Luteolin-mediated Kv1.3 K+ channel inhibition augments BCG vaccine efficacy against tuberculosis by promoting central memory T cell responses in mice

Dhiraj Kumar Singh; Ved Prakash Dwivedi; Shashi Prakash Singh; Anjna Kumari; Saurabh Kumar Sharma; Anand Ranganathan; Luc Van Kaer; Gobardhan Das

doi:10.1371/journal.ppat.1008887

Abstract

Despite the availability of multiple antibiotics, tuberculosis (TB) remains a major health problem worldwide, with one third of the population latently infected and ~2 million deaths annually. The only available vaccine for TB, Bacillus Calmette Guérin (BCG), is ineffective against adult pulmonary TB. Therefore, alternate strategies that enhance vaccine efficacy are urgently needed. Vaccine efficacy and long-term immune memory are critically dependent on central memory T (T_CM) cells, whereas effector memory T (T_EM) cells are important for clearing acute infections. Recently, it has been shown that inhibition of the Kv1.3 K⁺ ion channel, which is predominantly expressed on T_EM but not T_CM cells, profoundly enhances T_CM cell differentiation. We exploited this phenomenon to improve T_CM:T_EM cell ratios and protective immunity against Mycobacterium tuberculosis infection in response to BCG vaccination of mice. We demonstrate that luteolin, a plant-derived Kv1.3 K⁺ channel inhibitor, profoundly promotes T_CM cells by selectively inhibiting T_EM cells, and significantly enhances BCG vaccine efficacy. Thus, addition of luteolin to BCG vaccination may provide a sustainable means to improve vaccine efficacy by boosting host immunity via modulation of memory T cell differentiation.

Author summary

Bacillus Calmette Guérin (BCG) is not effective against adult pulmonary tuberculosis (TB). Inhibition of the Kv1.3 K⁺ ion channel by the antibiotic clofazimine has been shown to enhance BCG-induced immunity. However, clofazimine has limited efficacy and is associated with substantial side effects in treated patients. Therefore, we explored alternatives to clofazimine. Luteolin is a plant-based flavonoid that inhibits Kv1.3. We show that administration of luteolin during BCG vaccination enhances antigen-specific immunity by promoting the T central memory (T_CM) cell pool, which is critically important for long term host protection. Consequently, luteolin-mediated immune modulation enhances vaccine efficacy. As luteolin is a biologically safe food supplement, it could be easily applied during vaccination.

Figures

Citation: Singh DK, Dwivedi VP, Singh SP, Kumari A, Sharma SK, Ranganathan A, et al. (2020) Luteolin-mediated Kv1.3 K⁺ channel inhibition augments BCG vaccine efficacy against tuberculosis by promoting central memory T cell responses in mice. PLoS Pathog 16(9): e1008887. https://doi.org/10.1371/journal.ppat.1008887

Editor: David M. Lewinsohn, Portland VA Medical Center, Oregon Health and Science University, UNITED STATES

Received: February 18, 2020; Accepted: August 14, 2020; Published: September 21, 2020

Copyright: © 2020 Singh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: GD was funded by Department of Biotechnology (http://dbtindia.gov.in) grant number BT/PR6312/MED/29/605/2012. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Despite the availability of multiple effective antibiotics, tuberculosis (TB) has emerged as the greatest killer among all infectious diseases, with one third of the global population infected, and 10.4 million new cases and ~1.74 million deaths reported in 2016 [1]. Bacillus Calmette Guérin (BCG), the only usable TB vaccine currently available, is over a century old and fails to protect against adult pulmonary TB [2]. BCG elicits sufficient host protective T helper (Th) 1 (producers of IFN-γ) responses and exhibits efficacy against disseminated and meningeal TB in neonates. However, these cells become gradually exhausted and the host becomes again susceptible to Mycobacterium tuberculosis (M.tb) infection [3–5]. It is now increasingly clear that T effector memory (T_EM) cells with a Th1 cytokine production profile provide protection against acute TB infection, whereas T central memory (T_CM) cells provide vaccine efficacy by generating T_EM cells. Therefore, during the course of an ongoing infection, a wider pool of T_CM cells is desired to provide the host with a continuous supply of T_EM cells. The generation of T_EM cells is proportionate to bacterial burden and these cells rapidly produce copious amounts of IFN-γ, which promotes cellular immune responses and eliminates bacteria. T_EM cells, however, are terminally differentiated effector cells with low or no proliferative capacity [6–8]. Therefore, maintenance of long-term protective memory responses is thought to rely on T_CM cells with high proliferative capacity [9]. T_CM responses play a critical role in driving cell-mediated host immune responses and thereby the efficacy of TB vaccines [10,11]. Therefore, restoring host T_CM responses may enhance TB vaccine efficacy. Increasing the pool of T_CM (CD44^hiCD62L^hi or CD44^hiCCR7^hi) cells by concomitant regulation of T_EM (CD44^hiCD62L^lo or CD44^hiCCR7^lo) cells may be an effective strategy to develop long-lasting and robust recall responses.

In order to specifically modulate T_CM cells without altering T_EM cell responses, an intricate understanding of the differential physiology between these two T cell subsets is required. Kv1.3, a potassium ion channel, is predominantly expressed on T_EM cells and plays an important role in the maintenance and effector functions of these cells [12,13]. On the other hand, T_CM cells express higher levels of the KCa3.1 ion channel, which drives and maintains the activation of this memory T cell subset. In keeping with these findings, several independent studies have shown that knock-down of Kv1.3 results in differential modulation of T_CM vs. T_EM cells, to favor T_CM generation [13,14]. In fact, genetic deficiency of Kv1.3 has been shown to induce reversal of T_EM to T_CM cell ratios [13,14]. A recent study showed that inhibition of Kv1.3 K⁺ ion channels by the antibiotic clofazimine enhances the pool of T_CM cells, and these cells have the potential to continuously replace effector T cells at the site of infection, thereby improving host immunity [15]. Clofazimine, a well-known anti-leprotic drug, is reserved as a class 5 drug for TB and is currently only being used in long regimens to treat extremely drug-resistant (XDR) TB. The half-life of clofazimine in lungs is estimated at over 4 weeks, and this drug also accumulates in various organs [16]. Therefore, employing clofazimine during immunization of infants and adults is suboptimal.

In searching for a biologically safe alternative Kv1.3 inhibitor we explored 3,4,5,7-tetrahydroxyflavone, also known as luteolin, a plant-based flavonoid that inhibits Kv1.3 [17]. Luteolin has recently been employed as a food supplement and is considered safe for human use. It is a flavonoid found in many fruits, vegetables, and medicinal plants such as Reseda luteola L., Achillea millefolium L. and many others. Luteolin-rich herbal extracts have been used for a long time as traditional herbal remedies [18,19]. We observed that luteolin had mild bactericidal activity in vitro (S1 Fig), as reported [20]. Moreover, we also found that luteolin treatment significantly activated macrophages, as evidenced by increased expression of co-stimulatory molecules and improved bactericidal activity (S2A & S2B Fig). Taken together, these findings suggested that luteolin might have potent immunomodulatory effects, which along with selective enrichment of the T_CM pool may be highly beneficial in combating TB. Therefore, we performed a proof of concept study to explore the effects of luteolin on the memory T cell responses after BCG immunization, using the intraperitoneal route of administration to maintain homogeneity in dosage and to achieve higher bioavailability than the oral route [19,20].

Our results demonstrate that mice receiving luteolin during BCG vaccination exhibit superior host protection against TB, which was associated with potent Th1 and Th17 responses, and enrichment of CD44^hiCD62L^hi and CD44^hiCCR7^hi T_CM cells in both CD4⁺ and CD8⁺ compartments. Taken together, our findings reveal that specific immunomodulation of memory T cells during vaccination can enhance the efficacy of BCG vaccination and improve long-term host immunity.

Results

Luteolin biases T cells towards a T_CM phenotype in vitro

Although the ability of luteolin to inhibit the Kv1.3 K⁺ ion channel was reported over a decade ago [17], its capacity to modulate memory T cell responses has not yet been explored. To specifically evaluate this phenomenon, we stimulated naïve T cells in the presence of luteolin and found that it was indeed capable of significantly promoting T_CM responses in vitro both with and without prior T cell activation by anti-CD3 and anti-CD28 mAbs (Fig 1A & 1B). In addition, luteolin also enhanced CD4⁺ T cell activation as evidenced by increased prevalence of CD44⁺, CD25⁺ and CD69⁺ cells (Fig 1C) and proliferation (Fig 1D) measured by BrdU incorporation assay. These data suggest that luteolin selectively induces the T_CM cell population, which is considered the main driver of recall responses. As per the recent literature, the first exposure to a pathogen generates a primary effector T cell repertoire which then clears the primary infection and contracts to give rise to a T_CM cell pool. On subsequent challenge a secondary response is generated by the rapid proliferation of T_CM cells which give rise to secondary effector T cells that drive clearance of the pathogen. Although effector T cells are the main drivers of pathogen clearance, their rapid clonal expansion during a secondary immune response is chiefly driven by the T_CM pool [21, 22].

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Fig 1. Luteolin biases T cells towards a T_CM phenotype after in vitro activation with plate bound α-CD3 & soluble α-CD28 antibodies.

(A&B) Memory responses of CD4⁺ T cells in vitro. (C) T lymphocytes were activated in vitro with plate-bound α-CD3 & soluble α-CD28 antibodies in the presence or absence of luteolin and then stained with anti-CD4, -CD62L, -CD25, -CD44 and -CD69 mAbs. (D) T cell proliferation was monitored by BrdU incorporation post activation in vitro by staining with anti-CD4 and -BrdU antibodies. Data represented as the mean±STDEV values from data pooled from three experiments with five mice per experiment. Differences were considered significant at P<0.05 using one way ANOVA. *p<0.05, **p<0.005, ***p<0.0005.

https://doi.org/10.1371/journal.ppat.1008887.g001

Luteolin induces sustained T cell immunity in lung and spleen of BCG-immunized mice challenged with M.tb

To provide insight into the T cell response induced by luteolin in BCG-vaccinated mice, we analysed T lymphocytes from various experimental groups of mice: M.tb (sham-vaccinated, vehicle-treated followed by aerosol infection with M.tb strain H37Rv), BCG^IMM+M.tb (BCG-vaccinated, vehicle-treated followed by aerosol infection) and BCG^IMM+Luteolin+M.tb (BCG-vaccinated, luteolin-treated followed by aerosol infection) (Fig 2). Flow cytometric analysis (S3 Fig) revealed that CD4⁺ and CD8⁺ T cells in lungs and spleen of mice treated with luteolin were significantly increased as compared with mice immunized with BCG only (Fig 3A & 3B). Next, we determined the activation status of these cells by staining with anti-CD25, -CD44 and -CD69 antibodies. Fig 3C & 3D demonstrates that a large number of cells were indeed activated.

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Fig 2. Study design.

https://doi.org/10.1371/journal.ppat.1008887.g002

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Fig 3. Luteolin promotes T cell responses in BCG-immunized mice.

(A) Representative FACS plots and (B) combined data showing the proportion of CD4⁺ & CD8⁺ T lymphocytes in spleen and lung of mice of the indicated study groups at 60 days post-infection. (C) Representative FACS plots and (D) combined data showing activation profile of CD4⁺ T cells in spleen and lung of infected mice of the indicated study groups at 60 days post-infection. Data represented as the mean±STDEV values from data pooled from three experiments with five mice per experiment. Differences were considered significant at P<0.05 using one way ANOVA. *p<0.05, **p<0.005, ***p<0.0005.

https://doi.org/10.1371/journal.ppat.1008887.g003

Luteolin treatment during BCG vaccination promotes T_CM cells in mice challenged with M. tuberculosis

Long term immunity is mostly dependent on the total pool of T_CM cells. However, BCG primarily induces T_EM cells in the lung, which might contribute to its limited vaccine efficacy [23]. We determined the generation of T_CM cells in immunized and luteolin-treated mice (Fig 4). Phenotypic analyses revealed that CD44^hiCD62L^hi and CD44^hiCCR7^hi T_CM cells in both CD4⁺ (Fig 4A–4C) and CD8⁺ (Fig 4D–4F) compartments were significantly higher in the luteolin-treated mice than in any of the other experimental groups post infection. However, T cells in all experimental groups exhibited comparable memory phenotype with a stronger antigen-specific Th1 response in the luteolin treatment group before infection (S4 Fig). The nature of the effector memory response in these experimental groups correlated well with their bacterial burden. As we and others have previously reported that efficient pathogen clearance is attained by Th1 and Th17 cell responses [24,25], we next determined the production of representative cytokines by these cells using intracellular staining. We found that luteolin-treated mice generated significantly increased levels of IFN-γ- and IL-17-producing Th cells, but we found no significant differences in IL-4-producing cells (Fig 5A). IL-22 production was also found to be higher in the luteolin-treated group of mice (Fig 5A). These observations suggested that luteolin enhances BCG vaccine efficacy by increasing the T_CM:T_EM ratio of memory T cells with either a Th1 or Th17 phenotype as well as by induction of IL-22 (S5 Fig). Moreover, we also observed stronger polyfunctional Th cell responses (Fig 5B) in the lung of luteolin-treated mice (S6A & S6B Fig).

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Fig 4. Luteolin induces increased T_CM responses in BCG-immunized mice.

(A&D) Representative FACS plots, (B, C, E and F) combined data showing memory responses in the CD4⁺ T cell compartment (A-C) and CD8⁺ T cell compartment (D-F) in spleen and lung of infected mice of the indicated study groups at 60 days post-infection. Data represented as the mean±STDEV values from data pooled from three experiments with five mice per experiment. Differences were considered significant at P<0.05 using one way ANOVA. *p<0.05, **p<0.005, ***p<0.0005.

https://doi.org/10.1371/journal.ppat.1008887.g004

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Fig 5. M.tb antigen-specific cytokine response.

(A) Splenocytes from the indicated groups of mice were stimulated with M.tb H37Rv complete soluble antigen ex vivo, and the percentages of splenocytes producing cytokines were measured with flow cytometry. (B) Polyfunctional cytokine profiling from Th cells in the lung of the mice. Data represented as the mean±STDEV values from data pooled from three experiments (A) and two experiments (B) with five mice per experiment. Differences were considered significant at P<0.05 using one way ANOVA. *p<0.05, **p<0.005, ***p<0.0005.

https://doi.org/10.1371/journal.ppat.1008887.g005

Luteolin treatment enhances BCG vaccine-induced host protection against TB

Prior studies have shown that luteolin demonstrates its immunomodulatory activities by inhibiting Kv1.3 [17,26]. It was recently shown that functional blockade of the Kv1.3 channel can enhance antimycobacterial immunity by promoting T_CM responses [15,27]. Therefore, these observations prompted us to test the effects of luteolin on BCG-induced vaccine efficacy. For this purpose, we treated BCG-immunized mice with luteolin at 5 mg/kg body weight starting 1 day after immunization, for a period of 20 days. These mice were then rested for 1 month and challenged with M.tb strain H37Rv. M.tb pathogenic burden was estimated by Colony Forming Unit (CFU) determination in lung, spleen and liver at various time points. Luteolin-treated mice displayed increased host protection, as revealed by reduced bacterial burden in various organs (Fig 6A), and reduced macroscopic, granulomatous lesions in lung and spleen (Fig 6B). This was further confirmed by histological analyses, which revealed reduced numbers of granulomas (Fig 6C).

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Fig 6. Luteolin treatment of BCG-immunized mice confers improved protection against TB.

Mice immunized with BCG were treated with 5 mg/kg of luteolin or vehicle for 20 days and rested for an additional 30 days. These immunized mice and a control group of non-immunized mice were then infected with M.tb. Three randomly selected mice from various groups were euthanized at the indicated time points and lung, liver and spleen were harvested and homogenised. (A) CFUs from the lung, spleen and liver homogenates of M.tb infected mice. (B) Gross pathology of lungs and spleen at different time points after M.tb infection. Arrows indicate granulomatous lesions. (C) Lungs were dissected out and preserved in 10% neutral buffered Formalin (10% NBF). These preserved lungs were then processed for paraffin embedding, sectioning and staining with Hematoxylin and Eosin (H&E). Arrows indicate granulomatous lesions. Data represented as the mean±STDEV values from data pooled from three experiments with five mice per experiment. Differences were considered significant at P<0.05 using one way ANOVA. *p<0.05, **p<0.005, ***p<0.0005.

https://doi.org/10.1371/journal.ppat.1008887.g006

T cells from luteolin-treated, BCG-immunized mice confer improved protection against TB upon adoptive transfer

In order to accurately assess the role of luteolin-induced T_CM enrichment in boosting BCG-induced immunity and to rule out the other reported immunostimulatory functions of luteolin [19,28], we performed T cell adoptive transfer experiments. Congenic wild-type Thy1.2⁺ mice were immunized with BCG followed by luteolin treatment (5 mg/kg) for 20 days and then rested for 10 days. CD4⁺ T cells (10x10⁶) were adoptively transferred into γ-irradiated (sub-lethal dose of 800 rads/mice) Thy1.1⁺ congenic mice followed by infection with a low-dose aerosol challenge of M.tb strain H37Rv. Twenty days after infection, spleen and lungs were isolated from the surviving mice in each group for CFU estimation (Fig 7A & 7B) and antigen-specific intracellular cytokine responses (Fig 7A & 7C). Results showed that surviving recipient mice receiving T cells from luteolin-treated mice exhibited reduced CFUs (Fig 7B) and increased IFN-γ- and IL-17-producing, donor-derived (Thy1.2⁺) CD4⁺ T cells (Fig 7C). Since the cause of mortality in the adoptively transferred mice could not be objectively attributed to radiation toxicity or the infection, only mice surviving at the end of the study were used for CFU estimation and assessing T cell responses.

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Fig 7. Adoptively transferred T cells from luteolin-treated and BCG-immunized mice confer improved protection against TB.

(A) Experimental Layout: CD4⁺ T cells were isolated from congenic wild-type Thy1.2 mice immunized with BCG followed by luteolin treatment for 20 days and rested for 10 days. CD4⁺ T cells (10x10⁶) were adoptively transferred into γ-irradiated (sub-lethal dose of 800 rads/mice) Thy1.1 congenic mice followed by infection with M.tb. Twenty days after infection, spleen and lungs were isolated. (B) CFUs were estimated from lung homogenates of the different groups. (C) Splenocytes were challenged with M.tb complete soluble antigen ex vivo. T cells were then stained for the intracellular cytokines IL-4 vs. IFN-γ and IL-17. Data represented as the mean±STDEV values. Differences were considered significant at P<0.05 using one way ANOVA. Out of twelve mice within each group, only 5 mice in the BCG immunized control group and 6 mice in the luteolin control group survived on day 20. *p<0.05, **p<0.005, ***p<0.0005.

https://doi.org/10.1371/journal.ppat.1008887.g007

Discussion

Successful vaccine efficacy against M.tb infection is predominantly dependent on the induction of memory cells with a central memory phenotype (CD62L^hiCD44^hi) [2,10,11,24,29–31], whereas induction of cells with an effector memory phenotype (CD62L^loCD44^hi) is associated with active disease [32–34]. In this context, it has been shown that recombinant BCG vaccines with increased capacity to induce T_CM cells exhibit increased vaccine efficacy [11,35]. Several recombinant BCG vaccines with this property are in the development and testing pipeline [36–39].

Naïve T (CCR7⁺CD45RA⁺) and T_CM (CCR7⁺CD45RA^–) cells require antigen priming in lymph nodes before they migrate to inflammatory sites, whereas terminally differentiated T_EM cells (CCR7^–CD45RA^–) rapidly enter inflamed tissues, produce copious amounts of cytokines, and exhibit immediate effector function [40,41]. Terminally differentiated human CCR7^–CD45RA⁻T_EM cells have been reported to strongly upregulate Kv1.3 K⁺ ion channel expression. Resting (unstimulated) T cells of all subsets express about 200–400 Kv1.3 channels per cell along with very few calcium-activated K⁺ (IKCa1) channels. After activation, naïve T and T_CM cells upregulate IKCa1 (500–600 IKCa1 channels expressed per cell), whereas T_EM cells upregulate Kv1.3 (1,500–1,800 Kv1.3 channels expressed per cell) [42]. In fact, the Kv1.3^highIKCa1^low phenotype has been suggested as a specific functional marker for activated T_EM cell subsets belonging to both the CD4⁺ and CD8⁺ compartments [42]. Since T_EM cells expressed significantly higher Kv1.3 levels and lower IKCa1 levels than naïve T and T_CM cells post-activation, selective Kv1.3 channel blockade suppresses proliferation of T_EM cells without affecting naïve T or T_CM cells [42]. Selective inhibition of Kv1.3 channels therefore appears to be a potential approach for generating T_CM-driven long-term protective immunity against TB. While robust and rapid recall responses critically rely on the maintenance of memory T cells, this may also have detrimental effects such as depletion of the T_CM pool due to recurrent clonal expansion of T_EM cells during persistent infection by the same pathogen or due to recurrent exposure to related non-pathogenic species or strains [15,27]. The critical role of Kv1.3 in effector T cell function has already been explored as a target to suppress effector T cell functions in severe inflammatory diseases [26,43–45]. Kv1.3 loss of function in mice caused a delay in T_CM to T_EM progression by inhibiting cell cycle progression at the G2/M stage. This was mediated by up-regulation of SMAD3 phosphorylation, accelerating its translocation to the nucleus, where SMAD3 binds with the p21^cip1 promoter and subsequently suppresses expression of the cell cycle genes cyclin-dependent kinase (Cdk)1 and cyclin B1 [13]. Nevertheless, Kv1.3-deficient mice were otherwise healthy and developed a normal immune system, with similar proportions of T lymphocytes in the spleen and thymus and similar proliferative responses of splenocytes to challenge with Concanavalin A or anti-CD3 antibodies when compared to wild type control mice [46]. A recent study reported that blockade of Kv1.3 by addition of clofazimine during immunization of mice with BCG enhances the available pool of T_CM cells, which provides superior protection against pulmonary TB [15]. Clofazimine, despite being a very successful antileprosy drug, has some limitations for use in a vaccine setting, more specifically in infants. First of all, it is a class 5 reserved drug for TB and is currently only being used in long treatment regimens of extremely drug-resistant (XDR) TB. Further, the half-life of clofazimine in lungs is estimated at over 4 weeks, and it accumulates in various organs [16]. Employing clofazimine during immunization of infants and adults appears suboptimal. Therefore, in the current study we interrogated the efficacy of a biologically safe alternative Kv1.3 inhibitor, luteolin, which is gaining popularity as a food supplement and is available in various pediatric and adult formulations [47]. Our study shows that luteolin treatment during BCG vaccination effectively alters the T_CM:T_EM cell ratios, and demonstrates improved vaccine efficacy against M.tb infection compared with BCG alone. Luteolin generated an expanded T_CM pool that dictated efficient recall responses, which are considered imperative for vaccine efficacy and longevity of protective immune responses. T_CM cells are believed to be a perpetual source of T_EM cells, which are responsible for protection from infections by induction of a rapid effector response. Our adoptive transfer experiments clearly demonstrated the memory T cell-mediated protection and that these memory cells exhibit Th1 and Th17 phenotypes and are host-protective (Fig 7).

In summary, we have shown that luteolin alters the T_CM:T_EM cell ratio when used during BCG vaccination and thus exhibits improved vaccine efficacy by inducing enhanced T_CM responses and augmenting Th1 and Th17 responses. Whether luteolin can improve disease protection in individuals already immunized with BCG will be a topic for future investigation.

Material and methods

Ethics statement

Animal experiments performed were in accordance with the guidelines approved by the 53rd Meeting of the Institutional Animals Ethics Committee held on 11^th February, 2014 at International Centre for Genetic Engineering and Biotechnology (ICGEB) (Approval ID: ICGEB/AH/2014/01/RGP-13), New Delhi, India as well as guidelines issued by the Department of Biotechnology (DBT), Government of India. All mice used for experiments were ethically sacrificed by asphyxiation in carbon dioxide according to institutional and DBT regulations.

Mice

C57BL/6 mice that were Thy1.1⁺ or Thy1.2⁺ were initially purchased from The Jackson Laboratories (Bar Harbor, ME) and thereafter maintained at our specific pathogen-free animal facility at ICGEB. Mice used for infections were housed under barrier conditions in the Tuberculosis Aerosol Challenge Facility (TACF) of ICGEB and treated humanely as per the specified Animal Care protocols.

In vitro T cell stimulation

Splenocytes isolated from naïve C57BL/6 mice were enriched for T cells by nylon wool method [48] followed by 6 hours of rest to bring the cellular activity to basal levels. They were then stimulated with plate-bound α-CD3 (1 μg/ml) and soluble α-CD28 (2 μg/ml) antibody in the presence or absence (control) of luteolin for 48 hours. These cells were then collected and surface-stained for FACS analysis.

M.tb low-dose aerosol infection of mice

M.tb strain H37Rv (ATCC 27294; American Type Culture Collection, Rockville, MD) was a kind gift from the Colorado State University repository (Fort Collins, CO). Mouse infections were performed in accordance to the low-dose aerosol infection model using a Madison Aerosol Chamber (University of Wisconsin, Madison, WI) with the nebulizer pre-calibrated at ~200 CFU/mice. M.tb strain H37Rv was grown to mid-log phase (OD₆₀₀ ∼0.6) in Middlebrook 7H9 media (Difco, USA) with 0.1% Tween 80 (Sigma, USA), 0.2% glycerol and 10% Middlebrook albumin, dextrose and catalase (ADC) enrichment medium (Difco, USA). Bacteria were stored at −80°C in 20% glycerol stocks for further experiments. For aerosol infection, cultures were washed twice with PBS and made into a single cell suspension by passing through a 26-gauge syringe ten times followed by two passes through a 30-gauge syringe. Fifteen ml of the M.tb strain H37Rv single cell suspension (20X10⁶ cells per ml) was placed in the Nebulizer reservoir of the Madison Aerosol Chamber calibrated to deliver the desired CFUs of bacteria into the lungs of mice kept in the chamber in 15 minute cycles. At 24 hours after aerosol challenge 3 mice were euthanized for quantification of pathogen delivery to lungs by measuring CFUs in lung homogenates. Mice were found to be infected with ∼220 CFU of M.tb strain H37Rv in their lungs. The mice were maintained under BSL-3 containment thereafter.

Drug administration

Five mg/kg of luteolin (Sigma, USA) in 100 μl of PBS containing 5% DMSO v/v was administered intraperitoneally every day during the entire treatment phase, whereas controls were given vehicle only.

Quantification of pathogen burden by Colony Forming Units (CFU)

Randomly selected mice were sacrificed at different time points, organs were harvested, homogenized in 0.2 μm filtered PBS containing 0.05% Tween 80 and plated onto 7H11 Middlebrook plates containing 10% OADC supplement. One hundred-fold, one thousand-fold, and ten thousand-fold diluted lung cell homogenate and ten-fold and one hundred-fold diluted spleen and liver cell homogenates were plated in doublet on 7H11 plates and incubated at 37°C for ~21 days. CFUs were counted and pathogen burden in lung, liver and spleen was estimated.

Flow cytometry: Surface and intracellular staining

Spleens and lungs were isolated from mice and macerated by frosted slides in ice cold RPMI 1640 (Gibco, Invitrogen, UK) containing 10% FBS to prepare a single cell suspension. Red blood cells (RBCs) were lysed with RBC cell lysis buffer, incubated at room temperature for 3–5 minutes and washed with 10% RPMI 1640. The cells were counted and 1×10⁶ cells were used for surface staining. For intracellular staining 1×10⁶ cells were cultured per well in 12-well plates (Nunc, USA) and activated with M.tb H37Rv Complete Soluble Antigen (CSA) overnight, and 10 μg/ml Brefeldin A (eBiosciences, USA) was added during the last 6 hours of culture. Cells were washed twice with PBS and stained with antibodies directed against surface markers. After staining, cells were washed again with PBS and fixed with 100 μl fixation buffer (eBiosciences, USA) for 30 minutes, then re-suspended in 200 μl permeabilization buffer (eBiosciences, USA) and stained with fluorescently labelled anti-cytokine antibodies. Fluorescence intensity of fluorochrome-labelled cells was measured by flow cytometry (FACS Canto II, BD Biosciences, USA). FACS Diva was used for acquiring the cells and final data analysis was performed by Flow Jo (Tree star, USA).

Detection of cytokines

Cytokines in the serum of immunized, infected and infected and treated mice were assayed by Luminex microbead-based multiplexed assay using commercially available kits according to the manufacturer’s protocol (BioPlex, Bio-Rad, USA).

Antigen-specific degranulation assay

Splenocytes isolated from randomly selected mice in different groups were isolated as described above and 2X10⁶ splenocytes per well were cultured in RPMI 1640 (Gibco, Invitrogen, UK) containing 10% FBS in a 12-well plate (Nunc, USA) at 37^°C in 5% CO₂ for 4 hours to bring the cellular activity to basal levels. Splenocytes were then challenged with 20 μg/ml of H37Rv CSA and cultured for an additional 2 hours, after which 5 μl Monensin (Golgi-Stop, BD Biosciences) and 5 μl of anti-CD107A-FITC antibody (BD Biosciences) were added per well and cultured for an additional 4 hours at 37°C in 5% CO₂. These cells were then collected and surface-stained for FACS analysis.

BCG immunization experiments

Mice were either sham vaccinated or immunized subcutaneously with 1×10⁶ CFUs of BCG in 100 μl of sterile saline as described [31]. After one day of rest these mice were treated with either vehicle (saline) or luteolin at 5 mg/kg of body weight every day for a total of 20 days. Mice were subsequently rested for 30 days. Mice were then challenged via the aerosol route with M.tb strain H37Rv as above and organs were harvested for determination of bacterial burden and immune-profiling at different time points.

T Cell adoptive transfer experiments

For adoptive transfer experiments, Thy1.1⁺ mice were gamma-irradiated (4.6 rads/sec for 175 seconds) and rested for a day. CD4⁺ T cells were isolated using anti-CD4 beads over a magnetic column (MiltenyiBiotec, USA) from the lymph nodes of Thy1.2⁺ mice that were either naïve (Control) or had been previously immunized with BCG and treated with vehicle (BCG^IMM AT) or luteolin (BCG^IMM + Luteolin AT) for 20 days and then rested for an additional 10 days. These cells were then adoptively transferred into the irradiated recipient mice (10x10⁶ cells per mouse). After 4 days recipient mice were challenged with M.tb strain H37Rv through the aerosol route. Each group was comprised of 12 mice, of which 5 mice in the control group and BCG^IMM AT group and 6 mice in the BCG^IMM+Luteolin AT group survived on day 20 post-infection. The surviving mice were euthanized for CFU estimation or immunological studies.

Antibodies and reagents

We used the following antibodies: anti-CD3 (clone: 145-2C11)-PerCP-Cy5 or -APC, CD4 (clone: GK1.5, RM4-5)-FITC, -PerCP-Cy5 or -APC, CD8 (clone: 53–6.7)-FITC, -APC-H7, -PerCP-Cy5 or -APC, CD44 (clone: IM7)-APC, CD62L (clone: MEL-14)-PE, CD25 (clone: 3C7)-PE, -APC, FOXP3 (clone: MF23, R16-715)-APC, IFN-γ (clone: XMG1.2)-APC, IL-4 (clone: 11B11)-PE, IL-6 (clone: MPS-20F3)-PE, IL-10 (clone: JES5-16E3)-APC, IL-12 (clone: C15.6)-PE, IL-17 (clone: O79-289)-PE, IL-2 (clone: JES6-5H4)-PerCP or -FITC, IL-22 (clone: Poly5164)-PE, TNF-α (clone: MP6-XT22)-PE, TGF-β (clone: TW7-16B4)-APC (from Biolegend, USA), and CD69 (clone: H1.2F3)-PE (from eBiosciences, USA).

Statistical analysis

Data are represented as mean±STDEV derived from at least three independent experiments. Statistical analyses were conducted using IBM SPSS20 software. Significant differences between the group means were determined by One way ANOVA followed by Post-hoc analysis with Tukey’s correction for Multiple comparison except for data depicted in S2 & S4 Figs where unpaired t test was peformed (SPSS software). A value of p<0.05 was considered statistically significant.

Supporting information

S1 Fig. In vitro bactericidal activity of luteolin (100 μM/mL).

https://doi.org/10.1371/journal.ppat.1008887.s001

(TIF)

S2 Fig.

(A) Profiling of macrophage activation after luteolin treatment (25 μM/mL). (B) In vitro CFU assay in macrophages infected with M.tb and treated with luteolin.

https://doi.org/10.1371/journal.ppat.1008887.s002

(TIF)

S3 Fig. Gating strategies employed for immune profiling of T cells.

https://doi.org/10.1371/journal.ppat.1008887.s003

(TIF)

S4 Fig. Bar diagram to show the percentage of different subsets of memory T cells in Lungs after immunization and luteolin treatment prior to infection.

https://doi.org/10.1371/journal.ppat.1008887.s004

(TIF)

S5 Fig. Cytokine profiling from splenocytes of the respective experimental groups.

https://doi.org/10.1371/journal.ppat.1008887.s005

(TIF)

S6 Fig. Gating strategies employed for the profiling of antigen-specific cytokine responses in T cells from splenocytes.

https://doi.org/10.1371/journal.ppat.1008887.s006

(TIF)

Acknowledgments

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. We thank Prof. David Sherman (Seattle Institute of Biomedical Research, Seattle, WA) for providing Mycobacterium tuberculosis strain H37Rv (ATCC 27294). DKS is the recipient of a Senior Research Fellowship from the Council of Scientific and Industrial Research (CSIR), Government of India. VPD is the recipient of a DST-INSPIRE Faculty Fellowship, Department of Science and Technology, Government of India and of Early Career Research Award from Science and Engineering Research Board (SERB), Department of Science and Technology, Government of India.

References

1. Organization WH. Global tuberculosis report 2017. Geneva: World Health Organization; 2017. 157 p.
2. Colditz GA, Brewer TF, Berkey CS, Wilson ME, Burdick E, Fineberg HV, et al. Efficacy of BCG vaccine in the prevention of tuberculosis. Meta-analysis of the published literature. Jama. 1994;271(9):698–702. pmid:8309034
- View Article
- PubMed/NCBI
- Google Scholar
3. Masopust D, Ha SJ, Vezys V, Ahmed R. Stimulation history dictates memory CD8 T cell phenotype: implications for prime-boost vaccination. Journal of immunology. 2006;177(2):831–9. pmid:16818737
- View Article
- PubMed/NCBI
- Google Scholar
4. Wirth TC, Harty JT, Badovinac VP. Modulating numbers and phenotype of CD8+ T cells in secondary immune responses. European journal of immunology. 2010;40(7):1916–26. pmid:20411564
- View Article
- PubMed/NCBI
- Google Scholar
5. Wirth TC, Xue HH, Rai D, Sabel JT, Bair T, Harty JT, et al. Repetitive antigen stimulation induces stepwise transcriptome diversification but preserves a core signature of memory CD8(+) T cell differentiation. Immunity. 2010;33(1):128–40. pmid:20619696
- View Article
- PubMed/NCBI
- Google Scholar
6. Fearon DT, Manders P, Wagner SD. Arrested differentiation, the self-renewing memory lymphocyte, and vaccination. Science. 2001;293(5528):248–50. pmid:11452114
- View Article
- PubMed/NCBI
- Google Scholar
7. Lanzavecchia A. Lack of fair play in the T cell response. Nature immunology. 2002;3(1):9–10. pmid:11753400
- View Article
- PubMed/NCBI
- Google Scholar
8. Lanzavecchia A, Sallusto F. Progressive differentiation and selection of the fittest in the immune response. Nature reviews Immunology. 2002;2(12):982–7. pmid:12461571
- View Article
- PubMed/NCBI
- Google Scholar
9. Wherry EJ, Teichgraber V, Becker TC, Masopust D, Kaech SM, Antia R, et al. Lineage relationship and protective immunity of memory CD8 T cell subsets. Nature immunology. 2003;4(3):225–34. pmid:12563257
- View Article
- PubMed/NCBI
- Google Scholar
10. Sallusto F, Geginat J, Lanzavecchia A. Central memory and effector memory T cell subsets: function, generation, and maintenance. Annual review of immunology. 2004;22:745–63. pmid:15032595
- View Article
- PubMed/NCBI
- Google Scholar
11. Henao-Tamayo MI, Ordway DJ, Irwin SM, Shang S, Shanley C, Orme IM. Phenotypic definition of effector and memory T-lymphocyte subsets in mice chronically infected with Mycobacterium tuberculosis. Clinical and vaccine immunology: CVI. 2010;17(4):618–25. pmid:20107011
- View Article
- PubMed/NCBI
- Google Scholar
12. Li Z, Liu WH, Han S, Peng BW, Yin J, Wu YL, et al. Selective inhibition of CCR7(-) effector memory T cell activation by a novel peptide targeting Kv1.3 channel in a rat experimental autoimmune encephalomyelitis model. J Biol Chem. 2012;287(35):29479–94. pmid:22761436
- View Article
- PubMed/NCBI
- Google Scholar
13. Hu L, Gocke AR, Knapp E, Rosenzweig JM, Grishkan IV, Baxi EG, et al. Functional blockade of the voltage-gated potassium channel Kv1.3 mediates reversion of T effector to central memory lymphocytes through SMAD3/p21cip1 signaling. J Biol Chem. 2012;287(2):1261–8. pmid:22110135
- View Article
- PubMed/NCBI
- Google Scholar
14. Gocke AR, Lebson LA, Grishkan IV, Hu L, Nguyen HM, Whartenby KA, et al. Kv1.3 deletion biases T cells toward an immunoregulatory phenotype and renders mice resistant to autoimmune encephalomyelitis. Journal of immunology. 2012;188(12):5877–86. pmid:22581856
- View Article
- PubMed/NCBI
- Google Scholar
15. Singh DK, Dwivedi VP, Ranganathan A, Bishai WR, Van Kaer L, Das G. Blockade of the Kv1.3 K+ Channel Enhances BCG Vaccine Efficacy by Expanding Central Memory T Lymphocytes. The Journal of infectious diseases. 2016;214(9):1456–64. pmid:27571906
- View Article
- PubMed/NCBI
- Google Scholar
16. Swanson RV, Adamson J, Moodley C, Ngcobo B, Ammerman NC, Dorasamy A, et al. Pharmacokinetics and pharmacodynamics of clofazimine in a mouse model of tuberculosis. Antimicrob Agents Chemother. 2015;59(6):3042–51. pmid:25753644
- View Article
- PubMed/NCBI
- Google Scholar
17. Lahey TP, Rajadhyaksha V. Inhibition by 3-deoxyflavonoids of t-lymphocyte activation and therapies related thereto. Google Patents; 2005.
18. Jung HA, Jin SE, Min BS, Kim BW, Choi JS. Anti-inflammatory activity of Korean thistle Cirsium maackii and its major flavonoid, luteolin 5-O-glucoside. Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association. 2012;50(6):2171–9. pmid:22525859
- View Article
- PubMed/NCBI
- Google Scholar
19. Lopez-Lazaro M. Distribution and biological activities of the flavonoid luteolin. Mini reviews in medicinal chemistry. 2009;9(1):31–59. pmid:19149659
- View Article
- PubMed/NCBI
- Google Scholar
20. Bashyal P, Pandey RP, Thapa SB, Kang MK, Kim CJ, Sohng JK. Biocatalytic Synthesis of Non-Natural Monoterpene O-Glycosides Exhibiting Superior Antibacterial and Antinematodal Properties. ACS Omega. 2019;4(5):9367–75. pmid:31460026
- View Article
- PubMed/NCBI
- Google Scholar
21. Akondy RS, Fitch M, Edupuganti S, Yang S, Kissick HT, Li KW, et al. Origin and differentiation of human memory CD8 T cells after vaccination. Nature. 2017;552(7685):362–7. pmid:29236685
- View Article
- PubMed/NCBI
- Google Scholar
22. Youngblood B, Hale JS, Kissick HT, Ahn E, Xu X, Wieland A, et al. Effector CD8 T cells dedifferentiate into long-lived memory cells. Nature. 2017;552(7685):404–9. pmid:29236683
- View Article
- PubMed/NCBI
- Google Scholar
23. Orme IM. The Achilles heel of BCG. Tuberculosis (Edinb). 2010;90(6):329–32.
- View Article
- Google Scholar
24. Cruz A, Torrado E, Carmona J, Fraga AG, Costa P, Rodrigues F, et al. BCG vaccination-induced long-lasting control of Mycobacterium tuberculosis correlates with the accumulation of a novel population of CD4(+)IL-17(+)TNF(+)IL-2(+) T cells. Vaccine. 2015;33(1):85–91. pmid:25448107
- View Article
- PubMed/NCBI
- Google Scholar
25. Khader SA, Bell GK, Pearl JE, Fountain JJ, Rangel-Moreno J, Cilley GE, et al. IL-23 and IL-17 in the establishment of protective pulmonary CD4+ T cell responses after vaccination and during Mycobacterium tuberculosis challenge. Nature immunology. 2007;8(4):369–77. pmid:17351619
- View Article
- PubMed/NCBI
- Google Scholar
26. Chandy KG, Wulff H, Beeton C, Pennington M, Gutman GA, Cahalan MD. K+ channels as targets for specific immunomodulation. Trends in pharmacological sciences. 2004;25(5):280–9. pmid:15120495
- View Article
- PubMed/NCBI
- Google Scholar
27. Singh DK, Dwivedi VP, Ranganathan A, Bishai WR, Van Kaer L, Das G. Reply to Levis and Rendini. The Journal of infectious diseases. 2017;215(9):1488–9. pmid:28199694
- View Article
- PubMed/NCBI
- Google Scholar
28. Lin Y, Shi R, Wang X, Shen HM. Luteolin, a flavonoid with potential for cancer prevention and therapy. Current cancer drug targets. 2008;8(7):634–46. pmid:18991571
- View Article
- PubMed/NCBI
- Google Scholar
29. Becattini S, Latorre D, Mele F, Foglierini M, De Gregorio C, Cassotta A, et al. T cell immunity. Functional heterogeneity of human memory CD4(+) T cell clones primed by pathogens or vaccines. Science. 2015;347(6220):400–6. pmid:25477212
- View Article
- PubMed/NCBI
- Google Scholar
30. da Costa AC, Nogueira SV, Kipnis A, Junqueira-Kipnis AP. Recombinant BCG: Innovations on an Old Vaccine. Scope of BCG Strains and Strategies to Improve Long-Lasting Memory. Frontiers in immunology. 2014;5:152. pmid:24778634
- View Article
- PubMed/NCBI
- Google Scholar
31. Bhattacharya D, Dwivedi VP, Kumar S, Reddy MC, Van Kaer L, Moodley P, et al. Simultaneous inhibition of T helper 2 and T regulatory cell differentiation by small molecules enhances Bacillus Calmette-Guerin vaccine efficacy against tuberculosis. J Biol Chem. 2014;289(48):33404–11. pmid:25315774
- View Article
- PubMed/NCBI
- Google Scholar
32. Lenaerts A, Barry CE 3rd, Dartois V. Heterogeneity in tuberculosis pathology, microenvironments and therapeutic responses. Immunological reviews. 2015;264(1):288–307. pmid:25703567
- View Article
- PubMed/NCBI
- Google Scholar
33. Prezzemolo T, Guggino G, La Manna MP, Di Liberto D, Dieli F, Caccamo N. Functional Signatures of Human CD4 and CD8 T Cell Responses to Mycobacterium tuberculosis. Frontiers in immunology. 2014;5:180. pmid:24795723
- View Article
- PubMed/NCBI
- Google Scholar
34. Marin ND, Paris SC, Rojas M, Garcia LF. Functional profile of CD4+ and CD8+ T cells in latently infected individuals and patients with active TB. Tuberculosis (Edinb). 2013;93(2):155–66. pmid:23332142
- View Article
- PubMed/NCBI
- Google Scholar
35. Vogelzang A, Perdomo C, Zedler U, Kuhlmann S, Hurwitz R, Gengenbacher M, et al. Central memory CD4+ T cells are responsible for the recombinant Bacillus Calmette-Guerin DeltaureC::hly vaccine’s superior protection against tuberculosis. The Journal of infectious diseases. 2014;210(12):1928–37. pmid:24943726
- View Article
- PubMed/NCBI
- Google Scholar
36. Hess J, Miko D, Catic A, Lehmensiek V, Russell DG, Kaufmann SH. Mycobacterium bovis Bacille Calmette-Guerin strains secreting listeriolysin of Listeria monocytogenes. Proceedings of the National Academy of Sciences of the United States of America. 1998;95(9):5299–304. pmid:9560270
- View Article
- PubMed/NCBI
- Google Scholar
37. Grode L, Seiler P, Baumann S, Hess J, Brinkmann V, Nasser Eddine A, et al. Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille Calmette-Guerin mutants that secrete listeriolysin. The Journal of clinical investigation. 2005;115(9):2472–9. pmid:16110326
- View Article
- PubMed/NCBI
- Google Scholar
38. Kaufmann SH. Tuberculosis vaccines: time to think about the next generation. Seminars in immunology. 2013;25(2):172–81. pmid:23706597
- View Article
- PubMed/NCBI
- Google Scholar
39. Tameris M, Geldenhuys H, Luabeya AK, Smit E, Hughes JE, Vermaak S, et al. The candidate TB vaccine, MVA85A, induces highly durable Th1 responses. PloS one. 2014;9(2):e87340. pmid:24498312
- View Article
- PubMed/NCBI
- Google Scholar
40. Geginat J, Sallusto F, Lanzavecchia A. Cytokine-driven proliferation and differentiation of human naive, central memory, and effector memory CD4(+) T cells. The Journal of experimental medicine. 2001;194(12):1711–9. pmid:11748273
- View Article
- PubMed/NCBI
- Google Scholar
41. Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature. 1999;401(6754):708–12. pmid:10537110
- View Article
- PubMed/NCBI
- Google Scholar
42. Wulff H, Calabresi PA, Allie R, Yun S, Pennington M, Beeton C, et al. The voltage-gated Kv1.3 K(+) channel in effector memory T cells as new target for MS. The Journal of clinical investigation. 2003;111(11):1703–13. pmid:12782673
- View Article
- PubMed/NCBI
- Google Scholar
43. Beeton C, Pennington MW, Wulff H, Singh S, Nugent D, Crossley G, et al. Targeting effector memory T cells with a selective peptide inhibitor of Kv1.3 channels for therapy of autoimmune diseases. Molecular pharmacology. 2005;67(4):1369–81. pmid:15665253
- View Article
- PubMed/NCBI
- Google Scholar
44. Beeton C, Wulff H, Barbaria J, Clot-Faybesse O, Pennington M, Bernard D, et al. Selective blockade of T lymphocyte K(+) channels ameliorates experimental autoimmune encephalomyelitis, a model for multiple sclerosis. Proceedings of the National Academy of Sciences of the United States of America. 2001;98(24):13942–7. pmid:11717451
- View Article
- PubMed/NCBI
- Google Scholar
45. Beeton C, Wulff H, Standifer NE, Azam P, Mullen KM, Pennington MW, et al. Kv1.3 channels are a therapeutic target for T cell-mediated autoimmune diseases. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(46):17414–9. pmid:17088564
- View Article
- PubMed/NCBI
- Google Scholar
46. Koni PA, Khanna R, Chang MC, Tang MD, Kaczmarek LK, Schlichter LC, et al. Compensatory anion currents in Kv1.3 channel-deficient thymocytes. J Biol Chem. 2003;278(41):39443–51. pmid:12878608
- View Article
- PubMed/NCBI
- Google Scholar
47. Aziz N, Kim MY, Cho JY. Anti-inflammatory effects of luteolin: A review of in vitro, in vivo, and in silico studies. J Ethnopharmacol. 2018;225:342–58. pmid:29801717
- View Article
- PubMed/NCBI
- Google Scholar
48. Trizio D, Cudkowicz G. Separation of T and B lymphocytes by nylon wool columns: evaluation of efficacy by functional assays in vivo. Journal of immunology. 1974;113(4):1093–7.
- View Article
- Google Scholar

[ref1] 1. Organization WH. Global tuberculosis report 2017. Geneva: World Health Organization; 2017. 157 p.

[ref2] 2. Colditz GA, Brewer TF, Berkey CS, Wilson ME, Burdick E, Fineberg HV, et al. Efficacy of BCG vaccine in the prevention of tuberculosis. Meta-analysis of the published literature. Jama. 1994;271(9):698–702. pmid:8309034
View Article
PubMed/NCBI
Google Scholar

[3] View Article

[4] PubMed/NCBI

[5] Google Scholar

[ref3] 3. Masopust D, Ha SJ, Vezys V, Ahmed R. Stimulation history dictates memory CD8 T cell phenotype: implications for prime-boost vaccination. Journal of immunology. 2006;177(2):831–9. pmid:16818737
View Article
PubMed/NCBI
Google Scholar

[7] View Article

[8] PubMed/NCBI

[9] Google Scholar

[ref4] 4. Wirth TC, Harty JT, Badovinac VP. Modulating numbers and phenotype of CD8+ T cells in secondary immune responses. European journal of immunology. 2010;40(7):1916–26. pmid:20411564
View Article
PubMed/NCBI
Google Scholar

[11] View Article

[12] PubMed/NCBI

[13] Google Scholar

[ref5] 5. Wirth TC, Xue HH, Rai D, Sabel JT, Bair T, Harty JT, et al. Repetitive antigen stimulation induces stepwise transcriptome diversification but preserves a core signature of memory CD8(+) T cell differentiation. Immunity. 2010;33(1):128–40. pmid:20619696
View Article
PubMed/NCBI
Google Scholar

[15] View Article

[16] PubMed/NCBI

[17] Google Scholar

[ref6] 6. Fearon DT, Manders P, Wagner SD. Arrested differentiation, the self-renewing memory lymphocyte, and vaccination. Science. 2001;293(5528):248–50. pmid:11452114
View Article
PubMed/NCBI
Google Scholar

[19] View Article

[20] PubMed/NCBI

[21] Google Scholar

[ref7] 7. Lanzavecchia A. Lack of fair play in the T cell response. Nature immunology. 2002;3(1):9–10. pmid:11753400
View Article
PubMed/NCBI
Google Scholar

[23] View Article

[24] PubMed/NCBI

[25] Google Scholar

[ref8] 8. Lanzavecchia A, Sallusto F. Progressive differentiation and selection of the fittest in the immune response. Nature reviews Immunology. 2002;2(12):982–7. pmid:12461571
View Article
PubMed/NCBI
Google Scholar

[27] View Article

[28] PubMed/NCBI

[29] Google Scholar

[ref9] 9. Wherry EJ, Teichgraber V, Becker TC, Masopust D, Kaech SM, Antia R, et al. Lineage relationship and protective immunity of memory CD8 T cell subsets. Nature immunology. 2003;4(3):225–34. pmid:12563257
View Article
PubMed/NCBI
Google Scholar

[31] View Article

[32] PubMed/NCBI

[33] Google Scholar

[ref10] 10. Sallusto F, Geginat J, Lanzavecchia A. Central memory and effector memory T cell subsets: function, generation, and maintenance. Annual review of immunology. 2004;22:745–63. pmid:15032595
View Article
PubMed/NCBI
Google Scholar

[35] View Article

[36] PubMed/NCBI

[37] Google Scholar

[ref11] 11. Henao-Tamayo MI, Ordway DJ, Irwin SM, Shang S, Shanley C, Orme IM. Phenotypic definition of effector and memory T-lymphocyte subsets in mice chronically infected with Mycobacterium tuberculosis. Clinical and vaccine immunology: CVI. 2010;17(4):618–25. pmid:20107011
View Article
PubMed/NCBI
Google Scholar

[39] View Article

[40] PubMed/NCBI

[41] Google Scholar

[ref12] 12. Li Z, Liu WH, Han S, Peng BW, Yin J, Wu YL, et al. Selective inhibition of CCR7(-) effector memory T cell activation by a novel peptide targeting Kv1.3 channel in a rat experimental autoimmune encephalomyelitis model. J Biol Chem. 2012;287(35):29479–94. pmid:22761436
View Article
PubMed/NCBI
Google Scholar

[43] View Article

[44] PubMed/NCBI

[45] Google Scholar

[ref13] 13. Hu L, Gocke AR, Knapp E, Rosenzweig JM, Grishkan IV, Baxi EG, et al. Functional blockade of the voltage-gated potassium channel Kv1.3 mediates reversion of T effector to central memory lymphocytes through SMAD3/p21cip1 signaling. J Biol Chem. 2012;287(2):1261–8. pmid:22110135
View Article
PubMed/NCBI
Google Scholar

[47] View Article

[48] PubMed/NCBI

[49] Google Scholar

[ref14] 14. Gocke AR, Lebson LA, Grishkan IV, Hu L, Nguyen HM, Whartenby KA, et al. Kv1.3 deletion biases T cells toward an immunoregulatory phenotype and renders mice resistant to autoimmune encephalomyelitis. Journal of immunology. 2012;188(12):5877–86. pmid:22581856
View Article
PubMed/NCBI
Google Scholar

[51] View Article

[52] PubMed/NCBI

[53] Google Scholar

[ref15] 15. Singh DK, Dwivedi VP, Ranganathan A, Bishai WR, Van Kaer L, Das G. Blockade of the Kv1.3 K+ Channel Enhances BCG Vaccine Efficacy by Expanding Central Memory T Lymphocytes. The Journal of infectious diseases. 2016;214(9):1456–64. pmid:27571906
View Article
PubMed/NCBI
Google Scholar

[55] View Article

[56] PubMed/NCBI

[57] Google Scholar

[ref16] 16. Swanson RV, Adamson J, Moodley C, Ngcobo B, Ammerman NC, Dorasamy A, et al. Pharmacokinetics and pharmacodynamics of clofazimine in a mouse model of tuberculosis. Antimicrob Agents Chemother. 2015;59(6):3042–51. pmid:25753644
View Article
PubMed/NCBI
Google Scholar

[59] View Article

[60] PubMed/NCBI

[61] Google Scholar

[ref17] 17. Lahey TP, Rajadhyaksha V. Inhibition by 3-deoxyflavonoids of t-lymphocyte activation and therapies related thereto. Google Patents; 2005.

[ref18] 18. Jung HA, Jin SE, Min BS, Kim BW, Choi JS. Anti-inflammatory activity of Korean thistle Cirsium maackii and its major flavonoid, luteolin 5-O-glucoside. Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association. 2012;50(6):2171–9. pmid:22525859
View Article
PubMed/NCBI
Google Scholar

[64] View Article

[65] PubMed/NCBI

[66] Google Scholar

[ref19] 19. Lopez-Lazaro M. Distribution and biological activities of the flavonoid luteolin. Mini reviews in medicinal chemistry. 2009;9(1):31–59. pmid:19149659
View Article
PubMed/NCBI
Google Scholar

[68] View Article

[69] PubMed/NCBI

[70] Google Scholar

[ref20] 20. Bashyal P, Pandey RP, Thapa SB, Kang MK, Kim CJ, Sohng JK. Biocatalytic Synthesis of Non-Natural Monoterpene O-Glycosides Exhibiting Superior Antibacterial and Antinematodal Properties. ACS Omega. 2019;4(5):9367–75. pmid:31460026
View Article
PubMed/NCBI
Google Scholar

[72] View Article

[73] PubMed/NCBI

[74] Google Scholar

[ref21] 21. Akondy RS, Fitch M, Edupuganti S, Yang S, Kissick HT, Li KW, et al. Origin and differentiation of human memory CD8 T cells after vaccination. Nature. 2017;552(7685):362–7. pmid:29236685
View Article
PubMed/NCBI
Google Scholar

[76] View Article

[77] PubMed/NCBI

[78] Google Scholar

[ref22] 22. Youngblood B, Hale JS, Kissick HT, Ahn E, Xu X, Wieland A, et al. Effector CD8 T cells dedifferentiate into long-lived memory cells. Nature. 2017;552(7685):404–9. pmid:29236683
View Article
PubMed/NCBI
Google Scholar

[80] View Article

[81] PubMed/NCBI

[82] Google Scholar

[ref23] 23. Orme IM. The Achilles heel of BCG. Tuberculosis (Edinb). 2010;90(6):329–32.
View Article
Google Scholar

[84] View Article

[85] Google Scholar

[ref24] 24. Cruz A, Torrado E, Carmona J, Fraga AG, Costa P, Rodrigues F, et al. BCG vaccination-induced long-lasting control of Mycobacterium tuberculosis correlates with the accumulation of a novel population of CD4(+)IL-17(+)TNF(+)IL-2(+) T cells. Vaccine. 2015;33(1):85–91. pmid:25448107
View Article
PubMed/NCBI
Google Scholar

[87] View Article

[88] PubMed/NCBI

[89] Google Scholar

[ref25] 25. Khader SA, Bell GK, Pearl JE, Fountain JJ, Rangel-Moreno J, Cilley GE, et al. IL-23 and IL-17 in the establishment of protective pulmonary CD4+ T cell responses after vaccination and during Mycobacterium tuberculosis challenge. Nature immunology. 2007;8(4):369–77. pmid:17351619
View Article
PubMed/NCBI
Google Scholar

[91] View Article

[92] PubMed/NCBI

[93] Google Scholar

[ref26] 26. Chandy KG, Wulff H, Beeton C, Pennington M, Gutman GA, Cahalan MD. K+ channels as targets for specific immunomodulation. Trends in pharmacological sciences. 2004;25(5):280–9. pmid:15120495
View Article
PubMed/NCBI
Google Scholar

[95] View Article

[96] PubMed/NCBI

[97] Google Scholar

[ref27] 27. Singh DK, Dwivedi VP, Ranganathan A, Bishai WR, Van Kaer L, Das G. Reply to Levis and Rendini. The Journal of infectious diseases. 2017;215(9):1488–9. pmid:28199694
View Article
PubMed/NCBI
Google Scholar

[99] View Article

[100] PubMed/NCBI

[101] Google Scholar

[ref28] 28. Lin Y, Shi R, Wang X, Shen HM. Luteolin, a flavonoid with potential for cancer prevention and therapy. Current cancer drug targets. 2008;8(7):634–46. pmid:18991571
View Article
PubMed/NCBI
Google Scholar

[103] View Article

[104] PubMed/NCBI

[105] Google Scholar

[ref29] 29. Becattini S, Latorre D, Mele F, Foglierini M, De Gregorio C, Cassotta A, et al. T cell immunity. Functional heterogeneity of human memory CD4(+) T cell clones primed by pathogens or vaccines. Science. 2015;347(6220):400–6. pmid:25477212
View Article
PubMed/NCBI
Google Scholar

[107] View Article

[108] PubMed/NCBI

[109] Google Scholar

[ref30] 30. da Costa AC, Nogueira SV, Kipnis A, Junqueira-Kipnis AP. Recombinant BCG: Innovations on an Old Vaccine. Scope of BCG Strains and Strategies to Improve Long-Lasting Memory. Frontiers in immunology. 2014;5:152. pmid:24778634
View Article
PubMed/NCBI
Google Scholar

[111] View Article

[112] PubMed/NCBI

[113] Google Scholar

[ref31] 31. Bhattacharya D, Dwivedi VP, Kumar S, Reddy MC, Van Kaer L, Moodley P, et al. Simultaneous inhibition of T helper 2 and T regulatory cell differentiation by small molecules enhances Bacillus Calmette-Guerin vaccine efficacy against tuberculosis. J Biol Chem. 2014;289(48):33404–11. pmid:25315774
View Article
PubMed/NCBI
Google Scholar

[115] View Article

[116] PubMed/NCBI

[117] Google Scholar

[ref32] 32. Lenaerts A, Barry CE 3rd, Dartois V. Heterogeneity in tuberculosis pathology, microenvironments and therapeutic responses. Immunological reviews. 2015;264(1):288–307. pmid:25703567
View Article
PubMed/NCBI
Google Scholar

[119] View Article

[120] PubMed/NCBI

[121] Google Scholar

[ref33] 33. Prezzemolo T, Guggino G, La Manna MP, Di Liberto D, Dieli F, Caccamo N. Functional Signatures of Human CD4 and CD8 T Cell Responses to Mycobacterium tuberculosis. Frontiers in immunology. 2014;5:180. pmid:24795723
View Article
PubMed/NCBI
Google Scholar

[123] View Article

[124] PubMed/NCBI

[125] Google Scholar

[ref34] 34. Marin ND, Paris SC, Rojas M, Garcia LF. Functional profile of CD4+ and CD8+ T cells in latently infected individuals and patients with active TB. Tuberculosis (Edinb). 2013;93(2):155–66. pmid:23332142
View Article
PubMed/NCBI
Google Scholar

[127] View Article

[128] PubMed/NCBI

[129] Google Scholar

[ref35] 35. Vogelzang A, Perdomo C, Zedler U, Kuhlmann S, Hurwitz R, Gengenbacher M, et al. Central memory CD4+ T cells are responsible for the recombinant Bacillus Calmette-Guerin DeltaureC::hly vaccine’s superior protection against tuberculosis. The Journal of infectious diseases. 2014;210(12):1928–37. pmid:24943726
View Article
PubMed/NCBI
Google Scholar

[131] View Article

[132] PubMed/NCBI

[133] Google Scholar

[ref36] 36. Hess J, Miko D, Catic A, Lehmensiek V, Russell DG, Kaufmann SH. Mycobacterium bovis Bacille Calmette-Guerin strains secreting listeriolysin of Listeria monocytogenes. Proceedings of the National Academy of Sciences of the United States of America. 1998;95(9):5299–304. pmid:9560270
View Article
PubMed/NCBI
Google Scholar

[135] View Article

[136] PubMed/NCBI

[137] Google Scholar

[ref37] 37. Grode L, Seiler P, Baumann S, Hess J, Brinkmann V, Nasser Eddine A, et al. Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille Calmette-Guerin mutants that secrete listeriolysin. The Journal of clinical investigation. 2005;115(9):2472–9. pmid:16110326
View Article
PubMed/NCBI
Google Scholar

[139] View Article

[140] PubMed/NCBI

[141] Google Scholar

[ref38] 38. Kaufmann SH. Tuberculosis vaccines: time to think about the next generation. Seminars in immunology. 2013;25(2):172–81. pmid:23706597
View Article
PubMed/NCBI
Google Scholar

[143] View Article

[144] PubMed/NCBI

[145] Google Scholar

[ref39] 39. Tameris M, Geldenhuys H, Luabeya AK, Smit E, Hughes JE, Vermaak S, et al. The candidate TB vaccine, MVA85A, induces highly durable Th1 responses. PloS one. 2014;9(2):e87340. pmid:24498312
View Article
PubMed/NCBI
Google Scholar

[147] View Article

[148] PubMed/NCBI

[149] Google Scholar

[ref40] 40. Geginat J, Sallusto F, Lanzavecchia A. Cytokine-driven proliferation and differentiation of human naive, central memory, and effector memory CD4(+) T cells. The Journal of experimental medicine. 2001;194(12):1711–9. pmid:11748273
View Article
PubMed/NCBI
Google Scholar

[151] View Article

[152] PubMed/NCBI

[153] Google Scholar

[ref41] 41. Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature. 1999;401(6754):708–12. pmid:10537110
View Article
PubMed/NCBI
Google Scholar

[155] View Article

[156] PubMed/NCBI

[157] Google Scholar

[ref42] 42. Wulff H, Calabresi PA, Allie R, Yun S, Pennington M, Beeton C, et al. The voltage-gated Kv1.3 K(+) channel in effector memory T cells as new target for MS. The Journal of clinical investigation. 2003;111(11):1703–13. pmid:12782673
View Article
PubMed/NCBI
Google Scholar

[159] View Article

[160] PubMed/NCBI

[161] Google Scholar

[ref43] 43. Beeton C, Pennington MW, Wulff H, Singh S, Nugent D, Crossley G, et al. Targeting effector memory T cells with a selective peptide inhibitor of Kv1.3 channels for therapy of autoimmune diseases. Molecular pharmacology. 2005;67(4):1369–81. pmid:15665253
View Article
PubMed/NCBI
Google Scholar

[163] View Article

[164] PubMed/NCBI

[165] Google Scholar

[ref44] 44. Beeton C, Wulff H, Barbaria J, Clot-Faybesse O, Pennington M, Bernard D, et al. Selective blockade of T lymphocyte K(+) channels ameliorates experimental autoimmune encephalomyelitis, a model for multiple sclerosis. Proceedings of the National Academy of Sciences of the United States of America. 2001;98(24):13942–7. pmid:11717451
View Article
PubMed/NCBI
Google Scholar

[167] View Article

[168] PubMed/NCBI

[169] Google Scholar

[ref45] 45. Beeton C, Wulff H, Standifer NE, Azam P, Mullen KM, Pennington MW, et al. Kv1.3 channels are a therapeutic target for T cell-mediated autoimmune diseases. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(46):17414–9. pmid:17088564
View Article
PubMed/NCBI
Google Scholar

[171] View Article

[172] PubMed/NCBI

[173] Google Scholar

[ref46] 46. Koni PA, Khanna R, Chang MC, Tang MD, Kaczmarek LK, Schlichter LC, et al. Compensatory anion currents in Kv1.3 channel-deficient thymocytes. J Biol Chem. 2003;278(41):39443–51. pmid:12878608
View Article
PubMed/NCBI
Google Scholar

[175] View Article

[176] PubMed/NCBI

[177] Google Scholar

[ref47] 47. Aziz N, Kim MY, Cho JY. Anti-inflammatory effects of luteolin: A review of in vitro, in vivo, and in silico studies. J Ethnopharmacol. 2018;225:342–58. pmid:29801717
View Article
PubMed/NCBI
Google Scholar

[179] View Article

[180] PubMed/NCBI

[181] Google Scholar

[ref48] 48. Trizio D, Cudkowicz G. Separation of T and B lymphocytes by nylon wool columns: evaluation of efficacy by functional assays in vivo. Journal of immunology. 1974;113(4):1093–7.
View Article
Google Scholar

[183] View Article

[184] Google Scholar

Correction

Abstract

Author summary

Figures

Introduction

Results

Luteolin biases T cells towards a TCM phenotype in vitro

Luteolin induces sustained T cell immunity in lung and spleen of BCG-immunized mice challenged with M.tb

Luteolin treatment during BCG vaccination promotes TCM cells in mice challenged with M. tuberculosis

Luteolin treatment enhances BCG vaccine-induced host protection against TB

T cells from luteolin-treated, BCG-immunized mice confer improved protection against TB upon adoptive transfer

Discussion

Material and methods

Ethics statement

Mice

In vitro T cell stimulation

M.tb low-dose aerosol infection of mice

Drug administration

Quantification of pathogen burden by Colony Forming Units (CFU)

Flow cytometry: Surface and intracellular staining

Detection of cytokines

Antigen-specific degranulation assay

BCG immunization experiments

T Cell adoptive transfer experiments

Antibodies and reagents

Statistical analysis

Supporting information

S1 Fig. In vitro bactericidal activity of luteolin (100 μM/mL).

S2 Fig.

S3 Fig. Gating strategies employed for immune profiling of T cells.

S4 Fig. Bar diagram to show the percentage of different subsets of memory T cells in Lungs after immunization and luteolin treatment prior to infection.

S5 Fig. Cytokine profiling from splenocytes of the respective experimental groups.

S6 Fig. Gating strategies employed for the profiling of antigen-specific cytokine responses in T cells from splenocytes.

Acknowledgments

References

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Luteolin biases T cells towards a T_CM phenotype in vitro

Luteolin treatment during BCG vaccination promotes T_CM cells in mice challenged with M. tuberculosis