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. 2020 Mar 19;367(7):fnaa053. doi: 10.1093/femsle/fnaa053

High-polyphenol extracts from Sorghum bicolor attenuate replication of Legionella pneumophila within RAW 264.7 macrophages

Aubrey K Gilchrist 1, Dmitriy Smolensky 2, Tshegofatso Ngwaga 1, Deepika Chauhan 1, Sarah Cox 2, Ramasamy Perumal 3, Leela E Noronha 4, Stephanie R Shames 1,✉
PMCID: PMC8023677  PMID: 32188994

ABSTRACT

Polyphenols derived from a variety of plants have demonstrated antimicrobial activity against diverse microbial pathogens. Legionella pneumophila is an intracellular bacterial pathogen that opportunistically causes a severe inflammatory pneumonia in humans, called Legionnaires’ Disease, via replication within macrophages. Previous studies demonstrated that tea polyphenols attenuate L. pneumophila intracellular replication within mouse macrophages via increased tumor necrosis factor (TNF) production. Sorghum bicolor is a sustainable cereal crop that thrives in arid environments and is well-suited to continued production in warming climates. Sorghum polyphenols have anticancer and antioxidant properties, but their antimicrobial activity has not been evaluated. Here, we investigated the impact of sorghum polyphenols on L. pneumophila intracellular replication within RAW 264.7 mouse macrophages. Sorghum high-polyphenol extract (HPE) attenuated L. pneumophila intracellular replication in a dose-dependent manner but did not impair either bacterial replication in rich media or macrophage viability. Moreover, HPE treatment enhanced both TNF and IL-6 secretion from L. pneumophila infected macrophages. Thus, polyphenols derived from sorghum enhance macrophage restriction of L. pneumophila, likely via increased pro-inflammatory cytokine production. This work reveals commonalities between plant polyphenol-mediated antimicrobial activity and provides a foundation for future evaluation of sorghum as an antimicrobial agent.

Keywords: Sorghum polyphenol, Legionella, pneumophila, RAW 264.7, macrophages

Restriction of Legionella pneumophila intracellular replication in macrophages by sorghum polyphenols.

INTRODUCTION

Plant polyphenols are bioactive compounds that naturally function in plant defense against biotic and abiotic stressors. However, polyphenols can also promote human health through antioxidant, anticancer and antimicrobial activity (Daglia 2012; Park et al. 2012; Tomás-Barberán and Andrés-Lacueva 2012; Smolensky et al. 2018). Camellia sinensis (tea) polyphenols (catechins) have demonstrated antimicrobial activity against a variety of bacterial, viral and fungal pathogens (Reygaert 2014). Specifically, the tea polyphenol epigallocatechin gallate (EGCg) has broad antimicrobial activity against a variety of pathogens in vitro and within eukaryotic host cells (Matsunaga et al. 2001; Rogers et al. 2005; Reygaert 2014; Betts et al. 2019). With the increased prevalence of antibiotic resistance amongst bacterial pathogens, plant polyphenols are promising therapeutic options to combat bacterial infections.

Sorghum (Sorghum bicolor (L.) Monench) is a dryland cereal crop that produces bioactive polyphenols and performs well in arid climates, which is critical for sustainability in a continuously warming climate. Sorghum polyphenols have demonstrated antioxidant and anticancer activities (Park et al. 2012; Smolensky et al. 2018). Moreover, sorghum polyphenols have the ability to alleviate symptoms of colitis in addition to restoring microbiome diversity (Ritchie et al. 2015, 2017). Despite exploration into the health benefits and bioactivity of sorghum polyphenols, their antimicrobial potential has not been explored.

Legionella pneumophila is facultative intracellular bacterial pathogen that naturally inhabits freshwater environments where it parasitizes and replicates within unicellular protozoa (Isberg, O'Connor and Heidtman 2009). Inhalation of Legionella-contaminated aerosols from anthropomorphic fresh-water environments causes severe inflammatory pneumonia in immunocompromised individuals called Legionnaires’ Disease through uncontrolled bacterial replication within alveolar macrophages. To replicate within eukaryotic phagocytes, L. pneumophila employs a Dot/Icm type IV secretion system, which translocates hundreds of bacterial effector protein virulence factors directly into infected host cells (Ensminger 2016). Effector translocation by the Dot/Icm secretion system is essential for biogenesis of L. pneumophila’s replicative niche, the Legionella containing vacuole (LCV) and intracellular replication.

As an accidental pathogen of humans, L. pneumophila does not transmit between individuals and is highly susceptible to mammalian innate immune defenses. Specifically, L. pneumophila replication in macrophage is potently attenuated by proinflammatory cytokines, such as interferon (IFN)-γ and tumor necrosis factor (TNF) (Brieland et al. 1995; Skerrett et al. 1997; Shinozawa et al. 2002; Coers et al. 2007; Price et al. 2019). A previous study revealed that L. pneumophila is restricted in mouse macrophages by EGCg via increased inflammatory cytokine production (Matsunaga et al. 2001). The impact of EGCg on the macrophages was responsible for bacterial restriction since L. pneumophila in vitro replication was unaffected (Matsunaga et al. 2001). In the present study, we evaluated the impact of high-polyphenol extracts (HPE) from sorghum on L. pneumophila replication within macrophages. We discovered that HPE-treated macrophages were restrictive to L. pneumophila, which was not a consequence of macrophage cell death or direct antimicrobial restriction of L. pneumophila in vitro. However, treatment with HPE enhanced TNF and IL-6 secretion from L. pneumophila-infected macrophages, suggesting that sorghum polyphenols may elicit their antimicrobial activity by increasing inflammation.

MATERIALS AND METHODS

Bacterial strains, tissue culture, reagents and growth conditions

Legionella pneumophila Philadelphia-1 SRS43 wild-type, ∆flaA and dotA::Tn (Shames et al. 2017; Ngwaga et al. 2019) were cultured on supplemented charcoal–N (2-acetamido)-2-aminoethanesulfonic acid (ACES)-buffered yeast extract (CYE) and grown at 37°C as described previously (Shames et al. 2017). Isolated colonies from CYE agar plates were used to generate 48 h heavy patches of bacteria, which were then used for infection. Liquid cultures were grown at 37˚C in supplemented ACES-buffered yeast extract (AYE) as described (Feeley et al. 1979; Saito et al. 1981).

RAW 264.7 cells (ATCC) were maintained at 37°C/5% CO2 in Dulbecco's Modified Eagle Medium (DMEM; Gibco) supplemented with 10% heat-inactivated fetal bovine serum (HIFBS; Gibco). For infections and assays, cells were seeded and infected in low-serum media (2.5% HIFBS) where indicated. RAW cells were used from passages 4 to 15.

All chemicals were purchased from MilliporeSigma (St. Louis, MO) unless otherwise specified.

Generation of high-polyphenol sorghum bran extracts (HPE)

Sorghum accession PI570481 (tall and photo-period sensitive germplasm of Sudan origin) was obtained from Kansas State University, Agricultural Research Center, Hays, Kansas for its high phenolic content and previously demonstrated anti-cancer activity in tissue culture models and used for evaluation in this study (Smolensky et al. 2018). Total sorghum phenolic extraction was performed as previously described (Cox et al. 2019). In brief, sorghum was decorticated, and bran was ground in-house. Bran powder (10% w/v) was suspended in extraction solvent (vehicle), consisting of 70% (v/v) ethanol with 5% (w/v) citric acid. The samples were placed on a shaker at room temperature (20°C) for 2 h and then stored at −20°C overnight. The following day, the sample was centrifuged at 3000 r.c.f. for 10 min. The supernatant was collected as total phenolic extract and the pellet was discarded. The total phenol content of HPE was measured using gallic acid as a standard to be 49.6 mg Gallic acid equivalent (GAE)/g of sorghum bran which provided 4.9 mg GAE/ml phenolic content in the crude extract (Smolensky et al. 2018; Cox et al. 2019).

Quantification of L. pneumophila replication in vitro

Legionella pneumophila was cultured on CYE agar and 48 h heavy patches were used to subculture bacteria into supplemented AYE media (see above). All cultures were diluted to an OD600 of 0.5 and were grown in triplicates at 37°C with shaking. HPE-treated cultures were grown in the presence of 1.25 mg/mL HPE and vehicle control cultures were grown in a volume equivalent of vehicle [5% citric acid (w/v), 70% ethanol (v/v)]. At the indicated time points, OD600 was measured and bacteria were plated on CYE agar for colony forming unit (CFU) enumeration.

Infection of RAW 264.7 cells with Legionella pneumophila

RAW 264.7 cells were seeded in 24-well plates at 2 × 105 cells/well in low-serum media (DMEM + 2.5% HI FBS) one day prior to infection. Cells were infected with L. pneumophila at a multiplicity of infection (MOI) of 1 as described previously (Ngwaga et al. 2019) in the presence of HPE or vehicle as indicated. Plates were centrifuged at 250 r.c.f. to synchronize infection and incubated at 37°C/5% CO2 for 1 h. Media were aspirated and cells were washed gently 3 times with PBS and media were replaced with the same concentrations of HPE or vehicle. At the indicated time points, cells were lysed in sterile ultrapure water and bacteria were plated on CYE agar for CFU enumeration. Data are presented at either CFU per well at the indicated time point or fold replication at 48 h post-infection (normalization of CFU counts to counts at 1 h post-infection; day 0).

Cytotoxicity assay

To determine if sorghum HPE treatment impacted viability of RAW cells, cytotoxicity was quantified by lactate dehydrogenase (LDH) release assay using the Promega CytoTox™ 96 Non-Radioactive Cytotoxicity Assay according to manufacturers’ instructions. Briefly, RAW cells were seeded at 2.5 × 105/well in 24-well tissue culture plates in low-serum media one day prior to infection. Cells were infected with Lpn ∆flaA (MOI = 1) in the presence of 0.625 mg/mL HPE, 1.25 mg/mL HPE or volume equivalent of vehicle (1.25 µL/mL) for 1 h. Media were aspirated and cells were washed 3 times with sterile PBS followed by replenishing media in the presence or absence of HPE or vehicle. Lysis solution (1X) was added to control wells one hour prior to collecting supernatants. At 6 h post-infection, plates were centrifuged at 250 r.c.f. and supernatants were transferred to a sterile 96-well plate followed by addition of CytoTox™ reagent and incubation at room temperature in the dark for 30 min. Stop solution was added and absorbance was measured at 490 nm in a Victor 2 microplate reader (PerkinElmer). Background absorbance values were subtracted and % cytotoxicity was calculated by normalizing data to lysis buffer control (100% cytotoxicity).

Caspase 3 cleavage assay

RAW 264.7 cells were seeded at 1 × 106 in low-serum media in 6-well tissue culture plates one day prior to infection. Cells were treated with 1.25 mg/mL HPE or volume equivalent of vehicle and either infected with L. pneumophila at a MOI of 1 or left uninfected. As a control for induction of apoptosis, cells were treated with 10 µM staurosporine for 3 h (Yamaki et al. 2002). Cells were treated and/or infected for a total of 4 h followed by lysis in 120 µL of ice-cold RIPA buffer [10 mM Tris,(pH 7.5), 100 mM NaCl, 1 mM EDTA, 1% NP40 (v/v), 0.1% SDS (v/v), 0.5% sodium deoxycholate (w/v)] supplemented with fresh 1X ProBlock Gold Mammalian Protease Inhibitor Cocktail and 1X EDTA (GoldBio). Cells were scraped into pre-chilled 1.5 mL microcentrifuge tubes and lysates were clarified by centrifugation at 14 000 r.c.f. at 4°C for 10 min. Clarified lysates (60 µL) were added to 3X Laemmli sample buffer (30 µL) and boiled for 10 min followed by SDS-PAGE and western blot analysis.

MTT Cell Proliferation Assay

MTT Cell Proliferation Assay Kit (BioVision) was used according to manufacturers’ instructions. Briefly, RAW 267.4 cells were seeded at 1 × 104 cell/well in low-serum media a 96-well tissue culture plates one day prior to infection and treatment. Cells were either left uninfected or infected with Lpn ∆flaA (MOI = 1) and treated with 0.625 mg/mL, 1.25 mg/mL of HPE or volume equivalent of vehicle for 1 h, 24 h and 48 h. After 1 h, infected cells were washed and fresh medium with HPE or vehicle control were added back and cells were incubated for an additional 24 h or 48 h. At each time point, 50 µL of MTT Reagent was added per well and plates were incubated at 37°C for 4 h. MTT Solvent (150 µL/well) was added and plate was incubated at room temperature with shaking for 15 min. Absorbance at 590 nm was read in a Victor 2 plate reader.

SDS-PAGE and western blot

Boiled protein samples were loaded onto 12% SDS-PAGE gels and separated by electrophoresis. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes using a BioRad wet transfer cell. Membranes were blocked in blocking buffer [5% non-fat milk powder dissolved in tris-buffered saline, 0.1% Tween-20 (TBST)]. Primary antibodies [α-caspase 3 (#9662S; Cell Signaling Technology), α-β-actin (#4967S; Cell Signaling Technology)] were diluted in blocking buffer (1:1000) and incubated with membranes at 4°C overnight with rocking. Membranes were washed in TBST 3 × 10 min with rocking at room-temperature. Horseradish peroxidase (HRP)-conjugated secondary antibodies (goat α-mouse-HRP or goat α-rabbit-HRP; ThermoFisher) were diluted to 1:5000 in blocking buffer and incubated with membranes for 1–2 h at room temperature with rocking. Membranes were washed in TBST as above. Membranes were incubated with ECL substrate (GE Amersham) and imaged by chemiluminescence using an Azure Biosystems c300 Darkroom Replacer. For loading control (β-actin) blots, membranes were stripped with Thermo Scientific Restore Western Blot Stripping Buffer (ThermoFisher) according to manufacturers’ instructions.

Enzyme-linked immunosorbent assay (ELISA)

RAW 264.7 cells were seeded in 24-well tissue culture plates at 2.5 × 105 in low-serum media one day prior to infection. Cells were infected with L. pneumophila at a MOI of 1 or left uninfected and either treated with the indicated concentrations of HPE or volume equivalent of vehicle. One hour post-infected, media were aspirated and cells were washed 3X with sterile PBS. Media and HPE/vehicle treatments were replaced and cells were incubated for either 6 h, 24 h or 48 h. Lysates were harvested from cells and stored at −20°C until use. TNF and IL-6 were quantified using either the Mouse TNF-α or IL-6 ELISA MAX™ Kits (BioLegend) according to manufacturers’ instructions.

Statistical analysis

Statistical analysis was performed with GraphPad Prism software using unpaired Students t-test with a 95% confidence interval. For all experiments, data are presented as mean ± standard deviation of samples in triplicates.

RESULTS

High-polyphenol extracts (HPE) from Sorghum bicolor restrict Legionella pneumophila intracellular replication within RAW 264.7 cells

The polyphenol epigallocatchenin gallate (EGCg) from tea (Camellia sinensis) enhances restriction of L. pneumophila intracellular replication within mouse macrophages (Matsunaga et al. 2001; Rogers et al. 2005). Thus, we evaluated whether S. bicolor high-polyphenol extracts (HPE) would also be sufficient to restrict L. pneumophila intracellular replication. Flagellin (FlaA) from L. pneumophila activates the NLRC4 inflammasome, which results in bacterial restriction in mouse macrophages (Molofsky et al. 2006; Ren et al. 2006). To circumvent this issue, we utilized a flagellin deficient (∆flaA) L. pneumophila strain for infection experiments. Replication of L. pneumophila ∆flaA within mouse RAW 264.7 macrophages was quantified in the presence or absence of HPE. Initially, cells were treated with 1.25 mg/mL HPE or volume equivalent of vehicle (see Materials and Methods) and L. pneumophila intracellular replication was quantified by enumeration of colony forming units (CFU) for up to 48 h post-infection. Replication of L. pneumophila was significantly decreased within RAW cells treated with HPE compared to vehicle control (*P < 0.05, **P < 0.01, Fig 1A). To determine if growth attenuation by HPE was dose-dependent, we treated RAW 264.7 cells with increasing concentrations of HPE (0.625 mg/mL and 1.25 mg/mL) or vehicle control and quantified L. pneumophila intracellular replication. Compared to vehicle-treated cells, L. pneumophila intracellular replication with significantly attenuated in macrophages treated with 0.625 mg/mL (*P < 0.05, Fig 1B) and further decreased within cells treated with 1.25 mg/mL HPE (**P < 0.01, Fig 1B). Together, these data suggest that sorghum HPE treatment is sufficient to restrict L. pneumophila intracellular replication in a dose dependent manner.

Figure 1.

Figure 1.

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High-polyphenol sorghum extract (HPE) enhances bacterial killing by RAW 264.7 macrophages. (A) Quantification L. pneumophila ∆flaA recovered from RAW 264.7 macrophages treated with either HPE (1.25 mg/mL) or vehicle control. (B) Quantification L. pneumophila ∆flaA recovered from RAW 264.7 macrophages treated with either 1.25 mg/mL, 0.625 mg/mL or volume equivalent vehicle. (C) Quantification L. pneumophila dotA::Tn recovered from RAW 264.7 macrophages treated with either 1.25 mg/mL, 0.625 mg/mL or volume equivalent vehicle. Colony forming units (CFU) were enumerated at the indicated times post-infection. Data are presented as mean ± standard deviation (s.d.) of samples in triplicates. Asterisks denote statistical significance (**P < 0.01, *P < 0.05) by Students t-test compared to vehicle control. Data are representative of at least two independent experiments.

We subsequently evaluated whether HPE-mediated growth defects were restricted to replicating bacteria. RAW264.7 cells were treated with either vehicle control, 0.625 mg/mL, or 1.25 mg/mL HPE and infected with a strain of L. pneumophila with a loss-of-function mutation in dotA (dotA::Tn), which encodes a component of the Dot/Icm secretion system that is essential for effector translocation and thus bacterial intracellular replication. Although not replicating, the dotA::Tn strain persists within RAW 264.7 treated with vehicle control up to 48 h; however, this strain was cleared more rapidly from RAW 264.7 cells treated with HPE in a dose-dependent manner (Fig 1C). These data suggest that HPE-treatment enhances the microbicidal activity of RAW 264.7 cells.

Legionella pneumophila replication in vitro is not attenuated by sorghum HPE

We subsequently evaluated whether HPE has antimicrobial activity against L. pneumophila in vitro. Plate-grown L. pneumophila were sub-cultured into rich liquid medium in the presence of either 1.25 mg/mL HPE or vehicle control and L. pneumophila was quantified over 24 h by OD600 measurement or quantification of CFU (see Materials and Methods). Addition of 1.25 mg/mL HPE to rich medium did not impair L. pneumophila replication compared to vehicle control (Fig 2), suggesting that HPE is not microbicidal on its own. Thus, HPE-mediated restriction of L. pneumophila intracellular replication is likely not due to microbicidal activity of HPE.

Figure 2.

Figure 2.

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Sorghum HPE does not attenuate L. pneumophila replication in vitro. L. pneumophila was grown in the presence of 1.25 mg/mL HPE or volume equivalent of vehicle over 24 and (A) OD600 was measured or (B) CFU were quantified at the indicated times. Data are presented as mean ± s.d. of samples in triplicates. Data are representative of two independent experiments.

Attenuation of L. pneumophila intracellular replication is not due to HPE-mediated macrophage cell death

HPE was shown to induce death in immortalized carcinoma cell lines (Smolensky et al. 2018). Thus, we evaluated whether restriction of L. pneumophila by HPE was due to death of RAW 264.7 cells. To determine whether HPE was toxic to RAW 264.7 cells, we utilized a lactate dehydrogenase (LDH) release assay, which is used to quantify cell lysis. To determine if HPE was toxic to RAW 264.7 cells, cells were treated with 0.625 mg/mL or 1.25 mg/mL HPE or vehicle control and cytotoxicity was quantified by LDH release assay (see Materials and Methods). We observed low levels of cytotoxicity (< 10%) in both vehicle and HPE-treated cells (Fig 3A). To determine if RAW 264.7 cell viability in the presence of HPE was affected by L. pneumophila infection, cytotoxicity of HPE or vehicle treated cells infected with L. pneumophila was quantified. We found that there were no differences in viability of L. pneumophila-infected RAW 264.7 cells treated with HPE and vehicle treated cells compared to uninfected cells treated with vehicle alone (Fig 3A). Thus, HPE-mediated restriction of L. pneumophila intracellular replication is independent of host cell lysis.

Figure 3.

Figure 3.

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HPE does not impair RAW 264.7 cell viability. (A) RAW 264.7 cell viability was measured by LDH release assay after 6 h of treatment with either 0.625 mg/mL HPE, 1.25 mg/mL HPE or volume equivalent of vehicle in the presence or absence of L. pneumophila ∆flaA infection (MOI of 1), as indicated. Lysis solution was used as a control for 100% cytotoxicity. (B) RAW 264.7 cells were treated with 1.25 mg/mL HPE or volume equivalent of vehicle in the presence or absence of L. pneumophila ∆flaA (Lpn) infection (MOI of 1) for 4 h and abundance of full-length caspase 3 was visualized by western blotting (left panel) and quantified by densitometric analysis relative to the β-actin loading control (right panel). As a control for apoptosis, cells were treated with 10 µM staurosporine (STS) for 3 h. (C) Uninfected (left panel) and L. pneumophila infected (moi of 1; right panel) RAW cell viability quantified by MTT assay in the presence of 0.625 mg/mL, 1.25 mg/mL, or vehicle control. n.s. = not significant. Data shown are representative of at least two independent experiments.

Previous studies revealed that HPE can cause apoptosis of tumor cells. Initial stages of apoptosis are not accompanied by cell lysis and thus would not be detectable by LDH release assay. Therefore, to determine if HPE treatment caused RAW 264.7 cells to undergo apoptosis, we used western blot and densitometry to quantify full length caspase 3 compared to actin. We were unable to visualize the cleaved fragment of caspase 3; however, RAW 264.7 cells treated with staurosporine (STS), which induces apoptosis, contained decreased levels of full-length caspase 3 compared to untreated cells (Fig 3B), indicating that decreased abundance of full-length caspase 3 is evidence of apoptosis. Caspase 3 levels were unchanged in cells treated with HPE compared to the vehicle control and this was also not affected by L. pneumophila infection (Fig 3). Thus, HPE-mediated apoptosis of RAW 264.7 cells is likely not the reason for decreased L. pneumophila intracellular replication.

To further define the influence of HPE on RAW cell viability, we performed an MTT cell proliferation assay over 48 h. This assay measures conversion of MTT (3-[4,5-dimethylthiazol-2-y1]-2,5 diphenyl tetrazolium bromide) into formazan crystals, which occurs only in living, metabolically active cells (Meerloo, Kaspers and Cloos 2011). Thus, infected (MOI = 1) or uninfected RAW 264.7 cells were incubated in the presence of either vehicle control, 0.625 mg/mL HPE, or 1.25 mg/mL HPE and cell viability was quantified over 48 h (see Materials and Methods). HPE treatment did not impair viability of uninfected RAW cells relative to vehicle control at either concentration (Fig 3C). Importantly, HPE treatment did not impair RAW cell viability during Lpn infection compared to vehicle control over 48 h (Fig 3C). Together, our data demonstrate that HPE-mediated attenuation of L. pneumophila replication is not due to host cell death.

HPE treatment enhances tumor necrosis factor (TNF) and interleukin (IL)-6 secretion from L. pneumophila-infected macrophages

Previous work demonstrated that EGCg increased TNF, but not IL-6, secretion from L. pneumophila-infected macrophages (Matsunaga et al. 2001). We therefore hypothesized that sorghum polyphenols may also augment TNF secretion by L. pneumophila-infected macrophages. To test this hypothesis, we quantified TNF and IL-6 secretion from RAW cells infected with L. pneumophila (MOI of 1) and treated with the indicated concentrations of HPE or vehicle control for 6 h, 24 h or 48 h. Interestingly, we found HPE treatment resulted in a significant increase in TNF secretion from both L. pneumophila infected and uninfected RAW cells (*P < 0.05, **P < 0.01, Fig 4A). IL-6 secretion from uninfected RAW cells was significantly increased at 48 h post-treatment whereas L. pneumophila infected and HPE-treated RAW cells secreted more IL-6 at both 24 h and 48 h after treatment (*P < 0.05, **P < 0.01, Fig 4B). Thus, HPE enhances secretion of both TNF and IL-6 from RAW macrophages, and IL-6 secretion is further augmented by L. pneumophila infection.

Figure 4.

Figure 4.

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Influence of HPE on RAW 264.7 macrophages. ELISA to quantify (A) TNF and (B) IL-6 produced by RAW cells alone (left panel) or infected with L. pneumophila ∆flaA (moi of 1) (right panel) that were treated with either 0.625 mg/mL, 1.25 mg/mL HPE or vehicle for the indicated time points up to 48 h. Asterisks indicate statistical significance by Students’ t-test (*P < 0.05, **P < 0.01) compared to vehicle control. Data are shown as mean ± s.d. of samples in triplicates and are representative of three independent experiments.

DISCUSSION

In this study, we revealed that polyphenols from sorghum bran are capable of increasing macrophage restriction of L. pneumophila in culture. High-polyphenol extracts (HPE) attenuated L. pneumophila replication in a dose-dependent manner within macrophages but not in rich media in vitro. HPE treatment also resulted in enhanced clearance of the avirulent dotA::Tn strain, suggesting that HPE-treated macrophages actively eliminate intracellular L. pneumophila as opposed to stalling replication. We discovered that HPE enhances production of both TNF and IL-6 secretion from RAW 264.7 cells infected with L. pneumophila. Although IL-6 secretion was not increased by EGCg-treated macrophages, this may be due to cell line specific effects since the previous study was performed in the MH-S alveolar macrophage cell line (Matsunaga et al. 2001). However, these data together collectively suggest that upregulation of cytokine secretion is a potentially ubiquitous mechanism by which polyphenols attenuate L. pneumophila.

Sorghum polyphenols comprise a family of compounds with diverse chemical structures and biological activities. The major components of sorghum polyphenols are tannins, phenolic acids and flavonoids, all of which have documented health benefits (Awika and Rooney 2004). Our work demonstrates a role for polyphenols in bacterial restriction, but the specific component(s) of the extract responsible for this activity is unknown. Since EGCg is a flavonoid, it is possible that sorghum contains analogous flavonoids that function similarly since several sorghum genotypes produce relatively high levels of flavonoids compared to other crops (Dykes et al. 2009). This hypothesis is further supported by similar increases in cytokine secretion induced by sorghum and EGCg polyphenols. However, the component(s) of sorghum polyphenol extracts responsible for enhanced macrophage killing will be the subject of future investigation.

Sorghum polyphenols potently inhibit growth of cancer cells through generation of reactive oxygen species (ROS), apoptosis and cell cycle arrest (Smolensky et al. 2018). Viability of HT-29, HepG2 and Caco2 cells was significantly decreased following treatment with sorghum polyphenols (Awika et al. 2009; Smolensky et al. 2018). Sorghum polyphenols were also found to dampen symptoms of colitis in a mouse model of disease, indicating potential to alleviate colitis and potentially prevent colitis-associated cancer (Ritchie et al. 2017). However, sorghum HPE did not impair RAW cell replication or cause apoptosis or necrosis of RAW cells, which were derived from a murine leukemia virus-induced tumor (Raschke et al. 1978). Thus, sorghum polyphenols have distinct effects on epithelial cells and macrophages.

The antimicrobial properties of non-sorghum polyphenols have been documented in vitro and in vivo. The majority of these studies have focused on food-borne pathogens, but reveal the potential for natural and sustainable sources of antimicrobial compounds. While in vitro restriction of many bacterial, fungal and viral pathogens has been evidenced [reviewed in (Daglia 2012; Bouarab-Chibane et al. 2019; Othman, Sleiman and Abdel-Massih 2019)], polyphenols are also capable of specific inhibition of pathogen virulence. A striking example of this is the ability of tannic acid and n-propyl gallate to protect mice from Helicobacter pylori-mediated disease (Ruggiero et al. 2006). Ruggiero et al. discovered that administration of polyphenols to mice in drinking water decreased H. pylori-mediated gastritis and bacterial load in the stomachs of mice. Polyphenols from red wine and tea were also sufficient to inhibit the function of the H. pylori vacuolating toxin (VacA), demonstrating specific anti-virulence properties for these compounds (Tombola et al. 2003; Ruggiero et al. 2006). Moreover, infectivity and replication of the obligate intracellular pathogen Chlamydia pneumoniae was also inhibited by diverse plant polyphenols (Alvesalo et al. 2006).

Plant polyphenols have been demonstrated to modulate mammalian immunity [reviewed in (Ding, Jiang and Fang 2018)]. Namely, polyphenols have anti-inflammatory and antioxidant activity. The increase in TNF secretion from HPE-treated macrophages we observed is intriguing since other plant polyphenols suppress pro-inflammatory gene expression (Chan et al. 2018; Du et al. 2018). Interestingly, treatment with EGCg alone did not enhance TNF production by macrophages to the same extent as sorghum HPE after 24 h of treatment. Increased inflammatory cytokine production observed for sorghum polyphenols could be due to (1) the quantity of compound used; or (2) the mixture of polyphenols contained in our extract as compared to use of purified single polyphenols in other studies (Matsunaga et al. 2001; Du et al. 2018). It is also possible that the MH-S cells used in the previous study secrete less TNF downstream of pro-inflammatory stimuli compared to RAW cells. While this has not been directly evaluated, RAW cells secrete more TNF than primary mouse macrophages when stimulated with bacterial lipopolysaccharide (Berghaus et al. 2010). Whether sorghum HPE induces TNF production via the same mechanism as EGCg is unknown, but our work suggests that plant polyphenols can exert antimicrobial activity against intracellular pathogens via enhanced pro-inflammatory cytokine production.

Ultimately, our work provides the foundation for future investigation into use of sorghum polyphenols to combat bacterial infection. Whether sorghum HPE can restrict diverse microbial pathogens and/or L. pneumophila in animal models of infection will be the subject of future studies.

ACKNOWLEDGEMENTS

This work was funded by U.S. Department of Agriculture Research Service project # 3020–43440-001–00D and institutional start-up funds from Kansas State University (to SRS). Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

Conflicts of interests

None declared.

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