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\begin{document}

\title{Arthritis in systemic lupus erythematosus is characterized by local
IL-17A and IL-6 expression}



\author[1]{Natalie  Sippl}%
\author[1]{Francesca Faustini}%
\author[2]{Johan Rönnelid}%
\author[1]{Sara Turcinov}%
\author[1]{Karine Chemin}%
\author[1]{Iva  Gunnarsson}%
\author[1]{V Malmström}%
\affil[1]{Karolinska Institute}%
\affil[2]{Uppsala University}%


\vspace{-1em}



  
  \date{May 27, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Arthritis is a common clinical feature of systemic lupus erythematosus
(SLE) and is usually non-erosive as opposed to rheumatoid arthritis
(RA). While RA synovial pathology has been extensively studied, little
is known about the pathophysiology of lupus arthritis. Here, we aimed to
explore the cytokine and cellular compartments in synovial fluids of SLE
patients with arthritic manifestations. Acellular synovial fluid and
paired serum samples from SLE patients (n=17) were analyzed with
cytokine bead array for T helper associated cytokines. From two SLE
patients, synovial fluid mononuclear cells (SFMC) were analyzed by
multiparameter flow cytometry to dissect T cell, B cell, monocyte and
dendritic cell phenotypes. SLE-derived SFMC were further stimulated in
vitro to measure their capacity for producing IFN? and IL-17A. All
patients fulfilled the ACR 1982 classification criteria for SLE.
Clinical records were reviewed to exclude the presence of comorbidities
such as osteoarthritis or overlap with RA. IL-17A and IL-6 levels were
high in SLE synovial fluid. A clear subset of the synovial CD4+ T cells
expressed CCR6+, a marker associated with Th17 cells. IL-17-production
was validated amongst CD4+CCR6+ T cells following in vitro stimulation.
Furthermore, a strong IFN? production was observed in both CD4+ and CD8+
cells. Our study shows high IL-17A and IL-6 levels in synovial fluids of
patients with lupus arthritis. The Th17 pathway have been implicated in
several aspects of SLE disease pathogenesis and our data points to Th17
involvement also for lupus arthritis.%
\end{abstract}%



\sloppy


\textbf{TITLE PAGE}

\textbf{Arthritis in systemic lupus erythematosus is characterized by
local IL-17A and IL-6 expression}

Natalie Sippl\textsuperscript{1*}, Francesca
Faustini\textsuperscript{1*}, Johan R\selectlanguage{ngerman}önnelid\textsuperscript{2}, Sara
Turcinov\textsuperscript{1}, Karine Chemin\textsuperscript{1}, Iva
Gunnarsson\textsuperscript{1}, Vivianne Malmström\textsuperscript{1}

\textbf{Affiliations}

\textsuperscript{1} Division of Rheumatology, Department of Medicine,
Center of Molecular Medicine, Karolinska University Hospital Solna,
Karolinska Institute, Stockholm, Sweden

\textsuperscript{2} Department Immunology, Genetics and Pathology,
Uppsala University, Uppsala, Sweden.

* Natalie Sippl and Francesca Faustini contributed equally to this work

\textbf{Short Title:} Synovial IL-17A and IL6 expression in lupus
arthritis patients

\textbf{Correspondence to:} Vivianne Malmström, Center for Molecular
Medicine, L8:04, 171 76 Stockholm, Sweden. Email:
vivianne.malmstrom@ki.se

\textbf{Keywords:}

Systemic lupus erythematosus, arthritis, synovial fluid, cytokines, T
cells

\textbf{List of abbreviations}

SLE: systemic lupus erythematosus; LE: lupus erythematosus; RA:
rheumatoid arthritis ; SpA: spondyloarthritis; OA: osteoarthritis;
ARA/ACR: American Rheumatism Association/ American College of
Rheumatology; ASAS: assessment of spondyloarthritis international
society; SF: synovial fluid; PB: peripheral blood; SFMC/PBMC: synovial
fluid-/peripheral blood- mononuclear cells; ACPA: anti citrullinated
peptides antibodies; RF: rheumatoid factor; ANA: antinuclear antibodies;
CRP: C-reactive protein; CBA: cytokine bead array; tSNE: t-distributed
stochastic neighbor embedding; DMSO/FCS: dimethyl-sulfoxide/fetal calf
serum; Th: T helper cell; Th17: T-helper 17; T\textsubscript{PH}:
T-peripheral helper; Treg: T-regulatory cell; DC: dendritic cells; cDC:
classical dendritic cell; CD4/CD8/CD3/CD28/CD11c: CD- cluster of
differentiation; CCR4/CCR6: CCR- chemokine receptor; CXCR/CXCR3/CXCR5:
CXCR:C-X-C chemokine receptor; Eomes: Eomesodermin; HLA-DR: Human
Leucocyte Antigen-DR isotype; FoxP3:forkhead box P3; PD-1: programmed
cell death protein-1; GZMA: granzyme A; TNF: tumor necrosis factor;
IL-1\selectlanguage{greek}β/\selectlanguage{english}IL-2/IL-4/IL-6/IL-8/IL-10/IL-17A, IL: interleukin; IFN\selectlanguage{greek}γ:
\selectlanguage{english}interferon gamma; IgA/IgM: Immunoglobulin, isotype A/isotype M , IQR:
interquartile range

\textbf{Summary}

Arthritis is a common clinical feature of systemic lupus erythematosus
(SLE) and is usually non-erosive as opposed to rheumatoid arthritis
(RA). While RA synovial pathology has been extensively studied, little
is known about the pathophysiology of lupus arthritis. Here, we aimed to
explore the cytokine and cellular compartments in synovial fluids of SLE
patients with arthritic manifestations.

Acellular synovial fluid and paired serum samples from SLE patients
(n=17) were analyzed with cytokine bead array for T helper associated
cytokines. From two SLE patients, synovial fluid mononuclear cells
(SFMC) were analyzed by multiparameter flow cytometry to dissect T cell,
B cell, monocyte and dendritic cell phenotypes. SLE-derived SFMC were
further stimulated \emph{in vitro} to measure their capacity for
producing IFN\selectlanguage{greek}γ \selectlanguage{english}and IL-17A. All patients fulfilled the ACR 1982
classification criteria for SLE. Clinical records were reviewed to
exclude the presence of comorbidities such as osteoarthritis or overlap
with RA.

IL-17A and IL-6 levels were high in SLE synovial fluid. A clear subset
of the synovial CD4+ T cells expressed CCR6+, a marker associated with
Th17 cells. IL-17-production was validated amongst CD4+CCR6+ T cells
following \emph{in vitro} stimulation. Furthermore, a strong IFN\selectlanguage{greek}γ
\selectlanguage{english}production was observed in both CD4+ and CD8+ cells.

Our study shows high IL-17A and IL-6 levels in synovial fluids of
patients with lupus arthritis. The Th17 pathway have been implicated in
several aspects of SLE disease pathogenesis and

our data points to Th17 involvement also for lupus arthritis.

\textbf{MAIN MANUSCRIPT}

\textbf{Introduction}

Arthritis is a common manifestation in systemic lupus erythematosus
(SLE), observed in up to 90\% of patients \textsuperscript{1}. It often
occurs at disease onset and is part of classification
criteria\textsuperscript{2, 3}, but it is usually non-erosive, although
advanced imaging has revealed that chronic synovitis \textsuperscript{4}
and erosions \textsuperscript{5} are more common than previously
estimated.

Despite being frequent and causing a significant disease burden by
influencing quality of life \textsuperscript{6}, arthritis has received
less attention than other SLE manifestations, such as nephritis, that
directly impact morbidity and survival. While several studies have
investigated possible associations of arthritis with genetic risk
factors, immunological features, and autoantibodies \textsuperscript{6},
little is known of the local inflammatory cell phenotypes and cytokines.

In the 70ties and 90ties, descriptive studies of SLE synovial biopsies
have demonstrated microvascular changes, moderate proliferation of the
synovial lining layer as well as mononuclear cell infiltrates and fibrin
deposits\textsuperscript{7} in affected tissue. The synovial fluid was
found to typically contain less than 2000 leucocytes per microliter, and
to be characterized by lymphocytes and so called lupus erythematosus
(LE) cells, i.e. the presence of nucleic acids in the cytoplasm of
neutrophils and macrophages\textsuperscript{8}. Presence of anti-nuclear
antibodies (ANA) were also reported in synovial fluid of SLE
patients\textsuperscript{9}. In 2007, Toukap N et
al.\textsuperscript{10} analyzed the gene expression of synovial
biopsies and found that SLE patients display a distinct molecular
signature, with up-regulation of interferon inducible genes and
down-regulation of genes involved in extracellular matrix homeostasis.
However, the involvement of specific cell types or cytokines in lupus
arthritis still remains unknown.

In order to better understand SLE joint pathology, we analyzed cytokines
associated to T-cell responses and the cellular composition (when
present) of synovial fluid (SF) samples from SLE patients as compared to
paired peripheral blood samples and found an upregulation of IL-6 and
IL-17A in synovial fluid of SLE patients, as well as potential
pathogenic T cell subsets.

\textbf{Materials and Methods}

Patient material

SF was obtained from routine large joint aspiration from SLE, SpA and RA
patients fulfilling the 1982 ARA/ACR\textsuperscript{2},
ASAS\textsuperscript{11} and ACR classification
criteria\textsuperscript{12} respectively. Informed consent was given
prior to SF and blood sampling, as approved by the local ethics
committee.

Once collected, SF was centrifuged and stored as acellular at -80\selectlanguage{ngerman}°C. In
parallel, paired serum samples were also processed. The mononuclear cell
fractions from SF (when present) and peripheral blood were collected
following gradient density centrifugation over Ficoll-Hypaque and were
cryopreserved in DMSO/FCS in N\textsubscript{2}(l) until use.

SLE synovial fluid samples, once retrieved, were selected for inclusion
in the present study after revision of the electronic clinical charts,
in order to exclude biological material from patients with evidence of
joint involvement not primarily dependent on SLE. We therefore revised
clinical information to exclude primary osteoarthritis, overlap with
rheumatoid arthritis, crystal related arthritis, and other comorbidities
which could entail potential confounders.

From an initial list of 27 patients, we therefore selected 17 SLE
patients with joint involvement, excluding 10 patients (1 with SLE RA
overlap, 1 with onset of arthritis after the occurrence of a
haematological malignancy, 8 for evidence of primary OA or crystal
related arthritis). The clinical characteristics are listed in Table 1
(SLE patients used for the cytokine analysis) and Supplementary Tables
S1 (RA patients used for the cytokine analysis) and S2 (patient samples
for cell analysis).

Autoantibodies

IgA and IgM rheumatoid factor (RF) (Elia, Thermo Fisher Scientific,
Uppsala, Sweden) and anti-citrullinated peptide antibodies (ACPA;
CCPlus, EuroDiagnostica, Malmö, Sweden) were analyzed in sera and SF
from SLE patients according to the manufacturers' instructions.

Cytokine Bead Array

SF samples (n=17), matched serum samples (n=15) from SLE patients (Table
1), and SF samples from RA patients (n=10, Supplementary Table S1)
matched for storage time, were analyzed with cytokine bead array (CBA)
for T-helper-associated cytokines (BD Biosciences, San Jose, USA)
following the manufacturer's instructions. In brief, thawed serum
samples were diluted 1:2 with assay diluent and run-in duplicates in a
96-well plate. Cytokines tested were IL-17A, IFN\selectlanguage{greek}γ, \selectlanguage{english}IL-10, IL-6, IL-4,
IL-2 and TNF. The assay was run on a FACS Verse flow cytometer and
analyzed using the CBA software (BD Biosciences, San Jose, USA). Limits
of detections are displayed in Supplementary Fig. S2C.

Lymphocyte isolation and cell assays

SF mononuclear cells (SFMC) and paired peripheral blood samples (PBMC)
from two SLE, three SpA and two seronegative RA patients (matched for
age and sex, Supplementary Table S2), were analyzed by flow cytometry
panels for T cells and when possible for B cells, monocytes and
dendritic cells (Supplementary Table S4). Cryopreserved PBMC and SFMC
were thawed and stained with the corresponding antibody mixtures and
viability dye (Supplementary Table S4 and S5). For functional studies,
duplicates of 0.5\selectlanguage{ngerman}×10\textsuperscript{6} SFMC were stimulated with
anti\selectlanguage{english}-CD3/CD28 beads (1bead/cell, Dynabeads, Thermo-Fisher, Waltham, USA)
for 16h in 96\selectlanguage{english}-well flat\selectlanguage{english}-bottom plate. The last 4h brefeldin-A (5ug/ml,
Sigma Aldrich, St. Louis, USA) was added. Analyses were run on a
Fortessa LSR cell analyzer (BD Biosciences, San Jose, USA) and data were
processed by FlowJo software (BD Biosciences, San Jose, USA). Gating
strategies are displayed in Supplementary Fig. S1.

Statistical analysis

Continuous variables are expressed as median and interquartile range
(IQR), categorical variables as numbers and percentages. Non-parametric
statistics was used to explore differences and correlations as
appropriate. P-values \textless{}0.05 were deemed statistically
significant. Prism7 software (GraphPad, San Diego, USA) was used.

\textbf{Results}

Clinical characteristics

Clinical characteristics of the 17 SLE patients at the time of SF
sampling are summarized in Table 1. The patients were fairly
representative of a typical SLE population. Samples were collected at a
time of long disease history including long standing arthritis, often
polyarticular and which had occurred among the first disease
manifestations. Six of the patients (age at onset 28y, IQR 17-38) had
developed secondary knee osteoarthritis over time.

IL-17A and IL-6 are highly expressed in SLE SF

First, we wanted to investigate T-cell associated cytokines in SF of SLE
patients. Thus, SF from SLE patients (n=17) were tested for cytokine
content in comparison to paired serum samples (n=14) and SF from RA
patients (n=10), matched for storage time (Supplementary Table S2). In
SLE, IL-17A was detected in SF (44.9 pg/ml (10.7 - 405.2)) and matched
serum (37.5 pg/ml (22.4 - 94.2)) (Fig. 1A), although no statistically
significant difference was shown (\emph{p=0.4} ).

IL-6 was significantly higher in SLE-SF (771 pg/ml (108.5-2135)) as
compared to corresponding serum samples (1.7 pg/ml
(1.2-4.9),\emph{p=0.0006} ). Low concentrations of IL-10 could be
detected in 41\% of SLE SF samples (0 pg/ml (0-3.4)) and was higher in
serum (0.29 pg/ml (0.1-0.4)). Overall, the RA SF samples displayed
similar ranges of cytokines as SLE SF, with higher median values of IL-6
(3627 pg/ml (334.6-5000), p=0.11) and IL-10 (1.9 pg/ml (0-7.9), p=0.19)
and less IL-17A, (0 pg/ml (0-172), p=0.11) however without statistical
significance (Fig. 1A). The cytokine levels did not differ between SLE
patients with and without secondary osteoarthritis (data not shown).

Next, we explored the relationship of cytokine levels, in and between
compartments (Fig. 1B-C). In SLE SF, the levels of IL-6 correlated with
those of IL-10 (r=0.84, \emph{p=\textless{}0.0001} , CI=0.69-0.92) and,
weakly, with IL-17A (r=0.39, \emph{p=0.03} , CI=0.02-0.66) (Fig. 1B).
Also, IL-10 and IL-17A correlated weakly (r=0.43, \emph{p=0.01} ,
CI=0.07-0.69). When comparing serum and SF, levels of IL-6
correlated\emph{(r=0.7, p=0.0001)} but neither IL-17A nor IL-10 (Fig.
1C).

Cytokine analyses included also IFN-\selectlanguage{greek}γ, \selectlanguage{english}TNF, IL-2 and IL-4. In sera, IL-4
and IL-2 were higher compared to SF, however their concentrations were
consistently low (Supplementary Fig. 1A). Very low levels of IFN-\selectlanguage{greek}γ \selectlanguage{english}could
be detected in SF of four SLE patients. TNF levels were below the
detection limit in all samples (Supplementary Fig. 2B).

Subsequently, we investigated the presence of IgA and IgM RF in serum
and SF. These did not correlate with the cytokine levels nor with other
autoantibodies (data not shown). Interestingly, the concentration of RF,
when present, was mostly higher in serum compared to SF (Fig. 1D).

For one SLE patient, SF samples from six timepoints were available, the
first obtained approximately 15 years after disease onset. This patient
presented with a recurrent polyarthritis, but no evidence of secondary
OA could be tracked in the clinical records. High IL-6 levels were
repeatedly measured over a 10 years period, while IL-17A levels
fluctuated and IL-10 concentrations were low (Fig. 2A).

Cellular composition of SLE synovial fluid

Subsequently, we were interested in the cellular composition found in SF
of SLE patients. SFMC were available from two SLE patients, one with as
multiple timepoints as described above and another with cells from two
timepoints. These samples were compared with SFMC from three SpA and two
age and sex matched seronegative RA patients. All samples were
investigated for T-cell phenotypes and when possible also for B cell,
monocyte and dendritic cell subsets (Supplementary Table S4).

In SLE SF, lymphocytes consisted mainly of CD3+ T cells, with CD8+ cells
being more common in SF than in PB (Fig. 2B, Supplementary Fig. 3A). B
cells which were rare (\textless{}1\%) displayed a memory phenotype, for
one patient including age associated CD11c+ CD27+ B cells (Fig. 2B).

Examining the monocyte and dendritic cell (DC) subsets in SLE-SF,
64-67\% of the HLA-DR+ cells expressed CD14 without expressing CD16, a
sign for classical monocytes (Fig.2B, Supplementary Fig. S3C).
Non-monocyte cells represented \textasciitilde{}30\% of HLA-DR+ cells in
SF, and were mostly classical DC (CD11c+, cDC), in patient one mainly
cDC1 while the other exhibited equal proportions of cDC1 and cDC2.The
frequency of plasmacytoid DC varied between patients from 7-26\% (Fig.
2B, Supplementary Fig. S3C).

T helper17 signatures in SF of SLE patients

Focusing on T-cell phenotypes, we visualized the distribution by tSNE
analysis of the CD3+ T cells. As depicted in Fig. 2C, PBMC and SFMC
T-cell cluster in different directions. Within the CD4+ compartment, we
observed higher frequencies of CCR4+, CXCR5+, CCR6+ and similar levels
of CXCR3+ in SF compared to PB. The most prominent T-helper subset in SF
was CCR6+ cells, a marker for Th17 cells. 35-55\% of CD4+ T cells
expressed CCR6 (Fig. 2C, Supplementary Fig. 3D), with
\textasciitilde{}40\% co-expressing CCR4. Moreover, Tregs (CD25+
FoxP3+), Eomes+, Granzyme A+ (GZMA), were also enriched in SF as
compared to PB (Fig. 2C). PD1+HLA-DR+ cells were also abundant, and we
identified a distinct population of T-peripheral helper
(T\textsubscript{PH};
PD1\textsuperscript{high}HLA-DR\textsuperscript{high}) cells in SF, of
which \textasciitilde{}30\% co-expressed CCR6 (Fig. 3). In SF,
\textasciitilde{}70-80\% of CD8+ T cells expressed Eomes and GZMA and a
high proportion of CD8+ PD-1+ HLA-DR+/- cells were detected in all SF
samples (Fig. 2C, Supplementary Fig. S3D). CX3CR1 was not expressed on
CD4+ nor CD8+ T cells in SF. SF T-cell phenotypes in SLE, RA and SpA,
presented similar patterns.

From one SLE patient, SFMC from two timepoints were available
(Supplementary Fig.S3E). For most CD8+ and CD4+ subsets, similar
frequencies were detected with the exception of GZMA+ Eomes- T cells
which were more prominent at the first timepoint. In line with the other
SLE patient, CCR6+ CD4+ T cells was one of the most prominent subsets in
SF.

Since we observed both IL-17A and increased CCR6+ CD4+ T cells in SF of
SLE patients, we investigated the capacity of SF-derived T cells to
produce IL-17A. Following \emph{in vitro} stimulation of SFMCs from one
SLE and one SpA sample, intracellular IL-17A and IFN\selectlanguage{greek}γ \selectlanguage{english}production was
analyzed. In both samples, IL-17A production was induced and confined to
the CCR6+ CD4+ cells (Fig.4, Supplementary Fig.S4). We also observed
abundant IFN\selectlanguage{greek}γ \selectlanguage{english}production in IL-17-negative CD4+ T cells. Of note CD8+ T
cells also produced IFN\selectlanguage{greek}γ, \selectlanguage{english}but not IL-17A.

\textbf{Discussion}

We found that local joint inflammation in SLE is dominated by IL-6 and
IL-17. IL-6, a pleiotropic cytokine, can influence the function of
several immune cells such as B and T cells, orientating their fate
towards Th17 and T follicular helper phenotypes \textsuperscript{13}.
Its local expression in SLE inflamed large joints might also suggest a
more acute joint pathology and an effect on recruiting neutrophils.
Indeed, most of the SLE SF samples contained only neutrophils and became
acellular following the cell purification step. Previous studies
investigating IL-6 plasma concentration in relation to SLE arthritis
have shown that elevated IL-6 was associated with active ongoing
arthritis but not with CRP levels \textsuperscript{14, 15} and also a
correlation between IL-6 plasma level and joint
involvement\textsuperscript{15}. In our study, IL-6 SF levels also
correlated with IL-6 serum levels, albeit IL-6 concentration was much
lower in serum, suggesting a more relevant local role for this cytokine.

IL-17A, known to trigger the recruitment of neutrophils and monocytes to
the site of inflammation \textsuperscript{16} was abundant in SLE SF
samples, similarly to what has been observed in ankylosing
spondylitis\textsuperscript{17}. In recent years, several studies have
highlighted the possible implications of the IL-17 axis in SLE
pathogenesis\textsuperscript{18}. IL-17A has been found to be increased
in sera of SLE patients compared to healthy controls, expressed in
kidney tissue of patients with lupus nephritis and to predict an
unfavorable histopathological response to immunosuppressive
treatment\textsuperscript{19}. Current clinical trials are evaluating
IL-17A inhibition in refractory lupus nephritis
(https://www.clinicaltrials.gov/ct2/show/NCT04181762).

This is the first study that, to our knowledge, investigates the
cellular composition of SF in SLE. In most cases, the numbers of SF
mononuclear cells were low, thereby only the fluid could be preserved.
Still, we were able to analyze mononuclear cells from two synovial
fluids from SLE patients. Similar to other inflammatory
arthritides\textsuperscript{20, 21} an increase of Tregs could be
observed in SLE SF. We could also identify a
PD-1\textsuperscript{high}HLA-DR\textsuperscript{high}
T\textsubscript{PH} population in SF of SLE patients. This T-cell subset
has been described in ACPA+ RA patients and functions by promoting
B-cell responses and antibody production\textsuperscript{22}. Similarly,
PD1\textsuperscript{high}CXCR5- T\textsubscript{PH} have been described
in SLE where they appear to drive the dynamics of pathologic B-cell
responses, correlate with disease activity and are expressed in kidney
tissue in lupus nephritis\textsuperscript{23}.

In our study, CCR6+ CD4+ T cell subsets were abundant in SLE-SF. CCR6+
is a chemokine receptor expressed on Th17 cells and Th17 differentiation
is dependent on the cytokines IL-6, IL-1\selectlanguage{greek}β \selectlanguage{english}and TGF\selectlanguage{greek}β\selectlanguage{english}\textsuperscript{24}.
Th17 cells have previously been reported to increase during SLE flare
and to be reduced after successful treatment\textsuperscript{25}. Leipe
J et al.\textsuperscript{26} reported an increase of Th17 cells in
psoriatic arthritis and RA patients in peripheral blood and synovium,
with an enrichment of Th17 in SF in the affected joint. Similarly, CCR6+
cells were increased in SF of the two analyzed SLE patients compared to
peripheral blood.

Due to the increase of IL-17A and CCR6+ T cells in SF of SLE patients,
we stimulated SFMC with anti-CD3/CD28 beads to study their capacity to
produce cytokines. Thereby, we identified IL-17A producing T cells,
primarily represented by CCR6+CD4+ T cells. It is however possible that
other cells also could contribute to IL-17A production as both CD4 and
CD8, as well as \selectlanguage{greek}γδ \selectlanguage{english}T cells, innate lymphoid cells and mast cells have
been identified as IL-17A producers in psoriasis and psoriatic
arthritis\textsuperscript{27}.

While CCR6+ was one of the most prominent CD4+ T-cell subsets in SLE-SF,
the vast majority of SF CD8+ T cell expressed the transcription factor
Eomes and GZMA similar to observations in RA\textsuperscript{28}. GZMA
induces pro-inflammatory cytokines such as IL-6, IL-8, IL-1\selectlanguage{greek}β.
\selectlanguage{english}Altogether, our analysis suggests an involvement of T cells in the
pathogenesis of lupus arthritis.

The main limitation of our study is that it was conducted
retrospectively on historical material. Systematic assessment of joint
involvement and SLE disease activity were not performed at the time of
sampling, thus clinical associations could not be further explored.
Moreover, the sampling was performed only on large joints, which are
technically more accessible but imply some limitations, since SLE
arthritis more typically involves small joints. The long disease
duration in the SLE patients with ongoing and previous treatments could
also modify the SF composition. Being most often acellular or
paucicellular, a strength of our study is the inclusion of two cellular
samples of SLE SF.

From a clinical perspective, lupus arthritis is common already from the
time of diagnosis but is a manifestation often responding to treatment.
This may be a reason why arthritis has been given relatively less
attention from a research perspective. Our study cohort is admittedly
small but still represents one of the most comprehensive collections of
SF material in SLE. It is tempting to speculate that lupus arthritis is
less autoimmune in character as compared to rheumatoid arthritis and our
data lend support to such a theory by suggesting that it is part of a
systemic immune deviation towards Th17 and not joint specific. Indeed,
Th17 responses implicate the involvement of innate immunity through
neutrophils, cells that historically have been linked to SLE
pathophysiology as LE cells\textsuperscript{8}.

In conclusion, our study provided new exploratory insights into the
immunology of lupus arthritis. IL-17A and IL-6 levels were high in
synovial fluid of SLE patients. The presence of CCR6+ CD4+ T cells,
GZMA+ EOMES+ as well as PD1+ HLA-DR+ CD4+ and CD8+ T cells suggest a
pathogenic role of T cells in lupus arthritis. Our findings suggest that
SLE patients with arthritis may improve if treated with IL-17A blockade
for other clinical manifestations.

\textbf{References:}

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2. Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF, et al.
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erythematosus. Arthritis Rheum 1982; 25:1271-7.

3. Petri M, Orbai AM, Alarcon GS, Gordon C, Merrill JT, Fortin PR, et
al. Derivation and validation of the Systemic Lupus International
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erythematosus. Arthritis Rheum 2012; 64:2677-86.

4. Zayat AS, Mahmoud K, Md Yusof MY, Mukherjee S, D'Agostino MA, Hensor
EMA, et al. Defining inflammatory musculoskeletal manifestations in
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TABLE 1: Characteristics of the Systemic Lupus Erythematosus (SLE)
patients analyzed with the cytokine bead array\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
Clinical characteristic & \textbf{N=17}\tabularnewline
\midrule
\endhead
Gender (Females N (\%)) & 16 (94.1)\tabularnewline
Age at disease onset (years, M, (IQR) & 31(23-42.5)\tabularnewline
Age at SF sampling (years, M, (IQR) & 57 (48.5-63.2)\tabularnewline
Disease duration (years, M, (IQR) & 16 (6-34)\tabularnewline
Active smoking at sampling (N (\%)) & 6 (35.3)\tabularnewline
Organ involvement &\tabularnewline
Lupus Nephritis (N (\%)) & 3 (17.6)\tabularnewline
Cutaneous manifestations (N (\%)) & 6 (35.3)\tabularnewline
Neurolupus (N (\%)) & 3 (17.6)\tabularnewline
Serositis (N (\%)) & 2 (11.8)\tabularnewline
Secondary APS (N (\%)) & 3 (17.6)\tabularnewline
Treatment at sampling &\tabularnewline
Glucocorticoids (N (\%)) & 10 (58.8)\tabularnewline
Antimalarial (N (\%)) & 10 (58.8)\tabularnewline
Methotrexate (N (\%)) & 3 (17.6)\tabularnewline
Combined therapy\# (N (\%)) & 6 (35.3)\tabularnewline
No active treatment (N (\%)) & 2 (11.8)\tabularnewline
Autoantibodies &\tabularnewline
ANA (N (\%)) & 17 (100)\tabularnewline
anti-dsDNA (N (\%)) & 6 (35.3)\tabularnewline
RF (N (\%)) & 2 (11.8)\tabularnewline
anti-SSA/SSB (N (\%)) & 4 (23.5)\tabularnewline
anti-RNP (N (\%)) & 3 (17.6)\tabularnewline
anti-PL (N (\%)) & 9 (52.9)\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table legend}

*manifested during the course of the disease; \^{}dose range 2.5-15 mg
daily,

\textsuperscript{\#} combination of either GC and antimalarial, GC and
MTX, GC-MTX and antimalarial

\emph{Abbreviations} M: median, IQR: interquartile range, GC:
glucocorticoids, MTX: methotrexate; ANA: antinuclear antibodies;
Anti-dsDNA: anti-double stranded DNA; RF: rheumatoid factor;
Anti-SSA/SSB: anti-Sj\selectlanguage{ngerman}ögren's syndrome-related antibody A, B; Anti-RNP:
anti-ribonucleoprotein; aPL: antiphospholipid antibodies, \^{}mostly
anti-cardiolipin;

\textbf{FIGURES}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1:}

\textbf{Cytokine analysis of SLE SF samples} (A) IL-17A, IL-6 and IL-10
cytokine levels were measured in SLE SF samples (n=17), matched serum
samples (n= 14, combined with lines) and SF RA samples (n=10) and
significant difference was analyzed using Mann Whitney test. Bars
indicate median. (B) Spearman Correlations between IL17A, IL-6 and IL-10
levels in SF are displayed. (C) Spearman Correlation between IL-17A and
IL-6 levels in serum vs SF. (D) IgA and IgM RF levels in serum and SF
are shown. Red line indicates cut off: IgA RF = 18 IU/ml; IgM RF = 10
IU/ml)\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2:}

\textbf{Longitudinal Cytokine analysis and Cellular composition in SLE
SF} (A) Longitudinal cytokine analysis of SLE-SF. Change of medication
and the timepoint obtaining SFMC are indicated. (B) Proportions of cell
subsets in SF and matched PB. B-cell subsets: plasmablasts
(CD27++CD38++); na\selectlanguage{ngerman}ïve (IgD+CD27-), double positive (IgD+CD27+), memory
(IgD-CD27+), double negative (DN, IgD-CD27-). Monocyte subsets:
non-classical (CD14-C16+), intermediate (CD14+C16-+), classical
(CD14+C16-) and non-monocytes (CD14-CD16-). Dendritic Cell subsets:
classical DC (cDC, CD11c+), cDC1 (CD11c+CD1c+), cDC2 (CD11c+CD141+) and
pDC (CD11c-CD123+CD303+). Frequencies \textless{}4\% are not displayed.
(C) tSNE clustering of CD3+ cells from the two T-cell panels
(Supplementary Table S4) and CD4+ and CD8 T-cell subsets in paired PB
and SF (n=1)\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3:}

\textbf{T peripheral helper (TPH) cells in SF of SLE patients} (A)
Frequency of T\textsubscript{PH} cells in SF and serum samples in SLE
(n=2), SpA (n=3) and age and sex matched RA (n=2) patients. (B) Gating
plots of the SF samples of the two SLE patients.
T\textsubscript{PH}cells are defined as CD4+
PD-1\textsuperscript{high}HLA-DR\textsuperscript{high} cells gated by
quadrant gating. (C) Frequency of CCR6+ expression in
T\textsubscript{PH} cells in the three different diseases (D) SLE-SF
plots showing CCR6 gates after gating for CD4+
PD-1\textsuperscript{high} HLA-DR\textsuperscript{high} cells.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4:}

\textbf{T-cell stimulation of SFMC from SLE and SpA} Intracellular flow
cytometry dot plots of (A) CCR6 vs IL-17A and (B) IFN? vs IL-17A in
stimulated SFMC (n=1 SLE and n=1 SpA) The cells were stimulated over
night with anti-CD3/CD28 beads.

\textbf{Acknowledgements}

We would like to thank Susana Hernandez Machado, Gloria Rostvall and
Julia Norkko, for helping with sample collection and management and
Vijay Joshua for helping with the CBA software. This study was
conceived, designed and coordinated by NS, FF, KC, IG; VM. FF and ST
contributed to data collection and clinical assessments. Experimental
work was performed by NS in supervision of KC and VM. Rheumatoid Factor
data was obtained by JR. The data analysis and interpretation were
conducted by NS, FF, KC, VM, IG and the writing was drafted by FF and
NS. This work was supported by The King Gustaf V 80th Birthday Fund,
Stockholm County Council (ALF and for ST: combined clinical residency
and PhD training program), The Swedish Rheumatism Association, Ingegerd
Johansson's Foundation, and The Swedish research council.

\textbf{Disclosures}

The authors have no conflict of interest to disclose.

\textbf{Ethical statement:}

Our study complies with the Declaration of Helsinki and has been
approved by locally appointed ethical committee.

\selectlanguage{english}
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\end{document}

