CCL2 Responses to Mycobacterium tuberculosis Are Associated with Disease Severity in Tuberculosis Zahra Hasan 1 *, Jacqueline M. Cliff 2 , Hazel M. Dockrell 2 , Bushra Jamil 1 , Muhammad Irfan 1 , Mussarat Ashraf 1 , Rabia Hussain 1 1 The Aga Khan University, Karachi, Pakistan, 2 London School of Hygiene and Tropical Medicine, London, United Kingdom Abstract Background: Leucocyte activating chemokines such as CCL2, CCL3, and CXCL8 together with proinflammatory IFNc, TNFa and downmodulatory IL10 play a central role in the restriction of M. tuberculosis infections, but is unclear whether these markers are indicative of tuberculosis disease severity. Methodology: We investigated live M. tuberculosis- and M. bovis BCG- induced peripheral blood mononuclear cell responses in patients with tuberculosis (TB) and healthy endemic controls (ECs, n = 36). TB patients comprised pulmonary (PTB, n = 34) and extrapulmonary groups, subdivided into those with less severe localized extrapulmonary TB (L-ETB, n = 16) or severe disseminated ETB (D-ETB, n = 16). Secretion of CCL2, IFNc, IL10 and CCL3, and mRNA expression of CCL2, TNFa, CCL3 and CXCL8 were determined. Results: M. tuberculosis- and BCG- induced CCL2 secretion was significantly increased in both PTB and D-ETB (p,0.05, p,0.01) as compared with L-ETB patients. CCL2 secretion in response to M. tuberculosis was significantly greater than to BCG in the PTB and D-ETB groups. M. tuberculosis-induced CCL2 mRNA transcription was greater in PTB than L-ETB (p = 0.023), while CCL2 was reduced in L-ETB as compared with D-ETB (p = 0.005) patients. M. tuberculosis –induced IFNc was greater in L-ETB than PTB (p = 0.04), while BCG-induced IFNc was greater in L-ETB as compared with D-ETB patients (p = 0.036). TNFa mRNA expression was raised in PTB as compared with L-ETB group in response to M. tuberculosis (p = 0.02) and BCG (p = 0.03). Mycobacterium-induced CCL3 and CXCL8 was comparable between TB groups. Conclusions: The increased CCL2 and TNFa in PTB patients may support effective leucocyte recruitment and M. tuberculosis localization. CCL2 alone is associated with severity of TB, possibly due to increased systemic inflammation found in severe disseminated TB or due to increased monocyte infiltration to lung parenchyma in pulmonary disease. Citation: Hasan Z, Cliff JM, Dockrell HM, Jamil B, Irfan M, et al. (2009) CCL2 Responses to Mycobacterium tuberculosis Are Associated with Disease Severity in Tuberculosis. PLoS ONE 4(12): e8459. doi:10.1371/journal.pone.0008459 Editor: T. Mark Doherty, Statens Serum Institute, Denmark Received September 3, 2009; Accepted December 1, 2009; Published December 29, 2009 Copyright: ß 2009 Hasan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This investigation received financial support from the United Nations Children’s Fund (UNICEF)/United Nations Development Programme (UNDP)/ World Bank/World Health Organization (WHO) and was funded by the Special Programme for Training in Tropical Diseases Research, TDR, WHO. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: zahra.hasan@aku.edu Introduction Tuberculosis (TB) causes 1.8 million deaths annually with 9.27 million incident cases of which the majority (55%) are in Asia [1]. Although the primary disease remains at pulmonary sites, extrapulmonary disease is common especially in high TB burden settings [2,3] or where there is a high rate of human immunodeficiency virus (HIV) co-prevalence [4]. Protective immunity against Mycobacterium tuberculosis is depen- dent on the interplay between activated T cells, macrophages and other leucocytes. Proinflammatory cytokines such as, interferon gamma (IFN)-c, tumor necrosis factor-alpha (TNF)-a, interleukin (IL)-12 are essential for protective immunity against M. tuberculosis [5], [6]. IL-10 produced by macrophages is important in regulating the TH1 cytokine balance and down regulates proinflammatory responses [7]. Small molecular weight (8–10 kDa) chemotactic cytokines or, chemokines are responsible for regulating the migration, traffick- ing, homing and activation of monocytes, macrophages and other leucocytes. An effective granulomatous response is essential for the restriction of M. tuberculosis infection. TNFa which is essential for macrophage activation and granuloma formation [8,9] also influences the expression of chemokines by macrophages and mediates effective recruitment of leucocytes via the CC chemo- kines; CCL2 (monocyte chemoattractant protein (MCP)-1), CCL3 (macrophage inflammatory protein (MIP)- 1a), CCL4 (macro- phage inflammatory protein (MIP)- 1b), CCL5 (regulated on activation normal T cell expressed and secreted: RANTES) and CXC chemokines; CXCL8 (IL8), CXCL9 (monokine induced by IFNc: MIG) and CXCL10 (IFNc inducible 10kD protein: IP10) [10–13]. CCL2 and CCL3 are primarily secreted by monocytes, macrophages and dendritic cells. Responsiveness to CCL2 is dependent on its receptor CCR2, and CCL2 is a potent activator of cells which express CCR2 such as, monocytes, macrophages, CD4+ T cells and immature dendritic cells [14]. CCL2 is essential PLoS ONE | www.plosone.org 1 December 2009 | Volume 4 | Issue 12 | e8459