3.1. Cell Metabolic Activity and Migration
Cellular migration is of the utmost importance for several mechanisms, such as immune response, wound repair and tissue homeostasis [
21]. On the other hand, an aberrant cell migration leads to several pathologies such as the invasion of malignant cells into the surrounding tissue, with the formation of tumour metastasis, leading to a poorer prognostic for the patient [
22].
A patient with a chronic wound has a compromised quality of life due to excruciating pain and the constant need for medical care, resulting in a significant burden to the health system worldwide. Briefly, the physiological process of wound healing has several steps, such as haemostasis, inflammation, proliferation, and remodelling. The haemostasis starts as soon as the injury occurs and is characterized by vasoconstriction and blood clotting. This stage is of the utmost importance as it prevents excessive blood loss and provides the required matrix for cell migration. Upon the inflammation stage in which phagocytic cells are recruited, the proliferation stage starts. At this stage, several growth factors and cytokines are recruited by different cell types, such as keratinocytes. The final stage is characterized by the formation and organization of collagen bundles [
21]. If an arrest or delay occurs in any of these stages, particularly in the inflammation stage, it can lead to the formation of a chronic wound. It is known that keratinocytes have an important role in wound healing, particularly in re-epithelization [
23]. The non-migration of these cells to the injury site is associated with the clinical phenotype of chronic wounds.
Even though keratinocytes are not motile in normal steady-state condition, in the presence of signalling molecules, they can migrate to re-epithelize the wound site [
24]. Consequently, the enhancement of the keratinocyte migration rate is of the utmost importance for wound re-epithelization, particularly in the case of chronic wounds [
25].
On the other hand, cancer metastization or invasion into the surrounding tissue leads to a poorer prognostic for the patient, as in the case of invasive squamous cell carcinoma [
22]. Squamous cell carcinoma and basal cell carcinoma are keratinocyte carcinomas, particularly diagnosed in fair-skinned populations [
26,
27]. The decrease in cell migration might result in a less invasive tumour.
The ability of keratinocytes to migrate from one edge of the scratch, performed on a cell confluent monolayer, to its opposite edge was evaluated for a period of 24 h, in the presence and absence of the peptides designated by P188D. First, a preliminary cell viability study using fibroblasts demonstrated that the presence of 25 and 50 μg/mL of peptides resulted in 94.2 ± 0.77 and 92.4 ± 1.55% of viable cells, respectively. Therefore, 45 μg/mL was the chosen concentration for the following tests.
The effect of P188D peptide on keratinocyte migration was evaluated up to 24 h hours of contact. The presence of the peptide seems to decrease the keratinocyte migration rate in comparison to the control, this effect being clear at 24 h of contact (
Figure 1). Thus, it seems that the presence of the peptides negatively affects keratinocyte migration, which could be important to reduce the invasiveness of skin cancer cells.
The microbial degradation of a protein results in several peptides with different sizes, amino acid composition and sequence. It is known that these characteristics have a striking effect on several physiologic mechanisms [
28]. Therefore, to better understand the effect observed on keratinocyte migration, peptides were fractioned into smaller fractions using different percentages of acetonitrile 20%, 40% and 80% (20%, 40% and 80% fraction peptide). The final result was a solution with a narrowed peptides distribution. The influence of these fractions on the keratinocyte metabolic activity and migration was once again evaluated.
As can be seen in
Figure 2A,B, the presence of the 20% peptide fraction decreased the metabolic activity and migration rate of the keratinocytes, although it did not reach statistical significance. On the other hand, fractions 40 and 80% do not seem to have any effect on either mechanism. As these fractions did not induce significant changes in the keratinocyte proliferation and migration rate, an additional fractionation was performed, where peptides were separated according to their molecular weight, ranging from 9090 to 401 Da for the 20% fraction (P188D 20%) and from 8082 to 382 Da for the 40% fraction (P188D 40%) (
Table 1). Once again, the keratinocyte proliferation and migration rate were determined in contact with these new peptide fractions.
In this analysis, the results obtained were very distinct (
Figure 3) from the previous one (fraction 20%, 40% and 80%). Significant changes were observed in the metabolic activity of cells in the presence of peptides, particularly in the presence of 40% fraction peptides. Analysing
Figure 3A, it is clear that fractions P188D 20% P1, P188D 40% P1 seem to induce a higher metabolic activity of these cells; in contrast, P188D 40% P3 induced a decrease in the metabolic activity. Even though the results showed that sub-fraction P1 from 20% fraction peptide induced the highest metabolic activity in comparison to P2 or P3 from the same fraction, these differences did not reach statistical significance. On the other hand, significant changes were observed for the peptides from the 40% fraction, particularly P188D 40% P1, P3 and P5. While P1 significantly increased the metabolic activity, P3 decreased it.
All the peptide sub-fractions induced an increase in the keratinocyte migration rate (
Figure 3B), with significant differences. P188D 20%, P2 and P3 are significantly different, with the latter presenting a higher migration rate in comparison to the former. A similar result was observed for P188D 40% P2, wherein P188D 20% P2 and P188D 40% P2 are significantly different, with the latter inducing a higher migration rate. P188D 40 % P3 also significantly affects migration in comparison with P188D 20% P1 and P188D 20 % P3. It seems that the presence of these peptides has a more significant effect on the migratory ability of these cells than on their metabolic activity. In this case, the use of fractioned peptides is very important for skin reepithelization and therefore for the treatment of chronic wounds.
It is important to mention that resazurin assay is based on the capacity of living cells to transform resazurin to resorufin [
29]. This ability is correlated with cell viability and cell number/proliferation. The basis for this understanding is that the higher the conversion of the dye, the higher the cell number. Therefore, taking into consideration this fact, it is possible to interpret the results observed, as the peptides fraction has a higher influence on cellular migration than on proliferation.
There are different physiological or pathological conditions, such as wound repair that induce cells to shift their phenotype from proliferative to migratory in response to similar stimulus (reciprocal control of cell proliferation and migration). It is known that cytokines produced by cells present at the wound area can act as a chemoattractant for distant cells, leading to cell migration [
30]. However, upon their arrival, the cells switch from a migrating phenotype to a proliferating one [
30]. In this case, the wound inflicted on the cell monolayer may have induced cells to release chemoattractant, signalling cells to migrate to the injury site.
It has been demonstrated that keratin intermediate filaments regulate proliferation, migration, adhesion, and inflammatory phenotype of keratinocytes [
31].
Keratins 6, 16 and 17 are very important as they are considered barrier alarmins, rapidly inducing keratinocytes to migrate to the injury site [
31]. As in the wound healing process, keratinocytes suffer dramatic changes as they are required to migrate and proliferate during the healing phase. Their expression is founded in the epithelial remodelling phase until the barrier function is repaired, demonstrating the importance of these keratins in wound repair [
31]. Among others, the expression of keratin 6, 16, and 17 persist through the epithelial remodelling phase until the barrier function is restored [
32]. It is hypothesized that the peptides in the study may have a similar role to these keratins.
3.2. Quantification of TNF-α
Inflammation is a crucial process in several medical conditions, such as psoriasis and wound healing; therefore, the ability of these peptide fractions to influence the inflammatory response in terms of TNF-α release by macrophages was evaluated.
This assay had two distinct steps; the first one was to evaluate if the peptide fractions induced an inflammatory response in non-activated macrophages (0 mg/mL of LPS) and the second one was to evaluate if, in LPS pre-activated macrophages, the peptides would influence the release of the pro-inflammatory cytokine.
As it can be seen in
Figure 4A in non-activated macrophages, the peptide fraction P188D 40% induces a significantly high release of TNF-α in comparison to the control (cell culture media only), while the increase in TNF-α in the presence of P188D 20% is significantly lower and similar to the control (no statistically significant difference). In the presence of LPS pre-activated macrophages, the levels of TNF-α with or without the peptides fraction is similar. However, the amount of TNF-α released by peptide fraction P188D 40% was significantly reduced in the presence of LPS. As it is known, the maintenance of an inflammatory microenvironment favours tumorigenesis, particularly in skin cancers, such as the squamous cell carcinoma and basal cell carcinoma, in which there is an important inflammatory component [
33]. Even though these peptides are not able to decrease TNF-α release, they do not enhance it. This cytokine is important in the induction of a pro-inflammatory response and it seems that in activated macrophages the presence of these peptides will not contribute to enhancing inflammation, as in some of the traditional chemotherapy agents, such as cisplatin, paclitaxel, 5-fluorouracil, and doxorubicin [
34]. Therefore, the use of these peptides, such as 20% fraction peptides (P188D 20%), which seems to decrease the migration rate as well as the proliferation rate, can be an interesting alternative to the traditional chemotherapy agents.
The fractioned peptides from the last fractionation stage (chromatographic peaks by molecular weight) have a similar behaviour when in contact with pre-activated macrophages, where the values of this cytokine can be slightly higher than the negative control (DMEM) but never reaching statistical significance (
Figure 4B). On the other hand, the contact of the different fractions with the non-activated macrophages (without LPS) induced distinct results. The P188D 20% P1 and P2, and P188D 40% P3 induced a significantly higher release of TNF-α in comparison to the negative control and all other conditions. Again, these results suggest that these peptides can be used in medical conditions, where there is an inflammatory process involved, as they will not aggravate it, as in the case of cancer or wound healing. These peptides open the possibility to modulate the migration rate of the keratinocytes involved in both processes, to enhance or decrease it, depending on the patient’s needs.
Gao et al. [
35] demonstrated that the presence of hair keratins in a culture with rat neuronal Schwann cells increased proliferation and increased the number of cells passing through a trans-well pore. In the wound-healing experiment with primary Schwann cells, the damaged areas in the keratin group significantly decreased [
35]. The presence of keratins from chicken feathers, when fractioned, also resulted in an increase in the metabolic activity/proliferation, with the exception of one fraction P188D 40% P3. Our results also demonstrated that the peptide fraction stimulates cell proliferation in a wound-healing model similar to the one reported by Gao et al. [
35]. However, the keratins extracted from the chicken feathers did not seem to significantly affect the production of the inflammatory cytokine TNF-α, as described by Gao et al. [
35]. This difference may be due to the different experimental settings; while in the Gao et al. [
35] experimental model the keratin peptides are in contact with the cells prior to LPS activation, in our experimental model, the keratin peptides were added to the cell culture medium after LPS stimulation. Our experimental model allows the evaluation of these peptides as coadjutant molecules for the treatment of skin inflammatory diseases.
The current knowledge regarding macrophage activation in wound healing is the ability of these cells to shift from an M1 classical pro-inflammatory response (stimulated by TNF-α, LPS as an example) to an M2 (polarized by IL-4, for example) anti-inflammatory response activation pathway after microbial infection control [
36,
37,
38]. It has been described that a biomaterial made from human hair keratins elicit anti-inflammatory responses from naïve macrophages and polarize them towards M2 phenotype [
38]. Waters et al. [
39] described that keratin influences macrophage behaviour, although the mechanism behind it is extremely complex. Nevertheless, Waters et al. [
39] describes that, macrophages are altered when in contact with an immobilized keratin biomaterial surface, and that these changes appear to trend toward an anti-inflammatory phenotype. Even more importantly, it is described that the response of macrophages is dependent on keratin molecular weight. This fact is very important as it can explain the differences observed on the response of the macrophages to the chicken feather keratin hydrolysate.
As the results differ depending on the peptide fraction present, a proteomic study was performed to evaluate the main sequences of peptides present in the solution, as well as comparing it to the keratin structure present in chicken feathers.
3.3. Proteomics
In order to identify an amino-acid sequence that could be responsible for the results observed, the different fractions were analysed using the ProteinPilot™ software and a database containing the sequences of the proteins of interest (
Gallus gallus feather keratins, from Uniprot 09-2017). Peptide fractions of three proteins were detected, as described in
Table 2.
The proteomics analysis revealed the presence of peptides with different sequences and with homology to three distinct chicken keratin proteins: keratin 1, 3 and 4, as described in
Table 3,
Table 4,
Table 5 and
Table 6.
Analysing the fraction P188D 20% it was detected the presence of amino-acid sequences similar to the ones present in keratin 3 and 4 (
Table 3). The presence of the two keratins (3 and 4) seems to decrease the migration and metabolic activity of the keratinocytes. In the P188D 40% fraction only the presence of sequences related to keratin 4 were found (
Table 4). The loss of peptides with the homology to keratin 3 in P188D 40% results in keratinocytes with a behaviour similar to the control. It can be hypothesized that the presence of peptides with homology to keratin 3 may be responsible for cell migration and metabolic activity decrease. Keratins are known to be intermediate filament-forming proteins that are related to differentiation status [
40] and are known to be crucial for the motile processes, although is not fully clear [
41].
The proteomics analysis detected any peptides for the fractions P188D 40% P1, P4 and P5 related to chicken keratin feathers. In the case of fractions P188D 20% P1, 40% P2 and 40% P3, all the peptides present had homology to chicken feather keratin 4 with some modifications on the sequence resulting in changes in the protein, as can be seen in
Table 5 and
Table 6.
The peptides present in the fraction P188D 20% P2 had homology to feather keratin 1 and 4, while 20% P3 has a homology to the chicken feather keratin 3. The results obtained for migration or metabolic activity were not possible to correlate with the presence or absence of the peptides with homology to the keratin 3; this may be due to the loss of some of the peptide with particular sequences.
Nevertheless, this study demonstrates the ability to modulate keratinocyte migration and metabolic activity in the presence of different peptide sequences with homology to keratin.