Peripheral blood monocytes reveal an activated phenotype in pediatric uveitis

Accepted Manuscript Peripheral blood monocytes reveal an activated phenotype in pediatric uveitis

Karoline Walscheid, Lisa Neekamp, Arnd Heiligenhaus, Toni Weinhage, Dirk Holzinger, Carsten Heinz, Maren Kasper, Dirk Foell PII: DOI: Reference:

S1521-6616(17)30425-4 doi: 10.1016/j.clim.2017.09.014 YCLIM 7937

To appear in:

Clinical Immunology

Received date: Revised date: Accepted date:

8 June 2017 18 August 2017 14 September 2017

Please cite this article as: Karoline Walscheid, Lisa Neekamp, Arnd Heiligenhaus, Toni Weinhage, Dirk Holzinger, Carsten Heinz, Maren Kasper, Dirk Foell , Peripheral blood monocytes reveal an activated phenotype in pediatric uveitis. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Yclim(2017), doi: 10.1016/j.clim.2017.09.014

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Peripheral blood monocytes reveal an activated phenotype in pediatric uveitis Karoline Walscheid, MD 1,2, Lisa Neekamp2, Arnd Heiligenhaus, MD1,3, Toni Weinhage, PhD 2, Dirk Holzinger, MD 2, Carsten Heinz, MD 1,3, Maren Kasper, PhD 1, Dirk Foell, MD 2

T

Department of Ophthalmology and Ophtha Lab at St. Franziskus-Hospital Muenster,

SC RI P

1

Germany 2

Department of Pediatric Rheumatology and Immunology, University Hospital

Muenster, Germany

NU

Department of Ophthalmology, University of Duisburg-Essen, Germany

MA

3

Corresponding author

ED

Karoline Walscheid, MD, Department of Ophthalmology at St. Franziskus-Hospital, Hohenzollernring 74, 48145 Muenster, Germany

PT

Phone: +49-251-935-2711. Fax: +49-251-935-2719

CE

e-mail: [email protected]

AC

Financial disclosure: None of the authors has a financial or proprietary interest in any of the material or the method presented. Abstract Objective: To characterize peripheral blood monocytes in uveitis associated with juvenile idiopathic arthritis (JIAU). Methods: Peripheral blood monocytes from children with JIA (either with (n=18) or without uveitis (n=11)), idiopathic anterior uveitis (IAU; n=12) and healthy controls (n=11) were analyzed by flow cytometry.

ACCEPTED MANUSCRIPT Results: Percentage of CD14+CD86+ monocytes and CD86 expression on single cell level were significantly higher in all patient groups than in controls, whereas no major differences existed between patient groups. Frequency of CD39+ (p<0.05 all groups) and CD73+ monocytes (p=0.03 JIAU vs controls) was elevated in patients. Disease activity did not influence monocyte phenotypes, but in methotrexate-treated

T

patients numbers of CCR2+ monocytes were reduced and numbers of CD86+ and

SC RI P

CD39+ cells increased.

Conclusion: Monocytes from children with JIA U display a distinct phenotype when compared to cells from healthy children. Phenotypic changes seem to be neither

NU

arthritis- nor uveitis-dependent, but may be modified by treatment.

AC

CE

PT

ED

MA

Keywords: juvenile arthritis, uveitis, pathogenesis, monocytes, therapy

ACCEPTED MANUSCRIPT 1. Introduction Juvenile idiopathic arthritis (JIA) is the most common rheumatic disease in childhood and is frequently accompanied by a potentially sight-threatening chronic anterior uveitis. Children with oligoarticular subtype are at particularly high risk for ocular involvement (up to 45%) [1]. Disease management frequently requires systemic

SC RI P

T

immunomodulating therapy, with methotrexate (MTX) currently being the preferred conventional disease-modifying anti-rheumatic drug (DMARD) [2]. Despite the high clinical significance, pathogenesis of JIA-associated uveitis (JIAU) is still poorly understood. The disease is considered to be of an autoimmune nature,

NU

probably mediated by antigen-specific cells of the adaptive immune system (T and B cells) targeting ocular tissues, leading to a chronic inflammatory process that finally

MA

results in tissue destruction and visual loss [1].

Interaction of antigen-presenting cells of the innate immune system, as dendritic

ED

cells or monocytes/macrophages, with antigen-specific cells of the adaptive immune

PT

system has been found to be of major importance for the course of the inflammatory process in a well-characterized model for autoimmune uveitis (experimental

CE

autoimmune uveoretinitis, EAU) [3, 4]. Nevertheless, little is known about the role of

AC

the innate immune system in human uveitis, and particularly in pediatric patients. We recently demonstrated elevation of serum levels for S100A8/A9 and S100A12 proteins in JIA-associated and pediatric idiopathic anterior uveitis [5]. As these proteins are produced by neutrophils and monocytes upon activation, elevated serum levels suggest an important involvement of the innate immune system in the inflammatory process. Additionally, characterization of monocyte subsets in idiopathic intermediate uveitis, a different, but supposedly also autoimmune uveitis entity, revealed phenotypic differences between peripheral blood monocytes from uveitis patients (both children and adults) and from healthy control subjects, which

ACCEPTED MANUSCRIPT were modulated by systemic anti-inflammatory therapy [6]. We therefore sought to characterize

circulating

monocyte

phenotypes

in JIA-associated

uveitis

in

comparison with idiopathic anterior uveitis of childhood.

2. Materials and methods

SC RI P

T

2.1. Patients

Patients were recruited during 2014 and 2015 at the Department of Ophthalmology at St. Franziskus-Hospital Muenster, Germany (uveitis patients), and the Department of Pediatric Rheumatology and Immunology, University Hospital of Muenster,

NU

Germany (JIA patients without ocular involvement). All uveitis patients fulfilled the SUN criteria for anterior uveitis [7]. JIA patients were classified according to ILAR

MA

criteria, and either had oligo- (persistent or extended form) or polyarticular disease. Uveitis / arthritis patients either without systemic immunomodulating medication or

ED

receiving methotrexate (MTX) therapy were included in our study. All patients

PT

receiving MTX were treated with standard dosage calculated depending on body surface area (between 10 mg / week and 20 mg / week, administered either orally or

CE

subcutaneously). Topical, but no systemic corticosteroids were permitted. Disease

AC

activity was defined according to SUN criteria (active uveitis: ≥1+ anterior chamber cells), and JADAS (active arthritis: JADAS >1) scores [7, 8]. We included the following study groups: 1) Patients with JIA-associated uveitis (JIAU): Patients had a typical chronic course of anterior uveitis. No systemic disease besides JIA was present. All patients underwent ophthalmological examination according to current nationwide guidelines in Germany.

ACCEPTED MANUSCRIPT 2) Patients with idiopathic anterior uveitis (IAU): Patients had an established diagnosis of chronic IAU. No systemic immune-mediated inflammatory or infectious disease was present. 3) Patients with JIA without uveitis (JIA): These patients had an established diagnosis of JIA, without other systemic or ocular diseases. All patients underwent

SC RI P

T

frequent ophthalmological screening assessments in accordance with current recommendations and had at no time shown signs of ocular involvement. 4) Healthy pediatric controls: Children without ocular or inflammatory disease were recruited at the Clinic for Pediatric Surgery at St. Franziskus -Hospital Muenster,

NU

Germany, where they underwent routine surgical procedure for non-infectious or inflammatory indication. Children were preoperatively assessed for signs of infection

MA

by the surgeon and anesthesiologist, and parents were asked to complete a questionnaire asking for any chronic diseases, and for signs of inflammation (e.g.,

ED

respiratory tract infections) during the previous 6 weeks. Only children without any

PT

infectious, inflammatory, or chronic diseases were included in the study. Serum hsCRP levels were assessed in these patients, and all were within normal range.

CE

Informed consent was obtained from patients and parents. The study was per formed

AC

in accordance with the Declaration of Helsinki and was previously approved by the local ethics committee (University of Muenster, approval number 2014-397-f-S).

2.2. Samples A maximum of 7.5 ml (less volume from younger children) of peripheral venous blood was collected from each patient. Blood for flow cytometry analysis was collected in Lithium-Heparin tubes and was stored for a maximum of 2 hours at room

ACCEPTED MANUSCRIPT temperature (RT) until staining. Serum samples were centrifuged at 1000 x g for 10min immediately after acquisition, and aliquots were stored at -80°C until analysis.

2.3. Flow cytometry analysis 2.3.1. Antibodies & reagents

T

We used the following antibodies:

SC RI P

CD80 (clone 2D10.4), CD86 (IT2.2), CD39 (A1), PDL-1 (29E.2A3), PDL-2 (24F.10C12), B7-H4 (MIH43), CD73 (AD2) (all purchased from BioLegend, San Diego, USA); CD14 (c61D3), CD16 (eBioCB16) (all purchased from eBioscience,

NU

San Diego, USA), CX3CR1 (528728), CCR2 (48607), CD121b (34141) (all purchased from R&D Systems, Minneapolis, USA), CD124 (hIL4R-M57) (BD

MA

Biosciences, Heidelberg, Germany)

ED

2.3.2. Staining protocol

200µl of whole blood were stained for 30 min at room temperature with a

PT

combination of anti human CD14 and CD16 antibodies in combination with one or

CE

two of the following antibodies: CD121b, CX3CR1, CCR2, CD124, MHCII, CD80, CD86, CD73, PDL-1, PDL-2, B7-H4 or CD39. Afterwards, erythrocytes were lysed,

AC

and cells were fixed (using a fix/lysing solution; BD Biosciences, Heidelberg, Germany) for 10 min. Cells were washed twice with phosphate buffered saline (containing 2% fetal calf serum, 2mM EDTA, 0.05% NaN3) and were resuspended.

2.3.3. Analysis Samples were measured using a FACSCanto A cytometer (BD Biosciences, Heidelberg, Germany) and were analyzed with FlowJo software version 9.6.2 (Tree Star Inc., Ashland, USA). Percentage of positive cells and mean fluorescence

ACCEPTED MANUSCRIPT intensity (geometric mean, MFI) were documented. Monocytes were defined by morphological characteristics (based on forward/side scatter) and were further characterized by the expression of CD14 and CD16.

2.4. Statistical analysis

T

Data were tested for normality using the D´Agostino and Pearson omnibus normality

SC RI P

test. Differences in monocyte phenotypes between patient groups and controls were analyzed by Mann Whitney U-test. For analysis and figure preparation, we used GraphPad Prism Version 5.0a (GraphPad Software, Inc., San Diego, USA). P-values

NU

<0.05 were considered statistically significant. Due to the exploratory character of

MA

our study, p-values depicted were not corrected for multiple comparisons.

3. Results

ED

3.1. Patient data

PT

Patient data are displayed in table 1. Most JIA patients were diagnosed with oligoarticular subtype (n=26; 90%), and three with RF (rheumatoid factor)-negative

CE

polyarthritis (10%). 94% of patients were antinuclear antibody- (ANA) positive (of

AC

patients with known ANA status; not known in 5 patients ). Children in the control group were younger and were more frequently male than patients, which was due to the inclusion criterion of undergoing routine elective surgical procedures (mainly phimosis and hernia surgery).

3.2. Monocyte phenotyping 3.2.1. Costimulatory molecules

ACCEPTED MANUSCRIPT The percentage of CD14+CD86+ monocytes and the level of CD86 expression on single cell level (MFI) was significantly higher in all patient groups than in controls, whereas no significant differences were noted between the three patient groups (figure 1, only significant p-values shown). No differences were observed for the

T

expression of other costimulatory factors (CD80, PDL-1, PDL-2, B7-H4).

SC RI P

3.2.2. Regulatory molecules

The number of monocytes expressing the regulatory ectonucleotidases CD39 and CD73 was markedly elevated in all patient groups as compared to healthy controls

NU

(figure 1). However, in the JIA and IAU patient group, differences were significant

MA

only for CD39.

3.2.3. Reception of anti-inflammatory signals

ED

No significant differences between patients and/or controls were observed regarding the expression of the decoy receptor for IL-1 (CD121b, IL-1 receptor type II) or the

CE

PT

IL-4 receptor (CD124) (data not shown).

3.2.4. Migration-associated molecules

AC

The numbers of CCR2-expressing monocytes from all patient groups were slightly increased as compared to the control group , whereas the frequency of CX3CR1 positive monocytes were down-regulated. However, changes were not statistically significant (data not shown).

3.3. Subgroup analysis We then investigated the potential influence of clinical characteristics (systemic immunomodulating therapy and disease activity of arthritis and/or uveitis) on

ACCEPTED MANUSCRIPT monocyte phenotypes. Due to the small subgroup numbers, we were not able to perform multivariate regression analysis, and therefore we aimed for comparing the mean values of the different subgroups.

3.3.1. Disease activity

T

To describe a potential influence of uveitis-, or arthritis activity on the monocyte

SC RI P

phenotype, we divided each patient group into two subgroups, comparing patients with active (active uveitis and/or active arthritis) to those with clinically quiescent disease. As only two patients in the IAU group presented with active uveitis,

NU

statistical comparison was not performed in this patient group.

In both the JIA and the JIAU group, disease activity did not appear to be influencing

MA

monocyte phenotypes significantly. As an example, data for JIAU patients are displayed in figure 2. In one half of our JIAU patients, both ocular and joint disease

ED

was active (n=4), whereas the others (n=4) had active uveitis while arthritis was clinically quiescent. The graphical display of individual values did not indicate any

PT

distinct differences between those with active uveitis only and those in whom

CE

inflammation of both joints and eye was present (see figure 2), but due to the very small patient numbers of n=4 for both subgroups, statistical comparison was not

AC

performed.

3.3.2. Systemic therapy We then analyzed patient subgroups divided into those receiving and those without systemic methotrexate therapy. Data for the JIAU patient group are displayed in figure 2. In contrast to disease activity, MTX therapy indeed seemed to influence the expression of several markers on monocytes in this patient group: Patients receiving MTX had higher percentages of monocytes expressing CD39 and CD86 (both

ACCEPTED MANUSCRIPT p=0.008), whereas the numbers of cells expressing CCR2 was markedly lowered (p=0.03). In contrast, these effects were not observed in the JIA group. Again, statistical comparison was not performed in the IAU group, as only three of these

3.3.3. Combined analysis according to clinical characteristics

T

patients did not receive systemic therapy.

SC RI P

To increase sample numbers of subgroups, we then compared patient subgroups stratified by either systemic therapy or disease activity, but irrespective of the uveitis-, or arthritis diagnosis. We found a marked reduction in cells expressing CCR2 in

NU

patients receiving MTX, which was even more pronounced than when analyzing JIAU patients only (55.6±14.5% CD14+CCR2+ monocytes in patients without

MA

therapy vs 42.4±17.1% in MTX treated patients, p=0.03). CCR2+ monocytes were reduced in patients with clinically inactive (both uveitis and arthritis) disease

ED

(44±17.2% CD14+CCR2+ monocytes vs 52±16.8% in active disease), but this did not reach statistical significance (p=0.2). Numbers of CD80-, CD86- and CD73-

PT

expressing cells were only slightly elevated in patients under MTX therapy and in

AC

4. Discussion

CE

those patients with clinically quiescent uveitis and/or arthritis.

Our study demonstrates differential expression of molecules with both costimulatory and regulatory potential (CD86, CD39, CD73), as well as changes in CCR2expression on monocytes from patients with juvenile idiopathic arthritis and / or uveitis as compared to pediatric controls. Differences were noted especially in patients receiving MTX therapy.

ACCEPTED MANUSCRIPT Data on monocyte involvement in juvenile arthritis or uveitis are scarce. Elevated levels of monocyte-derived cytokines in sera and synovial fluid of patients with different JIA subtypes implicate a role for these cells in the course of disease, as does the good clinical response of oligoarticular JIA and JIAU to anti-TNFα treatment [9]. Elevated expression of monocyte -related genes in patients with

T

polyarticular JIA has been revealed by means of transcriptional profiling of PBMCs

SC RI P

[9], however, no systematic analyses of PBMC phenotypes in oligoarticular JIA , or pediatric uveitis have been performed yet, and we are therefore unable to discuss

NU

the data generated in our study in the context of literature.

As the differences observed were more pronounced in patients receiving MTX

MA

therapy, we studied the literature regarding the effect of MTX on different cell types. Wijngaarden et al. found that MTX treatment downregulated Fcγ-RI & -IIa expression

ED

on isolated monocytes, but did not affect expression of CD80, CD86 and MHCII [10],

PT

which is contrasting our results. However, it is conceivable that the effects described in vitro might differ from those we observe in patients on this medication. Peres et al.

CE

showed that regulatory T cells from rheumatoid arthritis patients expressed more CD39 if patients were responsive to MTX treatment as compared to those who were

AC

non-responders, and CD39-expression was induced by MTX treatment [11]. Figueiro et al. examined effect of MTX treatment on isolated glioblastoma cells and found enhancement of both CD39- and CD73-expression [12], a trend we observed in our collective as well. However, results are comparable only to some extent, as effects surely are depending on the cell types examined. We found significantly decreased levels of monocytes expressing CCR2 in patients receiving MTX therapy. Ellingsen et al. observed increased CCR2 expression on monocytes from untreated patients with active rheumatoid arthritis and found that a

ACCEPTED MANUSCRIPT decrease in CCR2 expression upon systemic MTX therapy was associated with good clinical response [13]. It has been shown that CCR2 is essential for migration of monocytes to inflamed tissues, whereas migration to non-inflamed tissues is CX3CR1-dependent [14]. A reduction of CCR2+ monocytes in MTX-treated patients might therefore indicate decreasing inflammatory activity in those patients; however,

T

reduction of this cell subset was marked, but not significant, in patients with clinically

SC RI P

inactive as opposed to active disease status and therefore, correlations cannot be made entirely clear.

Though the changes observed in our patients are pronounced, the meaning of these

NU

findings cannot be ultimately explained. Antigen presenting cells expressing CD86 interact with T cells via binding of CD86 to either CD28 (activating signal) or CTLA -4

MA

(inhibitory signal) expressed on T cells. Increased expression of CD86 could therefore lead to either costimulation or inhibition of T cells, depending on the

ED

expression level of CTLA-4 on the T cell surface [15]; the fact that expression of

PT

other costimulatory molecules (CD80, B7-H4, PDL-1, PDL-2) was not significantly different between patients and controls could implicate that in this setting, CD86

CE

might act in a more regulatory way. CD39 and CD73 are ectonucleotidases with antiinflammatory function, converting proinflammatory ATP

to anti-inflammatory

AC

adenosine by a two-step procedure and it has been shown that anti-inflammatory effects of MTX are at least in part mediated by action of CD39 and CD73 via this pathway[11]. It is therefore tempting to speculate that increase of CD39/73- and maybe even of CD86-positive monocytes, as well as a decrease in cells expressing CCR2 indicates a regulating mechanism leading to resolution of inflammation in those patients receiving MTX therapy.

ACCEPTED MANUSCRIPT Limitation to our study certainly is the small number of patients without systemic immunosuppressive medication, as the majority of the patients with both chronic uveitis and arthritis being in our care require systemic immunosuppressive drugs. Longitudinal monitoring of patients before and after initiation of systemic therapy could probably help to better understand influence of disease activity versus therapy

T

on cell subsets. Future studies could also address the question of drug-specificity of

SC RI P

the changes observed by comparing patients treated with MTX to those receiving other medications.

Additionally, we were probably including patients from different stages of disease,

NU

referring not only to apparent clinical activity of articular or ocular disease, but ranging from patients with long term stable arthritis but remitting form of uveitis to JIA

MA

patients just before imminent arthritis relapse or in remission from flare -up of uveitis

ED

with remaining subclinical inflammatory activity.

PT

5. Conclusion

Taken together, our data lead us to the conclusion that the differences in monocyte

CE

phenotype we observe are not specific for the respective disease entity, but

AC

represent changes due to autoimmune cell activation and regulating mechanisms in general, probably intensified by effects of MTX treatment. This may point to a systemic immune deviation that could in part contribute to the overlapping articular and ocular manifestations of idiopathic inflammatory arthritis and/or uveitis. We therefore suggest further investigations regarding changes in PBMC phenotypes in individual patients by follow-up analyses during the course of disease, especially assessing effects of systemic immunomodulating therapy.

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

MA

NU

SC RI P

T

[1] V. Kalinina Ayuso, N. Makhotkina, M. van Tent-Hoeve, J.D. de Groot-Mijnes, N.M. Wulffraat, A. Rothova, J.H. de Boer, Pathogenesis of juvenile idiopathic arthritis associated uveitis: the known and unknown, Survey of ophthalmology, 59 (2014) 517531. [2] M.J. Hawkins, A.D. Dick, R.J. Lee, A.V. Ramanan, E. Carreno, C.M. Guly, A.H. Ross, Managing Juvenile Idiopathic Arthritis-associated Uveitis, Survey of ophthalmology, (2015). [3] J.T. Rosenbaum, H.W. Kim, Innate immune signals in autoimmune and autoinflammatory uveitis, International reviews of immunology, 32 (2013) 68 -75. [4] F. Willermain, J.T. Rosenbaum, B. Bodaghi, H.L. Rosenzweig, S. Childers, T. Behrend, G. Wildner, A.D. Dick, Interplay between innate and adaptive immunity in the development of non-infectious uveitis, Progress in retinal and eye research, 31 (2012) 182-194. [5] K. Walscheid, A. Heiligenhaus, D. Holzinger, J. Roth, C. Heinz, C. Tappeiner, M. Kasper, D. Foell, Elevated S100A8/A9 and S100A12 Serum Levels Reflect Intraocular Inflammation in Juvenile Idiopathic Arthritis-Associated Uveitis: Results From a Pilot Study, Investigative ophthalmology & visual science, 56 (2015) 7653-7660. [6] K. Walscheid, T. Weinhage, D. Foell, C. Heinz, M. Kasper, A. Heiligenhaus, Phenotypic changes of peripheral blood mononuclear cells upon corticosteroid treatment in idiopathic intermediate uveitis, Clinical immunology, 173 (2016) 27-31. [7] D.A. Jabs, R.B. Nussenblatt, J.T. Rosenbaum, Standardization of uveitis nomenclatur e for reporting clinical data. Results of the First International Workshop, Am J Ophthalmol, 140 (2005) 509-516. [8] A. Consolaro, A. Ravelli, Defining criteria for disease activity states in juvenile idiopathic arthritis, Rheumatology (Oxford), (2015). [9] C. Macaubas, K. Nguyen, D. Milojevic, J.L. Park, E.D. Mellins, Oligoarticular and polyarticular JIA: epidemiology and pathogenesis, Nature reviews. Rheumatology, 5 (2009) 616-626. [10] S. Wijngaarden, J.A. van Roon, J.G. van de Winkel, J.W. Bijlsma, F.P. Lafeber, Downregulation of activating Fcgamma receptors on monocytes of patients with rheumatoid arthritis upon methotrexate treatment, Rheumatology (Oxford), 44 (2005) 729-734. [11] R.S. Peres, F.Y. Liew, J. Talbot, V. Carregaro, R.D. Oliveira, S.L. Almeida, R.F. Franca, P.B. Donate, L.G. Pinto, F.I. Ferreira, D.L. Costa, D.P. Demarque, D.R. Gouvea, N.P. Lopes, R.H. Queiroz, J.S. Silva, F. Figueiredo, J.C. Alves-Filho, T.M. Cunha, S.H. Ferreira, P. Louzada-Junior, F.Q. Cunha, Low expression of CD39 on regulatory T cells as a biomarker for resistance to methotrexate therapy in rheumatoid arthritis, Proceedings of the National Academy of Sciences of the United States of America, 112 (2015) 2509 2514. [12] F. Figueiro, C.P. de Oliveira, L.S. Bergamin, L. Rockenbach, F.B. Mendes, E.H. Jandrey, C.E. Moritz, L.F. Pettenuzzo, J. Sevigny, S.S. Guterres, A.R. Pohlmann, A.M. Battastini, Methotrexate up-regulates ecto-5'-nucleotidase/CD73 and reduces the frequency of T lymphocytes in the glioblastoma microenvironment, Purinergic Signal, 12 (2016) 303312. [13] T. Ellingsen, N. Hornung, B.K. Moller, J.H. Poulsen, K. Stengaard-Pedersen, Differential effect of methotrexate on the increased CCR2 density on circulating CD4 T lymphocytes and monocytes in active chronic rheumatoid arthritis, with a down regulation only on monocytes in responders, Annals of the rheumatic diseases, 66 (2007) 151-157.

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

MA

NU

SC RI P

T

[14] F. Geissmann, S. Jung, D.R. Littman, Blood monocytes consist of two principal subsets with distinct migratory properties, Immunity, 19 (2003) 71-82. [15] A.H. Sharpe, G.J. Freeman, The B7-CD28 superfamily, Nature reviews. Immunology, 2 (2002) 116-126.

MA

NU

SC RI P

T

ACCEPTED MANUSCRIPT

1.

Monocyte

phenotyping

ED

Figure

in

patients

and

controls.

PT

Expression of surface molecules on peripheral blood monocytes from patients with juvenile idiopathic arthritis (JIA), JIA-associated uveitis (JIAU), idiopathic anterior

CE

uveitis (IAU) and healthy pediatric controls. A - C: Percentage of monocytes positive

AC

for the respective surface marker; D - F: Mean fluorescence intensity (MFI) of the respective surface marker. Comparison of patient groups and controls by Mann Whitney U-test.

CE

PT

ED

MA

NU

SC RI P

T

ACCEPTED MANUSCRIPT

Figure 2. Monocyte phenotyping in subgroups of patients with juvenile idiopathic

AC

arthritis-associated uveitis. Expression of surface molecules on peripheral blood monocytes from patients with juvenile idiopathic arthritis-associated uveitis (JIAU) and healthy pediatric controls. JIAU patients were divided into subgroups according to clinical characteristics (systemic therapy, disease activity of either uveitis or arthritis). Comparison of patient groups by Mann Whitney U-test.

ACCEPTED MANUSCRIPT Table 1. Patient data. IAU 12 2 (16.7)

control 11 9 (81.8)

9.3 ± 5.2

13 ± 4.9

9.3 ± 4.0

4.7 ± 3.1

n.a. 5 (45.5) 5 (45.5) n.a. n.a. n.a. 8 (of 10) 1 (of 3) 0 (of 4) n.a. 6.3 ± 4.6

6 (33.3.) 4 (22.3) 13 (72.2)

2 (16.7) n.a. 9 (75)

15 (83.4) 10 (55.6)

11 (91.7) 8 (66.7)

8 (44.5) 16 (of 16) 0 (of 14) 0 (of 14) 5.5 ± 2.9 5.0 ± 3.1

6 (50) 10 (of 10) 1 (of 4) 0 (of 2) 6.1 ± 2.3 n.a.

n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.

T

JIAU 18 8 (44.5)

SC RI P

n male (n/%) age (mean±SD) uveitis activity (n/%) arthritis activity (n/%) Methotrexate therapy (n/%) topical steroids (n/%) uveitis complications (n/%) previous ocular surgery (n/%) ANA positive (n/*) HLA-B27 positive (n/*) RF positive (n/*) age at uveitis diagnosis (mean±SD) age at arthritis diagnosis (mean±SD)

JIA 11 3 (27.3)

JIA Juvenile idiopathic arthritis; JIAU Juvenile idiopathic arthritis-associated uveitis; IAU idiopathic anterior uveitis; n.a. not applicable; ANA antinuclear antibodies; RF

NU

rheumatoid factor; * number of patients in which the respective laboratory value was

AC

CE

PT

ED

MA

known

ACCEPTED MANUSCRIPT Highlights 

Blood monocytes from children with autoimmune uveitis display an activated phenotype.



Similar changes are observed in juvenile idiopathic arthritis (JIA) patients without uveitis. Cell phenotypes are comparable amongst the different disease entities.



Monocyte phenotypes distinctly differ between patients and healthy controls.



Phenotypic changes seem to be independent of uveitis or arthritis activity.

AC

CE

PT

ED

MA

NU

SC RI P

T

