Opposing roles of endothelial and leukocyte-expressed IL-7Rα in the regulation of psoriasis-like skin inflammation

The interleukin 7 receptor alpha chain (IL-7Rα) is predominately expressed by lymphocytes, and activation by its ligand IL-7 supports the development and maintenance of T cells and boosts T-cell mediated immunity. We recently reported that lymphatic endothelial cells (LECs) in dermal lymphatics also express IL-7 and its receptor chains (IL-7Rα and CD132) and that IL-7 supports lymphatic drainage. This suggested that activation of IL-7Rα signaling in lymphatics could exert inflammation-resolving activity, by promoting the clearance of excess tissue fluid. Here we investigated how the potentially opposing effects of IL-7Rα signaling in immune cells and in the lymphatic vasculature would affect the development and progression of psoriasis-like skin inflammation. We found that during acute and chronic skin inflammation mice with an endothelial-specific deletion of IL-7Rα (IL-7RαΔEC mice) developed more edema compared to control mice, as a consequence of impaired lymphatic drainage. However, systemic treatment of wild-type mice with IL-7 exacerbated edema and immune cell infiltration in spite of increasing lymphatic drainage, whereas treatment with IL-7Rα blocking antibody ameliorated inflammatory symptoms. These data identify IL-7Rα signaling as a new pathway in psoriasis-like skin inflammation and show that its pro-inflammatory effects on the immune compartment override its anti-inflammatory, drainage-enhancing effects on the endothelium.


Genotyping PCR
DNA from cultured LN LECs was extracted using the NucleoSpin Tissue kit (Machery-Nagel, Düren, Germany). PCRs were performed to detect cre-mediated recombination, as well as the WT sequence. The forward primer 5' GGT ACC TTG AGC TCA GAA ATT GG 3' (primer 1) or 5' ACA GTG GGG CTC TTT TAC GA 3' (primer 2) respectively was used with the reverse primers 5'GAG GTA GAT GGC CTG CTT TAG 3' (primer 3). A quantitative real-time PCR was performed using the primer pair for the WT sequence on a Fast Real-time PCR system (Applied Biosystems, Thermo Fisher).

Cloning of mIL-7-Fc
Murine IL-7 cDNA was amplified from a murine IL-7-containing pMD18 plasmid (Sino Biological Inc., Beijing, China) by PCR. To optimize expression of the mIL-7-Fc protein, the native secretion sequence was replaced by a secretion sequence more suitable for production in CHO-S cells (kindly provided by Dario Neri, ETH Zurich 1 ). In a first PCR we appended the first part of the secretion sequence to the N terminus with the forward primer: 5' TCC TCC TGT   TCC TCG TCG CTG TGG CTA CAG GTG TGC ACT CGG AGT GCC ACA TTA AAG  Biolabs, Ipswich, USA). The assembled sequence for mIL-7-Fc was digested with HINDIII and NOTI restriction enzymes (both from New England Biolabs) and gel-purified, and ligated into a HINDIII/NOTI digested pcDNA3.1 plasmid (Thermo Fisher, Waltham, USA). PCR, digestion, and ligation products were purified with a PCR cleanup and gel extraction kit (Machery-Nagel).
The plasmid containing the mIL-7-Fc sequence was transformed into TOP10 E. Coli (Thermo Fisher) by heat shock (30 minutes on ice, 30 seconds at 42°C, 2 minutes on ice) and single colonies were screened for the correct sequence. The plasmid containing the correct sequence was purified with a plasmid DNA purification kit (Machery-Nagel).

Expression and purification of mIL-7-Fc
Expression and purification of mIL-7-Fc in CHO cells was performed as described previously 2 .
In brief, CHO cells were transfected with plasmid containing mIL-7-Fc by Polyethyleniminemediated transient gene expression. CHO cells were incubated in PRO-CHO4 medium (Lonza, Basel, Switzerland) containing 2mM glutamine (Thermo Fisher), hydroxytryptamine (Thermo Fisher) and antibiotic antimycotic (Anti Anti, Thermo Fisher) for one week at 31°C in a shaking incubator at 120 rotations per minute. The protein was purified by running the CHO cell supernatant over a PD10 resin (GE Healthcare, Chicago, USA) followed by a protein A resin (Sino Biological). After 2 washing steps in buffer containing 100 mM NaCl (Sigma-Aldrich), 5 mM EDTA (Sigma-Aldrich), 1% polysorbate 20 (Sigma-Aldrich) and 100 mM NaCl (Sigma-Aldrich), 5 mM EDTA (Sigma-Aldrich) respectively, the protein was eluted from the protein A resin with 0.1 M triethylamine PH 9 (Sigma-Aldrich) and the PH was adjusted immediately after