Interleukin-1ß and Interleukin-6 Genetic Polymorphisms and Sickle Cell Disease: An Egyptian Study

Transcription

1 Interleukin-1ß and Interleukin-6 Genetic Polymorphisms and Sickle Cell Disease: An Egyptian Study MONA EL-GHAMRAWY, MD, PROFESSOR OF PEDIATRICS & PEDIATRIC HEMATOLOGY, CAIRO UNIVERSITY

2 Conflicts of Interest to Declare None

3 Outline Pathophysiology of SCD Interleukin-1ß and Interleukin-6 Interleukin-1ß and Interleukin-6 in SCD Therapeutic Implications

4 Life-threatening Heterogeneous clinical phenotype Hydroxyurea is NOT protective against all complications

5 Pathophysiology of SCD β-globin gene Glutamic acid Deoxygenated Hb polymers Most established genetic modifiers - HbF concentration - Co-inheritance of α-thalassaemia HbS (ɑ2βˢ2) Sickle erythrocyte Valine Low oxygen tension Fever/Infection Acidosis Dehydration ACS = acute chest syndrome; ASS = acute splenic sequestration; HbS = sickle cell haemoglobin; PHT = pulmonary hypertension; SCD = sickle cell disease. Hemolysis Inflammation Endothelial dysfunction Proliferative vasculopathy Hypercoagulability Vaso-occlusion Anemia, gallstones, priapism, stroke, leg ulcers, PHT Pain, ACS, joint necrosis, ASS, increased infections, multiorgan damage Inati A, et al, 2008;.

6 Pathophysiology of SCD Steinberg, 2016

7 Consequences of Sickling Vaso-occlusion ischemia reperfusion injury K + loss RBC dehydration Adhesion to WBCs, endothelium Hb polymerization Membrane damage Lipid peroxidation NF- B = nuclear factor kappa B; PS = protein S; ROS = reactive oxygen species; TF = tissue factor; WBC = white blood cells; XO = Xanthine oxidase. Increased inflammation NF- B activation Adhesive proteins Sickled RBCs Hemolysis NO scavenging ROS, XO PS exposure Activation of coagulation TF Thrombin Inflammatory cytokines Activation of WBCs, platelets Endothelial dysfunction Protein C and S Platelet activation Natarajan K, et al, 2010.

8 Outline Pathophysiology of SCD Interleukin-1ß and Interleukin-6 Interleukin-1ß and Interleukin-6 in SCD Therapeutic Implications

9 Interleukin-1 ß Member of IL-1 superfamily (11 members) Potent pro-inflammatory cytokine Mapped to long arm of human chromosome 2 Primarily produced by monocytes, macrophages, dendritic cells, B lymphocytes and natural killer cells Bhat et al., 2014

10 Cell proliferation Differentiation Apoptosis Juvenile idiopathic arthritis Rheumatoid arthritis Osteonecrosis Cardiovascular disease Schett et al., 2016 Chen et al, 2006

11 Interleukin-6 Pro-inflammatory & antiinflammatory functions Chromosome 7p21 Released from immune, mesenchymal, endothelial, tumor cells & fibroblasts Tanaka and Kashimoto, 2014

12 Anti-inflammatory Pro-inflammatory Pro-and Anti-inflammatory Activities of IL-6 IL-6 Membrane-Bound IL-6R Soluble IL-6R Activation of STAT3 leading to: Intestinal epithelial cell proliferation Inhibition of epithelial cell apoptosis Induction of hepatic phase response Activation of immune system: Recruitment of mononuclear cells Inhibition of T-cell apoptosis Inhibition of T-reg differentiation ADAM17 Rose John, 2012

13 Outline Pathophysiology of SCD Interleukin-1ß and Interleukin-6 Interleukin-1ß and Interleukin-6 in SCD Therapeutic Implications

14 Damaged endothelial cells may release cytokines and growth factors that not only augment sickle erythrocyte endothelial interactions, but also lead to further alteration of endothelial surfaces. Repeated damage-adhesion-damage events could conceivably cause vasoocclusion and smooth muscle cell hyperplasia seen in patients with sickle cell anemia.

15

16 IL-1β +3954C/T polymorphism is associated with osteonecrosis, elevated pulmonary arterial pressure and lower reticulocytic count.

17 Inflammation in SCD Treg frequency Th17 response Acute phase proteins & cytokines Zhang et al., 2016 Olenscki et al, 2016

18 Interleukin-1β and Interleukin-6 Genetic Polymorphisms and sickle cell disease: An Egyptian study Detect the frequency of IL-1ß (+3954 C/T) and IL-6 (-174 G/C) polymorphisms in a cohort of Egyptian SCD patients Study their possible impact on the clinical course of the disease 60.25% 39.7%

19 Interleukin-1β and Interleukin-6 Genetic Polymorphisms and sickle cell disease: An Egyptian study Cross sectional study, Pediatric Hematology and BMT Unit, CUCH Patients group; n= 84 steady state SCD Control healthy group; n=100 Non-Egyptians Non-steady state Mean age: ± 5.9 years (5-18 yrs) 60.25% Review medical records Clinical examination Laboratory testing Genotyping of IL-1ß (+3954 C/T) and IL-6 (-174 G/C) by PCR-RFLP

20 Interleukin-1β and Interleukin-6 Genetic Polymorphisms and sickle cell disease: An Egyptian study % IL-1β (+3954C>T) genotypes in SCD IL-6 (-174G>C) genotypes in SCD

21 Characteristics of SCD patients Parameter Genotype Hb-SS Hb-S/ß thalassemia 60 (71.5%) 24 (28.5%) Range SCD (n=84) Median VOC 69 (82.1 %) VOC/year* 4.1± Blood Units/year* 5.6 ± Transfusion dependency Frequent ( 4 times /year) Infrequent (1 3 times /year) Sporadic (infection, preoperative, occasional) 47 (55.9%) 19 (22.6%) 18 (21.4%) Splenectomy 23 (27.3%) Osteonecrosis 5 (5.9%) Priapism 5 (5.9%) Stroke & TIAs 5 (5.9%) Leg ulcer 3 (3.5%) VOC: Vaso-occlussive crisis TIAs: Transient ischemic attack * Expressed as mean ± SD

22 Characteristics of SCD patients Parameter Mean ± SD SCD (n=84) Range Hemoglobin (g/dl) 7.8 ± Reticulocytic count (%) 11.7 ± Total leucocytic count (x 10 3 /cm 3 ) 9.9 ± Platelets (x 10 3 /cm 3 ) 386 ± HbS (%) SS S/ß thalassemia HbF (%) SS S/ß thalassemia ± ± ± ± 8.67

23 Genotypic & Allelic Distribution of IL-1β C/T and IL G/C in SCD Patients & Controls Genotype Controls (n=100) SCD patients (n=84) OR (95% CI) P value (n-%) (n-%) IL-1β C/T Wild genotype CC 56/100 (56%) 39/84 (46.4%) 1 (Reference) Heteromutant CT 34/100(34%) 32/84 (38.1%) 1.19 ( ) Homomutant TT 10/100 (10%) 13/84 (15.5%) 1.64 ( ) 0.95 CT & TT 44/100 (44%) 45/84 (53.6%) 1.47 ( ) C allele ( ) T allele IL G/C Wild genotype GG 31/100 (31%) 25/84 (29.8%) 1 (Reference) Heteromutant GC 59/100 (59%) 49/84 (58.2%) 0.97 ( ) 0.12 Homomutant CC 10/100 (10%) 10/84 (12%) 1.21 ( ) 0.17 GC & CC 69/100 (69%) 59/84 (70.2%) 1.06 ( ) 0.26 G allele ( ) 0.11 C allele Combined genotypes analysis IL-1β/IL-6 CC/GG 15/100 (15%) 8/84 (9.5%) 1 (Reference) CC/ GC &/or CC 41/100 (41%) 31/84 (37.3%) 0.92 ( ) 0.45 CT & TT/ GG 16/100 (16%) 17/84 (20.2%) 1.56 ( ) CT &/or TT / GC &/or CC 28/100 (28%) 28/84 (33.3%) ) OR= Odds ratio, CI= Confidence interval

24 Interleukin-1β and Interleukin-6 Genetic Polymorphisms and sickle cell disease: An Egyptian study For IL G/C polymorphism, distribution of polymorphic variants (GC & CC) was significantly higher among patients with repeated attacks of VOC (p=0.023), especially severe attacks requiring hospitalization or emergency visits (p=0.03). For IL-1β polymorphisms, no significant differences between patients having wild genotype (CC) or polymorphic genotypes (CT & TT) regarding their gender, presenting symptoms, clinical or laboratory features. For Egyptian controls, the genotypic frequencies of IL-1β were close to that reported in Brazil, Poland & Netherlands. For IL G/C, it was close to that reported by UK Caucasians & Germans but far from that reported in Brazilians, Chinese, Indians and Polish populations VOC: Vaso-occlussion Vicari et al, 2015, Nemetz et al, 1999 Wujcicka et al, 2015, Fishman et al, 2015 Buchs et al, 2001 Olenscki et al, 2016

25 Interleukin-1β and Interleukin-6 Genetic Polymorphisms and sickle cell disease: An Egyptian study Limitations: Sample Size Association between serum IL-1 and IL-6 levels and the studied polymorphisms IL G/C polymorphism could be considered as a molecular predictor of repeated severe VOC in pediatric SCD patients VOC: Vaso-occlussion

26 Outline Pathophysiology of SCD Interleukin-1ß and Interleukin-6 Interleukin-1ß and Interleukin-6 in SCD Therapeutic Implications

27 Consequences of Sickling Disease Modifying agents Anti-adhesion therapies Vaso-occlusion ischemia reperfusion injury Adhesion to WBCs, endothelium Hb polymerization K + loss RBC dehydration Membrane damage Lipid peroxidation NF- B = nuclear factor kappa B; PS = protein S; ROS = reactive Antiinflammatory oxygen species; TF = tissue factor; WBC = white blood cells; XO = Xanthine oxidase. agents Increased inflammation NF- B activation Adhesive proteins Sickled RBCs Hemolysis NO scavenging ROS, XO PS exposure Activation of coagulation TF Thrombin Anti-coagulants Inflammatory cytokines Activation of WBCs, platelets Endothelial dysfunction Protein C and S Platelet activation Natarajan K, et al, 2010.

28 Upstream Targets HbF inducers Modulators of HbO2 affinity BM Transplantation Gene Therapy Anti-inflammatory Vasodilators Anti-thrombotic & antiplatelets Antiadhesives Anti-RBC dehydration agents Antioxidants Downstream Targets Costa & Conran,2016

29 Anti-IL-1ß Monoclonal Antibodies Canakinumab Phase II, placebocontrolled trial ongoing in pediatric and young adult patients with SCD Charles, 2016

30 In conclusion. SCD is considered as a chronic inflammatory condition, even under steady state. Association between genetic polymorphisms and inflammation may partly explain the phenotypic heterogeneity of SCD. Ethnic differences between studied populations may account for variations in reported genetic polymorphisms frequencies

31 In conclusion. Our study provides evidence of the possible role of IL-6, as an inflammatory marker, in the vaso-occlusive subphenotype of SCD and marking a more unfavorable disease phenotype. As genetic alterations of immune regulatory genes could play a pivotal role in disease pathogenesis and course, further studies are recommended for better characterization of patients prone for complications and for identification of novel molecular markers that could modify disease course and subsequently morbidity and mortality.

32 Acknowledgment Mervat M. Khorshied Ola M. Ibrahim Alaa A. Gad

33 Thank you