Impaired Plasmacytoid Dendritic Cell Innate Immune Responses in Patients with Herpes -Associated Acute Retinal Necrosis

This information is current as Nicolai A. Kittan, Antonio Bergua, Sabrina Haupt, Norbert of October 1, 2021. Donhauser, Philipp Schuster, Klaus Korn, Thomas Harrer and Barbara Schmidt J Immunol 2007; 179:4219-4230; ; doi: 10.4049/jimmunol.179.6.4219 http://www.jimmunol.org/content/179/6/4219 Downloaded from

References This article cites 57 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/179/6/4219.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on October 1, 2021 *average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Impaired Plasmacytoid Dendritic Cell Innate Immune Responses in Patients with Herpes Virus-Associated Acute Retinal Necrosis1

Nicolai A. Kittan,2* Antonio Bergua,2† Sabrina Haupt,* Norbert Donhauser,* Philipp Schuster,* Klaus Korn,* Thomas Harrer,‡ and Barbara Schmidt3*

Plasmacytoid dendritic cells (PDC), the main producers of type I IFNs in the blood, are important for the recognition and control of viral and bacterial infections. Because several induce IFN-␣ production, severe courses of herpes virus infections in nonimmunocompromised patients may be related to numerical or functional PDC deficits. To evaluate this hypothesis, PBMC and PDC were repeatedly isolated from nine patients with acute retinal necrosis (ARN), caused by herpes ؍ simplex or . The patients experienced meningitis/encephalitis and frequent infections in childhood (n and stress Downloaded from ,(4 ؍ infections (n ,(1 ؍ ocular surgery (n ,(2 ؍ recurrent herpes virus infections at unusual localizations (n ,(2 -The median percentage of isolated PDC was significantly lower in patients compared with 18 age .(6 ؍ around ARN (n matched healthy controls (p < 0.001), confirmed by FACS analysis using peripheral blood, and was extremely low during acute disease. PDC counts dropped in five controls suffering from respiratory infections or diarrhea. IFN-␣ production in PDC and PBMC exposed to different stimuli was significantly lower in patients than in controls (p < 0.05). Anergy to these stimuli was observed on four occasions, in particular during acute disease. PDC of patients showed up-regulated IFN /regulatory factor-7 mRNA levels and evidence of in vivo activation (CD80) and maturation (CD83) (p < 0.05). CD8؉ cell http://www.jimmunol.org These data support a risk factor model in which .(0.04 ؍ responses were significantly lower in patients vs controls (p numerical and functional deficits in PDC-mediated innate immune responses contribute to an impaired control of latent herpes virus infections and subsequent development of ARN. The Journal of Immunology, 2007, 179: 4219–4230.

cute retinal necrosis (ARN),4 first described in 1971 (1), despite antiviral therapy and vitrectomy with silicon oil instilla- is a rare inflammatory necrotic process affecting one or, tion. The most frequent causes are varicella zoster virus (VZV), A in some cases, both retinas in immunocompetent as well predominantly occurring in elderly individuals, and HSV 1 and 2, as immunocompromised patients (2). The patients present with associated with a history of encephalitis and meningitis in patients by guest on October 1, 2021 unspecific inflammatory symptoms such as a red eye and ocular older and younger than 25 years, respectively (4, 5). Triggering pain accompanied by blurred vision. Clinical signs are focal, well- events such as periocular trauma, neurosurgery, and high-dose cor- demarcated areas of necrosis in the peripheral retina, rapid cen- ticoids have been reported (6). tripetal progression, occlusive vasculopathy, and inflammatory re- A pioneering insight into the pathogenesis of ARN was pro- sponses in the vitreous body and anterior chamber (3). The vided by an early animal model in which HSV inoculation into the sequelae are irreversible retinal damage and severely reduced vi- anterior chamber of rabbits was followed by retinal necrosis of the sion or blindness due to necrosis of the retina, which often occurs uninoculated eye (7), later confirmed in mice (8). The virus spreads through synaptically connected nuclei and neurons to the contralateral, but not ipsilateral, optical nerve and retina. In T cell- *Institute of Clinical and Molecular Virology, German National Reference Centre for and NK-cell depleted mice, however, the virus spreads to both Retroviruses, †Department of Ophthalmology, and ‡Department of Internal Medicine III with Institute for Clinical Immunology, University Hospital Erlangen, University retinas and from the anterior chamber to the ipsilateral retina, re- of Erlangen-Nu¨rnberg, Erlangen, Germany spectively, confirming a role for both cells types in the control of Received for publication October 2, 2006. Accepted for publication June 29, 2007. virus infection (9–11). Notably, T infiltration of the The costs of publication of this article were defrayed in part by the payment of page brain and cytokine production cannot be detected until 1–2 days charges. This article must therefore be hereby marked advertisement in accordance after virus infection (12). The necrotic process seems to be driven with 18 U.S.C. Section 1734 solely to indicate this fact. by CD4ϩ cells, macrophages, polymorphonuclear cells, B cells, 1 This work was supported by the German Research Foundation (Grant SCHM 1702/ and the inflammatory cytokines TNF-␣ and IFN-␥ (13, 14). HSV-1 1-1; SFB466, Project A12; Grant SCHM1702/2-1) (to B.S.), the Graduate College GRK1071 (“Viruses of the Immune System”) (to N.A.K. and S.H.), and the “Akad- tegument proteins have been characterized as major targets for emie der Wissenschaften und Literatur zu Mainz.” T cells within the vitreous fluid (15). In addition, VZV-specific 2 N.A.K. and A.B. contributed equally to the work. delayed hypersensitivity was absent in a subset of patients with 3 Address correspondence and reprint requests to Dr. Barbara Schmidt, Institute of ARN (16). Clinical and Molecular Virology, German National Reference Centre for Retrovi- ruses, Schlossgarten 4, D-91054 Erlangen, Germany. E-mail address: baschmid@viro. Recently, plasmacytoid dendritic cells (PDC) have been identified med.uni-erlangen.de as major producers of type I IFNs in the blood (17, 18). Together with 4 Abbreviations used in this paper: ARN, acute retinal necrosis; VZV, varicella zoster myeloid dendritic cells (MDC), they play a crucial role in innate im- virus; PDC, plasmacytoid dendritic cell; MDC, myeloid dendritic cell; lin, lineage; mune defenses against microbial pathogens (19), including viruses IRF, IFN regulatory factor; IQR, interquartile range. such as HSV (20, 21). Besides a broad antiviral activity, type I IFNs Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 regulate early immune activation toward a cellular-based response, www.jimmunol.org 4220 IMPAIRED INNATE IMMUNITY IN ARN

thus bridging innate and adaptive immunity (22). Furthermore, PDC activate NK cells with subsequent lysis of infected cells (23, 24). PDC are recruited to varicella skin lesions (25) and to human cerebrospinal fluid under neuroinflammatory conditions (26). In addition, their ac- Sequence tivity in lymph nodes induces strong anti-HSV CTL (27), further em- Reference M15182 U73036 NM_002163 BC035343 AF259262 AY242128 phasizing the role of CD8ϩ cells in maintaining HSV latency (28, 29). Because it is still unclear why only selected individuals in a large population of HSV- and VZV-seropositive individuals are so severely 7 7 7 7 7 7 affected by these viruses, we hypothesized that PDC or related pop- –10 –10 –10 –10 –10 –10 ulations such as MDC or NK cells play a role in the onset and patho- 1 2 2 1 1 1 10 10 10 10 10 10

genesis of virus-induced ARN. Linear Range (copies/reaction) CXCR3 Materials and Methods and Recruitment of patients and controls From 1996 to 2004, eight patients were diagnosed with ARN and evidence of HSV-DNA or VZV-DNA in the vitreous body and/or cerebrospinal fluid at the Department of Ophthalmology (University Hospital, Erlangen, Ger- many). Between November 2004 and April 2006, five of these individuals

agreed to participate and were enrolled in our study (further referred to as 8, CCR7, TLR9,

P01, P03–P05, and P07, see Table I). Four additional patients (P02, P06, Downloaded from P08, P09) suffered from ARN during the observation period. The controls and were age-matched healthy volunteers in and around the Institute of Clinical and Molecular Virology (University Hospital, Erlangen, Germany). A questionnaire asked for specific circumstances, in particular infections, pre-

ceding or occurring around ARN, signs of acute or recurrent infection with GUS, IRF7 HSV or VZV, and clinical symptoms of other viral infections, e.g., warts, lymphadenopathy, exanthema, seizures. Patients and controls were also asked about bacterial infections (common cold, sinusitis, pneumonia, men- http://www.jimmunol.org/ ingitis), children’s diseases, and response to vaccinations. Information was Real-time PCR primers also collected about the susceptibility of family members to bacterial or 5 Ј -tgg tcc5 Ј -gat tgg gtc tga5 Ј -gcc gtc agc gtg ata tgg gtg gag aa-3 Ј tgc gct aaa gtt gg-3 Ј gg-3 Ј 5 Ј -cgc cac gac ttt gtt ttc tg-3 Ј 5 Ј -gaa ctg5 Ј -gct gct cca ggt5 Ј -acc ggc gtc acg acg gaa5 Ј -tga acc caa gac-3 Ј aga aca ctt-3 Ј 5 Ј -tgc ctt gcg acg cag atg5 Ј -tgg gtc gcc a-3 Ј cct acc caa5 Ј -ggt gca agg ctg-3 Ј aga tca ttg gct gct t-3 Ј ggg ttg-3 Ј tgg gca tga-3 Ј viral infections, hereditary or autoimmune diseases, and unclear causes of 5 Ј -ctg tca agg gca gta acc tgt tc-3 Ј death. This study was approved by the Ethical Committee of the Medical Faculty, University of Erlangen-Nu¨rnberg (No. 3299), and informed con- b sent was obtained from all participants. b Isolation and stimulation of PBMC and PDC PBMC were obtained from EDTA-containing blood using standard Ficoll gradient centrifugation (Biochrom). PDC were purified from PBMC in a by guest on October 1, 2021 IRF7_1652i_s: IRF8_964i_s: GUS_839i_as: IRF7_1787i_as: IRF8_1038i_as: CCR7_791i_s: CCR7_854i_as: TLR9_2023i_s: TLR9_2097i_as: CXCR3_421i_s: CXCR3_481i_as: two-step LS/MS column isolation procedure using the BDCA4 (ϭCD304) GUS_772i_s: cell isolation kit (Miltenyi Biotec) as described previously (30). Purity of isolated PDC was checked in selected donors using FITC-conjugated Abs against the PDC-specific lectin BDCA2 (31) (Miltenyi Biotec) and anti- CD4-PE (BD Biosciences). The viability of isolated PDC was above 85% as revealed by trypan blue staining. PBMC and PDC were cultivated in RPMI 1640 medium containing 10% heat-inactivated (56°C, 60 min) FCS (Invitrogen Life Technologies), supplemented with 50 mg/ml glutamine, 200 U/ml penicillin, 90 U/ml streptomycin, and 20 ng/ml IL-3 (R&D Sys- tems). PDC and PBMC were plated at a density of 104 cells/200 ␮l and 106 cells/500 ␮l in 96- and 24-well flat-bottom plates, respectively. PDC were stimulated immediately after isolation and PBMC were stimulated the fol- lowing day, using UV-irradiated supernatant from a clinical HSV-1 isolate (106 PFU/ml), CpG-A (ODN 2336, 1 ␮M; purchased from Coley Pharma- ceutical Group), a synthetic TLR7 agonist (S-27609, 5 ␮M; provided by 3M Pharmaceuticals), and LPS (1 ␮M; Sigma-Aldrich). PDC were stim- ulated in triplicates whenever sufficient cells were isolated. PBMC super- natants were harvested at 6 and 12 h after stimulation; cells were stored at Ϫ80°C after snap-freezing in liquid nitrogen. PDC supernatants were har- Outer primers vested after 24 h of stimulation and stored at Ϫ20°C. 5 Ј -gac aga ggc agg tga cgc g-3 Ј 5 Ј -caa tga5 Ј -tcc ata cca cag5 Ј -gct cgc ata gct tat ggc cca acc agg-3 Ј gct atc5 Ј -cgg cca tac aag taa ct-3 Ј 5 Ј -gat aac gg-3 Ј ggt tgg5 Ј -gat gat ctg gtt cgg gtg5 Ј -tcc gaa ctt tc-3 Ј gct gtt tct5 Ј -aac cac ggc g-3 Ј tgg tcg cac-3 Ј 5 Ј -agg gag atg act cca aga5 Ј -gga tca ttg cc-3 Ј agt gcc act tgt tga gc-3 Ј att act ggc tc-3 Ј agt gg-3 Ј Cytokine assays 5 Ј -ggc tgg tga att acc aga tc-3 Ј PMBC and PDC supernatants were analyzed for IFN-␣ 2a/2b using an ELISA module set (Bender Medsystems) according to the manufacturer’s recommendations. In general, 10 ␮l of cell culture supernatants were an- a alyzed unless values above the linear range required further dilution. Flow cytometry GUS_751a_s: IRF8_842a_s: All FACS determinations were performed on EDTA-anticoagulated GUS_937a_as: IRF7_1582a_s: IRF7_1813a_as: IRF8_1043a_as: CCR7_722a_s: CCR7_920a_as: TLR9_1761a_s: TLR9_2184a_as: CXCR3_134a_s: CXCR3_622a_as: blood within 4 h after collection. PDC and MDC counts were deter- mined as described previously (32). In brief, 2 ml of whole blood was washed with Dulbecco’s PBS supplemented with 1% FCS and 0.5 mM Outer primers for the generation of standards and real-time PCR primers for mRNA quantification of the housekeeping gene ␮ EDTA (Sigma-Aldrich), followed by incubation with 100 l of FcR- Gene s, Sense; as, antisense. Published by Izaguirre et al. (Ref. 34). GUS IRF8 IRF7 CCR7 TLR9 CXCR3 blocking reagent (Miltenyi Biotec) at 4°C for 10 min to reduce nonspecific a b

staining. Aliquots (100 ␮l) of this cell suspension were incubated with a Table I. The Journal of Immunology 4221 Downloaded from http://www.jimmunol.org/

FIGURE 1. FACS setting. a, Forward/side scatter plot with gating on PBMC (R1), lin marker/CD4 scatter plot with gating on CD4ϩlinϪ cells (R2), display of CD4ϩlinϪ PBMC on a CD11c/CD4 scatter plot with CD11cϪ cells identified as PDC (R3) and CD11cϩ cells identified as MDC (R4) as described previously (32). BDCA2/BDCA4 scatter plot with gating on double-positive PDC (R5) and CD16/CD161 scatter plot with gating on double-positive NK cells (R6). b, Up-regulation of CD83 on PDC, which were identified as BDCA4ϩ cells after subtracting CD11cϩCD14ϩ cells. c, Expression of IFN-␥ on unstimulated (left panels) and HSV-1-stimulated (right panels) CD4ϩ and CD8ϩ T and CD16ϩ NK cells using a tight lymphocyte gate (R7). by guest on October 1, 2021

FITC-conjugated Ab mixture against lineage (lin) markers CD3, CD14, free DNase I (Roche Diagnostics). After ammonium chloride precipitation, CD16, and CD20, anti-CD4-PE, and anti-CD11c-PE-Cy5. All mAbs were RNA was reverse-transcribed using random primers with and without purchased from BD Biosciences except for anti-BDCA2 and anti-BDCA4 SuperScript II (both Invitrogen Life Technologies) at 25°C for 10 min, (Miltenyi Biotec). The respective mouse IgG Abs were used as isotype 42°C for 50 min, and 75°C for 15 min. A 1/30 dilution of cDNA was controls. After staining for 20 min at 4°C, red cells were lysed in a buffer subjected to real-time PCR using the ABI Prism 7500 detection system ␮ ϫ containing 155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA. Pelleted (Applied Biosystems). The 50- l reaction contained 0.5 TaqMan Uni- white cells were resuspended in the FACS buffer described above, and versal PCR Master Mix (Applied Biosystems), 0.1ϫ SYBRGreen I Nu- 200,000 events were acquired using a three-color FACSCalibur with cleic Acid Gel Stain (Invitrogen Life Technologies), and 100 nM of the CellQuest 3.3 software (BD Biosciences). PDC and MDC were discrimi- respective real-time PCR primers (Table I). Except for IFN regulatory fac- nated by CD11c expression (Fig. 1a). Confirmatory staining of PDC was tor 7 (IRF7; Ref. 34), real-time PCR primers were searched using Primer performed using anti-BDCA2-FITC, anti-BDCA4-PE, and anti-CD14-PE- Express, version 2.0 (Applied Biosystems). Amplifications were conducted Cy5 (Immunotools) (Fig. 1a). CD4ϩ and CD8ϩ T cells were identified by at 50°C for 10 min and 45 cycles of 95°C for 15 s and 60°C for 1 min, staining with anti-CD3-FITC and anti-CD4-PE or anti-CD8-PE. NK cells followed by melting curve analysis. For the generation of standard curves, were stained with anti-CD16-FITC and anti-CD161-PE (Fig. 1a). Absolute larger PCR products from each gene were generated by conventional PCR differential white cell counts were provided by the Department of Trans- with outer primers (also Table I) and photometrically quantified after fusion Medicine and Hemostaseology (University Hospital, Erlangen, Ger- QIAquick PCR purification or QIAquick gel extraction (both Qiagen). All many). Cell counts were expressed as percentages (percent of PBMC) and samples were analyzed in triplicates with a coefficient of variation below absolute numbers (cells per microliter) by multiplying percentages with 30%. Values of the specific genes were normalized with respect to the absolute counts for mononuclear cells. housekeeping gene ␤-glucuronidase (GUS) (35). Expression of baseline surface markers on PDC and MDC was performed using anti-CCR7-FITC and anti-CXCR3-FITC (R&D Systems, Wiesbaden, Serologic testing Germany), anti-CD80-FITC (Immunotools), and anti-CD83-FITC (Becton Abs for HSV and VZV were tested using the HSV-1/2-IgG/-IgM ELISA -Serion), respecگDickinson). In this approach, PDC were identified as BDCA4-PE-positive (DiaSorin) and VZV-IgG/-IgM ELISA (Institut Virion ϩ cells with exclusion of CD14 , and MDC as cells positive for tively. The plasma samples were diluted until values within the linear range CD1c-PE (Miltenyi Biotec) and negative for CD14-PE-Cy5 and CD19-PE- of the assay were obtained. Type-specific Abs against HSV-1 and HSV-2 Cy5 (Immunotools). Up-regulation of PDC and MDC surface markers was were discriminated using the HerpeSelect 1 and 2 Immunoblot IgG (Focus evaluated after exposure of PBMC to HSV-1 for 20 h immediately after cell Diagnostics; distributed by Mikrogen). isolation. In this procedure, MDC were identified as described above, whereas PDC staining additionally excluded CD11cϩ cells to adjust for BDCA4 up- Determination of IFN-␥ production regulation on stimulated monocytes and MDC (33) (Fig. 1b). PBMC were plated in duplicates in 24-well flat-bottom plates at a density 6 Real-time PCR amplification of mRNAs of 1 ϫ 10 cells/500 ␮l. Cells were immediately stimulated after isolation using the UV-irradiated HSV-1 isolate described above and harvested after RNA was isolated from PBMC using the RNeasy Mini kit (Qiagen). 20 h. The IFN-␥ production was analyzed using the IFN-␥ secretion assay Genomic DNA was removed using QIAshredder and an on-column DNA detection kit (PE, human) according to the manufacturer’s recommenda- digestion (Qiagen), followed by incubation of the eluted RNA with RNase- tions (Miltenyi Biotec). In brief, cells were stained with FITC-conjugated 4222

Table II. Clinical characteristics of patients with ARN caused by herpes virus infectiona

Age at ARN (Age at Blood Draw, Diagnosis of Herpes Virus Herpes Virus Affections in Number of No. in Years) Infection Circumstances around ARN Clinical Symptoms in Childhood Adulthood Family Members Visits

P01 18 (21) HSV-DNAb (VBϩ , CSFϪ ) Severe respiratory infection Severe meningitis at 7 days Frequent HSV affections Sister with frequent herpes 4 immediately before (hospitalization for 2 mo); (one time per month) labialis ARN, treated with plantar warts, sinusitis, at unusual antibiotics tonsillitis, aphthosis localizations (finger, chest), but never herpes labialis P02c 25 HSV-2-DNAb (VBϩ , Uneventful Aphthosis Frequent herpes labialis na 1 CSF nd) P03 38 (44) HSV-DNAb (VBϩ , CSFϩ ) Stress Severe meningitis at 18 mo Pneumonia at 20 years; Brother corneal HSV ulcer, 4 (hospitalization for 3 mo); frequent herpes sister HSV infection at frequent sinusitis and labialis (one time per eyelid and herpes zoster, common cold month); severe two other siblings bronchitis at 35 years healthy (local corticoids for 2 years); HSV encephalitis at 37 years P04 38 (39) VZVϪ DNA (VBϩ , CSFϪ ) Stress Uneventful Healthy Healthy 4 P05 49 (51) VZV-DNA (VBϩ , CSFϪ ) Stress, common Plantar warts, Frequent herpes labialis Daughter frequent HSV 4 cold 3 wk common cold and (six times per year), affections at unusual prior to ARN sinusitis arthritis for many localizations (eyelid, years cheek) P06c 65 VZV-DNA (VBϩ , CSFϪ ) Weight loss and neck pain Uneventful Herpes zoster at Healthy 4

3 wk prior to ARN 20 years ARN IN IMMUNITY INNATE IMPAIRED P07 70 (73) VZV-DNA (VBϩ , Cataract surgery one year Uneventful Healthy Healthy 3 CSF nd) prior to ARN

P08c 72 VZV-DNA (VBϩ , Pneumonia 1 mo prior to Uneventful Healthy Healthy 2 CSF nd) ARN, treated with antibiotics P09c 73 VZV-DNA (VBϩ , Uneventful Uneventful Healthy Healthy 2 CSF nd)

a VB, Vitreous body; CSF, cerebrospinal fluid; nd, not done; na, not available. b Serologic evidence of infection with HSV-1 (P01, P03) and HSV-1/-2 (P02).

c These patients suffered from ARN during the observation period.

Downloaded from from Downloaded http://www.jimmunol.org/ by guest on October 1, 2021 1, October on guest by The Journal of Immunology 4223 Downloaded from http://www.jimmunol.org/

FIGURE 2. Numerical deficits in cells of the innate immune system in patients vs controls. a, Determination of isolated BDCA4ϩ cells as percentage of PBMC. refer by guest on October 1, 2021 (ءء) marks the samples from patients drawn during ARN; double asterisks (ء) Individual values for patients (E) and healthy controls (F). A single asterisk to samples of controls suffering from respiratory infections (n ϭ 4) or diarrhea (n ϭ 2). Horizontal bars indicate median values, which were significantly different in patients vs controls (p Ͻ 0.001, Mann-Whitney U test). b, Linear regression analysis of percentages of isolated BDCA4ϩ cells vs CD4ϩ linϪCD11cϪ cells obtained by FACS analysis on EDTA-anticoagulated blood; छ, patient samples; ࡗ, control samples. Calculations were performed using the Spearman rank correlation. Percentages of PDC determined as CD4ϩlinϪCD11cϪ cells (c) and BDCA2ϩBDCA4ϩCD14Ϫ cells (d), MDC as CD4ϩlinϪCD11cϩ cells (e), NK cells as CD16ϩCD161ϩ cells (f), CD3ϩCD4ϩ (g), and CD3ϩCD8ϩ T lymphocytes (h) in patients vs controls, obtained by FACS analysis of peripheral blood. Statistics were performed using the Mann-Whitney U test; n.s., not significant.

Abs to CD4, CD8, and CD16, PE-conjugated anti-IFN-␥, and counter- ables, and the ␹2 test for categorical variables. All statistical calculations stained with CD14-PE-Cy5. IFN-␥ production was evaluated using a tight assumed a two-sided significance at p values Յ0.05. lymphocyte gate (Fig. 1c). Results Statistics Characteristics of patients and controls Whenever more than one sample was analyzed from patients or controls, median values were used for statistical calculations. The Mann-Whitney U Five subjects suffered from ARN 0.5–6 years before study enroll- test was used for comparisons between two independent groups, the Spear- ment and sampling, four patients (P02, P06, P08, P09) were in- man rank correlation coefficient for correlations between continuous vari- cluded with acute disease (Table II). Subjects with ARN due to

Table III. Absolute countsa

Cells/␮l (Median, IQR)

Cell Type Patients Controls p Value

CD4ϩlinϪCD11cϪ PDC 5.51 (3.76–6.88) 8.49 (7.53–11.13) 0.007 CD4ϩlinϪCD11cϩ MDC 3.96 (3.48–7.07) 6.75 (5.56–8.42) 0.08 (NS) CD16ϩCD161ϩ NK cells 46 (30–53) 39 (22–47) 0.96 (NS) CD4ϩ T lymphocytes 777 (495–1025) 932 (773–1110) 0.21 (NS) CD8ϩ T lymphocytes 504 (317–694) 710 (517–852) 0.12 (NS) Total lymphocytes 2370 (1750–2500) 2280 (1960–2690) 0.34 (NS) Total granulocytes 3870 (3460–4840) 3720 (2540–4360) 0.33 (NS)

a Absolute counts of PDC, MDC, CD16ϩCD161ϩ NK cells, CD4ϩ and CD8ϩ T lymphocytes as well as total lymphocyte and granulocyte counts in patients and controls. NS, Not significant. 4224 IMPAIRED INNATE IMMUNITY IN ARN

FIGURE 3. Comparison of IFN-␣ production in PDC (a) and PBMC (b) obtained from patients and healthy controls. PDC and PBMC were exposed Downloaded from to different stimuli for 24 and 12 h, respectively. Dots represent values from individual subjects, box plots indicate median and interquartile ranges, whiskers show 10th and 90th percentiles. Values of p were obtained using the Mann-Whitney U test. Mock unstimulated cells; n.s., not significant.

HSV (P01, P02, P03) were younger than those with VZV-associated cold (P05), weight loss, and neck pain (P06). Four patients also had a retinopathy (P04–P09). Personal interviews revealed specific circum- medical history of severe and/or recurrent infections. These included stances around ARN in six patients, namely stress (P03, P04, P05), severe meningitis/encephalitis in early childhood (P01, P03), frequent http://www.jimmunol.org/ severe respiratory infection treated by antibiotics (P01, P08), common respiratory infections in childhood and recurrent herpes simplex by guest on October 1, 2021

FIGURE 4. Cell-type analyses in the peripheral blood and vitreous body of one patient with acute VZV-associated retinopathy (P08) (a) and another patient with subacute HSV-2-associated ARN (P02) (b). Forward/side scatter plots with gating on mononuclear cells (R1) (left panels), CD4/CD8 scatter plots (middle left panels), CD4/lin/CD11c scatter plots with gating on PDC (R3) and MDC (R4) (middle right panels), and CD19/CD20 scatter plots (right panels). Influx of NK cells into the vitreous body was not observed in any of the patients (data not shown). The Journal of Immunology 4225 Downloaded from http://www.jimmunol.org/

FIGURE 5. Real-time PCR quantification of mRNA levels of five genes involved in innate immune responses in relation to the housekeeping gene GUS using unstimulated PBMC from patients and controls. a, Standard curves, presented as mean and 95% confidence interval, obtained from four consecutive runs for each gene. Ct, threshold cycle; cop., copies. Quantification of mRNA of the IRF7 (b) and IRF8 (c), the CCR7 (d), TLR9 (e), and CXCR3 (f)in samples from patients and controls. Patient values are given as median if more than one sample was available for analysis. Horizontal bars indicate median values of all patient and control samples; n.s., not significant. by guest on October 1, 2021 infections in adulthood (P01, P02, P03, P05), herpes simplex 97.1% (IQR, 95.9–97.4%). On 14 of 19 patient visits, percentages encephalitis (P03) (36), and herpes zoster at a young age (P06) of isolated BDCA4ϩ cells were below 0.20%, compared with only (Table II). In the remaining two patients (P07, P09), there was 1 of 56 visits in controls ( p Ͻ 0.001) (Fig. 2a). Percentages of no apparent trigger for ARN and the medical history was un- isolated BDCA4ϩ cells were significantly lower in patients com- eventful with respect to infectious diseases; the only notable pared with controls (median, 0.15 vs 0.32%; IQR, 0.11–0.19% vs event was cataract surgery 1 year before ARN in patient P07. 0.25–0.40%; p Ͻ 0.001). When controls suffered from bacterial or Three patients (P01, P03, P05) reported recurrent HSV infec- viral infections, e.g., common cold, diarrhea, cough, and sore tions in family members, in two cases at unusual localizations throat, percentages of isolated BDCA4ϩ cells dropped below the (eyelid, cheek). lowest value observed when they were healthy on 5 of 6 occasions. None of the 18 controls reported serious infections similar to the FACS analyses of the peripheral blood were performed in parallel patients, and only 3 controls (C03, C04, C017) reported recurrent to the isolation of BDCA4ϩ cells on 39 occasions; they revealed a herpes labialis, albeit at a low frequency (once or twice a year). significant correlation of CD4ϩlinϪCD11cϪ cells and isolated Infrequent herpes labialis among family members was reported by BDCA4ϩ cells (r2 ϭ 0.54, p Ͻ 0.001) (Fig. 2b). Only one patient four controls (C02, C04, C07, C10). Control C09 suffered from (P03) had consistently higher percentages of CD4ϩlinϪCD11cϪ Hashimoto’s disease with evidence of anti-thyroid Abs 12 years cells compared with isolated BDCA4ϩ cells. The percentage of ago. Because PDC counts were reported to decline with age (37), isolated BDCA4ϩ cells significantly correlated with the percentage of patients and controls were matched for age (median, 51 years vs 42 BDCA2ϩBDCA4ϩCD14Ϫ cells obtained by FACS analyses of the years; range, 21–73 years vs 25–74 years; interquartile range peripheral blood (n ϭ 14; r2 ϭ 0.42; p ϭ 0.003). The latter also (IQR) 39–70 years vs 29–58 years; p ϭ 0.61, NS) and gender significantly correlated with the percentage of CD4ϩlinϪCD11cϪ (percentage of females, 33 vs 44%, p ϭ 0.92). cells (n ϭ 22; r2 ϭ 0.75; p Ͻ 0.001) (data not shown). Percentages of CD4ϩlinϪCD11cϪ cells in the peripheral blood Numerical PDC deficit in patients with ARN were significantly lower in patients vs controls (median, 0.23 vs BDCA4ϩ cells were isolated in 19 and 56 separate experiments 0.43%; IQR, 0.19–0.32% vs 0.31–0.55%; p ϭ 0.005), respectively from patients and controls, respectively. Patients were examined (Fig. 2c). This was also true for the percentages of on 2–3 occasions, controls on 1–10 occasions. For all subjects with BDCA2ϩBDCA4ϩCD14Ϫ cells (median, 0.20 vs 0.49%; IQR, 0.17– three or more isolation procedures (n ϭ 12), the median coefficient 0.29% vs 0.34–0.62%, p ϭ 0.014; Fig. 2d) and CD4ϩlinϪCD11cϩ of variation was 25.4% (IQR, 20.1–36.1%), similarly observed in MDC (median, 0.20 vs 0.32%; IQR, 0.17–0.24% vs 0.24–0.39%, patients and controls. The purity of isolated cells, determined as p ϭ 0.03, Fig. 2e). In contrast, no significant differences were found median percentage of BDCA2ϩ CD4ϩ cells in six controls, was for CD16ϩCD161ϩ NK cells ( p ϭ 0.56, Fig. 2f), CD3ϩCD4ϩ T 4226 IMPAIRED INNATE IMMUNITY IN ARN Downloaded from http://www.jimmunol.org/

FIGURE 6. Analysis of markers for immune stimulation. a, Expression of surface markers for migration (CCR7), activation (CD80), maturation (CD83), and endocytosis (BDCA2) on PDC in patients (n ϭ 8) vs controls (n ϭ 11). b, Up-regulation of CD80 and CD83 on PDC after exposure to UV-irradiated HSV-1 for 20 h. c, Amount of IFN-␣ in plasma samples of patients (n ϭ 9) and controls (n ϭ 18). d, Expression of the CXCR3 on PDC in patients (n ϭ 8) and controls (n ϭ 11). lymphocytes ( p ϭ 0.59, Fig. 2g), and CD3ϩCD8ϩ T lymphocytes P06 and P08 underwent vitrectomy within 1 day of admission and by guest on October 1, 2021 ( p ϭ 0.56, Fig. 2h). These data were confirmed using absolute cell diagnosis was confirmed by detection of VZV DNA in the vitreous counts (for details see Table III), except for CD4ϩlinϪCD11cϪ MDC body. FACS analyses of peripheral blood and vitreous humor in ( p ϭ 0.08, NS). In addition, no significant differences were found for both patients showed undetectable to very low PDC counts in both absolute lymphocyte and granulocyte counts between patients and compartments and an influx of CD8ϩ T lymphocytes into the vit- controls (Table III). reous body (Fig. 4a). PDC and PBMC of both patients were an- ergic toward all stimuli at this occasion (data not shown). Patient ␣ Functional abnormalities in IFN- production in patients P09 received systemic corticoids for 10 days before the diagnosis with ARN of VZV-associated ARN was finally confirmed by surgery, which The intra- and interassay variability of the IFN-␣ ELISA was de- may explain the relatively high PDC count on this occasion. After termined using a low (520 pg/ml) and a high positive control (4222 corticoid therapy was stopped, the PDC count decreased consid- pg/ml). Respective coefficients of variation were 4.9 and 7.1% in erably (Fig. 2a). Patient P02 was treated with oral for 6 quadruplicate analyses and 27.0 and 23.0% in seven consecutive wk because peripheral necrosis without infiltration of the vitreous runs. IFN-␣ production was determined for PDC and PBMC ob- body did not require immediate surgery. After deterioration of the tained from patients and controls in 19 and 20 as well as in 16 and 26 clinical situation, the patient finally received a diagnostic vitrec- separate experiments, respectively. IFN-␣ production was signifi- tomy, revealing the presence of HSV-2 DNA. FACS analysis of ϩ cantly lower in patients compared with controls after exposure of this subacute infection no longer revealed an influx of CD8 cells PDC to HSV-1 ( p ϭ 0.009), CpG-A ( p ϭ 0.016), and the TLR7 but PDC and B cells, suggesting a Th2-type reaction. agonist ( p ϭ 0.001) (Fig. 3a). Significant differences were also ob- served after exposure of PBMC to these stimuli for 6 h (data not Elevated levels of IRF7 mRNA in patients with ARN shown) and 12 h ( p ϭ 0.005, p ϭ 0.01, and p ϭ 0.008) (Fig. 3b). No To ensure sufficient representation of different RNA transcripts, we IFN-␣ production was noticed when PBMC were exposed to LPS, excluded samples with Ͻ100 copies/reaction of the housekeeping consistent with the finding that PDC do not express TLR4 (38). On gene GUS. This procedure left 15 samples from 9 patients and 15 four occasions, cells isolated from patient P01, P08, and P09 did not samples from 15 controls for subsequent analysis. GUS levels respond to any of the stimuli with IFN-␣ levels above 100 pg/ml. were comparable between patients and controls (copies per reac- tion; median, 1645 vs 1168; IQR, 1040–1722 vs 783-2073; p ϭ PDC depletion and functional anergy in acute VZV-associated 0.55). The mean percentage of contaminating genomic DNA was retinopathy 0.35%; 67 of 74 samples had genomic DNA levels below 1%. We Four patients were admitted to the Department of Ophthalmology studied five cellular transcripts which we suspected to be involved in after suffering from blurred vision for one to several days. Patients the pathogenesis of ARN. In this respect, IRF7 has been characterized The Journal of Immunology 4227

FIGURE 7. Analysis of humoral and cellular adap- tive immunity. a, Levels of IgG Abs for HSV and VZV in plasma samples of patients vs controls. b, Induction of IFN-␥ production in CD4ϩ and CD8ϩ T lymphocytes and CD16ϩ NK cells upon stimulation with HSV-1 for 20 h; n.s., not significant. Downloaded from http://www.jimmunol.org/

as key molecule in the induction of IFN production and is constitu- tively, data not shown). In addition, there was no evidence of a tively expressed in PDC (34). IRF8 was selected because knockout general immune activation as shown by similarly low IFN-␣ mice have a lower percentage of PDC compared with wild-type mice plasma levels in patients and controls, analyzed in 18 and 35 sam- (39); CCR7 was selected because it is up-regulated upon PDC stim- ples, respectively (Fig. 6c). Moreover, the migratory capacity of by guest on October 1, 2021 ulation and promotes migration to secondary lymphatic tissue (40). PDC was not significantly different between patients and controls, TLR9 was selected because reduced TLR9 transcripts were found in as evident from the expression of CXCR3 ( p ϭ 0.28, Fig. 6d). patients with common variable immunodeficiency (41) and CXCR3 was selected to evaluate the migratory capacity of immune cells in the No evidence of impaired humoral immunity in patients blood. The slope of all six standard curves was similar (Fig. 5a). After with ARN normalizing the values with respect to GUS, median levels of IRF7 To evaluate humoral immune responses, HSV and VZV IgG and mRNA were found to be significantly higher in unstimulated PBMC IgM Abs were determined in the plasma of patients and controls. of patients compared with controls ( p ϭ 0.05) (Fig. 5b). In contrast, IgM Abs were not detected in any of the control and patient the mRNA levels of IRF8 (Fig. 5c), CCR7 (Fig. 5d), TLR9 (Fig. 5e), plasma samples, in particular not during the acute episodes of and CXCR3 (Fig. 5f) were almost identical between patients and con- ARN. These data indicate that ARN resulted from herpes viral trols. The highest levels of IRF7, IRF8, and TLR9 mRNA in control reactivation and not primary infection in all patients of our PBMC were detected in the individual with a history of Hashimoto’s study. All patients and controls were seropositive for VZV, thyroiditis. whereas all nine patients and 11 of 18 controls were seroposi- Evidence of immune stimulation in PDC of patients with ARN tive for HSV. The levels of HSV-IgG and VZV-IgG were not significantly different between patients and controls ( p ϭ 0.54 and To evaluate PDC activation at baseline, expression of different p ϭ 0.98, respectively; Fig. 7a). The discrimination of HSV-1 and surface markers was compared between patients (n ϭ 8) and con- HSV-2 Abs revealed seropositivity for HSV-1 in P01 and P03 trols (n ϭ 15). Whereas no differences were found for the expres- and seropositivity for HSV-1 and HSV-2 in P02. sion of CCR7 ( p ϭ 0.87) and BDCA2 ( p ϭ 0.77), significantly ϭ higher expression levels of CD80 and CD83 (both p 0.04) were ϩ detected in PDC of patients compared with controls (Fig. 6a). Af- Impaired CD8 cell responses in patients with ARN ter exposure to UV-irradiated HSV-1, PDC activation and matu- To evaluate cellular adaptive immune responses, PBMC of pa- ration as evaluated by CD80 and CD83 expression was not sig- tients (n ϭ 9) and HSV-seropositive controls (n ϭ 11) were ex- nificantly different between patients (n ϭ 8) and HSV-seropositive posed to HSV-1 for 20 h and then analyzed for IFN-␥ production controls (n ϭ 11) ( p ϭ 0.07 and p ϭ 0.71, respectively, Fig. 6b). using FACS analysis. The increase in IFN-␥ production in stimu- The expression of MDC surface markers at baseline was compa- lated vs unstimulated cells from patients compared with controls rable between the two groups (CCR7, p ϭ 0.09; CD80, p ϭ 0.65; was considerably lower in CD4ϩ T lymphocytes and CD16ϩ NK CD83, p ϭ 0.90; data not shown). Furthermore, no significant cells, and significantly reduced in CD8ϩ CTLs of patients (me- differences were observed for the up-regulation of CD80 and dian, 0.22 vs 0.65%, IQR, 0.07–0.41% vs 0.41–1.34%, p ϭ 0.04; CD83 after HSV-1 stimulation ( p ϭ 0.71 and p ϭ 0.68, respec- Fig. 7b). The stimulation of an HSV-seronegative donor did not 4228 IMPAIRED INNATE IMMUNITY IN ARN result in IFN-␥ expression, indicating that the activity in HSV- It remains unclear what caused the activation of PDC in our seropositive donors was stimulus specific (data not shown). patients. It is tempting to speculate that the trigger was an ongoing subclinical herpes virus infection, which was not adequately con- Other immunologic tests trolled by the adaptive immune system of the patients. Our data None of the patients exhibited antinuclear Abs on Hep2 cells. One clearly show no evidence of impaired humoral immunity (Fig. 7a); patient (P04) showed a weak reactivity in a Borrelia spp. IgG- Ag presentation by PDC and MDC seems to work sufficiently, as ELISA, which however could not be confirmed by Western is evident from the up-regulation of costimulatory CD80 and blotting. CD83 after exposure to HSV-1 (Fig. 6b, and data not shown). In contrast, significantly lower CD8ϩ T lymphocyte IFN-␥ produc- Discussion tion upon HSV-1 exposure was found in patients compared with Our data strongly suggest that low PDC counts are associated with controls (Fig. 7b), indicating an impaired cellular immune control severe recurrent herpes virus infections, as evident from signifi- of HSV infections. In this respect, the important role of CD8ϩ cells cantly lower percentages of isolated BDCA4ϩ cells (Fig. 2a), in controlling and maintaining HSV-1 latency has been reported CD4ϩlinϪCD11cϪ cells (Fig. 2c), and BDCA2ϩBDCA4ϩCD14Ϫ (28, 29). However, alternatively, the low PDC counts in our stud- cells in the peripheral blood (Fig. 2d) in patients with ARN com- ies could impair an effect of priming CTLs or NK cells, which has pared with healthy controls. Notably, the PDC counts of our con- recently been described by others (23, 24). Following this hypoth- trol group were very similar to recently published data (32). Al- esis, elevated IRF7 levels and increased surface expression of though limited by the small number of patients with ARN, we CD80 and CD83 may serve as compensatory mechanism for low could also draw some conclusions on PDC dynamics. Thus, PDC PDC counts. counts showed substantial fluctuations over time in ARN patients A detailed insight into the pathogenesis of ARN was provided Downloaded from as well as in controls. A particularly low percentage of PDC was by animal models (7, 8). The neuronal spread of HSV inoculated observed in patients tested during acute disease who had not re- into the anterior chamber was followed by an infiltration of T ceived systemic corticoid therapy. Notably, low PDC values were lymphocytes (12), which, in concert with other cells and inflam- observed among the controls especially during episodes of acute matory cytokines, triggered the elimination of virus-infected cells infections and with one exception the PDC counts observed during (13, 14). We observed a similar influx of CD8ϩ T cells into the these episodes were the lowest found in the respective individuals vitreous body in the two cases of acute VZV-associated ARN (Fig. http://www.jimmunol.org/ (Fig. 2a). Similar data have recently been published for acute den- 4a). The speed of T cell recruitment to the site of infection and gue virus infection, in which an early decline of circulating PDC proper NK cell function seem to be crucial for limiting virus was predictive of severe disease (42). Whether low numbers of spread (10, 11), suggesting that low PDC and low NK cell counts circulating PDC are predictive of herpes virus-associated ARN or may have contributed to the occurrence of ARN in three patients just an associated symptom among others remains to be deter- with very low NK cell counts (P01, P06, P09). In this respect, it mined. Additional studies are required to clarify whether low PDC may be important to study IRF8 knockout mice as a model for counts occur in bacterial and immunologic uveitis as well. HSV reactivation, which have lower PDC counts compared with IFN-␣ production in response to different stimuli was also re- wild-type mice (48). In the patient with subacute HSV-2-associ- by guest on October 1, 2021 duced in patients compared with controls, both after stimulation of ated ARN (P02), the CD8ϩ cell influx was replaced by an influx of PBMC and PDC (Fig. 3). However, there was no clear functional B cells and PDC (Fig. 4b), suggesting a role for different cell defect as we originally suspected after complete anergy had been populations in the course of ARN (13). found in the first sample from P01, because all patients with an- Two patients (P01, P02) reported severe meningitis and/or en- ergic PBMC/PDC on one occasion showed IFN-␣ production on cephalitis in early childhood (Table II), reminiscent of perinatal another occasion. The overall reduced IFN-␣ production, and in HSV-2 infections which were followed by immediate or delayed particular the intermittent anergy, may play an important role in necrotic (49, 50). HSV-1 encephalitis preceding ARN was the pathogenesis of ARN, because a major role of type I, but not described by our group (36) and others (51–54). Elevated PDC type II IFNs in limiting HSV replication in the cornea and nervous counts in cerebrospinal fluid were detected in neuroinflammatory system was shown using mice with knockout mutations in IFN conditions, suggesting that PDC may contribute to the orchestra- receptors (43). tion of local immune responses in this immune-privileged com- Stimulation of PDC causes an activation of a downstream sig- partment (26). Our data show lack of PDC infiltration in acute naling cascade resulting in nuclear translocation of NF-␬B and episodes of ARN (Fig. 4a), which may indicate a defective innate type I IFN secretion (44). IRF7 mRNA, which is constitutively immune response contributing to the severity of ARN. However, expressed in PDC (34), was found significantly elevated in un- our data provide evidence for the presence of PDC in vitreous stimulated patient vs control PBMC (Fig. 5b), which may be in- humor in advanced stages of ARN (Fig. 4b). terpreted as evidence of a stimulated TLR/IFN pathway in vivo. In In conclusion, our data support a multifactorial model for the this respect, self- and cross-hyporesponsiveness have been re- development of ARN. Impaired control of latent HSV and/or VZV ported after TLR stimulation (45–47), suggesting that the inter- infection due to low PDC counts and function as well as reduced mittent anergy, which we observed in patient PBMC and PDC in CD8ϩ cell activity may lead to severe herpes virus reactivation vitro, may have been caused by preceding PDC stimulation in like ARN. This finding would be most apparent if acute events like vivo. In particular, maturation of PDC has been associated with a bacterial or viral infections, stress, ocular trauma, or corticosteroid reduced capacity of IFN-␣ production (30). This hypothesis is sup- treatment as well as factors in the personal histories of patients, ported by significantly higher expression of activation and matu- e.g., (neonatal) herpes virus meningitis or encephalitis, accumu- ration markers on patient PDC (Fig. 6a). PDC stimulation is fol- late. This risk factor model is supported by evidence of other lowed by their homing to lymphatic tissue (40), where they serve groups (4–6, 55). The family histories of the patients in our study as APCs (31). The depletion of CCR7-expressing PDC may ex- point to a—presumably genetically determined—defect in the plain why neither elevated mRNA levels (Fig. 5d) nor enhanced control of latent HSV and/or VZV infection. Notably, a case of expression of CCR7 (Fig. 6a) were found in patient PBMC and HSV-2-associated ARN was described in a patient with systemic PDC, respectively. lupus erythematosus (56). In this disease, PDC are depleted from The Journal of Immunology 4229 the peripheral blood into skin lesions (57), similar to PDC deple- 18. Cella, M., D. Jarrossay, F. Facchetti, O. Alebardi, H. Nakajima, A. Lanzavecchia, tions to varicella skin lesions (25). Systemic lupus erythematosus and M. Colonna. 1999. Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type I interferon. Nat. Med. 5: 919–923. patients show a characteristic granulocyte expansion in the periph- 19. Fitzgerald-Bocarsly, P. 2002. Natural interferon-␣ producing cells: the plasma- eral blood (58). This signature was found on 4 of 21 occasions in cytoid dendritic cells. BioTechniques (Suppl.): 16–19. 20. Lund, J., A. Sato, S. Akira, R. Medzhitov, and A. Iwasaki. 2003. Toll-like re- patients (see Fig. 4b), but on 4 of 32 occasions in controls as well ceptor 9-mediated recognition of -2 by plasmacytoid den- ( p ϭ 0.80). Altogether, naturally low numbers of circulating PDC dritic cells. J. Exp. Med. 198: 513–520. or induced migration of these cells from the peripheral blood to 21. Hochrein, H., B. Schlatter, M. O’Keeffe, C. Wagner, F. Schmitz, M. Schiemann, S. Bauer, M. Suter, and H. Wagner. 2004. Herpes simplex virus type-1 induces lymphatic tissue or skin may contribute to an impaired first line of IFN-␣ production via Toll-like receptor 9-dependent and -independent pathways. defense against infection and reactivation of latent viruses, as we Proc. Natl. Acad. Sci. USA 101: 11416–11421. suggest for herpes virus-associated ARN. 22. McKenna, K., A. S. Beignon, and N. Bhardwaj. 2005. Plasmacytoid dendritic cells: linking innate and adaptive immunity. J. Virol. 79: 17–27. 23. Gerosa, F., A. Gobbi, P. Zorzi, S. Burg, F. Briere, G. Carra, and G. Trinchieri. Acknowledgments 2005. The reciprocal interaction of NK cells with plasmacytoid or myeloid den- We thank Bernhard Fleckenstein and Friedrich E. Kruse for continuous dritic cells profoundly affects innate resistance functions. J. Immunol. 174: 727–734. support. We thank all patients and controls for participation in the study. The 24. Marshall, J. D., D. S. Heeke, C. Abbate, P. Yee, and G. Van Nest. 2006. Induction synthetic TLR7 agonist S-27609 was kindly provided by 3M Pharmaceuticals of interferon-␥ from natural killer cells by immunostimulatory CpG DNA is (St. Paul, MN), and LPS by A. Gessner, Institute for Clinical Microbiology, mediated through plasmacytoid-dendritic-cell-produced interferon-␣ and tumour ␣ Immunology and Hygiene (University of Erlangen-Nu¨rnberg, Erlangen, necrosis factor- . Immunology 117: 38–46. 25. Gerlini, G., G. Mariotti, B. Bianchi, and N. Pimpinelli. 2006. Massive recruitment Germany). Serologic tests were performed by the diagnostic services of the of type I interferon producing plasmacytoid dendritic cells in varicella skin le- Institute of Clinical and Molecular Virology (Erlangen, Germany). We sions. J. Invest. Dermatol. 126: 507–509.

thank Silke Bergmann and Kathrin Eismann for excellent technical assis- 26. Pashenkov, M., Y. M. Huang, V. Kostulas, M. Haglund, M. Soderstrom, and Downloaded from tance. Absolute differential white cell counts were kindly provided by the H. Link. 2001. Two subsets of dendritic cells are present in human cerebrospinal fluid. Brain 124: 480–492. Department of Transfusion Medicine and Hemostaseology (University 27. Yoneyama, H., K. Matsuno, E. Toda, T. Nishiwaki, N. Matsuo, A. Nakano, Hospital, Erlangen, Germany). S. Narumi, B. Lu, C. Gerard, S. Ishikawa, and K. Matsushima. 2005. Plasmacy- toid DCs help lymph node DCs to induce anti-HSV CTLs. J. Exp. Med. 202: Disclosures 425–435. 28. Divito, S., T. L. Cherpes, and R. L. Hendricks. 2006. A triple entente: virus, The authors have no financial conflict of interest. neurons, and CD8ϩ T cells maintain HSV-1 latency. Immunol. Res. 36: 119–126. 29. Khanna, K. M., A. J. Lepisto, V. Decman, and R. L. Hendricks. 2004. Immune http://www.jimmunol.org/ References control of herpes simplex virus during latency. Curr. Opin. Immunol. 16: 1. Urayama, A., N. Yamada, and T. Sasaki. 1971. Unilateral acute uveitis with 463–469. retinal periarteritis and detachment. Jpn. J. Clin. Ophthalmol. 25: 607–619. 30. Schmidt, B., B. M. Ashlock, H. Foster, S. H. Fujimura, and J. A. Levy. 2005. 2. Atherton, S. S. 2001. Acute retinal necrosis: insights into pathogenesis from the HIV-infected cells are major inducers of plasmacytoid dendritic cell interferon mouse model. Herpes 8: 69–73. production, maturation, and migration. Virology 343: 256–266. 3. Holland, G. N. 1994. Standard diagnostic criteria for the acute retinal necrosis syn- 31. Dzionek, A., Y. Sohma, J. Nagafune, M. Cella, M. Colonna, F. Facchetti, drome. Executive Committee of the American Uveitis Society. Am. J. Ophthalmol. G. Gunther, I. Johnston, A. Lanzavecchia, T. Nagasaka, et al. 2001. BDCA-2, a novel plasmacytoid dendritic cell-specific type II C-type lectin, mediates antigen 117: 663–667. ␣ ␤ 4. Ganatra, J. B., D. Chandler, C. Santos, B. Kuppermann, and T. P. Margolis. 2000. capture and is a potent inhibitor of interferon / induction. J. Exp. Med. 194: Viral causes of the acute retinal necrosis syndrome. Am. J. Ophthalmol. 129: 1823–1834. 32. Schmidt, B., S. H. Fujimura, J. N. Martin, and J. A. Levy. 2006. Variations in

166–172. by guest on October 1, 2021 5. Van Gelder, R. N., J. L. Willig, G. N. Holland, and H. J. Kaplan. 2001. Herpes plasmacytoid dendritic cell (PDC) and myeloid dendritic cell (MDC) levels in simplex virus type 2 as a cause of acute retinal necrosis syndrome in young HIV-infected subjects on and off antiretroviral therapy. J. Clin. Immunol. 26: patients. Ophthalmology 108: 869–876. 55–64. 6. Tran, T. H., D. Stanescu, L. Caspers-Velu, F. Rozenberg, C. Liesnard, 33. Dzionek, A., A. Fuchs, P. Schmidt, S. Cremer, M. Zysk, S. Miltenyi, D. W. Buck, A. Gaudric, P. Lehoang, and B. Bodaghi. 2004. Clinical characteristics of acute and J. Schmitz. 2000. BDCA-2, BDCA-3, and BDCA-4: three markers for dis- HSV-2 retinal necrosis. Am. J. Ophthalmol. 137: 872–879. tinct subsets of dendritic cells in human peripheral blood. J. Immunol. 165: 7. Von Szily, A. 1924. Experimental endogenous transmission of infection from 6037–6046. bulbus to bulbus. Klin. Monatsbl. Augenheilkd. 72: 593–602. 34. Izaguirre, A., B. J. Barnes, S. Amrute, W. S. Yeow, N. Megjugorac, J. Dai, 8. Whittum, J. A., J. P. McCulley, J. Y. Niederkorn, and J. W. Streilein. 1984. D. Feng, E. Chung, P. M. Pitha, and P. Fitzgerald-Bocarsly. 2003. Comparative ␣ Ocular disease induced in mice by anterior chamber inoculation of herpes sim- analysis of IRF and IFN- expression in human plasmacytoid and - plex virus. Invest. Ophthalmol. Vis. Sci. 25: 1065–1073. derived dendritic cells. J. Leukocyte Biol. 74: 1125–1138. 9. Azumi, A., and S. S. Atherton. 1994. Sparing of the ipsilateral retina after anterior 35. Loseke, S., E. Grage-Griebenow, A. Wagner, K. Gehlhar, and A. Bufe. 2003. ϩ ϩ ␣ chamber inoculation of HSV-1: requirement for either CD4 or CD8 T cells. Differential expression of IFN- subtypes in human PBMC: evaluation of novel Invest. Ophthalmol. Vis. Sci. 35: 3251–3259. real-time PCR assays. J. Immunol. Methods 276: 207–222. 10. Tanigawa, M., J. E. Bigger, M. Y. Kanter, and S. S. Atherton. 2000. Natural killer 36. Bergua, A., B. Schmidt, and M. Kuchle. 2001. [Fulminant acute retinal necrosis cells prevent direct anterior-to-posterior spread of herpes simplex virus type 1 in 10 months after ipsilateral herpes simplex virus encephalitis–a case report]. Klin. the eye. Invest. Ophthalmol. Vis. Sci. 41: 132–137. Monatsbl. Augenheilkd. 218: 737–740. 11. Archin, N. M., B. L. van den, L. Perelygina, J. M. Hilliard, and S. S. Atherton. 37. Shodell, M., and F. P. Siegal. 2002. Circulating, interferon-producing plasmacy- 2003. Delayed spread and reduction in virus titer after anterior chamber inocu- toid dendritic cells decline during human ageing. Scand. J. Immunol. 56: lation of a recombinant of HSV-1 expressing IL-16. Invest. Ophthalmol. Vis. Sci. 518–521. 44: 3066–3076. 38. Kadowaki, N., S. Ho, S. Antonenko, R. W. Malefyt, R. A. Kastelein, F. Bazan, 12. Archin, N. M., and S. S. Atherton. 2002. Infiltration of T-lymphocytes in the and Y. J. Liu. 2001. Subsets of human dendritic cell precursors express different brain after anterior chamber inoculation of a neurovirulent and neuroinvasive Toll-like receptors and respond to different microbial antigens. J. Exp. Med. 194: strain of HSV-1. J. Neuroimmunol. 130: 117–127. 863–869. 13. Zheng, M., and S. S. Atherton. 2005. Cytokine profiles and inflammatory cells 39. Tamura, T., P. Tailor, K. Yamaoka, H. J. Kong, H. Tsujimura, J. J. O’Shea, during HSV-1-induced acute retinal necrosis. Invest. Ophthalmol. Vis. Sci. 46: H. Singh, and K. Ozato. 2005. IFN regulatory factor-4 and -8 govern dendritic 1356–1363. cell subset development and their functional diversity. J. Immunol. 174: 14. Abe, T., M. Sato, Y. Saigo, and M. Tamai. 2003. Interferon ␥ expression and 2573–2581. clinical features in patients with acute retinal necrosis syndrome. Graefes Arch. 40. Cyster, J. G. 1999. Chemokines and cell migration in secondary lymphoid organs. Clin. Exp. Ophthalmol. 241: 982–987. Science 286: 2098–2102. 15. Verjans, G. M., M. E. Dings, J. McLauchlan, K. A. van Der, P. Hoogerhout, 41. Cunningham-Rundles, C., L. Radigan, A. K. Knight, L. Zhang, L. Bauer, and H. F. Brugghe, H. A. Timmermans, G. S. Baarsma, and A. D. Osterhaus. 2000. A. Nakazawa. 2006. TLR9 activation is defective in common variable immune Intraocular T cells of patients with herpes simplex virus (HSV)-induced acute deficiency. J. Immunol. 176: 1978–1987. retinal necrosis recognize HSV tegument proteins VP11/12 and VP13/14. J. In- 42. Pichyangkul, S., T. P. Endy, S. Kalayanarooj, A. Nisalak, K. Yongvanitchit, fect. Dis. 182: 923–927. S. Green, A. L. Rothman, F. A. Ennis, and D. H. Libraty. 2003. A blunted blood 16. Kezuka, T., J. Sakai, N. Usui, J. W. Streilein, and M. Usui. 2001. Evidence for plasmacytoid dendritic cell response to an acute systemic viral infection is asso- antigen-specific immune deviation in patients with acute retinal necrosis. Arch. ciated with increased disease severity. J. Immunol. 171: 5571–5578. Ophthalmol. 119: 1044–1049. 43. Leib, D. A., T. E. Harrison, K. M. Laslo, M. A. Machalek, N. J. Moorman, and 17. Siegal, F. P., N. Kadowaki, M. Shodell, P. A. Fitzgerald-Bocarsly, K. Shah, H. W. Virgin. 1999. Interferons regulate the phenotype of wild-type and mutant S. Ho, S. Antonenko, and Y. J. Liu. 1999. The nature of the principal type 1 herpes simplex viruses in vivo. J. Exp. Med. 189: 663–672. interferon-producing cells in human blood. Science 284: 1835–1837. 44. Beutler, B. 2004. Innate immunity: an overview. Mol. Immunol. 40: 845–859. 4230 IMPAIRED INNATE IMMUNITY IN ARN

45. Dobrovolskaia, M. A., A. E. Medvedev, K. E. Thomas, N. Cuesta, 51. Gain, P., C. Chiquet, G. Thuret, E. Drouet, and J. C. Antoine. 2002. Herpes V. Toshchakov, T. Ren, M. J. Cody, S. M. Michalek, N. R. Rice, and S. N. Vogel. simplex virus type 1 encephalitis associated with acute retinal necrosis syndrome 2003. Induction of in vitro reprogramming by Toll-like receptor (TLR)2 and in an immunocompetent patient. Acta Ophthalmol. Scand. 80: 546–549. TLR4 agonists in murine macrophages: effects of TLR “homotolerance” versus 52. Hadden, P. W., and C. J. Barry. 2002. Images in clinical medicine: herpetic “heterotolerance” on NF-␬B signaling pathway components. J. Immunol. 170: encephalitis and acute retinal necrosis. N. Engl. J. Med. 347: 1932. 508–519. 53. Maertzdorf, J., L. A. Van der, G. S. Baarsma, A. D. Osterhaus, and 46. Medvedev, A. E., A. Lentschat, L. M. Wahl, D. T. Golenbock, and S. N. Vogel. G. M. Verjans. 2001. Herpes simplex virus type 1 (HSV-1)-induced retinitis 2002. Dysregulation of LPS-induced Toll-like receptor 4-MyD88 complex for- following herpes simplex encephalitis: indications for brain-to-eye transmission mation and IL-1 receptor-associated kinase 1 activation in endotoxin-tolerant of HSV-1. Ann. Neurol. 49: 104–106. cells. J. Immunol. 169: 5209–5216. 54. Preiser, W., H. W. Doerr, S. Buxbaum, H. F. Rabenau, and H. Baatz. 2004. Acute 47. Yeo, S. J., J. G. Yoon, S. C. Hong, and A. K. Yi. 2003. CpG DNA induces self retinal necrosis six years after herpes simplex encephalitis: an elusive immune and cross-hyporesponsiveness of RAW264.7 cells in response to CpG DNA and deficit suggested by insufficient test sensitivity. J. Med. Virol. 73: 250–255. lipopolysaccharide: alterations in IL-1 receptor-associated kinase expression. 55. Verma, L., P. Venkatesh, G. Satpal, K. Rathore, and H. K. Tewari. 1999. Bilateral J. Immunol. 170: 1052–1061. necrotizing herpetic retinopathy three years after herpes simplex encephalitis fol- lowing pulse corticosteroid treatment. Retina 19: 464–467. 48. Tsujimura, H., T. Tamura, and K. Ozato. 2003. Cutting edge: IFN consensus 56. Rappaport, K. D., and W. M. Tang. 2000. Herpes simplex virus type 2 acute sequence binding protein/IFN regulatory factor 8 drives the development of type retinal necrosis in a patient with systemic lupus erythematosus. Retina 20: I IFN-producing plasmacytoid dendritic cells. J. Immunol. 170: 1131–1135. 545–546. 49. Kychenthal, A., A. Coombes, J. Greenwood, C. Pavesio, and G. W. Aylward. 57. Blomberg, S., M. L. Eloranta, B. Cederblad, K. Nordlin, G. V. Alm, and 2001. Bilateral acute retinal necrosis and herpes simplex type 2 encephalitis in a L. Ronnblom. 2001. Presence of cutaneous interferon-␣ producing cells in pa- neonate. Br. J. Ophthalmol. 85: 629–630. tients with systemic lupus erythematosus. Lupus 10: 484–490. 50. Landry, M. L., P. Mullangi, P. Nee, and B. R. Klein. 2005. Herpes simplex virus 58. Bennett, L., A. K. Palucka, E. Arce, V. Cantrell, J. Borvak, J. Banchereau, and type 2 acute retinal necrosis 9 years after neonatal herpes. J. Pediatr. 146: V. Pascual. 2003. Interferon and granulopoiesis signatures in systemic lupus er- 836–838. ythematosus blood. J. Exp. Med. 197: 711–723. Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021