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P038 In vivo demonstration of tmTNF reverse signaling: significance in the therapeutic response to anti-TNF agents during murine arthritis

Authors:

Abstract

Career situation of first and presenting author Post-doctoral fellow. Introduction Anti-TNF agents are widely used in rheumatoid arthritis (RA). Their effect on inflammation results from the neutralization of soluble TNF (sTNF), but also supposedly from the induction of reverse signaling through their binding to membrane TNF (tmTNF). Despite possible clinical relevance, reverse signaling has been described only in vitro but has not been proven in vivo. Objectives In this study we aim to demonstrate for the first time the existence of tmTNF reverse signaling in vivo and its importance in anti-TNF response during arthritis. Methods Triple transgenic mouse model (3TG), KO for TNFR1/TNFR2 and KI for tmTNF, thus secreting no sTNF was developed. To analyze reverse signaling, mice were injected either with etanercept (ETA, 10 mg/kg), an anti-mouse TNF antibody (MP6-XT22, rat IgG1, 10 mg/kg) or an anti-human IL17 antibody (secukinumab, SEC, 10 mg/kg) as a control. Daily clinical evaluation of K/BxN serum induced-arthritis was performed in 3TG as well as WT mice. Polarization of bone marrow-derived macrophages (BMDM) and cytokine production from non-arthritic WT and 3TG mice under the action of anti-TNF in vitro was evaluated by RT-qPCR, CBA and ELISA. Results In vivo, the administration of anti-TNF (ETA or MP6-XT22) decreased arthritic scores in WT mice (p=0.005) as well as in 3TG mice (p<0.001), unlike SEC which had no effect, proving that anti-TNF binding of tmTNF decreased arthritis. In vitro effect of anti-TNF on BMDM from WT as well as 3TG mice induced a decrease in the expression of genes specific of inflammatory macrophages (CD38, GpR18 and FpR2), and an increase in the expression of genes specific of alternative macrophages (Arg1, EgR2, c-Myc). We also observed an inhibition of the secretion of pro-inflammatory cytokines (IL12p70 and IL-6) and an early peak of IL-10 secretion demonstrating an effect of reverse signaling on macrophage polarization and activation. This suggested a switch in macrophage polarization as a probable mechanism for modulation of inflammation during K/BxN serum-induced arthritis. Conclusions Our work provides in vivo evidence for the involvement of reverse signaling in the anti-TNF-mediated modulation of arthritis. Reverse signaling is expected to result in the modulation of macrophage polarization from an inflammatory to an alternative functional phenotype in arthritic mice. Our data prompt us to consider new interpretation of the effects of anti-TNF in the treatment of RA. Disclosure of Interest None declared.
Methods Phenotypic analysis of peripheral blood B-lympho-
cytes was made by flow-cytometry in a cohort of SLE patients
treated with belimumab. SLE-disease activity was assessed by
SLEDAI-2K score. BAFF was tested by ELISA. SPSS was used
for statistical analysis.
Results The relative change of BAFF levels at 6 and 12
months from baseline showed linear correlation with the per-
centage of naïve B-cells (Pearson correlation=0.645,
p=0.044 and 0.639, p=0.002, respectively) and of transitional
B-cells (Pearson correlation=0.768, p=0.009 and 0.623,
p=0.055, respectively). The percentage and absolute number
of naïve B-cells showed a progressive decrease during time
(ANOVA, p=0.013 and p=0.001 respectively). In terms of
response prediction, a significant association of SLEDAI per-
centage improvement at 12 months with the decrease of total
number of B-cells within the first 6 months of therapy was
observed (Log regression r=0.707, p=0.05).
Conclusions BAFF inhibition induces B-cell number modifica-
tions in a SLE cohort. The reduction of total number of B-
cells within the first six months shows predictive value for
SLEDAI response after the first year of therapy.
REFERENCES
1. Vossenkämper A, Lutalo PM, Spencer J. Translational mini-review series on B cell
subsets in disease. Transitional B cells in systemic lupus erythematosus and Sjög-
rens syndrome: clinical implications and effects of B cell-targeted therapies. Clin
Exp Immunol 2011;167(1):714.
2. Boneparth A, Davidson A. B-cell activating factor targeted therapy and lupus.
Arthritis Res Ther 2012;14 (Suppl 4):S2.
3. Jacobi AM, Huang W, Wang T, Freimuth W, Sanz I, Furie R, et al. Effect of long-
term belimumab treatment on B cells in systemic lupus erythematosus. Arthritis
Rheum 2010;62:201210.
Acknowledgements The authors are grateful to Carla Bosio
and Alessandra Paletti, laboratory technicians at the Laboratory
of Rheumatology and Clinical Immunology in Brescia, for
their valuable collaboration. The authors wish to thank the
nurses of the Rheumatology and Clinical Immunology Unit in
Brescia for their support in blood collection.
Disclosure of Interest None declared.
P037 ANTI-CARBAMYLATED PROTEIN ANTIBODIES : ARE
THEY USEFUL FOR THE DIAGNOSIS OF RHEUMATOID
ARTHRITIS?
1
F Ponchel*,
1
LHunt,
2
M van Delft,
1
PEmery,
2
L Trouw.
1
LIRMM, University of Leeds,
Leeds, UK;
2
Rheumatology, LUMC, Leiden, Netherlands
10.1136/annrheumdis-2018-EWRR2019.29
Career situation of first and presenting author Resident.
Introduction The current EULAR-2010 diagnostic criteria for
Rheumatoid Arthritis (RA) relies heavily on the presence of
auto-antibodies. So far only anti-citrullinated peptide antibody
(ACPA) or rheumatoid factor (RF) are accounted for however,
novel auto-reactivities have been described notably anti-carbamy-
lated protein (anti-CarP) and anti-oxydized peptide antibodies.
Objectives To investigate the potential of anti-CarP antibodies
for the diagnosis of RA.
Methods 402 patients were recruited from an early arthritis
register (ethically approved) with a diagnosis recorded over 24
months. 95 healthy controls were included. All participants
provided written informed consent. An anti-CarP antibody
ELISA was performed using carbamylated FCS as target anti-
gen.
1
Results were expressed as aU/mL. Statistical analysis
used ROC and AUC analysis established using SPSS V22.
Results We first determined the 95% CI of the distribution of
ELISA results in HCs to define a negative/positive cut-off for
the test, established at >250 aU/mL.
195/402 patient were classified as RA at inclusion; 28
developed RA over time while 56 remained undifferentiated
arthritis; 83 had non persistent symptoms and 40 other
inflammatory joint diseases.
Anti-CarP ELISA results were clearly much higher in RA
(median 250 [IQR 25762], p<0.0001) with 49% of patients
being positive as well as in the group that progressed to RA
(25% positive, 82 [13235], p=0.001), compared to all other
EAC patients together (8% positive, 26 [580]) or HC (5%
positive, 41 [1106]).
In this particular cohort, anti-CarP had an odd ratio
OR=5.55 for predicting RA with a sensitivity SEN=91.5%
and positive predictive value PPV=87%, compared to ACPA
which had an OR=8.78 (SPE=92%, PPV=92%) and RF with
an OR=4.18 (SPE=85%, PPV 84%).
Titres were not associated with other variables (age, gender,
JC, CRP or DAS). A ROC analysis determined that linear
data (aU/mL) for anti-CarP were not better than a positive/
negative status at predicting RA.
A score counting the number of auto-antibodies (01-23)
had a high predictive value (AUC=0.840, p<0.0001 SEN/SPE
of 65%/95% and PPV/NPV of 94%/68%).
We then established that a combination of ACPA+antiCarP
was superior at predicting RA (OR=25.1 SEN/SPE=43%/
98.5%, PPV/NPV=97%/58%) than the ACPA+RF combination
(OR=11.9, SEN/SPE=54%/95%, PPV/NPV=94%/62%),
although less sensitive.
Conclusions Although RF is more frequently observed than
anti-CarP, it is also less specific for RA. A combination of
ACPA+anti CarP may prove more useful at classifying early
arthritis patients for RA than an ACPA+RF combination.
Future work needs to establish a standardised assay for anti-
CarP and replicate this data.
Disclosure of Interest None declared.
P038 IN VIVO DEMONSTRATION OF TMTNF REVERSE
SIGNALING: SIGNIFICANCE IN THE THERAPEUTIC
RESPONSE TO ANTI-TNF AGENTS DURING MURINE
ARTHRITIS
1
BRauwel*,
2
Nsimons,
1
KDiallo,
1
MBaron,
1,2
Y Degboe,
3
A kruglov,
4
S Nedospasov,
1,2
AConstantin,
1,2
ACantagrel,
1
J-L Davignon.
1
Inserm UMR 1043 CPTP;
2
CHU Purpan
service Rhumatologie, Toulouse, France;
3
German Rheumatism Research Centre, Berlin,
Germany;
4
Engelhardt Institute of Molecular Biology, Moscow, Russian Federation
10.1136/annrheumdis-2018-EWRR2019.30
Career situation of first and presenting author Post-doctoral fellow.
Introduction Anti-TNF agents are widely used in rheumatoid
arthritis (RA). Their effect on inflammation results from the
neutralization of soluble TNF (sTNF), but also supposedly
from the induction of reverse signaling through their binding
to membrane TNF (tmTNF). Despite possible clinical rele-
vance, reverse signaling has been described only in vitro but
has not been proven in vivo.
Objectives In this study we aim to demonstrate for the first
time the existence of tmTNF reverse signaling in vivo and its
importance in anti-TNF response during arthritis.
Methods Triple transgenic mouse model (3TG), KO for
TNFR1/TNFR2 and KI for tmTNF, thus secreting no sTNF
was developed. To analyze reverse signaling, mice were injected
Abstracts
A14 ARD 2019;78(Suppl 1):A1A83
on January 8, 2021 by guest. Protected by copyright.http://ard.bmj.com/Ann Rheum Dis: first published as 10.1136/annrheumdis-2018-EWRR2019.30 on 1 March 2019. Downloaded from
either with etanercept (ETA, 10 mg/kg), an anti-mouse TNF
antibody (MP6-XT22, rat IgG1, 10 mg/kg) or an anti-human
IL17 antibody (secukinumab, SEC, 10 mg/kg) as a control.
Daily clinical evaluation of K/BxN serum induced-arthritis was
performed in 3TG as well as WT mice. Polarization of bone
marrow-derived macrophages (BMDM) and cytokine production
from non-arthritic WT and 3TG mice under the action of anti-
TNF in vitro was evaluated by RT-qPCR, CBA and ELISA.
Results In vivo, the administration of anti-TNF (ETA or MP6-
XT22) decreased arthritic scores in WT mice (p=0.005) as
well as in 3TG mice (p<0.001), unlike SEC which had no
effect, proving that anti-TNF binding of tmTNF decreased
arthritis. In vitro effect of anti-TNF on BMDM from WT as
well as 3TG mice induced a decrease in the expression of
genes specific of inflammatory macrophages (CD38, GpR18
and FpR2), and an increase in the expression of genes specific
of alternative macrophages (Arg1, EgR2, c-Myc). We also
observed an inhibition of the secretion of pro-inflammatory
cytokines (IL12p70 and IL-6) and an early peak of IL-10
secretion demonstrating an effect of reverse signaling on mac-
rophage polarization and activation. This suggested a switch in
macrophage polarization as a probable mechanism for modula-
tion of inflammation during K/BxN serum-induced arthritis.
Conclusions Our work provides in vivo evidence for the
involvement of reverse signaling in the anti-TNF-mediated
modulation of arthritis. Reverse signaling is expected to result
in the modulation of macrophage polarization from an inflam-
matory to an alternative functional phenotype in arthritic
mice. Our data prompt us to consider new interpretation of
the effects of anti-TNF in the treatment of RA.
Disclosure of Interest None declared.
P039/O03 CIRCULATING FOLLICULAR HELPER T CELLS ARE
INCREASED IN SYSTEMIC SCLEROSIS AND PROMOTE
PLASMABLAST DIFFERENTIATION THROUGH THE IL-21
PATHWAY WHICH CAN BE INHIBITED BY RUXOLITINIB
1
L Ricard*,
1
VJachiet,
2
FMalard,
1
YYe,
1
N Stocker,
3
S Rivière,
4
PSenet,
4
J-B Monfort,
3
OFain,
2
M Mohty,
1
B Gaugler,
3
A Mekinian.
1
Centre de recherche Saint-Antoine, INSERM;
2
Hématologie;
3
Médecine Interne, pital Saint-Antoine;
4
Dermatologie, Hôpital Tenon,
Paris, France
10.1136/annrheumdis-2018-EWRR2019.31
Career situation of first and presenting author Student for a
master or a PhD
Introduction Systemic sclerosis (SSc) is an autoimmune disease
characterized by widespread fibrosis, microangiopathy and
autoantibodies. Follicular helper T (Tfh) cells
CD4
+
CXCR5
+
PD-1
+
cooperate with B lymphocytes to induce
the differentiation of plasmocytes secreting Immunoglobulins
(Ig). Circulating Tfh (cTfh) cells are increased in several auto-
immune diseases.
1
Tfh cells can infiltrate the skin of SSc
patients, and induce fibrosis in vitro.
2
Objectives The aim of this study was to perform a quantita-
tive and functional analysis of cTfh cells in SSc.
Methods Using flow cytometry, we analyzed cTfh cells from
52 SSc patients with no immodulotary treatment and 38
healthy controls (HC). In vitro coculture experiments of
sorted cTfh and B cells from 13 SSc patients and 6 HC were
performed for functional analysis. IgG and IgM production
were measured by ELISA. BCL-6, Blimp-1, CXCL13 and IL-
21 expression were analyzed by RTqPCR.
Results We observed that cTfh cell numbers are increased in
SSc patients compared with HC. Furthermore, the increase in
cTfh cells was more potent in patients with severe forms of
SSc such as diffuse SSc and in the presence of arterial pulmo-
nary hypertension. cTfh cells from SSc patients present an
activated Tfh phenotype, with high expression of BCL-6 and
increased capacity to produce IL-21 in comparison to HC. In
vitro, cTfh cells from SSc patients had higher capacity to stim-
ulate the differentiation of CD19
+
CD27
+
CD38
hi
B cells and
their secretion of IgG and IgM through the IL-21 pathway
than cTfh cells from HC. Blocking IL-21 or using the JAK1/2
inhibitor ruxolitinib reduced the Tfh cells' capacity to stimu-
late the plasmablasts and Ig production.
Conclusions Circulating Tfh cells are increased in SSc and cor-
relate with SSc severity. The IL-21 pathway or JAK1/2 block-
ade by ruxolitinib could be a promising strategy in the
treatment of SSc.
REFERENCES
1. Crotty S. T follicular helper cell differentiation, function, and roles in disease.
Immunity 2014;41(4):529-42.
2. Taylor DK, Mittereder N, Kuta E, Delaney T, Burwell T, Dacosta K, et al. T follicu-
lar helper-like cells contribute to skin fibrosis. Sci Transl Med 2018;10(431).
Acknowledgements This work was supported by the Aterhit
foundation and received grants from 'Groupe Francophone de
Recherche sur la Sclérodermie(GFRS).
Disclosure of Interest None declared
P040 THE EUROPEAN CONSENSUS FINDING STUDY GROUP
ON AUTOANTIBODIES 2017/18 INVESTIGATION.
CHARACTERISATION OF AUTOANTIBODY CONTENT IN A
NEW INTERNATIONAL REFERENCE STANDARD FOR
DENSE FINE SPECKLED 70KD (DFS70) AUTOANTIBODIES
1
J Rönnelid*,
2
M Blüthner,
3
C Dahle,
4
L Andrade,
5
E Feist,
6
D Hamann, on behalf of The
European Consensus Finding Study Group on autoantibodies (ECFSG)*.
1
Uppsala Univ,
Dept Immunol, Genet and Pathol, Uppsala, Sweden;
2
Laboratory Volkmann, Karlsruhe,
Germany;
3
Linköping Univ, Dept Clin Exp Med, Linköping, Sweden;
4
Dept Rheumatol, São
Paulo Univ, São Paulo, Brazil;
5
Charité, Dept Rheumatol Clin Immunol, Berlin, Germany;
6
Utrecht Univ, LTI Diagnostics, Utrecht, Netherlands
10.1136/annrheumdis-2018-EWRR2019.32
Career situation of first and presenting author Instructor.
Introduction The European Consensus Finding Study Group
on autoantibodies (ECFSG) a.k.a. the EULAR autoantibody
study group has been active for 30 years.
Objectives To reach consensus about autoantibody measure-
ments in clinical practice, and to evaluate upcoming autoanti-
body standard reagents concerning autoantibody content.
Methods ECFSG focus on evaluating difficult to interpret
serum samples, where differences between assays can be
clearly visible. Ten unknown samples are distributed yearly to
European laboratories, and analyzed broadly. Results are col-
lected with information about laboratory techniques used, and
discussed in relation to clinical information on the donating
patients during EWRR. The 2017/2018 investigation contained
nine patient samples, and a not yet launched pooled standard
for anti-dense fine speckled 70kD antibodies, an ANA reactiv-
ity with specific nuclear staining on HEp-2 cells that can be
confounded with homogenous ANA, but that is not associated
with autoimmune disease.
Results Acceptable consensus was reached for the clinical sam-
ples. Anti-DFS70 pattern was reported from 32/38
Abstracts
ARD 2019;78(Suppl 1):A1A83 A15
on January 8, 2021 by guest. Protected by copyright.http://ard.bmj.com/Ann Rheum Dis: first published as 10.1136/annrheumdis-2018-EWRR2019.30 on 1 March 2019. Downloaded from
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B-cell activating factor (BAFF), a member of the family of TNF-like cytokines, supports the survival and differentiation of B cells. The successful development of belimumab, a human antibody targeting soluble BAFF, has marked an important milestone in the development of biologic therapy for treatment of systemic lupus erythematosus (SLE), although much remains unknown regarding the clinical utility of BAFF inhibition in SLE and other autoimmune diseases. In the present review, we provide an overview of the knowledge concerning BAFF's role in murine and human B-cell development and maturation, as well as the clinical and mechanistic effects of BAFF inhibition in human SLE.
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Systemic sclerosis (SSc) is a debilitating inflammatory and fibrotic disease that affects the skin and internal organs. Although the pathophysiology of SSc remains poorly characterized, mononuclear cells, mainly macrophages and T cells, have been implicated in inflammation and fibrosis. Inducible costimulator (ICOS), which is expressed on a subset of memory T helper (TH) and T follicular helper (TFH) cells, has been shown to be increased in SSc and associated with disease pathology. However, the identity of the relevant ICOS⁺ T cells and their contribution to inflammation and fibrosis in SSc are still unknown. We show that CD4⁺ ICOS-expressing T cells with a TFH-like phenotype infiltrate the skin of patients with SSc and are correlated with dermal fibrosis and clinical disease status. ICOS⁺ TFH-like cells were found to be increased in the skin of graft-versus-host disease (GVHD)–SSc mice and contributed to dermal fibrosis via an interleukin-21– and matrix metalloproteinase 12–dependent mechanism. Administration of an anti-ICOS antibody to GVHD-SSc mice prevented the expansion of ICOS⁺ TFH-like cells and inhibited inflammation and dermal fibrosis. Interleukin-21 neutralization in GVHD-SSc mice blocked disease pathogenesis by reducing skin fibrosis. These results identify ICOS⁺ TFH-like profibrotic cells as key drivers of fibrosis in a GVHD-SSc model and suggest that inhibition of these cells could offer therapeutic benefit for SSc.
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OTHER ARTICLES PUBLISHED IN THIS MINI-REVIEW SERIES ON B CELL SUBSETS IN DISEASE B cells in multiple sclerosis: drivers of disease pathogenesis and Trojan horse for Epstein—Barr virus entry to the central nervous system? Clinical and Experimental Immunology 2012, 167: 1–6. Reconstitution after haematopoietic stem cell transplantation – revelation of B cell developmental pathways and lineage phenotypes. Clinical and Experimental Immunology 2012, 167: 15–25. Systemic lupus erythematosus (SLE) and Sjögren's syndrome are autoimmune disorders which are characterized by a disturbed B cell homeostasis which leads ultimately to dysfunction of various organs. One of the B cell subsets that appear in abnormal numbers is the population of transitional B cells, which is increased in the blood of patients with SLE and Sjögren's syndrome. Transitional B cells are newly formed B cells. In mice, transitional B cells undergo selection checks for unwanted specificity in the bone marrow and the spleen in order to eliminate autoreactive B cells from the circulating naive B cell population. In humans, the exact anatomical compartments and mechanisms of the specificity check-points for transitional B cells remain unclear, but appear to be defective in SLE and Sjögren's syndrome. This review aims to highlight the current understanding of transitional B cells and their defects in the two disorders before and after B cell-targeted therapies.
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To understand the effects of long-term BLyS inhibition in human systemic lupus erythematosus (SLE). Seventeen patients with SLE who were enrolled in a clinical trial of belimumab, a BLyS-specific inhibitor, plus standard of care therapy were studied. Phenotypic analysis of lymphocytes was performed using flow cytometry. Circulating antibody-secreting cells were enumerated using enzyme-linked immunospot assay. Serum was analyzed by enzyme-linked immunosorbent assay using an antibody that recognizes products of the V(H)4-34 gene. Lymphocyte counts, Ig levels, and anti-double-stranded DNA antibody levels were available as part of the clinical trial analyses. Samples were collected on days 0, 84, 168, 365, and 532 and after day 730. The total number of B cells started to decrease from baseline between days 84 and 168. This was due to a decrease in naive and transitional B cells. CD27+IgD+ memory B cells and plasmablasts decreased only after 532 days, whereas CD27+IgD- memory B cells were not affected, and there were no changes in T cells. Serum IgM levels began to decline between days 84 and 168, but there were no changes in serum levels of IgG, IgG anti-DNA antibodies, or V(H)4-34 antibodies during the study. SLE patients had more IgM-, IgG-, and autoantibody-producing B cells than did normal controls on day 0. There was only a modest decrease in the frequency of total IgM-producing, but not IgG-producing, cells on days 365 and 532, consistent with the phenotypic and serologic data. Our data confirm the dependence of newly formed B cells on BLyS for survival in humans. In contrast, memory B cells and plasma cells are less susceptible to selective BLyS inhibition.