The Different Immune Responses by Age Are due to the Ability of the Fetal Immune System to Secrete Primal Immunoglobulins Responding to Unexperienced Antigens.

Among numerous studies on coronavirus 2019 (COVID-19), we noted that the infection and mortality rates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) increased with age and that fetuses known to be particularly susceptible to infection were better protected despite various mutations. Hence, we established the hypothesis that a new immune system exists that forms before birth and decreases with aging. Methods: To prove this hypothesis, we established new ex-vivo culture conditions simulating the critical environmental factors of fetal stem cells (FSCs) in early pregnancy. Then, we analyzed the components from FSCs cultivated newly developed ex-vivo culture conditions and compared them from FSCs cultured in a normal condition. Results: We demonstrated that immunoglobulin M (IgM), a natural antibody (NAb) produced only in early B-1 cells, immunoglobulins (Igs) including IgG3, which has a wide range of antigen-binding capacity and affinity, complement proteins, and antiviral proteins are induced in FSCs only cultured in newly developed ex-vivo culture conditions. Particularly we confirmed that their extracellular vesicles (EVs) contained NAbs, Igs, various complement proteins, and antiviral proteins, as well as human leukocyte antigen G (HLA-G), responsible for immune tolerance. Conclusion: Our results suggest that FSCs in early pregnancy can form an independent immune system responding to unlearned antigens as a self-defense mechanism before establishing mature immune systems. Moreover, we propose the possibility of new solutions to cope with various infectious diseases based on the factors in NAbs-containing EVs, especially not causing unnecessary immune reaction due to HLA-G.

Different Features of Interleukin-37 and Interleukin-18 as Disase Activity Markers of Adult-Onset Still's Disease.

The aim of this study was to evaluate the usefulness of serum interleukin (IL)-37 and IL-18 as disease activity markers of adult-onset Still's disease (AOSD) and to compare their related clinical features. Forty-five patients with a set of high and subsequent low disease activity status of AOSD were enrolled. Modified Pouchot (mPouchot) score and serologic disease activity markers including levels of IL-37 and IL-18 were compared between high and low disease activity status. The relationships between disease activity parameters and differences in levels of cytokines according to each disease manifestation were evaluated in high disease activity status. mPouchot score and all disease activity markers including IL-37 and IL-18 significantly declined after treatment. Though both cytokines positively correlated with mPouchot score, the two did not correlate with each other in high disease activity status. IL-18 positively correlated with ferritin, AST, and LDH while IL-37 correlated better with CRP. The expression level of IL-37 was related to leukocytosis while IL-18 was related to pleuritis, pneumonitis, abnormal LFT, and hyperferritinemia. In addition, patients in the IL-18 dominant group presented with higher LDH levels and required a higher mean corticosteroid dose. In conclusion, IL-37 and IL-18 are disease activity markers reflecting different aspects of AOSD that can complement each other.

Study of immune-tolerized cell lines and extracellular vesicles inductive environment promoting continuous expression and secretion of HLA-G from semiallograft immune tolerance during pregnancy.

An immune reaction is a protector of our body but a target to be overcome for all non-self-derived medicine. Extracellular Vesicles (EVs), noted as a primary alternative to cell therapy products that exhibit immune rejection due to mismatching-major histocompatibility complex (MHC), were discovered to have excellent curative effects through the delivery of various biologically active substances. Although EVs are sure to incur immune reaction by immunogenicity due to alloantigens from their parental cells, their immune rejection is rarely known. Hence, to develop cell lines and EVs as medicines with no immune rejection, we noted the immune tolerance where the foetus, as semi-allograft, is perfectly protected from the maternal immune system. We designed the ex-vivo culture systems to simulate in-vivo environmental factors inducing extravillous trophoblast (EVT)-specific Human Leukocyte Antigen-G (HLA-G) expression and secretion of HLA-G-bearing EVs at the mother-foetus interface. Using them, we confirmed that immune-tolerized stem cells (itSCs) continuously expressing and secreting HLA-G like EVTs during pregnancy can be induced. Also, EVs secreted from itSCs are verified as immune-tolerized EVs (itSC-EVs) containing HLA-G and not causing immune rejection through various analytical methods. These findings can provide a new perspective on the local and extensive immune tolerance environment where HLA-G is expressed and secreted by pregnancy-related hormones and different biological conditions. Furthermore, they show the new way to develop itSCs-EVs-based therapeutics that are free from time, space, and donor limitation causing immune rejection. ABBREVIATIONS: CFSE: carboxyfluorescein succinimidyl ester; DC: dendritic cells; ELISA: enzyme-linked immunosorbent assay; EV: extracellular vesicles; EVT: extravillous trophoblast; FSH: follicle stimulating hormone; HA: hyaluronic acid; hCG: human chorionic gonadotropin; HLA-G: human leukocyte antigen G; iPSC: induced pluripotent stem cells; itSC-EVs: immune-tolerized extracellular vesicles from itSCs; itTBC-EVs: immune-tolerized extracellular vesicles from itTBCs; itSCs: immune tolerized stem cells; itTBCs: immune-tolerized trophoblast cells; LH: luteinizing hormone; MHC: major histocompatibility complex; MSC: mesenchymal stem cells; NK: natural killer cells; NTA: nanoparticle tracking analysis; PBMC: peripheral blood mononuclear cells; PHA: phytohemagglutinin; SP-IRIS: single particle interferometric reflectance imaging sensing; STB: syncytiotrophoblast.

IL-17A induces osteoblast differentiation by activating JAK2/STAT3 in ankylosing spondylitis.

BACKGROUND: IL-17A has recently emerged as a potential target that regulates the extensive inflammation and abnormal bone formation observed in ankylosing spondylitis (AS). Blocking IL-17A is expected to inhibit bony ankylosis. Here, we investigated the effects of anti IL-17A agents in AS. METHODS: TNFα, IL-17A, and IL-12/23 p40 levels in serum and synovial fluid from patients with ankylosing spondylitis (AS), rheumatoid arthritis (RA), osteoarthritis (OA), or healthy controls (HC) were measured by ELISA. Bone tissue samples were obtained at surgery from the facet joints of ten patients with AS and ten control (Ct) patients with noninflammatory spinal disease. The functional relevance of IL-17A, biological blockades, Janus kinase 2 (JAK2), and non-receptor tyrosine kinase was assessed in vitro with primary bone-derived cells (BdCs) and serum from patients with AS. RESULTS: Basal levels of IL-17A and IL-12/23 p40 in body fluids were elevated in patients with AS. JAK2 was also highly expressed in bone tissue and primary BdCs from patients with AS. Furthermore, addition of exogenous IL-17A to primary Ct-BdCs promoted the osteogenic stimulus-induced increase in ALP activity and mineralization. Intriguingly, blocking IL-17A with serum from patients with AS attenuated ALP activity and mineralization in both Ct and AS-BdCs by inhibiting JAK2 phosphorylation and downregulating osteoblast-involved genes. Moreover, JAK2 inhibitors effectively reduced JAK2-driven ALP activity and JAK2-mediated events. CONCLUSIONS: Our findings indicate that IL-17A regulates osteoblast activity and differentiation via JAK2/STAT3 signaling. They shed light on AS pathogenesis and suggest new rational therapies for clinical AS ankylosis.

Accelerated osteogenic differentiation of human bone-derived cells in ankylosing spondylitis.

Ankylosing spondylitis (AS) is characterized by excessive bone formation with syndesmophytes, leading to bony ankylosis. The contribution of osteoblasts to the pathogenesis of ankylosis is poorly understood. The aim of this study was to determine molecular differences between disease controls (Ct) and AS bone-derived cells (BdCs) during osteogenic differentiation with or without inflammation using AS patient serum. We confirmed osteoblastic differentiation of Ct and AS BdCs under osteogenic medium by observing morphological changes and measuring osteoblastic differentiation markers. Osteoblast differentiation was detected by alkaline phosphatase (ALP) staining and activity, and alizarin red and hydroxyapatite staining. Osteoblast-specific markers were analyzed by quantitative reverse-transcriptase-polymerase chain reaction, immunoblotting, and immunostaining. To examine the effects of inflammation, we added AS and healthy control serum to Ct and AS BdCs, and then analyzed osteoblast-specific markers. AS BdCs showed elevated basal intercellular and extracellular ALP activity compared to Ct. When osteoblast differentiation was induced, AS BdCs exhibited higher expression of osteoblast-specific marker genes and faster mineralization than Ct, indicating that these cells differentiated more rapidly into osteoblasts. ALP activity and mineralization accelerated when serum from AS patients was added to Ct and AS BdCs. Our results revealed that AS BdCs showed significantly increased osteoblastic activity and differentiation capacity by regulating osteoblast-specific transcription factors and proteins compared to Ct BdCs. Active inflammation of AS serum accelerated osteoblastic activity. Our study could provide useful basic data for understanding the molecular mechanism of ankylosis in AS.

Identification and characterization of human bone-derived cells.

This study was designed to identify and characterize primary bone-derived cells (BdCs) and investigate the potential role of osteoblast differentiation. Primary BdCs were isolated from surgical bone for comparative analysis with mesenchymal stem cells (MSCs) and fetal osteoblasts (FOBs) and for potential differentiation to mature osteoblasts. Using three different cells, we successfully cultivated human osteoblast differentiation and activity which were evaluated using microarray and biochemical methods. BdCs are more correlated to MSCs in bioinformatics result and similar with FOBs in gene expression. In particular, Osterix, osteoprogenitor marker, was high expressed in BdCs, while the expression in MSCs and FOBs were very low. Furthermore, BdCs exhibited a marked alkaline phosphatase (ALP) expression, early stage of osteogenic marker, and retained osteogenic properties and physiological changes into maturation as in FOBs. BdCs also showed an increase in bone morphogenic protein 2 (BMP2), osteopontin (OPN), and osteocalcin (OCN) mRNA expressions during differentiation. This study suggests that BdCs may be osteoprogenitor cells or undifferentiated preosteoblasts with strong capacity to differentiate toward mature osteoblasts.