Autophagic dysfunction in patients with Papillon-Lefèvre syndrome is restored by recombinant cathepsin C treatment

Elsevier Editorial System(tm) for Journal of Allergy and Clinical Immunology Manuscript Draft Manuscript Number: JACI-D-17-01273 Title: Autophagic dysfunction in Papillon Lefèvre is restored by recombinant Cathepsin C treatment Article Type: Original Article Section/Category: Atopic dermatitis and inflammatory skin disease Keywords: Papillon-Lefèvre; Cathepsin C; autophagy; lysosomal permeabilization Corresponding Author: Dr. Mario D. Cordero, Ph.D Corresponding Author's Institution: Institute of Nutrition and Food Technology "José Mataix Verdú", Department of Physiology, Biomedical Research Center, University of Granada First Author: Pedro Bullón Order of Authors: Pedro Bullón; Beatriz Castejón-Vega; Lourdes Román- Malo; Arturo Falcao; María Paz Jimenez-Guerrero; David Cotán; Tamara Y. Forbes-Hernandez; Alfonso Alfonso Varela-López; Francesca Francesca Giampieri; José L. Quiles; Maurizio Battino; José A. Sánchez-Alcázar; Mario D. Cordero, Ph.D Manuscript Region of Origin: SPAIN Abstract: Background: Cathepsin C (CatC) is a lysosomal enzyme involved in the degradation of intracellular proteins and associated with the autophagic process. Several mutations with loss of function in the Cathepsin C (CatC) gene have been shown to be the genetic mark of Papillon-Lefèvre syndrome (PLS), a rare autosomal recessive disease characterized by severe early-onset periodontitis, palmoplantar hyperkeratosis and increased susceptibility to infections. Deficiencies or dysfunction in other cathepsins such as B or D have been associated with autophagic and lysosomal disorders. Objectives: Here, we characterized the basis for autophagic dysfunction in PLS by analyzing skin fibroblasts from PLS patients with several mutations in CatC and reduced enzymatic activity. Methods: Skin fibroblasts were isolated from patients and characterized by genetic study. Authophagy flux was evaluated by accumulation of p62/SQSTM1 and bafilomycin assay and confirmed by visible autophagosome accumulation by Transmision Electron Microscopy. A recombinant CatC was produced by produced by produced by baculovirus system in insect cell cultures. Results: Our data showed metabolic alterations in the oxidative/antioxidative status, reduced oxygen consumption and a marked autophagic dysfunction with autophagosome accumulation. This was associated with lysosomal permeabilization, CatB release and NLRP3- inflammasome activation. A treatment with recombinant CatC (rCatC) improved cell growth, autophagic flux and partially restored lysosomal permeabilization. Conclusions: Our data provide a novel molecular mechanism of PLS. Dysfunctional autophagy as a secondary event resulting from insufficient lysosomal function in PLS could show a new therapeutic target. 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Akdis, Editor-in-Chief Journal of Allergy and Clinical Immunology September 06, 2017 Dear Editor, We would like to submit our manuscript: “Autophagic dysfunction in Papillon Lefèvre is restored by recombinant Cathepsin C treatment” to be considered for publication in Trends in Immunology, in form of an original contribution. Papillon-Lefèvre syndrome (PLS) is a rare autosomal recessive condition characterized by severe early-onset periodontitis and palmoplantar hyperkeratosis and where dermatological symptoms begin prior to 2 years of age and continue throughout life 2 accompanied by increased susceptibility to infections, furunculosis and pyoderma, or pyogenic liver abscess, among others. PLS results from mutations that inactivate or introduce loss of function in the Cathepsin C (CatC) gene. In this study, we have described for first time the implication of the authophagic dysfunction and lysosomal permeabilization in the pathophysiology of skin fibroblasts from PLS. Furthermore, we show a partial reconstitution of the pathological process after treatment with a recombinant CatC produced by produced by baculovirus system in insect cell cultures. In summary, we believe that our study considerably enriches the perception of pathogenic mechanisms of the dermatological alterations of PLS and propose new pharmacological targets. I hereby state: 1. That the submitting author has the written consent from all authors to submit the manuscript and that all authors accept complete responsibility for the contents of the manuscript 2. That the manuscript is not currently under consideration elsewhere and the work reported will not be submitted for publication elsewhere until a final decision has been made as to its acceptability by the Journal 3. That the manuscript is truthful original work Yours sincerely Dr. Mario D. Cordero Institute of Nutrition and Food Technology "José Mataix Verdú", Department of Physiology, Biomedical Research Center, University of Granada, 18100 Granada, Spain. +34-958-241-000 (ext. 20316), Email: [email protected]. Cover Letter to Editor only Bullon et al 1 Autophagic dysfunction in Papillon Lefèvre is restored by recombinant Cathepsin 1 C treatment 2 Pedro Bullón, MD, PhD1,2*, Beatriz Castejón-Vega, BSc1*, Lourdes Román-Malo, 3 PhD1,2, Arturo Falcao, BSc1, María Paz Jimenez-Guerrero, BSc3, David Cotán, PhD3, 4 Tamara Y. Forbes-Hernandez, PhD4, Alfonso Varela-López, PhD5, Antonio Pérez-5 Pulido, PhD6, Francesca Giampieri, PhD4, José L. Quiles, PhD5, Maurizio Battino, 6 PhD4, José A. Sánchez-Alcázar, MD, PhD3, Mario D. Cordero, PhD5, 7 7 1 Research Laboratory, Dental School, University of Sevilla, Sevilla, Spain 8 2 Dept. of Periodontology, Dental School, University of Sevilla, Spain. 9 3 Centro Andaluz de Biología del Desarrollo (CABD), Consejo Superior de 10 Investigaciones Científicas, Universidad Pablo de Olavide, Carretera de Utrera Km 1, 11 Sevilla, 41013, Spain 12 4 Dipartimento di Scienze Cliniche Specialistiche ed Odontostomatologiche, Sez. 13 Biochimica, Università Politecnica delle Marche, 60131, Ancona, Italy. 14 5 Department of Physiology, Institute of Nutrition and Food Technology "José Mataix", 15 Biomedical Research Center (CIBM), University of Granada, Armilla, Avda. del 16 Conocimiento s.n., 18100 Armilla, Spain. 17 6 Centro Andaluz de Biología del Desarrollo (CABD), Universidad Pablo de Olavide-18 CSIC-Junta de Andalucía. 19 7 Senior author of the study 20 * These authors contributed equally to this work. 21 Running Title: Autophagic dysfunction in Papillon Lefèvre 22 23 Corresponding Author: 24 Dr. Mario D. Cordero (Senior) 25 Institute of Nutrition and Food Technology "José Mataix Verdú", Department of 26 Physiology, Biomedical Research Center, University of Granada, 18100 Granada, 27 Spain. +34-958-241-000 (ext. 20316), Email: [email protected] 28 Manuscript Bullon et al 2 Dr. Maurizio Battino 29 Dipartimento di Scienze Cliniche Specialistiche ed Odontostomatologiche – Sez 30 Biochimica. Università Politecnica delle Marche 31 Via Ranieri, 65. 60100 Ancona, Italy, Email: [email protected] 32 33 34 35 Abstract 36 Background: Cathepsin C (CatC) is a lysosomal enzyme involved in the degradation of 37 intracellular proteins and associated with the autophagic process. Several mutations 38 with loss of function in the Cathepsin C (CatC) gene have been shown to be the genetic 39 mark of Papillon-Lefèvre syndrome (PLS), a rare autosomal recessive disease 40 characterized by severe early-onset periodontitis, palmoplantar hyperkeratosis and 41 increased susceptibility to infections. Deficiencies or dysfunction in other cathepsins 42 such as B or D have been associated with autophagic and lysosomal disorders. 43 Objectives: Here, we characterized the basis for autophagic dysfunction in PLS by 44 analyzing skin fibroblasts from PLS patients with several mutations in CatC and 45 reduced enzymatic activity. 46 Methods: Skin fibroblasts were isolated from patients and characterized by genetic 47 study. Authophagy flux was evaluated by accumulation of p62/SQSTM1 and 48 bafilomycin assay and confirmed by visible autophagosome accumulation by 49 Transmision Electron Microscopy. A recombinant CatC was produced by produced by 50 produced by baculovirus system in insect cell cultures. 51 Bullon et al 9 of five wells were utilized per condition in any given experiment. Data are expressed as 184 pMol of O2 consumed per minute normalized to 1000 cells (pMol O2/1000 cells/min). 185 Electron microscopy 186 Fibroblasts were fixed for 15 min in the culture plates with 1.5% glutaraldehyde in 187 culture medium, then for 30 min in 1.5% glutaraldehyde-0.1 M NaCacodylate/HCl, pH 188 7.4. They were then washed three times in 0.1 M NaCacodylate/HCl, pH 7.4 for 10 min 189 and post-fixed with 1% OsO4-H2O, pH 7.4 for 30 min. After dehydration in increasing 190 concentrations of ethanol, 5 min for each step: 50,70, 90 and three times 100%, 191 impregnation steps and inclusion were performed in Epon and finally polymerized at 192 60°C for 48 h. 60–80 nm sections were obtained using an ultramicrotome Leica ultracut 193 S (Leitz Microsystems, Wetzlar, Germany) and contrasted with uranyl acetate and lead 194 citrate. Observations were performed on a Zeiss LEO 906 E (Oberkochen, Germany) 195 transmission electron microscope. 196 Proliferation rate 197 Two hundred thousand fibroblasts were cultured in the absence or presence of rCatC 198 (0.25µg) for 24, 48, 72, and 120h. After discharging supernatant with dead cells, cell 199 counting was performed from 3 high power fields using an inverted microscope and a 200 40X objective. 201 Cathepsin B release 202 CatB redistribution from lysosomes/autophagolysosomes to the cytosol was assessed by 203 immunofluorescence techniques using antibodies against CatB and LAMP-I as a marker 204 of lysosomal/autophagolysosomal compartment. In control fibroblasts, CatB-specific 205 immunostainings reveal cytoplasmic puncta structures that are surrounded by 206 Bullon et al 10 lysosomal/autophagolysosomal membrane proteins such as LAMP-I. After lysosomal 207 permeabilization, the immunofluorescence detection of CatB reveals a diffuse staining 208 throughout the entire cell. 209 Galectin puncta 210 Lysosomal permeabilization was also detected by the galectin puncta assay as 211 previously described 12. 212 Recombinant Cathepsin C production 213 Sequence was designed from the literature and an external service (GeneArt Invitrogen) 214 was used for its synthesis inside a bacterial vector with ampicillin resistance gen. 215 Construct codifying for recombinant CatC was synthesized and cloned into a pBAC4x 216 baculovirus transfer vector under the control of the polyhedrin promoter. Sf21 cell line 217 was co-transfected using a linearized modified Autographa californica nuclear 218 polyhedrosis virus DNA and the created transfer plasmid was transferred by a 219 lipofectamin-mediated method as specified by the manufacturer (Invitrogen™). The 220 Sf21 cells were sown in 6 wells-plates (1×106 cells/plate). After attachment, the cells 221 were infected with the recombinant baculovirus at MOI of 5 for 72 h at 27 °C in 222 darkness. Based on titration of virus obtained by quantitative PCR and the amount of 223 virus required, the volumes were calculated to infect cells. The infected cells and culture 224 supernatant were harvested and separated by centrifugation at 800×g for 5min at 4ºC. 225 Cell pellet was homogenized in PBS 1X and protease inhibitor, 1 mM PMSF, then 226 sonicated with an ultrasonic processor Sonopuls HD2070 (Bandelin) for 3×10 s at 30% 227 potency. Once the clarified fractions were obtained by centrifugation with recombinant 228 proteins, these were used in cell cultures assays. 229 Statistical Analysis 230 Bullon et al 11 Data in the figures is given as mean ± SD. Data between different groups were analyzed 231 statistically by using ANOVA on Ranks with Sigma Plot and Sigma Stat statistical 232 software (SPSS for Windows, 19, 2010, SPSS Inc. Chicago, IL, USA). For cell-culture 233 studies, Student’s t test was used for data analyses. A value of p<0.05 was considered 234 significant. 235 Results 236 Genetic characterization shows homozygous and heterozygous mutations in CatC 237 with clinical phenotype 238 Three patients were selected for this study. Patient one was a 31-year-old man with 239 hyperkeratosis in the palmoplantar region and on his hands and moderate periodontitis 240 (Figure 1A). After genetic characterization, the patient was a compound heterozygote 241 for two nonsense CTSC mutations (c.96T>G and c401G>A, numbered according to 242 reference cDNA sequence NM_001814.4). The first mutation has been previously 243 described 13 and the second is a novel mutation. Both genetic changes were observed in 244 the mother and father respectively without phenotypic manifestation, while the brother 245 did not show any mutation. Patient 2 was a 21-year-old woman with hyperkeratosis in 246 the palmoplantar region and on her joints and moderate periodontitis (Figure 1B). After 247 genetic characterization, the patient was a compound homozygote for one nonsense 248 CTSC mutation, c.1286G>A, previously described 14. This genetic change was observed 249 in the mother and father in heterozigosis without phenotypic manifestation, while the 250 brother did not show any mutation. Patient 3 was a XX-year-old woman previously 251 described (patient L in Table 2, reference 2) with hyperkeratosis in the palmoplantar 252 region elbows and hands, and suffering from severe periodontitis that led to edentulism. 253 Bullon et al 12 The human CatC sequence is processed into a mature form and it produces a trimer 254 consisting of the exclusion domain, heavy chain and light chain, which are bound by 255 disulfide bonds. The active enzyme is composed by a tetramer of these heterotrimers 256 (Figure 1D, WT). Evaluation of the effect of the amino acid changes on the protein 257 structure of CatC showed the region colored in grey corresponding to the lost sequence 258 of the one allele. In patient 1, one of the cysteine of the disulfide bond is lost, and it 259 could affect the 3D structure of the exclusion domain with important consequences on 260 the function (Figure 1D, P1). In patient 2, the amino acids in the active site are all 261 conserved, not affecting the active site of the enzyme, but probably influencing the 262 structure, by the folding adopted by the light and heavy chains inducing a possible 263 effect in the function (Figure 1D, P2). In patient 3, only one amino acid in the active 264 site is conserved with a possible more affected function (Figure 1D, P3). Accordingly, 265 enzymatic activity of CatC was observed reduced in all patients (Figure 1E). 266 Fibroblasts from PLS patients show metabolic alterations 267 Because dermatological symptoms are an important clinical manifestation of PLS, we 268 evaluated the metabolic status of skin fibroblasts from PLS patients. Fibroblasts from 269 patients presented a reduced growth ratio and abnormal morphology (Figure 2A and 270 B). We also investigated the mitochondrial functionality on fibroblasts from PLS 271 patients by measuring the OCR values in control and PLS fibroblasts, exposed 272 sequentially to four modulators of oxidative phosphorylation (OXPHOS): oligomycin 273 (an inhibitor of F1Fo-ATPase or complex V), 2,4-DNP (uncoupling of the OXPHOS 274 electron transport chain) and antimycin/rotenone (complex I and III inhibitors 275 respectively) (Figure 2C). The basal OCR was markedly affected in fibroblasts from 276 PLS patients compared to controls (Figure 2C). The spare respiratory capacity (SRC) 277 of cells -an indicator of how close a cell is operating to its bioenergetics limitwas 278 Bullon et al 13 obtained by calculating the mean of OCR values after injection of 2,4-DNP minus the 279 basal respiration. Fibroblasts from PLS patients showed a significant decrease of SRC 280 compared to control cells (Figure 2D). These metabolic alterations were accompanied 281 by increased oxidative stress characterized by high levels of reactive oxygen species 282 (ROS) and mitochondrial superoxide production and reduced activity of the antioxidant 283 enzymes, superoxide dismutase and catalase (Figure S1A-D). 284 Autophagic flux is impaired in PLS 285 Degradative substrates are cleared intracellularly by a complex macro-autophagic 286 response 15. By Western blot and mRNA expression, levels of ATG12 and the 287 autophagy marker LC3-II were markedly increased in fibroblasts from PLS (Figure 3A-288 B). Furthermore, PLS showed an increment in p62/SQSTM1 accumulation, an 289 established marker of autophagic clearance (Figure 3A). Next, we performed 290 autophagic flux analysis using the Bafilomycin A1 (BafA1), a specific inhibitor of 291 vacuolar H+-ATPases and a blocker of autophagosome-lysosome fusion, to check for 292 autophagosome/autophagolysosomal formation. As expected, BafA1 treatment in 293 control cells led to a significant increase in the amount of LC3-II, suggesting that 294 autophagic flux is normal (Figure 3C). However BafA1 treatment in PLS fibroblasts 295 had a reduced effect on LC3-II expression levels (Figure 3C), indicating that 296 autophagic flux was impaired (Figure 3C). Electron microscopy analysis of fibroblasts 297 from the patients revealed abundant multilamellar bodies and increased autophagosome 298 number as indicated by the accumulation of double-membrane vesicle structures 299 (Figure 4). 300 Lysosomal permeability is associated to PLS 301 Bullon et al 14 Degradation of autophagic substrates/autophagosome takes place in the lysosomal 302 compartment by acidic proteases, such as cathepsins. Reduced autophagic flux with the 303 consequent autophagosome accumulation can be a consequence of reduced 304 autophagosome–lysosome fusion or inefficient lysosomal degradation 16. According to 305 this, we determined whether the impaired autophagic flux is associated to lysosomal 306 dysfunction. Because the cysteine Cats family includes CatB, C, H, K, and L 17, we 307 analyzed the level of other important Cat, CatB. Surprisingly, CatB protein levels were 308 increased in fibroblasts from patients with PLS (Figure 5A). Thus, we hypothesized 309 that lysosomal protease CatB may play an important role in the pathophysiology of PLS 310 through its release from the lysosomal/autophagolysosomal compartment to the cytosol. 311 In healthy fibroblasts, CatB signal colocalized with the lysosomal/autophagolysosomal 312 marker LAMP-1 indicating that it was located within lysosomes/autophagolysosomes 313 (Figure 5B). However, in PLS fibroblasts, the CatB signal was diffuse through the 314 cytosol and it did not colocalize completely with the LAMP-I marker suggesting 315 lysosome/autophagolysosome membrane permeabilization (LMP) (Figure 5B). This 316 LMP was also confirmed by the detection of galectin puncta at leaky 317 autophagolysosomes (Figure 6) as it has been described that galectin translocation to 318 phagosomal and lysosomal membranes is a marker of vacuole lysis and/or 319 permeabilization 18. Interestingly, this CatB release and lysosomal alteration were 320 accompanied by NLRP3-inflammasome activation (Figure 5C). 321 Recombinant CatC improves growth and induces partial restoration of autophagic 322 flux in fibroblasts 323 Because mutation in patients induces an important reduction of the enzymatic activity 324 of CatC, which is associated to an impairment of autophagic flux and lysosomal 325 dysfunction, we tested the effect of a recombinant CatC (rCatC) in fibroblasts from 326 Bullon et al 15 patients with PLS. First, the rCatC was produced by baculovirus system in insect cell 327 cultures according to the methods described in Material and methods section 19. Three 328 extracts were isolated and tested, supernatant and cellular extracts: insoluble and soluble 329 extracts. Soluble extract showed more enzyme concentration (tested by Dot Blot) of 330 rCatC (Figure S2). Immunoblot analysis of the purified rCatC revealed three 331 polypeptides with molecular masses of approximate 55 kDa, 25 kDa, and 7.8 kDa 332 (Figure S3). The amount of 55 kDa polypeptides was much larger than that of the other 333 two. Flatworm CatC expressed in insect cells also exhibited similar three bands of 55 334 kDa, 25 kDa, and 7.8 kDa polypeptides after purification. Enzymatic activity assay 335 showed increased enzymatic activity in the soluble extract (Figure S4). 336 After comparing three different doses, rCatC treatment in fibroblasts from a 337 representative patient showed increased ratio of cell growth in low doses of 0.25µg. 338 However, control fibroblasts showed and important growth decrement in a dose-339 dependent manner (Figure 7A-C). Enzymatic activity of CatC was observed to be 340 partially recovered in the fibroblasts from the patient after 120h of rCatC treatment 341 (Figure 7D). By Western blot analysis, levels of the autophagy marker LC3-II were 342 markedly increased in fibroblasts from control fibroblasts after rCatC treatment. No 343 significant changes concerning LC3-II were found in fibroblasts from PLS, taking into 344 account the increased basal levels of the patient (Figure 7E). Furthermore, the increased 345 level of p62/SQSTM1 accumulation in PLS were recovered by the rCatC treatment after 346 120h showing an autophagic clearance (Figure 7E). 347 rCatC improves lysosomal permeability associated to PLS 348 Because we previously showed lysosomal permeability associated to the 349 pathophysiology of the fibroblasts from PLS, the potential effect of the rCatC on this 350 Bullon et al 16 aspect was also explored. The increased levels of CatB protein previously observed 351 were decreased in fibroblasts from the patients with PLS after rCatC treatment (Figure 352 7E). Thus, partial co-colocalization of CatB signal with the LAMP-1 marker suggested 353 a reduction of LMP (Figure S5). This reduction of the LMP, was also confirmed after 354 no detection of galectin puncta at leaky autophagolysosomes (Figure 8). 355 Discussion 356 The dearth of knowledge about the molecular bases of PLS leads to a loss of therapeutic 357 tools. Previous works have been oriented to the description of new mutations and 358 several pathophysiological characteristics such as oxidative stress, neutrophilic function 359 impairment without deepening in molecular mechanisms. The present report provides 360 experimental evidence about metabolic and autophagic impairment in CatC-deficient 361 fibroblasts from PLS patients. First, we assessed metabolic changes in cultured 362 fibroblasts. As expected, a reduction of mitochondrial metabolic rate was observed. 363 This entailed a significant reduction in cell growth. Then, we provided experimental 364 evidence for the activation of autophagy in CatC-deficient fibroblasts. From an 365 ultrastructural and functional point of view, autophagy is characterized by the formation 366 of double-membraned vesicles called autophagosomes. Autophagic process is initiated 367 by the formation of a phagophore or nucleation membrane to which cytoplasmic content 368 is targeted. This phagophore is elongated and results in a new autophagosome which 369 engulfs material marked for degradation. Finally, mature autophagosomes fuses with 370 lysosomes, resulting in autophagolysosomes, and the content is degraded via catalytic 371 enzymes 15. Enhanced autophagy may be due to either increased autophagosome 372 formation or impaired clearance of the autophagosomes. Because the autophagosome is 373 an intermediate structure, the number of autophagosomes observed at any specific time 374 point is a result of the balance between the rate of their generation and the rate of their 375 Bullon et al 17 conversion into autophagolysosomes. As LC3-II overexpression can evoke either an 376 increment of autophagy or an impairment of the autophagic flux, these LC3-II changes 377 must be interpreted together with the p62/SQSTM1 levels 20. We observed an impaired 378 autophagic flux determined by expression of LC3-II and increased expression of 379 proteins involved in clearance pathways like p62/SQSTM1 and confirmed by BafA1 380 assay and TEM. Interestingly, this blockade of autophagosome fusion with lysosomes 381 or autophagic flux occurs in multiple lysosomal diseases like Niemann pick, Gaucher 382 and Pompe diseases 21-23. 383 Oxidative stress has been associated to the pathophysiology of PLS 2 and we observed 384 increased ROS production accompanied by reduced antioxidant defenses. Oxidative 385 stress is associated to the induction of lysosomal instability and membrane 386 permeabilization 24. Previously, reduction in some cysteine Cats family members like 387 CatL have been related to upregulate the CatB and with autophagy dysfunction 25 in a 388 compensatory transcriptional upregulation with increased enzymatic activation of CatB. 389 Furthermore, in a lysosomal disease such as Niemann pick disease, the expression of 390 mature protein forms for both CatB and CatD are upregulated with accumulation of 391 autophagosomes 26. Our findings show reduced enzymatic activity of CatC with a 392 compensatory upregulation of mature CatB proteins leading to a lysosomal 393 permeabilization. All these disturbances on the machinery of autophagy and lysosome 394 metabolism could explain several of the clinical phenotype characteristics of PLS. 395 Autophagy is a process not only involved in the degradation and recycling of 396 macromolecules including proteins, lipids and carbohydrates, for the synthesis of 397 essential components and as an energy supply, but also in the innate and adaptive 398 immunity against microbial invasion 7. Accordingly, it is not surprising that defects in 399 the autophagy process have been linked to a wide range of diseases increasing 400 Bullon et al 18 susceptibility to infections 7. Thus, many patients with PLS show increased 401 susceptibility to infections and periodontal disease 2, which probably could be 402 associated with autophagy dysfunction. Autophagy is also involved in the homeostasis 403 of the skin, which is the first line of defense against many different insults. So, 404 autophagy might have a critical role in the development and progression of skin 405 diseases. However, the exact mechanism by which autophagy dysfunction could be 406 involved in the development of skin diseases is still unknown. Nevertheless, several 407 skin diseases like vitiligo, psoriasis, hyperpigmentation or the skin alteration from 408 systemic lupus erythematosus, have been related autophagy defects 27 which might lead 409 to inflammatory cytokine production mediated by p62/SQSTM1, as reported for 410 psoriatic skin 28. Accordingly, in the present study a similar autophagy dysfunction with 411 p62/SQSTM1 in PLS was found, which has been associated with increased production 412 of inflammatory cytokines 29. Furthermore, the compensatory upregulation of mature 413 CatB found here was accompanied by a NLRP3-inflammasome activation. NLRP3-414 inflammasome complex is a molecular platform which activates innate immune 415 defenses through the maturation of proinflammatory cytokines (interleukins IL-1β and 416 IL-18). This complex is activated by a wide variety of danger signals such as potassium 417 efflux out of the cell, mitochondrial ROS, translocation of NLRP3 to the mitochondria, 418 cardiolipin, or the release of cathepsins, like CatB, into the cytosol after lysosomal 419 destabilization 30. So, inflammatory characteristics of PLS could be explained by 420 NLRP3-inflammasome activation. 421 Finally, the treatment and management of PLS is poor and is limited to oral approach 422 and dermatological damage treatment with emollients, salicylic acid and topical 423 steroids, with toxic effects in many cases 31. Therefore, it is important to have effective 424 pharmacological treatments for PLS based on the molecular etiology of the disease. 425 Bullon et al 25 control fibroblasts. Data represent the mean±SD of three separate experiments.*P < 569 0.05; **P < 0.01; **P < 0.001 between control and PLS patients. 570 Figure 3.- A. Autophagic protein expression levels of ATG12, LC3-I (top panels, top 571 band), LC3-II (top panels, bottom band), and p62 were determined in control and PLS 572 fibroblast cultures by Western blot analysis, as described in Materials and Methods. 573 ATG12 band represents the Atg12-Atg5 conjugated form. GAPDH was used as a 574 loading control. B. Impaired autophagic flux in PLS fibroblasts. Determination of LC3-575 II in the presence and absence of bafilomycin A1 in control (CTL) and PLS fibroblasts 576 (patients). Control and PLS fibroblasts were incubated with bafilomycin A1 (100 nM 577 for 12 h). Total cellular extracts were analyzed by immunoblotting with antibodies 578 against LC3. GAPDH was used as a loading control. For control cells, data are for 579 experiments on 2 different control cell lines. 580 Figure 4.- Ultrastructure of PLS fibroblasts. A. Control fibroblasts showing 581 mitochondria with typical ultrastructure. B. Laminar bodies (black arrows) and 582 autophagosome (white arrows) present in PLS fibroblasts. Scale bar 10 µm (low 583 magnification) and 2 µm (high magnification). 584 Figure 5.- Cathepsin B relases in PLS fibroblasts. A. Protein expression levels of 585 CatB B were determined in control and PLS fibroblast cultures by Western blotting, as 586 described in Materials and Methods. B. Immunofluorescence of CatB in control and 587 pathological cells. Note that in PLS fibroblasts CatB diffuses throughout the cytosol. C. 588 NLRP3-inflammasome protein expression levels of NLRP3 (top band) and Caspase 1 589 (intermediate bands), were determined in control and PLS fibroblast cultures by 590 Western blot analysis, as described in Materials and Methods. GAPDH was used as a 591 loading control. 592 Bullon et al 26 Figure 6.- Representative fluorescence images of fibroblasts from control and PLS. 593 Cells were fixed and stained with anti-Galectin-3 antibodies (green) and anti-LAMP-I 594 (red). Nuclei were stained with Hoechst 33342 (blue). Increased Galectin-3- puncta and 595 colocalization of Galectin-3 and LAMP-I puncta are shown in patients. 596 Figure 7.- A-C. Cell growth with rCatC determined in healthy and representative PLS 597 fibroblasts. D. Enzymatic activity of CatC in homogenate from skin fibroblasts after 598 120h of rCatC treatment. Data represent the mean – SD of three separate experiments. 599 *p < 0.01 between controls and control treated with rCatC; ap < 0.01 between PLS and 600 PLS treated with rCatC. E. Autophagic protein expression levels of LC3 and p62, CatB 601 were determined in control and representative PLS fibroblast cultures after rCatC 602 treatment by Western blot analysis, as described in Materials and Methods. GAPDH 603 was used as a loading control. For control cells, data are for experiments on 2 different 604 control cell lines. 605 Figure 8.- Representative fluorescence images of fibroblasts from control and PLS to 606 evaluate the effect of the rCatC in lysosomal permeabilization. Cells were fixed and 607 stained with anti-Galectin-3 antibodies (green) and anti-LAMP-I (red). Nuclei were 608 stained with Hoechst 33342 (blue). Increased Galectin-3- puncta and colocalization of 609 Galectin-3 and LAMP-I puncta are shown in patients. 610 611 612 613 614 615 Bullon et al 27 SUPPLEMENTARY DATA 616 Autophagic dysfunction in Papillon Lefèvre is restored by recombinant Cathepsin 617 C treatment 618 Pedro Bullón1,2*, Beatriz Castejón-Vega1*, Lourdes Román-Malo1, María Paz Jiménez-619 Guerrero3, David Cotán3, Tamara Y. Forbes-Hernandez4, Alfonso Varela-López5, 620 Antonio Pérez-Pulido6, Francesca Giampieri4, José L. Quiles5, Maurizio Battino4, José 621 A. Sánchez-Alcázar3, Mario D. Cordero5 622 1 Research Laboratory, Dental School, University of Sevilla, Sevilla, Spain 623 2 Dept. of Periodontology, Dental School, University of Sevilla, Spain. 624 4 Dipartimento di Scienze Cliniche Specialistiche ed Odontostomatologiche, Sez. 625 Biochimica, Università Politecnica delle Marche, Ancona, Italy. 626 5 Department of Physiology, Institute of Nutrition and Food Technology "José Mataix", 627 Biomedical Research Center (CIBM), University of Granada, Armilla, Avda. del 628 Conocimiento s.n., 18100 Armilla, Spain. 629 6 Centro Andaluz de Biología del Desarrollo (CABD), Universidad Pablo de Olavide-630 CSIC-Junta de Andalucía. 631 * These authors contributed equally to this work. 632 Running Title: Autophagic dysfunction in Papillon Lefèvre 633 634 Corresponding Author: 635 Dr. Mario D. Cordero 636 Institute of Nutrition and Food Technology "José Mataix Verdú", Department of 637 Physiology, Biomedical Research Center, University of Granada, 18100 Granada, 638 Spain. +34-958-241-000 (ext. 20316), Email: [email protected] 639 640 641 642 643 644 Bullon et al 28 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 Supplementary figure 1. Oxidative stress and oxidative damage levels in fibroblasts 665 from PLS patients. (A and B) Total ROS and mitochondrial ROS production were 666 analyzed in fibroblasts from control and PLS patients by flow cytometry as described in 667 Material and Methods. (C and D) Antioxidant enzymes SOD and catalase (CAT) 668 activities were also analyzed in fibroblasts from control and PLS patients as described 669 in Material and Methods. Data represent the mean±SD of three separate experiments.*P 670 < 0.05, **P < 0.01 and ***P < 0.001 between control and PLS patients. 671 672 673 *** ** *** *** *** *** *** *** *** *** *** *** Bullon et al 29 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 Supplementary figure 2. Detection of CatC protein in theof three different extracts 689 (cell extract and the soluble and insoluble extract) determined by Dot Blot. 690 691 692 693 694 695 696 697 698 699 700 701 SUPERNATANT 1:1 1:10 1:50 1:100 1:500 INSOLUBLE 1:1 1:10 1:50 1:100 1:500 SOLUBLE 1:1 1:10 1:50 1:100 1:500 Bullon et al 30 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 Supplementary figure 3. Immunoblot analysis of the insoluble extract revealed three 724 polypeptides with molecular masses of approximate 55 kDa, 25 kDa, and 7.8 kDa. 725 Super: supernatant, Insol: insoluble, Sol: soluble. 726 727 728 729 Blank Super Insol Sol 25 kda 55 kda Bullon et al 31 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 Supplementary figure 4. Enzymatic activity of celular and soluble extracts. 751 752 753 754 755 756 Neg Cel Sol 1:10 1:1 A B Mean n SD Bullon et al 32 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 Supplementary figure 5. Representative fluorescence images of fibroblasts from 780 control and PLS with and without rCatC. Cells were fixed and stained with anti-781 Galectin-3 antibodies (green) and anti-LAMP-I (red). Nuclei were stained with Hoechst 782 33342 (blue). Increased Galectin-3- puncta and colocalization of Galectin-3 and LAMP-783 I puncta are shown in patients. 784 785 Figure No. Click here to download high resolution image Figure No. Click here to download high resolution image