IFN-γ-Preconditioned Human Gingival-Derived Mesenchymal Stromal Cells Inhibit Plasmacytoid Dendritic Cells via Adenosine.

Plasmacytoid dendritic cells (pDCs) are vital players in antiviral immune responses because of their high levels of IFN-α secretion. However, this attribute has also implicated them as critical factors behind the immunopathogenesis of inflammatory diseases, and no currently available therapy can efficiently inhibit pDCs' aberrant activation. Mesenchymal stromal cells (MSCs) possess stromal immunomodulatory functionality, regulating immune cell activation through several mechanisms, including the adenosinergic (CD39/CD73/adenosine) pathway. The IFN-γ preconditioning of bone marrow MSCs improves their inhibitory properties for therapy applications; however, isolating human gingival tissue-derived MSCs (hGMSCs) is more accessible. These cells have shown better immunomodulatory effects, yet the outcome of IFN-γ preconditioning and its impact on the adenosinergic pathway has not been evaluated. This study first validated the immunoregulatory properties of primary-cultured hGMSCs, and the results showed that IFN-γ preconditioning strengthens CD39/CD73 coexpression, adenosine production, and the regulatory properties of hGMSC, which were confirmed by describing for the first time their ability to reduce pDC activation and their IFN-α secretion and to increase the frequency of CD73+ pDC. In addition, when CD73's enzymatic activity was neutralized in hGMSCs, adenosine production and the IFN-γ preconditioning effect were restrained. This evidence might be applied to design hGMSCs- and adenosine-based immunotherapeutic strategies for treating inflammatory disorders that are associated with pDC overactivation.

Detection of Asymptomatic Sickle Cell Hemoglobin Carriers and Fetal Hemoglobin Regulating Genetic Variants in African Descendants from Oaxaca, Mexico.

Sickle cell anemia has been classified as a noninfectious neglected tropical disease and, although not exclusively, affects African descendants more frequently. This study aimed to detect asymptomatic sickle cell hemoglobin carriers (HbAS) in marginalized and vulnerable populations during a public health screening in African descendants from Oaxaca, Mexico, and to validate an amplification refractory mutation system (ARMS)-PCR methodology to detect fetal-hemoglobin (HbF)-regulating genetic variants in BCL11A toward affordable routine association of single nucleotide variants (SNVs) with HbF concentrations. To this aim, hemoglobin variants were detected by acidic citrate agar and alkaline cellulose acetate electrophoreses. SNVs in the hemoglobin subunit beta gene (HBB) were identified by the ß-globin mutation detection assay (ß-GMDA) and ARMS-PCR, respectively, and validated by Sanger sequencing. The association between genotypes and HbF concentrations was evaluated using Spearman's correlation coefficient. The results obtained during a directed screening in 140 self-identified African descendants revealed 42 HbS-carriers (30%), of which 39 showed normal total hemoglobin concentrations (92.8%), only 3 presented anemia (7.2%), and 9 showed quantifiable HbF concentration (21.4%). As validated by Sanger sequencing, the designed ARMS-PCR efficiently detected homozygous and heterozygous variants in BCL11A. In a cohort of 42 heterozygous (HbAS) and 27 healthy (HbAA) individuals from the same population, only one SNV (rs766432) showed statistically significant association with increasing HbF concentration, and two new unrelated homozygous silent variants were identified. This study reveals the need to raise coverage of HbS screening in vulnerable populations and shows a feasible low-cost ARMS-PCR methodology to determine the presence of SNVs in quantitative trait loci affecting HbF.

LL-37 Triggers Antimicrobial Activity in Human Platelets.

Platelets play a crucial role in hemostasis and the immune response, mainly by recognizing signals associated with vascular damage. However, it has recently been discovered that the antimicrobial peptide LL-37 activates platelets in functions related to thrombus formation and inflammation. Therefore, this work aims to evaluate the effect of LL-37 on the activation of antimicrobial functions of human platelets. Our results show that platelets treated with LL-37 increase the surface expression of receptors (Toll-like receptors (TLRs) 2 and -4, CD32, CD206, Dectin-1, CD35, LOX-1, CD41, CD62P, and αIIbß3 integrins) for the recognition of microorganisms, and molecules related to antigen presentation to T lymphocytes (CD80, CD86, and HLA-ABC) secrete the antimicrobial molecules: bactericidal/permeability-increasing protein (BPI), azurocidin, human neutrophil peptide (HNP) -1, and myeloperoxidase. They also translate azurocidin, and have enhanced binding to Escherichia coli, Staphylococcus aureus, and Candida albicans. Furthermore, the supernatant of LL-37-treated platelets can inhibit E. coli growth, or platelets can employ their LL-37 to inhibit microbial growth. In conclusion, these findings demonstrate that LL-37 participates in the antimicrobial function of human platelets.

Morphological and Compositional Analysis of Neutrophil Extracellular Traps Induced by Microbial and Chemical Stimuli.

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of neutrophil extracellular traps (NETs). This study analyzes the morphological and compositional changes in NETs induced by microbial and chemical stimuli using standardized in vitro methodologies for NET induction and characterization with human cells. The procedures described here allow the analysis of NET morphology (lytic or non-lytic) and composition (DNA-protein structures and enzymatic activity), and the effect of soluble factors or cellular contact on such characteristics. Additionally, the techniques described here could be modified to evaluate the effect of exogenous soluble factors or cellular contact on NET composition. The applied techniques include the purification of polymorphonuclear cells from human peripheral blood using a double density gradient (1.079-1.098 g/mL), guaranteeing optimal purity and viability (≥ 95%) as demonstrated by Wright's staining, trypan blue exclusion, and flow cytometry, including FSC versus SSC analysis and 7AAD staining. NET formation is induced with microbial (Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans) and chemical (phorbol myristate acetate, HOCl) stimuli, and the NETs are characterized by DNA-DAPI staining, immunostaining for the antimicrobial peptide cathelicidin (LL37), and quantification of enzymatic activity (neutrophil elastase, cathepsin G, and myeloperoxidase). The images are acquired through fluorescence microscopy and analyzed with ImageJ.

Variability in Susceptibility to Type I Interferon Response and Subgenomic RNA Accumulation Between Clinical Isolates of Dengue and Zika Virus From Oaxaca Mexico Correlate With Replication Efficiency in Human Cells and Disease Severity.

Dengue and Zika viruses cocirculate annually in endemic areas of Mexico, causing outbreaks of different magnitude and severity every year, suggesting a continuous selection of Flavivirus variants with variable phenotypes of transmissibility and virulence. To evaluate if Flavivirus variants with different phenotypes cocirculate during outbreaks, we isolated dengue and Zika viruses from blood samples of febrile patients from Oaxaca City during the 2016 and 2019 epidemic years. We compared their replication kinetics in human cells, susceptibility to type I interferon antiviral response, and the accumulation of subgenomic RNA on infected cells. We observed correlations between type I interferon susceptibility and subgenomic RNA accumulation, with high hematocrit percentage and thrombocytopenia. Our results suggest that Flaviviruses that cocirculate in Oaxaca, Mexico, have variable sensitivity to the antiviral activity of type I interferons, and this phenotypic trait correlates with the severity of the disease.

Human Platelets Contain, Translate, and Secrete Azurocidin; A Novel Effect on Hemostasis.

Platelets play a significant role in hemostasis and perform essential immune functions, evidenced by the extensive repertoire of antimicrobial molecules. Currently, there is no clear description of the presence of azurocidin in human platelets. Azurocidin is a 37 kDa cationic protein abundant in neutrophils, with microbicidal, opsonizing, and vascular permeability-inducing activity. Therefore, this work aimed to characterize the content, secretion, translation, and functions of azurocidin in platelets. Our results show the presence of azurocidin mRNA and protein in α-granules of platelet and megakaryoblasts, and stimulation with thrombin, ADP, and LPS leads to the secretion of free azurocidin as well as within extracellular vesicles. In addition, platelets can translate azurocidin in a basal or thrombin-induced manner. Finally, we found that the addition of low concentrations of azurocidin prevents platelet aggregation and activation. In conclusion, we demonstrate that platelets contain, secrete, and translate azurocidin, and this protein may have important implications for hemostasis.

miRNAs Contained in Extracellular Vesicles Cargo Contribute to the Progression of Idiopathic Pulmonary Fibrosis: An In Vitro Aproach.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease. Lesions in the lung epithelium cause alterations in the microenvironment that promote fibroblast accumulation. Extracellular vesicles (EVs) transport proteins, lipids, and nucleic acids, such as microRNAs (miRNAs). The aim of this study was to characterize the differentially expressed miRNAs in the cargo of EVs obtained from the LL97 and LL29 fibroblast cell lines isolated from IPF lungs versus those derived from the CCD19 fibroblast cell line isolated from a healthy donors. We characterized EVs by ultracentrifugation, Western blotting, and dynamic light scattering. We identified miRNAs by small RNA-seq, a total of 1144 miRNAs, of which 1027 were known miRNAs; interestingly, 117 miRNAs were novel. Differential expression analysis showed that 77 miRNAs were upregulated and 68 were downregulated. In addition, pathway enrichment analyses from the Gene Ontology and Kyoto Encyclopedia of Genomes identified several miRNA target genes in the categories, cell proliferation, regulation of apoptosis, pathways in cancer, and proteoglycans in cancer. Our data reveal that miRNAs contained in EVs cargo could be helpful as biomarkers for fibrogenesis, diagnosis, and therapeutic intervention of IPF.

Proteomic Analysis Reveals Key Proteins in Extracellular Vesicles Cargo Associated with Idiopathic Pulmonary Fibrosis In Vitro.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, irreversible, and highly fatal disease. It is characterized by the increased activation of both fibroblast and myofibroblast that results in excessive extracellular matrix (ECM) deposition. Extracellular vesicles (EVs) have been described as key mediators of intercellular communication in various pathologies. However, the role of EVs in the development of IPF remains poorly understood. This study aimed to characterize the differentially expressed proteins contained within EVs cargo derived from the fibroblast cell lines LL97A (IPF-1) and LL29 (IPF-2) isolated from lungs bearing IPF as compared to those derived from the fibroblast cell lines CCD8Lu (NL-1) and CCD19Lu (NL-2) isolated from healthy donors. Isolated EVs were subjected to label-free quantitative proteomic analysis by LC-MS/MS, and as a result, 331 proteins were identified. Differentially expressed proteins were obtained after the pairwise comparison, including all experimental groups. A total of 86 differentially expressed proteins were identified in either one or more comparison groups. Of note, proteins involved in fibrogenic processes, such as tenascin-c (TNC), insulin-like-growth-factor-binding protein 7 (IGFBP7), fibrillin-1 (FBN1), alpha-2 collagen chain (I) (COL1A2), alpha-1 collagen chain (I) (COL1A1), and lysyl oxidase homolog 1 (LOXL1), were identified in EVs cargo isolated from IPF cell lines. Additionally, KEGG pathway enrichment analysis revealed that differentially expressed proteins participate in focal adhesion, PI3K-Akt, and ECM-receptor interaction signaling pathways. In conclusion, our findings reveal that proteins contained within EVs cargo might play key roles during IPF pathogenesis.

Structural differences of neutrophil extracellular traps induced by biochemical and microbiologic stimuli under healthy and autoimmune milieus.

Neutrophil extracellular traps (NETs) are networks of decondensed chromatin loaded with antimicrobial peptides and enzymes produced against microorganisms or biochemical stimuli. Since their discovery, numerous studies made separately have revealed multiple triggers that induce similar NET morphologies allowing to classify them as lytic or non-lytic. However, the variability in NET composition depending on the inducer agent and the local milieu under similar conditions has been scarcely studied. In this work, a comparative study was conducted to evaluate structural and enzymatic divergences in NET composition induced by biochemical (phorbol myristate acetate [PMA] and hypochlorous acid [HOCl]) and microbiologic (Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa) stimuli, along with the presence of plasma from healthy donors or patients with systemic lupus erythematosus (SLE). The results showed a differential composition of DNA and the antimicrobial peptide cathelicidin (LL37) and a variable enzymatic activity (neutrophil elastase, cathepsin G, myeloperoxidase) induced by the different stimuli despite showing morphologically similar NETs. Additionally, SLE plasma´s presence increased DNA and LL37 release during NET induction independently of the trigger stimulus but with no enzymatic activity differences. This work provides new evidence about NET composition variability depending on the inducer stimulus and the local milieu.

Silent red blood cell autoantibodies: Are they naturally occurring or an effect of tolerance loss for a subsequent autoimmune process?

Unexpected anti-red blood cell (RBC) alloantibodies are routinely investigated in immunohematology and blood banking since their existence in pregnant women may induce haemolytic disease of the foetus and newborn, and their presence in donors may induce haemolytic transfusion reactions or hyperacute rejection in solid organ transplantation. Unexpected anti-RBC alloantibodies may target antigens of the most blood types excluding the expected antibodies targeting the ABO antigens. Their incidence in humans was originally linked to alloimmunization events such as blood transfusions, transplants, or pregnancies. But later, many findings revealed their existence in pathogenic processes such as malignancies, infections, and autoimmune diseases; and usually (but not always) associated to autoimmune haemolytic anaemia (AIHA). Nevertheless, unexpected anti-RBC autoantibodies are also occasionally found in healthy individuals in the absence of AIHA and with no history of alloimmunization or the associated pathologic processes. Hence, they are generally known as non-clinically significant, are excluded for typification and called "silent red blood cell autoantibodies (SRBCAA)". This review highlights evidence related to genetic predisposition, molecular mimicry, immune dysregulation, and immune tolerance loss surrounding the existence of anti-RBC antibodies, describing the presence of SRBCAA as possible early witnesses of the development of autoimmune diseases.

Human platelets and megakaryocytes express defensin alpha 1.

Platelets are anucleate cells that have a role in several innate immune functions, including the secretion of proteins with antimicrobial activity. Several studies have demonstrated the ability of platelets to secrete thrombin-induced platelet microbicidal proteins and antimicrobial peptides, like hBD-1. However, the expression and secretion of defensins of the alpha family by platelets have not been fully elucidated. The aim of this study was to characterize the expression of defensin alpha 1 (DEFA1) in human platelets and megakaryocytes. Our data indicate that DEFA1 mRNA and protein are present in peripheral blood platelets and in the megakaryoblastic leukemia cell line (MEG-01). DEFA1 co-localize with α-granules of platelets and MEG-01 cells, and was also detected in cytoplasm of MEG-01 cells. The assay of our in vitro model of platelet-like particles (PLPs) revealed that MEG-01 cells could transfer DEFA1 mRNA to their differentiated PLPs. Furthermore, platelets secreted DEFA1 into the culture medium when activated with thrombin, adenosine diphosphate, and lipopolysaccharide; meanwhile, MEG-01 cells secreted DEFA1 when activated with thrombopoietin. Platelet's secreted DEFA1 can rebind to platelet's surface and have antibacterial activity against the gram-negative bacteria Escherichia coli. In summary, our data indicate that both, human platelets and megakaryocytes, can express and secrete DEFA1. These results suggest a new role of platelets and megakaryocytes in the innate immune response.

Irregular antibodies in no hemolytic autoimmune diseases are able to induce erythrophagocytosis.

Irregular antibodies are produced by alloimmunization because of pregnancies or blood transfusions. They are called "irregular" due to target erythrocyte antigens from "rare blood systems," those different from the ABO system. Irregular antibodies have been widely investigated in immunohematology since their presence in blood donors may lead to difficulties in blood typing and in blood cross-matching, or to induce hemolytic transfusion reactions. Nevertheless, their incidence and participation in the physiopathology of autoimmune diseases have not been thoroughly studied. In this work, we analyzed the presence and pro-hemolytic capabilities of irregular antibodies in patients with different autoimmune diseases lacking signs of hemolytic anemia, in comparison with healthy multiparous women. Five of 141 autoimmune patients (3.5 %) and two of 77 multiparous women (2.6 %) were positive. Although frequency was relatively low and similar in both populations, the targeted antigens were Kell (k, Kpb, Jsb) and Luth (Lub) in multiparous women, and the same plus Duffy (Fya), Kidd (Jka) and MNS (M, s) in autoimmune patients. Irregular antibodies from autoimmune patients did not induce complement-mediated hemolysis (intravascular), but they were able to induce macrophages-mediated phagocytosis (extravascular hemolysis) in vitro. It is the first approach exploring the presence of irregular antibodies associated with the loss of immune tolerance and demonstrating their hemolytic potential in autoimmune patients without hemolytic manifestations. The presence of irregular antibodies targeted to Duffy (Fya), Kidd (Jka) and MNS (M, s) antigens only in autoimmune patients suggests a loss of immune tolerance to these erythrocyte antigens.