Topical Diclofenac Reprograms Metabolism and Immune Cell Infiltration in Actinic Keratosis

Abstract

Background: Melanoma and squamous cell carcinoma of the skin are characterized by an altered glucose metabolism, but little is known about metabolic changes in precancerous skin lesions such as actinic keratosis (AK). Here, we studied the central carbon metabolism and immune cell infiltrate of actinic keratosis lesions before, under, and 4 weeks after treatment with topical diclofenac (Solaraze®). Methods: This study was designed as a prospective, randomized, controlled, monocentric investigation (ClinicalTrials.gov Identifier: NCT01935531). Myeloid and T cell infiltration was analyzed in skin biopsies from 28 patients by immunohistochemistry. Furthermore, immune cell activation was determined via quantitative real-time PCR (IFN-γ, IL-10, CSF1, TGF-β, IL-6). Glucose, amino acid and Krebs' cycle metabolism was studied by mass spectrometry prior, during and after treatment with topical diclofenac. Biopsies from sun-exposed, untreated, healthy skin served as controls. Results: Increased lactate and decreased glucose levels suggested accelerated glycolysis in pre-treatment AK. Further, levels of Krebs' cycle intermediates other than citrate and amino acids were elevated. Analysis of the immune infiltrate revealed less epidermal CD1a+ cells but increased frequencies of dermal CD8+ T cells in AK. Treatment with diclofenac reduced lactate and amino acid levels in AK, especially in responding lesions, and induced an infiltration of dermal CD8+ T cells accompanied by high IFN-γ mRNA expression, suggesting improved T cell function. Discussion: Our study clearly demonstrated that not only cancers but also pre-malignant skin lesions, like AK, exhibit profound changes in metabolism, correlating with an altered immune infiltrate. Diclofenac normalizes metabolism, immune cell infiltration and function in AK lesions, suggesting a novel mechanism of action.

Keywords: CD1a; CD8; actinic keratosis; diclofenac; lactate; metabolism.

Figures

Figure 1

Increased lactate and amino acid levels in actinic keratosis lesions. (A) qRT-PCR analysis of GLUT-1, LDHA, LDHB, COX1, and COX2 gene expression in actinic keratosis lesions (AK) and control biopsies (co) of sun-exposed, untreated, healthy skin in 28 patients. (B) Representative staining of GLUT-1 in actinic keratosis lesion and control biopsy. (C) Immunohistochemical staining of GLUT-1 in epidermis, dermis and skin appendages in actinic keratosis lesions and control biopsies. The selected pixels of the evaluated images were expressed as the percentage of the total area. (D–G) Determination of intratumor metabolite levels by mass spectrometry. The dark line indicates the median. *p < 0.05, **p < 0.01, ***p < 0.001 (Mann-Whitney test). Ala, alanine; Gln, glutamine; Asn, asparagine; Orn, ornithine; Gly, glycine; Arg, arginine.

Figure 2

Metabolic response to diclofenac in AK lesions. (A–H) Temporal changes in intratumor metabolite levels of (A) glucose, (B) lactate, (C) tryptophan, (D) glutamine, (E) alanine, (F) phenylalanine, (G) fumarate, and (H) citrate, in actinic keratosis lesions and control skin biopsies. Left graphs show all patients pre, on (diclo), and post treatment with diclofenac. Right graphs show non-responders (NR, n = 8) and responders (R, n = 12) to diclofenac on (diclo) and post treatment compared to controls (co). The dark lines indicate the median. *p < 0.05, **p < 0.01 (Kruskal-Wallis test).

Figure 3

Actinic keratosis induces changes in the skin immune infiltrate. (A–D) Immunohistochemical staining of (A) CD1a, (B) CD68, (C) CD4, and (D) CD8 in epidermis, dermis and skin appendages in actinic keratosis lesions (AK) and control biopsies (co) of sun-exposed, untreated, healthy skin in 28 patients. The selected pixels of the evaluated images were expressed as the percentage of the total area. (E) Representative stainings of CD1a (upper images), CD8 (middle images), and CD68 (lower images) in actinic keratosis lesions and control biopsies. (F) qRT-PCR analysis of CSF1, TGF-β, IFN-γ, IL-6, and IL-10 gene expression in actinic keratosis lesions and control skin biopsies. The dark lines indicate the median. *p < 0.05, **p < 0.01, ***p < 0.001 (Mann-Whitney test).

Figure 4

Diclofenac induced an increased immune response. (A–E) Immunohistochemical staining of (A,B) CD1a, (C,D) CD8, and (E,F) CD68 in epidermis and dermis in actinic keratosis lesions (AK) and control skin biopsies (co). The selected pixels of the evaluated images were expressed as the percentage of the total area. (G,H) qRT-PCR analysis of (G)IFN-γ and (H)IL-10 gene expression in actinic keratosis lesions and control skin biopsies. Left graphs show all patients pre, on (diclo) and post treatment with diclofenac. Right graphs show non-responders (NR, n = 8) and responders (R, n = 12) to diclofenac on (diclo) and post treatment compared to controls (co). The dark lines indicate the median. *p < 0.05, **p < 0.01, ***p < 0.001 (Kruskal-Wallis test).