A Novel Role for C5a in B-1 Cell Homeostasis.

B-1 cells constitute a unique subpopulation of lymphocytes residing mainly in body cavities like the peritoneal cavity (PerC) but are also found in spleen and bone marrow (BM). As innate-like B cells, they mediate first line immune defense through low-affinity natural IgM (nIgM) antibodies. PerC B-1 cells can egress to the spleen and differentiate into nIgM antibody-secreting plasma cells that recognize conserved exogenous and endogenous cellular structures. Homing to and homeostasis within the PerC are regulated by the chemokine CXCL13 released by PerC macrophages and stroma cells. However, the exact mechanisms underlying the regulation of CXCL13 and B-1 homeostasis are not fully explored. B-1 cells play important roles in the inflammatory response to infection, autoimmunity, ischemia/reperfusion injury, obesity, and atherosclerosis. Remarkably, this list of inflammatory entities has a strong overlap with diseases that are regulated by complement suggesting a link between B-1 cells and the complement system. Interestingly, up to now, no data exist regarding the role of complement in B-1 cell biology. Here, we demonstrate for the first time that C5a regulates B-1 cell steady-state dynamics within the peritoneum, the spleen, and the BM. We found decreased B-1a cell numbers in the peritoneum and the spleen of C5aR1-/- mice associated with increased B1-a and B1-b numbers in the spleen and high serum titers of nIgM antibodies directed against phosphorylcholine and several pneumococcal polysaccharides. Similarly, peritoneal B-1a cells were decreased in the peritoneum and splenic B-1a and B-1b cells were increased in C5aR2-/- mice. The decrease in peritoneal B-1 cell numbers was associated with decreased peritoneal CXCL13 levels in C5aR1-/- and C5aR2-/- mice. In search for mechanisms, we found that combined TLR2 and IL-10 receptor activation in PerC macrophages induced strong CXCL13 production, which was significantly reduced in cells from C5aR1- and C5aR2-deficient mice and after combined C5aR-targeting. Such stimulation also induced marked local C5 production by PerC macrophages and C5a generation. Importantly, peritoneal in vivo administration of C5a increased CXCL13 production. Taken together, our findings suggest that local non-canonical C5 activation in PerC macrophages fuels CXCL13 production as a novel mechanism to control B-1 cell homeostasis.

Monitoring C5aR2 Expression Using a Floxed tdTomato-C5aR2 Knock-In Mouse.

The biological significance of C5a receptor [(C5aR)2/C5L2], a seven-transmembrane receptor binding C5a and C5adesArg, remains ill-defined. Specific ligation of C5aR2 inhibits C5a-induced ERK1/2 activation, strengthening the view that C5aR2 regulates C5aR1-mediated effector functions. Although C5aR2 and C5aR1 are often coexpressed, a detailed picture of C5aR2 expression in murine cells and tissues is still lacking. To close this gap, we generated a floxed tandem dye (td)Tomato-C5aR2 knock-in mouse that we used to track C5aR2 expression in tissue-residing and circulating immune cells. We found the strongest C5aR2 expression in the brain, bone marrow, and airways. All myeloid-derived cells expressed C5aR2, although with different intensities. C5aR2 expression in blood and tissue neutrophils was strong and homogeneous. Specific ligation of C5aR2 in neutrophils from tdTomato-C5aR2 mice blocked C5a-driven ERK1/2 phosphorylation, demonstrating functionality of C5aR2 in the reporter mice. In contrast to neutrophils, we found tissue-specific differences in C5aR2 expression in eosinophils, macrophages, and dendritic cell subsets. Naive and activated T cells stained negative for C5aR2, whereas B cells from different tissues homogeneously expressed C5aR2. Also, NK cell subsets in blood and spleen strongly expressed C5aR2. Activation of C5aR2 in NK cells suppressed IL-12/IL-18-induced IFN-γ production. Intratracheal IL-33 challenge resulted in decreased C5aR2 expression in pulmonary eosinophils and monocyte-derived dendritic cells. In summary, we provide a detailed map of murine C5aR2 immune cell expression in different tissues under steady-state conditions and upon pulmonary inflammation. The C5aR2 knock-in mouse will help to reliably track and conditionally delete C5aR2 expression in experimental models of inflammation.

The association of mannose-binding lectin 2 polymorphisms with outcome in very low birth weight infants.

OBJECTIVES: Studies on the influence of mannose-binding lectin (MBL) deficiency on infection susceptibility in preterm infants have yielded controversial results. We investigated the association of genotype-based MBL levels with outcome in very-low-birth weight infants (VLBWI). METHODS: We genotyped 3 genetic variants of MBL2 (rs1800450, rs1800451, rs5030737) in 6878 VLBWI. MBL plasma levels were categorized as normal (wild type, A/A), low (heterozygotes, A/O) or undetectable (homozygotes, O/O). Primary outcome was the effect of genotype-based MBL2 levels on blood-culture proven and clinical sepsis during primary stay in hospital. We also evaluated burden of infection within 24 months after discharge. RESULTS: We found no association between MBL levels and sepsis risk in the whole cohort. Infants without measurable MBL levels born between 32 0/7 to 36 6/7 weeks of gestation, however, had a higher rate of Gram-negative sepsis than infants with normal or reduced MBL levels. In a follow-up investigation at 24 months (n = 1070 infants), infants without measurable MBL levels suffered more frequently from stomatitis and urinary tract infection. CONCLUSIONS: In a large cohort of VLBWI MBL2 deficiency had no major impact on infection risk unless children were born between 32 0/7 and 36 6/7 weeks of gestation.

Regulation and function of anaphylatoxins and their receptors in allergic asthma.

Allergic asthma is a disease of the airways driven by maladaptive T helper 2 (Th2) and Th17 immune response against harmless, airborne substances. The hallmarks of this disease are airway hyperresponsiveness (AHR), eosinophilic and neutrophilic airway inflammation and mucus overproduction. Distinct dendric cell (DC) subsets together with airway epithelial and pulmonary vascular endothelial cells play critical roles in allergen sensing and in driving T cell differentiation towards Th2 and Th17 effector or regulatory T cells (Treg). Previous studies suggested already a pivotal role for the anaphylatoxins (C5a, C3a) in the pathogenesis of allergic asthma. During sensitization for example it is described, that C3a promotes, whereas C5a protects from the development of maladaptive immunity during allergen sensitization. Here we will discuss the role of the anaphylatoxins (C3a, C5a) and their receptors during the pathogenesis of allergic asthma, and specifically in lung DC biology. We will also have a look on canonical and non-canonical complement activation and we will discuss novel concepts on how the adaptive immune system can regulate the function of ATRs also in the context of allergic asthma.

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.

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 protein­coupled 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 lectin­like 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 domain­containing 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.

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 alpha(4)beta(7)-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 beta7-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 beta7-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-gamma 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.