Lupus Skin Is Primed for IL-6 Inflammatory Responses through a Keratinocyte-Mediated Autocrine Type I Interferon Loop

Abstract

Cutaneous lupus erythematosus is a disfiguring and common manifestation in systemic lupus erythematosus, and the etiology of this predisposition for cutaneous inflammation is unknown. Here, we sought to examine the keratinocyte as an important source of IL-6 and define the mechanism for its increased production in cutaneous lupus erythematosus. Evaluation of discoid and subacute cutaneous lupus erythematosus lesions showed significant epidermal up-regulation of IL-6 compared with control via real-time PCR and immunohistochemistry. Keratinocytes from unaffected skin of lupus patients produced significantly more IL-6 compared with healthy control subjects after exposure to toll-like receptor 2, 3, or 4 agonists or exposure to UVB radiation. Pretreatment with type I interferons (IFN-α and IFN-κ) increased IL-6 production by control keratinocytes, and type I IFN blockade decreased IL-6 secretion by lupus keratinocytes. Secretion of keratinocyte-specific IFN-κ was significantly increased after toll-like receptor 2 and UVB treatment in lupus keratinocytes, and neutralization of IFN-κ decreased IL-6 production by lupus keratinocytes. Thus, lupus keratinocytes are primed for IL-6 hyperproduction in a type I IFN-dependent manner. Increased production of IFN-κ by lupus keratinocytes drives this response, indicating that IFN-κ may play a pathogenic role in cutaneous lupus erythematosus and serve as a target for treatment.

Figure 1. IL-6 is increased in CLE in both the epidermis and inflammatory infiltrate

(a). Expression of IL-6 and IFN-regulated gene MX1 in discoid (n=23) and subacute cutaneous lupus (SCLE) (n=21) biopsies was determined by real-time PCR and expressed as fold change over healthy controls. (b). Representative immunohistochemistry for IL-6 expression in healthy control (n=3), SCLE (n=5), and DLE (n=5) lesions. IL-6 staining is noted in the keratinocytes as well as in the dermal inflammatory infiltrate. Scale bar=50μm. (c). Cutaneous IL-6 upregulation via RT-PCR was significantly higher in Ro+ (n=14) than Ro− (n=16) patients. Data analyzed using Student’s t-test (a) or Mann-Whitney U test (c);*=p<0.05.

Figure 2. Lupus keratinocytes from unaffected skin produce increased IL-6 following treatment with TLR agonists or exposure to UV radiation

Lupus and control keratinocytes were treated with LPS (a), PolyI:C (b), or PTG (c) at indicated concentrations, or exposed to 50 mJ/cm² UVB radiation (d) and IL-6 production was measured via ELISA. Data represents mean +/− SEM IL-6 concentration for four lupus samples, repeated in duplicate-quadruplicate. *=p<0.05; **=p<0.01; ****=p<0.0001 via pared t-test.

Figure 3. Enhanced IL-6 production by lupus keratinocytes is maintained across passages

a). Lupus and control keratinocytes were treated with TLR 2 agonist peptidoglycan at a concentration of 10 ug/ml and IL-6 production was measured via ELISA at passages 3, 4, and 5. Data for each passage represents mean +/− SEM IL-6 concentration for four lupus samples, repeated in triplicate. b). Lupus and control keratinocytes were exposed to 50 mJ/cm2 UVB radiation and IL-6 production was measured via ELISA at passages 3, 4, and 5. Data for each passage represents mean +/− SEM IL-6 concentration for four lupus samples, repeated in duplicate-quadruplicate. *=p<0.05; **=p<0.01; ***=p<0.001 for comparison of SLE vs. control for each passage via pared t-test.

Figure 4. Type I IFN signaling stimulates increased production of IL-6 following TLR exposure and UV irradiation

Control keratinocytes were primed with or without 1000 U/ml IFNα (a, b) or 1000 U/ml IFNκ (c, d) for 24 hours, followed by stimulation with PTG (a, c) at described concentrations or 50 mJ/cm2 UVB irradiation (b, d). IL-6 production was measured via ELISA. Results represent mean+SEM for single experiment repeated in triplicate. *=p<0.05; **=p<0.01; ****=p<0.0001, via Student’s paired t-test.

Figure 5. Elevated IFNκ production drives hyperproduction of IL-6 in lupus keratinocytes

Lupus keratinocytes were pretreated with neutralizing IFNR ab (a) or IFNκ Ab (b) prior to stimulation with TLR 2 agonist, as described, and IL-6 concentration was measured via ELISA. (c, d) Lupus and control keratinocytes were stimulated with TLR 2 agonist (c) or UVB radiation (d) and IFNκ concentration was measured via ELISA. Data represents mean +/− SEM IL-6 for four lupus samples, repeated in triplicate. *=p<0.05; **=p<0.01; ***=p<0.001 via Student’s paired t-test.