CCL17 is produced by conventional dendritic cells, signals through CCR4 on regulatory T (T_reg) cells and drives atherosclerosis by suppressing T_reg functions through yet undefined mechanisms. Here we show that conventional dendritic cells from CCL17-deficient mice display a pro-tolerogenic phenotype and transcriptome that is not phenocopied in mice lacking its cognate receptor CCR4. In the plasma of CCL17-deficient mice, CCL3 was the only decreased cytokine/chemokine. We found that CCL17 signaled through CCR8 as an alternate high-affinity receptor, which induced CCL3 expression and suppressed T_reg functions in the absence of CCR4. Genetic ablation of CCL3 and CCR8 in CD4⁺ T cells reduced CCL3 secretion, boosted FoxP3⁺ T_reg numbers and limited atherosclerosis. Conversely, CCL3 administration exacerbated atherosclerosis and restrained T_reg differentiation. In symptomatic versus asymptomatic human carotid atheroma, CCL3 expression was increased, whereas FoxP3 expression was reduced. Together, we identified a non-canonical chemokine pathway whereby CCL17 interacts with CCR8 to yield a CCL3-dependent suppression of atheroprotective T_reg cells.

Upon activation the human bradykinin B₂ receptor (B₂R) acts as guanine nucleotide exchange factor for the G proteins G_q/11 and G_i. Thereafter, it gets phosphorylated by G protein-coupled receptor kinases (GRKs) and recruits β-arrestins, which block further G protein activation and promote B₂R internalization via clathrin-coated pits. As for most G protein-coupled receptors of family A, an intracellular helix 8 after transmembrane domain 7 is also predicted for the B₂R. We show here that disruption of helix 8 in the B₂R by either C-terminal truncation or just by mutation of a central amino acid (Lys-315) to a helix-breaking proline resulted in strong reduction of surface expression. Interestingly, this malfunction could be overcome by the addition of the membrane-permeable B₂R antagonist JSM10292, suggesting that helix 8 has a general role for conformational stabilization that can be accounted for by an appropriate antagonist. Intriguingly, an intact helix 8, but not the C terminus with its phosphorylation sites, was indispensable for receptor sequestration and for interaction of the B₂R with GRK2/3 and β-arrestin2 as shown by co-immunoprecipitation. Recruitment of β-arrestin1, however, required the presence of the C terminus. Taken together, our results demonstrate that helix 8 of the B₂R plays a crucial role not only in efficient trafficking to the plasma membrane or the activation of G proteins but also for the interaction of the B₂R with GRK2/3 and β-arrestins. Additional data obtained with chimera of B₂R with other G protein-coupled receptors of family A suggest that helix 8 might have similar functions in other GPCRs as well.

For most cell culture experiments, it is indispensable that the cells are firmly anchored to culture plates, withstanding rinsing steps that can create shear forces and tolerating temperature changes without detaching. For semi-adherent cells such as the common HEK 293 or PC-12 cells, this could so far be obtained by time-consuming plate pre-coating with cationic polymer solutions. We report here, that i) pre-coating with the cheaper poly-ethylenimine (PEI) works as well as the commonly used poly-D-lysine (PDL), but more importantly and novel ii) that simple direct addition of either PEI (1.5 μg/ml) or PDL (2 μg/ml) to the cell culture medium results in strongly anchored HEK 293 cells, indistinguishable from ones seeded on pre-coated plates. Therefore, the replacement of plate pre-coating by direct addition of either PEI or PDL gives comparable excellent results, but is highly labour-, time-, and cost-efficient. Moreover, we could show that addition of PDL or PEI also works similarly well in animal-free culture using human platelet lysate instead of fetal bovine serum. Interestingly, additional experiments showed that strong cell attachment requires only cationic polymers but not fetal bovine serum or human platelet lysate added to the medium.

Determinants for desensitization and sequestration of G protein‐coupled receptors often contain serine or threonine residues located in their C‐termini. The sequence context, however, in which these residues have to appear, and the receptor specificity of these motifs are largely unknown. Mutagenesis studies with the B₂ bradykinin receptor (B₂wt), stably expressed in HEK 293 cells, identified a sequence distal to N338 (NSMGTLRTSI, including I347 but not the basally phosphorylated S348) and in particular the TSI sequence therein, as a major determinant for rapid agonist‐inducible internalization and the prevention of receptor hypersensitivity. Chimeras of the noninternalizing B₁ bradykinin receptor (B₁wt) containing these B₂wt sequences sequestered poorly, however, suggesting that additional motifs more proximal to N338 are required. In fact, further substitution of the B₁wt C‐terminus with corresponding B₂wt regions either at C330(7.71) following putative helix 8 (B₁CB₂) or at the preceding Y312(7.53) in the NPXXY sequence (B₁YB₂) resulted in chimeras displaying rapid internalization. Intriguingly, however, exchange performed at K322(7.63) within putative helix 8 generated a slowly internalizing chimera (B₁KB₂). Detailed mutagenesis analysis generating additional chimeras identified the change of V323 in B₁wt to serine (as in B₂wt) as being responsible for this effect. The slowly internalizing chimera as well as a B₁wt point‐mutant V323S displayed significantly reduced inositol phosphate accumulation as compared to B₁wt or the other chimeras. The slow internalization of B₁KB₂ was also accompanied by a lack of agonist‐induced phosphorylation, that in contrast was observed for B₁YB₂ and B₁CB₂, suggesting that putative helix 8 is either directly or indirectly (e.g. via G protein activation) involved in the interaction between the receptor and receptor kinases.

Candida albicans yeast produces 10 distinct secreted aspartic proteases (Saps), which are some of the most important virulence factors of this pathogenic fungus. One of the suggested roles of Saps is their deregulating effect on various proteolytic cascades that constitute the major homeostatic systems in human hosts, including blood coagulation, fibrinolysis, and kallikrein-kinin systems. This study compared the characteristics of the action of all 10 Saps on human kininogens, which results in generating proinflammatory bradykinin-related peptides (kinins). Recombinant forms of Saps, heterologously overexpressed in Pichia pastoris were applied. Except for Sap7 and Sap10, all Saps effectively cleaved the kininogens, with the highest hydrolytic activity toward the low-molecular-mass form (LK). Sap1–6 and 8 produced a biologically active kinin—Met-Lys-bradykinin—and Sap3 was exceptional in terms of the kinin-releasing yield (>60% LK at pH 5.0 after 24 hours). Des-Arg1-bradykinin was released from LK by Sap9 at a comparably high yield, but this peptide was assumed to be biologically inactive because it was unable to interact with cellular B2-type kinin receptors. However, the collaborative actions of Sap9 and Sap1, −2, −4–6, and −8 on LK rerouted kininogen cleavage toward the high-yield release of the biologically active Met-Lys-bradykinin. Our present results, together with the available data on the expression of individual SAP genes in candidal infection models, suggest a biological potential of Saps to produce kinins at the infection foci. The kinin release during candidiasis can involve predominant and complementary contributions of two different Sap3- and Sap9-dependent mechanisms.