Ultraviolet Radiation Signaling through TLR4/MyD88 Constrains DNA Repair and Plays a Role in Cutaneous Immunosuppression This information is current as of September 25, 2021. Erin Harberts, Hua Zhou, Rita Fishelevich, Juan Liu and Anthony A. Gaspari J Immunol published online 25 February 2015 http://www.jimmunol.org/content/early/2015/02/24/jimmun ol.1402583 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 25, 2015, doi:10.4049/jimmunol.1402583 The Journal of Immunology

Ultraviolet Radiation Signaling through TLR4/MyD88 Constrains DNA Repair and Plays a Role in Cutaneous Immunosuppression

Erin Harberts,*,†,1 Hua Zhou,†,1 Rita Fishelevich,† Juan Liu,† and Anthony A. Gaspari*,†,‡

UV radiation (UVR) induces DNA damage, leading to the accumulation of mutations in epidermal keratinocytes and immunosup- pression, which contribute to the development of nonmelanoma skin cancer. We reported previously that the TLR4–MyD88 signaling axis is necessary for UV-induced apoptosis. In the dinitrofluorobenzene contact hypersensitivity model, UV-irradiated MyD88-deficient (MyD882/2) C57BL/6 mice had intact ear swelling, exaggerated inflammation, and higher levels of dinitrofluorobenzene-specific IgG2a compared with wild-type (WT) mice. Even with normal UV-induced, dendritic cell migration, DNA damage in the local lymph nodes was less pronounced in MyD882/2 mice compared with WT mice. Cultured, UV-irradiated Downloaded from WT APCs showed cleavage (inactivation) of the DNA damage–recognition molecule PARP, whereas PARP persisted in MyD882/2 and TLR42/2 APCs. Epidermal DNA from in vivo UV-irradiated MyD882/2 mice had an increased resolution rate of cyclobutane pyrimidine dimers. Both in vitro treatment of MyD882/2 APCs with and intradermal in vivo injections of PARP inhibitor, PJ-34, caused WT-level cyclobutane pyrimidine dimer repair. Lymphoblasts deficient in DNA repair (derived from a xeroderma pig- mentosum group A patient) failed to augment DNA repair after MyD88 knockdown after UVR, in contrast to lymphoblasts from a healthy control. These data suggest that interference with the TLR4/MyD88 pathway may be a useful tool in promoting DNA http://www.jimmunol.org/ repair and maintaining immune responses following UVR-induced damage. The Journal of Immunology, 2015, 194: 000–000.

uman skin is ubiquitously exposed to UV radiation CBPDs are the main type of DNA damage associated mainly (UVR). The longer wavelengths of UVA are able to with UVB irradiation. These dimers require the nucleotide excision H penetrate down through the epidermis to the dermis and repair (NER) complex to be excised and corrected; once this process associated connective tissue, whereas the shorter UVB wavelengths occurs, the integrity of the affected DNA is maintained (11). Another are absorbed by epidermal cells, primarily keratinocytes (KCs) (1). type of DNA damage–causing radiation, gamma irradiation, results UVR has both positive and negative effects on human health. It is in double-strand breaks that cannot be repaired using a comple-

responsible for the biosynthesis of vitamin D3, can stimulate the mentary template; it is associated with mutagenesis and chromo- by guest on September 25, 2021 production of photoprotective melanin (2–5), and is used thera- some abnormalities that often lead to cell death. Repair of both UV peutically to treat inflammatory skin diseases, such as psoriasis, and gamma irradiation damage was shown to be altered in the vitiligo, localized scleroderma, and atopic dermatitis (6–8). At the presence of endosomal TLR agonists (12, 13), implying that both same time, UVR has many negative effects, most notably that it is the NER and double-strand breaks repair complex may be affected directly absorbed by cellular DNA, resulting in cyclobutane py- by the status of TLR activation. In this study, we focus on DNA rimidine dimer (CBPD) mutations that can lead to carcinogenesis damage caused by UVR in the form of CBPDs, which requires NER of resident epithelial cells. Contributing to the carcinogenic effects machinery to be excised and repaired. of UVR is the fact that it is also a known potent immunosup- The NER pathway is responsible for excising and repairing pressant, rendering the recipient less able to stimulate an immune CBPD mutations after UVR, and it is a necessary mechanism that response against cancerous cells that may otherwise be destroyed is used to repair a wide range of DNA lesions (14). NER can be (9, 10). categorized into two subpathways: global genome repair (GGR) and transcription-coupled repair (15, 16). The removal of UV-induced *Department of Microbiology and Immunology, University of Maryland, Baltimore, CBPDs falls into the category of GGR, whereas transcription-coupled MD 21201; †Department of Dermatology, University of Maryland, Baltimore, MD repair ensures that an intact and accurate genome is maintained and 21201; and ‡Research Service, Baltimore Veterans Affairs Medical Center, Balti- more, MD 21201 passed on during cell division (17). GGR begins when a DNA 1E.H. and H.Z. contributed equally to this work. damage–sensing molecule, such as PARP or DDB, recognizes a DNA lesion. Xeroderma pigmentosum (XP) family are Received for publication October 9, 2014. Accepted for publication January 28, 2015. then recruited to the damaged bp. These XP proteins begin to re- This work was supported by Veterans Affairs Merit Award 1I01-Bx0004405-01A2 pair the lesion by unwinding the surrounding DNA, stabilizing the (to A.A.G.). now ssDNA, and excising the mutated bp. A DNA polymerase and Address correspondence and reprint requests to Dr. Anthony A. Gaspari, Department DNA ligase are recruited to fill in the necessary bp that are com- of Dermatology, University of Maryland, 419 West Redwood Street, Suite 240, plementary to the intact template strand (1). The importance of an Baltimore, MD 21201. E-mail address: [email protected] intact NER pathway is highlighted by XP patients (18); they are Abbreviations used in this article: CBPD, cyclobutane pyridimine dimer; CHS, con- tact hypersensitivity; DNFB, dinitrofluorobenzene; GGR, global genome repair; KC, deficient in one or more of the XP family proteins and present with keratinocyte; LC, Langerhans cell; LLN, local lymph node; NER, nucleotide excision multiple basal cell carcinomas and other skin malignancies at an repair; PAR, poly(ADP-ribose); PM, peritoneal macrophage; siRNA, small interfer- extremely young age (19, 20). Often, XP patients have to be com- ing RNA; UVR, UV radiation; WT, wild-type; XP, xeroderma pigmentosum. pletely protected from exposure to sunlight and other sources of Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 radiation to avoid skin cancers.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402583 2 IMMUNE MAINTENANCE AND DNA REPAIR AFTER UVR IN MyD882/2 MICE

Interestingly, it was found that mice deficient in the innate immune CA). CBPDs were measured in duplicate and in a random order using signaling pathway TLR4 are resistant to cutaneous UV immuno- an OxiSelect Cellular UV-Induced DNA Damage ELISA Kit (CPD) suppression (21). Expression of TLR2 and TLR4 also were shown (Cell Biolabs), according to the manufacturer’s instructions. Briefly, the extracted DNA was heat denatured and coated onto microtiter plates pro- to be increased in epidermal cells following UVR, and an observed vided by the manufacturer of the ELISA. UV-irradiated calf thymus DNA increase in immune signaling molecules, such as MAPK and was provided by the manufacturer, and it served as a set of internal standards NF-kB, is dependent on this TLR expression (22). Previously, we for each assay. The plates were probed for DNA damage using a mAb, found that the TLR4/MyD88 signaling pathway is necessary for followed by a biotinylated secondary reagent and subsequent color devel- opment. OD was measured at 450 nm. UV-induced apoptosis and that, without this intact pathway, UV- induced cell death is skewed to an inflammatory, necroptotic cell PARP-activity ELISA death (23). We hypothesized that, in addition to TLR4-deficient mice, PARP activity was measured in histone-coated strip wells using the High MyD88-deficient mice are resistant to UV-induced immunosup- Throughput (HT) Colorimetric PARP/Apoptosis ELISA Kit (Trevigen), pression and that this phenotype is a result of increased resolution following the manufacturer’s procedures. This ELISA measures the in- of UV-induced DNA damage to skin-resident APCs. corporation of biotinylated poly(ADP-ribose) (PAR) into histone proteins after samples are incubated with anti-PAR Ab and then HRP-conjugated secondary Ab (anti-mouse IgG-HRP). Absorbance was read at 450 nm. Materials and Methods PARP activity for each sample was calculated from a standard curve run in Experimental mice duplicates within each experiment. Mouse experiments were approved by the University of Maryland Insti- DNA dot blot tutional Animal Care and Use Committee. Fourteen- to sixteen-week-old female mice were used in all experiments. Wild-type (WT) C57BL/6 Whole-genomic DNA was extracted using a DNeasy Blood and Tissue Kit Downloaded from mice (The Jackson Laboratory; strain C57BL/6J; stock no. 000664), (QIAGEN), following the manufacturer’s protocol. For DNA dot blots, 500 MyD882/2 mice on a C57BL/6 background (The Jackson Laboratory; ng heat-denatured DNA was spotted onto a nitrocellulose membrane and strain B6.129P2(SJL)-Myd88tm1.1Defr/J; stock no. 009088), and TLR42/2 allowed to dry at room temperature for 1 h and then baked at 80˚C for 20 mice on a C57BL/6 background (The Jackson Laboratory; strain min. The membrane was probed with an anti-CBPD Ab (Cosmo Bio; cat. B6.B10ScN-Tlr4lps-del/JthJ; stock no. 007227) were housed in the Baltimore no. NMDND001) and developed using secondary Ab and reagents from Veterans Affairs Medical Center animal facility and used in the experi- the WesternBreeze Kit (Invitrogen; cat. no. WB7104), according to the ments described. Where indicated, hair was removed from the abdomen manufacturer’s protocol. Image density quantification was performed using of mice using electric clippers. When ears were exposed to UVR, mice Image-Pro Plus software version 4.5.1.29 (Media Cybernetics). We nor- http://www.jimmunol.org/ were anesthetized by i.p. injection of a ketamine (80 mg/kg)/xylazine malized DNA to the amount spotted onto membrane (500 ng, as measured (10 mg/kg) mixture, and ears were taped down to a surface so only the by NanoDrop ND1000 spectrophotometer) versus the OD, as measured dorsal side was exposed to UVR. All in vivo experiments were repeated by our image-analysis software. three times to assure reproducibility. Immunohistochemistry UV light source 2 2 Formalin-fixed WT and MyD88 / mouse ear biopsies were embedded in Groups of four mice at a time or cell culture plates with the media removed paraffin, sectioned, and stained with H&E. Ear sections were visualized were irradiated with a Panasol II Two-Foot Broad Band UVB light source, using a Nikon Eclipse E600 microscope, and the images were documented equipped with eight UVB broad band lamps (National Biologic). We using the SPOT imaging system (Diagnostic Instruments, Sterling Heights, calibrated the UVB dosing with National Biologic measuring devices—the MI). To harvest mouse epidermal sheets, ears were obtained 24 h after by guest on September 25, 2021 2 UVB 500C radiometer (measures output in the 290–320 nm range) and the irradiation of one side of the ear with 100 mJ/cm UVR. The irradiated UVA 400C radiometer (measures output in the 320–400 nm range)—to side was split from the control side and incubated with Dispase (BD determine the time necessary to deliver the desired doses of UVB. Cell Biosciences; cat. no. 354235) at 4˚C for 2 h. The epidermis was separated monolayers or animal skin were subjected to UVR at a distance of 12 in and fixed in ice-cold methanol for 20 min. The epidermal sheets were from the light source. stained with FITC-conjugated anti-mouse I-A/I-E clone M5/114.15.2 (BioLegend; cat. no. 107605) and mounted onto slides with DAPI- Contact sensitization model containing mounting medium for fluorescent visualization. Epidermal The dinitrofluorobenzene (DNFB) contact sensitization model was carried sheets were visualized using a Zeiss LSM700 microscope. out as previously described (24). Briefly, hair was removed from the ab- Cell culture and reagents domen of WT or MyD882/2 mice with electric clippers, and they were exposed to an immunosuppressive dose of UVR, 70 mJ/cm2, for four PMs obtained by thioglycollate administration in mouse abdomen were consecutive days (days 23 through 0). On the last day of UVR, day-0 and purified by short plastic adherence in culture media (DMEM supplemented day-1 mice were sensitized with 20 ml 0.5% DNFB on the abdomen. On with 20% FCS and 100 mg/ml Pen-Strep), washed three times, and cul- day 5, baseline ear thickness measurements were taken, and immune re- tured, as described, for experimental use. Primary human KCs were de- action was elicited by painting 20 ml 2% DNFB onto the ear. Twenty-four rived, as previously described, from neonatal foreskins (26). Use of human hours after elicitation, day-6 ear thickness was measured, and the amount samples was approved by the University of Maryland Medical School of swelling was determined by subtracting the baseline thickness. Institutional Review Board. KCs were cultured in Epi-Life growth medium supplemented with epidermal and pituitary extracts (Cascade Cytokine ELISA Biologics, Portland, OR). Lymph node single-cell suspensions were made A Mouse IFN-g ELISA kit was purchased from BioLegend (cat. no. 575309), by passing lymph node tissue through a 70-mm nylon cell strainer; cells and samples were run according to the manufacturer’s protocol for cell culture were washed twice with PBS and plated at a concentration of 1 million supernatants. DNFB-specific IgG2a serum ELISA was run as previously de- cells/ml RPMI 1640 supplemented with 10% FCS and 100 mg/ml Pen-Strep. scribed (25). Briefly, serum was collected from contact hypersensitivity (CHS) For some experiments, PJ-34 (Sigma-Aldrich; cat. no. P4365) or Dynabeads mice 21 d after initial sensitization. ELISA plates (Corning Glass Works) Mouse T-Activator CD3/CD28 (Life Technologies; cat. no. 11456D) was were coated with 10 mg trinitrophenyl-OVA or DNP-OVA (Sigma-Aldrich, added to cell culture medium at the indicated concentration. All in vitro St Louis, MO), and the plates were blocked with 0.25% BSA in Tween/PBS. experiments were repeated three times to assure reproducibility. Sera were plated in triplicate to allow for Ab binding. Unbound Ab was Small interfering RNA knockdown, RNA extraction, and washed off, and biotinylated anti-mouse IgG2a (2 mg/ml; BD Pharmingen, San Diego, CA) was added. O-phenylenediamine dihydrochloride (Sigma- quantitative PCR Aldrich) was added, according to the manufacturer’s instructions, for color Expression of MyD88 was knocked down in a pool of primary KCs from development. All ELISA plates were read on a Bio-Rad benchmark micro- three donors using small interfering RNA (siRNA; QIAGEN; cat. no. plate reader (Bio-Rad, Hercules, CA), and OD was read at 450 nm. SI00300909) transfected into the cells with an Amaxa Human Keratinocyte DNA-damage ELISA Nucleofector Kit, following the manufacturer’s instructions. Scrambled siRNA was used as a control (QIAGEN; cat. no. 1022076). RNA extraction, Genomic DNA was extracted and purified from mouse peritoneal macro- cDNA synthesis kit, and real-time PCR were run according to previously phages (PMs) and human KCs using a DNeasy Kit (QIAGEN, Valencia, published methods (27, 28). Relative levels of MyD88 mRNA were The Journal of Immunology 3 normalized to 18s mRNA. Quantitative real-time PCR was performed us- ing QPCR SYBR Green Supermix (QIAGEN). The following primers were used in the reactions: MyD88 (RefSeq accession no. NM_002468.4) (reference position: 979); 18s (RefSeq accession no. X03205.1) (reference position: 1447); IFN-g (RefSeq accession no. NM_008337.3); IL-12p40 (RefSeq accession no. NM_008352.2); IL-23p19 (RefSeq accession no. NM_031252.2); Foxp3 (RefSeq accession no. NM_054039.1); IL-4 (RefSeq accession no. NM_021283.1); IL-10 (RefSeq accession no. NM_010548.1); 18s (RefSeq accession no. X03205); XPA (RefSeq acces- sion no. NM_011728); XPC (RefSeq accession no. NM_009531.2); Xab2 (RefSeq accession no. NM_026653.1); Rad23b (RefSeq accession no. NM_009011.4); Ddb1 (RefSeq accession no. NM_015735); and Ddb2 (RefSeq accession no. NM_028119.4). All of these primers were purchased from SABiosciences (QIAGEN). Western blots concentrations in cell lysates made using RIPA buffer were mea- sured using a DC Protein Assay (Bio-Rad). For Western blots, 10 mg protein/lane was loaded onto a NuPAGE 10% Bis-Tris gel (Life Tech- nologies cat. no. NP0301), electrophoresed, and transferred to nitrocellu- lose membranes using the Novex mini-gel system (Invitrogen). Membranes were probed with one of the primary Abs—anti–b-actin polyclonal Ab (; cat. no. 4967), anti-MyD88 mAb (Cell Signaling; cat. no. D80F5), Downloaded from or anti-PARP mAb (Cell Signaling; cat. no. 46D11)—and developed using the WesternBreeze kit (Invitrogen; cat. no. WB7104 or WB7106), accord- ing to the manufacturer’s protocol. Statistical analysis Quantitative data were analyzed for statistically significant differences, between groups of n replicates, as described in the figure legends, using http://www.jimmunol.org/ GraphPad InStat software (GraphPad, La Jolla, CA). ANOVA analysis was performed for the quantitative data and p , 0.05 was considered signifi- cant. Data are mean 6 SD from three independent experiments.

Results 2/2 2/2 FIGURE 1. MyD88 mice are resistant to UVR-induced immune MyD88 mice are resistant to immunosuppressive effects 2/2 of UVR suppression. WT and MyD88 mice were studied using a standard CHS model (DNFB as the hapten, see Materials and Methods). (A) Ear thick- To determine how MyD88 induces UV-induced immunosuppression, ness was measured before and 24 h after elicitation, and the change in ear we studied MyD882/2 mice using the DNFB CHS model. Twenty- swelling induced by DNFB challenge is shown. (B) Ear sections from WT by guest on September 25, 2021 2 2 four hours after elicitation, both WT and MyD882/2 mice that were and MyD88 / sen and UV+sen groups were stained with H&E and ex- sensitized with DNFB exhibited a robust ear swelling reaction, amined for histologic evidence of inflammation. A representative histo- whereas mice elicited with DNFB only showed a low level of logic ear section from each group is depicted (n = 7). **p = 0.0096, p , baseline ear swelling. WT mice that were subjected to UVR before *** 0.0001. DNFB sensitization showed a significant (p , 0.0001) reduction in ear swelling compared with WT mice that were not exposed to shown). Single-cell suspensions made from LLNs of the same ir- 2 2 UVR. However, ear swelling in MyD882/2 mice that were exposed radiated mice showed that MyD88 / mice are capable of pro- to the same UV doses was not significantly diminished and was ducing significantly more (p = 0.0094) IFN-g when stimulated significantly (p = 0.0096) greater than the paired UVR WT group of with a mitogen compared with WT mice (Fig. 2C). Although WT 2 2 mice (Fig. 1A). H&E-stained ear sections from unirradiated and and MyD88 / mice have the same amount of APC emigration, 2 2 UV-irradiated MyD882/2 mice showed similar histologic mor- the IFN-g production capability of LLN cells from MyD88 / phology, with significant edema and cellular infiltrates. Although mice is higher. the ear sections from unirradiated WT mice showed similar histo- To further support this conclusion, serum was obtained from each logic signs of an intact immune response, the ear sections from UV- mouse on day 21 after sensitization, and DNFB-specific IgG2a, an irradiated WT mice showed limited edema and decreased histologic Ab response that is dependent on Th1 lymphocyte–derived IFN-g signs of inflammation (Fig. 1B). Thus, after acute exposures to four (21), was measured. There was significantly (p = 0.0127) less consecutive doses of UVR, MyD882/2 mice are resistant to im- DNFB-specific IgG2a in the serum of UV-irradiated WT mice than 2 2 mune suppression compared with WT mice. Further studies will be in the serum from UV-irradiated MyD88 / animals (Fig. 2D). necessary to determine the level of resistance of MyD882/2 mice DNFB is known to elicit primarily a Th1 response, which results in to immune suppression in the context of chronic UVR exposures. the production of IgG2a; as expected, a significant DNFB-specific It is known that UVR depletes epidermal Langerhans cells (LCs) IgG1 response was not observed (data not shown). However, there by two main mechanisms: by causing cell death and migration to the was a significant difference with regard to DNFB-specific IgG2a. 2 2 local lymph node (LLN) (29). To address the possibility that UVR We could not detect IFN-g in the serum of WT or MyD88 / mice is causing emigration of APCs to LLNs at different rates in WT and in the steady-state or following sensitization (S) and elicitation (E) MyD882/2 animals, epidermal sheets were stained for MHC class or UV+S+E (data not shown). II, which stains epidermal LCs (30). Twenty-four hours after 100 2 The microenvironment of the LLN promotes immune mJ/cm UVR, similar levels of APC emigration from the epidermis 2/2 were observed in WT and MyD882/2 mice (Fig. 2A, 2B). Exam- competence in UV-irradiated MyD88 mice ination of LC densities at 96 h after UVR exposures revealed A fundamental concept of UVR-induced cutaneous immune similar levels of depletion in WT and MyD882/2 mice (data not suppression is the arrival of APCs with DNA damage in the LLN, 4 IMMUNE MAINTENANCE AND DNA REPAIR AFTER UVR IN MyD882/2 MICE

FIGURE 2. UV-induced emigration of APCs from epidermis is maintained and DNA damage is de- creased in LLNs of MyD882/2 mice. (A) Epidermal sheets from ears subjected to 100 mJ/cm2 UVR or paired skin from the unirradiated side of the same ear were stained with MHC class-II Ab (clone M5/ 114.15.2). Exposure to UVR caused a similar de- pletion (emigration) of APCs from epidermal sheets 24 h after UVR in WT and MyD882/2 mice. Rep- resentative images are shown from each group (n = 3/group). (B) Quantification of APCs/field (0.01 mm2). Downloaded from (C) Single-cell suspensions from cervical lymph nodes harvested 24 h after ears were subjected to UVR and equal numbers of lymph node cells (105/well) were polyclonally stimulated with CD3/ CD28-coated beads for 72 h, and supernatants were analyzed for release of IFN-g using a commercially available ELISA (n = 3/group). Data are mean 6 SD. http://www.jimmunol.org/ (D) Using the same mice as in Fig. 1A, serum levels of DNFB-specific IgG2a were measured using an ELISA 21 d after sensitization with DNFB (see Materials and Methods). Data are mean 6 SD. *p , 0.05. by guest on September 25, 2021

which is thought to play a role in the generation of regulatory Increased repair of UV-induced DNA damage in both mouse T cells (31). Therefore, we examined whether UVR, skin-derived and human models of MyD88 deficiency 2/2 MyD88 APCs arrived in the LLN with less DNA damage The reduction in DNA damage in the LLNs of MyD882/2 mice led compared with UVR, skin-derived WT APCs. Our initial flow + + us to wonder whether this phenotype also can be observed as cytometry analyses of APC populations (MHC-II , CD11c ), be- increased resolution of CBPDs in UVR skin. To test this hy- fore or 24 h after UVR, were not significantly different in the LLN pothesis, serial skin biopsy specimens were taken from the ab- 2/2 of WT or MyD88 mice (data not shown). Twenty-four and domen of UV-irradiated WT and MyD882/2 mice. Immediately forty-eight hours after a single exposure to UVR, a significantly after being exposed to 70 mJ/cm2 UVR, both strains of mice , lower (p 0.01) amount of DNA damage was detected in the showed similar levels of CBPDs in epidermal DNA. However, 2/2 LLNs of MyD88 mice compared with WT mice, as measured 24 h after UVR, levels of CBPDs in epidermal DNA from with a quantitative DNA damage ELISA (Fig. 3A). Additionally, MyD882/2 mice were significantly (p , 0.05) lower than in WT the expression of a family of associated with sensing DNA epidermal DNA (Fig. 4A). These data suggest that the skin of damage (32) was elevated in the LLNs of UVR-irradiated WT mice without the MyD88 signaling pathway is able to resolve 2/2 mice compared with MyD88 mice (Fig. 3B). As a result of UVR-induced CBPDs more efficiently than is WT mouse skin. A fewer DNA-damaged APCs arriving in the LLN after UVB ex- similar phenotype of increased resolution of CBPDs was observed 2 2 posure in MyD88 / mice, there was a relatively higher level of in TLR42/2 mice (data not shown). expression of genes associated with immune competence (IFN-g, To confirm that this phenotype also can be observed in human IL-12, and IL-23) and a lower level of genes associated with im- skin cells, siRNA knockdown of MyD88 in human primary KCs mune suppression compared with WT mice (Fig. 3C). By dem- derived from surgical specimens from three healthy donors was onstrating that there is a functional Th1 skewing environment in performed. A .80% knockdown of MyD88 was achieved at both 2 2 the LLN of MyD88 / mice, these data provide mechanistic the mRNA and protein levels, as measured by RT-PCR at 24 h 2 2 insights as to why CHS is intact in MyD88 / mice compared with after transfection and as measured by Western blot at 48 h after WT mice. transfection, respectively (Fig. 4B). KCs were subjected to UVR The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

FIGURE 4. Increased DNA repair of UV-induced CBPDs in vivo and in vitro in the absence of an intact MyD88 signaling pathway. (A) Epi- dermal DNA was extracted from abdominal skin biopsies from WT and MyD882/2 mice (n = 3/group) that were subjected to 70 mJ/cm2 UVR. A CBPD-specific ELISA was used to measure resolution of DNA damage over time. UV-0 h represents skin removal immediately (seconds) after UVR exposure. Data are mean 6 SD of densitometry from the 0-h time point. *p , 0.05, **p , 0.01. (B) A pool of human primary KCs from three patient samples were transfected with either a MyD88-specific by guest on September 25, 2021 siRNA or a scrambled control siRNA. RT-PCR for MyD88 was performed on cDNA made from the transfected KCs 24 h after transfection. A .80% FIGURE 3. The LLN environment in MyD882/2 mice promotes im- knockdown of MyD88 mRNA was achieved. MyD88 transcript levels were mune competence. (A) Ears of WT and MyD882/2 mice (n = 4 mice/ normalized to 18s RNA. Western blot with lysates made 48 h after . C group) were irradiated or not with 100 mJ/cm2 UVR, whole-genomic DNA transfection. A 80% knockdown of MyD88 protein was achieved. ( ) was extracted from the cervical lymph nodes 24 and 48 h later, and CBPDs Forty-eight hours after transfection, the KCs were irradiated with the in- in genomic DNA were measured using a CBPD-specific ELISA (see dicated doses of UVR, and CBPD levels were measured by ELISA 24 h later (see Materials and Methods). Samples in which MyD88 was knocked Materials and Methods). In another set of mice, mRNA was extracted from 2 the LLN, and expression (normalized to 18s RNA) was assayed using down showed a reduction in CBPD 24 h after 25 mJ/cm UVB compared quantitative PCR. Data are mean 6 SD. (B) DNA damage–sensing genes. with control transfected KCs. A representative experiment of four repli- 6 (C) Cytokine genes in the LLN. Data are the fold-change relative to the cates with similar results is shown. Data are mean SD (n = 3/group). , LLN from unirradiated, control mice (WT or MyD882/2)(n = 2/group). *p 0.05, ANOVA. Data are mean 6 SD. **p , 0.01, ANOVA. repair is no longer necessary because the cell is dying (31). 2 2 48 h after transfection, DNA was extracted 24 h after UVR, and Consistent with our previous finding that cells of MyD88 / and 2 2 a DNA damage ELISA was run to evaluate levels of CBPD after TLR4 / mice do not undergo efficient apoptosis after UVR increasing doses of UVR. KCs with MyD88 knocked down compared with WT mice (23), we found that PARP is cleaved in exhibited significantly less CBPD 24 h after 25 mJ/cm2 UVR a time-dependent manner after UVR in WT PMs; however, this 2 2 compared with control transfected KCs (Fig. 4C). MyD88 siRNA- cleavage is significantly decreased in MyD88 / PMs (Fig. 5A). 2 2 treated human KCs, similar to MyD882/2 mice, are able to repair To determine whether PARP activity is maintained in MyD88 / UV-induced DNA damage at a faster rate than are paired control- PMs, we used a functional PARP assay (ELISA, see Materials and treated KCs. These data suggest that loss of the MyD88-dependent Methods) to compare PARP activity in PMs after increasing doses 2 2 signaling pathway has similar effects on DNA repair of UVR- of UVR. There was maintenance of PARP activity in MyD88 / induced damage in KCs and APCs. PMs after increasing doses of UVR (Fig. 5B), in contrast to WT PMs, which exhibited a dose-dependent loss of PARP activity 2/2 PARP is required for increased DNA repair in MyD88 mice after UVR exposure. To investigate whether the DNA damage– using an in vitro model recognition molecule PARP is necessary for the increased resolution DNA damage recognition is crucial for effective repair and reso- of dimers observed in MyD88-deficient models, the water-soluble lution of lesions. Damage-recognition molecules, such as PARP, PARP inhibitor, PJ-34, was used in PM culture experiments. In the are cleaved by caspases during apoptosis, a condition when DNA presence of PJ-34, MyD882/2 PMs and epidermal cells lost their 6 IMMUNE MAINTENANCE AND DNA REPAIR AFTER UVR IN MyD882/2 MICE ability to resolve CBPDs at a higher rate, whereas PJ-34 treatment dependent on CBPD recognition by PARP and repair by NER had no effect on the resolution of CBPDs in WT PMs or epidermal machinery. This repair is activated by UVR in the absence of the cells (Fig. 5C, 5D). Consistent with the DNA repair data, PJ-34 apoptosis-initiating TLR4/MyD88 signaling pathway. also interfered with the enhanced survival that was observed in MyD882/2 PMs, but it had no effect on the survival of WT PMs Discussion (data not shown). These data show that PARP is necessary for Apoptosis initiated by UVR has long been thought of as a protective increased DNA repair in vitro. mechanism that enables the skin to get rid of damaged cells in a way that does not cause potentially damaging inflammation (34). NER machinery is required for increased DNA repair in Apoptotic cell fragments cause immune-suppressive reactions in MyD88-deficient cells APCs (35, 36), but their role in UVR-induced immune suppres- In addition to damage recognition by PARP, we investigated sion has not been investigated thoroughly. We demonstrated pre- whether the observed enhanced resolution of CBPDs is dependent viously that cell death in the skin in vivo, as well as in cultured on NER machinery, the predominant repair pathway for CBPDs APCs in vitro, is nonapoptotic; instead, it proceeds by a nec- (33). It was shown that bone marrow–derived cells accumulate roptotic pathway (23). The immunosuppressive effects of UVR CBPDs after UVR similarly to epithelial cells that are routinely have far-reaching effects on the immune system; by allowing cells physiologically exposed to sunlight (29). Because bone marrow– to die without inflammation, apoptosis caused by signaling through derived cells respond similarly, we were able to use a lymphoblast MyD88 may substantially contribute to this impaired immune cell line from an XP patient who is deficient in XPA, one of the surveillance. To substantiate this hypothesis we carried out CHS 2 2 critical NER molecules, in our experiments. A .75% knockdown experiments in WT and MyD88 / mice and, as hypothesized, Downloaded from of MyD88 was achieved, by siRNA transfection, at both the found that MyD882/2 mice are resistant to UV-induced immuno- mRNA and protein levels in XP and WT lymphoblasts compared suppression (Fig. 1). These data are similar to the study in which with control transfected lymphoblasts (data not shown). Forty- TLR42/2 mice were demonstrated to be relatively resistant to the eight hours after transfection, the cells were irradiated with 50 immune-suppressive effects of UVB (21), suggesting that UVB mJ/cm2 UVR, and CBPDs were detected by DNA dot blot im- immune suppression via TLR4 signaling is mediated by a MyD88- 2/2 mediately following and 24 h after irradiation (Fig. 6A). As pre- dependent signaling pathway, because the phenotype of MyD88 http://www.jimmunol.org/ dicted, the WT cells with MyD88 knocked down showed an mice (resistance to UVR immune suppression) is identical to that increased resolution of CBPDs 24 h post-UVR; however, the of TLR42/2 mice. XPA-deficient cells did not have this phenotype of increased re- Work by Margret Kripke and colleagues in the 1970s found that pair. These DNA dot blot data were confirmed by a CBPD ELISA this UV-induced immunosuppression was a critical step in allowing (Fig. 6B). These data describe a pathway for DNA repair that is for the growth of cancerous tumors on the skin (37–39). UVR by guest on September 25, 2021 FIGURE 5. Increased resolution of CBPD is de- pendent on PARP. (A) PARP cleavage was measured by Western blot in cultured PMs before and 4 and 8 h after 25 mJ/cm2 UVR. Cells from WT mice exhibit a time-dependent cleavage of PARP after UVR. This cleavage is greatly diminished in MyD882/2 PMs. (B) Lysates were prepared from WT or MyD88 PMs 24 h after the indicated doses of UVB, and the diluted lysates were assayed for PARP functional activity by ELISA (see Materials and Methods). *p , 0.05. (C) PMs from WT and MyD882/2 mice were treated for 30 min with either media alone or media with 1 mg/ml PJ-34 (a water-soluble PARP inhibitor). Cells were exposed to 25 mJ/cm2 UVR, and DNA was extracted immediately after or 24 h after UVR. CBPDs were assayed using a specific ELISA (see Materials and Methods). Densitometry of CBPDs is graphed as the percentage of dimers detected immediately after UVR. A representative experiment of three replicates with similar results is shown. Data are mean 6 SD (n =3/ group). *p , 0.05, **p , 0.01, ANOVA. (D) Hair was removed from the abdomen of WT and MyD882/2 mice, which were then injected intradermally with 235 mg of PJ-34 in sterile saline or saline alone. Two hours after injection, mouse abdomens were irradi- ated with 70 mJ/cm2 UVR. Serial skin biopsies were taken immediately following and 24 h after irradia- tion. Epidermal DNA was extracted from the skin biopsies, and a CBPD ELISA was performed. Data are mean OD 6 SD (n = 4/group). *p , 0.05, ANOVA. The Journal of Immunology 7

FIGURE 6. Knockdown of MyD88 in cells from an XP group A (XPA) patient had no effect on resolution of CBPDs. Lymphoblasts from a healthy donor and an XPA patient were transfected with either MyD88 or control siRNA. A .75% knockdown of MyD88 was achieved (data not shown). Forty-eight hours after transfection, cells were irradiated with 50 mJ/cm2 UVR, and DNA was extracted immediately thereafter or 24 h later. (A) DNA dot blot of CBPDs 24 h after a single exposure (50 mJ/cm2) of UVB. No CBPDs were detected in unirradiated lymphoblasts (data not shown). (B) DNA dot blot data were confirmed using

a CBPD-specific ELISA. A decrease in CBPDs was Downloaded from detected 24 h after UVB in lymphoblasts from a normal donor with MyD88 siRNA compared with control (scrambled) siRNA. This difference was not observed in XPA cells. Data are mean OD 6 SD (n = 4 replicates/ group). *p , 0.05, ANOVA. http://www.jimmunol.org/

causes the proliferation of tumor-specific regulatory T cells (31), pressed (43). We demonstrate that a lack of MyD88 signaling and it damages the DNA of skin-resident APCs and causes them to results in a preserved systemic anti-hapten (DNP) response, be- by guest on September 25, 2021 migrate out of the skin to the LLN (40). In our model, we found cause the sera from UVR WT mice exhibited a significantly de- that skin-resident APCs migrated out of the epidermis at a similar creased anti-hapten Ab response, whereas this was relatively rate in WT and MyD882/2 mice (Fig. 2A); however, when probed intact in MyD882/2 mice (Fig. 2C). for DNA damage, the LLN of MyD882/2 mice presented with As discussed previously, TLR42/2 and MyD882/2 cells do not fewer CBPDs (Fig. 3A). Because the CBPDs were a result of undergo a typical apoptotic cell death after UVR; instead, they die direct UVR exposure, our data support the idea that migratory by necroptosis (44). This observation supports our findings that epidermal APCs from WT mice arrive in the LLN with DNA MyD882/2 mice are resistant to UV immunosuppression because damage, whereas APCs from MyD882/2 mice arrive in the LLN the apoptotic fragments released during classical UV-induced cell without DNA damage, because they repair such damage more death are known to contribute to UV-induced systemic immuno- efficiently. This idea is further supported by our observation that suppression. Not only is the irradiated organism systemically af- DNA damage–sensing genes are upregulated in the LLN of WT fected during UV-induced apoptosis, but at the cellular level, many mice but not MyD882/2 mice (Fig. 3B). The arrival of immune- proteins are routinely cleaved by caspase-3, including the DNA competent APCs in the LLN of MyD882/2 mice results in a mi- damage–recognition molecule PARP (45), which is considered a croenvironment that supports Th1 development, because both biomarker for apoptosis. IL-12 and IL-23 can further promote the repair of UVR-induced In our experimental systems, we found that PARP was not DNA damage (41, 42). cleaved (i.e., inactivated) after UVR in MyD882/2 PMs (Fig. 5A). Maintenance of Th1 responses by APCs that reach the LLN with PARP plays a role in the recognition of DNA damage, and it less DNA damage supports the integrity of CHS responses (Fig. 1), works synergistically with NER enzymes (46–48). Lysates derived which indicates intact skin immune surveillance after an acute from MyD882/2 (but not WT) PMs maintained functional PARP UVR exposure. Additionally, when stimulated with anti-CD3/anti- activity (ADP-ribosylate histones in vitro) (Fig. 5B). CD28, LLN cells from MyD882/2 mice were capable of producing We also found that increased resolution of CBPDs is observed significantly more IFN-g (Fig. 2C). Although IFN-g production is after UVR in both mouse and human cells without a functional suppressed in WT mice by UVR, it is augmented slightly in MyD88 pathway (Fig. 4). The DNA damage–recognition/repair MyD882/2 mice, which could be a consequence of the previously molecule PARP is cleaved and inactivated during apoptosis, and described inflammatory necroptotic cell death. These data sug- the TLR42/2/MyD882/2 cells die by necroptosis instead of ap- gest that there is increased DNA repair in MyD882/2 cells that optosis. Therefore, we hypothesized that the decrease in UV- may allow for the observed resistance to UV immunosuppression. induced DNA damage is due to increased DNA repair. PARP is We demonstrated previously that UVR suppresses the systemic able to recognize CBPDs and function as a recruiter for the NER immune response after hapten sensitization, in that the IgG2a machinery, which can come in and repair the DNA lesion (49). It (IFN-g–driven) anti-hapten (in this case DNP) response, is sup- is not surprising that we observed increased resolution of CBPDs 8 IMMUNE MAINTENANCE AND DNA REPAIR AFTER UVR IN MyD882/2 MICE in MyD88-deficient cells, which maintain full-length/functional 9. Pamphilon, D. H., A. A. Alnaqdy, and T. B. Wallington. 1991. Immunomodu- lation by ultraviolet light: clinical studies and biological effects. Immunol. Today PARP after UVR. If this increased resolution of CBPDs is de- 12: 119–123. pendent on full-length (active) PARP, when PARP is inhibited, 10. Kripke, M. L. 1991. Immunological effects of ultraviolet radiation. J. Dermatol. more CBPDs should be detected in UV-irradiated MyD88-deficient 18: 429–433. 11. Martin, C., M. Hurwitz, and S. Bowyer. 1991. Spectroscopic limits to an ex- cells. Using PJ-34, a water-soluble PARP inhibitor, we found that, tragalactic far-ultraviolet background. Astrophys. J. 379: 549–563. 2/2 in vitro (Fig. 5C), CBPDs occur in MyD88 cells at levels 12. 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