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Immune system needs microbial exposure - Re-Connecting Nature™ supports immune system by providing high biodiversity microbial exposure

Written by:

Johanna Kalmari

Uute Scientific Oy

MSc Johanna Kalmari received her degree in Molecular Biology and Gene Technology from the University of Eastern Finland. She has previously worked in the field of environmental biotechnology and currently works as a project manager at Uute Scientific Oy as well as a PhD student in Tampere University, Faculty of Medicine and Health Technology.

IIda Mäkelä

Uute Scientific Oy

MSc Iida Mäkelä has received her degree in Environmental Change and Global Sustainability from the University of Helsinki in 2021. She has worked as a part of the ADELE research group who look for nature-based solutions to the numerous health related problems today’s urban living creates. She is currently working as a Product Manager at Uute Scientific Oy and a PhD student in the Doctoral programme in Interdisciplinary Environmental Sciences at University of Helsinki.

Abstract
Biodiversity loss and urbanisation has increased simultaneously with immune-mediated diseases. The immune system needs constant stimulation from the environment to function properly and to distinguish harmful targets from harmless ones. In simple terms: kill pathogenic bacteria but do not kill your own cells. Exposure to environmental micro-organisms brings diversity to the skin and gut and helps our immune system suppress signaling that leads to inflammation. Re-Connecting Nature™ is a microbial extract from Finnish nature, that is designed as an ingredient for consumer products to give rich microbial exposure. This is especially important for those who are not in contact with nature on a daily basis. A new in vitro study indicates that Re-Connecting Nature™ treatment reduces the amount of pro-inflammatory cytokines released in an epidermis skin model. Our recent in vivo results support the in vitro findings and show that skin barrier function is improved after continuous use of a skin lotion containing Re-Connecting Nature™. These results are in line with the previous university studies, in which re-introducing natural microbial diversity to the skin of daycare children, improved their blood cytokine ratio for the attenuated inflammatory signaling.
Biodiversity and immune tolerance

Urbanisation and changes in our modern lifestyle have disconnected humans from naturally occurring biodiversity. The World Allergy Organization is concerned about the biodiversity loss that is related to this increased urbanisation. Loss of macro diversity  -e.g. plants – in cities leads to shrinking micro diversity (microbes) with poor human microbiota exposure. This leads to poor immune tolerance and finally inflammation and clinical diseases (1). The Finnish national allergy program from 2008-2018 changed its strategy from avoiding potential allergens to people tolerating them. This strategy managed to reduce the prevalence of allergy and asthma (2).

The Biodiversity Hypothesis states that contact with natural environments enriches the human microbiome, promotes immune balance and protects from allergies and inflammatory disorders (7). Humans have developed to co-exist with micro-organisms, and therefore contact with environmental microbes in everyday life induce immunoregulatory pathways and balance responses against foreign proteins, like allergens. The immune system has difficulties distinguishing signals of danger from non-danger, when the interplay between human and environmental microbiome is missing. For instance, the immune system may recognize pollen as a virus (4). Reduced biodiversity exposure and alterations in the gut and skin microbiota are associated with many conditions including elevated inflammatory responses like asthma, allergy, inflammatory bowel disease (IBD), type1 diabetes and obesity (1,5).

On a molecular level, continuous exposure to environmental microbes through the skin, gut and respiratory tract activates positive, immunoregulatory circuits, whereas inappropriate inflammatory responses are down regulated (5). The continuous stimulation of the immune system by environmental microbes leads to the generation of suppressive regulatory T-cells (Treg) necessary to down-regulate inflammation. In the absence of stimulation – antigens – these Treg populations are likely to decrease (3,6). The interactions of the microbes with their specific receptors on, and in, immune cells are necessary for the development and maintenance of epithelial cell integrity, tolerance, and tissue repair. Without sufficient microbial stimulation another path- a self-perpetuating system of an enhanced number of inflammatory mediators and microbes that tolerate it – is induced. The environment also modulates our immune system by epigenetic mechanisms where environmental factors activate or silence genes. This epigenetic modulation is especially important in early childhood, but changes can occur later as well (5).

The microbiome on our skin and gut is important to our overall immune system functioning and health. Overgrowth of some species while relative abundance of others decrease – dysbiosis – are contributors to diseases like atopic dermatitis (14). The microbiome on skin and in the gut includes both transient and resident microbes, and therefore at least part of the microbiome is in a dynamic interaction with the environment (1). Thus, the environment affects our microbiome, and it is indicated that atopic individuals have lower biodiversity around their homes and lower diversity of Gammaproteobacteria on their skin than healthy individuals (27). Some environmental microbes are particularly important for health and have anti-inflammatory properties. For example, Gammaproteobacteria, and its genus Acinetobacter, induce immunoregulation (8,14) and prevent allergic sensitisation and lung inflammation (10). Boreal forest floor origin Mycobacterium genus have anti-inflammatory properties and is associated with improved mood and learning while mitigating stress and depression (9,14).

There are positive results of intervention trials where direct contact with soil, enriched with added microbial biodiversity, created beneficial immunological changes (11-14). In a daycare study, 28-day contact with soil diversified skin and gut microbiota, increased Treg cell frequency and changed cytokine ratio leading to a decreased inflammatory state (12). In another, long-term, study with daycare children, the daycare environment was enriched with biodiverse forest floor vegetation and peat. This exposure enriched skin’s commensal microbiota and suppressed the potentially pathogenic bacteria as compared to a standard, urban daycare environment (14).

Microbial structures like lipoproteins are recognized by Pattern Recognition Receptors (PRR) that are found on various type of cells, including keratinocytes on skin (29,30). As these cells are recognized bacterial molecular structures, an immune response was also reported with heat inactivated microbes (10,28). Re-Connecting Nature™ is a patented and pioneering microbial extract developed at the Universities of Tampere and Helsinki. It contains over 600 bacterial species, including beneficial Acinetobacter and Mycobacterium, in safe form: inactivated but biochemically recognisable by our immune system to give the necessary natural exposure. Re-Connecting Nature™ is currently available in cosmetics provided by many manufacturers and also in textiles made by Ruskovilla®.

Re-Connecting Nature™ in a lotion created significant immuneological response in skin model

Uute Scientific Oy contracted it’s in vitro skin model study with a scientifically validated, commercially available, lab-grown skin. The objective was to study the effects of Re-Connecting Nature™ on skin microbiome, barrier proteins and cytokine secretion. Cytokines are signal molecules that regulate our immune responses, and are secreted by many types of cells including epithelial cells like keratinocytes. Some cytokines promote and some down-regulate inflammation responses with some having more complex roles. The test model included laboratory grown stratified epidermis, on which three skin commensal bacteria (S. epidermis, C. striatum. C. acnes) were grown. The defense test included pathogenic S. aureus. Re-Connecting Nature™ extract was used in four different concentrations (0,5%, 1%, 5%, 10%) in a cosmetic lotion. Controls included untreated and vehicle, i.e., just the lotion, treated skin organoids.

According to the microbial results, Re-Connecting Nature™ at low concentrations (0,5%, 1%) had a neutral effect on the growth of S. epidermis and C. acnes and a prebiotic (growth supporting) effect on the growth of C. striatum. At higher concentrations there was more interference, which may be due to limited number of microbes and short time exposure (24h) used in this model. At the defense test with S. aureus challenge, Re-Connecting Nature™ did not have any significant effect on the growth of tested microbes, apart from the highest concentration (10%), where significant inhibitive effect was seen on the growth of pathogenic S. aureus. In future, it would be interesting to study if there is an inhibitive effect on pathogens at the lower concentrations.

Histological results showed that Re-Connecting Nature™ has no negative effect on the important skin barrier proteins (Loriclin, Claudin, Filaggrin and Ki67) expression in the skin model, not even at high concentrations. Immunological results showed that Re-Connecting Nature™ treatments reduced the levels of cytokines released in the skin model. In the situation where only three commensal microbes were present, Re-Connecting Nature™ treatments significantly reduced five pro-inflammatory cytokine levels (IL-4, IL-12p70, IL-13, TNFα, IFNγ) compared to the vehicle control. In the defense test with S. aureus, Re-Connecting Nature™ treatments significantly reduced seven pro-inflammatory cytokine levels (IL-1β, IL-2, IL-4, IL-12p70, IL-13, TNFα, IFNγ) compared to the vehicle. Importantly, immunological responses were detected even with the lowest concentration (0,5%). Based on these results the recommended concentration to use Re-Connecting Nature™ in products varies between 0,5 – 4%.

Re-Connecting Nature™ in a lotion created significant immunological response in skin model

Uute Scientific latest study was performed by a partner using an in-vitro skin model. The target was to study the effects of Re-Connecting Nature™ on skin microbiome, barrier proteins and cytokine secretion. Cytokines are signal molecules that regulate our immune responses, and are secreted by many types of cells including epithelial cells like keratinocytes. Some cytokines promote and some down-regulate inflammation responses with some having more complex roles. The test model included laboratory grown stratified epidermis, on which three skin commensal bacteria (S. epidermis, C. striatum. C. acnes) were grown. The defense test included pathogenic S. aureus. Re-Connecting Nature™ extract was used in four different concentrations (0,5%, 1%, 5%, 10%) in a cosmetic lotion. Controls included untreated and vehicle (= just the lotion) treated skin organoids.

According to the microbial results, Re-Connecting Nature™ at low concentrations (0,5%, 1%) had a neutral effect on the growth of S. epidermis and C. acnes and a prebiotic (=growth supporting) effect on the growth of C. striatum. At higher concentrations there was more interference, which may be due to limited number of microbes and short time exposure (24h) used in this model. At the defense test with S. aureus challenge, Re-Connecting Nature™ did not  have any significant effect on the growth of tested microbes, apart from the highest concentration (10%), where significant inhibitive effect was seen on  the growth of pathogenic S. aureus. In future studies, it would be interesting to see if there is an inhibitive effect on pathogens at the lower concentrations.

Histological results showed that Re-Connecting Nature™ has no negative effect on the important skin barrier proteins (Loriclin, Claudin, Filaggrin and Ki67) expression in the skin model, not even at high concentrations. Immunological results showed that Re-Connecting Nature™ treatments reduced the levels of cytokines released in the skin model. In the situation where only three commensal microbes were present, Re-Connecting Nature™ treatments significantly reduced five pro-inflammatory cytokine levels (IL-4, IL-12p70, IL-13, TNFα, IFNγ) compared to the vehicle control. In defense test with S. aureus, Re-Connecting Nature™ treatments reduced significantly seven pro-inflammatory cytokine levels (IL-1β, IL-2, IL-4, IL-12p70, IL-13, TNFα, IFNγ) compared to vehicle. Importantly, immunological responses were detected even with the lowest concentration (0,5%). Based on these results the recommended concentration to use Re-Connecting Nature™ in products varies between 0,5 – 4%.

Re-Connecting Nature™enhanced skin barrier function in in vivo study

Uute Scientific Oy conducted an in vivo study (n=6), where three weeks of pretreatment with lotion containing Re-Connecting Nature™ strengthened the skin barrier against a chemical irritation.

A test lotion with 1% Re-Connecting Nature™ (RCN) and placebo – the same base lotion without the active ingredient – were used for the selected test areas for three weeks, 1-2 times per day. Test volunteers chose their forearms or legs as test areas, and these areas were situated symmetrically with the same body parts (e.g left arm RCN, right arm placebo). Side (right/left) selected for each lotion varied between volunteers. After three weeks of pretreatment with lotions, the test sites were treated with 3 % SLS (Sodium lauryl sulphate) patch for 24 hours. Applied measurements were transepidermal water loss TEWL (VapoMeter, Delfin Technologies Ltd.) and Erythema index (SkinColorCatch, Delfin Technologies Ltd.). Measurements were conducted at 4 different time points (skin baseline before irritation (day 0), and after irritation at timepoints of 2 days, 6 days and 13 days). Each measured timepoint contained a total of 12 test area measurements: 6 measurements for each treatment. Results show that lotion with Re-Connecting Nature™ protected skin against the chemical irritant as compared to placebo treatment.​ Transepidermal water loss (TEWL) and Erythema index were both higher at placebo site compared to RCN site after the irritation. TEWL results were statistically significant (Fig1, P-value < 0.05) indicating enhanced barrier function as result of RCN pretreatment.

Skin benefits of Re-Connecting Nature™

Based on the literature and in vitro and in vivo studies presented here, several possible skin benefits can be associated with products containing Re-Connecting Nature™ microbial extract. Re-Connecting Nature™ extract at recommended concentrations supports natural microbiome diversity, and it may prevent overgrowth of pathogenic S. aureus. The mechanisms, by which Re-Connecting Nature™ support the skin barrier function, can be detected by observing the reduced levels of inflammatory cytokines, especially IL-4, IL-13, TNFα and IFNγ, which are linked to the impaired function of the skin barrier in several studies (15-21). In atopy, skin barrier disruption occurs with overgrowth of S. aureus, and levels of IL-4 and IL-13 (18,31) are elevated. Furthermore, there is itching, which is at least partly mediated by IL-4 (22). Based on the literature and our in vitro and in vivo studies, Re-Connecting Nature™ has strong potential for relieving the symptoms of atopic skin and promoting the barrier function of normal skin.

Skin barrier disruption can occur without any clinical skin disorders. The function of the skin barrier begins to fail and the rate of its repair declines after age of 55. Low-grade inflammation has been observed in ageing skin (23). Psychological stress disrupts the skin barrier, which may be related to stress-induced pro-inflammatory cytokine secretion (16). Some cytokines, like TNFα and IFNg, also inhibits collagen synthesis (24,25). Reducing the amount of pro-inflammatory cytokines helps maintain normal skin barrier function and collagen production in aging skin and in skin under stress. Reducing the amount of IFNg may also lead to faster skin regeneration and repair (25). UV-radiation induced sunburn reactions are related to inflammatory events, like the production of TNFα and IL-1b (26). Lowering levels of pro-inflammatory cytokines may reduce the redness and irritation caused by sunburn.

The literature review links lowered levels of proinflammatory cytokines with several skin benefits. Combining these findings with our in vitro skin model study results, opened promising new avenues for the use of Re-Connecting Nature™ in different products to achieve these benefits. The in vivo study testing skin barrier function of people with normal/sensitive against chemical irritant further supported the cytokine data from in vitro skin model.  The benefits from regular use of Re-Connecting Nature™ in cosmetic application strengthened the barrier function of the skin in a relatively short time period of three weeks, resulting in higher resilience of skin against stress factors, such as a chemical irritant, used during in vivo study with participants. While creating skin benefits, we are helping to prevent immune-mediated diseases.

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