Analysis of Impairements in T Cells Subsets in First Degree Relatives of Patients with Type 1 Diabetes as Risk Markers for Disease Development

Beograd : [T. J. Miličić]

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MEDICINA - INTERNA MEDICINA, ENDOKRINOLOGIJA / MEDICINE - INTERNAL MEDICINE, ENDOCRINOLOGY

Prethodna istraživanja su ukazala na značajnu ulogu subpopulacija T limfocita, T helper 1 (Th1) i T helper 2 (Th2), fenotipski definisanih ekspresijom odgovarajućih hemokinskih receptora CXCR3 i CCR4, kao i T regulatorne (T reg) subpopulacije, u inicijalnoj fazi tipa 1 dijabetesa (T1D). Meñutim, povezanost promena u nivou CXCR3+ (Th1 asociranih), CCR4+ (Th2 asociranih) i CD25high (T reg asociranih) subpopulacija T memorijskih limfocita kao i citokina, njihovih liganda/medijatora funkcije, interferon-γ inducibilnog hemokina 10 (interferon-γ inducibile chemokine-IP-10) (Th1 asociranog), timusom i aktivacijom regulisanog hemokina (thymus and activation-regulated chemokine-TARC) (Th2 asociranog) i transformišućeg faktora rasta β (transforming growth factor β-TGFβ) (Treg asociranog), i rizika za ispoljavanje T1D, nije još uvek razjašnjena. Istovremeno, povezanost promena ovih imunoloških parametara i metaboličkih parametara u smislu nivoa insulinske sekrecije i insulinske senzitivnosti u ranim fazama razvoja T1D, nije do sada detaljnije analizirana. U tom smislu, cilj istraživanja je poreñenje nivoa (a) CXCR3+, CCR4+ i CD25high subpopulacija memorijskih T limfocita (b) hemokina/citokina IP-10, TARC, TGFβ i (c) insulinske sekrecije i insulinske senzitivnosti, izmeñu grupa zdravih prvih roñaka pacijenata sa T1D (PR), sa visokim i niskim rizikom za ispoljavanje bolesti (gde je rizik za T1D definisan prisustvom/odsustvom antitela na glutamat dekarboksilazu (GAD) i tirozin fosfatazu (IA-2), pacijenata sa novootkrivenim T1D (NT1D) u insulin zavisnom stanju na početku bolesti i u stanju kliničke remisije (KR), i kontrolnih ispitanika. Uključili smo PR sa visokim rizikom (GADA+, IA-2+) (N=17), PR sa niskim rizikom (GADA-, IA-2-) (N=34), pacijente sa NT1D (N=24), pacijente sa T1D u KR (N=10) i kontrolne ispitanike (N=18). Nivo CXCR3+, CCR4+ i CD25high T memorijskih limfocita je analiziran četvorobojnom imunofluoroscencijom i protočnom citometrijom. Nivo hemokina/citokina i antitela u serumu odreñeni su ELISA metodom. Prva faza insulinske sekrecije odreñivana je zbirom nivoa insulina u 1. i 3. minutu nakon IVGTT-a. Nivo insulinske senzitivnosti evaluiran je hiperinsulinemijskim euglikemijskim klampom. Rezultati ukazuju da je u PR sa visokim rizikom utvrñen povišen nivo CXCR3+ Th1 subpopulacije i IP-10 hemokina, i snižen nivo CCR4+ Th2 i CD25high T reg subpopulacije limfocita, što bi moglo sugerisati da je u PR rizik za ispoljavanje T1D povezan sa pojačanom aktivnošću Th1 i smanjenom aktivnošću Th2 i T regulatornog imunskog odgovora. Sa druge strane, početak T1D je povezan sa značajnim smanjenjem nivoa CXCR3+ i CCR4+ subsetova T memorijskih limfocita i TGFβ, i porastom nivoa IP-10 i TARC, reflektujući njihovu akumulaciju u pankreasna ostrvca i funkcionalno iscrpljivanje regulatorne subpopulacije T limfocita, što bi moglo ukazati da je nastanak bolesti moduliran na nivou ovih subsetova T memorijskih limfocita i hemokina...

Previous studies have reported an important role of T cells subpopulations, T helper 1 (Th1) and T helper 2 (Th2), characterized with expression of chemokine receptors CXCR3 and CCR4 on their surface, respectively, as well as T regulatory (T reg) subpopulations, in the initial phase of type 1 diabetes (T1D). However, the relationship among the impairments in the level of CXCR3+ (Th1 associated), CCR4+ (Th2 associated) and CD25high (T reg associated) T memory cells subpopulations, as well as cytokines, their ligands/mediators of function, interferon-γ inducibile chemokine-IP-10 (Th1 associated), thymus and activation-regulated chemokine-TARC (Th2 associated) and transforming growth factor β-TGFβ (Treg associated), and risk for T1D developing, has not yet been clarified. Additionally, relationship between the immunological and metabolic changes, regarding the insulin secretion and insulin sensitivity level, early in T1D development, is still controversial. Therefore, the aim of this study was to analyse the changes in (a) percentage of CXCR3+, CCR4+ T memory cell and CD25high T cells subsets (b) IP-10, TARC and TGFβ (c) insulin secretion and insulin sensitivity levels and in peripheral blood in the following groups of subjects: (1) 17 high-risk nondiabetic first degree relatives (hrFDRs) of patients with T1D (glutamate decarboxylase antibodies-GADA+, tyrosine phosphatase insulinoma antigen-2 antibodies-IA-2+); (2) 34 low-risk nondiabetic first degree relatives (lrFDRs) of patients with T1D (GADA-, IA-2-); (3) 24 recent-onset T1D patients and (4) 10 patients in clinical remission (5) 18 healthy, unrelated control subjects. The percentages of CXCR3+, CCR4+ and CD25high T memory cell subsets were analyzed in peripheral blood by using four-color immunofluorescence staining and flowcytometry. IP-10, TARC, TGFβ, GADA and IA-2 levels were determined by ELISA. Insulin secretion was evaluated by firstphase insulin response (FPIR) as insulin levels 1+3 min after IVGTT. Insulin sensitivity was tested by using euglycemic hyperinsulinemic clamp method (M value). Our results have demonstrated that hr FDRs, defined by the presence of the autoantibodies, showed higher levels of CXCR3+ T cell subset and IP-10 chemokine, both associated with Th1 response, together with lower level of CCR4+ Th2 and CD25high T reg cell subsets. In this study, complementary investigations imply that in FDRs, the risk of progression to T1D might be strongly influenced by enhanced activity of Th1 and diminished activity of Th2 and T reg autoimmune response. In addition, we demonstrated that the onset of T1D is characterized by the decreases in CXCR3+ Th1 and CCR4+ Th2 memory T subsets and TGFβ levels, and increases in IP-10 and TARC, presumably reflecting possible recruitment of those cells in pancreatic tissue and functionally exhaustion of T reg subpopulation...

tip 1 dijabetesa, prvi roñaci, hemokinski receptori, hemokini, Th1, Th2, T reg, insulinska sekrecija, insulinska senzitivnost

type 1 diabetes, first degree relatives, chemokine receptors, chemokines, Th1, Th2, Treg, insulin secretion, insulin sensitivity

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