Emerging role of C5aR2: novel insights into the regulation of uterine immune cells during pregnancy.

Pregnancy is a fascinating immunological phenomenon because it allows allogeneic fetal and placental tissues to survive inside the mother. As a component of innate immunity with high inflammatory potential, the complement system must be tightly regulated during pregnancy. Dysregulation of the complement system plays a role in pregnancy complications including pre-eclampsia and intrauterine growth restriction. Complement components are also used as biomarkers for pregnancy complications. However, the mechanisms of detrimental role of complement in pregnancy is poorly understood. C5a is the most potent anaphylatoxin and generates multiple immune reactions via two transmembrane receptors, C5aR1 and C5aR2. C5aR1 is pro-inflammatory, but the role of C5aR2 remains largely elusive. Interestingly, murine NK cells have been shown to express C5aR2 without the usual co-expression of C5aR1. Furthermore, C5aR2 appears to regulate IFN-γ production by NK cells in vitro. As IFN-γ produced by uterine NK cells is one of the major factors for the successful development of a vital pregnancy, we investigated the role anaphylatoxin C5a and its receptors in the establishment of pregnancy and the regulation of uterine NK cells by examinations of murine C5ar2-/- pregnancies and human placental samples. C5ar2-/- mice have significantly reduced numbers of implantation sites and a maternal C5aR2 deficiency results in increased IL-12, IL-18 and IFN-γ mRNA expression as well as reduced uNK cell infiltration at the maternal-fetal interface. Human decidual leukocytes have similar C5a receptor expression patterns showing clinical relevance. In conclusion, this study identifies C5aR2 as a key contributor to dNK infiltration and pregnancy success.

C5aR2 Deficiency Ameliorates Inflammation in Murine Epidermolysis Bullosa Acquisita by Regulating Fcγ Receptor Expression on Neutrophils.

Epidermolysis bullosa acquisita (EBA) is a rare blistering skin disease induced by autoantibodies directed against type VII collagen. The transfer of antibodies against murine type VII collagen into mice mimics the effector phase of EBA and results in a subepidermal blistering phenotype. Activation of the complement system, and especially the C5a/C5aR1 axis driving neutrophil activation, is critical for EBA pathogenesis. However, the role of the alternative C5a receptor, C5aR2, which is commonly thought to be more immunosuppressive, in the pathogenesis of EBA is still elusive. Therefore, we sought to delineate the functional relevance of C5aR2 during the effector phase of EBA. Interestingly, C5ar2-/- mice showed an attenuated disease phenotype, suggesting a pathogenic contribution of C5aR2 in disease progression. In vitro, C5ar2-/- neutrophils exhibited significantly reduced intracellular calcium flux, ROS release, and migratory capacity when activated with immune complexes or exposed to C5a. These functions were completely absent when C5ar1-/- neutrophils were activated. Moreover, C5aR2 deficiency lowered the ratio of activating and inhibitory FcγRs, impeding the sustainment of inflammation. Collectively, we show here a proinflammatory contribution of C5aR2 in the pathogenesis of antibody-induced tissue damage in experimental EBA.

Stability engineering of the Geobacillus stearothermophilus alcohol dehydrogenase and application for the synthesis of a polyamide 12 precursor.

The thermostable NAD(+)-dependent alcohol dehydrogenase from Geobacillus stearothermophilus (BsADH) was exploited with regard to the biocatalytic synthesis of ω-oxo lauric acid methyl ester (OLAMe), a key intermediate for biobased polyamide 12 production, from the corresponding long-chain alcohol. Recombinant BsADH was produced in Escherichia coli as a homogeneous tetrameric enzyme and showed high activity towards the industrially relevant substrate ω-hydroxy lauric acid methyl ester (HLAMe) with K M = 86 µM and 44 U mg(-1). The equilibrium constant for HLAMe oxidation to the aldehyde (OLAMe) with NAD(+) was determined as 2.16 × 10(-3) from the kinetic parameters of the BsADH-catalyzed forward and reverse reactions. Since BsADH displayed limited stability under oxidizing conditions, the predominant oxidation-prone residue Cys257 was mutated to Leu based on sequence homology with related enzymes and computational simulation. This substitution resulted in an improved BsADH variant exhibiting prolonged stability and an elevated inactivation temperature. Semi-preparative biocatalysis at 60 °C using the stabilized enzyme, employing butyraldehyde for in situ cofactor regeneration with only catalytic amounts of NAD(+), yielded up to 23 % conversion of HLAMe to OLAMe after 30 min. In contrast to other oxidoreductases, no overoxidation to the dodecanoic diacid monomethyl ester was detected. Thus, the mutated BsADH offers a promising biocatalyst for the selective oxidation of fatty alcohols to yield intermediates for industrial polymer production.