Biljana Smiljanovic

The multifaceted balance of TNF-α and type I/II interferon responses in SLE and RA: how monocytes manage the impact of cytokines

Many cytokines are involved in the pathogenesis of autoimmune diseases and are recognized as relevant therapeutic targets to attenuate inflammation, such as tumor necrosis factor (TNF)-α in rheumatoid arthritis (RA) and interferon (IFN)-α/γ in systemic lupus erythematosus (SLE). To relate the transcriptional imprinting of cytokines in a cell type- and disease-specific manner, we generated gene expression profiles from peripheral monocytes of SLE and RA patients and compared them to in vitro-generated signatures induced by TNF-α, IFN-α2a, and IFN-γ. Monocytes from SLE and RA patients revealed disease-specific gene expression profiles. In vitro-generated signatures induced by IFN-α2a and IFN-γ showed similar profiles that only partially overlapped with those induced by TNF-α. Comparisons between disease-specific and in vitro-generated signatures identified cytokine-regulated genes in SLE and RA with qualitative and quantitative differences. The IFN responses in SLE and RA were found to be regulated in a STAT1-dependent and STAT1-independent manner, respectively. Similarly, genes recognized as TNF-α regulated were clearly distinguishable between RA and SLE patients. While the activity of SLE monocytes was mainly driven by IFN, the activity from RA monocytes showed a dominance of TNF-α that was characterized by STAT1 down-regulation. The responses to specific cytokines were revealed to be disease-dependent and reflected the interplay of cytokines within various inflammatory milieus. This study has demonstrated that monocytes from RA and SLE patients exhibit disease-specific gene expression profiles, which can be molecularly dissected when compared with in vitro-generated cytokine signatures. The results suggest that an assessment of cytokine-response status in monocytes may be helpful for improvement of diagnosis and selection of the best cytokine target for therapeutic intervention.

Cell-Specific Type I IFN Signatures in Autoimmunity and Viral Infection: What Makes the Difference?

Gene expression profiling of peripheral blood mononuclear cells (PBMCs) has revealed a crucial role for type I interferon (IFN) in the pathogenesis of systemic lupus erythematosus (SLE). However, it is unclear how particular leucocyte subsets contribute to the overall type I IFN signature of PBMCs and whole blood samples.Furthermore, a detailed analysis describing the differences in the IFN signature in autoimmune diseases from that observed after viral infection has not been performed to date. Therefore, in this study, the transcriptional responses in peripheral T helper cells (CD4+) and monocyte subsets (CD16− inflammatory and CD16+ resident monocytes) isolated from patients with SLE, healthy donors (ND) immunised with the yellow fever vaccine YFV-17Dand untreated controls were compared by global gene expression profiling.It was striking that all of the transcripts that were regulated in response to viral exposure were also found to be differentially regulated in SLE, albeit with markedly lower fold-change values. In addition to this common IFN signature, a pathogenic IFN-associated gene signature was detected in the CD4+ T cells and monocytes from the lupus patients. IL-10, IL-9 and IL-15-mediated JAK/STAT signalling was shown to be involved in the pathological amplification of IFN responses observed in SLE. Type I IFN signatures identified were successfully applied for the monitoring of interferon responses in PBMCs of an independent cohort of SLE patients and virus-infected individuals. Moreover, these cell-type specific gene signatures allowed a correct classification of PBMCs independent from their heterogenic cellular composition. In conclusion, our data show for the first time that monocytes and CD4 cells are sensitive biosensors to monitor type I interferon response signatures in autoimmunity and viral infection and how these transriptional responses are modulated in a cell- and disease-specific manner.

Upregulation of Immunoproteasome Subunits in Myositis Indicates Active Inflammation with Involvement of Antigen Presenting Cells, CD8 T-Cells and IFNγ

Objective In idiopathic inflammatory myopathies (IIM) infiltration of immune cells into muscle and upregulation of MHC-I expression implies increased antigen presentation and involvement of the proteasome system. To decipher the role of immunoproteasomes in myositis, we investigated individual cell types and muscle tissues and focused on possible immune triggers. Methods Expression of constitutive (PSMB5, -6, -7) and corresponding immunoproteasomal subunits (PSMB8, -9, -10) was analyzed by real-time RT-PCR in muscle biopsies and sorted peripheral blood cells of patients with IIM, non-inflammatory myopathies (NIM) and healthy donors (HD). Protein analysis in muscle biopsies was performed by western blot. Affymetrix HG-U133 platform derived transcriptome data from biopsies of different muscle diseases and from immune cell types as well as monocyte stimulation experiments were used for validation, coregulation and coexpression analyses. Results Real-time RT-PCR revealed significantly increased expression of immunoproteasomal subunits (PSMB8/-9/-10) in DC, monocytes and CD8+ T-cells in IIM. In muscle biopsies, the immunosubunits were elevated in IIM compared to NIM and exceeded levels of matched blood samples. Proteins of PSMB8 and -9 were found only in IIM but not NIM muscle biopsies. Reanalysis of 78 myositis and 20 healthy muscle transcriptomes confirmed these results and revealed involvement of the antigen processing and presentation pathway. Comparison with reference profiles of sorted immune cells and healthy muscle confirmed upregulation of PSMB8 and -9 in myositis biopsies beyond infiltration related changes. This upregulation correlated highest with STAT1, IRF1 and IFNγ expression. Elevation of T-cell specific transcripts in active IIM muscles was accompanied by increased expression of DC and monocyte marker genes and thus reflects the cell type specific involvement observed in peripheral blood. Conclusions Immunoproteasomes seem to indicate IIM activity and suggest that dominant involvement of antigen processing and presentation may qualify these diseases exemplarily for the evolving therapeutic concepts of immunoproteasome specific inhibition.

Monocyte alterations in rheumatoid arthritis are dominated by preterm release from bone marrow and prominent triggering in the joint

Objective Rheumatoid arthritis (RA) accompanies infiltration and activation of monocytes in inflamed joints. We investigated dominant alterations of RA monocytes in bone marrow (BM), blood and inflamed joints. Methods CD14+ cells from BM and peripheral blood (PB) of patients with RA and osteoarthritis (OA) were profiled with GeneChip microarrays. Detailed functional analysis was performed with reference transcriptomes of BM precursors, monocyte blood subsets, monocyte activation and mobilisation. Cytometric profiling determined monocyte subsets of CD14++CD16−, CD14++CD16+ and CD14+CD16+ cells in BM, PB and synovial fluid (SF) and ELISAs quantified the release of activation markers into SF and serum. Results Investigation of genes differentially expressed between RA and OA monocytes with reference transcriptomes revealed gene patterns of early myeloid precursors in RA-BM and late myeloid precursors along with reduced terminal differentiation to CD14+CD16+monocytes in RA-PB. Patterns associated with tumor necrosis factor/lipopolysaccharide (TNF/LPS) stimulation were weak and more pronounced in RA-PB than RA-BM. Cytometric phenotyping of cells in BM, blood and SF disclosed differences related to monocyte subsets and confirmed the reduced frequency of terminally differentiated CD14+CD16+monocytes in RA-PB. Monocyte activation in SF was characterised by the predominance of CD14++CD16++CD163+HLA-DR+ cells and elevated concentrations of sCD14, sCD163 and S100P. Conclusion Patterns of less mature and less differentiated RA-BM and RA-PB monocytes suggest increased turnover with accelerated monocytopoiesis, BM egress and migration into inflamed joints. Predominant activation in the joint indicates the action of local and primary stimuli, which may also promote adaptive immune triggering through monocytes, potentially leading to new diagnostic and therapeutic strategies.

Genomic stratification by expression of HLA-DRB4 alleles identifies differential innate and adaptive immune transcriptional patterns - A strategy to detect predictors of methotrexate response in early rheumatoid arthritis

Effective drug selection is the current challenge in rheumatoid arthritis (RA). Treatment failure may follow different pathomechanisms and therefore require investigation of molecularly defined subgroups. In this exploratory study, whole blood transcriptomes of 68 treatment-naïve early RA patients were analyzed before initiating MTX. Subgroups were defined by serologic and genetic markers. Response related signatures were interpreted using reference transcriptomes of various cell types, cytokine stimulated conditions and bone marrow precursors. HLA-DRB4-negative patients exhibited most distinctive transcriptional differences. Preponderance of transcripts associated with phagocytes and bone marrow activation indicated response and transcripts of T- and B-lymphocytes non-response. HLA-DRB4-positive patients were more heterogeneous, but also linked failure to increased adaptive immune response. RT-qPCR confirmed reliable candidate selection and independent samples of responders and non-responders the functional patterning. In summary, genomic stratification identified different molecular pathomechanisms in early RA and preponderance of innate but not adaptive immune activation suggested response to MTX therapy.

A6.07 Tissue- and cell-specific transcriptomes indicate systemic nature of ra and revealed combinations of protein biomarkers relevant for disease characterisation in serum

Background and objectives Clinical signs and symptoms, radiographic changes and routine laboratory tests have indispensable roles in diagnosis of rheumatoid arthritis (RA). Nevertheless, a high degree of heterogeneity between RA patients and increasing options of treatment require the identification of objective criteria relevant for diagnosis and therapeutic stratification of patients. This study focused on global approaches in dissecting inflammation in RA including transcriptome analyses of synovial tissue, blood and bone marrow monocytes and proteome analyses of selected molecules in serum from long-lasting and early RA. Materials and methods Gene-expression profiling of synovial tissues, blood and bone marrow monocytes of RA and osteoarthritis (OA) patients were performed by Affymetrix microarrays. Based on transcriptome data, 28 molecules were selected for protein analyses by ELISA and multiplex immunoassays in sera from patients with long-lasting RA (n = 17) and OA (n = 16), early RA (n = 10) and healthy donors (n = 14). Results Transcriptome analyses of synovial tissues from RA and OA patients showed the most prominent differences between these two diseases and identified more than 1000 differentially expressed genes. More subtle differences were disclosed by gene-expression profiling of blood and bone marrow monocytes from RA and OA with 300 and 150 differentially expressed genes, respectively. From RA tissue- and cell-specific transcriptomes 28 genes were selected for protein analyses in serum from RA and OA patients including: chemokines (CXCL13, CCL18), adhesion molecules (VCAM1, ICAM1, E- and P-Selectins), enzymes (MMP3, A1AT), alarmins (S100P and S1008/9) and the soluble form of cell surface molecules (CD14, CD163). Out of 28 markers only 16 reached statistical significance to discriminate long-lasting RA from OA. A combination of 5 markers was able to correctly classify long-lasting RA. However, the same combination of markers identified only one-third of early RA patients. Conclusions Tissue- and cell-specific transcriptomes demonstrated the systemic nature of RA. Proteome analyses of serum from long-lasting RA patients confirmed transcriptome data and showed that molecular patterns determined by the combination of inflammatory and cell-specific markers are required for disease stratification. In early RA transcriptome data outperformed proteome data suggesting that focus on transcriptional alterations is more sensitive approach for disease management of early RA.

A10.6 Dissecting Disease-Specific Differences in RA and OA by Transcriptome Analyses of Synovial Tissue, Blood and Bone Marrow Monocytes

Background and Objectives Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with joint destruction. Joint inflammation is characterised by infiltration and activation of various immune cells. To avoid difficulties in sampling synovial tissue and to avoid fluctuation in cellular composition of leukocytes, which accompanies inflammation both in synovial tissue and blood, this study was focused on transcriptome analyses of synovial tissues, blood and bone marrow monocytes. The main aim was to analyse potential of blood monocytes in dissecting inflammation in RA and in reflecting inflammation that is evident in synovial tissue. Osteoarthritis (OA), which represents a non-inflammatory disease was used as control in this study. Materials and Methods Synovial tissues from 10 RA and 10 OA patients were used for gene-expression profiling by Affymetrix HG-U133A arrays. Blood and bone marrow monocytes, obtained from 8 RA and 8 OA patients undergoing hip replacement surgery, were utilised for gene-expression profiling by Affymetrix HG-U133Plus arrays. The BioRetis database was used for microarray analyses and generation of RA profiles. Results Transcriptome analyses of synovial tissues from RA and OA patients revealed more than 1000 differentially expressed genes. Increased expression of genes involved in chemotaxis (CCL13, CCL18, CXCL9, CXCL10, CXCL13), cell adhesion and activation (ICAM1, PECAM1, ITGAL, ITGB2, CD40, CD86) indicate to inflammation but also to infiltration of various cell types like monocytes/macrophages, NK, T- and B-cells. By comparing transcriptome of RA and OA monocytes, both from blood and bone marrow, it was obvious that monocytes were able to disclose differences between these two diseases. The RA disease-specific gene-expression profile was evident both in blood and bone marrow and it demonstrated only a minor overlap between these two bodies compartments. Altogether, a typical RA inflammatory profile disclosed in synovial tissues was greatly silenced in blood monocytes, and almost completely absent in bone marrow derived monocytes. Conclusions The RA gene-expression profile was the most specific and robust in synovial tissue, demonstrating the dominance of the inflammatory process in the joints. Nevertheless, the systemic nature of RA was also evident at the level of blood and bone marrow monocytes. Concerning that blood is a favourable and easily accessible material for diagnosis and that monocytes are able to exhibit disease-specific alterations, understanding monocyte response in different rheumatic diseases seems to be advantageous approach for biomarkers discovery. This approach should be essential for identifying the objective criteria relevant for disease and therapeutic stratification of patients with RA.

The type 1 interferon signature: facts, fads and fallacies

Background and objectives The type 1 interferon signature (IFN1) has been reported in systemic lupus erythematosus (SLE) and subsequently in many other diseases. It is deduced from the expression of a group of genes known as downstream events after IFN1 stimulation. Involving certain toll-like receptors and being related especially to an antiviral immune response, interpretation of these findings is difficult, in particular if such signatures are reported in bacterial infections as well. Therefore, the authors aimed for a better interpretation by comparing qualities and quantities of the IFN1 signature after stimulation and in various diseases. Materials and methods Transcriptomes of chronic inflammatory diseases, infections and defined stimulation experiments with IFN1 and other cytokines, which were generated by the authors or retrieved from the Gene Expression Omnibus were systematically investigated using the BioRetis analysis platform for primary analysis and Genesis for comparative visualisation. Results A typical IFN1 signature was identified by screening for differentially expressed genes before and after IFN1 treatment in patients or in vitro stimulation of immune cells with IFN1 compared to other cytokines. The pattern of these genes was compared with the pattern in transcriptomes from patients with different rheumatic diseases including rheumatoid arthritis (RA), spondyloarthritis, SLE, Sjögrens Syndrome, Myositis, Scleroderma and juvenile rheumatic diseases as well as with infectious diseases. Leading candidates related to IFN1 were IFIT3, IFI44L, IFI35, IFI44, MX1, MX2, OAS1, OAS2, OAS3 or SIGLEC1 already known from previous studies. These genes were characteristic for viral infections, SLE and other connective tissue diseases. Bacterial infections were positive only in few exceptions, suggesting co-infection with viruses. When observed in arthritic conditions, the diagnosis of RA was challenged. When comparing signatures induced in vitro, a much broader range of immune reactions was observed in early stages with substantial overlap between different cytokines. However, magnitude of change was still an indicator for specificity, suggesting that specific triggers and responses are always associated with additional more or less unspecific activation patterns. This may be a general rule to enable subsequently the development of a higher complexity of the immune response whenever necessary. Conclusions In summary, the IFN1 signature is certainly a prominent observation in the current literature. However, definitions for the IFN1 response have to be applied carefully in order to avoid misinterpretations by overestimation of minor or negligible amounts. Therefore, analysis should include comparisons with typical positive IFN1 signatures as reference and if possible with changes in other pathways relevant for differential diagnosis.

SAT0249 Reduction of monocyte activation by bowel cleanse and one week fasting suggests permanent pathogenetic triggering from the gut in rheumatoid arthritis

Background: Fasting can improve clinical disease activity in rheumatoid arthritis (RA) [1], but mechanism involved are not clear. Recently, we demonstrated that monocytes in RA express transcriptome patterns of increased myelopoiesis, premature egress from bone marrow and reduced circulation time as indicators of permanent activation of the innate immune response [2]. Objectives: We investigated the influence of bowel cleanse and fasting on monocyte subpopulations in the blood to determine the extent of microbiota and gut immunity related triggering of chronic inflammation in RA. Methods: RA patients (n=22) and controls (n=12, metabolic syndrome), who presented for fasting according to the Buchinger procedure (bowel cleanse with colonoscopy fluid), were analyzed for DAS28, CrP, differential blood count and high resolution cytometric phenotyping at baseline, day 3, day 7 (end of fasting) and day 10. ImmunoClust was applied for automated cell clustering [3]. Results: Disease activity was strikingly decreased after fasting in virtually all RA patients (DAS28 from 4.24 to 3.17, p<0.00005) with significant reduction already after 3 days (p<0.01). This was accompanied by a significant decline of CrP and ESR. Differential blood count revealed a slight decrease in total leukocytes and significant reduction of lymphocytes and eosinophils in RA. However, these blood changes were also observed but on a lower level in the metabolic controls. The most dominant and RA specific effect was a significant reduction of total monocytes when compared to RA baseline or to controls at day 10. Deep profiling of the monocyte compartment revealed reduced non-classical (CD14+CD16+) and intermediate (CD14++CD16+) monocytes prior to fasting in RA compared to controls and confirmed previous results [2]. Bowel cleanse and fasting induced a significant increase of these two monocyte subpopulations by absolute counts and even more by percentage of total monocytes. This indicates reduced recruitment to inflamed tissue and prolonged circulation with more cells differentiating from classical to non-classical monocytes in the blood [4]. The decrease of lymphocytes in RA patients after fasting was characterized by a dominant reduction of naive T-, B-cells and CD16- NK-cells along with a relative increase in memory lymphocytes and CD16+ NK-cells. These effects were also observed but less pronounced in controls. Conclusions: Bowel cleanse and fasting in RA induces a reduction of inflammation related to monocyte activation and turnover immediately within few days. Changes in the monocyte compartment were specific for RA compared to controls and dominated the immunological changes, suggesting that innate triggering mechanisms from gut and its microbiota are etiologically relevant in RA. References [1] Kjeldsen-Kragh J, et al. Lancet1991;338(8772):899. [2] Smiljanovic B, et al. Ann Rheum Dis2018;77(2):300. [3] Sörensen T, et al. Cytometry A2015;87(7):603. [4] Tak T, et al. Blood2017;130(12):1474. Acknowledgements: Technical assitance: Silvia Pade, Barbara Walewska Funding: German Federal Ministry of Education and Research grant ArthoMark (01EC1009A), Corona-Stiftung grant BioFast (S199/10063/2016). Disclosure of Interest: None declared

05.08 Increased turnover of monocytes in patients with rheumatoid arthritis identified by transcriptome and cytometric profiling

Background Targeting molecules involved in monocyte activation is an important treatment strategy for RA. In this study we aimed to determine monocyte maturation and activation from bone marrow (BM) via blood into synovial fluid (SF) by investigating monocytes transcriptomes and by cytometric profiling of classical (CD14++CD16-), intermediated (CD14++CD16+) and non-classical (CD14+CD16+) monocytes. Materials and methods CD14+ cells from BM and blood of RA and osteoarthritis (OA) patients were profiled with Affymetrix microarrays. A detailed functional analysis was performed with reference transcriptomes of BM precursors, monocyte blood subsets, monocyte activation and egress from BM induced by G-CSF (granulocyte colony-stimulating factor). Cytometric profiling of CD14, CD16, HLA-DR and CD163 expression were used to determine monocyte subsets and to follow their activation and differentiation in BM, blood and SF. Results Transcriptomes of RA-BM monocytes exhibited i) pronounce gene pattern of early myeloid precursors from BM and ii) weak gene pattern of late myeloid precursors from BM. Transcriptomes of RA blood monocytes demonstrated i) pattern of late myeloid precursors from BM and ii) reduced pattern of terminally differentiated CD14+CD16+ monocytes from blood. Cytometric profiling of BM, blood and SF monocytes in RA and OA showed that all three body compartments have their own distribution of monocyte subsets. BM was characterised with classical and intermediate subsets and both subsets showed decreased CD16 expression in RA when compared to OA. As expected, blood was characterised with three subsets, and RA blood showed decreased CD14 and HLA-DR expression on classical monocytes and reduced frequency of non-classical subset. In RA-SF, classical monocytes were absent, intermediate were most dominant and cell-phenotype with low CD16 expression but similar to non-classical monocytes was related to macrophages. Cell frequency of intermediate subset in SF positively correlated with inflammation (ESR; R>0.85) and showed the highest expression of HLA-DR, CD14, CD163. Conclusions Monocyte turnover is increased in RA and characterised with accelerated monocytopoiesis, faster BM egress and migration into inflamed joints. Permanent monocyte activation in the joint and their role in linking innate and adaptive immunity, which is targeted by biologics, emphasises their high diagnostic value and relevance for therapeutic stratification.