Allicin enhances antimicrobial activity of macrophages during Mycobacterium tuberculosis infection

Author’s Accepted Manuscript Allicin enhances antimicrobial activity of macrophages during Mycobacterium tuberculosis infection Ved Prakash Dwivedi, Debapriya Bhattacharya, Mona Singh, Ashima Bhaskar, Santosh Kumar, Parveen Sobia, Luc Van Kaer, Gobardhan Das www.elsevier.com/locate/jep

PII: DOI: Reference:

S0378-8741(18)31763-X https://doi.org/10.1016/j.jep.2018.12.008 JEP11634

To appear in: Journal of Ethnopharmacology Received date: 14 May 2018 Revised date: 24 October 2018 Accepted date: 7 December 2018 Cite this article as: Ved Prakash Dwivedi, Debapriya Bhattacharya, Mona Singh, Ashima Bhaskar, Santosh Kumar, Parveen Sobia, Luc Van Kaer and Gobardhan Das, Allicin enhances antimicrobial activity of macrophages during Mycobacterium tuberculosis infection, Journal of Ethnopharmacology, https://doi.org/10.1016/j.jep.2018.12.008 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 galley proof before it is published in its final citable 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.

Allicin enhances antimicrobial activity of macrophages during Mycobacterium tuberculosis infection

Ved Prakash Dwivedia1*, Debapriya Bhattacharyab,c1, Mona Singhb1, Ashima Bhaskard, Santosh Kumara, Parveen Sobiae, Luc Van Kaerf, Gobardhan Dasb* a

Immunology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India. b Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India. c Center of Biotechnology, Siksha 'O' Anusandhan University Kalinganagar, Ghatikia Bhubaneswar, Orissa, India d Signal Transduction Laboratory-1, National Institute of Immunology, New Delhi, India e College of Health Sciences, Laboratory of Medicine and Medical Sciences, University of KwaZulu-Natal, Durban, South Africa. f Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA. [email protected] [email protected] *

Corresponding author. Dr. Ved Prakash Dwivedi International Centre for Genetic Engineering and Biotechnology (ICGEB) Aruna Asaf Ali Marg New Delhi 110067 * Corresponding author. Prof. Gobardhan Das Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India. Abstract Ethnopharmacological relevance: The emergence of drug-resistant Mycobacterium tuberculosis (M.tb) strains has severely hampered global efforts towards tuberculosis (TB) eradication. The internationally accepted therapy “Directly Observed Treatment Short-course (DOTS)” is lengthy, and incorporates risks for the generation of drug-resistant M.tb variants. Multiple and extremely drug-resistant (MDR and XDR) variants of TB are now widespread throughout the globe, and totally drugresistant (TDR) strains have appeared. Therefore, new classes of antibiotics are urgently needed to combat these deadly organisms. Historically, garlic is known to kill mycobacterial strains, and its active compound, allicin, kills various microorganisms. Here we have shown 1

These authors contributed equally to the manuscript.

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that allicin not only reduced the bacterial burden in the lungs of mice infected with Mycobacterium tuberculosis (M.tb), but also induces strong anti-tubercular immunity. Materials and methods: In the present study, the anti-mycobacterial and immunomodulatory activity of garlic extract and its pure constituent allicin were demonstrated based on several in vitro and in vivo experiments in murine model of tuberculosis. Furthermore, the validation of study was done by immunoblots showing the modulation of MAPK and SAPK/JNK signaling by allicin in macrophages. Results: Here, we report that allicin/garlic extract exhibits strong anti-mycobacterial responses in vitro and in vivo against drug-sensitive, MDR and XDR strains of TB. In addition to direct killing, allicin also induced pro-inflammatory cytokines in macrophages. Moreover, allicin/garlic extract treatment in murine models of infection resulted in induction of strong protective Th1 response, leading to drastic reduction in mycobacterial burden. These results indicated that allicin/garlic extract has both antibacterial and immunomodulatory activity. Furthermore, garlic extract reversed the immune dampening effects of frontline anti-TB drugs. Conclusion: Allicin/garlic extract alone or as an adjunct to classical antibiotics holds great promise for treatment of drug-sensitive as well as drug-resistant TB. These results warrant further study and validation of allicin for treatment of TB.

Keywords: Mycobacterium tuberculosis, T cells, Allicin, adjunct therapy, garlic extract

1. Introduction Despite great scientific progress since its discovery, tuberculosis (TB) remains a leading cause of mortality in humans (WHO, 2015a; WHO, 2015b; Dye, 2014; WHO, 2016). One of the major hurdles in the clearance of Mycobacterium tuberculosis (M.tb) is the emergence of multiple drug-resistant (MDR) and extensively drug-resistant (XDR) strains. Lengthy antiTB treatment often leads to non-compliance and hence generation of resistant strains and latent TB infections (Fair and Tor, 2014; Shenoi and Friedland, 2009; Pinto and Menzies, 2011; Cohen et al., 2015; Tousif et al., 2014; WHO, 2013). Importantly, after completion of

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Directly Observed Treatment Short-course (DOTS), patients remain vulnerable to reactivation and re-infection of the disease, suggesting therapy-related immune impairment (Tousif et al., 2014). Therefore, an alternate therapeutic strategy that avoids these limitations is urgently needed to combat this deadly disease. Medicinal plant derived antimicrobial molecules may provide new drug candidates for treating drug-resistant microorganisms. Because of their vast chemical diversity and their use as a traditional medicine, natural products are important sources of new antimicrobials and immunomodulators. Garlic is one of the common food spices, which is used as an herbal medicine for prevention and treatment of a variety of diseases, including several infectious diseases (Rivlin, 2001; Rios and Recio, 2005; Ross et al., 2001). Garlic is a strong antibacterial agent and acts as an inhibitor of both Gram-positive and Gram-negative bacteria (Ankri and Mirelman, 1999; Bakri and Douglas, 2005; Reuter et al., 1996). The principal antimicrobial constituent of garlic has been identified as the oxygenated sulphur compound, thio-2-propene-1-sulfinic acid S-allyl ester, which is usually referred to as allicin (Curtis et al., 2004; Miron et al., 2000; Miron et al., 2002). In 1944, Rao et. al. reported that garlic extract in vitro is bacteriostatic at low concentrations, but at higher concentrations it is bactericidal (Rao et al., 1946). Other studies have also reported anti-mycobacterial activity of garlic extracts (Petrovska and Cekovska, 2010; Dini et. al., 2011; Sharifi-Rad et al., 2017). In this study, we have tried to extend the previous knowledge by investigating the effect of both garlic extract and allicin on different strains of M.tb in vitro, ex vivo and in in vivo murine model of tuberculosis. Firstly, we determined the minimal inhibitory concentration (MIC) of allicin on drug-sensitive and -resistant strains of M.tb and found a potent anti-mycobacterial activity against various strains, including MDR and XDR strains. Furthermore, allicin treatment drastically reduced the bacterial burden in macrophages in a dose-dependent manner, without affecting cell viability. Remarkably, we found that allicin significantly prevented internalization of M.tb by the macrophages, suggesting blockade of the adhesion molecule ICAM and these data were further supported by previous study by EW Son et al, where they showed that allicin inhibits ICAM-1 expression in human vascular endothelial cells via downregulation of the JNK signaling pathway (Son et al., 2006). We further tested the efficacy of allicin and garlic extract in a murine model of TB, which revealed a significant reduction of bacterial burden in the lungs, concomitant with strong anti-tubercular immune responses. These effects of allicin were dependent on SAPK/JNK signaling as evidenced by previous reports (Ling et al., 2014). Additionally, allicin and garlic extract significantly reduced antibiotic-induced toxicity during treatment. Therefore, allicin 3

or garlic extract warrants further exploration as a treatment of TB, either alone or in combination with currently available anti-TB drugs.

2. Materials and Methods 2.1. Ethics Statement Animal experiments were performed according to the guidelines approved by the Institutional Animal Ethics Committee of the International Centre for Genetic Engineering and Biotechnology (ICGEB) (Approval ID: ICGEB/IAEC/IMM-13/2007) (New Delhi, India) and the Department of Biotechnology guidelines (Government of India). All mice used for experiments were ethically sacrificed by asphyxiation in carbon dioxide according to institutional and Department of Biotechnology, Government of India, regulations. 4

2.2. Mice C57BL/6 (6–8 wks; 25-35 gram body weight) mice were provided by our institute (ICGEB, New Delhi, India). All animals were maintained in the animal facility of the ICGEB. 2.3. Bacteria M.tb strain H37Rv (ATCC number 25618) was initially a kind gift from the Colorado State University repository. All MDR and XDR strains were kind gifts from Dr KVS Rao, ICGEB, New Delhi. Bacilli were grown in 7H9 (Middlebrooks, DifcoTM, USA) medium supplemented with 10% ADC (albumin, dextrose, and catalase; DifcoTM) and with 0.05% Tween 80 and 0.2% glycerol, and cultures were grown to mid-log phase. Aliquots of the cultures in 20% glycerol were preserved at -80oC and these cryo-preserved stocks were used for infections. 2.4. Chemical and Reagents: Allicin (CAS 539-86-6; purity >98%)) was procured commercially from Santa-Cruz Biotechnology. All FACS antibodies used in this study were purchased from Biolegend, USA. PMA (Phorbol myristic acid) and ionomycin used for intracellular cytokine staining were purchased from Sigma-Aldrich, India. Fixation and permeabilization kit were obtained from BD Biosciences, USA. Alamar blue dye was purchased from Thermo Fisher Scientific. Reagents (7H11, 7H9, OADC) required for determination of bacterial burden or CFU were purchased from Difco, USA. Thioglycollate medium, for isolation of peritoneal macrophages, was purchased from Difco, USA. Other chemical and reagents used in this study have been mentioned in the respective sub-headings. 2.5. Preparation of Garlic extract: We purchased Allicin (CAS 539-86-6; purity >98%) from Santa-Cruz Biotechnology, Inc. Allicin was dissolved in autoclaved water to make 1mg/ml solution. Regarding garlic extract, garlic cloves (Allium sativum- Bulb: soft neck) were purchased from Vasant Kunj region of New Delhi and we obtained characterization certificate from National Botanical Research Institute (Specimen Number: NBRI-SOP-202), Lucknow, India (Figure S1 A & B). 100 gram of garlic cloves were taken, peeled off and grinded using 100 ml of sterile water. Garlic paste was filtered using muslin cloth; crude extract was collected in a beaker. These extracts were filtered and further centrifuged at 8000 rpm. Resultant supernatant collected and dried by using speed vac and dissolved in water to achieve final concentration of 1g/ml and stored at -200C for further use. 2.6. Thin Layer Chromatography (TLC)

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Samples were prepared and then applied to the TLC plate (TLC silica gel 60 F 254, aluminum sheets) usually 1-5 μl volumes to the origins of a TLC plate 2 cm above its bottom with the help of capillary tubes. TLC chamber was filled with 8 ml of hexane: isopropanol (3:1). Lid was closed and allowed run up to three fourth of the total TLC length. The plate was then removed and dried for 5-10 mins. TLC plate was then stained using iodine vapor (Biradar et al., 2014) (Figure S2). 2.7. High Performance Liquid Chromatography (HPLC) Allicin was dissolved in sterile water to make stock solution of 1mg/ml. Garlic bulbs were purchased from local market of New Delhi (Certificate attached as supplementary figure). 100 gm garlic bulbs were peeled and grinded thoroughly using 100ml of sterile water to obtain fine garlic juice, which was filtered by muslin cloth. Aqueous extract was further centrifuged at 8000 rpm. Resultant supernatant was collected (1gm/ml) and stored at -20ºC for further use. For HPLC it was further diluted to 100mg/ml. The presence of allicin in garlic extract was analyzed by HPLC (Waters 1525 HPLC) equipped with flexinject dual injector, photodiode array detector, and C18 reverse column (WATERS SPHERISORB 5um ODS2 (250 × 4.6 mm). The column, maintained at temperature 20ºC, was operated in isocratic mode (50:50 Acetonitrile: water) at a flow rate of 1 ml/min. The UV detector was set at 210400 nm. Since the machine has manual injector and as per the instrument protocol only specific loop volume (100µl) of material could be injected not less or more than that. Hence we loaded only 100 µl of sample in instrument. 100ul of standard allicin was injected manually and allowed to run for 30 mins followed by cleaning of the column with the same solvent system for 60 mins (Figure S3). After cleaning, 100ul of aqueous garlic extract was injected manually with same runtime of 30 mins (Wallock-Richards et al., 2014) (Figure S4). 2.8. Alamar Blue assay Bacilli were grown until mid log phase (OD600 0.6 to 0.8) and added to a 96-well plate containing two-fold serial dilutions of allicin at final OD600 of 0.01. After incubation at 37oC for 5 days 1X alamar blue (Thermo Fisher Scientific) was added in each well and a color change from blue to pink was observed after 16 h of incubation. Wells containing only media and no bacilli served as solvent control group and wells having only bacilli without any drug were treated as negative control group. 2.9. Drug Treatment We divided mice in four groups. (i) First group mice were taken as infected control, (ii) second group were infected and treated with isoniazid, (iii) third group were infected and 6

treated with allicin or garlic extract while (iv) fourth groups were infected and treated with allicin or garlic extract along with standard anti-tuberculosis drug isoniazid. 2mg/kg of Allicin (Santa-Cruz Biotechnology, USA) and 5 mg/kg of garlic extract in 100 µl of PBS was administered intraperitoneally every day during the entire treatment phase. INH (5 mg/kg) was given ad libitum in the drinking water during therapy (Singh et al., 2016; Dwivedi et al., 2017). 2.10. M.tb infection of mice and estimation of Colony Forming Units (CFU) Mice were infected with M.tb H37Rv via the aerosol route using a Madison aerosol chamber (University of Wisconsin, Madison, WI) with its nebulizer pre-calibrated to deposit a total of ∼110 bacilli to the lungs of each mouse as previously described (Bhattacharya et al., 2014a; Singh et al., 2016, Bhattacharya et al., 2014b; Dwivedi, et al., 2012; Tousif et al., 2014; Chatterjee et al., 2011). Briefly, mycobacterial stocks recovered from a −80°C freezer were quickly thawed and subjected to light ultra sonication to obtain a single cell suspension. The concentration of bacterial cell suspension was determined by taking OD600. OD600 of 0.6 corresponds to 10X107 bacilli. Fifteen ml of the bacterial cell suspension (10×106 cells per ml) was placed in the nebulizer of the Madison aerosol chamber pre-calibrated to deliver via aerosol route the desired number of CFUs to the lungs of animals placed inside the chamber. Three randomly selected mice were sacrificed at various time points and organs were harvested, homogenized in 0.2 μm filtered PBS containing 0.05% Tween 80 and plated onto 7H11 Middlebrooks (Difco, USA) plates containing 10% oleic acid, albumin, dextrose and catalase (OADC) (Difco, USA). Undiluted, ten-fold diluted and one hundred-fold diluted lung and spleen cell homogenates were plated in duplicate on the above 7H11 plates and incubated at 37°C for 21–28 days. Colonies were counted and CFU were estimated (Bhattacharya et al., 2014a; Singh et al., 2016, Bhattacharya et al., 2014b; Dwivedi, et al., 2012; Tousif et al., 2014; Chatterjee et al., 2011). 2.11. Isolation of mouse peritoneal macrophages Six- to 8-week-old female C57BL/6 mice were given a 2 ml i.p. injection of thioglycollate medium (4%). Five days post injection macrophages were obtained by peritoneal lavage. Macrophages were washed once with cold PBS and suspended in cold RPMI-1640. Cell viability was determined by Trypan blue exclusion method. The numbers of macrophages in this suspension were determined using a haemocytometer chamber and seeded at a density of 2X105 per cm2 in RPMI-1640 with 10% heat-inactivated fetal calf serum. Macrophages were

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infected with M.tb strains at a MOI of 1:10 as described earlier (Rahman et al., 2015; Dwivedi et al., 2017). 2.12. Determination of Cytotoxicity Monolayers of macrophages in 12-well tissue culture plates (2.0 x 105 cells per cm2) were grown and incubated at 37oC in 5% CO2 for 6 h, different concentrations of allicin were added to culture wells in triplicate at a final volume of 100 μl, and incubated for 48 h. Thereafter, the medium was replaced with fresh RPMI containing 1 mg/ml of propidium iodide. Cells were incubated at 37oC for 15 min and then washed with PBS and acquired and analyzed with a FACS Canto II flow cytometer (BD Biosciences, USA). 2.13. FACS Analysis For surface staining cells were harvested and washed with PBS and stained with fluorescently conjugated monoclonal antibodies anti-CD4 (clone GK1.5)-PerCpCy5.5, anti-CD8 (clone SK1)-APC, anti-CD45RB (clone C363-16A)-PE, anti-CD11c (clone N418)-PE, and antiCD11b (clone M1/70)-APC (Biolegend, USA). For intracellular cytokine staining, cells were treated with 50 ng/ml PMA (Sigma-Aldrich) and 500 ng/ml ionomycin (Sigma-Aldrich) in the presence of 1 μg/ml brefeldin-A (Biolegend, USA) added during the last 6 h of culture. Cells were washed twice with PBS and re-suspended in a permeabilization buffer (Cytofix/Cytoperm kit; BD), and stained with the following fluorescently conjugated monoclonal antibodies: anti-IFN-γ (clone XMG1.2)-APC, anti-IL-4 (clone GK1.5)-PE, antiIL-17 (clone TC11)-PE, anti-IL-1 (clone JAMA 147)-PE, anti-IL-12 (clone C15.6)-APC, anti-IL-TNF-a (clone MP6-XT22)-PE, anti-IL-6 (clone MP5-20F3)-PE, and anti-IL-10 (clone JES5-16E3)-PE (Biolegend, USA). Fluorescence intensity was measured by flow cytometry (FACS Canto II; BD) and data were analyzed with Flow Jo (Tree star, USA). 2.14. Western blotting Peritoneal macrophages derived from C57BL/6 mice were infected with H37Rv and treated with allicin (30 g/ml) for 1 h. Whole cell lysate was prepared using lysis buffer (20 mMTris-HCl; pH 8.0, 500 mM NaCl, 0.25% Triton X 100, 1 mM EDTA, 1 mM PMSF, 1mM dithiothreitol, 1X Protease inhibitor cocktail [88266; Thermo fisher scientific]). Samples were electrophoresed on a 10% SDS-polyacrylamide gel and electroblotted onto nitrocellulose membranes. Blots were blocked for 1 h in 5% BSA in PBST (PBS with 0.1% Tween 20), and p38 MAPK, phospho p38 MAPK, SAPK/JNK and phospho SAPK/JNK proteins were detected with respective antibodies at a dilution of 1:1000 as recommended by the manufacturer (Cell Signaling Technologies, USA). Goat anti-rabbit immunoglobulin G-

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conjugated horseradish peroxidase (diluted 1:2000) was used as a secondary antibody (Cell Signaling Technologies, USA). Immunoblotting for GAPDH was carried out to confirm equal loading. 2.15. Confocal Microscopy For confocal microscopic studies, the infection was carried out with GFP tagged Mycobacterium H37Rv. Peritoneal macrophages were seeded on coverslips and prior to infection cells were treated with allicin or ICAM inhibitor for overnight and infection were done with an MOI of 1:10 as described above. The coverslips were then washed with PBS and macrophages were treated with 100µg/ml gentamycin to kill the extracellular bacteria and fixed with 2.5 % paraformaldehyde for 20 minutes followed by a wash with PBS. The cells were then permeabilized by treatment with 0.1% NP-40 and the coverslips were mounted on glass slides using DAPI containing mounting agent for and nucleus staining. Cells were then observed using 488 nm (green) and 405nm (violet) lasers on Olympus Fluoview FV1000 and photographs were captured. Percentage of cells infected in each experimental groups were calculated by counting the number of infected and uninfected cells.We checked the bacterial load by counting the number of bacteria invaded in per infected cells. 2.16. Histology Lungs of infected animals were harvested and fixed in 10% buffered formalin, and H&E staining was performed on 5-μm-thick paraffin-embedded tissues. For each mouse in each group, a minimum of 5 fields was screened to detect the granulomas. A total of approximately 6*5 (30) fields were studied to calculate the granuloma score. The images given are representative of all the images. The image size (scan field size) depends on the objective magnification lens used. 2.17. Statistical analysis All data were derived from at least three independent experiments. Significant difference was determined by t test analysis (Graph pad prism). A value of p<0.05 was accepted as an indication of statistical significance. 3. Results 3.1. Allicin kills intracellular M.tb in macrophages. To assess the anti-mycobacterial activity of allicin we performed an alamar blue assay. We treated drug-sensitive and -resistant strains of M.tb with allicin for 5 days and determined the bacterial survival via alamar blue. Interestingly, we found that allicin exhibits potent antimycobacterial activity against drug-sensitive as well as several MDR and XDR variants of 9

TB (Figure 1 A&B). To examine the effect of allicin-mediated killing of intracellular M.tb in macrophages, we performed in vitro colony forming unit (CFU) assays of bacteria isolated from infected macrophages. We infected peritoneal macrophages with M.tb strain H37Rv, treated them with increasing concentrations of allicin (i.e. 10, 20, 30 & 40 g/ml) and determined CFUs at various time points. We found that allicin treatment significantly reduces the bacterial burden (Figure 1C) in macrophages. To examine toxicity of allicin on host cells, we performed cell viability assays and found no effect on cell viability (Figure 1D). Collectively, these data suggested that even at lower concentration allicin has potent antimycobacterial activity. 3.2. Allicin inhibits M.tb uptake by macrophages. Next, we elucidated the effect of pre-treatment of allicin on M.tb infection in macrophages. We isolated thioglycollate elicited peritoneal macrophages from C57BL/6 mice and treated them with allicin overnight. We also used an ICAM-1 inhibitor as a positive control, as we have previously shown that ICAM-1 inhibitor blocks M.tb invasion in macrophages (Bhalla et al., 2015). Macrophages were infected with a GFP-expressing recombinant M.tb strain and examined under confocal microscopy at 4 h post infection. Interestingly, we observed that allicin treatment inhibited the cells to be infected the percentage of cells infected as well as the internalization of M.tb in the macrophages and this effect was even more prominent in allicin treatment as compared to the ICAM-1 inhibitor (Figure 2A,B & C). 3.3. Garlic extract perturbs mycobacterial survival in mice. We assessed the in vivo efficacy of allicin and garlic extract against M.tb in a murine infection model. We infected C57BL/6 mice with a low dose (~110 CFU) of M.tb H37Rv through the aerosol route. At various time points after infection we harvested lungs for determination of bacterial loads. We found that garlic extract treatment drastically reduced the bacterial burden and no viable M.tb bacilli were observed 60 days post treatment. Additionally, a combination of isoniazid and garlic extract exhibited additive effects and cleared M.tb within 45 days post treatment, whereas isoniazid treatment alone was effective in more than 60 days (Figure 3A). These data were further strengthened by histological analyses of the lungs, which revealed reduced numbers of granulomas after garlic extract treatment than all experimental groups (Figure 3B). Furthermore, we also did the CFU assay with allicin and observed comparable bacterial reduction as garlic extract (Figure 3 C). Next, we investigated the effect of garlic extract on infection with MDR and XDR strains, using the same conditions used for drug-sensitive strains. Treatment with garlic

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extract dramatically reduced the bacterial burden (Figure 3D), prevented skin lesions and extended survival beyond 90 days until the end of the experiment, whereas sham-treated animals exhibited severe skin lesions and died within 15 days post infection with MDR (Figure 3E) and XDR (Figure 3F) M.tb strains. These observations suggest that garlic extract is a potent anti-mycobacterial agent, either directly inhibiting bacterial growth or also via immunomodulation. 3.4. Profiling of immune responses in garlic extract-treated M.tb infected mice. To determine if garlic extract exhibits immunomodulatory properties, we infected animals, treated them with garlic extract, and determined immune parameters at various time points. Immuno-phenotypic experiments suggested that garlic extract treatment has modest effect on the prevalence of CD4+ and CD8+ T cells but comparatively higher than that of isoniazid treatment (Figure 4A&B). We also observed that treatment with garlic extract reverses the adverse effect of isoniazid on T cell (Figure 4A&B). We did not observe any changes in the numbers of macrophages (CD11b+ cells) and dendritic cells (CD11c+ cells) after garlic extract or isoniazid treatment (Figure 4C&D). Next, we performed intracellular cytokine profiling for innate cytokines from the spleens of infected and garlic extract-treated mice, and observed significantly enhanced IL-12 production compared to control and isoniazid treatment (Figure 4E), whereas levels of IL-10 and TNF-were comparable in all experimental groups (Figure 4E). To investigate the polarization of T helper (Th) cell subsets, we performed intracellular staining of signature cytokines produced by Th1, Th2 and Th17 cells. We found that garlic extract treatment enhanced the frequency of IFN-γproducing T cells than control and isoniazid treatment, whereas no significant differences were observed for IL-4- and IL-17-producing T cells in all groups (Figure 4F). Collectively, our data suggest that garlic extract promotes the generation of protective Th1 immune responses against M.tb infection. 3.5. Allicin inhibits activation of MAPK and SAPK/JNK pathways in macrophages. From the above experiments, we concluded that allicin/garlic extract is not only a potent antimycobacterial agent but also exhibits immunomodulatory properties. Therefore, we investigated intracellular levels of pro- and anti-inflammatory cytokines in macrophages infected with M.tb and treated with allicin. Interestingly, we found that allicin treatment significantly induced levels of IL-1and reduced levels of TNF-. However, the levels of other cytokines such as IL-6, IL-10, IL-12 and TGF- were comparable between the experimental groups (Figure 5A). To explore the underlying mechanism, we investigated

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effects of allicin on MAPK and SAPK/JNK signaling, which are critically important for the induction of pro- and anti-inflammatory cytokines, respectively. Peritoneal macrophages derived from C57BL/6 mice were infected with M.tb and treated with allicin (30 μg/ml) for one hour, and activation status of MAPK and SAPK/JNK pathways was investigated via western blotting (Figure 5B). Relative to infected and untreated macrophages, an increase in the phosphorylation of SAPK/JNK was observed in allicin-treated macrophages. However, activation of p38-MAPK was significantly inhibited. Notably, non-phosphorylated p38 MAPK, SAPK/JNK and control house-keeping GAPDH levels were unaltered following allicin treatment (Figure 5B). Previously, Son et al have shown that allicin treatment for more than 20 hours downregulates gamma IR-induced ICAM-1 expression via inhibition of both AP-1 activation and the JNK pathway in human umbilical vein endothelial cells (HUVECs) (Son et al., 2006), however, in our study we treated murine derived primary macrophages with allicin for an hour and checked the signaling. This data is further strengthened by Xu et al 2014 where they showed a significant increase in JNK phosphorylation upon treatment with allicin as early as 15 min (Ling et al., 2014).

4. Discussion Current therapy of TB is lengthy and consists of multiple antibiotics, which not only poses risks for the generation of drug-resistance but also impairs host protective immune responses, resulting in enhanced vulnerability to reinfection and disease reactivation (Tousif et al., 2014). Drug-resistant TB is spreading at an alarming rate, and nearly all countries throughout the globe are under the threat from MDR and XDR TB. Most importantly, TDR strains have already appeared. Therefore, new classes of therapeutics are urgently needed to combat this deadly organism. Recently, others and we reported that a combination of antibiotics and 12

immunomodulators provides superior efficacy as a therapeutic modality for TB (Tousif et al., et al., 2014; Bhattacharya et al., 2014a; Singh et al., 2016). Therefore, compounds that possess both antibiotic and host-protective immunomodulatory effects might hold great promise for TB therapy. Plant-derived compounds are a vast resource, yet they have not been well explored. Previous studies have shown that garlic extract has anti-mycobacterial activities (Rivlin, 2001; Rios and Recio, 2005; Ross et al., 2001; Ankri and Mirelman, 1999; Bakri and Douglas, 2005, Rao et al., 1946; Hasan et al., 2007; Su et al., 2008), and that its active compound allicin also exhibits various immunomodulatory activities (Ankri and Mirelman, 1999; Miron et al., 2000; Miron et al., 2002). We extended these studies and found that allicin as well as garlic extract can kill drug-sensitive as well as drug-resistant (both MDR and XDR) TB. We further noticed that allicin kills M.tb residing in macrophages without altering survival of the host. Therefore, allicin/garlic extract is a promising option for treating drug-resistant TB (Fig. 1). TB is a disease of poor neighborhoods, and in rural settings patients often do not go to health care facilities. Therefore, we tested if a crude extract of garlic, the active compound of which is allicin, can be used for treating MDR and XDR TB. Our findings suggest that allicin or garlic extract drastically reduces the bacterial burden in murine model of susceptible as well as drug resistant tuberculosis (Fig. 3). Moreover, combination therapy with INH and garlic extract/allicin further reduced the bacillary load. A dynamic balance between distinct Th cell subsets dictates the outcome of infection, development of latent infection, or acute disease progression (Rahman et al., 2015; Rook, 2007; Hawn et al., 2013). It has been well documented that IFN--producing T helper cells (Th1) induce robust protective immune responses against M.tb, whereas IL-4-producing T helper cells (Th2) promote disease progression by inhibiting host-protective Th1 cells (Rahman et al., 2015; Rook, 2007; Hawn et al., 2013). Therefore, simultaneous redirection of the host immune response is required to selectively induce the IFN--producing Th1 cell subset for improved protection against M.tb, and to concomitantly inhibit Th2 cells (Rahman et al., 2015; Rook, 2007). M.tb successfully inhibits IL-12 production in susceptible hosts and thus inhibits the development of Th1 responses (Rahman et al., 2015; Hawn et al., 2013). In addition, it has clearly been shown that M.tb not only prevents Th1 responses, but also facilitates Th2 responses that counterregulate host-protective Th1 responses (Rahman et al., 2015). Considering the superior treatment efficacy of combination therapy with antibiotics and an immunomodulator, we sought to investigate immune responses altered by garlic extract in vivo. Our results indicated

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that garlic extract elevated protective pro-inflammatory cytokines and has a modest effect on anti-inflammatory cytokines, which is a desirable outcome for optimal efficacy of TB treatment. Furthermore, garlic extract minimized the immune dampening effects of isoniazid (Fig 4). To further dwell into the mechanism behind the obtained results, intracellular levels of proand anti-inflammatory cytokines in infected macrophages were analyzed after allicin treatment. Interestingly, we found that allicin induced pro-inflammatory responses, but not at excessive levels. Notably, allicin selectively inhibited TNF-L-10 whereas IL-1and IL12 levels were moderately elevated (Fig 4). Although inflammatory responses are required for host protection against M.tb infection, excessive inflammation is detrimental and may be fatal (Toblin et al., 2012; Juffermans et al., 2000). Consistent with this notion, addition of steroids along with conventional antibiotics yields improved treatment efficacy in certain cases of TB (Toblin et al., 2012; Juffermans et al., 2000). Consistent with these results, we found that treatment with allicin selectively induces Th1 responses, which are known to exert host-protective immune response. It is well established that IL-1 plays an important role in host protection against M.tb infection (Yamada et al., 2000; Cooper et al., 2011; Saha et al., 1994). Based on this notion, together with our finding that allicin differentially regulates TNF- and IL-1 in infected macrophages, we hypothesized that the number of bacteria in macrophages dictates the magnitude of inflammatory responses and differential cytokine regulation. Interestingly, we found that allicin dramatically inhibited M.tb invasion in macrophages (Fig 2). This finding prompted us to measure ICAM-1 expression, as we have previously shown that inhibition of ICAM-1 dramatically reduces M.tb invasion in macrophages (Bhalla et al., 2015). Nevertheless, previous reports established that simultaneous up-regulation of ICAM-1 and down-regulation of co-stimulatory molecules is one of the main immune evasion mechanisms adopted by M.tb (Narayan and Sonika, 2010). Therefore, allicin/garlic extract exerts several important functions, including the inhibition of mycobacterial invasion; direct killing of M.tb, and modulation of host protective immune responses. Furthermore, allicin enhances the activation of the SAPK/JNK pathway in infected macrophages, which is critically important for the induction of pro-inflammatory as well as anti-inflammatory cytokines and these results were in agreement with the published literature where it has been shown that allicin induces IL1 production through SAPK/JNK pathway (Fig 5) (Ling et al., 2014). Moreover, allicin treatment inhibited the phosphorylation of p38

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MAPK, which may presumably be the cause of TNF- down-regulation that creates profound inflammation (Fig 5) (Dwivedi et al., 2017; Schorey and Cooper, 2003). The activation of the SAPK/JNK pathway in macrophages provides protective host immunity during M.tb infection and produces various effector molecules that show anti-mycobacterial activity (Dwivedi et al., 2017; Schorey and Cooper, 2003). Taken together, our results clearly indicate that garlic extract not only kills drug susceptible strains of M.tb but also restricts the growth of various drug-resistant strains in vivo. These results warrant further study and validation of garlic extract for treatment of TB.

Conclusion Our data indicates that garlic extract successfully kills drug resistant variants of tuberculosis and also possess immunomodulatory effects. Furthermore, it reverses the immune dampening effects of frontline anti-TB drugs. Therefore, addition of garlic extract along with conventional antibiotics treatment will be tremendously helpful in treating susceptible as well as drug resistant TB.

Acknowledgements We acknowledge the support of the DBT-supported Tuberculosis Aerosol Challenge Facility at the International Centre for Genetic Engineering and Biotechnology (ICGEB, New Delhi, India) and their staff in accomplishing this work. VPD and AB and are the recipients of a DST-INSPIRE Faculty Fellowship. VPD is also the recipient of an Early Career Research Award (ECRA), Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Government of India. DB is the recipient of Senior Research Associateship of the Council of Scientific and Industrial Research (CSIR), Government of India. We acknowledge financial support from the Department of Science and Technology (DST) and the Department of Biotechnology (DBT), Govt. of India.

Author contributions Ved Prakash Dwivedi, Debapriya Bhattacharya, Mona Singh, Ashima Bhaskar, Santosh Kumar and Parveen Sobia performed the experiments. Ved Prakash Dwivedi, Debapriya Bhattacharya, Ashima Bhaskar, Luc Van Kaer and Gobardhan Das wrote the manuscript.

Conflict of interest

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Fig 1: Allicin mediates killing of M.tb in macrophages without affecting cell viability. (A&B) Anti-mycobacterial activity of allicin as assessed by Alamar Blue assay for different mycobacterial strains (H37Rv, SDR [BND], MDRs [1934, 2261, 2287, 2549, BND], and XDR [MCY-85). (C) Bacterial survival in macrophages infected with H37Rv either untreated or treated with different concentrations of allicin. (D) Propidium iodide assay for cell viability following treatment of macrophages with graded concentrations of allicin. The red line represents untreated macrophages. Numbers depict the percentage of cells stained with propidium iodide. The results shown are representative of four independent experiments. Error bars indicate mean ± S.D (n=3). Statistical significance was calculated by comparing control vs Allicin treatment. p value * ≤ 0.05. Fig 2: Allicin treatment makes macrophages more resistant to Mycobacterium tuberculosis infection. (A) Confocal microscopy of macrophages infected with H37Rv and treated with ICAM inhibitor (positive control) or allicin. (B) Percentage of macrophages infected with H37Rv and treated with ICAM inhibitor or allicin. Dots indicating the number of fields captured. The results shown are representative of three independent experiments. Error bars indicate means ± S.D (n=3). Statistical significance was calculated by comparing control vs Allicin treatment. p value * ≤0.05. Fig 3: In vivo effect of garlic extract on mice infected with Mycobacterium tuberculosis. (A) CFU from the lung homogenates of mice that were infected with H37Rv and treated with garlic extract and/or isoniazid. (B) Histology pictures to show effects of garlic extract on infection. Arrows indicate granulomatic lesions in the lung sections of M.tb infected mice. (C) CFU from the lung homogenates of mice that were infected with H37Rv and treated with allicin and/or isoniazid. (D) CFU from the lung homogenates of mice that were infected with MDR and XDR strains of M.tb and treated with garlic extract. (E) Percent survival of the mice infected with MDR and treated with garlic extract. (F) Percent survival of mice infected with XDR and treated with garlic extract. The results shown are means ± S.D (n=6 mice within each group). Statistical significance was calculated by comparing control vs garlic extract treatment. p value * ≤ 0.05. GE: Garlic extract, INH: isoniazid. Fig 4: Profiling of immune cells and their responses from mice infected with Mycobacterium tuberculosis and treated with garlic extract. (A&B) FACS data to show the percentage of CD4+ and CD8+ T cells and CD45Rcells from the spleens of mice infected with H37Rv and treated with garlic extract and/or isoniazid. (C&D) FACS data to show the percentage of dendritic cells (CD11c+) and macrophages (CD11b+) from the spleens

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of mice infected with H37Rv and treated with garlic extract and/or isoniazid. (E) FACS analysis to show the percentage of cells expressing intracellular cytokines IL-10, IL-12 and TNF- from the spleens of H37Rv infected mice treated with GE and/or INH. (F) FACS analysis to show the percentage of cells expressing intracellular cytokines IFN-γ, IL-4 and IL-17 of CD4+ T cells isolated from the spleens of H37Rv infected mice, treated with garlic extract and/or isoniazid. The results shown are representative of three independent experiments. Error bars indicate means ± S.D (n=3). Statistical significance was calculated by comparing H37Rv infected vs H37Rv + garlic extract treatment. p value * ≤ 0.05. GE: Garlic extract, INH: isoniazid. Fig 5: Allicin inhibits p38 MAPK and activates SAPK/JNK signaling in macrophages. (A) Representative bar diagram to show the FACS data of intracellular cytokines from macrophages infected with H37Rv and treated with allicin. (B) Peritoneal macrophages were infected with H37Rv and treated with 20 μg/ml allicin for 1 hour. Phosphorylation of p38 MAPK and SAPK/JNK was assessed in whole cell lysate of macrophages uninfected or infected with H37Rv with and without allicin treatment by western blot. The results shown are representative of three independent experiments. Statistical significance was calculated by comparing infected vs allicin treatment. p value * < 0.05. Supplementary Fig 1: Characterization of Garlic cloves used for the study. (A) Garlic cloves collected from New Delhi, India (B) Authorization certificate from National Botanical Research Institute (NBRI), Lucknow, India for the Garlic cloves used for the study. Supplementary Fig 2: Thin Layer Chromatography (TLC) analysis of Allicin present in Garlic. Supplementary Fig 3: High Performance Liquid Chromatography (HPLC) of the Allicin. Supplementary Fig 4: High Performance Liquid Chromatography (HPLC) of the garlic extract.

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