Mechanisms of control of allergic skin inflammation by T regulatory cells - Most patients with Atopic dermatitis (AD) have a skin barrier defect. This allows cutaneous sensitization to antigens that leads to allergic skin inflammation (ASI). In a minority of patients, AD arises from an immune deficiency. Patients with DOCK8 deficiency and WASP deficiency have a normal skin barrier, but impaired Treg cell function. Dock8-/- mice developed exaggerated ASI following application of ovalbumin (OVA) to tape stripped skin. Adoptive transfer experiments demonstrate that defective Tregs underlie their exaggerated ASI. We propose to use the DOCK8 deficiency model to study of the pathways that drive AD independently of skin barrier defects and determine the mechanisms by which Tregs restrain ASI. DOCK8 activates the GTPase CDC42. CDC42 activates the Ser/Thr kinase PAK2 as well as WASP, which drives actin filaments branching. Preliminary data show that AD is more severe in DOCK8- deficient than WASP-deficient patients. Similarly, ASI is more severe in Dock8-/- than in Wasp-/- mice. Tregs in OVA sensitized skin and their FOXP3 expression are decreased in Dock8-/-, but not Wasp-/-, mice. DOCK8 deficient induced Tregs (iTregs) have reduced stability in vitro particularly in the presence of IL-4. Tregs from Dock8-/, but not Wasp-/-, mice, as well as WT Tregs treated with PAK inhibitors, have decreased levels of pY-STAT5 following IL-2 stimulation due to accelerated dephosphorylation. This suggests that the DOCK8-PAK2 axis, but not the DOCK8-WASP axis, is essential to maintain pY-STAT5 levels in Tregs AlphaFold Multimer modeling predicts a direct interaction between STAT5B and the tyrosine phosphatase SHP2, abolished by mimicking PAK2-mediated serine phosphorylation of STAT5B. We will test the overall hypothesis that in DOCK8 deficiency, loss of WASP activation, which disrupts the cortical branched actin cytoskeleton and Treg cell interaction with target cells, and loss of PAK2 activation, which reduces the levels of IL-2 driven pY-STAT5 and Treg stability, synergize to promote ASI. We will test the following hypotheses 1) Reduced IL-2 driven STAT5 phosphorylation in DOCK8 deficient Tregs impairs their stability in a Th2 milieu and ability to restrain ASI. 2) Defective WASP activation and defective PAK activation independently impair Treg ability to suppress ASI in DOCK8 deficiency. 3) Serine phosphorylation of pY-STAT5 by IL-2 driven DOCK8 mediated activation of PAK2 promotes Treg stability by inhibiting the docking of SHP2 to STAT5. Many patients with AD remain resistant to current therapies. Understanding the roles of DOCK8 and associated proteins as well as the regulation of the STAT5 signaling axis in Treg function and the molecular pathogenesis of AD could inform novel therapies in AD, particularly those designed to boost Treg cells.
