Pleural CD14+ monocytes/macrophages of healthy adolescents show a high expression of metallothionein family genes.

Nowadays laparoscopic interventions enable the collection of resident macrophage populations out of the human cavities. We employed this technique to isolate pleural monocytes/macrophages from healthy young adults who underwent a correction of pectus excavatum. High quality CD14+ monocytes/macrophages (plMo/Mφ) were used for RNA-sequencing (RNA-seq) in comparison with human monocyte-derived macrophages (MDM) natural (MDM-0) or IL-4-polarized (MDM-IL4). Transcriptome analysis revealed 7166 and 7076 differentially expressed genes (DEGs) in plMo/Mφ relative to natural MDM-0 and polarized MDM-IL4, respectively. The gene set enrichment analysis, which was used to compare RNA-seq data from plMo/Mφ with single-cell (scRNA-seq) data online from human bronchial lavage macrophages, showed that plMo/Mφs are characterized by a high expression of genes belonging to the metallothionein (MT) family, and that the expression of these genes is significantly higher in plMo/Mφ than in MDM-0 or MDM-IL4. Our results provide additional insights on high MTs-expressing macrophage subsets, which seem to be present not only in bronchial lavage of healthy adults or in pleural exudates of lung cancer patients but also in pleural fluid of healthy young adults. Macrophage subsets expressing high MTs may have specific roles in lung defense, repair, and homeostasis, and require further investigations.

Distinct metabolic responses to heme in inflammatory human and mouse macrophages - Role of nitric oxide.

Activation of inflammation is tightly associated with metabolic reprogramming in macrophages. The iron-containing tetrapyrrole heme can induce pro-oxidant and pro-inflammatory effects in murine macrophages, but has been associated with polarization towards an anti-inflammatory phenotype in human macrophages. In the current study, we compared the regulatory responses to heme and the prototypical Toll-like receptor (TLR)4 ligand lipopolysaccharide (LPS) in human and mouse macrophages with a particular focus on alterations of cellular bioenergetics. In human macrophages, bulk RNA-sequencing analysis indicated that heme led to an anti-inflammatory transcriptional profile, whereas LPS induced a classical pro-inflammatory gene response. Co-stimulation of heme with LPS caused opposing regulatory patterns of inflammatory activation and cellular bioenergetics in human and mouse macrophages. Specifically, in LPS-stimulated murine, but not human macrophages, heme led to a marked suppression of oxidative phosphorylation and an up-regulation of glycolysis. The species-specific alterations in cellular bioenergetics and inflammatory responses to heme were critically dependent on the availability of nitric oxide (NO) that is generated in inflammatory mouse, but not human macrophages. Accordingly, studies with an inducible nitric oxide synthase (iNOS) inhibitor in mouse, and a pharmacological NO donor in human macrophages, reveal that NO is responsible for the opposing effects of heme in these cells. Taken together, the current findings indicate that NO is critical for the immunomodulatory role of heme in macrophages.

Split-Combine Click-SELEX Reveals Ligands Recognizing the Transplant Rejection Biomarker CXCL9.

Renal rejection is a major incidence in patients after kidney transplantation and associated with allograft scarring and function loss, especially in antibody-mediated rejection. Regular clinical monitoring of kidney-transplanted patients is thus necessary, but measuring donor-specific antibodies is not always predictive, and graft biopsies are time-consuming and costly and may come up with a histological result unsuspicious for rejection. Therefore, a noninvasive diagnostic approach to estimate an increased probability of kidney graft rejection by measuring specific biomarkers is highly desired. The chemokine CXCL9 is described as an early indicator of rejection. In this work, we identified clickmers and an aptamer by split-combine click-SELEX (systematic evolution of ligands by exponential enrichment) that bind CXLC9 with high affinity. The aptamers recognize native CXCL9 and maintain binding properties under urine conditions. These features render the molecules as potential binding and detector probes for developing point-of-care devices, e.g., lateral flow assays, enabling the noninvasive monitoring of CXCL9 in renal allograft patients.

An Antibody-Aptamer-Hybrid Lateral Flow Assay for Detection of CXCL9 in Antibody-Mediated Rejection after Kidney Transplantation.

Chronic antibody-mediated rejection (AMR) is a key limiting factor for the clinical outcome of a kidney transplantation (Ktx), where early diagnosis and therapeutic intervention is needed. This study describes the identification of the biomarker CXC-motif chemokine ligand (CXCL) 9 as an indicator for AMR and presents a new aptamer-antibody-hybrid lateral flow assay (hybrid-LFA) for detection in urine. Biomarker evaluation included two independent cohorts of kidney transplant recipients (KTRs) from a protocol biopsy program and used subgroup comparisons according to BANFF-classifications. Plasma, urine and biopsy lysate samples were analyzed with a Luminex-based multiplex assay. The CXCL9-specific hybrid-LFA was developed based upon a specific rat antibody immobilized on a nitrocellulose-membrane and the coupling of a CXCL9-binding aptamer to gold nanoparticles. LFA performance was assessed according to receiver operating characteristic (ROC) analysis. Among 15 high-scored biomarkers according to a neural network analysis, significantly higher levels of CXCL9 were found in plasma and urine and biopsy lysates of KTRs with biopsy-proven AMR. The newly developed hybrid-LFA reached a sensitivity and specificity of 71% and an AUC of 0.79 for CXCL9. This point-of-care-test (POCT) improves early diagnosis-making in AMR after Ktx, especially in KTRs with undetermined status of donor-specific HLA-antibodies.

Alpha1-antitrypsin counteracts heme-induced endothelial cell inflammatory activation, autophagy dysfunction and death.

Free heme toxicity in the vascular endothelium is critical for the pathogenesis of hemolytic disorders including sickle cell disease. In the current study, it is demonstrated that human alpha1-antitrypsin (A1AT), a serine protease inhibitor with high binding-affinity for heme, rescues endothelial cell (EC) injury caused by free heme. A1AT provided endothelial protection against free heme toxicity via a pathway that differs from human serum albumin and hemopexin, two prototypical heme-binding proteins. A1AT inhibited heme-mediated pro-inflammatory activation and death of ECs, but did not affect the increase in intracellular heme levels and up-regulation of the heme-inducible enzyme heme oxygenase-1. Moreover, A1AT reduced heme-mediated generation of mitochondrial reactive oxygen species. Extracellular free heme led to an increased up-take of A1AT by ECs, which was detected in lysosomes and was found to reduce heme-dependent alkalization of these organelles. Finally, A1AT was able to restore heme-dependent dysfunctional autophagy in ECs. Taken together, our findings show that A1AT rescues ECs from free heme-mediated pro-inflammatory activation, cell death and dysfunctional autophagy. Hence, A1AT therapy may be useful in the treatment of hemolytic disorders such as sickle cell disease.

A Potential Late Stage Intermediate of Twin-Arginine Dependent Protein Translocation in Escherichia coli.

The twin-arginine translocation (Tat) system transports folded proteins across membranes of prokaryotes, plant plastids, and some mitochondria. According to blue-native polyacrylamide gel electrophoresis after solubilization with digitonin, distinct interactions between the components TatA, TatB, and TatC result in two major TatBC-containing complexes in Escherichia coli that can bind protein substrates. We now report the first detection of a TatABC complex that likely represents the state at which transport occurs. This complex was initially found when the photo cross-linking amino acid p-benzoyl-l-phenylalanine (Bpa) was introduced at position I50 on the periplasmic side of the first trans-membrane domain of TatC. Cross-linking of TatCI50Bpa resulted in TatC-TatC-cross-links, indicating a close proximity to neighboring TatC in the complex. However, the new complex was not caused by cross-links but rather by non-covalent side chain interactions, as it was also detectable without UV-cross-linking or with an I50Y exchange. The new complex did not contain any detectable substrate. It was slightly upshifted relative to previously reported substrate-containing TatABC complexes. In the absence of TatA, an inactive TatBCI50Bpa complex was formed of the size of wild-type substrate-containing TatABC complexes, suggesting that TatB occupies TatA-binding sites at TatCI50Bpa. When substrate binding was abolished by point mutations, this TatBCI50Bpa complex shifted analogously to active TatABCI50Bpa complexes, indicating that a defect substrate-binding site further enhances TatB association to TatA-binding sites. Only TatA could shift the complex with an intact substrate-binding site, which explains the TatA requirement for substrate transport by TatABC systems.

HLA class II antibodies induce necrotic cell death in human endothelial cells via a lysosomal membrane permeabilization-mediated pathway.

Antibody-mediated rejection (AMR) is the major cause of allograft loss after solid organ transplantation. Circulating donor-specific antibodies against human leukocyte antigen (HLA), in particular HLA class II antibodies are critical for the pathogenesis of AMR via interactions with endothelial cells (ECs). To investigate the effects of HLA class II antibody ligation to the graft endothelium, a model of HLA-DR antibody-dependent stimulation was utilized in primary human ECs. Antibody ligation of HLA class II molecules in interferon-γ-treated ECs caused necrotic cell death without complement via a pathway that was independent of apoptosis and necroptosis. HLA-DR-mediated cell death was blocked by specific neutralization of antibody ligation with recombinant HLA class II protein and by lentiviral knockdown of HLA-DR in ECs. Importantly, HLA class II-mediated cytotoxicity was also induced by relevant native allele-specific antibodies from human allosera. Necrosis of ECs in response to HLA-DR ligation was mediated via hyperactivation of lysosomes, lysosomal membrane permeabilization (LMP), and release of cathepsins. Notably, LMP was caused by reorganization of the actin cytoskeleton. This was indicated by the finding that LMP and actin stress fiber formation by HLA-DR antibodies were both downregulated by the actin polymerization inhibitor cytochalasin D and inhibition of Rho GTPases, respectively. Finally, HLA-DR-dependent actin stress fiber formation and LMP led to mitochondrial stress, which was revealed by decreased mitochondrial membrane potential and generation of reactive oxygen species in ECs. Taken together, ligation of HLA class II antibodies to ECs induces necrotic cell death independent of apoptosis and necroptosis via a LMP-mediated pathway. These findings may enable novel therapeutic approaches for the treatment of AMR in solid organ transplantation.