Peanut lipids display potential adjuvanticity by triggering a pro‐inflammatory response in human keratinocytes

Currently, the earliest cellular and molecular signals driving allergic sensitization to peanuts are not fully understood, even though peanut allergens have been studied extensively. Meanwhile, lipids contained within allergen sources are emerging as players in the pathogenesis of allergies. Exposure of infants to peanut oil-containing lotions was described as a risk factor for the development of peanut allergy (1). This article is protected by copyright. All rights reserved.


S U P P O R T I N G I N F O R M A T I O N
Additional Supporting Information may be found online in the supporting information section at the end of the article. DOI: 10.1111/all.13475

Peanut lipids display potential adjuvanticity by triggering a pro-inflammatory response in human keratinocytes
To the Editor, Currently, the earliest cellular and molecular signals driving allergic sensitization to peanuts are not fully understood, even though peanut allergens have been studied extensively. Meanwhile, lipids contained within allergen sources are emerging as players in the pathogenesis of allergies. Exposure of infants to peanut oil-containing lotions was described as a risk factor for the development of peanut allergy. 1 There is evidence that only peanut extracts containing lipids were able to induce sensitization. 2,3 How this occurs at the molecular level is still unknown. Moreover, in order to induce epicutaneous sensitization to purified peanut allergens, tape-stripping of the skin is commonly performed. 4 This procedure damages the skin and induces release of inflammatory cytokines in keratinocytes (KC). 5 If crude peanut extract containing both allergens and lipids, but not purified peanut allergens alone, can sensitize mice via intact skin, 1,3 we wondered whether this effect might be due to the presence of peanut lipids (PNL). Therefore, we hypothesized that PNL might represent an adjuvant for peanut allergens in sensitization via the skin. To address this issue, we exposed human primary KC, major components of the outer layer of the skin, to PNL containing all major lipid classes ( Figure S1) in the presence or absence of purified and functionally active peanut major allergens Ara h 1 or Ara h 2 ( Figure S2), to study the induced immune response. Food-processing modifications might contribute to influence the immune response; thus, roasted peanuts were used as source of PNL and allergens.
Details for materials and methods are provided in this article's online supporting information (Data S1).
First, we measured mRNA levels of IL-8, IL-6, TNF-a, and IL-1b in KC. We found that PNL, but not allergens alone, induced higher mRNA levels of these inflammatory mediators compared with the control (Figure 1). This effect persisted when PNL were coadminis- Interestingly, the combination of PNL and Ara h 1 or Ara h 2 inhibited PNL-induced IL-10 release. Peanut allergens did not alter the capacity of PNL to induce pro-inflammatory cytokines. However, via the inhibition of PNL-induced IL-10 release, they may contribute to extend the duration of inflammation and promote an environment suitable for the orchestration of Th2 immune responses leading to sensitization.
We also measured higher COX-2 mRNA and protein levels in KC stimulated with PNL compared with the control (Figure 1; Figure S3). COX-2 is essential for the induction of in vivo allergic inflammation in C57BL/6 mice 8 and responsible for prostaglandin synthesis, inflammatory mediators with a key role in skin allergic inflammation. HO-1 mRNA levels ( Figure 1), but not proteins (data not shown), were also increased upon PNL treatment. HO-1 is highly expressed in KC and is increased in response to stress and inflammatory stimuli. 9 The capacity of PNL to activate human KC was further supported by our results demonstrating stress-related morphological changes appearing after 24 hours of PNL treatment in the presence or absence of allergens ( Figure S4) without affecting cell viability ( Figure S5).
Endothelin-1 (ET-1), a peptide causing pruritus in mice and humans, is induced in KC in atopic dermatitis lesions and in response to house-dust-mite. 10 PNL, but not allergens, enhanced the ET-1 production (Figure 2), suggesting that PNL could promote itching and eventually skin barrier damage.
Our data demonstrated that KC were able to recognize PNL as exogenous stimuli that directly triggered the production of inflammatory mediators. Such mediators were shown to cause barrier  Table S1. LETTERS TO THE EDITOR | 1747 disruption, facilitate allergen penetration and initiate cutaneous inflammation. 5,6 Interestingly, allergens coadministered with PNL were not able to modify (neither inhibit nor synergize) this effect of PNL, suggesting that the capacity of PNL to activate the innate immune system is maintained in the presence of the allergens, as in crude peanut extract. 2,3 In contrast, peanut allergens coadministered with PNL inhibited the production of PNL-induced IL-10, likely produced by KC as a compensatory mechanism. Altogether, this might indicate that PNL combined with allergens are necessary to prolong the pro-inflammatory milieu induced by PNL which might contribute to favor allergic sensitization.
Here, we show that purified PNL activate KC and promote a pro-inflammatory response similar to the effect of tape-stripping, 5 proposing a potential role for PNL as adjuvant for peanut allergens.
Indeed, without the PNL-induced pro-inflammatory milieu, the allergens were unable to elicit any response in KC.