To Cytokine or Not to Cytokine

To Cytokine or Not to Cytokine 1 LEWIS H. KULLER, MD, DRPH PROFESSOR EMERITUS THURSDAY, MAY 7, 2015

Definition of Cytokine 2 Taber s Cyclopedic Medical Dictionary, 22 nd ed, 2013

Acute Phase Response The acute phase response is the name given to a characteristic pattern of alterations in the concentrations of plasma proteins that occurs following a wide variety of different forms of inflammation. 3 Kuller LH et al. Relation of C-reactive protein and coronary heart disease in the MRFIT nested case-control study. Am J Epidemiol 1996;144:537-47

Cytokines are Produced by Several Inflammatory and Vascular Cells IL-12 and IL-18, produced by macrophages (Mac), are potent inducers of IFN-γ and promote the differentiation of naïve T cells into proatherogenic Th1 cells. Macrophage-derived cytokines activate SMC and EC to produce an array of proinflammatory mediators. On the other hand, the antiinflammatory cytokines IL-10 and TGF-β, also produced by macrophages, promote antiatherogenic Treg cell differentiation. Other antiinflammatory mediators with potent antiatherogenic properties include IL-33, IL- 1ra, and IL-18 binding protein (IL-18BP). In naive CD4+ T cells from mice, Th17 cells are induced in response to a combination of IL-6 or IL-21 and TGF-β and require induction of the transcription factor RORγt. In human (h), Th17 differentiation requires TGF-β, IL-1β, and IL-6. Ait-Oufella H et al. Recent advances on the role of cytokines in atherosclerosis. Arterioscler Thromb 4 Vascular Biol 2011;31:969-79

5 Psaty B et al.

Characteristics of Innate and Adaptive Immune Responses 6 Weyand CM et al. T-cell immunity in acute coronary syndromes. Mayo Clin Proc 2001;76:1011-20

Feedforward Loop Between Innate and Adaptive Immune Systems in Systemic Autoimmunity 7 Factors of the innate system activate and promote adaptive components, which feedback into the innate arm to promote immune activation. Central components of the innate system that feed into the adaptive system in systemic autoimmune diseases include type 1 interferon and other cytokines, MHC II, and apoptotic debris, while important factors from the adaptive system promoting innate responses encompass proinflammatory cytokines, autoantibodies, and immune complexes. BAFF=B-cell activating factor (TNFSF13B). IFNα=interferon α. IL=interleukin. LTα= lymphotoxin α. TGF β=transforming growth factor β. TNFα=tumour necrosis factor α. Wahren M et al. Autoimmune rheumatic diseases 3. Immunopathogenic mechanisms of systemic autoimmune disease. Lancet 2013;382:819-31

Sato K. Helper T cell diversity and plasticity-possible role in cardiovascular disease. Circ J 2014;78:2843-4 8

Pathophysiologic Relevance of Cytokines in Chronic Liver Disease 9 Most types of cells in the liver, including Kupffer cells, hepatocytes, and stellate cells, either synthesize or respond to cytokines. In the early phase of chronic liver disease, specific agents such as viruses, ethanol, and toxins may stimulate the production of cytokines. In the late phase, endotoxin may be the key agent stimulating cytokine production. Clinical features of chronic liver disease that are mediated by cytokines include cachexia, cholestasis, fibrosis, synthesis of acutephase proteins, and hypergammaglobulinemia. Whereas proinflammatory cytokines such as tumor necrosis factor α (TNF-α) and interleukin- 6 are mainly involved in cholestasis and the synthesis of acute-phase proteins, transforming growth factor β (TGF-β) released by activated Kupffer cells and hepatocytes may be one of the critical cytokines involved in fibrosis. In patients with progressive liver disease, the balance between proinflammatory and antiinflammatory cytokines may be shifted toward the proinflammatory axis, thus the counteracting antiinflammatory cytokines are unable to control inflammation and fibrosis. Tilg H, et al. Cytokines in alcoholic and nonalcholic steatohepatitis. N Engl J Med 2000;343:1467-76

Changes in IL-1a, IL-6, CRP by Weight Loss Categories 10 Wong E et al. Epidemiology of cytokines. The Women On the Move through Activity and Nutrition (WOMAN) Study. Am J Epidemiol 2008;168:443-53

Wong E et al. Epidemiology of cytokines. The Women On the Move through Activity and Nutrition (WOMAN) Study. Am J Epidemiol 2008;168:443-53

Number of Split Samples Above 25% Difference by Cytokine (WHI BA20 RA Study), N=153 12 Cytokine N % IL-1B 33 21 IL-2 60 39 IL-4 68 44 IL-5 37 18 IL-6 30 20 IL-7 23 15 IL-8 23 15 IL-10 32 20 IL-12 41 26 IL-13 40 26 IL-17 67 44 IL-18 33 22 TNF-a 51 33 MIP-18 17 11 G-CSF 42 27 MCP-1 26 17 GM-CSF 12 8

The CVs for 40 blinded duplicates, 20 placed within the same batch, and 20 placed across different batches. Results are shown separately for 67 Bio-Rad markers (A C) and 97 Millipore markers (D F) across 3 specimen types heparin plasma, serum, and EDTA plasma.(d F) across 3 specimen types serum, heparin plasma, and EDTA plasma. Chaturvedi AK et al. Evaluation of multiplexed cytokine and inflammation marker measurements: a methodologic study. Cancer Epidemiol Biomarkers Prevention 2011;20:1902-11

Median Values of Cytokines by RA+/- Cytokine (pg/ml) Likely RA Neg Likely RA (n=742) P value (n=2135) IL1B 1.53 1.92 <0.0001 IL2 3.31 6.18 <0.0001 IL4 1.61 2.00 <0.0001 IL5 3.03 3.57 <0.0001 IL6 6.13 9.30 <0.0001 IL7 6.91 7.82 <0.0001 IL8 8.49 9.36 <0.0001 IL10 2.41 2.76 <0.0001 IL12 12.7 16.9 <0.0001 IL13 2.44 3.21 <0.0001 IL17 0.35 2.85 <0.0001 GCSF 146.8 159.0 0.0005 MCP_1 14.5 18.4 <0.0001 MIP_1B 41.9 45.1 0.0014 IFN-Y 42.1 55.1 <0.0001 Birru, et al. WHI BA20 RA ms proposal. Associations of HLA-DR Shared Epitope Alleles and Serum Cytokines Among Postmenopausal women with and without Anti-Cyclic Citrullinated Peptide Antibodies 14

Comparison of Extremes of IL6 (1Q vs. 4Q) Among anti- CCP+ or DMARD+ at Baseline (WHI BA20 RA Study) IL6 1Q IL6 4Q P value N % N % BMI 25 53 43.1 70 56.9 25.1-30 62 56.4 48 43.6 30.1-35 29 46.0 34 54.0 >35 25 59.5 17 40.5 Smoking Never 79 56.8 60 43.2 Ever 89 44.7 110 55.3 Joint Pain Not severe 138 53.9 118 46.1 Health Severe 31 38.3 50 61.7 Good/excellen t 15 134 55.4 108 44.6 Fair/poor 36 37.9 59 62.1 0.109 0.028 0.014 0.004

Comparison of Extremes of IL6 (1Q vs. 4Q) Among anti- CCP+ or DMARD+ at Baseline (WHI BA20 RA Study) 16 IL6 1Q IL6 4Q P value N % N % anti-ccp - + 72 98 86.8 11 13.3 38.1 159 61.9 <.0001 RF - + 97 91.5 9 8.5 31.2 161 68.8 <.0001 73 Age 60 73 56.2 57 43.9 61-69 72 51.8 67 48.2 >69 25 35.2 46 64.8 # of shared epitopes 0 89 64.5 49 35.5 1 66 46.2 77 53.9 0.015 <.0001 2 9 19.6 37 80.4

Cytokines by CHD Case Control Among Anti-CCP+ Participants (WHI BA20 RA Study) 17 Cytokines (pg/ml) No CHD Median CHD Median P-value IL-1B 2.32 2.18 0.956 IL-2 10.00 11.06 0.480 IL-4 3.21 2.45 0.362 IL-5 4.42 4.26 0.997 IL-6 15.35 13.43 0.626 IL-7 9.25 9.24 0.610 IL-8 11.08 11.05 0.814 IL-10 3.03 3.02 0.862 IL-12 20.84 24.65 0.269 IL-13 4.13 3.91 0.704 G-CSF 180.09 178.03 0.847 MCP-1 19.70 18.76 0.991 MIP-1 40.84 44.99 0.752 TNF-a 39.08 35.26 0.976 IFH-g 71.31 66.53 0.884 IL-17 4.45 4.25 0.901 Kuller LH et al. WHI Rheumatoid Arthritis BA20 data

Cytokines by Alive vs Dead for Anti-CCP+ Rheumatoid Arthritis Participants (Excluding Women with Baseline CVD or Cancer) Cytokine (pg/ml) Median Alive at 2009 (n=461) 25 th Percentile 75 th Percentile Median Deceased by 2009 (n=78) 25 th Percentile 75 th Percentile P-value IL-1B 2.0 1.3 3.8 2.5 1.6 6.6 0.070 IL-2 7.6 3.3 27.7 11.0 4.4 47.6 0.076 IL-4 2.0 1.1 3.2 2.2 1.5 3.6 0.185 IL-5 3.6 2.6 5.2 3.9 3.0 5.5 0.266 IL-6 11.0 6.2 25.5 13.0 8.4 49.5 0.028 IL-7 7.8 5.6 11.2 8.1 6.3 11.2 0.310 IL-8 9.4 6.9 12.2 9.6 8.0 13.3 0.034 IL-10 2.9 2.1 4.2 3.2 2.4 5.4 0.045 IL-12 17.8 10.9 36.2 22.2 14.9 66.9 0.015 Kuller LH et al. WHI Rheumatoid Arthritis BA20 data 18

Median a Cytokine Levels at Baseline by Anti-CCP and RF Status Among Participants in the WHI RA Study RF- (n=157) RF+ (n-613) 19 RF- (n=1912) RF+ (n=302) 16 15.2 Median Cytokine Level pg/ml 14 12 10 8 6 4 2 0 1.6 2.4 6.3 3.2 2.5 0.8 IL-1b* IL-6* IL-10 IL-17 WBC Count Anti-CCP+ (n=770) b 4.2 5.5 6.2 1.5 1.8 6.1 7.5 2.4 2.7 0.4 2.3 5.8 5.37 IL-1b* IL-6* IL-10 IL-17 WBC Count Anti-CCP- (n=2214) c *p=<.01 a Wilcoxon test of median values. b Information on RF status was missing for 2 women; information on cytokine levels was missing for 2 women. c Information on RF status was missing for 2 women; information on cytokine levels was missing for 3 women. Kuller LH, et al. Am J Epidemiol 2014;179:917-26

Cox Model, Covariate = Age, excluding RA at FU only or CVD at BL CHD Death 3 2.77 2.5 2.31 2.49 Age-Adjusted HR 2 1.5 1 1.17 1.06 1.73 1.56 1.05 1.04 1.23 1.24 1.14 1.12 1 1.53 2.09 0.5 0 RF+ IL6* RF+ IL6* RF+ IL6* RF+ IL6* Anti-CCP+ or DMARD+ Anti-CCP+ Anti-CCP- and DMARD- Anti-CCP- and DMARD+ *4Q (vs IL6 1Q) Kuller LH et al. WHI Rheumatoid Arthritis BA20 data 20

CHD Event by IL-6 excluding those with RA at follow up only and those with CVD at baseline Weighted Age-Adjusted Rate/ 1000 person-years 10 9 8 7 6 5 4 3 2 1 0 6.51 N=1718 # of events 109 Anti-CCP-/Baseline DMARD- 4.1 N=1478 # of events 78 8.86 N=1288 # of events 123 4.23 N=605 # of events 31 Q1 Q2 Q3 Q4 Kuller LH et al. WHI Rheumatoid Arthritis BA20 data 21

Schematic mechanism of platelet and clotting activation elicited by bacteria in pneumonia. The dotted lines indicate inhibition; the solid lines indicate activation 22 Journal of Thrombosis and Haemostasis; Volume 12, Issue 9, pages 1391-1400, 23 JUL 2014 DOI: 10.1111/jth.12646 http://onlinelibrary.wiley.com/doi/10.1111/jth.12646/full#jth12646-fig-0002

Risk of Death Associated with Biomarker Levels at Study Entry 23 Biomarker Type of Analysis <25 th Percentile (Ref) 25 th -49 th Percentile 50 th -74 th Percentile 75 th Percentile OR associated with One IQR Higher Biomarker Level after Log10 Transformation OR (95% CI) P- value OR (95% CI) P- value OR (95% CI) P-value OR (95% CI) P-value hscrp (µg/ml) No. a Adjusted 16/45 1.0 (ref.) 9/42 0.6 (0.2-1.5) 0.50 20/28 2.5 (0.9-7.2) 0.08 40/55 3.1 (1.2-8.0) 0.02 2.3 (1.4-3.7) 0.001 IL-6 (pg/ml) No. Adjusted 8/48 1.0 (ref.) 10/41 1.0 (0.3-3.6) 0.98 26/48 4.5 (1.4-14.2) 0.01 40/29 12.4 (3.6-42.0) <0.0001 4.1 (2.3-7.3) <0.0001 D-dimer (µg/ml) No. Adjusted 8/51 1.0 (ref.) 22/54 8.3 (1.9-36.8) 0.005 18/40 12.6 (2.4-65.1) 0.003 37/25 41.2 (7.5-225.6) <0.0001 5.3 (2.6-10.9) <0.0001 Kuller LH et al. Inflammatory and coagulation biomarkers and mortality in patients with HIV infection. PLoS Med 2008;5:e203

Relationship Between Plasma Interleukin-6 Levels by Genotype in Groups Stratified by (a) Demographics, (b) Presence of Metabolic Abnormalities, and (c) Lifestyle Parameters 24 Jiang CQ et al. Interleukin-6 receptor gene polymorphism modulates interleukin-6 levels and the metabolic syndrome: GBCS-CVD. Obesity 2010;18:1969-74

Alterations of the T-cell repertoire during chronological aging 25 Thymic involution with age impairs production of new naïve T cells. Exposure to antigens throughout life results in the reduction of the size of the naïve reserve and the expansion of the memory compartment. Clonal expansion of memory cells along with antigen-independent homeostatic expansion help to maintain the number of circulating T cells. The rate of homeostatic expansion of naïve T cells increases late in life as a compensatory mechanism for the overall depletion of the naïve compartment. Clonal expansion of memory cells and homeostatic expansion of both naïve and memory cells cause telomere erosion that shortens the proliferative lifespan of T cells. Expansion of the memory compartment involves accumulation of oligoclonal T cells rather than the maintenance of overall TCR-repertoire diversity. As depicted, 65 years is arbitrarily set to demarcate young and old age categories, and is seemingly the age at which slopes of telomere length and thymic output are minimal. Vallejo AN. Immune remodeling: lessons from repertoire alterations during chronological aging and in immune-mediated disease. Trends Mol Med 2007;13:94-102

Age- and Sex-Standardized Prevalence of Individual Infections 26 Zajacova A et al. Socioeconomic and race/ethnic patterns in persistent infection burden among U.S. adults. J Gerontol A Biol Sci Med Sci 2009;64A:272-9

Changes in TREC+ cells with age 27 The percentage of sj-trec positive cells undergoes a dramatic age-dependent decline from young to middle-aged subjects and centenarians. Kruskal Wallis test among the three groups revealed a P < 0.0001. No significant difference was observed between young and middle-aged donors. Note that only the values from individuals with detectable TREC levels (all young subjects, 10 out of 13 middle-aged donors, 4 out of 25 centenarians) are shown here. Nasi M et al. Thymic output and functionality of the IL-7/IL-7 receptor system in centenarians: implications for the neolymphogenesis at the limit of human life. Aging Cell 2006;5:167-75

28 McElhaney JE, et al. Immunosenescence: what does it mean to health outcomes in older adults? Curr Opin Immunol 2009;21:418-24

Schematic diagram for the cardiovascular inflammation reduction trial (CIRT) 29 Ridker PM. Testing the inflammatory hypothesis of atherothrombosis: scientific rationale for the cardiovascular inflammation reduction trial (CIRT). J Thromb Haemost 2009;7(Suppl1):332-9

Balancing the IL-1β system and its contributions to human disease. MWS indicates Muckle-Wells syndrome; NOMID, neonatal-onset multisystem inflammatory disease 30 Ridker PM et al. Interleukin-1β inhibition and the prevention of recurrent cardiovascular events: Rationale and design of the canakinumab anti-inflammatory thrombosis outcomes study (CANTOS). Am Heart J 2011;162:597-605

31 Ridker PM et al. Interleukin-1β inhibition and the prevention of recurrent cardiovascular events: Rationale and design of the canakinumab anti-inflammatory thrombosis outcomes study (CANTOS). Am Heart J 2011;162:597-605

Activation of T cells requires two signals T cell activation occurs only after interaction between T cell receptor (TCR) and antigen in the context of MHC (signal 1) plus CD28 costimulation (signal 2). Sharma P et al. The future of immune checkpoint therapy. Science 2015;348:56-61 32