Effects of serotype and species dependency of bacterial lipopolysaccharides in human melanocytes from lightly and darkly-pigmented skin

Highlights

• •This study represents a direct comparison of different serotypes (O55:B5, O26:B6 and O128:B12) of lipopolysaccharide (LPS) from species Escherichia coli and LPS from another species Porphyromonas gingivalis.
• •Effects were examined on primary human melanocytes from lightly pigmented (LP) and darkly pigmented (DP) donor skin.
• •None of LPSs altered melanin production in both cells after 48 h exposure.
• •LPS serotype O26:B6 showed the highest potency in the induction of IL-6 cytokine that was dependent on the pigmentation phenotype, while other serotypes and species were less effective or ineffective.

Abstract

Lipopolysaccharides (LPS) alter melanin synthesis and induce cytokine secretion in melanocytes; however, effects of different serotypes or species of LPS have been rarely addressed. Herein, LPS from serotypes (O55:B5, O26:B6, O128:B12) of E Coli and P gingivalis (LPS-PG) was examined in human melanocytes of lightly-pigmented (LP) and darkly-pigmented (DP) donors. Results showed no effect of any LPS on melanin production in both cells. B6 and B12 elicited higher levels of IL-6 than B5 in LP cells while B6 was more potent than B12 in DP cells. B5 and PG did not show any effect on IL-6 in both cells.

Melanocytes have been recognized as specialized skin cells which not only participate in the production of biopolymeric pigment melanin and their transfer to keratinocytes, but also act as immunocompetent cells participating in immune function and regulation of inflammation and infection [1,2]. Bacterial endotoxins (lipopolysaccharides; LPS) are known inducers of inflammatory response in immune cells by the activation of toll-like receptors 4 (TLR). The structure of LPS consists of three key parts: lipid A, an oligosaccharide component, and a glycan polymer structure called as O-antigen [3]. The O-antigen is responsible for conferring varied serotypes in LPS.

Neutrophils treated with LPS from Escherichia Coli (serotypes O55:B5, O127: B8, O128: B12, O111: B4, and O26: B6) were shown to selectively release extracellular traps (ETs) for the serotype O128: B12 [4]. A recent report documented the differential potencies of three LPS types (E. coli O111:B4; Salmonella enterica serotype enteritidis; Salmonella enterica serotype typhimurium) in the induction of cytokine secretion in human skin organ cultures [5]. In another study [6], where LPS serotypes 055: B5, 0111: B4 and 0127: B8 were studied, the authors reported variability in body temperature of rats that was attributable to different structures of the LPS serotypes. Furthermore, LPS serotypes 0111: B4 and 0127: B8 that were studied in female rats demonstrated differences in edema formation and albumin extravasation [7]. Differential inflammatory responses were reported in mice where different LPS serotypes (O111: B4, O55: B5, O127: B8 and O128: B12) were administered [8]. Taken together, these studies indicate that the inflammatory profile of LPS is dependent on serotypes. Moreover, the finding that immune cells such as neutrophils and macrophages are responsible for drawing the pigment cell melanocytes to skin wounds leading to hyperpigmentation [9], prompted to consider if melanocytes might also produce different levels of melanin or cytokines in response to different serotypes of LPS.

In the present study, LPS derived from E. coli of three different serotypes (O55: B5, O26: B6, O128: B12) and another species, Porphyromonas gingivalis (PG), a Gram-negative periodontopathic bacterium found in periodontal pockets of individuals with chronic periodontitis, was evaluated for effects on melanogenesis in primary human melanocytes derived from donors with different melanin contents. Use of LPS-PG is significant particularly as melanocytes also occur in the oral cavity.

Different LPSs (description in Table 1, Supplementary material) were evaluated for any cytotoxicity to HEM-DP and HEM-LP cells; results showed that in HEM-DP cells, LPS from PG was nontoxic over the range 1–100 µg/mL. LPS from EC showed serotype-dependent cytotoxicity where serotypes B6 and B12 showed cytotoxicity; B12 significantly diminished viability by 24% at 10 µg/mL and 95% at 100 µg/mL, respectively, in DP cells. At the concentration of 100 µg/mL, B6 showed similar cytotoxicity to B12 with diminution in viability by 96% (Fig. 1A). B5 was nontoxic at all concentrations.

Fig. 1.

Viability of (A) HEM-DP cells and (B) HEM-LP cells treated with different concentrations of LPS from PG and EC serotypes (B5, B6, and B12) for a duration of 48 h. One-way ANOVA by Tukey's test; Data are mean ± SD of at least two independent experiments for (A) and three independent experiments for (B). Relative levels of intracellular melanin in (C) HEM-DP cells and (D) HEM-LP cells treated with nontoxic concentrations of different LPS for 48 h. One-way ANOVA by Tukey's test; Data are mean ± SD of at least three independent experiments for (C) and (D). Fold-change in levels of cytokine IL-6 secreted in supernatants of culture of (E) HEM-DP cells and (F) HEM-LP cells treated with nontoxic concentrations of different LPS. One-way ANOVA by Tukey's test; Data are mean ± SD of triplicates from one experiment for (E) and mean ± SD of two independent experiments each run in duplicates for (F); *p < 0.05; **p < 0.01 #p < 0.0001.

In HEM-LP cells, the cytotoxicity profile was similar for both species and serotype, except that B12 at 10 µg/mL was nontoxic to LP cells (Fig.1B). Altogether, these results revealed that LPS from EC serotype B6 and B12 are cytotoxic in contrast to B5 at the highest concentration of 100 µg/mL in melanocytes from both pigmentation levels. LPS from EC serotype B12 at 10 µg/mL was excluded from further experiments of melanin and cytokine assays as we chose to include only nontoxic concentrations for further analysis.

HEM-DP and HEM-LP cells used in this study have been validated for their morphology and melanin contents where DP cells had a 1.34-fold higher melanin content than LP cells (Fig. S1). All the four LPSs showed no alterations in intracellular melanin levels as compared to control group either in HEM-DP cells (Fig. 1C) or HEM-LP cells (Fig. 1D). The results of absence of any change in melanin levels by EC serotype B6 are in line with a previous study [10] where authors showed that B6 did not alter melanin contents of either LP or DP cells after a 24 h exposure and was nontoxic up to a concentration of 50 µg/mL. Notably, there is no report on the effects of other serotypes on melanin contents of these cells. Strikingly, the role of LPS in inducing melanogenesis via MAP kinase signaling pathway in primary human melanocytes has been described previously [11] where the authors reported increase in melanin content by 25% in primary human melanocytes exposed to LPS from EC serotype B6 at 30 μg/mL. However, in this study, the authors used a long-term exposure of 5d vs. 48 h exposure used in current study.

LPS from PG was ineffective in stimulating IL-6 cytokine secretion in DP (Fig. 1E) as well as LP cells (Fig. 1F). LPS from EC serotype B5 similarly showed no effect at any concentration in IL-6 levels in either DP cells or LP cells. However, LPS from EC serotype B6 significantly induced 1.81-fold and 2.27-fold increases at concentration of 1 and 10 µg/mL, respectively, in DP cells (Fig. 1E), and significantly induced 1.52-fold and 1.57-fold increase at 1 and 10 µg/mL, respectively, in LP cells (Fig. 1F). On the other hand, LPS from serotype B12 significantly induced a 1.45-fold increase in IL-6 in DP cells at 1 µg/mL (Fig. 1E), while in LP cells, it significantly induced a 1.60-fold and 1.68-fold increase at 1 and 10 µg/mL, respectively (Fig. 1F). Altogether, in contrast to B5 that was inactive, the other two serotypes B6 and B12 showed a robust induction of IL-6 levels in both cells, with IL-6 levels significantly higher by B6 as compared to B12 in DP cells, which was a striking finding.

The results of significant induction of IL-6 by B6 serotype in both LP and DP melanocytes are in contrast to a previous study that reported no effect of LPS (B6 serotype) at any concentration on IL-6 levels after a 24 h exposure in melanocytes from moderately-pigmented Asian donor [12]. Interestingly, another study that used LPS serotype B6 highlighted the differential responses of LP and DP cells to LPS [10] with higher levels of IL-6 in LP cells as compared to DP cells. In addition, the aforementioned study [10] demonstrated that mRNA levels of TLR4 were significantly higher in DP cells as compared to LP cells, which might partly explain why IL-6 levels were higher in DP cells observed in this study. These discrepancies might be attributable to the different melanocyte cell types and donor variation as well as different times of exposure. Rationale for use of high concentrations of LPS (100 µg/mL) in this study was due to the fact that these concentration ranges have been found to increase pigmentation in melanocytes previously [11]. Moreover, the exact concentration at the site of infection in skin for LPS is unknown. Melanocytes are known to express multiple TLRs including TLR4 and TLR2 and their adapter molecule CD14 [13]. LPS from PG has been shown to interact with both TLR2 and TLR4 [14], unlike LPS from EC which interacts via TLR4. Furthermore, differences in the functional and binding affinities of different LPSs might explain the selective responses observed in the various serotypes, although it has been shown that LPS from PG is less effective in inducing cytokine release than LPS from EC [15]. Moreover, although no changes in melanin contents were noted in this study after an acute exposure of LPSs, the result of overproduction of IL-6 cytokine is indicative of inflammation and might be a causative factor in pathogenesis of vitiligo, an autoimmune skin disorder, characterized by higher IL-6 levels and melanocyte death [16]. Suppression of viabilities of DP and LP cells by higher concentrations of LPS serotypes B6 and B12 might be attributable to an enhanced production of TNF-α (tumor necrosis factor) that induced cytotoxicity and might cause melanocyte apoptosis, although further studies will be necessary to examine the mechanisms.

Although no changes were found in melanin production in both melanocytes after treatment with different LPSs in the duration of 48 h, longer durations representing chronic exposure might be needed to elicit increase in pigmentation as noted in some previous studies that have employed LPS serotype B6. Also, other EC serotypes B4 and B8 that were not evaluated in this study, along with serotypes of LPS from PG merit further investigation. Unlike EC, the serotypes for PG are not easily available commercially, hence were not evaluated, although future studies to identify if any specific serotype of PG might show activity on melanocytes could be informative especially, since melanocytes in the oral cavity (that bear some similarity to epidermal melanocytes) are exposed to this pathogen in chronic periodontitis. As only a single cytokine was evaluated in this study, further studies to expand on the secretion profile of other cytokines and chemokines and examine whether there might be a serotype-dependent response in both types of melanocytes will be interesting. Lastly, in this study, experiments were conducted using melanocytes from a single donor for LP and DP; the donor-dependent variation in response to inflammation should be explored in future studies utilizing multiple donors from both pigmentation phototypes and validate the results of serotype-specific effects. Furthermore, study with more cell lines of each pigmentation type will be necessary in future for validating findings obtained in this study.

In summary, results of this pilot study showed that neither the species (EC vs. PG) nor the serotype (B5, B6 and B12) elicited any alterations in cellular levels of melanin after a 48 h duration, although the EC serotype B6 was significantly more potent than B12 in DP cells but was similarly potent to B12 in LP cells in inducing IL-6 secretion. The B5 serotype showed no effect on cytokine levels in any cell type. In conclusion, the findings of this study suggest that researchers might need to be careful in the selection of LPS for in vitro studies when using melanocytes from different ethnicities and take into consideration their serotypes that affects the inflammatory profile. This can in turn reduce variability with use of different LPSs and increase reproducibility across studies, especially as LPS can induce pigmentation and inflammation but the origin of melanocyte pigmentation type is critical.

Funding

This research received no external funding.

Declaration of Competing Interest

The author declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.