Age-related changes in expression and function of Toll-like receptors in human skin

Abstract

Toll-like receptors (TLRs) initiate innate immune responses and direct subsequent adaptive immunity. They play a major role in cutaneous host defense against micro-organisms and in the pathophysiology of several inflammatory skin diseases. To understand the role of TLRs in the acquisition of immunological competence, we conducted a comprehensive study to evaluate TLR expression and function in the developing human skin before and after birth and compared it with adults. We found that prenatal skin already expresses the same spectrum of TLRs as adult skin. Strikingly, many TLRs were significantly higher expressed in prenatal (TLRs 1-5) and infant and child (TLRs 1 and 3) skin than in adult skin. Surprisingly, neither dendritic cell precursors in prenatal skin nor epidermal Langerhans cells and dermal dendritic cells in adult skin expressed TLRs 3 and 6, whereas the staining pattern and intensity of both TLRs in fetal basal keratinocytes was almost comparable to those of adults. Stimulation of primary human keratinocytes from fetal, neonatal and adult donors with selected TLR agonists revealed that the synthetic TLR3 ligand poly (I:C) specifically, mimicking viral double-stranded RNA, induced a significantly enhanced secretion of CXCL8/IL8, CXCL10/IP-10 and TNFα in fetal and neonatal keratinocytes compared with adult keratinocytes. This study demonstrates quantitative age-specific modifications in TLR expression and innate skin immune reactivity in response to TLR activation. Thus, antiviral innate immunity already in prenatal skin may contribute to protect the developing human body from viral infections in utero in a scenario where the adaptive immune system is not yet fully functional.

Fig. 1.

TLR expression profile of human skin before and after birth. (A) Embryonic (9-11 weeks EGA), fetal (12-13 weeks EGA) and adult (18-45 years) skin specimens were investigated (n=6-9 donors/group) for the indicated TLR expression levels using quantitative RT-PCR. Reactions were executed in duplicates and data were normalized to the housekeeping gene B2M. (B) Epidermis was separated from the dermis of skin samples from infants (3-10 months), children (5-12 years) and adults (18-31 years) (n=4-8 donors/group) using ammonium thiocyanate. Indicated TLR expression levels were determined using quantitative RT-PCR. Reactions were executed in duplicates and data were normalized to the housekeeping gene GAPDH. Bars represent the mean of investigated groups. The x- and y-axis indicate selected age groups and relative mRNA expression, respectively. *P<0.05, **P<0.01, ***P<0.001. (C) Immunofluorescence double labeling of cryostat sections from embryonic, fetal and adult skin samples (n=3 donors/group) revealed CD1c⁺TLR3⁻TLR6⁻ dendritic cells in prenatal and adult skin (inserts). The arrows denote epidermal LCs and the arrowheads identify DDCs. Nuclear counterstain was performed with DAPI. Scale bars: 100 μm in C; 20 μm in inserts.

Fig. 2.

Bacterial stimuli and TLR ligands induce distinct immune responses in fetal, neonatal and adult keratinocytes. (A) Primary keratinocytes from fetal, neonatal and adult skin were cultured in 12-well plates to ~75% confluence and stimulated in triplicates for 24 hours with the indicated bacterial stimuli and ligands at concentrations described in the Materials and methods. IL1α was used as positive control, cell culture medium (untreated) and TSB as negative controls. (B) CXCL8 (C) CXCL10 and (D) TNFα levels in supernatants were quantified by ELISA. Experiments were performed as three biological replicates/age group (different donors) and the data represent the average (mean±s.e.m.) of these donors. *P<0.05, **P<0.01, ***P<0.001. (E) Western blot analysis of untreated primary keratinocytes showed an almost equal expression of TLR3 and TLR6 proteins in all indicated age groups. Stimulation of keratinocytes with poly (I:C) did not affect TLR3 expression, whereas TLR6 expression was upregulated in fetal and neonatal, but not in adult, keratinocytes.

Fig. 3.

Poly (I:C) activates differential expression of chemokines, cytokines and angiogenic factors in fetal keratinocytes in comparison with neonates and adults. Keratinocytes from fetal, neonatal and adult donors (n=3 donors/group) were cultured in KGM and stimulated with poly (I:C) or left untreated (negative control) for 24 hours. Supernatants were collected, pooled and analysed for the indicated chemokines, cytokines and angiogenic factors using commercially available protein arrays.

Fig. 4.

Expression pattern of TLRs 3 and 6 in skin equivalents generated from fetal, neonatal and adult keratinocytes. (A) Cryostat sections of skin equivalents generated with keratinocytes from the indicated age groups (n=3 donors/group), and adult breast skin (positive control), were stained for TLR 3 and 6 protein expression using immunohistochemistry. (B) Immunofluorescence double labeling revealed K10⁻TLR3⁺TLR6⁺ basal keratinocytes (red) and K10⁺TLR3⁻TLR6⁻ suprabasal keratinocytes (green). Nuclei were stained with Hoechst dye (blue). Scale bars: 50 μm in A,B (immunohistochemistry); 40 μm in B (immunofluorescence).