Interleukin-6, Interleukin-13 and Interferon-γ as Potential Biomarkers for Treatment Failure in Pulmonary Tuberculosis Akshay Gupte 1 , Pavan Kumar 2 , Sriram Selvaraju 2 , Mandar Paradkar 3 , Vandana Kulkarni 3 , Neeta Pradhan 3 , Rewa Kohli 3 , Nishi Suryavanshi 3 , Kannan Thiru 2 , Luke Hanna 2 , Kavitha Dhanasekaran 2 , Sri Vijay Bala Yogendra 4 , Rahul Lokhande 3 , Subhash Babu 2 , Jonathan Golub 1 , Nikhil Gupte 1 , Bruno Andrade 5 , Vidya Mave 1 , Padmapriyadarasini Chandrasekaran 2 , Amita Gupta 1 1 Johns Hopkins University School of Medicine, Baltimore, USA; 2 National Institute for Research in Tuberculosis, Chennai, India; 3 Byramjee Jeejeebhoy Government Medical College, Pune, India; 4 Johns Hopkins University India Office, Pune, India; 5 Instituto Brasileiro para Investigação da Tuberculose, Salvatore, Brazil 1 World Health Organization. Global TB Report; Geneva, Switzerland, 2016. 2 Mitchison DA. Assessment of new sterilizing drugs for treating pulmonary tuberculosis by culture at 2 months [letter]. Am Rev Respir Dis 1993; 147: 1062–63. 3 Horne DJ, Royce SE, Gooze L, et al. Sputum monitoring during tuberculosis treatment for predicting outcome: systematic review and meta-analysis. The Lancet Infectious diseases 2010; 10(6): 387-94. References Data in this manuscript were collected as part of the Regional Prospective Observational Research for Tuberculosis (RePORT) India Consortium. This project has been funded in whole or in part with Federal funds from the Government of India’s (GOI) Department of Biotechnology (DBT), the Indian Council of Medical Research (ICMR), the United States National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (NIAID), Office of AIDS Research (OAR), and distributed in part by CRDF Global. The contents of this publication are solely the responsibility of the authors and do not represent the official views of the DBT, the ICMR, the NIH, or CRDF Global. Any mention of trade names, commercial projects, or organizations does not imply endorsement by any of the sponsoring organizations. AG was supported by NIH Research Training Grant # D43 TW009340 funded by the NIH Fogarty International Center, NINDS, NIMH, NHBLI and NIEHS. Acknowledgements • Tuberculosis (TB) is the leading infectious killer worldwide with over 10.4 million incident cases and 1.7 million deaths in 2016 1 . • While culture conversion by 2 months of anti-tuberculosis treatment (ATT) is widely used as a surrogate marker for microbiological response, recent clinical trials have shown suboptimal performance of 2 month culture in predicting unfavorable treatment outcomes, particularly treatment failure 2-3 . • Novel biomarkers predictive of unfavorable treatment outcomes are needed for the early identification and risk-stratification of TB cases. • The objective of this study was to identify systemic inflammatory markers associated with treatment failure in newly diagnosed adult pulmonary TB (PTB) cases in India. Background Methods Results Study population: • We randomly selected 30 new adult (>18 years) drug-sensitive PTB cases within 1 week of ATT initiation from the ongoing CTRIUMPH study in Pune and Chennai, India. • Participants were prospectively evaluated at 0 weeks (<7 days since ATT initiation), 8 weeks and 24 weeks for plasma concentrations of 20 cytokines linked to the host immune response in TB. • Treatment failure was defined as Mycobacterium tuberculosis growth on liquid or solid culture between 17 and 24 weeks of ATT. Cytokine analysis: • Group A (host immune response in TB): INF-γ, TNF-α, IL-1β, IL-4, IL-6, IL-10, CXCL-10, IL-12, IL-13 and IL-17 • Group B (lung tissue destruction and fibrosis): MMP-1, MMP-3, MMP- 7, TIMP-1, TIMP-2, TIMP-3, TIMP-4, TGFβ-1, TGFβ-2 and TGFβ-3 • Cytokine concentrations were evaluated, in duplicates, using multiplex ELISA according to manufacturer protocols (BioRad Inc). Statistical analysis: • Cytokine concentrations were log 2 transformed and z-score normalized for analysis. • Differentially expressed cytokines by duration of ATT and treatment failure were identified using non-parametric tests. • P-values were adjusted for multiple comparisons using the Benjamini- Hochberg procedure and a 10% false-discovery rate. Results Table 1. Baseline characteristics of study participants Figure 1. Differentially expressed markers compared to baseline concentrations (i.e. vs week 0) Figure 2. Differential cytokine expression by treatment outcomes Figure 3. Differential cytokine expression by individual participants and treatment failure at week 0 • No significant differences between those selected for inflammatory analysis compared to those not selected but part of the full cohort. • Notable exception being the absence of recurrence and deaths Conclusion • Overexpression of circulating IL-6, IL-13 and IFN-γ at treatment initiation may be associated with treatment failure among drug- sensitive pulmonary tuberculosis cases. • Well powered validation studies should be undertaken to evaluate the performance of these biomarkers, individually or in combination, for predicting unfavorable tuberculosis treatment outcomes. Contact: Akshay Gupte, PhD, MBBS, MSPH Center for Clinical Global Health Education Division of Infectious Diseases Johns Hopkins University School of Medicine Email: [email protected] Baseline characteristics Cohort (n=317) Sub-cohort (n=30) p-value Age (years), median (IQR) 40 (27-50) 36 (28-50) 0.87 Male sex, n (%) 203 (64) 20 (74) 0.40 BMI (kg/m 2 ), median (IQR) 18 (16-20) 18 (16-20) 0.98 Ever-smoking, n (%) 124 (39) 9 (31) 0.87 HIV coinfection, n (%) 19 (6) 2 (7) 0.68 Diabetes mellitus, n (%) 88 (28) 7 (26) 0.40 Cavitation, n (%) 122 (45) 10 (43) 0.99 Treatment outcomes, n (%) Failure 35 (12) 4 (14) 0.99 Recurrence 14 (4) 0 - Death 6 (2) 0 - Fold-difference Figure 4. Absolute cytokine concentrations at week 0 comparing treatment failures vs cures Discussion Key findings: • TIMP-4 and TNF-α were overexpressed at week 8 and week 24 following ATT initiation, respectively. • Relative to week 0 concentrations; IL1-β, IL-6 and MMP-7 concentrations declined at week 8; CXCL-10, TGF-β1, TGF-β2, TIMP-1 and TIMP-3 concentrations declined by week 24; and TGF-β3 concentrations declined at both week 8 and week 24. • None of the participants who failed treatment had HIV coinfection or diabetes. • Participants who failed treatment had significantly higher plasma concentrations of IL-6, IL-13 and IFN-γ at ATT initiation compared to those who were cured, however this difference was not statistically significant at 8 and 24 weeks of ATT. Limitations : • Limited sample size of n=4 failures. • We could not identify cytokines associated with recurrence or death. Future direction: • We plan to conduct well powered validation studies measuring the performance of IL-6, IL-13 and IFN-γ as predictive markers for unfavorable treatment outcomes and lung injury.