NF-κB p65 and c-Rel control epidermal development and immune homeostasis in the skin

Abstract

Psoriasis is an inflammatory skin disease in which activated immune cells and the pro-inflammatory cytokine TNF are well-known mediators of pathogenesis. The transcription factor NF-κB is a key regulator of TNF production and TNF-induced pro-inflammatory gene expression and both the psoriatic transcriptome and genetic susceptibility further implicate NF-κB in psoriasis etiopathology. However, the role of NF-κB in psoriasis remains controversial. We analyzed the function of canonical NF-κB in the epidermis using CRE-mediated deletion of p65 and c-Rel in keratinocytes. In contrast to animals lacking p65 or c-Rel alone, mice lacking both subunits developed severe dermatitis after birth. Consistent with partial histological similarity to human psoriasis, this condition could be prevented by anti-TNF treatment. Moreover, regulatory T cells in lesional skin played an important role in disease remission. Our results demonstrate that canonical NF-κB in keratinocytes is essential for the maintenance of skin immune homeostasis and is protective against spontaneous dermatitis.

Introduction

Psoriasis is a chronic inflammatory skin disease characterized by epidermal hyperplasia, altered keratinocyte differentiation and inflammatory infiltrate (1). It remains unclear whether the primary defect fomenting psoriasis lesion development affects keratinocytes or immune cell function. In murine models, forced expression of inflammatory cytokines in keratinocytes produces lesions with characteristics of human psoriasis (2–4). Constitutive activation of several transcription factors that regulate the expression of inflammatory cytokines, such as STAT-3 or Nuclear Factor of kappa light polypeptide gene enhancer in B-cells (NF-κB) in keratinocytes or immune cells, can also drive cutaneous inflammation (5, 6).

The transcription factor NF-κB is a complex formed by dimerization of its subunits, p65 (RelA), RelB, c-Rel, p50 and p52 (7). The canonical NF-κB pathway culminates in activation of dimers of p65, c-Rel, and p50 subunits. Genome wide association studies (GWAS) suggest a link between psoriasis and the NF-κB pathway (8) that is supported by mouse models. Ablation of the NF-κB inhibitor IκBα produces cutaneous inflammation and keratinocyte proliferation (6, 9). However, deletion of IKKβ, which mediates canonical NF-κB activation, produces a fulminant psoriasis-like disease in mice (10). Expression of an IκBα super-repressor in keratinocytes also results in a similar, though less severe phenotype (11). These phenotypes are driven by TNF, as IKKβ-deficient mice lacking TNFR1 or treated with anti-TNF Abs, do not develop the disease (10, 12, 13). Given IκBα has NF-κB-independent functions in keratinocyte development (14), and IKKβ has direct effects on ERK and STAT1 activation (15–17), it is unclear whether the cutaneous inflammation in these mice is fully attributable to defective NF-κB activation. To directly examine the role of NF-κB in skin, we deleted RelA and c-Rel in keratinocytes. Mice lacking both subunits developed dermatitis with some psoriasiform characteristics, which was prevented by TNF blockade. Spontaneous remission was promoted by Foxp3⁺ regulatory T cells (Tregs). These results highlight a crucial role for canonical NF-κB in the maintenance of skin homeostasis.

Materials and Methods

Animals

K14^cre and Rela^fl/fl and c-Rel^fl/fl mice (18–20) were kept in specific pathogen-free conditions in the animal care facility and experiments were conducted under IACUC approval at Columbia University (New York, NY).

Flow Cytometry

Spleen and lymph nodes cells were isolated by mechanical desegregation in PBS+3% FBS. Whole skin was minced and digested 3hrs at 37°C in DMEM (Gibco) with 1mg/ml collagenase-IV (Sigma) and 1mg/ml DNase I (Sigma) and strained. Cell suspensions were stained using antibodies from EBioscience: TCR-β (H57), CD4 (RM4.5), CD8 (53-6.7), CD19 (Ebio1D3), CD90.2 (53-2.1), CD45 (30F11), CD44 (IM7), NK1.1 (PK136), TCR-γδ (EbioGL3), CD11c (N418), CD11b (M1/70), IL-33R (ST2), CD25 (PC61, 7D4), and Foxp3 (FJK16s). Foxp3 staining was performed using the eBioscience kit. Cells were acquired on a LSR II (BD Biosciences) and analyzed with FlowJo.

mRNA expression

For qRT-PCR, frozen tissues were dissociated using Lysing Matrix D tubes (MP). Total RNA was isolated using Trizol and reverse transcribed by Superscript III (LifeTechnologies). qPCR with SYBR Green (Quanta Biosciences) was performed on a CFX96 or 384 (Bio-Rad), all values are relative to GAPDH. Primers sequences available upon request.

Histology

Ear or skin specimens were fixed with 4% neutral-buffered formalin for 4 days, transferred to 70% ethanol and embedded in paraffin. 5 μM sections were cut, deparaffinized, stained with hematoxylin and eosin (H&E) or TUNEL, imaged on an Axio M2 (Zeiss) and processed using AxioVision and ImageJ. Epidermal thickness was measured on at least 15 random fields per specimen; mean thickness is shown.

Statistical analyses

Statistical significance was calculated using the two-tailed unpaired Student t-test.

Results and Discussion

The role NF-κB in skin biology and pathophysiology remains ambiguous. Although mice lacking p65, cRel and TNF exhibit defects in epidermal differentiation (21), it is not clear if this is the result of a keratinocyte intrinsic requirement for NF-κB. Therefore, we crossed mice carrying either floxed Rela or c-Rel alleles to a transgenic mouse expressing Cre under the control of the keratin 14 (K14) promoter. K14^creRela^fl/fl (K14^ΔRela) and K14^crec-Rel^fl/fl (K14^Δc-Rel) pups were born at Mendelian ratios and displayed reduced expression of Rela and c-Rel mRNA, respectively, in the epidermis (Supplemental Fig. 1A). Histological analyses showed normal epidermal thickness and keratinocyte differentiation (data not shown), indicating deletion of Rela or c-Rel did not affect skin development. We next performed DNFB-induced contact-hypersensitivity (CHS). K14^ΔRela and K14^Δc-Rel mice exhibited increased ear swelling and TNF and IFN-γ expression compared to littermate controls (Supplemental Fig. 1C-F). Consistent with a recent study using mice lacking RelA in the epidermis (22), these data suggest RelA and c-Rel have a non-redundant, keratinocyte intrinsic, immunoregulatory role in skin.

Spontaneous dermatitis in mice lacking RelA and c-Rel in keratinocytes

It has previously been reported that RelA has a growth inhibitory role in keratinocytes (23) and prevents keratinocyte differentiation (24). However, no changes were observed in epidermal differentiation upon keratinocyte specific deletion of RelA. Therefore, to assess whether they are redundant in epidermal development we deleted RelA and c-Rel in keratinocytes (DKO mice). That leads to a full ablation of RelA and minimal residual c-Rel in DKO epidermis (Supplemental Fig 1A, B). DKO pups were born at expected Mendelian ratios but exhibited visible skin lesion from D5, which spread rapidly and covered most of the body by D12 (Fig. 1A). Early lesions were well-demarcated, scattered, rigid, scaly plaques without edematous or exudative reaction. H&E staining revealed hyperkeratosis and focal parakeratosis: features of psoriatic lesions. Epidermal thickening and dermal and epidermal mononuclear infiltrates were observed (Fig. 1B, E). DKO mice exhibited apoptotic loci in all layers of the epidermis (Fig. 1F).

Figure 1. Epidermal deletion of Rela and c-Rel drives psoriasis-like inflammation.

Representative pictures (A, C) and H/E staining on skin sections (B, D) of WT and DKO 12 (B, C) and 30 (C, D) days after birth. Bars=100μm, original magnification= 20×. In (B), right panels are a 40× magnification of DKO samples. (E) Mean +/− SEM epidermal thickness from 3–6 mice/group/time point. (F) TUNEL staining on d16 ventral skin. Arrows show TUNEL⁺ nuclei. Loricrin (G) and Keratin 10 (H) mRNA expression in whole skin 2 days after birth. (I) Epidermis was isolated from 14-days old WT and DKO mice and expression of S100A8, S100A9, Defb3, IL22, IL24 and IL1b was determined by q-RT-PCR. All data are from 3 or more independent experiments.*p<0.05, **p<0.01, ***p<0.001, n.s. non-significant.

No lethality was observed, and, in >90% of the mice, the skin lesions gradually resolved, and dorsal epidermal thickness permanently normalized by D30 (Fig. 1C-E). Disease re-occurrence was not observed in any animal. However, the abdominal skin retained gross and histological lesions (Fig. 1D, E). Strikingly, mRNA coding for calcium-binding proteins S100A8 and S100A9, Defensin B3, as and cytokines IL-22, IL-24 and IL-1β, were strongly up-regulated in the epidermis of DKO mice (Fig. 1I) as they are in human psoriasis. Expression of the differentiation markers Loricrin and Keratin 10 were decreased in the skin of DKO mice (Fig. 1G, H). However, skin barrier function remained intact in DKO mice, as assessed by a Toluidine Blue skin barrier assay (data not shown).

To further explore skin immune homeostasis, we performed flow cytometry on D16 tissues. We observed a 2-fold increase in the proportion of CD45⁺ leukocytes among total skin cells in DKO mice (Fig. 2A), which correlated with the mononuclear cell infiltration (Fig 1B). TCR-β⁺ T cells were increased in the skin of DKO mice (Fig. 2B). The CD4⁺Foxp3⁺ Treg proportion and number were dramatically enhanced, comprising up to 60% of the skin-infiltrating CD4⁺ T cells compartment (Fig. 2C-D). We observed a 3-to 5-fold increase in the numbers of ILC2, which have been implicated in inflammatory responses in the skin (25), in both LN and skin of DKO mice, compared to WT controls (Fig. 2E-F). K14 is expressed by medullary thymic epithelial cells (mTECs) (26) which are important for Treg selection and deletion of autoreactive T cells (27). However, no changes were observed in mTEC or T-cell subsets in the thymus of D7 DKO mice (Supplemental Fig. 2). Thus, the dermatitis in DKO mice is likely due to a keratinocyte intrinsic function of NF-κB. Together these results indicate that canonical NF-κB in keratinocytes is required for their optimal differentiation, as well as maintenance of immune homeostasis in the skin.

Figure 2. Skin leukocyte infiltration in DKOmice.

Spleen, LN (inguinal) and whole skin from D16 mice were analyzed by flow cytometry. (A) % of CD45⁺ (leukocytes) among total live cells. (B) Numbers of CD45⁺TCR-β⁺ cells in 10⁶ live cells. Representative dot plot (C, gated on CD45⁺TCR-β⁺CD4⁺ cells). (D) Numbers of CD45⁺TCR-β⁺CD4⁺Foxp3⁺ cells in 10⁶ live cells. Representative dot plot (E), gated on CD45⁺TCR-β⁻TCR-γδ⁻CD19⁻CD11c⁻CD11b⁻NK1.1⁻ (Lin⁻) cells. (F) Numbers of Lin⁻ CD25⁺ST2⁺ cells in 10⁶ live cells; mean +/− SEM. Data representative or cumulative of at least 3 independent experiments. *p<0.05, n.s. non-significant.

Dermatitis in DKO mice can be prevented by TNF-blockade

TNF contributes to chronic inflammatory diseases including psoriasis and anti-TNF treatments are a first-line treatment for moderate to severe psoriasis (28). Therefore, we asked whether TNF mediated the dermatitis of DKO mice. TNF mRNA was increased in the skin of DKO mice 7 days after birth (Fig. 3A). Injection of an anti-TNF mAb prior to the appearance of the symptoms, strongly reduced skin lesions, epidermal thickness, leukocyte infiltration and apoptosis compared to vehicle-treated animals (Fig. 3B-D and data not shown). These data definitively establish that TNF can drive psoriasifom dermatitis independent of activation of the canonical NF-κB pathway in keratinocytes.

Figure 3. TNF-blockade inhibits dermatitis.

(A) Tnf mRNA expression in the whole skin 7 days after birth, assessed by RT-qPCR. (B-D) WT and DKO mice were injected intra-peritoneally with 10 mg/kg anti-TNF (XT3.11, BioXCell), or vehicle (Ctrl) every other day from D4 to D12 after birth. Mice were sacrificed at D15. (B) Representative pictures of mice. (C) Representative H/E staining of ventral skin sections. Bars=100μm, original magnification= 20×. (D) Mean +/− SEM epidermal thickness. Dots represent individual mice, bars indicate mean. All data are from 1 of 2 independent experiments. *p<0.05, n.s. non-significant.

Regulatory T cells play a protective role in the remission of dermatitis

In contrast to K14^creIKK2^fl/fl mice (10, 13), DKO mice spontaneously begin to recover 3 weeks after birth. This led us to explore the mechanism of “remission”. Because we observed a massive Treg expansion in DKO skin prior to remission, we tested whether Treg contributed to remission by injecting an anti-CD25 Ab. This protocol achieved a 50% reduction of Foxp3⁺ T cells 2 days after treatment (Fig. 4A). Treg depleted DKO mice exhibited worsened pathology, with increased skin immune infiltrate (Fig. 4B-D). These results suggest Treg were necessary for disease recovery, and highlight the role of Treg in skin immune homeostasis.

Figure 4. Treg depletion inhibits recovery of DKO mice.

3 week-old DKO mice received a single intra-peritoneal injection of 150ug anti-CD25 mAb (PC61.5, BioXCell) or vehicle (Ctrl). (A) Representative Foxp3 staining in CD4⁺TCR-β⁺CD45⁺ gated cells. Numbers indicate the percentage gated. (B) Representative pictures of mice. (C) Representative H/E staining of ventral skin sections. Bars=100μm, original magnification= 20×. (D) Mean +/− SEM epidermal thickness. Dots represent individual mice, bars indicate mean. All data are representative or cumulative of 2 independent experiments *p<0.05, n.s. non-significant.

It has been proposed that the pathogenesis of psoriasis may follow a two-step model. First, environmental and/or genetic factors drive keratinocyte dysfunction and production of chemokines or inflammatory cytokines; in turn, activation of immune cells such as DC and macrophages may trigger a strong T-cell-dependent inflammatory response, leading to increased proliferation of epidermal cells and clinical symptoms. Here we show that perturbation of the canonical NF-κB in the epidermis can trigger cell death, immune infiltration and hyperkeratosis. These data indicate that NF-κB supports skin immune homeostasis and may prevent uncontrolled TNF-dependent leukocyte recruitment and activation.

Non-Standard Abbreviations

DNFB

2,4-dinitrofluorobenzene

IKK

Inhibitor of Kappa B Kinase

mTEC

medullary Thymic Epithelial Cells

TSLP

Thymic Stromal Lymphopoietin