s2fieldsofstudy: Biology Medicine

text:
Spontaneous resistance to phage NPV1 confers increased daptomycin susceptibility in Enterococcus faecalis OG1RF

Enterococcus faecalis is a Gram-positive opportunistic pathogen that inhabits the gastrointestinal tract. Because of the high frequency of antibiotic resistance among Enterococcus clinical isolates, interest in using phage to treat enterococcal infections and to decolonize high-risk patients for antibiotic-resistant Enterococcus is rising. Bacteria can evolve phage resistance, but there is little published information on these mechanisms in E. faecalis. In this report, we identified genetic determinants of resistance to phiNPV1 and their effects on E. faecalis susceptibilities to daptomycin and sodium chloride-induced osmotic stress. We found that loss-of-function mutations in epaR confer phiNPV1 resistance by blocking phage adsorption. We attribute the inability of the phage to adsorb to the loss of an extracellular polymer in strains with inactivated epaR. Phage-resistant epaR mutants exhibited increased daptomycin and osmotic stress susceptibility. Our results demonstrate that spontaneous resistance to phiNPV1 comes at a cost in E. faecalis OG1RF, resulting in concomitant increased susceptibilities to daptomycin and sodium chloride-induced osmotic stress.

decreased daptomycin susceptibility (24). Because of these results, we investigated the 137 daptomycin MIC of our OG1RF mutants ( Figure 5). We found that OG1RF-C, OG1RFΔepaR, 138 and OG1RFΔepaR complemented with the OG1RF-C epaR allele were each significantly more 139 susceptible to daptomycin than OG1RF. Interestingly, deletion of bgsB also conferred increased 140 daptomycin susceptibility ( Figure 5). This was complemented by expression of the wild-type 141 bgsB allele in cis, but not by expression of the OG1RF-C bgsB allele in cis ( Figure 5). Finally, 142 daptomycin susceptibility was substantially altered in the OG1RF∆epaR∆bgsB mutant, with 3 of 143 6 experimental trials resulting in an MIC below the level of detection of the E-test strip (<0.016 144 µg/mL; a value of 0.008 µg/mL was used for these data points in statistical analysis). Without 145 complete data regarding the MIC of OG1RF∆epaR∆bgsB, we did not quantitatively determine 146 whether there is a synergistic relationship between bgsB and epaR regarding daptomycin 147 susceptibility.
148 149 epaR protects OG1RF from osmotic stress. The epa gene cluster was up-regulated when E. 150 faecalis V583 was grown with 6.5% sodium chloride supplementation, indicating that the Epa 151 polymer has a role in osmotic stress response (25). As such, we investigated the effect of 152 sodium chloride on our epaR mutants. We tested our mutants for their tolerance for sodium 153 chloride stress using BHI agar supplemented with sodium chloride at concentrations of 0%, 154 2.5%, 5%, and 7.5%. Overnight cultures in stationary phase were serially diluted and spotted on difference in CFU compared to the wild-type ( Figure 6). However, when cells were cultured in 160 fresh BHI broth 1 h prior to spotting, a CFU difference was observed for OG1RF∆bgsB with 161 7.5% sodium chloride ( Figure S2). These data suggest that both epaR and bgsB play roles in 162 sodium chloride-induced osmotic stress response.

164
EpaR constitutes a major mutational pathway for ϕNPV1 resistance. We isolated 9 165 spontaneous ϕNPV1-resistant mutants of OG1RF and sequenced the epaR region of these 166 mutants. All 9 mutants have non-synonymous substitutions in epaR (Table 3). Since we know 167 that mutations in epaR affect daptomycin susceptibility, we also determined the daptomycin MIC 168 of these ϕNPV1-resistant strains and found that all were more significantly more susceptible to 169 daptomycin than the wild-type ( Figure S3).

171
Most of the EpaR sequence (amino acid positions 35-458 of 484 total) is a predicted sugar 172 transferase domain (TIGR03025; E-value: 2.45e -137 ). This domain consists of a conserved C-173 terminal region responsible for the sugar transferase activity (pfam02397) and a variable N-174 terminal region with predicted flippase activity. All epaR mutations in spontaneously ΦNPV1-175 resistant OG1RF strains occur in the C-terminal region of the sugar transferase domain (Table   176 3). We identified other proteins containing the same predicted sugar transferase domain as is indispensable for ϕNPV1 adsorption to E. faecalis OG1RF, and that inactivating mutations in 187 epaR constitute a major pathway for ϕNPV1 resistance in this strain background. We also found 188 that inactivating mutations in epaR and bgsB result in increased susceptibilities to daptomycin 189 and sodium chloride stress. Our results show that resistance to ϕNPV1 comes at a cost.

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Adsorption to the host is the first step to a productive phage infection. For a tailed phage particle

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The epa gene cluster is involved in the synthesis of a cell wall rhamnose polysaccharide 204 referred to as the enterococcal polysaccharide antigen (Epa). There is precedence for cell wall cluster consists of 18 core genes (epaA to epaR) and a set of strain-variable genes that occur 208 downstream (20, 29). Unfortunately, the Epa structure has not been determined (30), which is a 209 critical gap in knowledge about the enterococcal cell surface.

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The connection between the epa gene cluster and ϕNPV1 resistance was first investigated by

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In summary, in this study we characterized a mechanism for spontaneous ϕNPV1 resistance in 243 E. faecalis OG1RF. The major advance of this study is that we determined that phage 244 resistance comes at a cost to E. faecalis OG1RF; specifically, increased daptomycin 245 susceptibility. E. faecalis OG1RF is a natively daptomycin-sensitive strain that became more 246 sensitive as a result of phage resistance. We did not assess whether ϕNPV1 treatment could 247 reverse resistance in daptomycin-resistant clinical isolates. This is an exciting area for future 248 research.

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Bacterial strains, media, and bacteriophages. A complete list of bacterial strains and 252 bacteriophage used in this study can be found in Table 1. E. faecalis strains were cultured in 253 brain heart infusion (BHI; Difco) at 37°C without agitation. E. coli strains were cultured in LB 254 broth at 37°C with shaking at 225 rpm unless otherwise stated. Plates of the appropriate media 255 were made by adding 1.5% agar to the broth prior to autoclaving. For MIC testing, Muller Hinton 256 media supplemented with 1.5% agar (MHA) was used. Phages were stored in phage buffer as when required for selection. 5-bromo 4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) was 259 used at 120 µg/mL and 40 µg/ml for E. faecalis and E. coli, respectively. The upper soft agar for 260 phage assays was M17 medium supplemented with 0.75% agar, while the lower layer was BHI 261 supplemented with 1.5% agar.  Table S1.  Table S1. The approximately 2.0 kb product was purified and digested with BamHI 292 and EcoRI. The digested product was ligated to plasmid pLT06 through restriction sites added 293 on the primers (highlighted in red in Table S1). The ligation product was then purified and 294 electroporated and propagated in E. coli EC1000. OG1RF was made electrocompetent using 295 the glycine method (36) (3% glycine) and transformed with 1 µg of the plasmid. OG1RF 296 transformants were screened for successful transformation and subsequently inoculated in BHI 297 supplemented with Cm at 30°C. The culture was diluted 1:100 in BHI and incubated at 30°C for

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For some experiments, overnight cultures were subcultured into fresh BHI broth for 1 h prior to 328 serial dilution and spotting.

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Phage adsorption assay. An overnight bacterial culture was diluted 1:5 in fresh BHI broth. The 331 culture was then equilibrated at 37°C for 20 min in a water bath. ϕNPV1 was added at an M.O.I 332 of 10 -3 . After 15 min, a 1 mL aliquot was centrifuged at 16.6 x g for 1 min at room temperature. determined as follow:     being phage buffer rather than 50% acetic acid. phiNPV1 was added at a PFU of 10 5 and the 556 mixture was incubated for 15 minutes at 37°C. The PFU was evaluated with phage spot assay.


extfieldsofstudy: Chemistry Medicine

text:
High-fluence low-power laser irradiation promotes odontogenesis and inflammation resolution in periodontitis by enhancing stem cell proliferation and differentiation

Periodontitis can exert a severe impact on the life of patients, and the use of stem cell therapy for this disease is promising. The inflammatory response consequent to periodontitis can promote stem cell proliferation. Activated inflammation triggers inhibitory cytokine secretion, thus reducing inflammation subsequent to stem cell activation. High-fluence low-power laser irradiation (HF-LPLI) has the ability to regulate stem cell function through its effect on inflammation. Thus, the aim of the present study was to examine whether HF-LPLI is able to activate stem cells to promote regeneration in periodontitis by promoting inflammation resolution, as well as to evaluate the underlying mechanism of action if an effect is observed. Stem cells were treated with HF-LPLI following inflammation activation. Reverse transcription-quantitative polymerase chain reaction and EdU assay were used to evaluate cell proliferation and differentiation. Flow cytometry and immunofluorescence were also used to detect the ability of HF-LPLI to regulate the surrounding inflammatory environment. Animal models of periodontal disease were treated with stem cells and HF-LPLI, and regeneration was detected by hematoxylin and eosin staining and in vivo imaging. It was observed that HF-LPLI promoted inflammation resolution by reducing the excessive inflammatory response, and finally stimulated stem cell proliferation and differentiation. Furthermore, in vivo results revealed that stem cells treated with HF-LPLI induced bone regeneration. HF-LPLI stimulated stem cell proliferation and differentiation by promoting inflammation resolution subsequent to stem cell activation, providing a new strategy for the clinical treatment of periodontitis.

Introduction
Periodontitis is a chronic inflammation of supporting tissues caused by specific bacteria adhering to and growing on the surfaces of the teeth, which can even cause dental pulp defects and numerous systemic diseases, including pneumonia and cancer (1)(2)(3). Available treatments, such as gingivectomy, orthodontic therapy and periodontal splint fixation, are associated with high recurrence rate and pain (4,5). Recently, stem cell regeneration appears to provide a new direction in the treatment of periodontitis (6). dental pulp stem cells (dPScs) and periodontal ligament stem cells (PLScs) represent the basis for an effective treatment due to their regenerative ability (7). Short treatment time, absence of pain and no recurrence are the most marked advantages of using these stem cells (8)(9)(10). However, local inflammation not only blocks the normal physiological cell activities, but also damages the cells (7). Therefore, enhancing the treatment effect associated with the understanding of the mechanism is of utmost importance, while improving the inflamed microenvironment and/or promoting rapid cell proliferation and differentiation may represent potential therapeutic strategies.
Periodontitis is characterized by neutrophils and macrophages (MΦs) that infiltrate the tissues surrounding the teeth and mediate inflammation, representing the main reason for the worsening of the microenvironment, causing damage and bone resorption (11). MΦs can be divided into M1 and M2, depending on their metabolism (12). M1 secret pro-inflammatory cytokines and chemokines, while M2 secret cytokines that decrease inflammation, including interleukin (IL)-10 and transforming growth factor (TGF)-β (12,13). Previous studies have reported that certain inflammatory factors or a specific local microenvironment promote stem cell proliferation (14,15). Therefore, promoting MΦ transformation from M1 to M2 can reduce inflammation and achieve a therapeutic effect.
High-fluence low-power laser irradiation (HF-LPLI) serves an important role in regulating cell proliferation, differentiation, apoptosis and other cell physiological activities (16). It has been demonstrated that HF-LPLI can also serve a role in regulating inflammation when used for an appropriated period of time (17). HF-LPLI light sources include the He-Ne laser and the GaAs semiconductor laser. In addition, this technique has been reported to promote inflammation resolution, which in turn is associated with myeloperoxidase activity, as well as cyclooxygenase (cOX)-1 and cOX-2 gene expression (17).
Therefore, in the present study, it was hypothesized that HF-LPLI may also promote inflammation resolution by inducing neutrophil apoptosis and MΦ reprogramming subsequent to appropriate inflammation activation of stem cells, as well as consequent improvement of osteogenic differentiation, thereby enhancing the efficacy of stem cell treatment to combat periodontitis. Following in vitro validation of this hypothesis and the associated mechanism of action by several techniques, an animal periodontitis model was designed. Stem cell therapy was used, while HF-LPLI was subsequently administered. Finally, the difference in cell proliferation and osteogenic differentiation was evaluated between the control and experimental groups.

Materials and methods
DPSC and PLSC isolation, identification and culture. dPScs were obtained from dental pulp tissue explants. The third molars of adult patients (age, 16-25 years) were obtained from the department of Stomatology at the Liaocheng People's Hospital (Liaocheng, china). All the patients included were informed of the condition and agreed to participate in the research. Teeth were first cleaned by normal saline containing 3% antibiotic-antimycotic solution (Gibco; Thermo Fisher Scientific, Inc.), and then sterilized dental fissure burs were used to expose the pulp chamber. Subsequently, dental pulp tissues were dissected into fragments (<0.5 mm), placed into a 6-cm dish containing Dulbecco's modified Eagle's medium (dMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 20% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and incubated at 37˚C with 5% cO 2 for 2-3 weeks. dPScs were routinely passaged, and third passage cells were used for further experiments. An inverted phase contrast microscope (Nikon corporation, Tokyo, Japan) was used to observe the cell morphology (18,19).
PLScs were obtained from teeth from the same hospital. Initially, the tooth was washed by normal saline containing 3% antibiotic-antimycotic solution (Gibco; Thermo Fisher Scientific, Inc.). Pulp tissues were separated from the surface of the tooth. In total, 1 g/l collagenase type I and 2.4 g/l dispase (Gibco; Thermo Fisher Scientific, Inc.) were used to digest the tissues for 1 h at 37˚C. Samples were then centrifuged at 400 x g for 4 min at 4˚C by TD5Z Multi-frame Centrifuge (Jintan changzhou Instrument Factory, changzhou, china), and the pellet was collected. cells were resuspended in dMEM containing 20% FBS. cells at the sixth passage were used in subsequent experiments (20,21).
Flow cytometry. DPSCs and PLSCs were identified by flow cytometry. The cell suspension was prepared using an icy buffer (PBS), the cell concentration was adjusted to 5x10 5 cells/ml. Next, samples were centrifuged at 1,500 x g for 3 min at 37˚C and the appropriate fluorescent-labeled antibodies were added to each sample. Anti-STRO-1 antibody was used in dPScs (cat. no. ab214086; 1 µg/ml), cluster of differentiation (cd)44 antibody was used in PLScs (cat. no. ab157107; 1 µg/ml), and cd133 antibody was used in both cell samples (cat. no. ab19898; 1 µg/ml; all purchased from Abcam (cambridge, MA, USA). Subsequent to antibody addition, the samples were centrifuged at 1,500 x g for 3 min at 37˚C, followed by the addition of 0.5 ml formaldehyde and incubated for 24 h in -4˚C. Rabbit Anti-Rat IgG H&L (horseradish peroxidase; cat. no. ab6734; Abcam) was added as seconded antibody and incubated for 8 h in -4˚C. Samples were kept at 4˚C until the end of the experimental analysis. Flow cytometry (Bd LSRFortessa™ cell analyzer; Bd Biosciences, San Jose, cA, USA) was finally used to identify the cells (18)(19)(20)(21).
The two types of stem cells were cultured into 6-wells plates (5x10 4 cells/well). cells were divided into four groups, including the control, MΦ, MΦ+HF-LPLI, and the MΦ followed by HF-LPLI groups. In these groups, the concentration of MΦ was 1x10 4 cells/well. In the MΦ group, the cells were co-cultured with MΦ for 6 h and without HF-LPLI treated. In the MΦ+HF-LPLI group, stem cells were treated with HF-LPLI and simultaneously co-cultured with MΦ. In the MΦ followed by HF-LPLI group, stem cells were treated with HF-LPLI subsequent to co-culturing with MΦ for 6 h.
Neutrophil and MΦ isolation and culture. Male Sprague dawley (Sd) rats (age 6-8 weeks old, 250-280 g, n=10) were obtained from the Laboratory Animal center of Taishan Medical University (Taian, china). Sd rats were cultured in 37˚C, relative humidity 45% and 12-h light/dark environment with low fat diet and free to water. The rats were then subjected to an intraperitoneal injection of 1 ml 4% thioglycollate (Guangzhou SWAN chemical co., Ltd., Guangzhou, china; cat. no. 2365-48-2). After 24 h, 10 ml cold dulbecco's phosphate-buffered saline containing 100 U/ml penicillin was injected into the peritoneum. Peritoneal lavage fluids were mixed thoroughly and collected. Neutrophils from the fluid were spun and re-suspended with pre-warmed serum-free RPMI-1640 medium containing 0.05% bovine serum albumin (WelGENE, Inc., Pohang, Korea). cells were plated into a 6-well plate (5x10 6 cells/well) (22), and divided into the control and lipopolysaccharide (LPS) group for apoptosis detection by flow cytometry. In the LPS group, cells were cultured in medium containing 1 µg/ml LPS (Sigma-Aldrich; Merck KGaA, darmstadt, Germany) which was used to create an inflammatory culture condition.
Rat MΦs were purchased from the Shanghai Institutes for Biological Sciences of the chinese Academy of Sciences (Shanghai, china). cells were cultured in RPMI-1640 medium (WelGENE, Inc.) containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin, followed by plating in a 6-well plate (4x10 6 cells/well) and incubation at 37˚C in 5% CO 2 for 48 h. MΦs were divided into three groups, including the control, LPS and HF-LPLI groups, for immunofluorescence detection. In the HF-LPLI group, HF-LPLI treatment was applied following 1 µg/ml LPS incubation for 24 h.
HF-LPLI application. In the present study, a He-Ne laser with output power at 632.8 nm and a 10.5-mW red laser were used for HF-LPLI application. For in vitro experiments, samples were directly treated under 20 J/cm 2 HF-LPI for 1 h. Furthermore, oral radiation was performed by optical fiber, 20 J/cm 2 for 1 h to the first molars and their periodontal tissue with other teeth covered by aluminum foil paper (16).
Apoptosis detection by flow cytometry. Neutrophils and MΦs cultured in vitro were divided into the control and LPS groups. The supernatant of cells was collected into a 15 ml centrifuge tube. EGTA-free trypsin was added to digest the neutrophils, and trypsin was added to the corresponding centrifuge tube. Next, PBS was added to wash down the cells attached on the tube. Samples were centrifuged at 1,000 x g for 3 min, and then PBS was used to wash the cells twice, following by further centrifugation at 1,000 x g for 3 min. Subsequently, Annexin V was added according to the manufacturer's protocol described in the FITc Annexin V Apoptosis detection kit (Bd Biosciences). Flow cytometry (Bd LSRFortessa™ cell analyzer; Bd Biosciences) was finally used to evaluate apoptosis (23)(24)(25).
Caspase-3 activity analysis. The caspase-3 activity of neutrophils was detected by a caspase-3 Activity Assay kit (Beyotime Institute of Biotechnology, Haimen, china) according to the manufacturer's protocol. Briefly, samples at 0, 2, 4 and 6 h following HF-LPLI treatment were ground and then incubated in lysis buffer on ice for 15 min. The mixture was centrifuged at 16,000 x g for 10 min at 4˚C, followed by the addition of 2 mmol Ac-dEVd-pNA (Sigma-Aldrich; Merck KGaA) for 1 h. Absorbance was measured at 405 nm using a microplate reader (Thermo Fisher Scientific, Inc.). The caspase-3 activity of each sample was calculated according to the standard curve and normalized to the total protein concentration (26,27).
Immunofluorescence analysis. MΦs were seeded into 6-well plates at a density of 5x10 4 per well and incubated for 48 h, and then were washed three times with PBS, fixed with 4% paraformaldehyde for 15 min and subsequently treated with 0.5% Triton X-100 for 20 min at room temperature. Sufficient dilution of primary polyclonal anti-rat cd86 (cat. no. MA110293; 1:1,000) or cd163 (cat. no. AB-2716934; 1:1,000) antibodies against M1 and M2 (Thermo Fisher Scientific, Inc.) was added to each slide, and incubated overnight at 4˚C. The slides were washed three times with PBS/Tween-20 for 3 min each time and then treated with secondary antibodies at 20-37˚C in the dark for 1 h. Next, samples were incubated with dAPI (Beyotime Institute of Biotechnology) for 5 min. Slides were dried using an absorbent paper, mounted with liquid sealing agents containing anti-fluorescence quenching reagents and observed under a fluorescent microscope for image acquisition (28,29).
NO concentration detection. dAF-FM dA (Beyotime Institute of Biotechnology) was used to detect the NO concentration in adherent neutrophils. dAF-FM dA was diluted to 1:1,000 using the dilution buffer provided in the kit. Next, samples were incubated in an incubator for 20 min. cells were collected, treated with 5 µmol/l DAF-FM DA, incubated at 37˚C for 20 min and subjected to flow cytometry analysis (30).
A total of 7 scalp needles were used to draw blood from the caudal vein of the Sd rats, then centrifuged 2,000 x g for 2 min at 37˚C to obtain the rat serum. In order to detect the NO concentration in the rat serum, a Nitric Oxide Assay kit (Sigma-Aldrich; Merck KGaA) was used. The assay was conducted according to the protocol provided in the kit, and NO was measured using the Thermo Scientific™ SPEcTRONIc™ 200 spectrophotometer (Thermo Fisher Scientific, Inc.) (31).
EdU assay. In order to detect the proliferation and vitality of cells, click-iT™ EdU Flow cytometry Assay kit (Alexa Fluor™ 488; Thermo Fisher Scientific, Inc.) was used according to the manufacturer's protocol. Briefly, cells were washed three times in PBS containing 0.05% Triton X-100 prior to EdU-dNA detection. For nuclear staining (blue), stem cell samples were treated with 1 µg/ml dAPI in PBS containing 0.1% Triton X-100. Next, the samples were incubated for 1 h at 37˚C in the dark. Newly proliferating nucleotides were stained red by 2X EdU solution according to the manufacturer's protocol. Then, the samples were visualized under a fluorescence inverted microscopy (VMF400I, Suzhou Jing Tong Instrument co., Ltd. Suzhou, china) (35)(36)(37).
ELISA. The MΦ supernatant was collected from the three MΦ groups cultured in vitro, including the control, LPS and HF-LPLI cell groups. In addition, serum samples from the hearts of rats in the control, periodontitis, and 1 or 3-week HF-LPLI treatment groups were obtained by 2,000 x g for 15 min at 37˚C. Subsequently, 100 µl Standards and 100 µl samples were added into the corresponding reaction plate well. Subsequent steps were conducted according to the protocol provided by the MitoBiogenesis ™ In-cell ELISA kit (colorimetric; cat. no. ab110217; Abcam). Finally, the optical density was measured at 450 nm (38,39). Animal experiment. Male Sd rats (age 6-8 weeks old, 250-280 g) were obtained from the Laboratory Animal center in Taishan Medical University (N=24 in total, n=8 for a group). Sd rats were housed at 37˚C, relative humidity 45% and dark environment with low fat diet and free to water. All animal procedures were performed according to the Guide for the care and Use of Laboratory Animals (18), following the ARRIVE guidelines (41), and were approved by the Institutional clinical Experiments committee of the Liaocheng People's Hospital (Liaocheng, china) and the Animal care committee. Rats were divided into three groups of 8 rats each, including the control, periodontitis and HF-LPLI groups. Subsequent to gingival peeling and high-sugar feeding, all rats received azithromycin (10 mg/500 ml; Shanghai Yingrui Biopharma co., Ltd., Shanghai, china) for 4 days, followed by a 7-day antibiotic-free period. Next, rats were anesthetized with 10% chloral hydrate. A 0.2-mm wire was placed in the dentogingival area of both mandibular first molars in the periodontitis and HF-LPLI groups for 48 h, while the wire was placed and immediately removed in the control group. All rats were fed with the same food (at 8:00 a.m. and 8:00 p.m.). Rats in the HF-LPLI groups underwent surgery, followed by a 1-week 20-J/cm 2 HF-LPLI treatment for 1 h/day after local injection of 1x10 5 /ml stem cells in 100 µl, followed by the in vivo bioluminescence imaging and H&E staining (42,43). The Height of bone regeneration was calculated as 8 mm-(a1 + a2 + a3)/3. In the formula, a1, a2 and a3 were three random height measurements of residual defect height following bone regeneration.
In vivo bioluminescence imaging. Sd rats were divided into three groups, including the control (6 weeks), HF-LPLI/6-week and HF-LPLI/12-week groups, and were analyzed at the corresponding time point. Rats were anesthetized with 10% chloral hydrate and treated with fluorescein that was injected through the tail vein. At 60 min post-infection images were obtained after intraperitoneal injection of d-luciferin (150 mg/kg body weight; Thermo Fisher Scientific, Inc.), followed by 50 µl d-luciferin (0.75 mg in PBS) through the oral cavity. The IVIS Lumina image system (Xenogen corporation, Alameda, cA, EUA) was used to capture the images (44).
Hematoxylin and eosin (H&E) staining. Periodontal tissue ~200x200 µm fragments from the animal model were dewaxed by xylene, washed with an alcohol gradient and then soaked in distilled water. Next, tissues were stained with Harris hematoxylin solution for 5 min. Subsequent to rinsing, sections underwent color separation in 0.5% hydrochloric-alcohol solution for 5 sec, followed by washing two times with 95% ethyl alcohol for 5 min each, two times with absolute ethyl alcohol for 5 min each and xylene + ethyl alcohol (1:1) for 5 min. Finally, samples were subjected to xylene cleaning and neutral gum sealing, and followed by visualization under light microscope (Nikon corporation, Tokyo, Japan) (35)(36)(37).
Statistical analysis. Statistical analysis was performed using the SPSS software package (version 13; SPSS, Inc., chicago, IL, USA). The results are expressed as the mean ± standard deviation, and the analysis of variance test was used to evaluate the statistical significance between two groups. P<0.05 was considered to indicate a statistically significant when the.

HF-LPLI induces MΦ transformation and neutrophil apoptosis.
The inflammatory reaction is one of the causes of  ). Furthermore, it also demonstrated neutrophil apoptosis increased subsequent to HF-LPLI treatment, ranging between 7.28 and 18.1% (Fig. 1A).
In addition, HF-LPLI induced a time-dependent increase of caspase-3 activation, which was 2.8±0.17 times higher than the initial value at 6 h (Fig. 1B). This further demonstrated the role of HF-LPLI in promoting apoptosis. In fact, the MΦ apoptosis was also tested following HF-LPLI treatment, and the results indicated that HF-LPLI had no effect on MΦ apoptosis (data not shown). In the pre-experiments, it was observed that 6 h was the best time period for inflammation to promote the function of stem cells (data not shown). In the tests conducted in the present study, all the related experimental design selected inflammation treatment to last 6 h (data not shown). Immunofluorescence analysis detected no significant differences in the morphology of different types of MΦs, while the reprogramming of MΦs was confirmed. The LPS group exhibited an increase in the number of cd86-M1 (red staining), while the HF-LPLI-treated group mainly exhibited green staining, corresponding to cd163-M2 (Fig. 1c). Following HF-LPLI treatment, the number of cd86-M1 decreased from 73.21±2.23% to 18.29±1.47% (P<0.05), while cd163-M2 increased from 9.26±1.07% to 58.88±1.97% (Fig. 1d). In addition, dAF-FM IL-6 and tumor necrosis factor α (TNF-α) levels significantly decreased following HF-LPLI treatment, while the levels of anti-inflammatory cytokines, including IL-10 and TGF-β, secreted by M2 were increased ( Fig. 1E and F).

HF-LPLI maintains the stem cell properties of DPSCs and
PLSCs. RT-qPcR was used to detect the mRNA expression levels of genes associated with the reprogramming of stem cells, including Sox2, Oct4 and Klf4, prior to and following HF-LPLI treatment. The results indicated that Sox2, Oct4 and Klf4 expression levels in dPScs and PLScs in the control group treated with HF-LPLI slightly increased as compared with those in cells without HF-LPLI treatment, although a significant difference was not observed (P>0.05; Fig. 2A and B). When stem cells were co-cultured with MΦs or neutrophils, the expression of these three genes was significantly decreased (P<0.05), while HF-LPLI treatment inhibited this effect and increased the gene levels. More specifically, Oct4 expression in PLSCs was decreased to a value of 4.3±0.2 and 4.2±0.2 when stem cells were co-cultured with MΦs or neutrophils, respectively, while this level was 10.2±0.3 in the culture of stem cells without inflammatory cells, and HF-LPLI treatment was able to raise this level to 6.0~8.0±0.4 to a value of <3 ( Fig. 2A). In DPSCs, inflammatory cells exhibited a marked effect on Sox2 expression, which was decreased from 6.2±0.4 to a value of <3. However, HF-LPLI attenuated this effect, increasing the Sox2 expression to ~5 (Fig. 2B).

HF-LPLI improves DPSC and PLSC proliferation.
EdU assay results demonstrated that stem cell proliferation was not evident when these cells were cultured alone, while their proliferation was evident when co-cultured with MΦs. When stem cells were co-cultured with MΦs and simultaneously treated with HF-LPLI, the HF-LPLI treatment did not increase the promotion of stem cell proliferation induced by inflammatory cells. However, after co-culture for 6 h followed by treatment with HF-LPLI, the stem cell proliferation was greatly improved, as shown by the EdU results (Fig. 3A), and the difference with the other experimental groups and the control group was statistically significant (P<0.05). The number of EdU-positive cells in the MΦ group was 13±1 and 19±1 for dPScs and PLScs, respectively, while the number of cells was 5±1 and 6±1 in the control group, respectively. When HF-LPLI treatment was performed simultaneously with the MΦ co-culture in the stem cells, the number of EdU-positive cells was 8±1 or 7±1 for dPScs and PLScs, respectively. However, the number of proliferating cells was 21±2 or 29±2, respectively, when HF-LPLI treatment was performed at 6 h after MΦ co-culture (Fig. 3B).

HF-LPLI promotes DPSC odontoblastic differentiation.
The present study next focused on the establishment of the HF-LPLI ability to induce cell differentiation. ALP, OcN and Runx2 gene expression levels were evaluated in cells obtained after 2 weeks of proliferation, as described earlier.
The results demonstrated that the relative expression levels of the three genes in the MΦ group were significantly increased to ~2-3±0.2 (P<0.05; Fig. 4A). However, gene expression was inhibited to nearly 1.3±0.2 when co-culture with MΦs and HF-LPLI treatment were performed simultaneously, while gene expression was promoted when HF-LPLI was performed after 6 h of MΦ co-culture. In addition, OcN expression in dPScs reached 4.7±0.8 (Fig. 4A). Stem cells in the different MΦ and HF-LPLI groups were then subjected to alizarin red S staining to identify the calcium salt components in the cells. A significant increase in the calcium ion composition was found in the MΦ followed by HF-LPLI treatment group (Fig. 4B). For alizarin red S staining, the absorbance at 570 nm was ~0.6 at 2 weeks and 0.9 at 4 weeks in the group with MΦ co-culture followed by HF-LPLI treatment, while it was <0.5 in the control and MΦ co-culture alone groups (Fig. 4c).

HF-LPLI reduces the infiltration of inflammatory cells and release of inflammatory factors in periodontitis rats.
A large number of neutrophils and MΦs were detected in the inflammatory sites in periodontitis rats during immunofluorescence analysis. After 1 week of HF-LPLI treatment, the number of neutrophils and MΦs in the surrounding tissues decreased significantly from 131±6 and 119±5 to 44±3 and 52±4, respectively. After 3 weeks of detection, further improvement in the number of inflammatory cells was achieved, which were inhibited to 9±2 and 11±2, respectively. cd11b-labeled neutrophils and cd86-labeled MΦs were abundant in the surrounding tissues of periodontitis rats, while HF-LPLI application significantly decreased the staining for the inflammatory cells. Furthermore, this effect was found to be enhanced with time ( Fig. 5A and B).  IL-1β, IL-6, TNF-α and NO levels in the blood of rats belonging to different groups were subsequently examined, and significantly increased plasma concentrations of pro-inflammatory cytokines in rats with periodontitis were detected, as compared with the control group. The levels observed in the periodontitis rats were 173±12.6 pg/ml for IL-1β, 1,237±94.1 pg/ml for IL-6, 496±17.2 pg/ml for TNF-α and 0.02±0.004 µmol/ml for NO (Fig. 6A-d). After 1-week HF-LPLI treatment, these concentrations were significantly decreased, with IL-6 reduction to a level of 733±62.3 pg/ml representing the best result (Fig. 6B). After 3 weeks of treatment, the concentration of pro-inflammatory cytokines reached approximately the normal level. Taken together, these results indicated that appropriate HF-LPLI treatment was able to decrease the level of pro-inflammatory cytokines.
HF-LPLI induces dentin repair and tertiary dentin in a rodent model. The results were further confirmed in the animal model by evaluating the effectiveness of treating a dental defect (3x3x8 mm) with stem cells (Fig. 7A). The results of in vivo imaging demonstrated the presence of significant inflammation in the oral cavity of the animal model, and the inflammation was reduced after 6 weeks of treatment with stem cells followed by HF-LPLI (Fig. 7B). In addition, H&E staining results revealed that the pulp defect was successfully reconstructed in the rat model, and cell proliferation was clearly time-dependent (Fig. 7C). Calcification observed under a microscope indicated the occurrence of osteogenic differentiation. A statistically significant difference in osteogenesis was observed among the periodontitis animal model, the 6-week treatment and 12-week treatment groups (P<0.05; Fig. 7d), suggesting that HF-LPLI treatment was effective in vivo.

Discussion
dPScs and PLScs have previously been used in the treatment of periodontal tissue for regeneration studies (45); however, the improvement of their therapeutic effect and the underlying mechanism of their action remain under debate. In order to identify a new strategy for the application of dPScs and PLScs in the treatment of periodontitis, HF-LPLI was used in the current study to induce cell orientation and to identify the potential mechanism of promoting inflamma-tion reduction by enhancing neutrophil apoptosis and MΦ reprogramming.
In the experiments of the present study, HF-LPLI was found to promote neutrophil apoptosis and MΦ reprogramming, which may via the inactivation of Akt/GSK3β signaling pathway (17). Neutrophil apoptosis reduced the inflammatory response induced by periodontitis, while the transformation of MΦs reduced the secretion of pro-inflammatory cytokines and increased the release of anti-inflammatory cytokines, such as IL-10 and TGF-β. M1 MΦs secrete ROS, RNS, TNF-α, IL-1, IL-12, IL-23 and other chemokines, which are mainly involved in the inflammatory response and host immune functions, and cause inflammatory damage to normal tissues. Under the action of IL-4, IL-13, IL-10 and TGF-β, the M1 MΦs are polarized to M2, which secrete TGF-β, VEGF, EGF and other factors, particularly at the late stage of inflammation, and promote the repair of trauma and fibrosis (46). Thus, the transformation of M1 to M2 can effectively promote the regression of inflammation. Notably, the current study findings reported that HF-LPLI promotes neutrophil apoptosis, but did not promote MΦ apoptosis, and the mechanism involved in this effect warrants further investigation. This mechanism by which HF-LPLI promotes neutrophil apoptosis and MΦ reprogramming led to the maintenance of stem cell viability and increased the expression of stemness-associated genes. The proliferation of stem cells was guaranteed at an early stage of the treatment i.e., the stemness of the cells was well maintained allowing for unhindered proliferation. These results suggested a key role of HF-LPLI in the treatment of periodontitis.
As shown by EdU proliferation assay, stem cell proliferation was not significantly different from that in MΦ+HF-LPLI simultaneously-treated groups when stem cells were co-cultured with MΦs to create the inflammatory environment and subjected to HF-LPLI simultaneous treatment. However, when HF-LPLI treatment was applied 6 h after co-culture with MΦs, cell proliferation was signifi-cantly enhanced. These results suggested that inflammation obtained by HF-LPLI treatment for a certain period of time served a role in starting and promoting the proliferation of stem cells, while excessive inflammatory response was reduced by HF-LPLI treatment at 6 h, improving the microenvironment and further promoting stem cell proliferation. Gene expression and alizarin red S staining demonstrated similar results, further indicating that HF-LPLI promoted differentiation following activation by appropriate inflammation. The results also revealed that MΦ followed by HF-LPLI induced a better bone differentiation at 4 weeks, particularly when dPScs were used, which achieved a better differentiation result compared with PLScs. In the in vivo experiments, after induced periodontitis was treated with stem cells, no treatment was administered to the model to induce inflammation in 6 h. Proper inflammation promoted the proliferation of stem cells and was beneficial to subsequent differentiation. Subsequently, HF-LPLI was used to reduce the number of inflammatory cells and inhibited the release of inflammatory cytokines. The in vivo bioluminescence imaging revealed that, after 6 weeks of HF-LPLI treatment, the inflammation was reduced. As soon as inflammation was inhibited, H&E staining indicated tissue regeneration due to stem cell proliferation, and further osteogenic differentiation. Therefore, stem cells appeared to successfully achieve periodontitis treatment. At 12 weeks, the best treatment effect was obtained, which was markedly greater than the effect at 6 weeks, suggesting a better osteogenic differentiation after an additional 6 weeks.
In conclusion, HF-LPLI may represent a novel strategy for improving the effect of stem cells treatment. However, how to use HF-LPLI for achieving the best effect in the treatment of periodontitis should be confirmed. Furthermore, the treatment dose and the time required to maximize an anti-inflammatory effect, as well as the effect of HF-LPLI on human periodontitis require additional experiments.


hiPSC hepatocyte model demonstrates the role of unfolded protein response and inflammatory networks in α1-antitrypsin deficiency

Graphical abstract

Background & Aims: a 1 -Antitrypsin deficiency (A1ATD) is an autosomal recessive disorder caused by mutations in the SER-PINA1 gene. Individuals with the Z variant (Gly342Lys) retain polymerised protein in the endoplasmic reticulum (ER) of their hepatocytes, predisposing them to liver disease. The concomitant lack of circulating A1AT also causes lung emphysema. Greater insight into the mechanisms that link protein misfolding to liver injury will facilitate the design of novel therapies. Methods: Human-induced pluripotent stem cell (hiPSC)derived hepatocytes provide a novel approach to interrogate the molecular mechanisms of A1ATD because of their patient-specific genetic architecture and reflection of human physiology. To that end, we utilised patient-specific hiPSC hepatocyte-like cells (ZZ-HLCs) derived from an A1ATD (ZZ) patient, which faithfully recapitulated key aspects of the disease at the molecular and cellular level. Subsequent functional and ''omics" comparisons of these cells with their genetically corrected isogenic-line (RR-HLCs) and primary hepatocytes/human tissue enabled identification of new molecular markers and disease signatures. Results: Our studies showed that abnormal A1AT polymer processing (immobilised ER components, reduced luminal protein mobility and disrupted ER cisternae) occurred heterogeneously within hepatocyte populations and was associated with disrupted mitochondrial structure, presence of the oncogenic protein AKR1B10 and two upregulated molecular clusters centred on members of inflammatory (IL-18 and Caspase-4) and unfolded protein response (Calnexin and Calreticulin) pathways. These results were validated in a second patient-specific hiPSC line. Conclusions: Our data identified novel pathways that potentially link the expression of Z A1AT polymers to liver disease. These findings could help pave the way towards identification of new therapeutic targets for the treatment of A1ATD. Lay summary: This study compared the gene expression and protein profiles of healthy liver cells and those affected by the inherited disease a 1 -antitrypsin deficiency. This approach identified specific factors primarily present in diseased samples which could provide new targets for drug development. This study also demonstrates the interest of using hepatic cells generated from human-induced pluripotent stem cells to model liver disease in vitro for uncovering new mechanisms with clinical relevance.
Introduction a 1 -Antitrypsin (A1AT) is a 52 kDa protein encoded by the SER-PINA1 gene synthesised primarily by hepatocytes. 1 Secreted into the blood stream, it acts to control the function of neutrophil elastase, particularly in the lung. 2 A1AT also exerts antiapoptotic and anti-inflammatory properties during inflammation and hepatic injury. Most people carry the wild-type M allele, while the rarer Z variant (found in 1-3% of the population), is associated with the most common and severe form of clinically significant A1AT deficiency (A1ATD). 3 The Z allele is caused by a Glu342Lys mutation in exon 5 of the SERPINA1 gene, leading to conformational instability within the protein. 4 Approximately 70% of synthesised Z A1AT is degraded by intracellular quality control mechanisms, 15% is secreted whilst the remaining 15% accumulates in hepatocytes as ordered polymers. These polymers are associated with neonatal hepatitis, cirrhosis and hepatocellular carcinoma. Furthermore, the significant reduction in circulating plasma A1AT levels leads to uncontrolled proteolytic activity within the lung and development of early onset panlobular emphysema. 5 Despite the recognition of A1ATD over 50 years ago, 6 the detailed molecular mechanisms linking A1AT polymer accumulation to the development of liver disease remain poorly understood. This has been hampered by the availability of primary human hepatocytes expressing wild-type and mutant forms of A1AT capable of surviving in culture long-term. Although the use of animal models and artificial A1AT-expressing cell systems have contributed significantly to improved understanding of the disease processes, 7-9 multiple gene copies, interference of endogenous animal antiproteases, lack of pathological features post-polymer accumulation, and the absence of endogenous promoters to activate gene expression represent challenges for translating new findings in these model systems to man.
The advent of human-induced pluripotent stem cells (hiPSCs) 10 has provided an exciting platform to address these obstacles. Indeed, PiZZ hiPSCs (ZZ-hiPSCs) derived from patients with A1ATD were shown to differentiate into hepatocyte-like cells (ZZ-HLCs) that recapitulate the modified posttranslational processing and secretion kinetics of mutant A1AT. 11,12 Furthermore, patient-specific HLCs were genetically corrected to erase the disease signature (RR-HLCs) 13 and so generated a perfect control cell line with which to perform mechanistic studies.
Using these tools, we first established the suitability of HLCs to study the mechanisms of A1ATD by benchmarking their gene expression, protein synthesis and metabolic activity against primary hepatocytes. Then, by comparing the transcriptome and endoplasmic reticulum (ER)-enriched proteome of ZZ-and RR-HLCs, we validated the platform's efficiency for identifying novel biomarkers and pathways involved in mediating the disease processes that may be of immediate clinical relevance.

Materials and methods
hiPSC culture and hepatic differentiation All hiPSC lines used in this study have were derived as previously described 11 under Addenbrooke's Hospital Ethics reference number 08/H0311/201; R&D No. A091485. These cells were maintained at 37°C in humidified incubators supplemented with 5% v/v carbon dioxide and were differentiated using our established hepatic differentiation protocol with minor modifications: 14 Cells were differentiated at 5% oxygen and two splitting steps allowed for scale-up and prolonged maturation of differentiating cells (Fig. S1A). During the splitting steps on day 8 and day 25, cells were washed with PBS and dissociated with TrypLE TM (Life Technologies, 12563-029) for 20-45 min at 37°C. Harvested cells were centrifuged at 100 Â g for 3 min. Cells were resuspended in RPMI (day 8 split) or HepatoZYME-SFM (day 25) (Thermo Fisher, 17705-021). Cells were counted and resuspended in RPMI and Activin (day 8 split) or HepatoZYME-SFM (Thermo Fisher, 17705-021), OSM and HGF (day 25 split), supplemented in each case with ROCK inhibitor (1 lL/ml) to a concentration of 210,500 cells/cm 2 .

Primary hepatocyte procurement
Commercially plated human primary hepatocytes from two donors were purchased from Biopredic International (France).
The cells exhibited viabilities between 88-93% after isolation, fulfilled the manufacturer's requirements of Phase I and II-dependent enzyme activities and were cultured for 48 h in hepatocyte medium prior to harvesting. Fresh human primary hepatocytes were kindly provided by Dr. Roque Bort with approval of the hospital's ethics committee (Instituto de Investigación Sanitaria La Fe, Valencia, Spain). Upon isolation, hepatocytes were snap frozen and the resulting cell pellets were directly lysed for RNA extraction.

RNA-seq
Library preparation of RNA samples was performed with the Illumina TruSeq RNA Sample Preparation V2 assay. Samples were multiplexed on two lanes with 40 bp (HLCs) or 75 bp (PiZZ/PiMM) read lengths, single strand and single-end (HLCs) or paired-end (PiZZ/PiMM) reads and generated $16.5 million reads per sample on the Illumina HiSeq 2000. Bioinformatic analysis were carried out following standard procedures 11 and of raw data are available on arrayexpress accession number E-MTAB-6781.

Results
hiPSC-derived hepatocyte-like cells are suitable for modelling human disease We have previously generated disease specific hiPSCs (ZZ-hiPSCs) 11 and created an isogenic wild-type line (RR-hiPSC) using genome editing. 13 Once differentiated, ZZ-derived hepatocyte-like cells (ZZ-HLC) displayed intracellular polymer retention whilst corrected cells (RR-HLC) secreted normal levels of A1AT. Thus, RR-HLC represent the ideal control for molecular studies, since phenotypic differences observed between both cell types should exclusively be attributable to the misfolding of mutant A1AT in HLC-ZZs and not to variability between hiPSCs lines of different genetic backgrounds. 15 To further validate this hypothesis, we compared the hepatic function of ZZ-HLCs and RR-HLCs using primary hepatocytes freshly isolated, cryopreserved or freshly plated as a positive control. Of note, we applied an optimised protocol 14 allowing HLCs to differentiate for an extended period of time, increasing their functional repertoire (Fig. S1A-C). As expected, HLCs expressed a diversity of hepatic markers including A1AT, ALB and HNF4a at levels equivalent to primary hepatocytes ( Fig. 1A-C). They also displayed CYP3A4 expression and activity although at significantly lower levels than primary cells (Fig. 1A, Fig. S1B). Conversely, HLCs showed expression of foetal markers CYP3A7 and AFP at higher levels ( Fig. 1A) thereby confirming their foetal identity. 16 More importantly, ZZ-HLCs and RR-HLCs expressed comparable levels of hepatic markers (Fig. 1A). These observations were confirmed by immunostaining and DELFIA Ò , showing these markers to be expressed at equivalent levels in both cell types with the exception of A1AT, which was synthesised in lower concentrations into ZZ-HLCs culture media ( Fig. 1B-C). To confirm this observation, A1AT trafficking in ZZ-HLCs was assessed by growing HLCs in the presence of radioactively labelled [ 35 S] Cys/Met and by subsequently tracking its transition from the intracellular to the extracellular space over the course of 4 h. The band intensities of these pulse-chase experiments revealed a slower rate of glycoprotein maturation (e.g. sialic acid residue incorporation) and trafficking of A1AT to the extracellular space in ZZ-HLCs (Fig. 1D). Whilst wild-type A1AT was almost fully secreted after 4 h in RR-HLCs, mutant A1AT in ZZ-HLCs was mostly degraded or retained intracellularly during the same time period. 17 Taken together, these results demonstrate that ZZ-HLCs and RR-HLCs have reproducible biological characteristics in vitro, except for the processing of A1AT, and therefore provide a suitable control-study pair for in-depth characterisation of the disease pathophysiology of A1ATD.

ZZ-HLCs abnormally process A1AT polymer in a heterogenous, ER-dependent manner
To investigate the impact of A1AT protein accumulation on cellular trafficking, we studied the hepatic ER -the site at which mutant A1AT proteins aggregate -at a single cell level. ZZ-and RR-HLCs were transfected with the ER marker, green fluorescence protein (GFP)-KDEL, that has been shown to co-localise with A1AT polymers. 9 Transfections of HLCs with the GFP-KDEL construct yielded a population of RR-HLCs which all contained normal, reticular ER morphologies ( Fig. 2A). In contrast, ZZ-HLCs frequently displayed large ER inclusions. We classified these as inclusions of small, medium or large size and quantified the percentage of cells containing each sub-type (Fig. 2B). Overall, 40% of ZZ-HLCs showed inclusions, whilst the remaining 60% of cells exhibited a normal ER. We next analysed the effect of intracellular inclusions on ER architecture using ''fluorescence loss in photobleaching" (FLIP) and ''fluorescence recovery after photobleaching" (FRAP) to track live, GFP-KDEL transfected ZZ-HLCs and RR-HLCs. 18 This allowed an evaluation of the cell's ER connectivity (FLIP) and luminal protein mobility (FRAP) by monitoring the diffusion rates of inert fluorescent probes throughout different ER morphologies. Repeated photobleaching of a pre-defined fluorescent region resulted in complete depletion of fluorescence in inclusion-free ZZ-and RR-HLCs,    while neighbouring cells were unaffected (Fig. 3A). Of particular note was the small, consistent recovery of fluorescence in between the bleaching events, suggesting rapid diffusion of unbleached KDEL-fluorophores back to the area of interest. These observations reinforced our previous results obtained on transformed cell lines 19 by showing that in wild-type HLCs, ER proteins can sample distant compartments of the ER by diffusion when enclosed by connected, tubular ER luminae. In contrast, in inclusion-bearing ZZ-HLCs, FLIP within large inclusions resulted in the localised loss of KDEL-GFP fluorescence with no recovery of fluorescence after bleaching events. Furthermore, neighbouring inclusions were not affected, indicating walled-off ER cisternae with no or restricted ability for protein diffusion or interactions between them. These observations were further confirmed using another fluorescent protein chimera, the cytoplasmic domain of an ER signal anchor membrane protein (CytERM). 20 FLIP performed on ZZ-HLCs with large inclusions transfected with fluorescent CytERM resulted in complete loss of fluorescence of the inclusion's ER membrane, whilst neighbouring inclusions were unaffected (Fig. 3B). Finally, fragmentation and isolation of ER inclusions were confirmed using Z-stack images captured across ZZ-HLCs transfected with the fluorescent ER membrane protein (Fig. 3C). The mobility of ER proteins was then quantified using FRAP assays. 18 The pre-bleach fluorescence level of a selected region of interest inside the cytoplasm was normalised to 100% and a single bleaching event was performed within the region of interest. Diffusion rates of unbleached KDEL-GFP molecules from adjacent areas into the bleached region were determined by monitoring the restoration of fluorescent levels into the region of interest. We observed that the patterns of fluorescent recovery were similar within regions of reticular ER in RR-and ZZ-HLCs (Fig. 4A). Mapping the percentage of fluorescent rescue to the area of bleaching showed almost immediate recovery at approximately 60%, which was further restored to nearly 90% in RR-HLCs. In contrast, inclusion-bearing ZZ-HLCs showed recovery of only 30-40% of the original KDEL-GFP fluorescence. Regression analysis to reveal the rates of recovery, i.e. the slope of the respective fluorescent recovery curves, confirmed faster diffusion rates of proteins in areas of connected reticular ER compared to the rates seen in areas with disrupted ER morphologies (RR-HLC: 2.92 ± 0.75, ZZ-HLC no inclusions: 3.99 ± 1.03, ZZ-HLC with inclusions 1.23 ± 0.22). There appeared to be no or very little recovery of fluorescence in bleaching of larger inclusions, suggesting a size-dependent isolation of inclusions (Fig. 4B).
To quantify the percentage of total fluorescent molecules that contribute to the fluorescent recovery of the photobleached area during the assayed time frame, we assessed the mobile fractions associated with individual ER morphologies. These showed that protein viscosities of reticular ER morphologies were significantly lower than that of fractionated ER sites (p ≤0.0001) (Fig. 4C). In summary, these data showed that polymer accumulation in A1ATD reduced ER protein diffusion rates suggestive of immobilised ER components, reduced protein mobilities and/or disrupted ER cisternae in inclusion-bearing ZZ-HLCs. 21 Transcriptome and proteome analyses suggest ZZ-HLCs acquire mitochondrial abnormalities and are poised for malignancy Given the abnormalities in protein mobility observed in the ER, we hypothesised that polymer accumulation in ER inclusions could be associated with specific proteins inhibiting A1AT polymer degradation and eliciting stress responses. [22][23][24] To investigate this hypothesis, we performed proteomic analyses on ER fractions isolated from ZZ-and RR-HLCs (Fig. 5A). Three independent batches of ZZ-and RR-HLCs were fractionated into their subcellular ER fractions and analysed by label-free mass spectrometry. We identified 2,683 unique proteins in the ZZand RR-HLC ER proteomes, which were enriched most significantly for the ER (adjusted p value: 7.1-91) and mitochondria (adjusted p value: 1.6-172). Among these proteins, 14 were exclusively identified in all three batches of ZZ-HLCs whilst three proteins were present only in the RR-HLCs (Fig. 5B). By gene ontology (GO) enrichment analysis, these 17 proteins with strict distribution patterns displayed a diversity of functions involved in intracellular trafficking, extracellular matrix organisation and enzymatic activity. Five of these proteins (29%) were attributed to mitochondrial localisation and oxidoreductase activity: GLUD2, GFER, OXCT1, EARS2 and ADCK3 (COQ8A). In order to further understand the implications of A1AT protein accumulation on a whole-cell scale, genome-wide RNA sequencing (RNA-seq) was performed on ZZ-HLCs and RR-HLCs. Statistical analysis revealed 1,675 genes with significantly different expression (false-discovery rate (FDR) <5%), the majority of which were protein-coding genes (93.4%) (Fig. 5C). Of the 1,675 differentially expressed genes, 959 were upregulated in ZZ-HLCs (958 of them with a fold change >1.5), while 716 genes were downregulated in ZZ-HLCs (715 of them with a fold change <À1.5). A total of 319 genes with differential transcript expression were also detected in the mass spectrometry dataset (Fig. 5D). We then combined both proteomic and transcriptomic data sets by considering differential abundances of proteins and genes with log-fold changes >2 or <À2. Candidate markers that fulfilled these parameters included 18 upregulated and 15 downregulated genes/proteins (Fig. 5E)  CYP1A1). Of note, the low correlation of data points seen under these conditions may be explained by the comparison of wholecell transcriptomes with subcellular ER-specific proteomes. We next focused on examining the absolute gene expression of the assayed genes and considering ZZ-HLCs as the reference. We found that 20 of the 21 most highly-expressed and differentially upregulated genes in ZZ-HLCs were of mitochondrial origin (Fig. 5F). The majority of their functional repertoire was relevant to oxidoreductase activity and mitochondrial ATP synthesis in the electron transport chain and included NADH dehydrogenases, ATP synthases, cytochromes b and c. The only nonmitochondrial gene in the very highly-expressed, upregulated gene cluster was fibrinogen gamma chain (FGG), upregulation of which has previously been associated with hepatic inflammation 25 and hepatocellular carcinoma. 26 Other molecules with reported oncogenic associations were also found in the highlyexpressed, upregulated cluster and included H19, 27 SPARC 28 and SERPINE1. 29 Furthermore, GO over-representation analyses on all highly-expressed, upregulated genes revealed that the five most enriched and significant GO terms were tied to extracellular matrix genes associated with fibrosis and cirrhosis (p values <0.0001), a hallmark of liver disease in A1ATD (Fig. 5G). This included collagen-related genes (e.g. COL1A1, COL1A2, COL3A1, COL4A1 and COL5A1) and genes whose products are involved in the assembly and remodelling of the extracellular matrix such as ADAM metallopeptidases (e.g. ADAM19), fibrillin (e.g. FBN1), fibronectin (e.g. FN1), laminins (e.g. LAMB1), thrombospondin (e.g. THBS1) and matrix-associated proteins encoded by SPARC and WNT5A. Taken together, these analyses uncovered highly relevant proteins involved in the response to SERPINA1 mutations.

Validation of discoveries from the hiPSC-derived hepatocyte platform
We concluded that overall, several new areas of A1AT-deficient biology in HLCs were identified and warranted follow-up. The most significant of which were: i) A1AT polymers accumulated in a heterogenous manner within ZZ-HLC hepatocyte populations, ii) specific proteins associated with predisposition to malignancy were found to be highly upregulated in ZZ-HLCs, iii) mitochondrial biology in ZZ-HLCs was abnormally affected, and iv) ZZ-HLCs displayed activation of important inflammatory and unfolded protein response pathways. To validate the relevance of these findings, we next assessed the accumulation of A1AT polymers in liver tissue from individuals with PiZZ A1ATD 30 (Fig. 6A). While the majority of hepatocytes exhibited no polymer accumulation, those displaying characteristic A1AT inclusions were mainly located at the periphery of hepatic lobules, thereby validating the cell-to-cell heterogeneity of polymer formation observed in ZZ-HLCs ( Fig. 2A). We next compared genes differentially expressed in HLCs (ZZ-HLCs vs. RR-HLCs) and primary hepatocytes (PiZZ vs. PiMM) and found 80 upregulated genes common to both datasets (Fig. 6B). One of these 80 genes (AKR1B10) had a related enzyme product (AKR1B1) present exclusively in the ER-enriched proteome dataset of ZZ-HLCs (Fig. 5B) and a log 2 -fold change of 3.6 and 3.8 in differential gene expression between ZZ/RR-HLCs and PiZZ/PiMM hepatocytes respectively. The clinical significance of this molecule was assessed by immunostaining paraffinembedded liver sections from three patients with PiZZ whose clinical backgrounds (age, sex and severity of disease) matched those of the original hiPSC line donor. Samples stained positively for AKR1B10 in clusters of cells that were also positive for polymers of A1AT (Fig. 6C). Having demonstrated the potential clinical relevance of the data generated from our in vitro model for biomarker discovery, we next sought to explore physiological pathways that may underpin A1AT pathogenesis by focusing on GO terms associated with unfolded protein binding. Utilising STRING v9.1, we surveyed known and predicted protein-protein interactions encoded by those genes upregulated with the highest confidence view (score = 0.900) in ZZ-HLCs. We found evidence of gene network clusters indicating inflammatory (e.g. IL18) and cellular stress responses, (e.g. calnexin (CANX), calreticulin (CALR), BiP/GRP78 (HSPA5), ERdj5 (DNAJC10), ERdj6 (DNAJC3), DNAJB11, GRP94 (HSP90B1) and GRP170 (HYOU1) (Fig. S2). The differential gene expression profile of ZZ-HLCs not only appeared to mirror components of the A1AT-deficient pathology that have previously been identified (e.g. CANX and CALR), but also uncovered new genetic networks. Of particular interest were those genes linking misfoldinginduced inflammatory responses to mitochondrial abnormalities, since our electron microscopy studies demonstrated ZZ-HLC populations contained markedly abnormal mitochondrial phenotypes (Fig. 6D), as also previously described in human tissue and HLCs. 31 We sought to validate the importance of these genes by examining differential gene expression at the nexus of inflammatory (IL-18 and CASP4) and unfolded protein (CANX and CALR) clusters in a second hiPSC cell model of A1ATD derived from a different patient in a different laboratory. 32 All four genes were also found to be upregulated in PiZZ vs. PiMM cells, suggesting their importance for future investigations (Fig. S3). Taken together, these data demonstrated that the biomarkers and putative new mechanisms identified by our approach could be clinically relevant for a broad number of A1AT-deficient patients.

Discussion
Despite the classification of A1ATD as a monogenetic disease, the underlying molecular mechanisms causing liver pathology are poorly understood. hiPSCs offer an appealing model to interrogate such questions, but have until now been hampered by concerns over whether in vitro signatures reflect the human disease. To address this, our study characterised the molecular and cellular signatures of ZZ-HLCs in comparison to human tissue. We first determined that, as in patients, only a subset ($40%) of ZZ-HLCs contained A1AT polymers and that these polymers are contained within the ER but physically isolated from the rest of the ER lumen. Of note, Tafaleng et al. previously utilised ZZ-HLCs and ZZ liver samples to establish the presence of globular inclusions and dilated ER. 12 Our studies elaborate on these observations by using ZZ/RR-HLCs to provide real-time proof of physical ER fractionation and absent functional connection between abnormal ER structures. Importantly, our analyses of liver samples from patients with A1ATD also suggest that the disease could be regionalised as these globular inclusions were mainly located in the lobule periphery. Thus, disease progression mechanisms could be associated with liver zonation. To further shed light on the molecular mechanisms governing this specific type of intracellular A1AT retention, we then compared genome-wide RNA-seq and ER-specific proteomic datasets of ZZ-HLCs vs. RR-HLCs. The foremost dysregulated pathways, both on a transcriptomic and proteomic level, were associated with known pathways of the misfolded protein response. In addition, our analyses also revealed novel molecular proteins and pathways important in chaperone function, inflammation, mitochondrial dysfunction and malignancy, which we validated in human tissue. Despite the persistent molecular and functional similarities of HLCs with foetal hepatocytes, 16 this work has shown the adequacy of ZZ-HLCs to replicate the molecular pathways of PiZZ hepatocytes. Their developmental immaturity offers the opportunity to study the emergence, early onset and progression of A1ATD, even at stages in which the disease is still asymptomatic in most patients. 33 A good example of this is the photobleaching assays (Figs. 3, 4), which clearly demonstrated that ZZ-HLCs have significant loss of protein mobility, ER luminal communication and in some cases complete disruption of ER cisternae into isolated units. Whilst it has previously been demonstrated that even small deviations in protein diffusion rates can lead to biologically relevant changes, 34 our observations offer a novel insight into the subcellular anatomy of how this occurs in A1ATD. Such understanding is likely to be of clinical significance since cells harbouring fragmented ER inclusions have been shown to be more prone to apoptotic cell death and rely on replacement by globule-devoid hepatocytes with enhanced cell division rates. 35,36 However, whether A1AT polymer sequestration into globules is a protective cellular response or signals a state of injury in this setting (or even both) 36,37 is currently not known. Further in vitro studies, combined with human histological specimens and clinical data may allow us to deconstruct this problem by examining whether there is a link between the severity and number of ER inclusions, and an association between the severity and progression of liver disease. In a similarly important and clinically relevant manner, early identification of A1AT-deficient patients who are likely to progress to cancerous states represents an unmet but urgently required clinical challenge. Whilst AKR1B10 has been associated with hepatocellular carcinoma, 38,39 its co-localisation within cells containing polymers (Fig. 6C) suggests an association of this protein with A1ATD. The presence of AKR1B10 may therefore be a direct consequence of polymerised protein accumulation, characterising a subset of hepatocytes primed towards malignancy, making AKR1B10 a potential tissue biomarker for hepatocellular carcinoma risk stratification. Other proteins with known liver/cancer associations such as cysteine cathepsin family member CTSO 40,41 were also identified in our ZZ-HLC cells and may similarly provide prognostic and mechanistic insights into why individuals with A1ATD are at increased risk of tumourigenicity. 42 Understanding molecular pathways linking the polymerisation of Z A1AT to hepatic disease is central to the design of future therapeutics. Our genome and proteome analyses (Fig. 5) identified differentially regulated genes known to have an association with A1ATD (e.g. proteins of the ER-associated degradation machinery) as well as novel factors required to recognise and bind mutant glycoproteins, retro-translocate them from the ER lumen to the cytosol and tag them for proteasomal degradation. With the reassurance provided by this biological baseline, we also identified molecules that could offer insights into the pathophysiology of disease using transcriptomics and proteomics. The twenty highest-expressed, differentially upregulated genes in ZZ-HLCs were all of mitochondrial origin and associated with tasks of meeting higher energy demands. This manifested as physical disruption of the mitochondria (Fig. 6D) which although described previously, 43 is not yet comprehensively characterised with respect to its key molecular participants. 31 Our data provide new insight into the link between ER misfolding and mitochondrial disruption. Of particular interest, ER-stress response proteins ARMET (MANF), 44 OASIS (CREB3L1), 45 quality control chaperone proteins (HSP90, 46 Calreticulin, 47 Calnexin 48 ) and proinflammatory chemokines, cytokines and receptors (''inflammasome") known to mediate cytotoxic stress and inflammation (IL-1, IL-18, Caspase-4, TNF), 49,50 were upregulated in ZZ-HLCs. Interestingly, of the five most highly upregulated genes in ZZ-HLCs reported by Wilson et al., CASP4 and CFH also appeared in our list of 1,675 significant genes differentially regulated between ZZ-HLCs and RR-HLCs (FDR < 5%). 32 We therefore propose these targets, known also to mediate inter-organelle communication, as potentially being pivotal to disease and worthy of further research. Finally, this work also indicates that a more direct drug screening approach to target these novel pathways in A1ATD is merited.


extfieldsofstudy: Biology Medicine

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Induced pluripotent stem cell line from an atopic dermatitis patient heterozygous for c.2282del4 mutation in filaggrin: KCLi001-A

We have generated an induced pluripotent stem cell (iPSC) line KCLi001-A (iOP118) from a female atopic dermatitis (AD) patient, heterozygous for the loss-of-function mutation c.2282del4 in the filaggrin gene (FLG). Epidermal keratinocytes were reprogrammed using non-integrating Sendai virus vectors. The entire process of derivation and expansion of AD-iPSCs were performed under xeno-free culture conditions. Characterization of KCLi001-A line included molecular karyotyping, mutation screening using restriction enzyme digestion and Sanger sequencing, while pluripotency and differentiation potential were confirmed by expression of associated markers in vitro and by in vivo teratoma assay.

A B S T R A C T
We have generated an induced pluripotent stem cell (iPSC) line KCLi001-A (iOP118) from a female atopic dermatitis (AD) patient, heterozygous for the loss-offunction mutation c.2282del4 in the filaggrin gene (FLG). Epidermal keratinocytes were reprogrammed using non-integrating Sendai virus vectors. The entire process of derivation and expansion of AD-iPSCs were performed under xeno-free culture conditions. Characterization of KCLi001-A line included molecular karyotyping, mutation screening using restriction enzyme digestion and Sanger sequencing, while pluripotency and differentiation potential were confirmed by expression of associated markers in vitro and by in vivo teratoma assay.
Resource Resource utility Generation of a library of human iPSC lines with the most common variants in the FLG gene can be efficiently used to construct highly specific in vitro 3D skin models (Petrova et al., 2014) for drug discovery towards novel personalized therapies in AD.

Resource details
AD or eczema is an incurable, non-contagious, extensive inflammatory and extremely pruritic chronical cutaneous disorder. AD is one of the most common skin diseases which affects up to 20% of children and approximately 3% of adults worldwide, while its (caption on next page) prevalence is continuously increasing, particularly in underdeveloped countries (Asher et al., 2006). Several loss-of-function mutations within FLG exon 3, including c.2282del4 variant, are considered to be the most significant risk factors for atopic dermatitis in the European population (Palmer et al., 2006). The epidermal keratinocytes derived from a female AD patient who is heterozygous for c.2282del4, were reprogrammed into iPSCs following previously established protocol with genome non-integrating Sendai virus (SeV) vectors (Miere et al., 2016a). Three weeks post-transduction colonies with a typical morphology of pluripotent stem cells appeared and were selected to establish feeder-free iPSC clones (Fig. 1A). After ten passages, the elimination of the SeV vectors was confirmed in the KCLi001-A cell line by RT-PCR using specific primers (Fig. 1B). The clones were screened with restriction enzyme digestion and we have verified that the AD-related mutation (NM_002016.1:c.2282del4) was retained in the iPSCs. This finding was also confirmed independently by Sanger sequencing (Fig. 1C). Endogenous expression of pluripotency-related molecular markers (TRA-1-60, TRA-1-81, OCT4, NANOG) in the iPSCs was assessed by double immunofluorescence technique (Fig. 1D). Furthermore, undifferentiated colonies were also positive for alkaline phosphatase (AP) (Fig. 1D). Differentiation capacity of the KCLi001-A cells into three germ layers was determined by specific immunofluorescence staining of AFP (liver, endoderm), ACTA2 (cardiac muscle, mesoderm), and TUBB3 (neurons, ectoderm) in vitro ( Fig. 1E), as well as in vivo through a teratoma formation assay. All three germ layers, ectoderm, mesoderm, and endoderm, were present in the teratoma, as demonstrated by immunohistochemical analysis (Fig. 1F).
Since our aim is to use the line for modeling AD in vitro, we tested differentiation of the KCLi001-A iPSCs into epidermal keratinocytes (Petrova et al., 2014). The cells expressed keratinocyte-specific markers -keratins 14 and 18 (KRT14, KRT18), and isoform of TP63 known as ΔNp63 within three weeks in culture as expected (Fig. 1G).
Examination of the genomic integrity of our AD-iPSC line using array CGH after more than twenty passages showed a normal female karyotype (46, XX), whereas smaller imbalances have not been excluded (submitted in archive with journal). Taken together, these results prove that we have successfully produced a stable AD patient specific iPSC line which can provide a powerful tool for: 1) developing the first iPSC-derived 3D in vitro AD-human skin equivalents (HSE); 2) deciphering the molecular mechanisms of the disease; 3) innovative drug screening platform in atopic dermatitis. (Table 1). Fig. 1. Characterization of KCLi001-A line. A, The colonies display typical morphology of pluripotent stem cells under feeder-dependent and feeder-free conditions. B, The absence of 181 bp positive SeV band at 45 and 60 days of culture indicates that the cells are SeV free. β-actin 455 bp band serves as an internal control. C, The line is heterozygous for c.2282del4 mutation in FLG as indicated with restriction enzyme digestion and Sanger sequencing of PCR product spanning the mutation site. Mutation generates a site for DraIII that is not present in wildtype allele. The enzyme digestion cuts 811 bp product in mutated allele into 667 bp and 134 bp, whereas 811 bp remains intact in wildtype. hESC line KCL038 is used as a negative control. D, Pluripotency markers. Alkaline phosphatase activity (AP) was restricted to the iPSC colony (green) growing on AP-negative feeder cells. Both iPSC colony and feeders are positive for actin (phalloidin) staining (red). iPSC colonies are positive for POU5F1 (red), TRA-1-81 (green), NANOG (red) and TRA-1-60 (green) pluripotency markers. E, Spontaneous differentiation in vitro. The cells can differentiate into all three germ layers as demonstrated with markers specific for ectoderm (TUBB3), endoderm (AFP) and mesoderm (ACTA2). Nuclei are visualized with Hoechst 33342. F, Spontaneous differentiation in vivo. Gross anatomy and staining for human-specific MTCO2 marker iPSC indicated that the teratoma is incapsulated and did not invade surrounding tissues. Teratoma contained cells from all three germ layers as demonstrated with markers specific for ectoderm (TUBB3, GFAP), endoderm (AFP, GATA4) and mesoderm (AB-PAS, DESMIN). G, Directed differentiation into keratinocytes. The cells expressed keratinocyte-specific markers KRT14, KRT18, TP63 (ΔNp63) in time-dependent manner.

Epidermal keratinocytes reprogramming
Patient keratinocytes of passage 3 were transduced with genome integration-free SeV virus kit (CytoTune 2.0, Life Technologies) as described (Miere et al., 2016a). Clonal selection of fully reprogrammed cells was performed manually by picking individual clones with hESClike appearance ( Table 1). The iPSCs under feeder-free culture conditions were maintained on Matrigel (BD Biosciences) in TeSR2 medium (STEMCELL Technologies).

FLG mutation verification
Genomic DNA was extracted from KCLi001-A cells using DNeasy Blood & Tissue Kits (Qiagen) and samples were verified independently by restriction enzyme digestion and Sanger sequencing using the primers described in Table 2. A 311 bp sequence in exon 3 of FLG was amplified and Eurofins Genomics provided DNA sequencing service. Mutation c. 2282del4 creates a new DraIII site, which was used to screen short, highly specific 811 bp FLG gene fragment, as described in more detail previously (Palmer et al., 2006).

Reverse transcription PCR analysis of SeV vectors
Total RNA was isolated from the cells 7, 45 and 60 days posttransduction (RNeasy mini Kit, Qiagen). SeV specific primers were used to assess the presence of remaining Sendai virus vectors (Table 2). RT-PCR for the detection of SeV transgenes was carried out using the Su-perScript IV First-strand cDNA synthesis reaction kit (Invitrogen).

Pluripotency markers
The pluripotency status of KCLi001-A line was evaluated by immunostaining for three germ layer markers in spontaneously differentiated cells (Table 1) as previously described (Petrova et al., 2014).

Alkaline phosphatase activity
Emerging iPSCs were analyzed for alkaline phosphatase activity by AP Live Stain (Thermo Fisher). After live staining, iPSCs were washed and fixed, and cytoskeletal actin filaments have been contrasted by labelling with rhodamine phalloidin (Molecular Probes).

Spontaneuous differentiation into three germ layers
To test the differentiation capacity of our iPSC line, in vitro embryonic body formation, as well as in vivo conventional teratoma assay were assessed (Table 1), as previously described (Petrova et al., 2014).

Molecular karyotyping
Array comparative genomic hybridization (aCGH) and short tandem repeat (STR) analysis of 17 STR loci were conducted at Viapath Genetics Centre.

Mycoplasma contamination detection
The absence of mycoplasma contamination was detected using LookOut® Mycoplasma PCR Detection Kit (Sigma-Aldrich).