Immunology Letters 123 (2009) 60–71 Contents lists available at ScienceDirect Immunology Letters journal homepage: www.elsevier.com/locate/ Molecular events in the activation of B cells and macrophages by a non-microbial TLR4 agonist, G1-4A from Tinospora cordifolia Rashmi Raghu a , Deepak Sharma a , Rupal Ramakrishnan b , Shazia Khanam c , Gajanan J. Chintalwar d , Krishna Balaji Sainis a,∗ a Radiation Biology & Health Sciences Division, Bio-Medical Group, Bhabha Atomic Research Centre, Modular Laboratories, Trombay, Mumbai 400085, India b Department of Tumor Immunology, H. Lee Moffitt Cancer Center and Research Institute, 12909 Magnolia Drive, Tampa, FL 33612-9497, USA c Tata Institute of Fundamental Research, Colaba, Mumbai 400005, India d Bio-Organic Division, Bhabha Atomic Research Centre, Modular Laboratories, Trombay, Mumbai 400085, India article info Article history: Received 28 November 2008 Received in revised form 10 February 2009 Accepted 10 February 2009 Available online 20 February 2009 Keywords: B cell proliferation TLR4 Phagocytosis Splenomegaly NF-B abstract G1-4A, a polysaccharide from an Indian medicinal plant Tinospora cordifolia, was recently shown to protect mice against septic shock by modulating the proinflammatory cytokines. G1-4A also activated B cells polyclonally. The present report describes in detail the molecular events associated with G1-4A-induced immunomodulation in vitro and in vivo. G1-4A treatment led to an increase in the CD69 expression in lymphocytes. G1-4A-induced proliferation of B cells was completely inhibited by PI3K inhibitor Ly294002, mTOR inhibitor rapamycin and NF-B inhibitor plumbagin. Akt, ERK and JNK were activated by G1-4A which finally resulted in the activation of IKK, degradation of IB- and translocation of NF-B to the nucleus. Administration of G1-4A to mice led to splenomegaly and an increase in the numbers of T cells, B cells and macrophages. This increase in spleen cellularity was due to in vivo proliferation of lymphocytes and upregulation of anti-apoptotic genes. Anti-TLR4-MD2 complex antibody inhibited G1-4A-induced B cell proliferation and degradation of IB- suggesting that TLR-4 was a receptor for G1-4A on B cells. Activation of RAW 264.7 macrophages by G1-4A was found to be dependent on ERK and NF-B-mediated signals. The phagocytosis index in peritoneal exudate cells (PEC) isolated from G1-4A treated mice was significantly higher as compared to that in PEC from control mice. G1-4A administration also increased the number of CD11b + cells in the PEC without an increase in the total number of PEC. Thus the present understanding of the molecular mechanism of action of G1-4A, a novel non-microbial TLR4 agonist, will pave the way for its application as an immunomodulator and adjuvant. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Many pathogens have devised ways to subvert the activa- tion of leukocytes. Mycobacterium tuberculosis evades the human immune response by inhibiting the IFN- signaling pathway [1]. Salmonella inhibits phagosome lysosome fusion in macrophages [2]. The human immunodeficiency virus (HIV) interferes with the Fc- R signaling in human macrophages and inhibits phagocytosis [3]. Under such conditions, an external agent may be useful for the acti- vation of these cells. Many such agents have been identified from microbial origin like CpG DNA [4], LPS [5] and monophosphoryl lipid A [6]. These agents activate the immune system either by activat- ing phagocytes (LPS, CpG DNA) or by directly acting as polyclonal mitogens (LPS). However, these microbial products have limited ∗ Corresponding author. Tel.: +91 22 25593638/25593781; fax: +91 22 25505326/25505151. E-mail address: kbsainis@barc.gov.in (K.B. Sainis). application due to their toxicity. Plant derived polysaccharides have attracted widespread attention as ideal immunomodulators due to their therapeutic properties and relatively low toxicity and side effects as compared to those derived from microbial origin [7,8]. Crude extracts and purified fractions derived from certain parts of the Indian medicinal plant, Tinospora cordifolia (family Menis- permaceae), have been shown to exhibit creditable medicinal properties like general tonic, antipyretic, anti-inflammatory and prevention of complement disorders and protection against tumors [8,9]. It has also been shown to protect against abdominal infec- tions by enhancing the phagocytic efficiency and intracellular bactericidal activity of macrophages and neutrophils in Escherichia coli-induced peritonitis [10]. Sainis et al. earlier showed the polyclonal B cell mitogenic activ- ity of the dry stem crude extract (DSCE) of T. cordifolia [11,12]. Activity-based purification of DSCE resulted in an acetone pre- cipitated fraction which was further purified by gel permeation chromatography on Sephacryl S-400. This has been referred to as Tc-1, DSCE sc (aq) or G1-4A [11–14]. G1-4A augmented humoral 0165-2478/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2009.02.005