Kim C. M. Santegoets

Increased Frequency and Compromised Function of T Regulatory Cells in Systemic Sclerosis (SSc) Is Related to a Diminished CD69 and TGFβ Expression

Background Regulatory T cells (Tregs) are essential in the control of tolerance. Evidence implicates Tregs in human autoimmune conditions. Here we investigated their role in systemic sclerosis (SSc). Methods/Principal Findings Patients were subdivided as having limited cutaneous SSc (lcSSc, n = 20) or diffuse cutaneous SSc (dcSSc, n = 48). Further subdivision was made between early dcSSc (n = 24) and late dcSSc (n = 24) based upon the duration of disease. 26 controls were studied for comparison. CD3+ cells were isolated using FACS and subsequently studied for the expression of CD4, CD8, CD25, FoxP3, CD127, CD62L, GITR, CD69 using flow cytometry. T cell suppression assays were performed using sorted CD4CD25highCD127- and CD4CD25lowCD127high and CD3+ cells. Suppressive function was correlated with CD69 surface expression and TGFβ secretion/expression. The frequency of CD4+CD25+ and CD25highFoxP3highCD127neg T cells was highly increased in all SSc subgroups. Although the expression of CD25 and GITR was comparable between groups, expression of CD62L and CD69 was dramatically lower in SSc patients, which correlated with a diminished suppressive function. Co-incubation of Tregs from healthy donors with plasma from SSc patients fully abrogated suppressive activity. Activation of Tregs from healthy donors or SSc patients with PHA significantly up regulated CD69 expression that could be inhibited by SSc plasma. Conclusions/Significance These results indicate that soluble factors in SSc plasma inhibit Treg function specifically that is associated with altered Treg CD69 and TGFβ expression. These data suggest that a defective Treg function may underlie the immune dysfunction in systemic sclerosis.

TLR2 promotes Th2/Th17 responses via TLR4 and TLR7/8 by abrogating the Type I IFN amplification loop

TLR2 plays an important role in the removal of Gram-positive bacteria; contrastingly, it also appears to have important protective effects against unrestrained inflammation and subsequent organ injury during infection and autoimmunity. We hypothesized that TLR2 tunes the phenotype of dendritic cells (DCs) activated through other TLRs, thereby fulfilling a crucial role in the modulation of the immune response. TLR2 potently inhibited TLR4- and TLR7/8-induced cytokine production by human DCs. The inhibitory effect of TLR2 on the release of TNF-α but not of IL-12p70 was mediated by PI3K. TLR2 inhibits the production of IL-12p70 by dampening the type 1 IFN amplification loop. When DCs were triggered with the potent synergistic combination of LPS (TLR4) and R848 (TLR7/8) in conjunction with a TLR2 ligand, a clear shift to more Th2- and Th17-prone responses in the naive and memory T cell subpopulations was observed. This shift in T cell responses was inherent to the inability of TLR2-stimulated DCs to produce IL-12p70 and was dependent on the production of IL-1 and IL-6.

The inhibitory Fc gamma IIb receptor dampens TLR4-mediated immune responses and is selectively up-regulated on dendritic cells from rheumatoid arthritis patients with quiescent disease.

Rheumatoid arthritis (RA) is a common autoimmune disease leading to profound disability and premature death. Although a role for FcγRs and TLRs is accepted, their precise involvement remains to be elucidated. FcγRIIb is an inhibitory FcR important in the maintenance of tolerance. We hypothesized that the inhibitory FcγRIIb inhibits TLR responses on monocyte-derived dendritic cells (DC) and serves as a counterregulatory mechanism to dampen inflammation, and we surmised that this mechanism might be defective in RA. The expression of the inhibitory FcγRIIb was found to be significantly higher on DCs from RA patients having low RA disease activity in the absence of treatment with antirheumatic drugs. The expression of activating FcγRs was similarly distributed among all RA patients and healthy controls. Intriguingly, only DCs with a high expression of FcγRIIb were able to inhibit TLR4-mediated secretion of proinflammatory cytokines when stimulated with immune complexes. In addition, when these DCs were coincubated with the combination of a TLR4 agonist and immune complexes, a markedly inhibited T cell proliferation was apparent, regulatory T cell development was promoted, and T cells were primed to produce high levels of IL-13 compared with stimulation of the DCs with the TLR4 agonist alone. Blocking FcγRIIb with specific Abs fully abrogated these effects demonstrating the full dependence on the inhibitory FcγRIIb in the induction of these phenomena. This TLR4-FcγRIIb interaction was shown to dependent on the PI3K and Akt pathway.

Toll-like receptors in rheumatic diseases: Are we paying a high price for our defense against bugs?

In the last decade Toll‐like receptor (TLR) research has led to new insights in the pathogenesis of many rheumatic diseases. In autoimmune diseases like systemic lupus erythematosus, rheumatoid arthritis and systemic sclerosis TLR signaling is likely to be involved in tolerance breakthrough and chronic inflammation via combined Fc gamma receptors and TLR recognition of immune complexes. Furthermore, inflammatory diseases like psoriatic arthritis and gout also show more and more evidence for TLR involvement. In this review we will discuss the involvement of TLR signaling in several rheumatic diseases and stress their similarities and differences based on recent findings.

Soluble immune complexes shift the TLR-induced cytokine production of distinct polarized human macrophage subsets towards IL-10.

Background Costimulation of murine macrophages with immune complexes (ICs) and TLR ligands leads to alternative activation. Studies on human myeloid cells, however, indicate that ICs induce an increased pro-inflammatory cytokine production. This study aimed to clarify the effect of ICs on the pro- versus anti-inflammatory profile of human polarized macrophages. Materials and Methods Monocytes isolated from peripheral blood of healthy donors were polarized for four days with IFN-γ, IL-4, IL-10, GM-CSF, M-CSF, or LPS, in the presence or absence of heat aggregated gamma-globulins (HAGGs). Phenotypic polarization markers were measured by flow cytometry. Polarized macrophages were stimulated with HAGGs or immobilized IgG alone or in combination with TLR ligands. TNF, IL-6, IL-10, IL-12, and IL-23 were measured by Luminex and/or RT-qPCR. Results HAGGs did not modulate the phenotypic polarization and the cytokine production of macrophages. However, HAGGs significantly altered the TLR-induced cytokine production of all polarized macrophage subsets, with the exception of MΦIL-4. In particular, HAGGs consistently enhanced the TLR-induced IL-10 production in both classically and alternatively polarized macrophages (M1 and M2). The effect of HAGGs on TNF and IL-6 production was less pronounced and depended on the polarization status, while IL-23p19 and IL-12p35 expression was not affected. In contrast with HAGGs, immobilized IgG induced a strong upregulation of not only IL-10, but also TNF and IL-6. Conclusion HAGGs alone do not alter the phenotype and cytokine production of in vitro polarized human macrophages. In combination with TLR-ligands, however, HAGGs but not immobilized IgG shift the cytokine production of distinct macrophage subsets toward IL-10.

Fcγ receptor expression on splenic macrophages in adult immune thrombocytopenia

Splenic macrophages play a key role in immune thrombocytopenia (ITP) pathogenesis by clearing opsonized platelets. Fcγ receptors (FcγR) participate in this phenomenon, but their expression on splenic macrophages and their modulation by treatment have scarcely been studied in human ITP. We aimed to compare the phenotype and function of splenic macrophages between six controls and 24 ITP patients and between ITP patients according to the treatments they received prior to splenectomy. CD86, human leucocyte antigen D‐related (HLA‐DR) and FcγR expression were measured by flow cytometry on splenic macrophages. The major FcγR polymorphisms were determined and splenic macrophage function was assessed by a phagocytosis assay. The expression of the activation markers CD86 and HLA‐DR was higher on splenic macrophages during ITP compared to controls. While the expression of FcγR was not different between ITP and controls, the phagocytic function of splenic macrophages was reduced in ITP patients treated with intravenous immunoglobulin (IVIg) within the 2 weeks prior to splenectomy. The FCGR3A (158V/F) polymorphism, known to increase the affinity of FcγRIII to IgG, was over‐represented in ITP patients. Thus, these are the first results arguing for the fact that the therapeutic use of IVIg during human chronic ITP does not modulate FcγR expression on splenic macrophages but decreases their phagocytic capabilities.

Interactions among myeloid regulatory cells in cancer

Mounting evidence has accumulated on the critical role of the different myeloid cells in the regulation of the cancerous process, and in particular in the modulation of the immune reaction to cancer. Myeloid cells are a major component of host cells infiltrating tumors, interacting with each other, with tumor cells and other stromal cells, and demonstrating a prominent plasticity. We describe here various myeloid regulatory cells (MRCs) in mice and human as well as their relevant therapeutic targets. We first address the role of the monocytes and macrophages that can contribute to angiogenesis, immunosuppression and metastatic dissemination. Next, we discuss the differential role of neutrophil subsets in tumor development, enhancing the dual and sometimes contradicting role of these cells. A heterogeneous population of immature myeloid cells, MDSCs, was shown to be generated and accumulated during tumor progression as well as to be an important player in cancer-related immune suppression. Lastly, we discuss the role of myeloid DCs, which can either contribute to effective anti-tumor responses or play a more regulatory role. We believe that MRCs play a critical role in cancer-related immune regulation and suggest that future anti-cancer therapies will focus on these abundant cells.

Macrophage dysfunction in Psoriatic Arthritis

The pathogenesis of Psoriatic Arthritis (PsA) remains poorly understood. The underlying chronic inflammatory immune response is thought to be triggered by unknown environmental factors and might arise due to an impaired (innate) immune function by Dendritic Cells (DCs) [1]. Anti-inflammatory CD163+ type 2 macrophages (mf-2) are thought to have important functions in restoring immune homeostasis during an inflammatory response. Mf-2 are present in PsA synovium at high numbers. Why the immune response in PsA goes awry, despite the presence of these mf-2, is still largely undetermined.

Impaired dendritic cell proinflammatory cytokine production in psoriatic arthritis

Objective: The pathogenesis of psoriatic arthritis (PsA) remains poorly understood. The underlying chronic inflammatory immune response is thought to be triggered by unknown environmental factors potentially arising from a defective immune function. We undertook this study to determine whether an impaired acute inflammatory response by dendritic cells (DCs) might compromise the clearance of bacteria and predispose to chronic inflammation. Methods: We determined cytokine production by DCs from healthy controls and from patients with rheumatoid arthritis, PsA, and psoriasis in response to Mycobacterium tuberculosis, Mycobacterium avium paratuberculosis, and a range of other bacteria and Toll-like receptor (TLR) ligands. Phenotypic differences involved in cellular responses against (myco) bacteria were determined by quantitative polymerase chain reaction and flow cytometry. Results: The secretion of proinflammatory cytokines by PsA DCs was impaired upon in vitro challenge with mycobacteria and TLR-2 ligands. This impairment was associated with elevated serum levels of C-reactive protein. The expression of TLR-2 and other receptors known to mediate mycobacterial recognition was unaltered. In contrast, the intracellular TLR inhibitors suppressor of cytokine signaling 3 and A20 were more highly expressed in DCs from PsA patients. PsA DCs further demonstrated up-regulated levels of ATG16L1, NADPH oxidase 2, and LL37, which are molecules implicated in the immune response against intracellular bacteria. Conclusion: Our findings indicate that DCs from PsA patients have a disordered immune response toward some species of (myco) bacteria. This might predispose to impaired immune responses to, and in turn impaired clearance of, these bacteria, setting the stage for the chronic inflammation of joints, entheses, skin, and the gut.

Antigen-Presenting Cells and their Fcγ and Toll-Like Receptors: Leading Suspects in Autoimmunity

Antigen-presenting cells (APCs) play an important role in the development of autoimmune diseases. These cells recognize pathogen associated molecular patterns but also endogenously produced ligands through toll-like receptors (TLRs). Aberrant activation of these receptors and the following intracellular signaling pathways can induce the deleteri- ous production of pro-inflammatory cytokines. In genetically predisposed individuals this might lead to a breach in toler- ance and eventually autoimmunity. IgG and IgG immune complexes (ICs), which are abundantly present in autoimmune diseases like systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and systemic sclerosis (SSc) are recognized by APCs via Fc gamma receptors (Fc Rs) and can also modulate their activation state. Upon their uptake specific antigens present in ICs are capable of stimulating APCs via their intracellular TLRs, increasing their capability to induce (autoreac- tive) T and B cell responses. This underscores their likely role in the generation and maintenance of autoimmunity. By fo- cusing on three autoimmune diseases, SLE, RA and SSc, we will illustrate the importance of TLRs and Fc Rs in the pathogenesis of autoimmune diseases.