C5a drives airway constriction and inflammation during the effector phase of allergic asthma, mainly through the activation of C5a receptor 1 (C5aR1). Yet, C5aR1 expression on myeloid and lymphoid cells during the allergic effector phase is ill-defined. Recently, we generated and characterized a floxed green fluorescent protein (GFP)-C5aR1 knock-in mouse. Here, we used this reporter strain to monitor C5aR1 expression in airway, pulmonary and lymph node cells during the effector phase of OVA-driven allergic asthma. C5aR1 reporter and wildtype mice developed a similar allergic phenotype with comparable airway resistance, mucus production, eosinophilic/neutrophilic airway inflammation and Th2/Th17 cytokine production. During the allergic effector phase, C5aR1 expression increased in lung tissue eosinophils but decreased in airway and pulmonary macrophages as well as in pulmonary CD11b+ conventional dendritic cells (cDCs) and monocyte-derived DCs (moDCs). Surprisingly, expression in neutrophils was not affected. Of note, moDCs but not CD11b+ cDCs from mediastinal lymph nodes (mLN) expressed less C5aR1 than DCs residing in the lung after OVA challenge. Finally, neither CD103+ cDCs nor cells of the lymphoid lineage such as Th2 or Th17-differentiated CD4+ T cells, B cells or type 2 innate lymphoid cells (ILC2) expressed C5aR1 under allergic conditions. Our findings demonstrate a complex regulation pattern of C5aR1 in the airways, lung tissue and mLN of mice, suggesting that the C5a/C5aR1 axis controls airway constriction and inflammation through activation of myeloid cells in all three compartments in an experimental model of allergic asthma.
Abstract 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.
Abstract Background A close association between obesity and asthma has been described. The nature of this association remains elusive, especially with respect to allergic asthma. Controversial findings exist regarding the impact of short‐term high‐fat diet (HFD) feeding on the development of allergic asthma. Objective To delineate the impact of short‐term HFD feeding on the development of experimental allergic asthma. Methods Female C57BL/6JRJ mice were fed with a short‐term HFD or chow diet (CD) for 12 weeks. Allergic asthma was induced by intraperitoneal OVA/alum sensitization followed by repeated OVA airway challenges. We determined airway hyperresponsiveness (AHR) and pulmonary inflammation by histologic and flow cytometric analysis of immune cells. Furthermore, we assessed the impact of HFD on dendritic cell (DC)‐mediated activation of T cells. Results Female mice showed a mild increase in body weight accompanied by mild metabolic alterations. Upon OVA challenge, CD‐fed mice developed strong AHR and airway inflammation, which were markedly reduced in HFD‐fed mice. Mucus production was similar in both treatment groups. OVA‐induced increases in DC and CD4 + T‐cell recruitment to the lungs were significantly attenuated in HFD‐fed mice. MHC‐II expression and CD40 expression in pulmonary CD11b + DCs were markedly lower in HFD‐fed compared to CD‐fed mice, which was associated in vivo with a decreased T helper (Th) 1/17 differentiation and Treg formation without impacting Th2 differentiation. Conclusions/clinical relevance These findings suggest that short‐term HFD feeding attenuates the development of AHR, airway inflammation, pulmonary DC recruitment and MHC‐II/CD40 expression leading to diminished Th1/17 but unchanged Th2 differentiation. Thus, short‐term HFD feeding and associated metabolic alterations may have protective effects in allergic asthma development.
Fibrillin-1 and fibrillin-2 constitute the backbone of extracellular filaments, called microfibrils. Fibrillin assembly involves complex multistep mechanisms to result in a periodical head-to-tail alignment in microfibrils. Impaired assembly potentially plays a role in the molecular pathogenesis of genetic disorders caused by mutations in fibrillin-1 (Marfan syndrome) and fibrillin-2 (congenital contractural arachnodactyly). Presently, the basic molecular interactions involved in fibrillin assembly are obscure. Here, we have generated recombinant full-length human fibrillin-1, and two overlapping recombinant polypeptides spanning the entire human fibrillin-2 in a mammalian expression system. Characterization by gel electrophoresis, electron microscopy after rotary shadowing, and reactivity with antibodies demonstrated correct folding of these recombinant polypeptides. Analyses of homotypic and heterotypic interaction repertoires showed N- to C-terminal binding of fibrillin-1, and of fibrillin-1 with fibrillin-2. The interactions were of high affinity with dissociation constants in the low nanomolar range. However, the N- and C-terminal fibrillin-2 polypeptides did not interact with each other. These results demonstrate that fibrillins can directly interact in an N- to C-terminal fashion to form homotypic fibrillin-1 or heterotypic fibrillin-1/fibrillin-2 microfibrils. This conclusion was further strengthened by double immunofluorescence labeling of microfibrils. In addition, the binding epitopes as well as the entire fibrillin molecules displayed very stable properties.
Homocystinuria, a disorder originating in defects in the methionine metabolism, is characterized by an elevated plasma concentration of homocysteine. Most patients have a defect in the cystathionine-beta-synthase, the key enzyme in the conversion of homocysteine to cysteine. Many abnormalities in the connective tissue of patients with homocystinuria resemble those seen in Marfan syndrome, caused by mutations in fibrillin-1. These observations led to the hypothesis that the structure and function of fibrillin-1 is compromised in patients with homocystinuria. To test this hypothesis we produced recombinant human fibrillin-1 fragments spanning the central portion of the molecule (8-Cys/transforming growth factor-beta binding domain 3 to calcium binding EGF domain 22) and extensively analyzed the potential of homocysteine to modify structural and functional properties of these proteins. Circular dichroism spectroscopy revealed moderate changes of their secondary structures after incubation with homocysteine. Equilibrium dialysis demonstrated a number of high affinity calcium binding sites in the tandemly repeated calcium binding epidermal growth factor-like domains 11-22. Calcium binding of homocysteine-modified fragments was completely abolished. Incubation of the recombinant proteins with homocysteine rendered the analyzed calcium binding EGF domains as well as the 8-Cys/transforming growth factor-beta binding domain 3 significantly more susceptible to proteolytic degradation. Furthermore, data were obtained demonstrating that homocysteine can covalently modify fibrillin-1 via disulfide bonds. These data strongly suggest that structural and functional modifications as well as degradation processes of fibrillin-1 in the connective tissues of patients with homocystinuria play a major role in the pathogenesis of this disorder.
Abstract Eosinophils are considered a homogenous cell population, contributing to the allergic asthma phenotype by promoting mucus hypersecretion and airway hyperresponsiveness. Here, we identified a previously unrecognized vacuolated CD11cdim eosinophilic population (vEOS) which accmulates exclusively in the lung tissue and in mediastinal lymph nodes but not in the airways in a house dust mite-mediated allergic asthma model and an IL-33 mediated pulmonary inflammation. In addition to the vEOS, we found classical SiglecF+CD11c− eosinophils (EOS) in the airways and the lung. Both cell types shared typical structural features of eosinophils and expressed CCR3. In contrast to EOS, vEOS expressed higher levels of the CD11a, CD11b and CD18 integrins. Immunofluorescence microscopy revealed that vEOS were located exclusively near arteries and airways, where they were in close contact with T cells. Moreover, vEOS took up antigen and expressed costimulatory molecules more efficiently than EOS. Consequently, in co-culture experiments with OVA-TCR transgenic T cells, vEOS and dendritic cells (DCs) drove T cell proliferation. However, in the presence of vEOS, T cells divided more frequently than in co-culture with DCs. Further, T cell activation in response to co-culture with vEOS together with DCs markedly enhanced IL-17 but not IL-13 production. Collectively, our data demonstrate that vEOS are an important subset of eosinophils, which differ from EOS in their location and their ability to activate T cells. Further, they suggest that cross-talk between vEOS and DCs might be a crucial step for the induction of Th17 cells in allergic asthma. Thus, vEOS may serve as an important target for Th17-associated steroid-resistant asthma.
Even though genetic predisposition has proven to be an important element in Parkinson's disease (PD) etiology, monozygotic (MZ) twins with PD displayed a concordance rate of only about 20% despite their shared identical genetic background.We recruited 5 pairs of MZ twins discordant for idiopathic PD and established skin fibroblast cultures to investigate mitochondrial phenotypes in these cellular models against the background of a presumably identical genome. To test for genetic differences, we performed whole genome sequencing, deep mitochondrial DNA (mtDNA) sequencing, and tested for mitochondrial deletions by multiplex real-time polymerase chain reaction (PCR) in the fibroblast cultures. Further, the fibroblast cultures were tested for mitochondrial integrity by immunocytochemistry, immunoblotting, flow cytometry, and real-time PCR to quantify gene expression.Genome sequencing did not identify any genetic difference. We found decreased mitochondrial functionality with reduced cellular adenosine triphosphate (ATP) levels, altered mitochondrial morphology, elevated protein levels of superoxide dismutase 2 (SOD2), and increased levels of peroxisome proliferator-activated receptor-gamma coactivator-α (PPARGC1A) messenger RNA (mRNA) in skin fibroblast cultures from the affected compared to the unaffected twins. Further, there was a tendency for a higher number of somatic mtDNA variants among the affected twins.We demonstrate disease-related differences in mitochondrial integrity in the genetically identical twins. Of note, the clinical expression matches functional alterations of the mitochondria. ANN NEUROL 2021;89:158-164.
