Autophagy-based unconventional secretion of HMGB1 by keratinocytes plays a pivotal role in psoriatic skin inflammation

The precise mechanism through which macroautophagy/autophagy affects psoriasis is poorly understood. Here, we found that keratinocyte (KC) autophagy, which was positively correlated with psoriatic severity in patients and mouse models and could be inhibited by mitogen-activated protein kinase (MAPK) family inactivation. The impairment of autophagic flux alleviated psoriasisform inflammation. We also found that an autophagy-based unconventional secretory pathway (autosecretion) dependent on ATG5 (autophagy related 5) and GORASP2 (golgi reassembly stacking protein 2) promoted psoriasiform KC inflammation. Moreover, the alarmin HMGB1 (high mobility group box 1) was more effective than other autosecretory proteins in regulating psoriasiform cutaneous inflammation. HMGB1 neutralization in autophagy-efficient KCs eliminated the differences in psoriasiform inflammation between <i>Krt14^+/+-Atg5^f/f</i> KCs and <i>Krt14^Cre/+-atg5^f/f</i> KCs, and conversely, recombinant HMGB1 almost completely restored psoriasiform inflammation in <i>Krt14^Cre/+-atg5^f/f</i> KCs <i>in vivo</i>. These results suggest that HMGB1-associated autosecretion plays a pivotal role in cutaneous inflammation. Finally, we demonstrated that <i>Krt14^Cre/+-hmgb1^f/f</i> mice displayed attenuated psoriatic inflammation due to the essential crosstalk between KC-specific HMGB1-associated autosecretion and γδT cells. Thus, this study uncovered a novel autophagy mechanism in psoriasis pathogenesis, and the findings imply the clinical significance of investigating and treating psoriasis.<b>Abbreviations:</b> 3-MA: 3-methyladenine; ACTB: actin beta; AGER: advanced glycosylation end-product specific receptor; Anti-HMGB1: anti-HMGB1 neutralizing antibody; Anti-IL18: anti-IL18 neutralizing antibody; Anti-IL1B: anti-IL1B neutralizing antibody; ATG5: autophagy related 5; BAF: bafilomycin A₁; BECN1: beclin 1; CASP1: caspase 1; CCL: C-C motif chemokine ligand; CsA: cyclosporine A; ctrl shRNA: lentivirus harboring shRNA against control; CXCL: C-X-C motif chemokine ligand; DCs: dendritic cells; DMEM: dulbecco's modified Eagle's medium; ELISA: enzyme-linked immunosorbent assay; EM: electron microscopy; FBS: fetal bovine serum; <i>GORASP2</i> shRNA: lentivirus harboring shRNA against <i>GORASP2</i>; GORASP2/GRASP55: golgi reassembly stacking protein 2; GR1: a composite epitope between LY6 (lymphocyte antigen 6 complex) locus C1 and LY6 locus G6D antigens; H&E: hematoxylin and eosin; HMGB1: high mobility group box 1; <i>HMGB1</i> shRNA: lentivirus harboring shRNA against <i>HMGB1</i>; IFNG/IFN-γ: interferon gamma; IL17A: interleukin 17A; IL18: interleukin 18; IL1A/IL-1α: interleukin 1 alpha; IL1B/IL-1β: interleukin 1 beta; IL22/IL-22: interleukin 22; IL23A: interleukin 23 subunit alpha; IL23R: interleukin 23 receptor; IMQ: imiquimod; ITGAM/CD11B: integrin subunit alpha M; ITGAX/CD11C: integrin subunit alpha X; IVL: involucrin; KC: keratinocyte; KD: knockdown; KO: knockout; <i>Krt14^+/+-Atg5^f/f</i> mice: mice bearing an <i>Atg5 flox</i> allele, in which exon 3 of the <i>Atg5</i> gene is flanked by two loxP sites; <i>Krt14^+/+-Hmgb1^f/f</i>: mice bearing an <i>Hmgb1</i> flox allele, in which exon 2 to 4 of the <i>Hmgb1</i> gene is flanked by two loxP sites; <i>Krt14^Cre/+-atg5^f/f</i> mice: keratinocyte-specific <i>atg5</i> knockout mice generated by mating <i>Atg5-floxed</i> mice with mice expressing <i>Cre</i> recombinase under the control of the promoter of <i>Krt4; Krt14^Cre/+-hmgb1^f/f</i> mice: keratinocyte-specific <i>hmgb1</i> knockout mice generated by mating <i>Hmgb1-floxed</i> mice with mice expressing <i>Cre</i> recombinase under the control of the promoter of <i>Krt14; Krt14-Vegfa</i> mice: mice expressing 164-amino acid <i>Vegfa</i> splice variant recombinase under the control of promoter of <i>Krt14</i>; LAMP1: lysosomal associated membrane protein 1; LDH: lactate dehydrogenase; LORICRIN: loricrin cornified envelope precursor protein; M5: TNF, IL1A, IL17A, IL22 and OSM in combination; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK: mitogen-activated protein kinase; MKI67: marker of proliferation Ki-67; MTT: thiazolyl blue tetrazolium bromide; NFKB/NF-κB: nuclear factor kappa B; NHEKs: primary normal human epidermal keratinocytes; NS: not significant; OSM: oncostatin M; PASI: psoriasis area and severity index; PtdIns3K: class III phosphatidylinositol 3-kinase; qRT-PCR: quantitative RT-PCR; RELA/p65: RELA proto-oncogene, NF-kB subunit; rHMGB1: recombinant HMGB1; rIL18: recombinant interleukin 18; rIL1B: recombinant interleukin 1 beta; S100A: S100 calcium binding protein A; SQSTM1/p62: sequestosome 1; T17: IL17A-producing T; TCR: T-cell receptor; <i>tcrd</i> KO mice: <i>tcrd</i> (T cell receptor delta chain) knockout mice, which show deficient receptor expression in all adult lymphoid and epithelial organs; TLR: toll-like receptor; TNF/TNF-α: tumor necrosis factor; WOR: wortmannin; WT: wild-type; γδT17 cells: IL17A-producing γδ T cells.