60 SKIN MICROBIOME
communicates with the skin’s barrier and immunity cells. The interplay among these three layers,
completed through cellular networking, fundamentally determines the skin’s stress responses. By optimizing the skin’s immunity functions and delving deeper into the frontier of immunocosmetic research, or products that boost the skin’s natural defence mechanisms, the skin’s barrier can be mended and, thereby, the overall health of the skin’s microbiome can be enhanced. So, now that the skin’s microbiome has been discussed, what about the other side of this partnership, the immunity cells?
The role of dendritic cells Dendritic cells are responsible for the commencement of immune responses and are the most abundant antigen-presenting leukocytes. Their precursors travel from the bone marrow into the bloodstream in an inactive state, constantly surveying their environment. Simultaneously, these precursors are
increasing their capture of antigens, or molecules that cause the body to initiate an immune response, even in areas of the body where there is no inflammation. This is how dendritic cells prepare for pathogens. Ultimately, when pathogens do enter the body, these previously inactive dendritic cells are able to effectively detect these invaders due to the previously collected antigens. Signals are then sent out for the pathogens to
rapidly leave the skin’s tissue. During this process, the dendritic cells are going through maturation, which includes functional and phenotypical alterations.6,7
They are no longer surveying the
body’s parameter capturing antigens but are now producing pro-inflammatory cytokines, which regulate growth, differentiation, and transportation of the immune cells to the sites of infection.7 Once the dendritic cells travel to the body’s
lymph nodes, they present their antigens to T-cell zones, or white blood cells that are also developed in the bone marrow to fight infection. Once T-cells have been activated by an infected antigen-presenting cell, they are now able to kill any target cell that is infected with the same pathogen.8
By initiating this immune response,
the microbiome and skin barrier are effectively being restored. Currently, the microbiome landscape in
the personal care industry remains uncharted, offering a prime opportunity for brands to establish themselves as pioneers in microbiome modulation. This fuelled the innovation for an assay that would allow brands to have a role in this microbiome space. Since dendritic cells are able to be propagated in vitro from bone marrow, or various sources, using a multitude of growth factors such as granulocyte macrophage- colony stimulating factors (GM-CSF), an assay was developed based on the modulation of the immune-response capacity of dendritic cells. The Dermal Microbiome-Immunology Assay is
designed to assess the effect of active ingredients to alter dendritic cell paracrine signaling molecules to kill detrimental organisms found on the skin, and, therefore, promote commensal microbiota. This addresses the common misconception that a reduction in all bacteria is good.
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S.aureus
P.aeruginosa S.epidermidis
Figure 1: Time kill results for dendritic cells treated with an antimicrobial-free yeast-derived active inoculated with the tested microorganism populations across time intervals. Values indicate the percent of microorganisms killed
However, commensal bacteria, or ‘good’
bacteria, make up most of the skin’s microbiome and actually benefit the host. For that reason, when performing this assay, we do not want to see a reduction in all bacteria, just the pathogenic category. Table 1 describes the microorganisms that are examined in this assay, along with their importance. Monocytes are differentiated into dendritic
cells according to the manufacturer’s protocol and are cryopreserved. To generate conditioned media, dendritic cells are thawed and cultured in a flask with complete media for 24 hours to ensure cell viability. The cells are then harvested, resuspended in 2 mL of complete media, and seeded in a six-well fibronectin plate. After allowing the dendritic cells to attach for
24 hours, the complete media is replaced with 5 mL of either the complete media, which acted as the control variable, or differing concentrations of an antimicrobial-free cosmetic active diluted with complete media. It is also important to note that the dendritic cell generation medium is free of antimicrobial agents we well. Then, after the dendritic cells are exposed to experimental conditions for 24 hours, the conditioned media was harvested and placed at -80° Celsius until the time kill test.
Time kill test For the time kill test, conditioned media samples are thawed, and each condition is added into three individual containers. Each container is inoculated with one of the three test organisms presented in Table 1. The inoculum concentration for each organism is standardized using the 0.5 McFarland turbidity standard and is further diluted to yield approximately 106
microoganisms/mL.
The amount of each inoculum added to each sample was no more than 1.0% of the product volume; this is to ensure that the composition is not altered. Serial dilutions from each container are performed to enumerate the surviving microorganisms using the plate count technique.
The activity of the test material inoculated
is evaluated at specific time intervals of 30 seconds, one, five, ten, and 30 minutes after the inoculation to quantitatively determine the number of viable microorganisms remaining after the incubation time. The results of this study are read two days later. The area under the curve (AUC) is calculated
by adding the areas under the line between each consecutive absorbance measurement using the following equation:
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TABLE 2: TIME KILL TEST RESULTS: PERCENTAGE REDUCTION IN VIABLE ORGANISMS AFTER INOCULATION AND SAMPLING TIME INTERNALS
Microorganism Sample S. aureus
CM (Control) 0.01% active 0.1% active 1.0% active
P.aeruginosa
CM (Control) 0.01% active 0.1% active 1.0% active
S.epidermidis
CM (Control) 0.01% active 0.1% active 1.0% active
Concentration 2.5 x 106 Inoculum seconds
80.5% 99.9% 99.9% 99.9%
4.1 x 106
73.5% 99.9% 99.9% 99.9%
1.2 x 106
38.3% 23.2% 25.3% 22.3%
30 minute
81.2% 99.9% 99.9% 99.9%
78.1% 99.9% 99.9% 99.9%
56.1% 38.1% 36.1% 43.1%
1 minutes
81.0% 99.9% 99.9% 99.9%
81.2% 99.9% 99.9% 99.9%
60.2% 42.1% 35.1% 45.3%
5 minutes
83.3% 99.9% 99.9% 99.9%
83.3% 99.9% 99.9% 99.9%
75.2% 43.3% 42.4% 46.2%
10 minutes
Human opportunistic pathogen with antibiotic strains
Human opportunistic pathogen and used to study antibiotic resistance and pathogenesis
An element of normal skin microbiota, nonpathogenic, and supports dermal barrier integrity
CUMULATIVE TIME KILL TEST RESULTS
120% 100% 80% 60% 40% 20% 0%
30
Microorganism Growth Inhibition (AUC)
CM 0.01% 0.1% 1.0% CM 0.01% 0.1% 1.0% CM 0.01% 0.1% 1.0%
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