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An Investigation into the Immunomodulatory Activities of Sutherlandia frutescens in Healthy Mice

Sutherlandia frutescens is a medicinal plant that has been traditionally used in southern Africa for cancers, infections, and inflammatory conditions. We recently published experiments demonstrating that an aqueous extract of S. frutescens possessed potent immune-stimulatory activity. This work was carried out with murine macrophages, an immune cell type that plays a pivotal role in host defense from infection and in shaping host inflammatory and immune responses. Here, we conducted a series of follow-up experiments to explore the impact of consuming S. frutescens on host response to bacterial challenge using healthy mice. We found that feeding mice a diet containing S. frutescens failed to significantly alter host response to systemic infection by either a gram-positive or gram-negative bacterium (i.e., L. monocytogenes and E. coli, respectively). In contrast to the in vitro observations, we found no evidence that S. frutescens consumption stimulated in vivo inflammatory responses; instead, consumption of S. frutescens tended to diminish in vivo inflammatory responses. Several possible reasons for this are discussed.


Introduction
Recently we used a murine macrophage cell line and primary murine macrophages to demonstrate that extracts of S. frutescens have potent immune-stimulatory activities. [1] Specifically, short-term treatment with a crude aqueous extract of S. frutescens or with a polysaccharideenriched fraction of S. frutescens increased macrophage production of reactive oxygen species (ROS) and nitric oxide (NO). ROS are a group of highly reactive free radical and non-radical molecules, including superoxide (O 2 • -), hydrogen peroxide (H 2 O 2 ), hydroxyl radicals (OH•), hypochloric acid (HOCl), and peroxynitrite (ONOO-). [2] NO has been recognized as a versatile mediator of immune responses. [3] Both ROS and NO play critical roles in host defense to infection. [4][5][6] Additionally, the aqueous extract and polysaccharides-enriched fractions of S. frutescens activated NF-κB signaling in murine macrophages resulting in an increased period, mice were randomly assigned into three dietary treatment groups: 0, 0.25 or 1.0% (wt/ wt) ground S. frutescens leaf powder in AIN-93G basal rodent diet. [13] Experimental diets The botanical-containing diets were made using the ground powder of the vegetative parts of Sutherlandia frutescens purchased from Big Tree Nutraceutical (Fish Hoek, South Africa) and authenticated as described previously. [1] According to previous reports, the most commonly used dose of S. frutescens in humans is 2.5 g dry materials (leaf) per day. [14] For a 70 kg adult human, this dose of S. frutescens intake would be equivalent to 35.7 mg/kg per day. Therefore, we used 36 mg/kg per day to calculate the human equivalent dose for experimental diets in our mouse feeding studies. The human equivalent dose was calculated using a formula that adjusts for differences in metabolic body size and is described in detail elsewhere. [15,16] For mice this dose was estimated to be 411 mg/kg body weight per day. The average body weight of our healthy adult mice was~25 g, therefore we estimated that each mouse would need to consume 11 mg S. frutescens to achieve the dosage of 411 mg/kg BW per day. Food consumption for these mice was expected to be 4-5 g/d based on our previous experience and reports in the literature, thus to deliver~11 mg of S. frutescens per day, the diets should contain approximately 0.25% (g/100 g) of the ground S. frutescens material. We also prepared a diet containing four times this dosage (i.e., 1.0%) of S. frutescens to account for unexpected and unknown differences between mice and humans relative to the uptake and metabolism of putative bioactive compounds in S. frutescens. All diets were nutritionally complete [13] (i.e., AIN-93G diets) and identical, except for the presence of S. frutescens and the small amount of cornstarch that was removed to account for the addition of this botanical ( Table 1).

In vivo bacterial challenges
To assess the impact of dietary S. frutescens on host-resistance from infection by L. monocytogenes we used healthy male and female BALB/c. This strain of mice is known to be susceptible to infection from L. monocytogenes. [17] After 4 weeks of consuming the experimental diets, all mice received an intravenous injection with~10 4 cfu of L. monocytogenes (EGD strain) in 0.2 mL of sterile, endotoxin-free phosphate-buffered saline.
To investigate the impact of dietary S. frutescens on host response to an E. coli infection, male and female C57BL/6 mice were used. After consuming one of the experimental diets for 4 weeks, mice were injected intraperitoneally with~10 8 colony-forming units (cfu) of E. coli (K-12 strain) in 1 mL of sterile PBS.
Following bacterial challenge, mice were monitored twice daily for clinical symptoms of illness using a standard scoring sheet designed for monitoring and recording pain/discomfort in rodents and approved by the Animal Care and Use Committee of the University of Missouri. Any mice showing signs of pain or distress (e.g., ruffled fur, shallow breathing, hunched back) were immediately and humanely euthanized. As expected, no mice in our experiments experienced clinical symptoms requiring premature termination, however, we did record three premature deaths during the E. coli challenge experiments (i.e., female mice in CONTROL and 0.25% SF groups and a male mouse in the 1.0% SF group) and one unexpected death during the L. monocytogenes experiments (i.e., a female mouse in the 0.25% SF group).

Sickness behavior
The impact of S. frutescens on spontaneous locomotor activity before and during an infection was measured by mini-emitters as described by Spiers et al. [18] Representative mice (n = 4 of each sex) within each diet treatment group were lightly anesthetized and mini-emitters were surgical implanted subcutaneously along the spine just below the scapulas. Following a 4-day post-surgery recovery period, activity data were recorded every 5 seconds with accumulated events for every 20 min period for a 3-day period prior to and 3 days or 36 hours following the challenge with L. monocytogenes or E. coli, respectively, following the bacterial challenge.

Measurement of host clearance of the bacterial challenge
We determined the number of L. monocytogenes present in the spleen and liver of mice 3 days following the challenge. This timeframe best reflects the capacity of the innate immune system to control the cell-to-cell spread and proliferation of this intracellular pathogen. [19,20] Therefore, 3 days following the challenge with L. monocytogenes mice were humanely killed and the liver and spleen from each mouse was aseptically collected and weighed. One hundred milligram portions of each tissue were transferred to 1.5 mL sterile centrifuge tubes containing 0.2 mL of sterile PBS and 100 mg of 0.5mm zirconium oxide beads. Tubes were kept on ice until processed for bacterial load determination as described below.
The bacterial load in the liver and spleen was determined by a fluorescence-based microplate assay. [21] The assay is based on the reduction of the non-fluorescent dye resazurin to a fluorescent product, resorufin, by bacterial enzyme activity. Liver and spleen samples were collected and then homogenized using a Bullet Blender TM (Next Advance, Inc., New York, United States of America) according to the manufacturer's instructions and described elsewhere. [22] One hundred microlitersof the homogenates were diluted to 1.0 mL (10-fold) with PBS containing 0.2% saponin, then 7 μL of these diluted homogenates were added to wells of a black 384 well plate in triplicate. Each well contained 50 μL of 0.01% resazurin in LB medium. Resorufin fluorescence was measured every 20 minutes for 10 hours. The time required to reach 8000 RFU (T 8K ) was determined for each sample and cfu/mg tissue calculated using the standard curve. A suspension containing 1x10 9 cfu/mL of L. monocytogenes or E. coli was prepared, and serial 10-fold dilutions of this suspension were done in ice-cold LB medium containing 0.01% resazurin to create standards in the range 1x10 2 to 1x10 8 cfu/mL. Fifty microliters of these standards were added in triplicate to a pre-chilled black 384 well plate and fluorescence (excitation, 530 nm; emission, 590 nm) was read every 20 min for 10 h using a fluorescence plate reader (BioTek Synergy HT). A standard curve was then constructed that relates the log of the cfu/well to the time required to reach a standard fluorescence of 8000 relative fluorescence units (RFU). The limit of detection for this assay was approximately 4x10 3 cfu/g tissue. For liver samples that were below the limit of detection in the fluorescence assay, cfu/mg tissue was calculated using a traditional microbiological plate count approach. For these liver samples only, 100 μL of the diluted homogenate was plated on TSAY (tryptic soy agar + yeast extract) plates. Plates were incubated overnight at 37°C and colonies counted manually the next day.

In vivo host inflammatory response
In order to assess the impact of S. frutescens consumption on host inflammatory response, BALB/c and C57BL/6 mice were fed with experimental diets as described previously, followed by an intraperitoneal injection with L. monocytogenes (EGD strain, 5 x 10 6 cfu/mouse) and LPS (from E. coli 026:B6, 20 μg/mouse), respectively. Mice were anesthetized 2 hr post-LPS injection or 24 hrs. after L. monocytogenes challenge and blood was drawn by cardiac puncture. After allowing the blood to clot on ice for 30 minutes, serum was separated by centrifugation (1000× g, 20 min.) and stored at -80°C for later analyses of inflammatory cytokines/chemokines as described below.

Isolation and stimulation of peritoneal macrophages
Thioglycolate-elicited peritoneal macrophages were isolated from healthy mice as described previously. [23] Crude (i.e., unfractionated) peritoneal cells were re-suspended in Dulbecco's Modified Eagle's Medium (DMEM) with 5% FBS, and seeded into a 96-well plate at a density of 1×10 5 cells/well. Cells were cultured in the incubator at 37°C with 5% CO 2 for 3 h. The nonadherent cells were removed by washing with PBS, leaving behind a monolayer of macrophages (typically > 95% purity).
We used a commercial synthetic diacylated lipoprotein (i.e., Pam2CSK, Invivogen, San Diego, CA, USA) to mimic L. monocytogenes infection for these ex vivo experiments, while LPS served to model E. coli infection. The production of inflammatory cytokines/chemokines by elicited cells was also evaluated. We conducted dose-response experiments to determine the optimal concentration for each of these inflammatory stimuli in our assay. The concentrations used (i.e., 5 ng of Pam2CSK or 100 ng of LPS per mL of culture medium) provided a robust, but sub-maximal stimulation. Primary macrophages were co-cultured with LPS or Pam2CSK for 24 h, and the cell culture medium was harvested, stored at -80°C for later cytokines and chemokines quantitation.

Statistical analyses
All data were analyzed by one-way analysis of variance (ANOVA) using SAS 9.3 software (SAS Institute Inc., Cary, NC, USA). The post-comparisons were performed using Tukey's multiple comparison tests. A p < 0.05 was considered to indicate statistical significance. Data are presented as means ± SEM, except on those occasions when unequal variances existed between independent trials or assays, then LSmeans ± pooled SEM are reported.

Host resistance to bacterial infection
After consuming diets with 0.25% or 1.0% S. frutescens for~4 weeks, the average body weight of female and male mice (both BALB/c and C57BL/6 strains) were not significantly different within sexes from that of control mice (see S1 and S2 Tables, respectively). Challenging mice with L. monocytogenes resulted in a loss of 5 to 10% of their body weight during the next few days, regardless of which experimental diet the mice were consuming. Similarly, consumption of S. frutescens had no discernable impact on the loss of body weight in mice following an E. coli challenge. Additionally, there were no significant differences across diet treatment groups in liver or spleen weight following either bacterial challenge.
The impact of consuming S. frutescens containing diets on host defense against infection from two commonly studied bacterial pathogens is shown in Figs 1 and 2. In contrast to our expectations, there was little indication that consumption of S. frutescens enhanced host clearance of either of these bacteria during the early stages post-challenge. We did however, note a trend (p < 0.08) for a modestly lower burden (~0.5 log 10 ) of L. monocytogenes in the livers of male BALB/c mice fed the 0.25% S. frutescens diets compared with the mice fed the control (0% SF) or 1% S. frutescens diets (Fig 1C). Consumption of S. frutescens appeared to have no noticeable impact on the burden of L. monocytogenes in the spleens of male mice, nor on bacterial burden in the livers and spleens of the female BALB/c mice (Fig 1A, 1B and 1D). Additionally, there were no statistically significant differences in E. coli burden in livers or spleens of female or male C57BL/6 mice fed either S. frutescens-containing diets compared to the control diet (Fig 2A-2D).

Impact of infection on spontaneous activity (i.e., sickness behavior)
We implanted mini-emitters in a separate group of mice to monitor the impact of S. frutescens consumption on overall wellbeing of the mice immediately prior to and during bacterial challenges. The spontaneous activity (i.e., random locomotion within the cage) of four individual female mice in each dietary treatment group before and after infection with either L. monocytogenes or E. coli is shown in Fig 3 and Fig 4, respectively). Not surprisingly, spontaneous locomotor activity dramatically declined following the infectious challenge with either bacterial pathogen. Dietary S. frutescens consumption appeared to have no discernable impact on this activity either before or during the infection. While data from female mice are shown, similar results were observed for male mice similarly challenged with L. monocytogenes or E. coli (see S1 and S2 Figs). After consuming diets for~4 wks, all mice received an intravenous injection with~10 4 cfu of L. monocytogenes (EGD strain) in 0.2 mL of sterile, endotoxin-free phosphate-buffered saline. Liver and spleen samples were harvested three days after challenge and colony-forming units (cfu) were determined by a fluorescence-based microplate assay of tissue homogenates. Each symbol represents the data from a single mouse with the median and intra-quartile range shown. In vivo production of cytokines and chemokines Systemic infection with L. monocytogenes in previously healthy BALB/c mice induces the production of numerous cytokines and chemokines quantified from blood samples collected 24 h post-challenge (Table 2). Monocyte chemotactic protein-1 (MCP-1, CCL2), interferon(IFN)-γ, and interleukin-6 (IL-6) were the dominant factors detected in the blood of L. monocytogenesinfected mice. We observed no significant differences in the measured cytokines and chemokines based on the experimental diet consumed prior to and 24 hours following the infectious challenge. To evaluate the impact of S. frutescens intake on the inflammatory response in healthy mice we challenged them with lipopolysaccharide (LPS) from E. coli. Mice received an intraperitoneal injection with 20 μg of LPS followed by blood collection 2 hours later. We observed that consumption of S. frutescens by healthy C57BL/6 mice tended (p < 0.09) to reduce by nearly  50% the production of tumor necrosis factor-alpha (TNF-α) in response to a LPS challenge ( Table 3). As can be seen in Table 3, however, we failed to detect any other diet-induced differences in the circulating cytokines, chemokines, or growth factors quantified. We also did not observe any significant impact of S. frutescens consumption on basal or background cytokine/ chemokine levels in mice injected with vehicle (i.e., PBS) alone (see S1 Table).

Ex vivo cytokines and chemokines production
Primary peritoneal macrophages isolated from mice fed S. frutescens produced significantly less IL-1α following stimulation with LPS compared to macrophages isolated from mice fed the control diet (S4 Table). In contrast, ex vivo production of other cytokines/chemokines by LPS-or Pam2CSK-stimulated macrophages were not significantly impacted by dietary S. frutescens (S4 and S5 Tables).

Discussion
With most botanicals for which immune-modulating activity is claimed the evidence of their activity is limited to actions on isolated immune cells (i.e., in vitro). Previously, we reported  that a hot-water extract made from the medicinal plant S. frutescens potently activated murine macrophages, resulting in increased production of ROS and NO. [1] Production of ROS and NO by innate immune cells are early and critical components of host defense against infection from bacterial pathogens. [4,5] We found that this immune-stimulating activity was associated with a polysaccharide-enriched fraction of this aqueous extract. Zhang et al. [24] found that pectin-type polysaccharides isolated from Lessertia frutescens (an alternative name for Sutherlandia frutescens) stimulated innate immune cells via complement fixation. The complement cascade is a part of the host's immune response that promotes host clearance of microbes and damaged cells. There exists a large body of research showing that numerous botanical polysaccharides possess in vitro immune-stimulatory activity. [9] In the present study, however, we investigated the potential benefits of unfractionated vegetative parts of this botanical in response to bacterial infection. To our knowledge this is the first investigation to examine in vivo and ex vivo host anti-bacterial activity associated with the consumption of S. frutescens.
In the present study, we found that feeding healthy mice diets containing S. frutescens had little impact on the host's immune response to either of the bacterial pathogens we tested. In light of the results from our prior in vitro experiments, we designed the present experiments to focus on the early or innate elements of the host's immune response to infection. Following the following challenge, following sera isolation, specific cytokines and chemokines concentrations were determined using a commercial multiplex beads-based assay system. Data shown are from 12 mice from each diet group (i.e., 6 mice per diet treatment group in each of two independent trials), with values representing the means ± SEM. Values for T-cell derived cytokines (i.e., IL-2, IL-4, IL-5, IL-7, IL-9, IL-13, IL-15, and IL-17) tended to be very low in concentration; were not significantly impacted by the diet treatment, thus were not reported. b The impact of the diet intervention/treatment was tested in SAS by ANOVA, using contrast with interaction between the two independent trials/experiments with the main effect p-value shown in the 4 th column.
c Abbreviations (refer to intravenous administration of L. monocytogenes, these gram-positive bacterium are rapidly taken up by phagocytes, primarily in the spleen and liver. [17] Intracellular pathogens, such as L. monocytogenes, successfully replicate within these phagocytes unless the generation of ROS and NOS are stimulated. [12] Whether and to what extent the host is able to prevent replication and spread of this intracellular pathogen during the early stages of infection (e.g., 3 days postchallenge) is highly dependent upon the production of a variety of host factors, including: TNF-α, [25] IFN-γ, [26] IL-6 [27] and MCP-1 [28]. Therefore, we expected to find evidence that this botanical modified the early production of these host factors and that mice fed diets containing S. frutescens would contain fewer of these bacteria. However, when we sampled the blood just 24 hours post-challenge with L. monocytogenes we found no indication that consumption of S. frutescens had altered the production of these key cytokines and chemokines. Consistent with this result was our failure to observe a significant difference in the number of L. monocytogenes bacterium in either the spleen or liver of mice fed diets containing S. frutescens. Similar to our results examining in vivo host response to a L. monocytogenes infection, we found that dietary S. frutescens failed to modulate the host response to a challenge with a common gram-negative organism, E. coli. Yet, we did find some indication that consumption of S. frutescens might be having a discernable impact on in vivo production of a key pro-inflammatory mediator, tumor necrosis factor-α (TNF-α). When otherwise healthy mice were fed a diet containing S. frutescens they showed a trend (i.e., p < 0.09) to produce nearly 50% less TNF-α following an injection of E. coli endotoxin (i.e., LPS). One might expect that any treatment that reduces in vivo production of this inflammatory mediator would be associated with notable changes in sickness behavior. [29] To examine this possibility we used voluntary locomotion as a surrogate for sickness behavior as described elsewhere. [29] Yet, we did not observed any indication that S. frutescens feeding affected voluntary locomotion prior to and during an active infection from either E. coli or during an infection from L. monocytogenes. We measured many cytokines and chemokines that play important roles in shaping the early immune response to infection, yet ex vivo production of IL-1α was the only one that was significantly affected by dietary S. frutescens consumption. Recently, Africa and Smith [30] reported that treatment of co-cultures of human monocytes and endothelial cells with an aqueous extract of S. frutescens greatly diminished the production of IL-1 in response to stimulation with HIV-1 antigens. That a recent human clinical trial found that consumption of milled vegetative parts of S. frutescens in capsules might promote tuberculosis, [31] is consistent with what would be expected if host production of TNF-α and IL-1β was diminished. [32,33] The present study was limited in a number of ways. First, the amount of S. frutescens included in the experimental diets may have been too low to elicit an observable effect. While we sought to use human equivalent dosing calculations derived from the research literature we may have been too conservative in setting our diet inclusion levels at 0.25 and 1.0%, by weight. It is possible that inclusion of more S. frutescens may have had a greater impact on in vivo host immune/inflammatory responses. Excessive production of inflammatory mediators such as ROS, NOS, and cytokines can result in tissue damage and even death. [34,35] Consistent with this was our preliminary findings that mice fed a diet containing 2% (wt/wt) of ground leaves from S. frutescens experienced lower survival rates (i.e., 17%, 1 of 6 mice survived) than mice fed a diet devoid of this botanical following an LD 50 E. coli challenge.
A second limitation emerged based on the contradictory findings generated in vitro with this botanical. We have reported that ethanolic extracts of S. frutescens possess potent antiinflammatory activities [36], while aqueous extracts of S. frutescens stimulate macrophage inflammatory responses. [1] Therefore, inclusion of unfractionated botanical in the diet may have failed to noticeably modify host response to bacterial challenge due to a cancelling effect of two or more components with opposite or contradictory activities. In an attempt to address this possibility, we conducted a small follow-up experiment where we administered by oral gavage either an aqueous or ethanolic extract of S. frutescens to a limited number of mice. After 7 days, half of the mice in each group were challenged with LPS or Pam2CSK and blood was collected 2 hours later. However, we found no discernible effect of either extract of S. frutescens on in vivo production of inflammatory cytokines and chemokines.
Third, our experimental design was limited in that we only included single time points for the assessment of bacterial clearance and circulating inflammatory cytokines and chemokines. It is possible that S. frutescens may have had a significant impact on host responses at different times after the immune challenges. Another obvious limitation of our study was that we only examined host response to a single gram-negative bacterium and single gram-positive bacterium (i.e., E. coli and L. monocytogenes, respectively). Host response to other pathogens, bacterial as well as viral, can vary tremendously. In short, our failure to observe significant modulation of host response to these two pathogens cannot exclude the possibility that treatment with S. frutescens might not provide benefits for host immunity against other infectious agents.

Conclusions
In conclusion, this study is the first to examine whether oral consumption of unfractionated vegetative parts of S. frutescens can modify the host immune response in vivo. We found that host clearance of a gram-positive and a gram-negative bacterium from mice was not significantly altered by S. frutescens intake. S. frutescens exhibited only a modest impact on the production of a couple of select inflammatory cytokines. Whether such changes occur in humans, and if they do, whether they would provide clinically relevant benefits to individuals using this medicinal plant remains uncertain.
Supporting Information S1 Fig. Impact of Dietary S. frutescens on Spontaneous Activity (i.e., sickness behavior) of Male Mice After an Experimental Infection with either L. monocytogenes. Healthy male BALB/c weanling mice were fed experimental diets containing one of three doses of S. frutescens (i.e., 0, 0.25 or 1% by wt) for 3-4 wks. Mice were housed in pairs in a vivarium with a 12 hr light:dark cycle at a room temperature between 22-25°C and a relative humidity of 50-60% for the entirety of the study. One of each pair of mice had a mini-emitter surgically implanted under the skin along their spine between their shoulders. At~1 week post-surgery mice received an injection of 10 4 cfu of L. monocytogenes, EGD strain. Each mouse's movement/ activity was recorded every 5 seconds and reported as accumulated events for every 20 minute period. Each tick mark on the X-axis represents an hour. Data shown represent the mean activity (#events) of four mice in each diet treatment group (n = 4/diet treatment group). (TIF) S2 Fig. Impact of Dietary S. frutescens on Spontaneous Activity (i.e., sickness behavior) of Male Mice After an Experimental Infection with E. coli. Healthy male C57BL/6 weanling mice were fed experimental diets containing one of three doses of S. frutescens (i.e., 0, 0.25 or 1% by wt) for 3-4 wks. Mice were housed in pairs in a vivarium with a 12 hr light:dark cycle at a room temperature between 22-25°C and a relative humidity of 50-60% for the entirety of the study. One of each pair of mice had a mini-emitter surgically implanted under the skin along their spine between their shoulders. At~1 week post-surgery mice received an injection of 10 8 cfu of E. coli K12 strain. Each mouse's movement/activity was recorded every 5 seconds and reported as accumulated events for every 20 minute period. Each tick mark on the X-axis represents an hour. Data shown represent the mean activity (#events) of four mice in each diet treatment group (n = 4/diet treatment group). (TIF) S1 Table. Impact of Dietary S. frutescens on Body, Liver, and Spleen Weight of Mice Following an L. monocytogenes challenge. Healthy female and male BALB/c weanling mice were fed experimental diets containing one of three doses of S. frutescens (i.e., 0, 0.25 or 1% by wt) for 3-4 wks. At~7 wk of age, mice were weighed (i.e., pre-challenge), then injected intravenously with~10 4 cfu of L. monocytogenes, EGD strain. Three days following the challenge, mice were re-weighed (i.e., post-challenge), then humanely killed for the collection of liver and spleen, which were weighed and then homogenized for the subsequent enumeration of bacteria. All values are expressed as means ± SEM (n = 15-16 per dietary treatment group). (DOCX) S2 Table. Impact of Dietary S. frutescens on Body, Liver, and Spleen Weight of Mice Following an E. coli challenge. Healthy female and male C57Bl/6 weanling mice were fed experimental diets containing one of three doses of S. frutescens (i.e., 0, 0.25 or 1% by wt) for 3-4 wks. At~7 wk of age, mice were weighed (i.e., pre-challenge), then injected intravenously with 10 8 cfu of E. coli, K12 strain. Two days following the challenge, mice were re-weighed (i.e., post-challenge), then humanely killed for the collection of liver and spleen, which were weighed and then homogenized for the subsequent enumeration of bacteria. All values are expressed as means ± SEM (n = 15-16 per dietary treatment group). (DOCX) S3 Table. Serum Cytokines and Chemokines from Mice 2 hours Following an Injection with Vehicle (PBS). For this study, male C57Bl/6 mice were fed experimental diets containing one of three doses of S. frutescens (0, 0.25 and 1% SF by wt) for 3 wks. Mice were injected at 1 min intervals with 1.0 mL of sterile PBS (i.e., vehicle controls for LPS challenged mice). Two hours following this injection blood was collected from mice and allowed to clot for 30 minutes at room temperature. Serum was collected by centrifugation and stored at -80°C until assayed for cytokines/chemokines using a commercial multiplex kit. Data shown are from eight mice from each diet treatment group (n = 8/trt); values represent means ± SEM (pg/mL). Diet intervention/treatment, as tested by ANOVA, failed to significantly affect any measured parameter (i.e., p > 0.05). (DOCX) S4 Table. Ex Vivo LPS-induced Inflammatory Cytokine and Chemokine Production by Primary Macrophages Isolated from Mice Fed Differing Levels of S. frutescens. For this experiment, male C57BL/6 mice were fed experimental diets containing one of three doses of S. frutescens (0, 0.25 and 1% by wt) for 3-4 wks. Peritoneal macrophages were isolated 3 days following intraperitoneal injection with sterile thioglycolate broth. Adherent cells (i.e., >95% macrophages) were co-cultured with 100 ng/mL of LPS (from E. coli 0111:B4) and 24 h cell later culture supernatants were collected and subsequently diluted 10-fold in FBS-free DMEM, then analyzed for specific cytokines and chemokines using a commercial multiplex beadsbased assay system. Data shown are from twelve mice from each diet treatment group (n = 11-12/trt); all values are LSmeans ± pooled SEM, expressed in pg/mL, unless otherwise indicated. (DOCX) S5 Table. Ex Vivo Pam2CSK-induced Inflammatory Cytokine and Chemokine Production by Primary Macrophages Isolated from Mice Fed Differing Levels of S. frutescens. For this experiment, male C57BL/6 mice were fed experimental diets containing one of three doses of S. frutescens (0, 0.25 and 1% by wt) for 3-4 wks. Peritoneal macrophages were isolated 3 days following intraperitoneal injection with sterile thioglycolate broth. Adherent cells (i.e., >95% macrophages) were co-cultured with 5 ng of Pam2CSK and 24 h later cell culture supernatants were collected and subsequently diluted 10-fold in FBS-free DMEM, then analyzed for specific cytokines and chemokines using a commercial multiplex beads-based assay system. Data shown are from twelve mice from each diet treatment group (n = 12/trt); all values are LSmeans ± pooled SEM, expressed in pg/mL, unless otherwise indicated.