Mikael Brisslert

Carbamylation-Dependent Activation of T Cells: A Novel Mechanism in the Pathogenesis of Autoimmune Arthritis

The posttranslational modification of proteins has the potential to generate neoepitopes that may subsequently trigger immune responses. The carbamylation of lysine residues to form homocitrulline may be a key mechanism triggering inflammatory responses. We evaluated the role of carbamylation in triggering immune responses and report a new role for this process in the induction of arthritis. Immunization of mice with homocitrulline-containing peptides induced chemotaxis, T cell activation, and Ab production. The mice also developed erosive arthritis following intra-articular injection of peptides derived from homocitrulline and citrulline. Adoptive transfer of T and B cells from homocitrulline-immunized mice into normal recipients induced arthritis, whereas systemic injection of homocitrulline-specific Abs or intra-articular injection of homocitrulline-Ab/citrulline-peptide mixture did not. Thus, the T cell response to homocitrulline-derived peptides, as well as the subsequent production of anti-homocitrulline Abs, is critical for the induction of autoimmune reactions against citrulline-derived peptides and provides a novel mechanism for the pathogenesis of arthritis.

The Human Immunomodulatory CD25+ B Cell Population belongs to the Memory B Cell Pool

We have shown that human CD20+25+ B cells display immunomodulatory properties. The aim of this study was to investigate if CD25+ B cells are found within the CD27 memory B cell population, and to analyse pattern of their cytokine production.

Phenotypic and functional characterization of human CD25+ B cells

We demonstrate that humans have a phenotypically and functionally distinct subset of B lymphocytes that express the interleukin (IL)‐2 receptor (IL‐2R) α‐chain, cluster of differentiation (CD) 25. We found that one‐third of the circulating CD20+ B cells expressed CD25 and, using fluorescence‐activated cell sorter (FACS) analysis, that these cells were significantly larger and more granulated than B cells not expressing CD25. The simultaneous expression of the other two subunits (CD122 and CD132) and the proliferative responses of cells expressing CD25 to IL‐2 suggested that, in addition to CD25, functional IL‐2 receptors were expressed on this cell population. CD25 expression on B cells was selectively up‐regulated by Toll‐like receptor 2 (TLR2), TLR4, and TLR9 ligands but not by a TLR3 ligand or Epstein–Barr virus (EBV) stimulation. Blockade of the nuclear factor (NF)‐κB pathway completely abolished CD25 up‐regulation by these B cells. Interestingly, CD25+ B cells expressed significantly higher levels of surface immunoglobulins but lacked the ability to secrete immunoglobulin (Ig), as compared with CD25− B cells. Furthermore, CD25+ B cells performed significantly better as antigen‐presenting cells in allogeneic mixed lymphocyte reactions (MLR), which may be a result of their expression of high levels of the costimulatory molecules CD27 and CD80. Finally, blocking of CD25 on B cells led to an almost total abrogation of MLR. Our results indicate that CD25+ B cells have distinct phenotypic and functional properties, including the ability to contribute to antigen presentation, which is linked to their expression of CD25. Finally, the differential regulation of CD25 expression via selective TLR ligands suggests a role for CD25+ B cells in bridging innate and acquired immune responses.

Ethanol prevents development of destructive arthritis

Environmental factors are thought to play a major role in the development of rheumatoid arthritis. Because the use of ethanol is widespread, we assessed the role of ethanol intake on the propensity to develop chronic arthritis. Collagen type II-immunized mice were given water or water containing 10% (vol/vol) ethanol or its metabolite acetaldehyde. Their development of arthritis was assessed, as well as the impact of ethanol on leukocyte migration and activation of intracellular transcription factors. Mice exposed daily to this dose of ethanol did not display any liver toxicity, and the development of erosive arthritis was almost totally abrogated. In contrast, the antibody-mediated effector phase of collagen-induced arthritis was not influenced by ethanol exposure. Also, the major ethanol metabolite, acetaldehyde, prevented the development of arthritis. This antiinflammatory and antidestructive property of ethanol was mediated by (i) down-regulation of leukocyte migration and (ii) up-regulation of testosterone secretion, with the latter leading to decreased NF-κB activation. We conclude that low but persistent ethanol consumption delays the onset and halts the progression of collagen-induced arthritis by interaction with innate immune responsiveness.

Mice Chronically Fed High-Fat Diet Have Increased Mortality and Disturbed Immune Response in Sepsis

Background Sepsis is a potentially deadly disease that often is caused by gram-positive bacteria, in particular Staphylococcus aureus (S. aureus). As there are few effective therapies for sepsis, increased basic knowledge about factors predisposing is needed. Methodology/Principal Findings The purpose of this study was to study the effect of Western diet on mortality induced by intravenous S. aureus inoculation and the immune functions before and after bacterial inoculation. Here we show that C57Bl/6 mice on high-fat diet (HFD) for 8 weeks, like genetically obese Ob/Ob mice on low-fat diet (LFD), have increased mortality during S. aureus-induced sepsis compared with LFD-fed C57Bl/6 controls. Bacterial load in the kidneys 5–7 days after inoculation was increased 10-fold in HFD-fed compared with LFD-fed mice. At that time, HFD-fed mice had increased serum levels and fat mRNA expression of the immune suppressing cytokines interleukin-1 receptor antagonist (IL-1Ra) and IL-10 compared with LFD-fed mice. In addition, HFD-fed mice had increased serum levels of the pro-inflammatory IL-1β. Also, HFD-fed mice with and without infection had increased levels of macrophages in fat. The proportion and function of phagocytosing granulocytes, and the production of reactive oxygen species (ROS) by peritoneal lavage cells were decreased in HFD-fed compared with LFD-fed mice. Conclusions Our findings imply that chronic HFD disturb several innate immune functions in mice, and impairs the ability to clear S. aureus and survive sepsis.

Short- and long-term effects of anti-CD20 treatment on B cell ontogeny in bone marrow of patients with rheumatoid arthritis

IntroductionIn the present study we evaluated changes in the B cell phenotype in peripheral blood and bone marrow (BM) of patients with rheumatoid arthritis (RA) following anti-CD20 treatment using rituximab.MethodsBlood and BM samples were obtained from 37 patients with RA prior to rituximab treatment. Ten of these patients were resampled 1 month following rituximab, 14 patients after 3 months and the remaining 13 patients were included in the long-term follow up. B cell populations were characterized by CD27/IgD/CD38/CD24 expression.ResultsOne and three months following rituximab BM retained up to 30% of B cells while circulation was totally depleted of B cells. Analysis of the remaining BM B cells showed prevalence of immature and/or transitional B cells (CD38++CD24++) and CD27+IgD- memory cells, while IgD+ cells were completely depleted. A significant reduction of CD27+ cells in BM and in circulation was observed long after rituximab treatment (mean 22 months), while levels of naive B cells in BM and in circulation were increased. The levels of rheumatoid factor decline after rituximab treatment but returned to baseline levels at the time of retreatment.ConclusionsAnti-CD20 treatment achieves a depletion of IgD+ B cells shortly after the treatment. At the long term follow up, a reduction of CD27+ B cells was observed in blood and BM. The prolonged inability to up-regulate CD27 may inhibit the renewal of memory B cells. This reduction of CD27+ B cells does not prevent autoantibody production suggesting that mechanisms regulating the formation of auto reactive clones are not disrupted by rituximab.

Geranylgeranyltransferase type I (GGTase-I) deficiency hyperactivates macrophages and induces erosive arthritis in mice

RHO family proteins are important for the function of inflammatory cells. They are modified with a 20-carbon geranylgeranyl lipid in a process catalyzed by protein geranylgeranyltransferase type I (GGTase-I). Geranylgeranylation is viewed as essential for the membrane targeting and activity of RHO proteins. Consequently, inhibiting GGTase-I to interfere with RHO protein activity has been proposed as a strategy to treat inflammatory disorders. However, here we show that mice lacking GGTase-I in macrophages develop severe joint inflammation resembling erosive rheumatoid arthritis. The disease was initiated by the GGTase-I-deficient macrophages and was transplantable and reversible in bone marrow transplantation experiments. The cells accumulated high levels of active GTP-bound RAC1, CDC42, and RHOA, and RAC1 remained associated with the plasma membrane. Moreover, GGTase-I deficiency activated p38 and NF-κB and increased the production of proinflammatory cytokines. The results challenge the view that geranylgeranylation is essential for the activity and localization of RHO family proteins and suggest that reduced geranylgeranylation in macrophages can initiate erosive arthritis.

The tumour-associated glycoprotein podoplanin is expressed in fibroblast-like synoviocytes of the hyperplastic synovial lining layer in rheumatoid arthritis

IntroductionActivated fibroblast-like synoviocytes (FLSs) in rheumatoid arthritis (RA) share many characteristics with tumour cells and are key mediators of synovial tissue transformation and joint destruction. The glycoprotein podoplanin is upregulated in the invasive front of several human cancers and has been associated with epithelial-mesenchymal transition, increased cell migration and tissue invasion. The aim of this study was to investigate whether podoplanin is expressed in areas of synovial transformation in RA and especially in promigratory RA-FLS.MethodsPodoplanin expression in human synovial tissue from 18 RA patients and nine osteoarthritis (OA) patients was assessed by immunohistochemistry and confirmed by Western blot analysis. The expression was related to markers of synoviocytes and myofibroblasts detected by using confocal immunofluoresence microscopy. Expression of podoplanin, with or without the addition of proinflammatory cytokines and growth factors, in primary human FLS was evaluated by using flow cytometry.ResultsPodoplanin was highly expressed in cadherin-11-positive cells throughout the synovial lining layer in RA. The expression was most pronounced in areas with lining layer hyperplasia and high matrix metalloproteinase 9 expression, where it coincided with upregulation of α-smooth muscle actin (α-sma). The synovium in OA was predominantly podoplanin-negative. Podoplanin was expressed in 50% of cultured primary FLSs, and the expression was increased by interleukin 1β, tumour necrosis factor α and transforming growth factor β receptor 1.ConclusionsHere we show that podoplanin is highly expressed in FLSs of the invading synovial tissue in RA. The concomitant upregulation of α-sma and podoplanin in a subpopulation of FLSs indicates a myofibroblast phenotype. Proinflammatory mediators increased the podoplanin expression in cultured RA-FLS. We conclude that podoplanin might be involved in the synovial tissue transformation and increased migratory potential of activated FLSs in RA.

A unique population of IgG-expressing plasma cells lacking CD19 is enriched in human bone marrow.

Specific serum antibodies mediating humoral immunity and autoimmunity are provided by mature plasma cells (PC) residing in the bone marrow (BM), yet their dynamics and composition are largely unclear. We here characterize distinct subsets of human PC differing by CD19 expression. Unlike CD19(+) PC, CD19(-) PC were restricted to BM, expressed predominantly IgG, and they carried a prosurvival, distinctly mature phenotype, that is, HLA-DR(low)Ki-67(-)CD95(low)CD28(+)CD56(+/-), with increased BCL2 and they resisted their mobilization from the BM after systemic vaccination. Fewer mutations within immunoglobulin VH rearrangements of CD19(-) BMPC may indicate their differentiation in early life. Their resistance to in vivo B-cell depletion, that is, their independency from supply with new plasmablasts, is consistent with long-term stability of this PC subset in the BM. Moreover, CD19(-) PC were detectable in chronically inflamed tissues and secreted autoantibodies. We propose a multilayer model of PC memory in which CD19(+) and CD19(-) PC represent dynamic and static components, respectively, permitting both adaptation and stability of humoral immune protection.

Vaccination response to protein and carbohydrate antigens in patients with rheumatoid arthritis after rituximab treatment

IntroductionRheumatoid arthritis (RA) is frequently complicated with infections. The aim of our study was to evaluate vaccination response in patients with RA after B-cell depletion by using rituximab.MethodsInfluenza (Afluria) and pneumococcal polysaccharides (Pneumo23) vaccines were given 6 months after rituximab (post-RTX group, n = 11) or 6 days before rituximab treatment (pre-RTX group; n = 8). RA patients never exposed to RTX composed the control group (n = 10). Vaccine-specific cellular responses were evaluated on day 6 after vaccination, and vaccine-specific humoral responses, on day 21.ResultsOn day 6 after vaccination, formation of influenza-specific B cells was lower in post-RTX group as compared with the pre-RTX group and controls (P = 0.04). Polysaccharide-specific B cells were found in 27% to 50%, being equally distributed between the groups. On day 21, the impairment of humoral responses was more pronounced with respect to influenza as compared with the pneumococcal vaccine and affected both IgG and light-chain production. Total absence of influenza-specific IgG production was observed in 55% of the post-RTX group.ConclusionsRTX compromises cellular and humoral vaccine responses in RA patients. However, repeated RTX treatment or previous anti-tumor necrosis factor (anti-TNF) treatment did not accentuate these defects.