H Leiss

Tumor necrosis factor-inhibiting therapy preferentially targets bone destruction but not synovial inflammation in a tumor necrosis factor-driven model of rheumatoid arthritis.

OBJECTIVE To investigate how tumor necrosis factor (TNF)-inhibiting therapy affects bone destruction and inflammation in a TNF-driven mouse model of rheumatoid arthritis. METHODS In order to evaluate the influence of TNF on osteoclastogenesis in vitro, different concentrations of TNF were added to spleen cell-derived monocytes in the absence or presence of different concentrations of RANKL. In addition, the effects of TNF inhibition on osteoclast precursors as well as local bone destruction in vivo were assessed by treating TNF-transgenic mice with different doses of adalimumab. RESULTS TNF stimulated osteoclastogenesis mainly by increasing the number of osteoclast precursor cells in vitro. This TNF effect was independent of the presence of RANKL. In the hTNF-transgenic mouse model of destructive arthritis, low-dose TNF-inhibiting therapy with adalimumab had no effect on synovial inflammation but significantly inhibited local bone destruction and the generation of osteoclasts. This inhibition was accompanied by a reduction in the number of c-Fms-positive osteoclast precursor cells in the bone marrow and a reduction of the osteoclast precursor pools in the blood and inflamed synovial membrane of hTNF-transgenic mice. CONCLUSION Low-dose TNF-inhibiting therapy significantly reduces bone erosions by reducing the number of circulating and joint-invading osteoclast precursors. This effect is uncoupled from its antiinflammatory action.

Regulatory T cell-deficient scurfy mice develop systemic autoimmune features resembling lupus-like disease

IntroductionScurfy mice are deficient in regulatory T cells (Tregs), develop a severe, generalized autoimmune disorder that can affect almost every organ and die at an early age. Some of these manifestations resemble those found in systemic lupus erythematosus (SLE). In addition, active SLE is associated with low Treg numbers and reduced Treg function, but direct evidence for a central role of Treg malfunction in the pathophysiology of lupus-like manifestations is still missing. In the present study, we characterize the multiorgan pathology, autoantibody profile and blood count abnormalities in scurfy mice and show their close resemblances to lupus-like disease.MethodsScurfy mice have dysfunctional Tregs due to a genetic defect in the transcription factor Forkhead box protein 3 (Foxp3). We analyzed skin, joints, lung and kidneys of scurfy mice and wild-type (WT) controls by conventional histology and immunofluorescence (IF) performed hematological workups and tested for autoantibodies by IF, immunoblotting and enzyme-linked immunosorbent assay. We also analyzed the intestines, liver, spleen and heart, but did not analyze all organs known to be affected in scurfy mice (such as the testicle, the accessory reproductive structures, the pancreas or the eyes). We transferred CD4+ T cells of scurfy or WT mice into T cell-deficient B6/nude mice.ResultsWe confirm previous reports that scurfy mice spontaneously develop severe pneumonitis and hematological abnormalities similar to those in SLE. We show that scurfy mice (but not controls) exhibited additional features of SLE: severe interface dermatitis, arthritis, mesangioproliferative glomerulonephritis and high titers of anti-nuclear antibodies, anti-double-stranded DNA antibodies, anti-histone antibodies and anti-Smith antibodies. Transfer of scurfy CD4+ T cells (but not of WT cells) induced autoantibodies and inflammation of lung, skin and kidneys in T cell-deficient B6/nude mice.ConclusionOur observations support the hypothesis that lupus-like autoimmune features develop in the absence of functional Tregs.

Pristane-induced lupus as a model of human lupus arthritis: evolvement of autoantibodies, internal organ and joint inflammation

Objective Arthritis is frequently seen in human lupus, but rarely in lupus models. Pristane-induced lupus (PIL) can be induced in various mouse strains such as BALB/c and C57BL/6. We herein characterize clinical and histological features of arthritis in the context of systemic lupus and provide a prudent comparison with models of rheumatoid arthritis (RA). Methods A total of 57 BALB/c mice received pristane (PIL group) and were analyzed for serum autoantibodies (anti-chromatin-, -histone, -Sm, -dsDNA), as well as for clinical features and histopathology of joints, lungs and kidneys. Joint pathology was quantified by image analysis and tissue cytometry. Ten C57BL/6 mice (Bl/6-PIL) and historical groups of two different RA models were analyzed accordingly. Results In BALB/c PIL, clinical arthritis started at three months, occurred finally in 79% of PIL (but not in controls, p < 0.001) and correlated with areas of inflammation, erosion, cartilage damage, osteoclast numbers and total severity score (for all: r > 0.7, p < 0.001). After eight months, 58% of PIL (but no controls, p < 0.001) had mild-erosive arthritis. In contrast to RA, the most frequent inflammatory cell type of the pannus was granulocytes (17.7%), PIL had lower numbers of osteoclasts, erosions rarely affected both layers of the cortical bone and there was no progression to complete joint destruction (even after one year of observation). Serum autoantibodies (auto-abs) preceded arthritis and became significantly elevated in all PIL; affected joints showed increased deposits of IgG (and IgM) within the inflammatory tissue, indicative of an ab-mediated process. PIL mice with arthritis also showed signs of pulmonary (100%) and renal (46%) lupus. In contrast to BALB/c, Bl/6-PIL mice did not develop any signs of arthritis. Conclusion PIL in BALB/c mice is characterized by severe organ involvement, typical autoabs and by a mild-erosive arthritis with similarities to, but also with distinct differences from, RA. PIL may help to study arthritis along with other key features of systemic lupus erythematosus after therapeutic interventions or in knock-out models based on a BALB/c but not on a C57BL/6 background.

MicroRNA 155-deficiency leads to decreased autoantibody levels and reduced severity of nephritis and pneumonitis in pristane-induced lupus

Objective We herein examine the role of endogenous miR155 in the development of systemic manifestations in pristane induced lupus. Materials and methods Systemic lupus in miR155-deficient and wild type mice was induced upon injection of pristane and analyzed after 8 months, PBS-injected mice served as controls. Glomerulonephritis and pneumonitis were quantified using the kidney biopsy score and a newly adapted histomorphometric image analysis system; lung tissue was further analyzed by tissue cytometry. Serum levels of anti-dsDNA, anti-histone and anti-chromatin antibodies were measured by ELISA. Frequencies of B cells, activated and regulatory CD4+ T cells as well as Th1, Th2, Th17 cells were measured by flow cytometry. RT-qPCR was used to measure expression levels of interferon-signature and T-cell subset related as well as miR155-associated genes. Results After induction of lupus, miR155-deficient mice had significant less pulmonary involvement (perivascular inflammatory area in mm2/mm2 lung area 0.00092±0.00015 vs. 0.0027±0.00075, p = 0.0347) and renal disease (glomerular activity score 1.95±0.19 vs 3±0.26, p = 0.0029) compared to wild types. MiR155-deficient mice had significantly lower serum levels of disease-associated auto-antibodies and decreased frequencies of activated CD4+CD25+ (Foxp3-) cells. Upon restimulation, CD4+ cells showed a less pronounced Th2 and Th17 and a slightly decreased Th1 response in mir155-deficient mice. Pristane-treated wild types showed significantly up-regulated expression of genes related to the INF-signature (MX1, IP10, IRF7, ISG15). Conclusions MiR155-deficient mice had less severe organ involvement, lower serum auto-antibody levels, a less prominent T cell response and lower expressions of genes jointly responsible for disease development. Thus, antagonizing miR155 might be a future approach in treating SLE.

Non-classical monocytes as mediators of tissue destruction in arthritis

Objectives Bone destruction in rheumatoid arthritis is mediated by osteoclasts (OC), which are derived from precursor cells of the myeloid lineage. The role of the two monocyte subsets, classical monocytes (expressing CD115, Ly6C and CCR2) and non-classical monocytes (which are CD115 positive, but low in Ly6C and CCR2), in serving as precursors for OC in arthritis is still elusive. Methods We investigated CCR2−/− mice, which lack circulating classical monocytes, crossed into hTNFtg mice for the extent of joint damage. We analysed monocyte subsets in hTNFtg and K/BxN serum transfer arthritis by flow cytometry. We sorted monocyte subsets and analysed their potential to differentiate into OC and their transcriptional response in response to RANKL by RNA sequencing. With these data, we performed a gene ontology enrichment analysis and gene set enrichment analysis. Results We show that in hTNFtg arthritis local bone erosion and OC generation are even enhanced in the absence of CCR2. We further show the numbers of non-classical monocytes in blood are elevated and are significantly correlated with histological signs of joint destruction. Sorted non-classical monocytes display an increased capacity to differentiate into OCs. This is associated with an increased expression of signal transduction components of RANK, most importantly TRAF6, leading to an increased responsiveness to RANKL. Conclusion Therefore, non-classical monocytes are pivotal cells in arthritis tissue damage and a possible target for therapeutically intervention for the prevention of inflammatory joint damage.

THU0233 In vitro induced regulatory t-cells can ameliorate severity of pristane induced lupus (PIL)

Background Pristane induced lupus (PIL) is a well-established murine model for environmentally induced systemic lupus erythematosus (SLE). Mice develop specific autoantibodies and show symptoms of SLE including arthritis, diffuse proliferative immune complex glomerulonephritis and haemorrhagic pulmonary capillaritis. Objectives To investigate the therapeutic effects of in vitro -induced regulatory T cells (iTreg) in the murine model of PIL. Methods BALB/c mice were injected i.p. with either 0.5ml of pristane (PIL) or PBS (controls). Naive CD4+ thymocytes were sorted and cultured and cell suspensions with >80% of CD4+FoxP3+ cells (iTreg) were injected intravenously (i) once when PIL was induced (5x106 iTreg (iTreg-single)) or (ii) every 4 weeks (1x106 iTreg, iTreg-rep). Animals were monitored for paw swelling and grip strength. After 8 months histological analysis evaluated for cartilage degradation, number of osteoclasts and the extent of inflammation and bone erosion. Glomerulonephritis and pneumonitis were quantified using the kidney biopsy score and a newly adapted histomorphometric image analysis system; inflammatory tissue was further analyzed by tissue cytometry. Serum levels of anti-dsDNA, anti-histone and anti-chromatin antibodies were measured by ELISA. Results Monthly injections of 1x106 iTreg reduced the clinical as well as the histological severity of PIL-arthritis, seen by a higher mean grip strength (2.96±0.02 vs. 2.73±0.06, p<0.01), less mean paw swelling (0.04±0.02 vs. 0.36±0.07, p<0.01) and retardation of the symptom onset (Figure A). 62% of PIL-mice and 33% of iTreg-rep mice had erosive arthritis. There was a significant reduction of arthritis severity in all histological parameters (inflammatory area in mm2 0.19±0.06 vs. 0.69±0.11; erosive area in mm2 0.01±0.01 vs. 0.07±0.02; number of osteoclasts 2±1.13 vs. 9.14±2; cartilage degradation in mm2 0.06±0.01 vs. 0.19±0.03, all p<0.01, Figure B). The single boost of 5x106 iTreg could not prevent joint manifestations. However, a slight retardation in “loss of grip strength” and a significantly less erosive area was observed. In regards to the cellular composition of the inflammatory tissue in paws, a significantly increased relative amount of CD4+Foxp3+ cells was seen in the iTreg-rep group compared to the PIL group (in % 5.2±2.3 vs. 0.6±0.2, p<0.01). Repeatedly injected mice (iTreg-rep) had significant less pulmonary involvement (perivascular inflammatory area in mm2/mm2 lung area 0,003±0,001 vs. 0,007±0,001, p<0.01) and renal disease (glomerular activity score 1,17±0,31 vs. 2,43±0,39, p<0.05) compared to PIL (Figure C). Corresponding, iTreg-rep mice had significantly lower serum levels of disease-associated auto-antibodies (Figure D). Conclusions Repeated injections of iTreg ameliorate the clinical and histological severity of PIL- manifestations. A single boost of iTreg at the time of disease induction does not prevent manifestations, but retards the onset of symptoms. Thus iTreg have significant positive effects on PIL, which may have consequences for future approaches in treating SLE. Disclosure of Interest None declared

AB0136 In Vitro Induced Regulatory T-Cells Can Reduce Severity of Lupus Arthritis

Background Even though joint involvement is a major burden in patients suffering from systemic lupus erythematosus (SLE), it is not focus of scientific research and treatment strategies continue to be limited. Studying lupus arthritis and possible treatment options can be done with the murine model of Pristane-induced lupus (PIL). Objectives We herein investigate if in vitro induced regulatory T-cells (iTreg) are capable of ameliorating PIL arthritis and help us evaluate possible new treatment options. Methods BALB/c mice were injected i.p. with either 0.5ml of pristane (PIL-group) or PBS (controls). Naive CD4+ thymocytes were sorted and cultured and cell suspensions with >80% of CD4+FoxP3+ cells (iTreg) were injected intravenously (i) once when PIL was induced (5x106 iTreg, iTreg-boost), or (ii) every 4 weeks (1x106 iTreg, iTreg-rep). Animals were monitored for paw swelling and grip strength. After 8 months histological analysis evaluated for cartilage degradation, number of osteoclasts and the extent of inflammation and bone erosion. In addition, the cellular composition of the inflammatory tissue was determined by a cell-identification algorithm for nuclear segmentation (HistoQuest). Serum levels of anti-dsDNA, anti-histone and anti-chromatin antibodies were measured by ELISA. Results Monthly injections of 1x106 iTreg reduced the clinical as well as the histological severity of PIL-arthritis, seen by a higher mean grip strength (2.964±0.024 vs. 2.732± 0.063, p<0.01), less mean paw swelling (0.044±0.020 vs. 0.360 ±0.069) and retardation of the symptom onset. 62% of PIL-mice and 33% of iTreg-rep mice had erosive arthritis. There was a significant reduction of arthritis severity in all histological parameters (inflammatory area 0.188±0.0574 vs. 0.688±0.113; erosive area 0.011 ±0.009 vs. 0.069±0.017; number of osteoclasts 2.000± 1.125 vs. 9.143±1.999; cartilage degradation 0.059±0.004 vs. 0.187±0.033). The single boost of 5x106 iTreg could not prevent joint manifestation. However, a slight retardation in “loss of grip strength” and a significantly less erosive area was seen. In regards to the cellular composition of the inflammatory tissue, a significantly increased relative amount of Foxp3 cells was seen in the iTreg-rep group compared to the PIL group (5.2±2.3 vs. 0.6±0.2). Corresponding to the reduced severity in joint involvement, the iTreg-group had significantly lower serum levels of antibodies. Conclusions Repeated injections of iTreg ameliorate the clinical and histological severity of PIL- arthritis. A single boost of iTreg at the time of disease induction does not prevent joint manifestation, but retards the onset of symptoms and progression of erosive bone degradation. Thus, iTreg have significant positive effects on PIL arthritis, which may have consequence for future therapeutic considerations. Disclosure of Interest None declared

AB0135 Decreased Lupus Manifestations in Pristane-Induced Microrna 155-Deficient Mice

Background Deregulation of endogenous miR155 was observed in many autoimmune conditions, including systemic lupus erythematosus (SLE). Objectives We herein examined the role of miR155 in the development of systemic manifestations in mice with pristane-induced lupus (PIL). Methods MiR155-deficient and C57/Bl6 mice were injected with pristane or PBS as control and analyzed after 8 months. Glomerulonephritis and pneumonitis were quantified by using the composite kidney biopsy score (KBS) and by analyzing the numbers of affected lung-vessels and the area of the inflammatory lung-infiltrate, respectively. Specimens were stained with B220 (B), CD3 (T), Neu7/4 (neutrophils) and F4/80 (macrophages) and analyzed by cell-identification algorithms for nuclear segmentation (HistoQuest). Serum levels of anti-dsDNA, anti-histone and anti-chromatin antibodies were measured by ELISA. Frequencies of B cells, activated and regulatory CD4+ T cells as well as Th1, Th2, Th17 cells were measured by flow cytometry. Quantitative real-time polymerase chain reaction was used to measure expression levels of interferon-signature and T-cell subset related as well as miR155-associated genes. Results All pristane-injected mice showed signs of pneumonitis, while controls did not. Compared to wild types treated with pristane, similarly treated knockouts showed significantly decreased perivascular inflammatory area with B cells being the most prominent inflammatory cell. Wildtypes had a more severe renal involvement in the KBS than knockouts. Corresponding with reduced organ involvement, miR155 deficient mice had significantly lower serum levels of anti-dsDNA antibodies, anti-chromatin and anti-histone antibodies and decreased frequencies of activated CD4+CD25+(Foxp3–) cells. Interestingly, also frequencies of CD4+CD25+Foxp3+ regulatory T cells were lower in these mice. Upon restimulation, CD4+ cells showed a more pronounced Th2 response in wild types, but no significant differences in Th1 and Th17 phenotype. Regarding INF-signature and T-cell subset activation, pristane-treated wild types showed significantly up-regulated gene-expression patterns whereas equivalently treated mutants showed the same levels as PBS-treated controls. Conclusions MiR155 deficiency in PIL mice did not prevent disease, but was associated with less severe organ involvement, lower serum auto-abs levels, lower frequencies of Th2 cells, whereas lower expressions of genes jointly responsible for disease development may be one key mechanism. Thus, antagonisation of miR155 might be a future approach in treating SLE. Disclosure of Interest None declared

A1.33 LY6C resident monocytes with osteoclastogenic potential arise before clinical onset of arthritis

Introduction Bone erosions and systemic bone loss in rheumatoid arthritis patients results from an increased activity of osteoclast, which are derived from precursor cells of the myeloid lineage. Although there is much known about the mechanisms regulating the formation and activation of mature osteoclasts, the identity of an osteoclast precursor population in and its regulation by inflammatory cytokines during arthritis is poorly understood. Methods HTNFtg mice were clinically scored once per week for grip strength and swelling. In addition, blood was collected every week starting on week 4. Mice were sacrificed at week 10 - blood, spleen and bone marrow were collected for flow cytometry analysis. CCR2-/- mice were crossed into hTNFtg mice and histological analysis was performed. Different monocyte subsets were Facs-sorted and cultured in the presence of RANKL and MCSF to induce osteoclasts. Results We show that during TNF-driven arthritis CD11b+CD115+ cells are elevated in blood before the onset of clinical symptoms and remain elevated throughout. These cells are also elevated in spleen and bone marrow during arthritis. In blood, these cells can be separated by their expression of Ly6C into inflammatory monocytes (CD115+Ly6Chigh) and resident monocytes (CD115+ Ly6Clow). Interestingly, especially resident monocytes are elevated preclinical. Upon sorting resident and inflammatory monocyes from blood, we demonstrate that only resident monocytes are able to form multinucleated TRAP+ osteoclasts, but inflammatory monocytes do not. In order to further investigate the role of these monocyte subsets in the development of arthritic bone destruction and osteoclast formation we used CCR2 deficient mice, which lack circulating inflammatory monocytes and crossed them into hTNFtg animals. In line with our in vitro data, hTNFtg mice lacking CCR2 (i.e. inflammatory monocytes) showed no reduction in the amount of joint destruction but even enhanced local bone erosion and osteoclast generation. Conclusion CD115+ CD11b+ cells, especially Ly6C- resident monocytes with osteoclastogenic potential, increase during inflammatory arthritis. Elevated numbers of these cells can be detected before clinical onset of disease and therefore may provide a biomarker for erosive inflammatory arthritis and even a possible target for therapeutically intervention.

A3.10 Characterisation of CD4+ T cell response and effects of regulatory T cells in pristane induced lupus (PIL)

Introduction CD4+ T cells and the Th1 subset in particular play a pivotal role in SLE. Regulatory T cells (Treg) are essential for maintaining peripheral tolerance, but their number and function in SLE is decreased. We herein characterise CD4+ T cell and Treg homeostasis as well as the effects of intravenous administration of exogenous Treg on disease severity in a model of induced systemic lupus. Methods Mice were injected ip. with 0.5ml of pristane or PBS as control and killed after 8 months. Cell suspensions with CD4+ FoxP3+ cells (iTreg) were injected intravenously: when PIL was induced (5 x 10e6, iTreg-boost) or every 4 weeks (1 x 10e6, iTreg-repeated). Animals were monitored for clinical signs of arthritis and in order to analyse and compare disease severity, histological features of arthritis and pneumonitis were quantified by an image analysis system (Osteomeasure®). Lymphocytes were isolated from granulomas (ectopic lymphoid tissue within the peritoneum), lymphnodes (LN) and spleens and were analysed separately for each mouse by FACS. Results Clinically, PIL presented involvement of inner organs; most frequently affected were the lungs (100% pneumonitis) and the joints, whereas 52% out of the PIL group and 36% of the Treg-treated groups developed clinical arthritis. Monthly iTreg significantly decreased clinical signs of arthritis and histological lung and joint parameters. Out of 6 mice treated, 4 (66%) did not show any signs of arthritis at all. One-shot iTreg did not prevent joint manifestations or pneumonitis, but seemed to have a retarding effect indicated by a delayed onset of clinical symptoms and by a significantly decreased erosive area. Intraperitoneal granuloma typical for PIL appeared to be the hotspots of inflammation showing a significantly elevated Teff/Treg ratio of 1.3. Upon re-stimulation, CD4+ cells showed a pronounced Th1 response (27% IFNγ producers) compared to LN and spleens from both PIL and HC (with Th1 percentages ranging from 9-16%), but also frequencies of Th2 and Th17 cells were elevated in PIL, again with the highest yield in granuloma. iTreg reduced the Teff/Treg ratio in PIL granuloma to 0.7. Conclusion Repeated injections of exogenously induced Tregs reduce the Teff/Treg ratio as well as the severity of pneumonitis and arthritis. A single boost of Tregs even when applied before the clinical onset cannot decrease all parameters, but appears to retard onset of symptoms and progression. Thus, iTreg have significant effects on lupus symptoms when applied repeatedly, which may have consequence for future therapeutic considerations.