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MicroRNAs (miRNAs) play an important role in regulating fibrogenesis in the liver. The current study examined the ability of microRNA-214 (miR-214) level in liver and serum samples obtained from patients with BA to predict progressive liver fibrosis in patients with biliary atresia (BA). We examined miR-214 level in relation to conventional markers of liver fibrosis, with liver and serum samples from BA patients. Fifty-two patients with BA who underwent Kasai portoenterostomy and four control patients underwent liver biopsy. In 28 patients with BA, blood samples were collected to analyze circulating serum miR-214. MiR-214 levels in liver tissue were significantly upregulated in patients with BA who had severe liver fibrosis (F3–4) compared to those with none to mild fibrosis (F0–2), whereas suppressors-of-fused homolog (Sufu) mRNA levels were significantly suppressed in F3–4. Serum miR-214 levels were significantly higher in patients with F3–4 compared with F0–2. Area under the curve analysis showed that the serum miR-214 cut-off level for predicting F3–4 was 0.805 (p = 0.0046). Hepatic overexpression of miR-214 is associated with progression of liver fibrosis in patients with BA, and the circulating miR-214 level may serve as a non-invasive predictor of liver fibrosis.

Cellular xenogeneic rejection by the innate immune system is a major immunological obstruction that needs to be overcome for the successful clinical use of xenografts. Our focus has been on macrophage-mediated xenogeneic rejection, since suppressing macrophage function has considerable potential for practical applications in the area of xenotransplantation. We report herein on an investigation of the suppressive effect of human CD177 (hCD177) against macrophage-mediated xenogeneic rejection. Wild type swine aortic endothelial cell (SEC) and an SEC transfectant with hCD177 (SEC/hCD177) were co-cultured with macrophages, and the degree of cytotoxicity was evaluated by WST-8 assays, and phagocytosis was examined using Calcein-AM labeling methods. The expression of anti/pro-inflammatory cytokines was evaluated by RT-qPCR and the phosphorylation of SHP-1 on macrophages in co-culture was evaluated by Western blotting. The result of cytotoxicity assays indicated that hCD177 suppressed M1 macrophage-mediated xenogeneic rejection (vs. SEC, p 0.0001). Similarly, the result of phagocytosis assays indicated that hCD177 suppressed it (vs. SEC, p 0.05). In addition, hCD177 significantly suppressed the expression of IL-1β, a pro-inflammatory cytokine, in M1 macrophages (vs. SEC, p 0.01). Luciferase assays using THP1-Lucia NF-kB also showed a significant difference in NF-kB activation (vs. SEC, p 0.001). In addition, hCD177 was found to induce the phosphorylation of SHP-1 in M1 macrophages (vs. SEC, p 0.05). These findings indicate that hCD177 suppresses M1 macrophage-mediated xenogeneic rejection, at least in part via in the phosphorylation of SHP-1.

Although xenografts are one of the most attractive strategies for overcoming the shortage of organ donors, cellular rejection by macrophages is a substantial impediment to this procedure. It is well known that macrophages mediate robust immune responses in xenografts. Macrophages also express various inhibitory receptors that regulate their immunological function. Recent studies have shown that the overexpression of inhibitory ligands on porcine target cells results in the phosphorylation of tyrosine residues on intracellular immunoreceptor tyrosine-based inhibitory motifs on macrophages, leading to the suppression of xenogenic rejection by macrophages. It has also been reported that myeloid-derived suppressor cells, a heterogeneous population of immature myeloid cells, suppress not only NK and cytotoxic T lymphocyte cytotoxicity but also macrophage-mediated cytotoxicity. This review is focused on the recent findings regarding strategies for inhibiting xenogenic rejection by macrophages.

MicroRNAs (miRNAs) play an important role in regulating fibrogenesis in the liver. The current study examined the ability of microRNA-214 (miR-214) level in liver and serum samples obtained from patients with BA to predict progressive liver fibrosis in patients with biliary atresia (BA).We examined miR-214 level in relation to conventional markers of liver fibrosis, with liver and serum samples from BA patients. Fifty-two patients with BA who underwent Kasai portoenterostomy and four control patients underwent liver biopsy. In 28 patients with BA, blood samples were collected to analyze circulating serum miR-214.MiR-214 levels in liver tissue were significantly upregulated in patients with BA who had severe liver fibrosis (F3-4) compared to those with none to mild fibrosis (F0-2), whereas suppressors-of-fused homolog (Sufu) mRNA levels were significantly suppressed in F3-4. Serum miR-214 levels were significantly higher in patients with F3-4 compared with F0-2. Area under the curve analysis showed that the serum miR-214 cut-off level for predicting F3-4 was 0.805 (p = 0.0046).Hepatic overexpression of miR-214 is associated with progression of liver fibrosis in patients with BA, and the circulating miR-214 level may serve as a non-invasive predictor of liver fibrosis.

β-1,4-acetyl-galactosaminyltransferase 2 (β4GalNT2)-knockout (KO) pigs have been produced and reveal less antigenicity to both humans and nonhuman primates (NHP). In this study, we checked the antibody response of human and NHP sera to pig cells with or without this gene. The β4GalNT2-KO porcine endothelial cell (PEC), clone #11, was first established using the plasmid pX330 expressing hCas9 and sgRNA for β4GalNT2. The glycoantigen feature on the PEC was then studied. The Sda antigen, synthesized by β4GalNT2, was slightly ascertained on wild-type (WT)-PEC, and it became null in clone #11. The PEC response to lectins was also assessed, such as Dolichos biflorus agglutinin, soybean agglutinin, and Wisteria floribunda agglutinin. All of these lectins reduced the binding reaction to clone #11 as compared with WT-PEC. Next, several human and cynomolgus sera were checked for their natural antibody reaction to both WT-PEC and clone #11. In addition, human monocyte-mediated PEC phagocytosis was assessed. However, the reduction in phagocytosis to clone #11 was not significant. Human sera showed less reactivity to the changes in antigenicity of PEC by knocking out the β4GalNT2 than cynomolgus sera.

Purpose The delayed rejection caused by strong cell-mediated innate and adaptive xenogeneic immune responses continues to be a major obstacle. Therefore, suppressing macrophage function could be effective in avoiding this type of rejection. In this study, the suppression of T-cell immunoglobulin and ITIM domain (TIGIT) function against macrophage-mediated xenogeneic rejection was investigated. Material and methods Naive porcine aortic endothelial cell (PAEC) and PAEC transfectant with TIGIT (PAEC/TIGIT) were co-cultured with M1 macrophages, and the degree of cytotoxicity was determined by a counting beads assay. The anti/pro-inflammatory gene expression was determined by RT-PCR and the phosphorylated SHP-1 in the macrophages after co-culturing with PAEC or PAEC/TIGIT was evaluated by western blotting. Results CD155 was expressed at essentially equal levels on both M1 and M2 macrophages, whereas TIGIT was highly expressed on M2 macrophages but not in M1 macrophages. TIGIT on PAEC significantly reduced the cytotoxicity of M1 macrophages but no significant suppression of phagocytosis was detected. TIGIT also caused a decrease in the expression of pro-inflammatory cytokines, namely TNFα, IL-1β and IL-12 in M1 macrophages. Furthermore, PAEC/TIGIT caused a significant increase in phosphorylated SHP-1 in M1 macrophages compared to PAEC. Conclusion The findings of this study indicate that TIGIT suppresses xenogeneic M1 macrophage-induced cytotoxicity, probably at least in part, via the phosphorylation of SHP-1. In addition, the reduced expression of some pro-inflammatory cytokines, namely TNFα, IL-1β and IL-12, was observed in M1 macrophages that had been cultured with PAEC/TIGIT.

Innate immunity plays a major role in xenograft rejection. However, the majority of immunosuppressants focus on inhibiting acquired immunity and not innate immunity. Therefore, a novel immunosuppressant suitable for use in conjunction with xenografts continues to be needed. It has been reported that prenylated quinolinecarboxylic acid-18 (PQA-18), a p21-activated kinase 2 (PAK2) inhibitor, exerts an immunosuppressive function on T cells. Hence, the possibility exists that PQA-18 might be used in conjunction with xenografts, which prompted us to investigate the efficacy of PQA-18 on macrophages compared with Tofacitinib, a janus kinase (JAK) inhibitor. Initial experiments confirmed that PQA-18 is non-toxic to swine endothelial cells (SECs) and human monocytes. Both PQA-18 and Tofacitinib suppressed macrophage-mediated cytotoxicity in both the differentiation and effector phases. Both PQA-18 and tofacitinib suppressed the expression of HLA-ABC by macrophages. However, contrary to Tofacitinib, PQA-18 also significantly suppressed the expression of CD11b, HLA-DR and CD40 on macrophages. PQA-18 significantly suppressed CCR7 expression on day 3 and on day 6, but Tofacitinib-induced suppression only on day 6. In a mixed lymphocyte reaction (MLR) assay, PQA-18 was found to suppress Interleukin-2 (IL-2)-stimulated T cell proliferation to a lesser extent than Tofacitinib. However, PQA-18 suppressed xenogeneic-induced T cell proliferation more strongly than Tofacitinib on day 3 and the suppression was similar on day 7. In conclusion, PQA-18 has the potential to function as an immunosuppressant for xenotransplantation.

Portoenterostomy is the standard treatment for biliary atresia (BA) that reduces jaundice in two thirds of cases. However, progressive liver fibrosis is common, leading to cirrhosis in most patients. Autotaxin is a new marker for the progression of hepatic fibrosis. We examined the relationship between serum autotaxin levels and liver histological findings in patients with BA.BA patients with native livers were identified in our hospital. Patients underwent protocol liver biopsies every 1 to 5 years, and liver fibrosis was evaluated based on the METAVIR score. Serum autotaxin levels were compared with the last available pathological findings.Thirty-five patients were included and the median age was 10.6 years. Serum autotaxin levels was median 1.6 mg/L. The mean autotaxin level was 1.08 mg/L in F0, 1.07 mg/L in F1, 0.95 mg/L in F2, 2.17 mg/L in F3, and 2.50 mg/L in F4; it was significantly higher in F4 than in F0-F2 (P0.0024). For predicting cirrhosis (F4) and advanced liver fibrosis (≥F3), autotaxin had the almost same areas under the curve (AUCs 0.78 and 0.90, respectively) as well as M2BPGi.Autotaxin levels could be used to evaluate the status of native liver fibrosis.

Supplemental Digital Content is available in the text., Background. Neutrophil-induced tissue damage contributes to the rejection in xenotransplantation. Therefore, suppressing neutrophil function could be effective in suppressing xenogeneic rejection. In a previous study, we demonstrated that the ectopic expression of human cluster of differentiation 31 (CD31) on porcine endothelial cells (PEC) significantly suppressed neutrophil-mediated cytotoxicity through the homophilic binding of CD31. Cluster of differentiation 177 (CD177) was recently reported to be a high-affinity heterophilic binding partner for CD31 on endothelial cells. Thus, we hypothesized that human CD177 on PEC might induce a stronger suppression in neutrophil-mediated cytotoxicity compared with CD31. In this study, the inhibitory function of human CD177 on PEC in neutrophil-mediated cytotoxicity was investigated. Methods. PEC were transfected with a cloning plasmid containing cDNA inserts that encoded for hCD177 and hCD31 genes. Neutrophil-induced cytotoxicity was evaluated by flow cytometry after coculturing with PEC or PEC/CD177 in the presence of phorbol 12-myristate 13-acetate. To elucidate the mechanisms responsible for hCD177-induced suppression, the phosphorylation of src homology region 2 domain containing phosphatase 1 was measured by immunoblot analysis. Results. Human CD177 on PEC induced a significant reduction in neutrophil-induced cytotoxicity. In addition, CD177 on PEC induced a significant increase in the phosphorylation of src homology region 2 domain-containing phosphatase 1 in neutrophils and suppressed NETosis. Conclusions. These findings suggest that human CD177 suppresses neutrophil-mediated cytotoxicity through the inhibition of NETosis.

Background Innate immunity by natural killer (NK) cells, macrophages, and neutrophils cause severe rejections in xenotransplantation. Therefore, the development of strategies for suppressing macrophages has considerable potential in practical applications of xenotransplantation. Recently, we found that human CD31 on swine endothelial cells (SECs) suppresses neutrophil-mediated xenogeneic rejection through homophilic binding. Since a significant amount of CD31 is expressed not only on neutrophils but also on macrophages, we studied the function of human CD31 in macrophage-mediated cytotoxicity. Methods SECs and hCD31-transfected SECs (SEC/hCD31) were co-cultured with macrophages and cytotoxicity by macrophages was evaluated with water-soluble tetrazolium salt, or WST-8, assay. To confirm whether or not inhibitory signals are induced by hCD31 homophilic binding, the phosphorylation of the enzyme SHP-1 was investigated with Western blotting. Results No suppression of cytotoxicity was induced in macrophages that had been co-cultured with SEC/CD31. However, phosphorylation of SHP-1 was induced in macrophages that had been co-cultured with SEC/hCD31. Conclusions Human CD31 on SEC may induce not only inhibitory signals but also activation signals via the binding to other receptors for hCD31.

Although xenografts are one of the most attractive strategies for overcoming the shortage of organ donors, cellular rejection by macrophages is a substantial impediment to this procedure. It is well known that macrophages mediate robust immune responses in xenografts. Macrophages also express various inhibitory receptors that regulate their immunological function. Recent studies have shown that the overexpression of inhibitory ligands on porcine target cells results in the phosphorylation of tyrosine residues on intracellular immunoreceptor tyrosine-based inhibitory motifs on macrophages, leading to the suppression of xenogenic rejection by macrophages. It has also been reported that myeloid-derived suppressor cells, a heterogeneous population of immature myeloid cells, suppress not only NK and cytotoxic T lymphocyte cytotoxicity but also macrophage-mediated cytotoxicity. This review is focused on the recent findings regarding strategies for inhibiting xenogenic rejection by macrophages.