Cytotoxicity Effect of YSH on THP-1 (IC50) SRB

To determine the effect of Yemen Sidr honey on THP-1 cells, the cells were treated with different concentration of YSH (0.3, 0.6, 1.25, 2.5, 5, and 10%), and relative cell number

examined using an SRB assay (Tetrazolium hydroxide-Neutral Red-Sulforhodamine B). The viability of THP-1 cells was reduced in a dose-dependent manner (Fig 3.1A). (When YSH activity was compared to the effect against cancer cells (Figure 2.1A, D), THP-1 cell were less affected). The IC50 for YSH-induced THP-1 cell proliferation was determined to be 6.5% at 48 h.

Therefore, THP-1 cells treated with 1% YSH maintained a high cell viability (Fig 3.1B) and therefore cells were treated with 1% YSH for the rest of the study to induce behavioural changes in the differentiation of THP-1 cells.

A B

Figure 3.1: The concentration-dependent effect of Yemen Sidr honey treatment on THP-1 cells. Cells were treated with freshly prepared YSH at different concentrations to determine the half maximal inhibitory concentration (IC50). (A) An IC50 of 6.5% for cell viability was

determined using an SRB assay. The results were presented as the mean + SEM of three

independent experiments. (B) The cell proliferation assay was performed using the Incucyte live cell imaging count apparatus and software in cells treated with 1% YSH (red) or media control (yellow).

3.2.2 Effect of treatment with YSH on cell cycle progression on THP-1 cells.

To determine if there was a change in the cell cycle progression of THP-1 cells in

response to treatment with YSH, cells were exposed to 1% or 2.5% YSH or 1% sugar for 24, 48, and 72 h and then analyzed for DNA content using flow cytometry. There was no significant induction of cell cycle arrest or apoptosis in response to treatment with YSH at different time points. Treated cells were compared to controls (non-treated cells and 1% sugar). A total of 10,000 events were analyzed by flow cytometry following propidium iodide staining. Results are expressed as the percentage of total cells in each phase of the cell cycle and show no differences in the proportion of cells in the Sub-G1 peak or in G1, S or G2M.

Figure 3.2: Cell cycle analysis of THP-1 cells treated with YSH by flow cytometry.

Flow cytometry profiles of THP-1 cells treated with 1 or 2.5 % YSH or 1% sugar for (24 - 72 h.

The percentage of cells in each phase of the cell cycle was estimated by gating for the fluorescent intensity corresponding to the amount of DNA in each event with respect to non-treated cells. The X-axis represents the DNA content (PI staining) and the Y-axis corresponds to the number of cells.

3.2.3 Macrophage morphology, and polarization after activation of THP-1 cells

To generate a model of macrophage polarization, we used PMA-differentiated human THP-1 monocytes. THP-1 cells were treated with phorbol ester (PMA) for 3 - 5 days as indicated and the transformation from monocyte to macrophage was expected to have been completed as indicated by the changes in cell morphology that were observed. Representative images of the cell morphology before and after treatment are shown (Fig. 3.3A). To assess the morphological change in cell shape from the typical spherical monocyte phenotype to the amoeboid, adherent macrophage phenotype, cells were examined daily using the Incucyte live imaging system (Phase), until their shape was no longer considered spherical. The cells shown in Fig. 3.3A, were all at this macrophage stage. Control experiments were performed to confirm that the THP-1 cells treated with PMA showed the monocyte to-macrophage differentiation and the cells were characterized for increased expression of the CD68 cell surface macrophage marker using flow cytometry analysis (Fig. 3.3B). Untreated THP-1 cells do not express significant levels of CD68, but after 3 days of treatment with PMA, the cells were positive for CD68 staining. The PMA-differentiated human THP-1 monocytes (designated M0 macrophages), were treated with LPS to generate M1 (inflammatory) polarized macrophages or treated with IL-4 to generate M2

polarized macrophages, which represent the two opposite polarized states. The M1 macrophages showed cellular elongation in cell morphology, while the M2 macrophages showed a more rounded morphology (Fig. 3.3A). The differentiated THP-1 cells were also tested for expression of the surface markers CD80 (M1 phenotype) and CD163 (M2 phenotype) using flow cytometry:

increased expression of CD80 was seen on the M1 macrophages and increased expression of

CD163 was seen on the M2 macrophages (Fig. 3.3 C, D). Taken together, these finding indicated the successful polarization of THP-1 monocyte-like cells into M1 and M2-polarized

macrophages.

Macrophages are not proliferative cells unlike the monocyte precursor. The THP-1 cells show reduction in proliferation after receiving the activating treatment (PMA), which is also an indicator of the change in phenotype as these cells differentiate. Cells that received treatment with PMA were compared with the non-treated control cells (monocytes) using Incucyte live imaging counting. The data in (Fig. 3.3F) shows that PMA-activation generated a significantly less proliferative cell population than untreated cells.

Figure 3.3: Detection of macrophage activation, M1/M2 polarization, and proliferation.

(A). Morphology of THP-1 monocytes, (non-differentiated THP-1 cells), THP-1 cells treated with PMA and LPS (M1), and THP-1 cells treated with PMA and IL-4 (M2) were examined using the Incucyte imaging system (100×). (B) the expression of the CD68 macrophage cell surface marker (green) on untreated cells or THP-1 cells treated with PMA was measured by flow cytometry. (C, D) Flow cytometry of THP-1-derived macrophages treated with LPS or IL-4 for cell surface expression of CD80, the marker for M1 polarized macrophages and CD163, the marker for M2 polarized macrophages. Data are presented the mean % of cells, ±SEM. All results show the relative change compared to untreated THP-1 cells (p < 0.05). (E) Incucyte proliferation assay showing macrophage counts in THP-1 cells treated in the absence (yellow) or presence of PMA (red) for 3 days.

E

3.2.4 Effects of honey on M1 and M2 polarization surface marker expression

Having shown that THP-1 cells can be used as a model of macrophage polarization, we examined the role of honey on this differentiation. PMA-differentiated human THP-1 monocytes were treated with LPS (M1), IL-4 (M2), or honey with and without LPS and the cells examined for expression of the M1 and M2 cell surface markers. In addition, undifferentiated THP-1 were treated with honey and the expression of the cell surface antigens examined. It was hypothesized that the honey treatment may be acting as an immune activator by causing the differentiation of monocytes into macrophages. When treated with 1% YSH, THP-1 cells maintained high viability, yet displayed characteristics which would indicate successful differentiation to a macrophage phenotype as indicated by the increase in CD68 expression (Fig. 3.4A).

Treatment of THP-1-derived macrophages with YSH for 24 h, enhanced the expression of the CD80 cell surface marker that is associated with the pro-inflammatory M1 macrophage subtype in all conditions of honey treatment compared to controls. However, treatment with YSH for 24 h only weakly enhanced the expression of the CD163 a marker associated with the M2 repair subtype suggesting YSH may have an anti-cancer potential. However, when the THP-1-derived macrophages were treated for an extended period of time of 48 h, the level of the M2 marker was increased on those cells that were also treated with LPS (Fig.3.4A), which may at least partially explain the ability of honey to promote wound healing.

Figure 3.4: Effects of honey on M1 and M2 polarization surface marker expression.

(A, B) Cells were exposed to 1% YSH under different condition for 24 h, and 48 h. The cell surface expression of CD68, CD80, and CD163 were compared by flow cytometry. Macrophage cell populations were electronically gated according to their forward/side scatter

properties. Gated cells were analyzed for CD68 positivity, and then the CD68⁺ cells were then examined for CD80 (M1) or CD163 (M2) expression. The green histograms represent the

fluorescent profile of THP-1 cells stained for the indicated cell surface marker, whereas the purple histograms represent the fluorescent profile of cells stained with the isotope-matched control antibody, or untreated THP-1 cells that stained with the cell surface marker antibody.

Quantification of the flow cytometry assay was analyzed using EPIC Flow software, and statistical analysis for the relative number of positive cells is presented in (C, D). Data are presented as the mean ± SD from three independent experiments. All results show the relative change compared to untreated THP-1 cells (p < 0.05). YSH enhanced the expression of the cell surface markers associated with the pro-inflammatory M1 macrophage subtype, but had a weaker effect on enhancing markers associated with the M2 repair subtype suggesting YSH may have an anti-cancer potential.

3.2.5 The effect of YSH on secretion of cytokines by THP-1-derived macrophages.

Treatment of THP-1 cells with YSH (in the presence or absence of LPS and PMA

treatment) at a concentration of 1% (w/v) induced or stimulated the release of TNF-, IL-1, and IL-6 when compared to the untreated THP-1 cells or the THP-1 cells treated with PMA (THP-1-derived macrophages) (Fig 3.5). The levels of TNF-, IL-6 , and IL-1 released into the media peaked at 24 h following treatment with honey. The control THP-1 cells treated with sugar showed only baseline levels of TNF-, IL-1, and IL-6 production (5 pg/ml), which were the same as those from untreated cells.

Figure 3.5: Time course effect of 1 (w/v) YSH on TNF-α released from THP-1 cell.

THP-1 cells were treated with YSH (H, grey) or THP-1 cells treated with PMA and honey (PMA+H, blue) or PMA, LPS, and honey (PMA+L+H, orange) and incubated for 6, 12, and 24 h. Conditioned media was obtained, after removing the cells by centrifugation, and subjected to ELISA to measure secreted TNF- release.

THP-1-derived macrophages activated by treatment with pro-inflammatory stimuli, such as bacterial lipopolysaccharide (LPS) also increased the secretion of a variety of cytokines. To analyse the immuno-modulatory effects of honey further, the production of the proinflammatory cytokines, TNF-α, IL-1, and IL-6, and anti-inflammatory cytokines, IL-10 and TGF-, were measured in the cell culture supernatants of undifferentiated and PMA-differentiated THP-1 cells treated with 1% YSH for 6 - 24 hours in the presence and absence of LPS (100 ng/ml). The YSH incubation times were chosen based on those shown to stimulate maximal cytokine production in THP-1 cells. Treatment of these cells with 1% (w/v) YSH induced a significant increase in the release of TNF-, IL-1, and IL-6 (Fig 3.6, 3.7, and 3.8), compared to untreated THP-1 cells or cells treated with a sugar solution which did not, (sugar not shown) (Fig 3.6). The amount of TNF-α spontaneously released by the untreated and the PMA-differentiated THP-1 cell line was

barely detectable. (Note that the PMA-treated cells had been media changed after 3 days of treatment and before the other activators were added.) When YSH was added to the PMA-differentiated macrophages, there was a significant increase in the amount of TNF-α secreted.

The levels of TNF- were further increased when the cells were also treated with 100 ng/ml LPS. Results show that LPS and YSH each significantly (p<0.001) increased the expression of TNF-α compared to non-treated control monocytes.Treatment of THP-1 cells with only YSH induced the release of TNF-α, compared to controls, which was further increased by

approximately 25% when the THP-1 cells had been previously differentiated by treatment with PMA . Yet the data suggest that in the absence of LPS, all of the tested YSH honey samples have pro-inflammatory activity.

The data in (Fig 3.7) shows that YSH treatment of PMA-activated macrophages had the most significant effect (p<0.001) on increasing the expression of IL-1β compared to controls and other YSH-treated conditions. In addition, YSH treatment of both THP-1 and

PMA-differentiated THP-1 cells induced secretion of higher levels of IL-1 than untreated THP-1 cells. The expression of IL-1β in LPS-activated macrophages had an additive increase following the addition of YSH at the 24h time point. Cells treated with only YSH induced the production IL-6, while treatment with only LPS further increased IL-6 production by 2 fold after 24 h (Fig 3.8). On the other hand, YSH treatment inhibited the LPS-stimulated production of IL-6, corroborating the idea that some honeys might exhibit an anti-inflammatory effects.

Figure 3.6: YSH induced the release of TNF-α from undifferentiated and PMA-differentiated THP-1 cells.

THP-1 cells were left unstimulated (NT) or were stimulated for 3 days with PMA as controls.

PMA-differentiated THP-1treated samples were stimulated for 6 - 24 h with 1% (w/v) YSH, 100 ng/mL LPS, or YSH in combination with LPS. Undifferentiated THP-1 samples were treated with YSH only, as indicated. TNF-α release was measured by ELISA and presented as mean + SD of three replicates. Significant differences between the samples and controls (p ≤ 0.05).

Figure 3.7: Effect of YSH on IL-1β production in THP-1 cells.

The effect of 1% YSH (H) and 100 ng/ml LPS (L), separately and in combination, on IL-1β expression after 6 - 24 h treatment of THP-1 cells (Not) or THP-1-derived macrophages (PMA).

Results were calculated for individual experiments from the same cell population. Error bars show mean ± SD of three individual experiments.

Figure 3.8: Effect of honey on IL-6 production in THP-1 cells.

The effect of 1% YSH (honey) and 100 ng/ml LPS, separately and in combination, on IL-6 expression after 6 - 48 h treatment of THP-1 cells (NT) or THP-1-derived macrophages (PMA).

Results were calculated for individual experiments from the same cell population. Error bars show mean ± SD of three replicated experiments for 6 - 24 h, and two replicates for 48 h.

To further analyse the immuno-modulatory effects of YSH, the production of the anti-inflammatory cytokines, TGF-B and IL-10, in undifferentiated and PMA-differentiated THP-1 cells was measured. The level of secreted IL-10 was increased in YSH-treated THP-1 cells compared to untreated THP-1 cells (p<0.001) after 6 h (Fig 3.9). YSH or LPS treatment of activated THP-1 cells had approximately the same effect on IL-10 expression levels, but when the cells were treated with both YSH and LPS there was a significant (p<0.001) additive effect on IL-10 expression. These results may suggest that honey may have an immunosuppressive effect in the presence of local or systemic pathologic inflammation. On the other hand, without inflammation, honey may enhance local or systemic immune responses as in the presence of LPS treatment, YSH enhanced IL-10.

The results in (Fig 3.10) show that YSH treatment for 48 h does not have a significant effect on TGF-β expression compared to untreated controls, however, the results shows that treatment with both LPS and YSH for 48 h each significantly (p<0.001) increased the expression of TGF-β compared to the expression in PMA-treated control monocytes. In LPS-activated macrophages, treatment with YSH for 24 h also increased the expression of TGF-β compared with that caused by YSH only, and this expression level was still significantly (p<0.001) higher than in PMA-activated THP-1 cells. Taken together, expression of TGF-β was observed to be significantly increased when THP-1-derived macrophages were treated with either honey or LPS for 48 h. However, while treatment with YSH induced less TGF-β expression than the positive control, treatment with PMA and IL-4, caused its expression to be significantly higher than in the untreated control.

Figure 3.9: Effect of YSH on IL-10 in THP-1 cells.

The effect of 1% YSH (honey) and 100 ng/ml LPS, separately and in combination, on IL-10 expression by THP-1 cells (Not) or THP-1-derived macrophages (PMA) after 24 – 48 h

treatment. Error bars show the mean ± SEM of two individual experiments. p<0.001 analysed by t-tests multiple pairwise comparisons with the untreated monocytes and PMA -treated samples.

Figure 3.10: Effect of honey on TGF-β in THP-1 cells.

The effect of 1% YSH, and 100 ng/ml LPS, separately and in combination, on treatment of THP-1 cells (not) or THP-1-derived macrophages (PMA) on TGF-B expression after 24 – 48 h treatment. Error bars show the mean ± SD for three individual experiments. p<0.001 analysed by t-tests for multiple pairwise comparisons with the untreated monocytes and PMA-treated

samples.

3.2.6 Effect of heat treatment on Honey activity.

It is important to determine whether the YSH component(s) that are responsible for the immuno-stimulatory effect is heat stable or labile, as this will further indicate whether the immuno-stimulatory activity of honey is due to endogenous heat labile LPS or other heat labile components, such as proteins. Therefore, the aim of the study was to determine whether the heat-treated YSH were able to elicit a cytokine response from monocytic (THP-1) cells. The YSH samples were each heated at 80°C for 30 min, and used to treat the cells in parallel to the unheated YSH samples.

The ability of heated YSH to induce TNF-α and IL- release from cells was reduced by 35–50% compared to unheated YSH controls indicating that the immuno-stimulatory activity is mostly heat stable (Fig 3.11). Interestingly, only the secretion of IL-6 was reduced significantly when the YSH was heated. However, the quantified levels of heat labile LPS in the YSH cannot explain the activities observed. In addition, the level of anti-inflammatory cytokines IL-10 and TGF-β weren’t affected by the treatment with honey that had been headed, and had the same expression results as the cells treated with unheated honey (Fig 3.12).

Figure 3.11: The immunostimulatory activities of YSH are partially heat labile.

THP-1 cells or (honey) or PMA-differentiated THP-1 cells (PMA+LPS+Honey) were treated with 1% (w/v) of YSH, honey that had been unheated (black bars) or heat-treated (orange bars)

at 80° C for 30 min. The levels of (A) IL-1, (B) TNF-α, and (C) IL-6, release was measured by ELISA and plotted as mean + SD of three individual experiments.

Figure 3.12: The anti-inflammatory activities of YSH are partially heat labile.

THP-1 (honey) and PMA-differentiated THP-1 cells (PMA+LPS+honey) were treated with 1%

(w/v) of YSH that had been unheated (black bars) or heat-treated (orange bars) at 80°C for 30 min. (A, B) TGF-β ,IL-10 release was measured by ELISA and plotted as mean + SD of three individual experiments for TGF-β, and two for IL-10.

4.0 Discussion

Collectively, the findings in this chapter point to an enhancement of the M1 profile of macrophages by treatment with Yemeni Sidr honey (YSH) for 24 - 48 h. Particularly, these results include identification of M1 macrophage types distinguished by flow-cytometry-based cell surface markers, and by an increase in the ratio of M1/M2 cytokine profile. In these studies, the THP-1 cell line was used to model different subtypes of macrophages and to determine the effect of treatment with honey on macrophage differentiation. THP-1 cells treated with phorbol ester for 3 days were shown to undergo a change in cell shape and to stop proliferating consistent with differentiation to macrophages. These THP-1-derived macrophages also expressed the macrophage-specific cell surface marker CD68 but expressed low levels of cytokines. THP-1 cells differentiated into the M1 subtype of macrophage by treatment with PMA and

lipopolysaccharide (LPS) for 3-5 days were shown to express the macrophage marker CD68 and relatively high levels of the M1 cell surface marker CD80 (47% positive) but low levels of the M2 cell surface marker CD163 (only 7% positve). There is also a relative change in the shape of the cells as compared to the untreated THP-1 cells. Further, the THP-1-derived M1 macrophages were also shown to express relatively high levels of the pro-inflammatory cytokines, TNF-, IL-1 and IL-6 but not significant levels of the anti-inflammatory cytokines IL-10 and TGF-

which is consistent with published characterization of M1 polarized macrophages in vitro and in vivo. THP-1 cells were differentiated into the M2 subtype of macrophage by treatment with PMA and the Th2 helper cell cytokine, IL-4, for 3-5 days. These cells were confirmed to be M2-like because they expressed high levels of the macrophage marker, CD68, relatively high levels of the M2 cell surface marker, CD163 (50% positive), and relatively low levels of the M1 cell type marker CD80 (1% positive). The M2 polarized THP-1 macrophages were also shown to

secrete significant levels of TGF-βI. These results show that THP-1 cells can be differentiated into both M1 and M2 type macrophages which means that they are suitable model system to study macrophage polarization.

Treatment of THP-1-derived macrophages with YSH was shown to impact macrophage polarization and cytokine expression. The THP-1 cells were treated with 1% YSH which was shown not to affect cell growth or increase cell death. The relative cell survival decreases as the concentration of honey increases and shows and IC50 of around 6.5% honey. As hypothesized,

Treatment of THP-1-derived macrophages with YSH was shown to impact macrophage polarization and cytokine expression. The THP-1 cells were treated with 1% YSH which was shown not to affect cell growth or increase cell death. The relative cell survival decreases as the concentration of honey increases and shows and IC50 of around 6.5% honey. As hypothesized,