAMP and mTORC1 signaling-dependent manner; Belongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family. CDC2/CDKX subfamily | 3.30 | 0.73 |
CSF3 | Granulocyte colony-stimulating factor; Granulocyte/macrophage colony-stimulating factors are cytokines that act in hematopoiesis by controlling the production, differentiation, and function of 2 related white cell populations of the blood, the granulocytes and the monocytes-macrophages. This CSF induces granulocytes; Belongs to the IL-6 superfamily | 3.40 | 0.62 |
IL16 | Pro-interleukin-16; Interleukin-16 stimulates a migratory response in CD4+ lymphocytes, monocytes, and eosinophils. Primes CD4+ T-cells for IL-2 and IL-15 responsiveness. Also induces T-lymphocyte expression of interleukin 2 receptor. Ligand for CD4; Interleukins | 3.4 | 0.37 |
IL2 | Interleukin-2; Produced by T-cells in response to antigenic or mitogenic stimulation, this protein is required for T-cell proliferation and other activities crucial to regulation of the immune response. Can stimulate B-cells, monocytes, lymphokine- activated killer cells, natural killer cells, and glioma cells; Interleukins | 3.47 | 0.40 |
FLT3LG | Fms-related tyrosine kinase 3 ligand; Stimulates the proliferation of early hematopoietic cells by activating FLT3. Synergizes well with a number of other colony stimulating factors and interleukins; Endogenous ligands | 3.50 | 0.60 |
IL11 | Interleukin-11; Cytokine that stimulates the proliferation of hematopoietic stem cells and megakaryocyte progenitor cells and induces megakaryocyte maturation resulting in increased platelet production. Also promotes the proliferation of hepatocytes in response to liver damage. Binding to its receptor formed by IL6ST and either IL11RA1 or IL11RA2 activates a signaling cascade that promotes cell proliferation. Signaling leads to the activation of intracellular protein kinases and the phosphorylation of STAT3; Belongs to the IL-6 superfamily | 3.51 | 0.14 |
CCL20 | C-C motif chemokine 20; Acts as a ligand for C-C chemokine receptor CCR6. Signals through binding and activation of CCR6 and induces a strong chemotactic response and mobilization of intracellular calcium ions. The ligand-receptor pair CCL20-CCR6 is responsible for the chemotaxis of dendritic cells (DC), effector/memory T-cells and B- cells and plays an important role at skin and mucosal surfaces under homeostatic and inflammatory conditions | 3.60 | 0.51 |
ICAM1 | Intercellular adhesion molecule 1; ICAM proteins are ligands for the leukocyte adhesion protein LFA-1 (integrin alpha-L/beta-2). During leukocyte trans- endothelial migration, ICAM1 engagement promotes the assembly of endothelial apical cups through ARHGEF26/SGEF and RHOG activation; CD molecules | 3.60 | 0.06 |
MIF | Macrophage migration inhibitory factor; Pro-inflammatory cytokine. Involved in the innate immune response to bacterial pathogens. The expression of MIF at sites of inflammation suggests a role as mediator in regulating the function of macrophages in host defense. Counteracts the anti- inflammatory activity of glucocorticoids. Has phenylpyruvate tautomerase and dopachrome tautomerase activity ( |
3.60 | 0.10 |
CHI3L1 | Chitinase-3-like protein 1; Carbohydrate-binding lectin with a preference for chitin. Has no chitinase activity. May play a role in tissue remodeling and in the capacity of cells to respond to and cope with changes in their environment. Plays a role in T-helper cell type 2 (Th2) inflammatory response and IL-13-induced inflammation, regulating allergen sensitization, inflammatory cell apoptosis, dendritic cell accumulation and M2 macrophage differentiation. Facilitates invasion of pathogenic enteric bacteria into colonic mucosa and lymphoid organs | 3.76 | 0.003 |
CCL17 | C-C motif chemokine 17; Chemotactic factor for T-lymphocytes but not monocytes or granulocytes. May play a role in T-cell development in thymus and in trafficking and activation of mature T-cells. Binds to CCR4; Belongs to the intercrine beta (chemokine CC) family | 3.80 | 0.24 |
CCL3 | C-C motif chemokine 3; Monokine with inflammatory and chemokinetic properties. Binds to CCR1, CCR4, and CCR5. One of the major HIV-suppressive factors produced by CD8+ T-cells. Recombinant MIP-1-alpha induces a dose-dependent inhibition of different strains of HIV-1, HIV-2, and simian immunodeficiency virus (SIV); Belongs to the intercrine beta (chemokine CC) family | 3.80 | 0.63 |
CCL19 | C-C motif chemokine 19; May play a role not only in inflammatory and immunological responses but also in normal lymphocyte recirculation and homing. May play an important role in trafficking of T-cells in thymus, and T-cell and B-cell migration to secondary lymphoid organs. Binds to chemokine receptor CCR7. Recombinant CCL19 shows potent chemotactic activity for T-cells and B-cells but not for granulocytes and monocytes. Binds to atypical chemokine receptor ACKR4 and mediates the recruitment of beta-arrestin (ARRB1/2) to ACKR4; Belongs to the intercrine beta (chemokine CC) family | 3.90 | 0.23 |
IL34 | Interleukin-34; Cytokine that promotes the proliferation, survival and differentiation of monocytes and macrophages. Promotes the release of proinflammatory chemokines, and thereby plays an important role in innate immunity and in inflammatory processes. Plays an important role in the regulation of osteoclast proliferation and differentiation, and in the regulation of bone resorption. Signaling via CSF1R and its downstream effectors stimulates phosphorylation of MAPK1/ERK2 AND MAPK3/ERK1; Belongs to the IL-34 family | 4.10 | 0.42 |
IL1RN | Interleukin-1 receptor antagonist protein; Inhibits the activity of interleukin-1 by binding to receptor IL1R1 and preventing its association with the coreceptor IL1RAP for signaling. Has no interleukin-1 like activity. Binds functional interleukin-1 receptor IL1R1 with greater affinity than decoy receptor IL1R2; however, the physiological relevance of the latter association is unsure; Endogenous ligands | 4.23 | 0.28 |
IL23A | Interleukin-23 subunit alpha; Associates with IL12B to form the IL-23 interleukin, a heterodimeric cytokine which functions in innate and adaptive immunity. IL-23 may constitute with IL-17 an acute response to infection in peripheral tissues. IL-23 binds to a heterodimeric receptor complex composed of IL12RB1 and IL23R, activates the Jak- Stat signaling cascade, stimulates memory rather than naive T- cells and promotes production of proinflammatory cytokines. IL-23 induces autoimmune inflammation | 4.30 | 0.24 |
TFF3 | Trefoil factor 3; Involved in the maintenance and repair of the intestinal mucosa. Promotes the mobility of epithelial cells in healing processes (motogen) | 4.36 | 0.21 |
CXCL10 | C-X-C motif chemokine 10; Chemotactic for monocytes and T-lymphocytes. Binds to CXCR3; Belongs to the intercrine alpha (chemokine CxC) family | 4.40 | 0.36 |
TFRC | Transferrin receptor protein 1; Cellular uptake of iron occurs via receptor-mediated endocytosis of ligand-occupied transferrin receptor into specialized endosomes. Endosomal acidification leads to iron release. The apotransferrin-receptor complex is then recycled to the cell surface with a return to neutral pH and the concomitant loss of affinity of apotransferrin for its receptor. Transferrin receptor is necessary for development of erythrocytes and the nervous system (By similarity) | 4.45 | 0.16 |
TNFRSF8 | Tumor necrosis factor receptor superfamily member 8; Receptor for TNFSF8/CD30L. May play a role in the regulation of cellular growth and transformation of activated lymphoblasts. Regulates gene expression through activation of NF- kappa-B | 4.47 | 0.44 |
CD14 | Monocyte differentiation antigen CD14; Coreceptor for bacterial lipopolysaccharide. In concert with LBP, binds to monomeric lipopolysaccharide and delivers it to the LY96/TLR4 complex, thereby mediating the innate immune response to bacterial lipopolysaccharide (LPS). Acts via MyD88, TIRAP and TRAF6, leading to NF-kappa-B activation, cytokine secretion and the inflammatory response | 4.70 | 0.32 |
TNF | Tumor necrosis factor; Cytokine that binds to TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. It is mainly secreted by macrophages and can induce cell death of certain tumor cell lines. It is potent pyrogen causing fever by direct action or by stimulation of interleukin-1 secretion and is implicated in the induction of cachexia, Under certain conditions it can stimulate cell proliferation and induce cell differentiation. Impairs regulatory T-cells (Treg) function in individuals with rheumatoid arthritis via FOXP3 dephosphorylation | 4.70 | 0.60 |
THBS1 | Thrombospondin-1; Adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions. Binds heparin. May play a role in dentinogenesis and/or maintenance of dentin and dental pulp (By similarity). Ligand for CD36 mediating antiangiogenic properties. Plays a role in ER stress response, via its interaction with the activating transcription factor 6 alpha (ATF6) which produces adaptive ER stress response factors (By similarity) | 5.30 | 0.21 |
CCL2 | C-C motif chemokine 2; Chemotactic factor that attracts monocytes and basophils but not neutrophils or eosinophils. Augments monocyte anti-tumor activity. Has been implicated in the pathogenesis of diseases characterized by monocytic infiltrates, like psoriasis, rheumatoid arthritis or atherosclerosis. May be involved in the recruitment of monocytes into the arterial wall during the disease process of atherosclerosis; Belongs to the intercrine beta (chemokine CC) family | 5.70 | 0.23 |
FUT4 | Alpha-(1,3)-fucosyltransferase 4; May catalyze alpha-1,3 glycosidic linkages involved in the expression of Lewis X/SSEA-1 and VIM-2 antigens; CD molecules | 6.30 | 0.59 |
IL10 | Interleukin-10; Inhibits the synthesis of a number of cytokines, including IFN-gamma, IL-2, IL-3, TNF, and GM-CSF produced by activated macrophages and by helper T-cells; Belongs to the IL-10 family | 6.69 | 0.33 |
TDGF1 | Teratocarcinoma-derived growth factor 1; GPI-anchored cell membrane protein involved in Nodal signaling. Cell-associated TDGF1 acts as a Nodal coreceptor in cis. Shedding of TDGF1 by TMEM8A modulates Nodal signaling by allowing soluble TDGF1 to act as a Nodal coreceptor on other cells. Could play a role in the determination of the epiblastic cells that subsequently give rise to the mesoderm | 7.04 | 0.69 |
BSG | Basigin; Plays an important role in targeting the monocarboxylate transporters SLC16A1, SLC16A3, SLC16A8, and SLC16A11 to the plasma membrane. Plays pivotal roles in spermatogenesis, embryo implantation, neural network formation and tumor progression. Stimulates adjacent fibroblasts to produce matrix metalloproteinases (MMPS). Seems to be a receptor for oligomannosidic glycans. |
7.15 | 0.01 |
IL33 | Interleukin-33; Cytokine that binds to and signals through the IL1RL1/ST2 receptor which in turn activates NF-kappa-B and MAPK signaling pathways in target cells. Involved in the maturation of Th2 cells inducing the secretion of T-helper type 2-associated cytokines. Also involved in activation of mast cells, basophils, eosinophils and natural killer cells. Acts as a chemoattractant for Th2 cells, and may function as an “alarmin,” that amplifies immune responses during tissue injury; Interleukins | 7.30 | 0.09 |
CXCL11 | C-X-C motif chemokine 11; Chemotactic for interleukin-activated T-cells but not unstimulated T-cells, neutrophils, or monocytes. Induces calcium release in activated T-cells. Binds to CXCR3. May play an important role in CNS diseases which involve T-cell recruitment. May play a role in skin immune responses; Belongs to the intercrine alpha (chemokine CxC) family | 8.90 | 0.12 |
ANGPT1 | Angiopoietin-1; Binds and activates TEK/TIE2 receptor by inducing its dimerization and tyrosine phosphorylation. Plays an important role in the regulation of angiogenesis, endothelial cell survival, proliferation, migration, adhesion and cell spreading, reorganization of the actin cytoskeleton, but also maintenance of vascular quiescence. Required for normal angiogenesis and heart development during embryogenesis. After birth, activates or inhibits angiogenesis, depending on the context | 10.12 | 0.29 |
IGFBP3 | Insulin-like growth factor-binding protein 3; IGF-binding proteins prolong the half-life of the IGFs and have been shown to either inhibit or stimulate the growth promoting effects of the IGFs on cell culture. They alter the interaction of IGFs with their cell surface receptors. Also exhibits IGF-independent antiproliferative and apoptotic effects mediated by its receptor TMEM219/IGFBP-3R | 11.7 | 0.07 |
FGF7 | Fibroblast growth factor 7; Plays an important role in the regulation of embryonic development, cell proliferation and cell differentiation. Required for normal branching morphogenesis. Growth factor active on keratinocytes. Possible major paracrine effector of normal epithelial cell proliferation; Belongs to the heparin-binding growth factors family | 12.1 | 0.54 |
PLAUR | Urokinase plasminogen activator surface receptor; Acts as a receptor for urokinase plasminogen activator. Plays a role in localizing and promoting plasmin formation. Mediates the proteolysis-independent signal transduction activation effects of U-PA. It is subject to negative-feedback regulation by U-PA which cleaves it into an inactive form; CD molecules | 12.9 | 0.09 |
CCL5 | C-C motif chemokine 5; Chemoattractant for blood monocytes, memory T-helper cells and eosinophils. Causes the release of histamine from basophils and activates eosinophils. May activate several chemokine receptors including CCR1, CCR3, CCR4, and CCR5. One of the major HIV-suppressive factors produced by CD8+ T-cells. Recombinant RANTES protein induces a dose-dependent inhibition of different strains of HIV-1, HIV-2, and simian immunodeficiency virus (SIV). | 13.10 | 0.12 |
IL31 | Interleukin-31; Activates STAT3 and possibly STAT1 and STAT5 through the IL31 heterodimeric receptor composed of IL31RA and OSMR. May function in skin immunity. Enhances myeloid progenitor cell survival |
13.3 | 0.34 |
IL18 | Interleukin-18; Augments natural killer cell activity in spleen cells and stimulates interferon gamma production in T-helper type I cells; Belongs to the IL-1 family | 13.6 | 0.10 |
EGF | Pro-epidermal growth factor; EGF stimulates the growth of various epidermal and epithelial tissues |
14.4 | 0.28 |
C5 | Complement C5; Activation of C5 by a C5 convertase initiates the spontaneous assembly of the late complement components, C5-C9, into the membrane attack complex. C5b has a transient binding site for C6. The C5b-C6 complex is the foundation upon which the lytic complex is assembled; C3 and PZP like, alpha-2-macroglobulin domain containing | 16.20 | 0.13 |
VEGFA | Vascular endothelial growth factor A; Growth factor active in angiogenesis, vasculogenesis and endothelial cell growth. Induces endothelial cell proliferation, promotes cell migration, inhibits apoptosis and induces permeabilization of blood vessels. Binds to the FLT1/VEGFR1 and KDR/VEGFR2 receptors, heparan sulfate and heparin. NRP1/Neuropilin-1 binds isoforms VEGF-165 and VEGF-145. Isoform VEGF165B binds to KDR but does not activate downstream signaling pathways, does not activate angiogenesis and inhibits tumor growth | 17.2 | 0.03 |
An analysis conducted using the STRING database revealed that most of the differentially regulated factors were connected in a network with a PPI enrichment
We next sought confirmation about the influence of T2D on immune cells by assessing the phenotype of different cell populations in the BM, spleen, and peripheral blood from 10-week-old db/db and control age-matched wt/db mice (
Higher frequency and activated state of T lymphocytes in diabetic mice.
We also extended the analysis beyond lymphocytes to macrophages, identified as CD11b+F4/80+ cells; they were more abundant in db/db mice at the level of BM (9.25 ± 0.89 vs. 5.77 ± 0.58% in wt/db,
Next, we assessed the specific subsets of CD4+ and CD8+ cells using immunostaining for the marker of activation CD25. No difference between db/db and wt/db mice was observed regarding CD4+ cells (data not shown). In contrast, as shown in
CD4+ and CD8+ cells were further categorized into naïve and memory phenotypes based on the expression CD44 and CD62L, with the CD44−CD62Lhigh population being considered naïve, CD44highCD62high population considered TCM, and the CD44highCD62L− population considered TEM (
Increased central to peripheral memory cell gradient in diabetic mice.
Naïve cells were increased in BM of db/db mice (CD4+: 2.70-fold,
Type 2 diabetes on central memory cells did not show differences between groups. Nonetheless, db/db mice manifested a positive gradient between the BM and peripheral blood (CD4+: 2.98-fold,
Having shown the activation and redistribution of different classes of immune cells in db/db mice, we next examined if this was associated with an alteration of the cytokines content in the BM. Of 40 factors tested, 32 were upregulated in the BM of diabetic mice compared with the wt/db controls. Of these, 22 were uniquely expressed in db/db mice (
Cytokine and chemokines differentially modulated in BM of db/db mice.
Ccl2 | C-C motif chemokine 2; Chemotactic factor that attracts monocytes, but not neutrophils; Belongs to the intercrine beta (chemokine CC) family!!break (148 aa) |
Ccl12 | C-C motif chemokine 12; Chemotactic factor that attracts eosinophils, monocytes, and lymphocytes but not neutrophils. Potent monocyte active chemokine that signals through CCR2. Involved in allergic inflammation and the host response to pathogens and may play a pivotal role during early stages of allergic lung inflammation; Belongs to the intercrine beta (chemokine CC) family (104 aa) |
Cccl11 | Eotaxin; In response to the presence of allergens, this protein directly promotes the accumulation of eosinophils (a prominent feature of allergic inflammatory reactions), but not lymphocytes, macrophages or neutrophils; Belongs to the intercrine beta (chemokine Cnterleukin-4; Participates in at least several B-cell activation processes as well as of other cell types. It is a costimulator of DNA-synthesis. It induces the expression of class II MHC molecules on resting B-cells. It enhances both secretion and cell surface expression of IgE and IgG1. It also regulates the expression of the low affinity Fc receptor for IgE (CD23) on both lymphocytes and monocytes. Positively regulates IL31R |
Il4 | Interleukin-4; Participates in at least several B-cell activation processes as well as of other cell types. It is a costimulator of DNA-synthesis. It induces the expression of class II MHC molecules on resting B-cells. It enhances both secretion and cell surface expression of IgE and IgG1. It also regulates the expression of the low affinity Fc receptor for IgE (CD23) on both lymphocytes and monocytes. Positively regulates IL31R |
Ccl3 | 3C-C motif chemokine 3; Monokine with inflammatory, pyrogenic and chemokinetic properties. Has a potent chemotactic activity for eosinophils. Binding to a high-affinity receptor activates calcium release in neutrophils; Belongs to the intercrine beta (chemokine CC) fa |
Il16 | Pro-interleukin-16; Interleukin-16 stimulates a migratory response in CD4+ lymphocytes, monocytes, and eosinophils. Primes CD4+ T-cells for IL-2 and IL-15 responsiveness. Also induces T-lymphocyte expression of interleukin 2 receptor. Ligand for CD4 (1322 aa) |
Timp1 | Metalloproteinase inhibitor 1; Metalloproteinase inhibitor that functions by forming one to one complexes with target metalloproteinases, such as collagenases, and irreversibly inactivates them by binding to their catalytic zinc cofactor. Acts on MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, and MMP16. Does not act on MMP14 (By similarity). Also functions as a growth factor that regulates cell differentiation, migration and cell death and activates cellular signaling cascades via CD63 and ITGB1. Plays a role in integrin signaling; Belongs to the protease |
Csf1 | Macrophage colony-stimulating factor 1; Cytokine that plays an essential role in the regulation of survival, proliferation and differentiation of hematopoietic precursor cells, especially mononuclear phagocytes, such as macrophages and monocytes. Promotes the release of proinflammatory chemokines, and thereby plays an important role in innate immunity and in inflammatory processes. Plays an important role in the regulation of osteoclast proliferation and differentiation, the regulation of bone resorption, and is required for normal bone development. Required for normal male |
Csf2 | Granulocyte-macrophage colony-stimulating factor; Cytokine that stimulates the growth and differentiation of hematopoietic precursor cells from various lineages, including granulocytes, macrophages, eosinophils and erythrocytes (141 aa) |
Ccl4 | C-C motif chemokine 4; Monokine with inflammatory and chemokinetic properties (92 aa) |
Cxcl13 | C-X-C motif chemokine 13; Strongly chemotactic for B-lymphocytes, weakly for spleen monocytes and macrophages but no chemotactic activity for granulocytes. Binds to BLR1/CXCR5. May play a role in directing the migration of B-lymphocytes to follicles in secondary |
Tnf | Tumor necrosis factor; Cytokine that binds to TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. It is mainly secreted by macrophages and can induce cell death of certain tumor cell lines. It is potent pyrogen causing fever by direct action or by stimulation of interleukin-1 secretion and is implicated in the induction of cachexia, Under certain conditions it can stimulate cell proliferation and induce cell differentiation (235 aa) |
Il17a | Interleukin-17A; Ligand for IL17RA. The heterodimer formed by IL17A and IL17F is a ligand for the heterodimeric complex formed by IL17RA and IL17RC (By similarity). Involved in inducing stromal cells to produce proinflammatory and hematopoietic cytokines (B) |
Il1b | Interleukin-1 beta; Potent proinflammatory cytokine. Initially discovered as the major endogenous pyrogen, induces prostaglandin synthesis, neutrophil influx and activation, T-cell activation and cytokine production, B-cell activation and antibody production, and fibroblast proliferation and collagen production; Belongs to the IL-1 family (269 aa) |
Il2 | Interleukin-2; Produced by T-cells in response to antigenic or mitogenic stimulation, this protein is required for T-cell proliferation and other activities crucial to regulation of the immune response. Can stimulate B-cells, monocytes, lymphokine- activated killer cells, n |
Cxcl1 | Growth-regulated alpha protein; Has chemotactic activity for neutrophils. Contributes to neutrophil activation during inflammation (By similarity). Hematoregulatory chemokine, which, |
Gmcsf3 | Granulocyte colony-stimulating factor; Granulocyte/macrophage colony-stimulating factors are cytokines that act in hematopoiesis by controlling the production, differentiation, and function of 2 related white cell populations of the blood, the granulocytes and the monocytes-macrophages. This CSF induces granulocytes; Belongs to the IL-6 superfamily (208 aa) |
Trem | Triggering receptor expressed on myeloid cells 1; Stimulates neutrophil and monocyte-mediated inflammatory responses. Triggers release of pro-inflammatory chemokines and cytokines, as well as increased surface expression of cell activation markers. Amplifier of inflammatory responses that are triggered by bacterial and fungal infections and is a crucial mediator of septic shock (By similarity) (230 aa) |
Ccl5 | C-C motif chemokine 5; Chemoattractant for blood monocytes, memory T-helper cells and eosinophils. Causes the release of histamine from basophils and activates eosinophils. May activate several chemokine receptors including CCR1, CCR3, CCR4, and CCR5. May also be an agonist of the G protein-coupled receptor GPR75. Together with GPR75, may play a role in neuron survival through activation of a downstream signaling pathway involving the PI3, Akt and MAP kinases. By activating GPR75 may also play a role in insulin secretion by islet cells (91 aa) |
Cxxl10 | C-X-C motif chemokine 10; In addition to its role as a proinflammatory cytokine, may participate in T-cell effector function and perhaps T-cell development; Belongs to the intercrine alpha (chemokine CxC) family (98 aa) |
Cxcr3 | C-X-C chemokine receptor type 3; Receptor for the C-X-C chemokine CXCL9, CXCL10, and CXCL11 and mediates the proliferation, survival and angiogenic activity of mesangial cells through a heterotrimeric G-protein signaling pathway. Probably promotes cell chemotaxis |
Cxcl2 | C-X-C motif chemokine 2; Chemotactic for human polymorphonuclear leukocytes but does not induce che |
Ccl1 | C-C motif chemokine 1; Cytokine that is chemotactic for neutrophils; Belongs to the intercrine beta (chem) |
Cxcl9 | C-X-C motif chemokine 9; May be a cytokine that affects the growth, movement, or activation state of cells that participate in immune and inflammatory response (126 aa) |
Il23r | Interleukin-23 receptor; Associates with IL12RB1 to form the interleukin-23 receptor. Binds IL23 and mediates T-cells, NK cells and possibly certain macrophage/myeloid cells stimulation probably through activation of the Jak-Stat signaling cascade. IL23 functions in innate and adaptive immunity and may participate in acute response to infection in peripheral tissues. IL23 may be responsible for autoimmune inflammatory diseases and be important for tumorigenesis (By similarity) (659 aa) |
Il7 | Interleukin-7; Hematopoietic growth factor capable of stimulating the proliferation of lymphoid progenitors. It is important for proliferation during certain stages of B-cell maturation (154 aa) |
Altogether the above findings show a state of inflammation in patients and mice with T2D. This involves activation of adaptive immunity, as evidenced by the increase in CD25+ T cells and corroborated by the upregulation of a multitude of inflammation-associated mediators.
We next investigated the effect of the
Effect of abatacept on immune cells of T2D mice.
Examining the cytokine and chemokine production using a Proteome Profiler Mouse Cytokine Array, we did not find any major differences in the BM between abatacept and control-treated animals (
Cytokine and chemokine changes induced by Abatacept.
Il-27 (Ebi3) | 3.98 | Il27—Interleukin-27 subunit alpha; Associates with EBI3 to form the IL-27 interleukin, a heterodimeric cytokine which functions in innate immunity. IL-27 has pro- and anti-inflammatory properties, that can regulate T- helper cell development, suppress T-cell proliferation, stimulate cytotoxic T-cell activity, induce isotype switching in B-cells, and that has diverse effects on innate immune cells. Among its target cells are CD4 T-helper cells which can differentiate in type 1 effector cells (TH1), type 2 effector cells (TH2) and IL17 producing helper T-cells (TH17) |
Ccl4 | 3.45 | C-C motif chemokine 4; Monokine with inflammatory and chemokinetic properties |
Il-13 | 2.75 | Interleukin-13; Cytokine. Inhibits inflammatory cytokine production. Synergizes with IL2 in regulating interferon-gamma synthesis. May be critical in regulating inflammatory and immune responses (By similarity). Positively regulates IL31RA expression in macrophages |
Il-23 | 2.65 | Interleukin-23 subunit alpha; Associates with IL12B to form the IL-23 interleukin, a heterodimeric cytokine which functions in innate and adaptive immunity. IL-23 may constitute with IL-17 an acute response to infection in peripheral tissues. IL-23 binds to a heterodimeric receptor complex composed of IL12RB1 and IL23R, activates the Jak- Stat signaling cascade, stimulates memory rather than naive T- cells and promotes production of proinflammatory cytokines. IL-23 induces autoimmune inflammation and thus may be responsible for autoimmune inflammatory diseases |
Il-2 | 2.30 | Interleukin-2; Produced by T-cells in response to antigenic or mitogenic stimulation, this protein is required for T-cell proliferation and other activities crucial to regulation of the immune response. Can stimulate B-cells, monocytes, lymphokine- activated killer cells, natural killer cells |
Ccl5 | 2.21 | C-C motif chemokine 5; Chemoattractant for blood monocytes, memory T-helper cells and eosinophils. Causes the release of histamine from basophils and activates eosinophils. May activate several chemokine receptors including CCR1, CCR3, CCR4, and CCR5. May also be an agonist of the G protein-coupled receptor GPR75. Together with GPR75, may play a role in neuron survival through activation of a downstream signaling pathway involving the PI3, Akt and MAP kinases |
Ccl17 | 2.13 | Chemokine (C-C motif) ligand 17 |
CXCL12 | 2.11 | Stromal cell-derived factor 1; Chemoattractant active on T-lymphocytes, monocytes, but not neutrophils. Activates the C-X-C chemokine receptor CXCR4 to induce a rapid and transient rise in the level of intracellular calcium ions and chemotaxis. Also binds to atypical chemokine receptor ACKR3, which activates the beta-arrestin pathway and acts as a scavenger receptor for SDF-1. Acts as a positive regulator of monocyte migration and a negative regulator of monocyte adhesion via the LYN kinase. Stimulates migration of monocytes and T- lymphocytes through its receptors, CXCR4 |
Cxcl9 | 2.07 | C-X-C motif chemokine 9; May be a cytokine that affects the growth, movement, or activation state of cells that participate in immune and inflammatory response |
Il-4 | 2.0 | Interleukin-4; Participates in at least several B-cell activation processes as well as of other cell types. It induces the expression of class II MHC molecules on resting B-cells. It enhances both secretion and cell surface expression of IgE and IgG1. It also regulates the expression of the low affinity Fc receptor for IgE (CD23) on both lymphocytes and monocytes. Positively regulates IL31RA expression in macrophages |
Il-6 | 0.02 | Interleukin-6; Cytokine with a wide variety of biological functions. It is a potent inducer of the acute phase response. Plays an essential role in the final differentiation of B-cells into Ig- secreting cells Involved in lymphocyte and monocyte differentiation. Acts on B-cells, T-cells, hepatocytes, hematopoietic progenitor cells and cells of the CNS. Required for the generation of T(H)17 cells. Also acts as a myokine. It is discharged into the bloodstream after muscle contraction and acts to increase the breakdown of fats and to improve insulin resistance |
We further analyzed the abundance of APCs. Abatacept decreased the number of DC in peripheral blood (3.3 ± 0.4 vs. 7.3 ± 1.2% in vehicle,
Effect of abatacept on naïve and memory T cells of T2D mice.
Body weight and the weight of heart and spleen were similar between groups given vehicle or abatacept (
Effect of abatacept on metabolic and cardiac endpoints.
Recent evidence indicates that T cells play key roles in the progression of T2D.
In the present study, we provide novel proof-of-concept evidence for the activation of adaptive immunity in the BM of patients and mice with T2D. The studied human diabetic cohort had an increased frequency of BM T lymphocytes. Both CD4+ and CD8+ cells abundantly expressed CCR7, which, together with the observed high levels of CCL19 (CCR7 receptor ligand), suggests that the local BM environment was reshaped to favor immune cell recruitment from the circulation. Moreover, we demonstrated that the diabetic BM contains TCM cells that have undergone recent activation. Most importantly, BM T cells showed a clear upregulation of typical activation markers. Several features of the adaptive immunity activation seen in patients were confirmed in db/db mice. Finally, we proved that the treatment with abatacept, which interferes with the co-stimulation signaling mechanism, inhibited the activation of adaptive immunity, and improved cardiac function in db/db mice.
Abundant fat depots of overweight/obese patients reportedly contain autoreactive T cells that exert a cytotoxic effect on adipocytes and fuel adipose inflammation (
The co-signaling factor CTLA4 exerts competitive inhibition of T-cell activation (
The clinical indication of abatacept is to treat active rheumatoid arthritis, but seminal data are supporting its potential in the treatment of Type 1 diabetes and cardiovascular disease. In TrialNet study for people newly diagnosed, those who took Abatacept showed 59% higher insulin production and prolonged insulin production (
In the present study, we have shown the activation of adaptive immunity in diabetic BM and the possibility to rescue immune and cardiac abnormalities using the clinically available, immunomodulatory drug abatacept. The compound is not risk free and the use in diabetes requires additional investigation assessing safety and optimal risk-to-benefit context. Regarding limitations, we could not perform analyses of peripheral blood cells in the human study because the ethical license covered the investigation of BM leftovers only. The focus of the present study was on specific populations of lymphocytes, which were found generally increased/activated. This change implies a relative reduction in other BM cell populations expressing the CD45 marker, an aspect that was not assessed in the present study but documented previously by us and others (
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
The studies involving human participants were reviewed and approved by Bristol NHS. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by UK Home Office.
MS performed the flow cytometry studies and wrote the draft of the paper. NS and AB were involved in patient recruitment and sample collections. AT was responsible for the
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This manuscript has been released as a preprint at BioRxiv 2020.07.03.187088; doi:
antigen-presenting cells
bone marrow
BM mononuclear cells
cardiac output
central memory T cells
CC-chemokine receptor 7
dendritic cells
end diastolic volume
effector memory T cells
non-diabetic
hematopoietic stem/progenitor cells
stroke volume
terminally differentiated T memory cells
type 2 diabetes
visceral adipose tissue
phosphate buffered saline
room temperature
ethylenediaminetetraacetic acid
room temperature
flow cytometry staining
Roswell Park Memorial Institute
red blood cell
severe acute respiratory syndrome coronavirus 2.