PAIN HYPERSENSITIVITY IN SLURP1 AND SLURP2 KNOCKOUT MOUSE MODELS OF HEREDITARY PALMOPLANTAR KERATODERMA.

SLURP1 and SLURP2 are both small secreted members of the Ly6/u-PAR family of proteins and are highly expressed in keratinocytes. Loss of function mutations in SLURP1 lead to a rare autosomal recessive Palmoplantar Keratoderma (PPK), Mal de Meleda (MdM), which is characterized by diffuse, yellowish palmoplantar hyperkeratosis. Some individuals with MdM experience pain in conjunction with the hyperkeratosis that has been attributed to fissures or microbial superinfection within the affected skin. By comparison, other hereditary PPKs such as Pachyonychia congenita (PC) and Olmsted syndrome (OS) show prevalent pain in PPK lesions. Two mouse models of MdM, Slurp1 knockout and Slurp2X knockout, exhibit robust PPK in all four paws. However, whether the sensory experience of these animals including augmented pain sensitivity remains unexplored. In this study, we demonstrate that both models exhibit hypersensitivity to mechanical and thermal stimuli as well as spontaneous pain behaviors in males and females. Anatomical analysis revealed increased paw pad skin epidermal innervation and substantial alterations in palmoplantar skin immune composition in Slurp2X knockout mice. Primary sensory neurons innervating hind paw glabrous skin from Slurp2X knockout mice exhibit increased incidence of spontaneous activity and mechanical hypersensitivity both in vitro and in vivo. Thus, Slurp knockout mice exhibit polymodal PPK-associated pain that is associated with both immune alterations and neuronal hyperexcitability, and might therefore be useful for the identification of therapeutic targets to treat PPK-associated pain.Significance Statement Palmoplantar keratodermas (PPKs) are rare human skin disorders associated with thickening of the skin on the palms and soles. Pain is a common feature of some PPKs, yet the causes of PPK-associated pain are not understood. Here we show that two mouse models of one PPK, SLURP1 knockout mice and SLURP2 knockout mice, exhibit enhanced pain sensitivity and increased activity of pain-associated sensory neurons. These mouse lines will therefore be of value in defining causes of pain in PPKs and possibly developing improved therapies for that pain.

Distinct, age-dependent TLR7/8 signaling responses in porcine gamma-delta T cells.

Gamma-Delta T cells are a prominent subset of T cells in pigs. However, developmental changes, antigen recognition, cell migration, and their contributions to pathogen clearance remain largely unknown. We have recently shown that porcine γδ T cells express Toll-like receptors (TLRs), and that TLR7/8 stimulation can function as a co-stimulatory signal that complements cytokine-induced signals to enhance INFγ production. Nonetheless, the signaling pathways behind this increased cytokine responsiveness remained unclear. Here, we analyzed the signaling pathways by measuring cellular kinase activity and selective inhibition, confirming that the TLR7/8 expression by γδ T cells is indeed functional. Moreover, TLR downstream signaling responses showed a distinct age-dependency, emphasizing the importance of age in immune function. While the TLR7/8 co-stimulation depended on activation of IRAK1/4, p38 and JNK in adult-derived γδ T cells, γδ T cells from young pigs utilized only p38, indicating the existence of an alternative signaling pathway in young pigs. Overall, this data suggests that porcine γδ T cells could be able to recognize viral RNA through TLR7/8 and subsequently support the survival and activation of the adaptive immune response by cytokine production.

Co-stimulation by TLR7/8 ligand R848 modulates IFN-γ production of porcine γδ T cells in a microenvironment-dependent manner.

Gamma-Delta (γδ) T cells represent a prominent lymphocyte subset in pigs. Their role and function, however, remains largely unknown. Toll-like receptors (TLR) are key receptors for the recognition of pathogens, but so far, it is unknown if porcine γδ T cells express TLRs and therefore have the innate ability to recognize pathogens through pattern recognition receptors. In this study, we compared γδ T cells in different age groups of pigs and investigated the functional relevance of TLR7/8 expression. We found that the major γδ T cell phenotype shifts from CD2-CD8α-/dimCD27+ in young pigs to CD2+CD8αhighCD27- in 3-year-old pigs impacting their ability to produce IFN-γ upon cytokine and TLR stimulation. Furthermore, we report that stimulation with TLR7/8 ligand R848 increased IFN-γ production in purified γδ T cells upon co-stimulation with IL-2 and IL-12. However, the effect of R848 as a co-activator of γδ T cells was abrogated by the addition of monocytes or within PBMCs, suggesting that γδ T cells respond to multiple direct and indirect stimulations. Thus, our results indicate that γδ T cells express TLRs, are modulated by TLR7/8 ligand R848 and have subset-specific responses.