Christian M. Karsten

Anti-inflammatory activity of IgG1 mediated by Fc galactosylation and association of FcγRIIB and dectin-1

Complement is an ancient danger-sensing system that contributes to host defense, immune surveillance and homeostasis. C5a and its G protein–coupled receptor mediate many of the proinflammatory properties of complement. Despite the key role of C5a in allergic asthma, autoimmune arthritis, sepsis and cancer, knowledge about its regulation is limited. Here we demonstrate that IgG1 immune complexes (ICs), the inhibitory IgG receptor FcγRIIB and the C-type lectin–like receptor dectin-1 suppress C5a receptor (C5aR) functions. IgG1 ICs promote the association of FcγRIIB with dectin-1, resulting in phosphorylation of Src homology 2 domain–containing inositol phosphatase (SHIP) downstream of FcγRIIB and spleen tyrosine kinase downstream of dectin-1. This pathway blocks C5aR-mediated ERK1/2 phosphorylation, C5a effector functions in vitro and C5a-dependent inflammatory responses in vivo, including peritonitis and skin blisters in experimental epidermolysis bullosa acquisita. Notably, high galactosylation of IgG N-glycans is crucial for this inhibitory property of IgG1 ICs, as it promotes the association between FcγRIIB and dectin-1. Thus, galactosylated IgG1 and FcγRIIB exert anti-inflammatory properties beyond their impact on activating FcγRs.

Hydroxycarboxylic acid receptor 2 mediates dimethyl fumarate’s protective effect in EAE

Taken orally, the drug dimethyl fumarate (DMF) has been shown to improve functional outcomes for patients with MS; however, it is unclear how DMF mediates a protective effect. DMF and, more so, its active metabolite, monomethyl fumarate, are known agonists of the hydroxycarboxylic acid receptor 2 (HCA₂), a G protein-coupled membrane receptor. Here, we evaluated the contribution of HCA₂ in mediating the protective effect afforded by DMF in EAE, a mouse model of MS. DMF treatment reduced neurological deficit, immune cell infiltration, and demyelination of the spinal cords in wild-type mice, but not in Hca2⁻/⁻ mice, indicating that HCA₂ is required for the therapeutic effect of DMF. In particular, DMF decreased the number of infiltrating neutrophils in a HCA₂-dependent manner, likely by interfering with neutrophil adhesion to endothelial cells and chemotaxis. Together, our data indicate that HCA₂ mediates the therapeutic effects of DMF in EAE. Furthermore, identification of HCA₂ as a molecular target may help to optimize MS therapy.

The immunoglobulin, IgG Fc receptor and complement triangle in autoimmune diseases

Immunoglobulin G (IgG)-mediated activation of complement and IgG Fc receptors (FcγRs) are important defense mechanisms of the innate immune system to ward off infections. However, the same mechanisms can drive severe and harmful inflammation, when IgG antibodies react with self-antigens in solution or tissues, as described for several autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, and immune vasculitis. More specifically, IgG immune complexes (ICs) can activate all three pathways of the complement system resulting in the generation of C3 and C5 cleavage products that can activate a panel of different complement receptors on innate and adaptive immune cells. Importantly, complement and FcγRs are often co-expressed on inflammatory immune cells such as neutrophils, monocytes, macrophages or dendritic cells and act in concert to mediate the inflammatory response in autoimmune diseases. In this context, the cross-talk between the receptor for the anaphylatoxin C5a, i.e. C5ar1 (CD88) and FcγRs is of major importance. Recent data suggest a model of bidirectional regulation, in which CD88 acts upstream of FcγRs and sets the threshold for FcγR-dependent effector responses by regulating the ratio between activating and inhibitory FcγRs. Vice versa, FcγR ligation can either amplify or block C5aR-mediated effector functions, depending on whether IgG IC aggregate activating or inhibitory FcγRs. Further, complement and FcγRs cooperate on B cells and on follicular dendritic cells to regulate the development of autoreactive B cells, their differentiation into plasma cells and, eventually, the production of autoantibodies. Here, we will give an update on recent findings regarding this complex regulatory network between complement and FcγRs, which may also regulate the inflammatory response in allergy, cancer and infection.

The CD46-Jagged1 interaction is critical for human TH1 immunity

CD46 is a complement regulator with important roles related to the immune response. CD46 functions as a pathogen receptor and is a potent costimulator for the induction of interferon-γ (IFN-γ)-secreting effector T helper type 1 (TH1) cells and their subsequent switch into interleukin 10 (IL-10)-producing regulatory T cells. Here we identified the Notch family member Jagged1 as a physiological ligand for CD46. Furthermore, we found that CD46 regulated the expression of Notch receptors and ligands during T cell activation and that disturbance of the CD46-Notch crosstalk impeded induction of IFN-γ and switching to IL-10. Notably, CD4+ T cells from CD46-deficient patients and patients with hypomorphic mutations in the gene encoding Jagged1 (Alagille syndrome) failed to mount appropriate TH1 responses in vitro and in vivo, which suggested that CD46-Jagged1 crosstalk is responsible for the recurrent infections in subpopulations of these patients.

Monitoring and Cell-Specific Deletion of C5aR1 Using a Novel Floxed GFP-C5aR1 Reporter Knock-in Mouse

Many of the biological properties of C5a are mediated through activation of its receptor (C5aR1), the expression of which has been demonstrated convincingly on myeloid cells, such as neutrophils, monocytes, and macrophages. In contrast, conflicting results exist regarding C5aR1 expression in dendritic cells (DCs) and lymphoid lineage cells. In this article, we report the generation of a floxed GFP-C5aR1 reporter knock-in mouse. Using this mouse strain, we confirmed strong C5aR1 expression in neutrophils from bone marrow, blood, lung, and spleen, as well as in peritoneal macrophages. Further, we show C5aR1 expression in lung eosinophils, lung- and lamina propria–resident and alveolar macrophages, bone marrow–derived DCs, and lung-resident CD11b+ and monocyte-derived DCs, whereas intestinal and pulmonary CD103+ DCs stained negative. Also, some splenic NKT cells expressed GFP, whereas naive NK cells and B2 cells lacked GFP expression. Finally, we did not observe any C5aR1 expression in naive or activated CD4+ Th cells in vitro or in vivo. Mating the floxed GFP-C5aR1 mouse strain with LysMCre mice, we were able to specifically delete C5aR1 in neutrophils and macrophages, whereas C5aR1 expression was retained in DCs. In summary, our findings suggest that C5aR1 expression in mice is largely restricted to cells of the myeloid lineage. The novel floxed C5aR1 reporter knock-in mouse will prove useful to track C5aR1 expression in experimental models of acute and chronic inflammation and to conditionally delete C5aR1 in immune cells.

C5a receptor signalling in dendritic cells controls the development of maladaptive Th2 and Th17 immunity in experimental allergic asthma

The pathways underlying dendritic cell (DC) activation in allergic asthma are incompletely understood. Here we demonstrate that adoptive transfer of ovalbumin-pulsed wild-type (wt) but not of C5a receptor-deficient (C5aR−/−) bone marrow (BM)-derived DCs (BMDCs) induced mixed T helper type 2 (Th2)/Th17 maladaptive immunity, associated with severe airway hyperresponsiveness, mucus production, and mixed eosinophilic/neutrophilic inflammation. Mechanistically, antigen uptake, processing, and CD11b expression were reduced in C5aR−/− BMDCs. Further, interleukin (IL)-1β, -6, and -23 production were impaired resulting in reduced Th17 cell differentiation, associated with accelerated activated T-cell death in vitro and in vivo. Surprisingly, we found an increased frequency of CD11bhiCD11cintGr1+F4/80+ cells, expressing arginase and nitric oxide synthase in C5aR−/− BM preparations. Intratracheal administration of ovalbumin-pulsed wt DCs and sorted CD11bhiCD11cintGr1+F4/80+ C5aR−/− cells reduced Th2 immune responses in vivo. Together, we uncover novel roles for C5aR in Th17 differentiation, T-cell survival, and differentiation of a DC-suppressor population controlling Th2 immunity in experimental allergic asthma.

DC within the pregnant mouse uterus influence growth and functional properties of uterine NK cells

The vascular addressins mucosal addressin cell adhesion molecule‐1, P‐selectin and ICAM‐1 permit α4β7‐integrin‐expressing DC, especially those of the myeloid lineage (CD11c+CD11b+ DC), to access the pregnant mouse uterus. Injection of blocking monoclonal antibodies against mucosal addressin cell adhesion molecule‐1 in P‐selectin−/− mice or experimental approaches with β7‐integrin−/− or ICAM‐1−/− mice revealed that limited access or absence of CD11c+CD11b+ DC at the maternal/fetal interface negatively affects the frequency, size and functional properties of uterine NK (uNK) cells. Adoptive transfer of DC obtained from WT mice into β7‐integrin−/− mice abrogates these effects and emphasizes the importance of DC in uNK cell differentiation. Interestingly, those implantation sites lacking CD11c+CD11b+ DC are characterized by decreased IL‐15 and IL‐12 mRNA and/or protein levels. Chronic administration of IL‐15 in these mice gives rise to uNK cell numbers and size comparable to those of WT mice, whereas additional injection of IL‐12 positively affects the IFN‐γ expression of uNK cells. Real‐time RT‐PCR and protein arrays performed with isolated uterine DC underline the role of DC as a source of IL‐15 and IL‐12 in the pregnant mouse uterus.

Old dogs-new tricks: immunoregulatory properties of C3 and C5 cleavage fragments.

The activation of the complement system by canonical and non‐canonical mechanisms results in the generation of multiple C3 and C5 cleavage fragments including anaphylatoxins C3a and C5a as well as opsonizing C3b/iC3b. It is now well appreciated that anaphylatoxins not only act as pro‐inflammatory mediators but as immunoregulatory molecules that control the activation status of cells and tissue at several levels. Likewise, C3b/iC3b is more than the opsonizing fragment that facilitates engulfment and destruction of targets by phagocytes. In the circulation, it also facilitates the transport and delivery of bacteria and immune complexes to phagocytes, through a process known as immune adherence, with consequences for adaptive immunity. Here, we will discuss non‐classical immunoregulatory properties of C3 and C5 cleavage fragments. We highlight the influence of anaphylatoxins on Th2 and Th17 cell development during allergic asthma with a particular emphasis on their role in the modulation of CD11b+ conventional dendritic cells and monocyte‐derived dendritic cells. Furthermore, we discuss the control of anaphylatoxin‐mediated activation of dendritic cells and allergic effector cells by adaptive immune mechanisms that involve allergen‐specific IgG1 antibodies and plasma or regulatory T cell‐derived IL‐10 production. Finally, we take a fresh look at immune adherence with a particular focus on the development of antibacterial cytotoxic T‐cell responses.

Truncated and Full-Length Thioredoxin-1 Have Opposing Activating and Inhibitory Properties for Human Complement with Relevance to Endothelial Surfaces

Thioredoxin (Trx)-1 is a small, ubiquitously expressed redox-active protein with known important cytosolic functions. However, Trx1 is also upregulated in response to various stress stimuli, is found both at the cell surface and secreted into plasma, and has known anti-inflammatory and antiapoptotic properties. Previous animal studies have demonstrated that exogenous Trx1 delivery can have therapeutic effects in a number of disease models and have implicated an interaction of Trx1 with the complement system. We found that Trx1 is expressed in a redox-active form at the surface of HUVEC and acts as an inhibitor of complement deposition in a manner dependent on its Cys-Gly-Pro-Cys active site. Inhibition occurred at the point of the C5 convertase of complement, regulating production of C5a and the membrane attack complex. A truncated form of Trx1 also exists in vivo, Trx80, which has separate nonoverlapping functions compared with the full-length Trx1. We found that Trx80 activates the classical and alternative pathways of complement activation, leading to C5a production, but the inflammatory potential of this was also limited by the binding of inhibitors C4b-binding protein and factor H. This study adds a further role to the known anti-inflammatory properties of Trx1 and highlights the difference in function between the full-length and truncated forms.

Selectin Platelet Plays a Critical Role in Granulocyte Access to the Pregnant Mouse Uterus under Physiological and Pathological Conditions

Abstract Leukocyte recruitment to the pregnant mouse uterus is associated with highly regulated patterns of expression of vascular adhesion receptors. One striking observation is the localized expression of mucosal vascular addressin cell adhesion molecule (MADCAM1) and selectin, platelet (SELP, formerly P-selectin) by maternal vessels in the vascular zone (VZ) during the first half of pregnancy. From midgestation onwards, endothelial cells lining the maternal vessels of the VZ in addition express vascular cell adhesion molecule-1 (VCAM1). The predominant cell population within these vessels is monocyte-like cells. Granulocytes and low numbers of lymphocytes are also present. Murine fetal trophoblast cells are almost devoid of adhesion molecules, including SELP. In contrast, spontaneous abortions of allogeneic pregnancies are characterized by dramatic upregulation of SELP on maternal VZ vessels and on fetal trophoblast cells. Upregulation of SELP is associated with a dramatic influx of highly activated granulocytes, which infiltrate the vessels and tissue of the VZ and the trophoblast. The majority of the activated granulocytes within the trophoblast undergo nuclear fragmentation, which can be detected by TUNEL staining. To demonstrate that SELP is involved in the recruitment of granulocytes to the pregnant uterus, we undertook long-term in vivo inhibition studies using a monoclonal antibody to inhibit the contribution of SELP to leukocyte trafficking to the decidua. In addition, the pregnant uteri of syngeneic Selp−/− × Selp−/− mice were investigated and compared to the controls. Our results clearly demonstrate the importance of SELP for granulocyte access to the pregnant mouse uterus under physiological and pathological conditions.