Mycobacterium indicus pranii (MIP) mediated host protective intracellular mechanisms against tuberculosis infection: Involvement of TLR-4 mediated signaling

Accepted Manuscript Mycobacterium indicus pranii (MIP) mediated host protective intracellular mechanisms against tuberculosis infection: Involvement of TLR-4 mediated signaling Subrata Majumdar PII:

S1472-9792(16)30010-5

DOI:

10.1016/j.tube.2016.09.027

Reference:

YTUBE 1531

To appear in:

Tuberculosis

Received Date: 7 January 2016 Revised Date:

28 September 2016

Accepted Date: 29 September 2016

Please cite this article as: Majumdar S, Mycobacterium indicus pranii (MIP) mediated host protective intracellular mechanisms against tuberculosis infection: Involvement of TLR-4 mediated signaling, Tuberculosis (2016), doi: 10.1016/j.tube.2016.09.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Mycobacterium indicus pranii (MIP) mediated host protective intracellular mechanisms

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against Tuberculosis infection: Involvement of TLR-4 Mediated Signaling.

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ADDRESS OF THE CORRESPONDING AUTHOR:

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**Dr. Subrata Majumdar

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Professor

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Division of Molecular Medicine

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Bose Institute,

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P-1/12, CIT Scheme VII- M, Kolkata-700 054, India.

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Telephones: 2355-9416 / 9544 /9219. FAX: (91) (33) 2355-3886

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E-mail:[email protected].

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Summary:

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Mycobacterium tuberculosis infection inflicts the disease Tuberculosis (TB), which is fatal if left

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untreated. During M. tuberculosis infection, the pathogen modulates TLR-4 receptor down-

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stream signaling, indicating the possible involvement of TLR-4 in the regulation of the host

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immune response. Mycobacterium indicus pranii (MIP) possesses immuno-modulatory

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properties which induces the pro-inflammatory responses via induction of TLR-4-mediated

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signaling. Here, we observed the immunomodulatory properties of MIP against tuberculosis

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infection. We have studied the detailed signaling mechanisms employed by MIP in order to

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restore the host immune response against the in vitro tuberculosis infection. We observed that in

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infected macrophages MIP treatment significantly increased the TLR-4 expression as well as

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activation of its downstream signaling, facilitating the activation of P38 MAP kinase. MIP

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treatment was able to activate NF-κB via involvement of TLR-4 signaling leading to the

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enhanced pro-inflammatory cytokine and NO generation in the infected macrophages and

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generation of protective immune response. Therefore, we may suggest that, TLR4 may represent

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a novel therapeutic target for the activation of the innate immune response during Tuberculosis

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infection.

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Keywords:

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TLR-4, Mycobacterium indicus pranii, MAP Kinase, Cytokine, Tuberculosis

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1. Introduction:

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M. tuberculosis is an obligate intracellular pathogen that establishes itself within the host through

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immunosuppression of the host protective arsenals [1]. Tuberculosis infection inhibits antigen

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specific T-cell responses within the host by abrogating the macrophage and T-cell function.

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These pathogens use several mechanisms to suppress macrophage activation in order to evade

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our host immune response. M. tuberculosis impairs free radical (super oxide and nitric oxide)

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generation [2] and interleukin-12 - a host protective cytokine [3] production from macrophages.

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In contrast, the disease-promoting cytokines, transforming growth factor β (TGF-β) and

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interleukin (IL)-10 are enhanced in tuberculosis infection [4]. Thus, host protection as well as

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disease-progression depend on the IL-12 to IL-10 (IL-12: IL-10) ratio, which is primarily

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regulated by the reciprocal signaling through extracellular stress regulated kinase (ERK) 1/2 and

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p38 mitogen-activated protein kinase (MAPK).

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Innate immunity synchronizes the inflammatory response to pathogens and the involvement of

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Toll-like receptors (TLRs) to this response is becoming extensively recognized [5]. TLRs are

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triggered by pathogen-associated molecular pattern molecules (PAMPs), which are characteristic

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of various groups of pathogens [6]. Activation of TLR4 signaling leads to upregulation of

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MyD88 - IRAK 1 interaction, which, in turn, promotes TRAF6 activation and NF-kB nuclear

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translocation. The nuclear translocation of NF-kB culminates in the induction of

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proinflammatory responses [7].

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Chemotherapeutic approaches against Tuberculosis (TB) with first line antibiotics have shown

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moderate success due to severe side effects and emergence in the drug-resistant strains [8]. A

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new immunotherapeutic strategy has been employed by a number of groups where the use of

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different TLR agonists, against the diseases have been shown to be successful [9, 10].

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In addition, the use of different bacteria to potentiate the host immune response against different

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disease is of interest. As an example, BCG vaccination reduces the risk of developing childhood

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tuberculosis [11]. In addition, heat-killed suspension of M. vaccae (SRL172) is efficacious

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against other diseases and induces potent anti-tuberculosis responses [12, 13]. Moreover

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immunotherapy with SRL172 in cancer patients has significant impact on the overall disease

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outcome [14].

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In the present study, we have described the use of a novel immunomodulator (MIP) that relieved

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the immune system from the suppression induced by M. tuberculosis that is responsible for high

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mortality worldwide. MIP, previously known as Mycobacterium w is a saprophytic bacterium

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which stimulates cell mediated immune responses in leprosy patients [15]. Following

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biochemical, molecular and phylogenic analysis, it has been shown that MIP is closely related to

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M. avium intracellulare [16]. Interestingly, MIP is able to retain its immunologic potential also

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in the heat killed form and share antigens with Mycobacterium leprae and M. tuberculosis. MIP

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treatment, together with chemotherapy, increases bacterial clearance along with the reduction of

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the recovery time of leprosy patients [17, 18]. In addition, MIP treatment is able to enhances

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immunity to other diseases, e.g. HIV [19] psoriasis [20]. Moreover, in Tuberculous Pericarditis

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patients, MIP treatment has been shown to be effective [21]. Despite these observations, the

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mechanism by which MIP enhances anti-tuberculosis responses is not known.

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Currently, several trial are on going to study the efficacy of MIP with respect to tuberculosis

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[22]. Therefore, it is imperative to study the mechanisms by which MIP functions as an

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immunomodulator. In this study, we have examined the immunomodulatory potential of MIP

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against tuberculosis. We observed that in infected macrophages MIP treatment induced TLR4

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expression. MIP treatment was able to activate NF- kB via involvement of TLR-4 signaling

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leading to the enhanced pro-inflammatory cytokine and NO generation in the infected

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macrophages. During M. tuberculosis infection, the pathogen modulates TLR-4 receptor down-

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stream signaling, indicating the possible involvement of TLR- 4 in the regulation of the host

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immune response. MIP possesses immuno-modulatory properties which induces the pro-

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inflammatory responses via activation of TLR-4-mediated signaling. Here, we found that

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treatment of M. tuberculosis-infected macrophages with MIP caused a significant increase in the

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TLR-4 expression as well as activation of its downstream signaling, facilitating the activation of

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MAP kinase P38. All these events culminated in the up-regulation of proinflammatory response.

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This study demonstrated that MIP conferred protection against tuberculosis via involvement of

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TLR-4 signaling.

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2. Materials and methods:

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2.1. Preparation of peritoneal macrophages: Peritoneal macrophages were isolated as

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described elsewhere [4]. Briefly, mouse macrophages were isolated by peritoneal lavage 48hrs

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after intra-peritoneal injection of sterile 4% thioglycolate broth (DIFCO) from C57BL/6 mice.

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The peritoneal macrophages were collected by using the ice cold sterile PBS for infusing the

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peritoneal cavity. The macrophages were cultured in a 37°C incubator with 5% CO2 in DMEM

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(Sigma Adrich) which contained 10% heat-inactivated FBS (Gibco,Brl), 2 mmol/l glutamine and

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100 U/ml penicillin and streptomycin (Sigma Aldrich). On the basis of morphologic criteria,

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more than 85% of the remaining adherent cells were macrophages.

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2.2. M. tuberculosis H37Rv and MIP (Mycobacterium indicus pranii): M. tuberculosis H37Rv

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(ATCC 25177) were grown in shaker flasks with Middlebrook 7H9 medium (BD Difco, NJ,

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USA) containing 0.02% glycerol, 0.05% Tween 80 and 10% albumin-dextrose complex

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enrichment (BD Difco, NJ, USA). Bacteria were harvested during the mid-log growth phase by

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centrifugation at 2,500 g for 15 min. The bacteria were then washed twice using the centrifugal

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washing method and suspended in saline at the desired concentration [1]. MIP (Mycobacterium

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indicus pranii) was a gift from Dr B. M. Khamer (Cadila Pharmaceuticals Limited, Gujarat,

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India).

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2.3. Cytotoxicity assay with MTT method:

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Macrophages in 96-well tissue culture plates (Tarson) incubated with MIP (103–108 cells/mL),

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were cultured in DMEM supplemented with 10% FCS for 48 h. The medium was replaced with

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fresh DMEM (without phenol red) containing 1 mg/mL MTT. Cells were incubated at 37°C for 3

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h, the untransformed MTT was removed and 50 mL of 0.04 M HCl-isopropanolic solution was

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added to each well. After 15 min incubation, the absorbance was measured on an automatic plate

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reader (Thermolab System Multiskan Ex) at a reference wavelength of 690 nm and test

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wavelength of 650 nm.

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2.4. Infection and Treatment of macrophages: The adherent cell populations were infected

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with live M. tuberculosis H37Rv at a MOI (multiplicity of infection) of 1:10 (macrophage:

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Mycobacteria). After 3h of infection the extracellular bacteria were removed from the cells by

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washing it properly. Then the infected macrophages were treated with MIP.

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2.5. Quantification of viable Mycobacteria by Colony Forming Unit (CFU) count: The

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infected and treated macrophages were lysed with 0.5% SDS solution [4]. This lysate was

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serially diluted and plated on Middlebrook 7H10 containing Oleic acid-ADC in triplicate.

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Colony forming units (CFU) were counted after 21 days of incubation at 37°C. Data expressed

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as CFU in terms of Mean ± standard deviation (SD).

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2.6. Nitrite assay: Nitrite level in culture was measured using the Nitric Oxide Colorimetric

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Assay kit (Boehringer Mannheim Biochemicals) [23]. Briefly, cell-free supernatants were

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collected from differently treated macrophages, and nitrite levels were estimated in accordance

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with the manufacturer’s instructions. Data were expressed in micromoles of nitrite.

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2.7. Measurement of cytokine release by sandwich ELISA: Cytokines were measured from

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infected cell supernatants as described elsewhere. Briefly, cell-free supernatants were collected

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from differently treated macrophages, and the levels of cytokines were measured using mouse

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IL-10, IFN-γ, IL-12, TNF-α (BD) and TGF-β (eBioscience) ELISA sets [4].

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2.8. Isolation of RNA: Total RNA extracted from differently treated macrophages (TRI reagent;

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Sigma). For cDNA synthesis, 1 µg of total RNA from each sample was reverse-transcribed using

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the Revert Aid

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amplified with respective primers (listed in Table 1) and 0.5 unit Taq DNA polymerase

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(Fermentas) in 50 µl reaction volume under the following conditions: initial activation step

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(2min at 95°C) and cycling step (denaturation for 30 s at 94°C, annealing for 30 s at 58°C, and

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extension for 1 min at 72°C for 35 cycles), using Perkin Elmer Gen Amp PCR system 2400.

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PCR amplified products were subsequently size fractioned on 1.5% agarose gel, stained with

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ethidium bromide and visualized under UV-light.

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2.9. Preparation of cell lysate and immunoblot analysis: Cell lysates from differently treated

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macrophages were prepared as described elsewhere [24]. Briefly Equal amounts of protein (50

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ug) were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and

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immunoblotting was performed as described elsewhere [25]. Immunoblotting was performed to

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detect the expression of TLR-4, TRAF-6, NF-κB, phosphorylated or dephosphorylated forms of

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(Abcam) p38MAPK and ERK-1/2 (Santacruz).

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M-MuLV Reverse Transcriptase (Fermentas). cDNA from each sample was

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2.10. Co-immunoprecipitation: In co-immunoprecipitation studies, the lysates of differently

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treated macrophages were incubated with either anti-TLR4 antibody (Santa Cruz Biotechnology

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Inc., USA) or Myd88 antibody or IRAK-1 antibody. The complexes were then captured with

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immobilized Protein A agarose beads. Finally, the sample mixtures were separated using 10%

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SDS PAGE and the blots were developed with anti-MyD 88, IRAK-1 or IRAK-M (Abcam)

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antibodies respectively to detect the TLR4–and its downstream molecules interactions that has

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been described elsewhere [26].

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2.11. Preparation of nuclear and cytoplasmic extracts

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The nuclear and cytoplasmic extracts were prepared from differently treated macrophages as

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described elsewhere [27]. Briefly, sedimented cells were resuspended in hypotonic buffer [10

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mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.2 mM PMSF, and 0.5 mM DTT] and

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allowed to swell on ice for 10 min. Cells were homogenized in a homogenizer. The nuclei were

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separated by spinning at 3300g for 5 min at 4ºC. The supernatant was used as the cytoplasmic

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extract. The nuclear pellet was extracted in nuclear extraction buffer {20 mM HEPES (pH 7.9),

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0.4 M NaCl,1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 0.5 mM PMSF, and 0.5 mM DTT}

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for 30 min on ice and centrifuged at 12,000g for 30 min. The supernatant was used as nuclear

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extract.

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2.12. Electrophoretic Mobility Shift Assay (EMSA)

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NF-κB specific oligonucleotide 5'-TAGTTGAGGGCACTTTCCCAGG-3' from the NF-κB/Rel

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A DNA binding domain in murine IkB light chain gene enhancer (synthesized from SIGMA)

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were labeled with 32P with Klenow using γ-32P dATP. Nuclear extracts (15 μg per sample) were

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incubated with 3 X 105 cpm

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(containing 12.5 mM HEPES pH 7.9, 10% glycerol, 5 mM MgCl2, 50 mM KCl, 1mM EDTA, 1

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mM DTT, 300 mg/ml BSA) and 44 mg Salmon sperm DNA for 20 min. For cold competition,

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10- and 100-fold excess unlabeled probe was incubated for 15 min at room temperature with the

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above mixture before addition of the labeled probe. Shift complexes were resolved in 6%

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acrylamide gels at 4°C in 0.5X TBE. Dried gels were autoradiographed [28].

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2.13. Flow Cytometry: Expression of TLR-4 on surface of macrophages was analyzed by flow

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cytometry as described elsewhere [4]. Briefly, adherent differently treated macrophages were

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P labeled probe (0.2 ng DNA) in the presence of binding buffer

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harvested and washed twice in ice-cold fluorescence-activated cell sorter (FACS) buffer [PBS

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containing 10% (w/v) BSA and 0.1% (w/v) sodium azide]. PE-labelled anti-TLR-4 antibody

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(SantaCruz Biotechnology Inc., USA) was used for the detection of cell surface TLR-4.

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Expression of cell surface molecules was evaluated and analyzed with a FACS Verse flow

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cytometer (Becton Dickinson).

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2.14. Statistical analysis: The experiments were performed at least 3 times, and the data were

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presented as means±SD. One way ANOVA followed by Tukeys Post hoc test was employed to

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assess the significance of the differences between the mean values of different experimental

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groups (GraphPad InStat 3.1). A value of P< 0.05 was considered to be significant while the

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value of P <0.001 was considered to be highly significant.

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Result:

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3.1. Determination of the non-cytotoxic dose of MIP

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The cytotoxic effect of MIP was studied in murine peritoneal macrophages, by MTT method.

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Murine peritoneal macrophages were infected with M. tuberculosis (macrophage: bacteria ratio

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of 1:10) for 3h. Uninfected and M. tuberculosis infected peritoneal macrophages were treated

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with different doses of MIP ranging from 103 to 108 cells/ml (Figure 1A). Treatment of

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uninfected and infected macrophages with MIP at doses of 106cells/ml, 107cells/ml and

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108cells/ml resulted in the reduction of 10%, 25% and 40% cell survivability respectively. We

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then assessed the efficacy of MIP in reducing the intracellular bacterial survival in peritoneal

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macrophages. Treatment of peritoneal macrophages with 106cells/ml MIP for 24hr showed

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nearly 90% (p<0.001) reduction in the CFU counts during MIP treatment (Figure 1B). For the

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remainder of ex vivo experiments described below analyzing the impact of MIP on innate

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resistance of macrophages to M. tuberculosis we utilized MIP at a dose of 106cells/ml.

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3.2. MIP up-regulated the TLR4 expression in M. tuberculosis-infected macrophages:

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In the previous result we observed that MIP treatment was able to down-regulate the bacterial

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survival inside the host macrophages. Therefore, we aimed to study the detailed signaling

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mechanisms involved in the MIP mediated reduction of bacterial survival. M. tuberculosis

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infection was associated with severe impairment of host macrophage function due to the down-

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regulation of TLR4 expression as well as its downstream signaling [29]. Therefore, we studied

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whether MIP treatment could restore the TLR4 expression in infected macrophages. C57BL/6-

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derived peritoneal macrophages were infected with M. tuberculosis (1:10 ratio) followed by

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treatment with MIP for the detection of TLR4 expression. PCR (Figure 2A), western blot (Figure

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2B) and (Figure 2C) FACS analyses showed that during infection the TLR-4 expression was

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down regulated (~2-fold) in infected macrophages whereas MIP treatment significantly up-

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regulated TLR4 expression (~1.2 fold). Therefore, from this study we suggested that MIP

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treatment was able to up-regulate the TLR4 expression during the course of infection.

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3.3. Activation of TLR4 signaling by MIP treatment during M. tuberculosis infection.

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It is known that TLR-4 activation depends on the association of TLR-4 with MyD88, an adaptor

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molecule located immediate downstream of TLR-4, and this event is crucial for the initiation of

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further signals. Therefore, we studied the activation of TLR4 downstream signaling. The cell

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lysates from differently treated macrophages were subjected to immunoprecipitation with either

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anti-TLR-4, anti-MyD88 or anti-IRAK-1 antibodies and the blots were probed with anti-MyD88,

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anti-IRAK-1 or anti- IRAK-M antibodies (Figure 3A, 3B and 3C) respectively. To ensure equal

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input for the IP products, were re-probed with respective Abs. Co-immunoprecipitation studies

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showed a strong selective association between TLR-4/MyD88 and MyD88/IRAK-1 in MIP

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treated infected macrophages compared to untreated infected macrophages. On the other hand,

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IRAK-M, a negative regulator of TLR-4 signaling, [30] did not show any significant association

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with IRAK-1 in MIP treated infected sets (Figure 3C).

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Furthermore, the expression of IRAK-M was abrogated in MIP treated infected macrophages

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(Figure 3D). TRAF-6 which plays a crucial role in TLR induced NF-κB activation [31], was

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enhanced in MIP treated infected sets compared to untreated infected macrophages (Figure 3D).

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Moreover, MIP treatment significantly up-regulated IKK-α expression whereas it down-

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regulated IκB-α expression in infected macrophages compared with untreated infected

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macrophages (Figure 3D).

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Moreover, we checked the NF-κB p65 expression in both infected and treated macrophages. MIP

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treatment led to enhanced NF-κB p65 expression in infected macrophages which was

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downregulated in the untreated infected macrophages (Figure 3D). In addition, we have studied

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the expression of NF-κB p65 in the cytosolic and nuclear extracts isolated from differently

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treated macrophages where MIP treatment led to enhanced NF-κB expression in the in the

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nuclear fraction of infected macrophages (Figure 3E). We further studied the NF-κB p65 nuclear

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translocation in the infected macrophages during MIP treatment. MIP treatment significantly

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enhanced NF-κB p65 nuclear translocation in the infected macrophages (Figure 3F). Therefore,

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we may assume that the enhanced expression of IKK complex in infected macrophages led to the

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activation and subsequent translocation of NF-κB p65 during MIP treatment.

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3.4. Regulation of MAP Kinase by MIP during H37Rv infection

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In addition to activation of TLR-4 down-stream signaling, we next aimed to study the effect of

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MIP in the regulation of MAP Kinase. We observed enhanced P38 MAPK phosphorylation in

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the MIP treated infected macrophages as compared to the control and untreated infected

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macrophages (Figure 4A). On the contrary, the phospho ERK 1/2 expression was down-

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regulated in the MIP treated infected macrophages as compared to the untreated infected

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macrophages (Figure 4B). Therefore, we suggested that MIP treatment reciprocally regulated

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the MAP Kinases during the course of infection.

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3.5. MIP Enhanced Th1 Promoting Cytokine Production from Tuberculosis-infected

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Macrophages

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The host-protective anti-tuberculosis response is associated with IL-12-dependent Th1 response.

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However, Tuberculosis-infected macrophages augment Th2 response [1]. Therefore, we

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evaluated whether MIP treatment of infected macrophages induced a Th1-promoting cytokine

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response. Indeed, IL-12 production was significantly up-regulated in MIP-treated uninfected (~7-

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fold) and infected macrophages (~5.5-fold) as compared with untreated infected macrophages

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(Figure 5A, 5F). A similar result was observed with IFN-γ (~2.2 and 1.8-fold) and TNF-α (~1.8

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and 1.6-fold) as well (Figure 5B-5C, 5F). In contrast, IL-10 (~3.5-fold) as well as TGF-β (~3-

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fold) levels were significantly less in MIP-treated infected macrophages as compared with

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untreated infected macrophages both at the protein and mRNA level (Figure 5D-5E, 5F). These

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results suggested that MIP up-regulated the Th1-promoting cytokines in infected macrophages.

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Moreover, blocking of TLR-4, ERK1/2 or P38 by using specific inhibitors significantly

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influenced the MIP mediated cytokine generation in infected macrophages. Inhibition of TLR-4

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(IL-12: ~4-fold, p<0.001; IFN-γ: ~2-fold, p<0.05; TNF-α: ~2-fold, p<0.05) or P38 (IL-12:~5-

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fold, p<0.001; IFN-γ: ~1.8-fold, p<0.001; TNF-α: ~2-fold, p<0.001) significantly reduced the

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expression and generation of proinflammatory cytokines whereas augmented the anti-

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inflammatory cytokine generations (For TLR-4 inhibition- IL-10: ~3.5-fold, p<0.001; TGF-β:

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~2.2-fold, p<0.001 and for

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p<0.001) as compared with MIP treated infected macrophages. On the other hand, inhibition of

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ERK1/2 further augmented the effect of MIP in inducing the proinflammatory cytokine

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generation both at the mRNA and protein level. Therefore, these results suggested that the MIP

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mediated alteration in the cytokine profile solely dependent on the involvement of TLR-4 and

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P38 MAP Kinase.

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3.6. MIP Enhanced NO Production by Tuberculosis infected Macrophages

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The reactive nitrogen species are the important bactericidal molecules [32] Therefore, we wanted

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to examine whether MIP contributed in the induction of NO in the infected macrophages. It was

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observed that treatment of infected macrophages with MIP increased nitrite production by nearly

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2.5-fold in comparison with the untreated control (Figure 6A). These results corroborated with

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the significantly increased iNOS mRNA expression in MIP treated infected macrophages (Figure

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6B). Moreover, inhibition of TLR-4 or P38 prior to MIP treatment led to significant reduction of

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P38 inhibition- IL-10:~3.5-fold, p<0.001; TGF-β: ~2.2-fold,

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both the iNOS expression as well as NO generation (For TLR-4 inhibition- ~2-fold, p<0.001 and

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for P38 inhibition- ~2.2-fold, p<0.001) in the infected macrophages. However, inhibition of

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ERK1/2 further augmented the MIP mediated iNOS expression and NO generation. Therefore,

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these results suggested that MIP treatment enhanced the production of bactericidal molecules

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along with the pro-inflammatory cytokines by involving TLR-4 and P38 MAP Kinase.

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4. Discussion:

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Emergence of drug-resistant bacterial strains limits the effectiveness of the currently available

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drugs. Therefore, an alternative therapeutic approach seems to be a pressing need. Among the

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alternative therapies, immunotherapy is one of the most promising options. Therefore, the

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present study has been undertaken to investigate the underlying mechanisms of MIP mediated

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protection against M. tuberculosis induced pathogenesis. Here we have evaluated the effect of

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MIP on the regulation of signal transduction events primarily involving TLR and MAPK

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signaling.

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Protection against infectious diseases is primarily regulated by the cell-mediated immune

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responses where cytokines play a major role [4, 33]. Interestingly, MIP is known to have

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proinflammatory cytokine evoking properties through the induction of TLR-4 [34]. However,

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during infection IL-12 is downregulated which indicates the possible involvement of TLR and

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cytokine in the regulation of immune response against M. tuberculosis infection.

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In the present study, MIP was found to render significant protection against M. tuberculosis

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infection of murine macrophages in vitro. MIP treatment significantly upregulated the TLR-4

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expression in M. tuberculosis infected macrophages (Figure 2). TLR-4 played an important role

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in the generation of effective immunity against tuberculosis [35, 36]. Interestingly, TLR-4 and

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MyD88 association initiated a chain of signaling cascades involving the recruitment of different

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kinases, mainly IRAK-1 and IRAK-4. IRAKs, including IRAK-4, recruited IRAK-1 association

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with MyD88 which led to the activation and phosphorylation of IRAK-1 [37, 38]. IRAK-1, in

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association with MyD88, further interacted with other intermediary proteins such as TRAF-6,

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and this led to the activation of IKK complex [38]. Hence, our previous results prompted us to

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study whether MIP treatment could induce TLR-4 downstream signaling in infected

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macrophages.

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MIP treatment of M. tuberculosis infected macrophages led to successful initiation of TLR-4

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down-stream signaling via TLR-4 and MyD88 association (Figure 3) which resulted in selective

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activation or deactivation of intermediate signaling molecules ultimately resulting in nuclear

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translocation of the transcription factor NF-kB p65 (Figure 3). These associations were

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eventually involved in the activation of MAP kinase P38 (Figure 4) and ultimately led to the

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production of host protective proinflammatory cytokines (Figure 5) by the infected macrophages.

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These results suggested that MIP had the ability to establish a host-protective Th1 immune

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response in tuberculosis-infected macrophages by activating the TLR4 signaling pathway. There

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is ample evidence that the iNOS pathway is crucial for the killing of bacteria [39]. Here, we

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showed that MIP treatment augmented iNOS expression and NO generation (Figure 6) in M.

352

tuberculosis infected macrophages, resulting in bacterial killing. Moreover, NF-kB plays a

353

central role in the regulation of genes involved in proinflammatory cytokines production as well

354

as inflammatory mediators such as nitric oxide generation [40, 41]. Here we observed that

355

blocking of TLR4 or MAP Kinases have tremendous impact on the MIP mediated cytokine as

356

well as NO generation. These results clearly suggested that MIP has the ability to establish host

357

protective Th1 immune response in M. tuberculosis infected macrophages via utilizing TLR-4.

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From the detailed observations regarding the mechanism of MIP mediated protection against M.

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tuberculosis induced pathogenesis, we suggest that, MIP is capable of initiating the TLR-4

360

mediated signaling in M. tuberculosis infected macrophages. Moreover, MIP leads to generation

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of protective effector response by altering the profile of impaired MAPK signaling during

362

Tuberculosis. Thus these findings provide crucial cues in understanding the immunotherapeutic

363

role of MIP in rendering protection against Tuberculosis. Thus, TLR4 may represent a novel

364

therapeutic target for the activation of the innate immune response, not only for the treatment of

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tuberculosis but also for the treatment of other chronic infectious diseases.

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Acknowledgements

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We are thankful to The Director, Bose institute for providing the research facilities. We are

368

grateful to the Council of Scientific and Industrial Research, New Delhi, India for providing

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Senior Research Fellowship to Shibali Das.

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Funding: This work was supported by Council of Scientific and Industrial Research (CSIR),

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New Delhi and Bose Institute, DST funded institute, Govt. of India

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Competing interests: None declared.

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Ethical approval: Not required.

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Transparency declarations: None to declare.

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References:

378

[1]. Das S, Banerjee S, Majumder S, Chowdhury BP, Goswami A, Halder K, Chakraborty U,

379

Pal NK, Majumdar S. Immune subversion by Mycobacterium tuberculosis through CCR5

ACCEPTED MANUSCRIPT

380

mediated

signaling:

involvement

381

10.1371/journal.pone.0092477.

of

IL-10.

PLoS

One

2014;9:e92477.

doi:

[2]. Majumder N, Bhattacharjee S, Bhattacharyya (Majumdar) S, Dey R, Guha P, Pal NK,

383

Majumdar S. Restoration of impaired free radical generation and proinflammatory

384

cytokines by MCP-1 in mycobacterial pathogenesis. Scand J Immunol 2008;67:329–39.

385

doi: 10.1111/j.1365-3083.2008.02070.x.

RI PT

382

[3]. Madan-Lala R, Sia JK, King R, Adekambi T, Monin L, Khader SA, Pulendran B,

387

Rengarajan J. Mycobacterium tuberculosis impairs dendritic cell functions through the

388

serine hydrolase Hip1. J Immunol 2014;192:4263-72. doi:10.4049/jimmunol.1303185.

389

[4]. Das S, Bhattacharjee O, Goswami A, Pal NK, Majumdar S. Arabinosylated

390

lipoarabinomannan (Ara-LAM) mediated intracellular mechanisms against tuberculosis

391

infection: involvement of protein kinase C (PKC) mediated signaling. Tuberculosis

392

(Edinb) 2015;95:208-16. doi: 10.1016/j.tube.2014.11.007.

394

M AN U

TE D

393

SC

386

[5]. Underhill DM, Ozinsky A. Toll-like receptors: key mediators of microbe detection. Curr Opin Immunol 2002;14:103-10.

[6]. Netea MG, Van der Meer JW, Kullberg BJ. Toll-like receptors as an escape mechanism

396

from the host defense. Trends Microbiol. 2004 Nov;12(11):484-8. PubMed PMID:

397

15488388.

AC C

EP

395

398

[7]. Kawahara T, Kuwano Y, Teshima-Kondo S, Kawai T, Nikawa T, Kishi K, Rokutan K.

399

Toll-like receptor 4 regulates gastric pit cell responses to Helicobacter pylori infection. J

400 401 402

Med Invest. 2001 Aug;48(3-4):190-7. PubMed PMID: 11694959.

[8]. Joshi JM. Tuberculosis chemotherapy in the 21 century: Back to the basics. Lung India 2011;28:193-200. doi: 10.4103/0970-2113.83977.

ACCEPTED MANUSCRIPT

[9]. Rai PK, Chodisetti SB, Nadeem S, Maurya SK, Gowthaman U, Zeng W, Janmeja

404

AK,Jackson DC, Agrewala JN. A novel therapeutic strategy of lipidated promiscuous

405

peptide against Mycobacterium tuberculosis by eliciting Th1 and Th17 immunity of host.

406

Sci Rep. 2016;6:23917. doi: 10.1038/srep23917.

RI PT

403

[10]. Coler RN, Bertholet S, Pine SO, Orr MT, Reese V, Windish HP, Davis C, Kahn M,

408

Baldwin SL, Reed SG. Therapeutic immunization against Mycobacterium tuberculosis is

409

an effective adjunct to antibiotic treatment. J Infect Dis. 2013;207:1242-1252. doi:

410

10.1093/infdis/jis425.

SC

407

[11]. Trunz BB, Fine P, Dye C. Effect of BCG vaccination on childhood tuberculous

412

meningitis and miliary tuberculosis worldwide: a meta-analysis and assessment of cost-

413

effectiveness. Lancet. 2006 ;367:1173-80. PubMed PMID: 16616560.

415

[12]. Grange JM, Bottasso O, Stanford CA, Stanford JL. The use of mycobacterial adjuvantbased agents for immunotherapy of cancer. Vaccine. 2008;26:4984–90.

TE D

414

M AN U

411

[13]. Yang XY, Chen QF, Li YP, Wu SM. Mycobacterium vaccae as adjuvant therapy to

417

anti-tuberculosis chemotherapy in never-treated tuberculosis patients: a meta-analysis.

418

PLoS One. 2011;6:e23826. doi: 10.1371/journal.pone.0023826.

EP

416

[14]. Stanford JL, Stanford CA, O’Brien ME, Grange JM. Successful immunotherapy with

420

Mycobacterium vaccae in the treatment of adenocarcinoma of the lung. Eur J Cancer.

421 422 423

AC C

419

2008;44:224–7.

[15]. Talwar GP. Towards development of a vaccine against leprosy. Introduction. Lepr India 1978;50:492–7.

ACCEPTED MANUSCRIPT

424

[16]. Saini V, Raghuvanshi S, Talwar GP, Ahmed N, Khurana JP, Hasnain SE, Tyagi AK,

425

Tyagi AK. Polyphasic taxonomic analysis establishes Mycobacterium indicus pranii as a

426

distinct species. PLoS One. 2009; 4:e6263. [17]. Zaheer SA, Mukherjee R, Ramkumar B, Misra RS, Sharma AK, Kar HK, Kaur H, Nair

428

S, Mukherjee A, Talwar GP. Combined multidrug and Mycobacterium w vaccine therapy

429

in patients with multibacillary leprosy. J Infect Dis. 1993; 167:401–10.

RI PT

427

[18]. Sharma P, Mukherjee R, Talwar GP, Sarathchandra KG, Walia R, Parida SK, Pandey

431

RM, Rani R, Kar H, Mukherjee A, Katoch K, Benara SK, et al. Immunoprophylactic

432

effects of the antileprosy Mw vaccine in household contacts of leprosy patients: clinical

433

field trials with a follow up of 8–10 years. Lepr Rev. 2005; 76:127–43.

436 437

M AN U

435

[19]. Kharkar R. Immune recovery in HIV with Mycobacterium w. J Indian Med Assoc. 2002;100:578–9.

[20]. Rath N, Kar HK. Efficacy of intra dermal heat-killed Mycobacterium w in psoriasis: a

TE D

434

SC

430

pilot study. Int J Dermatol. 2003;42:756–7. [21]. Mayosi BM, Ntsekhe M, Bosch J, Pandie S, Jung H, Gumedze F, Pogue J, Thabane L,

439

Smieja M, Francis V, Joldersma L, Thomas KM, Thomas B, Awotedu AA, Magula NP,

440

Naidoo DP, Damasceno A, Chitsa Banda A, Brown B, Manga P, Kirenga B, Mondo C,

441

Mntla P, Tsitsi JM, Peters F, Essop MR, Russell JB, Hakim J, Matenga J, Barasa AF,

443 444

AC C

442

EP

438

Sani MU, Olunuga T, Ogah O, Ansa V, Aje A, Danbauchi S, Ojji D, Yusuf S; IMPI Trial Investigators. Prednisolone and Mycobacterium indicus pranii in tuberculous pericarditis. N Engl J Med. 2014;371:1121-30. doi: 10.1056/NEJMoa1407380.

ACCEPTED MANUSCRIPT

445

[22]. Katoch K, Singh P, Adhikari T, Benara SK, Singh HB, Chauhan DS, Sharma VD,

446

Lavania M, Sachan AS, Katoch VM. Potential of Mw as a prophylactic vaccine against

447

pulmonary tuberculosis. Vaccine. 2008;26:1228–34. [23]. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR.

449

Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem

450

1982;126:131-38.

RI PT

448

[24]. Majumdar S, Kane LH, Rossi MW, Volpp BD, Nauseef WM, Korchak HM. Protein

452

kinase C isotypes and signal-transduction in human neutrophils: selective substrate

453

specificity of calcium-dependent beta-PKC and novel calcium-independent nPKC.

454

Biochim Biophys Acta 1993;1176:276-86.

M AN U

SC

451

[25]. Ghosh S, Bhattacharyya S, Sirkar M, Sa GS, Das T, Majumdar D, Roy S, Majumdar S.

456

Leishmania donovani suppresses activated protein 1 and NF-kappaB activation in host

457

macrophages via ceramide generation: involvement of extracellular signal-regulated

458

kinase. Infect Immun 2002;70:6828-38.

TE D

455

[26]. Bhattacharya P, Bhattacharjee S, Gupta G, Majumder S, Adhikari A, Mukherjee A,

460

Majumdar SB, Saha B, Majumdar S. Arabinosylated lipoarabinomannan-mediated

461

protection in visceral leishmaniasis through up-regulation of toll-like receptor 2

462

signaling: an immunoprophylactic approach. J Infect Dis 2010;202:145-55. doi:

AC C

463

EP

459

10.1086/653210.

464

[27]. Yaron A, Gonen H, Alkalay I, Hatzubai A, Jung S, Beyth S, Mercurio F, Manning AM,

465

Ciechanover A, Ben-Neriah Y. Inhibition of NF-kappa-B cellular function via specific

466

targeting of the I-kappa-B-ubiquitin ligase. EMBO J 1997;16:6486-94.

ACCEPTED MANUSCRIPT

[28]. Ghosh S, Bhattacharyya S, Sirkar M, Sa GS, Das T, Majumdar D, Roy S, Majumdar S.

468

Leishmania donovani suppresses activated protein 1 and NF-kappaB activation in host

469

macrophages via ceramide generation: involvement of extracellular signal-regulated

470

kinase. Infect Immun 2002;70:6828-38.

RI PT

467

[29]. Rocha-Ramírez LM, Estrada-García I, López-Marín LM, Segura-Salinas E, Méndez-

472

Aragón P, Van Soolingen D, Torres-González R, Chacón-Salinas R, Estrada-Parra S,

473

Maldonado-Bernal C, López-Macías C, Isibasi A. Mycobacterium tuberculosis lipids

474

regulate cytokines, TLR-2/4 and MHC class II expression in human macrophages.

475

Tuberculosis (Edinb) 2008;88:212-20. doi:10.1016/j.tube.2007.10.003.

477

M AN U

476

SC

471

[30]. Kobayashi K, Hernandez LD, Galán JE, Janeway CA Jr, Medzhitov R, Flavell RA. IRAK-M is a negative regulator of Toll-like receptor signaling. Cell 2002 ;110:191-202. [31]. He L, Wu X, Siegel R, Lipsky PE. TRAF6 regulates cell fate decisions by inducing

479

caspase 8-dependent apoptosis and the activation of NF-kappaB. J Biol Chem 2006

480

;281:11235-49.

TE D

478

[32]. Lindgren H, Stenmark S, Chen W, Tärnvik A, Sjöstedt A. Distinct roles of reactive

482

nitrogen and oxygen species to control infection with the facultative intracellular

483

bacterium Francisella tularensis. Infect Immun 2004;72:7172-82.

EP

481

[33]. Das S, Halder K, Goswami A, Chowdhury BP, Pal NK, Majumdar S.

485

Immunomodulation in host-protective immune response against murine tuberculosis

486 487

AC C

484

through regulation of the T regulatory cell function. J Leukoc Biol 2015;98:827-36. doi:10.1189/jlb.3A0315-114R.

488

[34]. Adhikari A, Majumder S, Banerjee S, Gupta G, Bhattacharya P, Majumdar SB, Saha B,

489

Majumdar S. Mycobacterium indicus pranii (Mw)-mediated protection against visceral

ACCEPTED MANUSCRIPT

490

leishmaniasis: involvement of TLR4 signalling. J Antimicrob Chemother 2012;67:2892-

491

902. doi: 10.1093/jac/dks315. [35]. Branger J, Leemans JC, Florquin S, Weijer S, Speelman P, Van Der Poll T. Toll-like

493

receptor 4 plays a protective role in pulmonary tuberculosis in mice.Int Immunol

494

2004;16:509-16.

RI PT

492

[36]. Abel B, Thieblemont N, Quesniaux VJ, Brown N, Mpagi J, Miyake K, Bihl F, Ryffel

496

B. Toll-like receptor 4 expression is required to control chronic Mycobacterium

497

tuberculosis infection in mice. J Immunol 2002;169:3155-62.

499

[37]. Li S, Strelow A, Fontana EJ, Wesche H. IRAK-4: a novel member of the IRAK family

M AN U

498

SC

495

with the properties of an IRAK-kinase. Proc Natl Acad Sci U S A 2002;99:5567-72. [38]. Takeda K, Akira S. TLR signaling pathways. Semin Immunol 2004;16:3-9. Review.

501

[39]. Herbst S, Schaible UE, Schneider BE. Interferon gamma activated macrophages kill

502

mycobacteria by nitric oxide induced apoptosis. PLoS One 2011;6:e19105. doi:

503

10.1371/journal.pone.0019105.

505

[40]. Lawrence T. The nuclear factor NF-kappaB pathway in inflammation. Cold Spring Harb Perspect Biol 2009;1:a001651. doi: 10.1101/cshperspect.a001651.

EP

504

TE D

500

[41]. Hatano E, Bennett BL, Manning AM, Qian T, Lemasters JJ, Brenner DA. NF-kappaB

507

stimulates inducible nitric oxide synthase to protect mouse hepatocytes from TNF-alpha-

508 509

AC C

506

and Fas-mediated apoptosis. Gastroenterology 2001;120:1251-62.

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Figure legends:

511

Figure 1: Determination of the non-cytotoxic dose of MIP. (A)Murine peritoneal

512

macrophages cultured in DMEM followed by infection with M. tuberculosis (macrophage:

513

bacteria ratio of 1:10) for 3h and treated with MIP (103-108cells/ml). After 48h of incubation, a

514

cell viability assay was performed using the MTT method (spectrophotometric reading of the

515

MTT-formazan formed was read at 650 nm and data are expressed as the percentage of viable

516

cells). The experiment was repeated 3 times, yielding similar results and data were expressed as

517

mean ± SD. **indicates P<0.05 and ***indicates P<0.001. (B)In another set of experiment,

518

murine macrophages, isolated from C57BL/6 mice, were cultured with DMEM media followed

519

by infection with M. tuberculosis bacteria (macrophage: bacteria ratio of 1:10) for 3h.

520

Macrophages were treated with MIP (106cells/ml). After 24h of incubation, differently treated

521

macrophages were lysed. The respective lysates were serially diluted and plated on Middle brook

522

7H10 with Oleic acid-ADC in triplicate. After 21 days, colonies appeared in each plate. Data are

523

represented in log10CFU /ml as mean ± SD. ***P <.001 compared to that of the untreated

524

infected macrophages.

525

Figure 2: MIP up-regulated the TLR-4 expression in M. tuberculosis-infected macrophages.

526

(A)Murine macrophages were cultured and then infected with Mycobacterium tuberculosis

527

H37Rv (Multiplicity of Infection = 1:10) for 3h. Macrophages were treated with MIP (106

528

cells/ml) for another 3hrs. Changes in messenger RNA (mRNA) expression of TLR-4 and

529

GAPDH were determined by semi quantitative RT-PCR. (B)In a separate set, the infected and

530

treated macrophages were lysed and subjected to Western blot with anti-TLR-4 and anti-GAPDH

531

antibody. (C) Infected macrophages were analyzed by flow cytometry for TLR-4 (PE)

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expression. Data represented here are from one of the three independent experiments, all of

533

which yielded similar results.

534

Figure 3: Activation of TLR-4 signaling by MIP treatment during M. tuberculosis infection.

535

Macrophages were treated and infected as described above. After 24 h of incubation, cell lysates

536

were subjected to immuno-precipitation with either anti-TLR4 or anti-MyD88 or IRAK-

537

1antibodies, and the blots were probed with (A) anti-MyD88, (B) IRAK-1 and (C) IRAK-M

538

antibodies respectively. To ensure equal input for the IP products, were re-probed with

539

respective Abs. (D) In a separate set of experiment, the infected and treated macrophages were

540

lysed and subjected to Western blot with anti-IRAK-M, TRAF-6, IKK-α, IκB-α, NF-κB p65 and

541

anti-GAPDH antibody. Data represented here are from one of the three independent experiments,

542

all of which yielded similar results. (E)In another experimental set, uninfected or infected

543

macrophages treated as above for 30 min, followed by western blot assay using differently

544

treated cytosolic and nuclear extracts with anti-NF-κB p65, anti-GAPDH or anti-Lamin ab. Data

545

represented here are from one of the three independent experiments, all of which yielded similar

546

results. (F) In another experimental sets uninfected or infected macrophages treated as above for

547

1h, followed by electrophoretic mobility shift assay using differently treated nuclear extracts

548

with labeled NF-κB probe to analyze the nuclear translocation and DNA-binding of NF-κB in

549

control and infected macrophages. Normal macrophages stimulated with LPS (1µg/ml) served as

550

a positive control. The autoradiograms are representative of 3 independent experiments that had

551

identical results.

552

Figure 4: Regulation of MAP Kinase by MIP during H37Rv infection. (A, B) Murine

553

macrophages were infected with M. tuberculosis as described in figure 3 legend for 3h and then

554

treated with MIP. After 45min of incubation, cell lysates were subjected to SDS PAGE, and the

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blots were probed with phospho and dephospho anti-P38 and ERK1/2 antibody. Data represented

556

here are from one of three independent experiments, all of which yielded similar results.

557

Figure 5: MIP Enhanced Th1 Promoting Cytokine Production from Tuberculosis-infected

558

Macrophages. (A-E) Murine macrophages were either treated with TLR-4 si-RNA, Control si-

559

RNA, and PD (ERK1/2 inhibitor) (50µM) or SB (P38 inhibitor) (20 µM) for 1h followed by M.

560

tuberculosis infection for 3h. The macrophages were then treated with MIP and incubated for

561

24h and then assayed for the levels of IL-12, IFN-γ, TNF-α, IL-10 and TGF-β in the culture

562

supernatant by ELISA. ELISA data are expressed as means ± standard deviations of values from

563

triplicate experiments that yielded similar observations. ***P <0.001 and **P <0.05 compared to

564

that of the MIP treated infected macrophages. (F) In a separate set of experiment, the infected

565

macrophages were treated with MIP as described above. After 3h of treatment, RNA was

566

isolated and semi quantitative RT-PCR analyses for IL-12, IFN-γ, TNF-α, IL-10, TGF-β and

567

GAPDH were done. Data represented here are from one of the three independent experiments, all

568

of which yielded similar results. (Inset) In a separate set of experiment, the macrophages were

569

pretreated with either TLR4 siRNA or control si-RNA and then infected and treated with MIP as

570

described above. After 3h of treatment, RNA was isolated and semi quantitative RT-PCR

571

analyses for TLR-4 and GAPDH were done. Data represented here are from one of the three

572

independent experiments, all of which yielded similar results.

573

Figure 6: MIP Enhanced NO Production by Tuberculosis infected Macrophages. Murine

574

macrophages were either treated with TLR-4 si-RNA, Control si-RNA and PD (ERK1/2

575

inhibitor) (50µM) or SB (P38 inhibitor) (20 µM) for 1h followed by M. tuberculosis infection for

576

3h. The macrophages were then treated with MIP and incubated for 48h and then assayed for the

577

levels of nitrite generation in the culture supernatant by Griess reagent as described in Methods

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(A). Data are expressed as means ± standard deviations of values from triplicate experiments that

579

yielded similar observations. ***P <0.001 and **P <0.05 compared to that of the MIP treated

580

infected macrophages. In a separate experimental set, the macrophages were pretreated with

581

inhibitors and then infected for 3h as described above. The macrophages were then treated with

582

MIP for another 3h. RNA was isolated and semi quantitative RT-PCR analyses for iNOS and

583

GAPDH were done (B). Data represented here are from one of the three independent

584

experiments, all of which yielded similar results.

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List of primers: Target genes and

Primer sequences

promoters Forward: 5′-TGTGTCCGTCGTGGATCTGA-3′

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GAPDH

Reverse: 5′-CCTGCTTCACCACCTTCTTGA-3′ IL-10

Forward: 5′-CGGGAAGACAATAACTG-3′

Reverse: 5′-CATTTCCGATAAGGCTTGG-3′

Forward: 5′-CAACATCAAGAGCAGTAGCAG-3′

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IL-12

Reverse: 5′-TACTCCCAGCTGACCTCCAC-3′

Forward: 5′ -ACACTGCATCTTGGCTTTGCAGCT-3′

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IFN-γ

Reverse: 5′-TGAGCTCATTGAATGCTTGGCGCT-3′ iNOS

Forward: 5′-GAGATTGGAGTTCGAGACTTCTGTG-3′ Reverse: 5′-TGGCTAGTGCTTCAGACTTC-3′

TGF-β

Forward: 5′ -AAGGGAAAGCATGAATGGAGCGCT-3′ Reverse: 5′-TCAAGCTCTTTGCCTTGCCCTGAA-3′ Forward: 5′-CAAGAACATAGATCTGAGCTTCAACCC-3′

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TLR4

Reverse: 5′-GCTGTCCAATAGGGAAGCTTTCTAGAG-3′ TNF-α

Forward: 5′-ACAAAGGTGCCGCTAACCACATGT-3′

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Reverse: 5′-ATGCTGCTGTTTCAGTCGAAGGCA-3′

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