Inducible nitric oxide synthase regulates macrophage polarization via the MAPK signals in concanavalin A-induced hepatitis.

INTRODUCTION: Acute liver inflammatory reactions contribute to many health problems; thus, it is critical to understand the underlying pathogenic mechanisms of acute hepatitis. In this study, an experimental in vivo model of concanavalin A (ConA)-induced hepatitis was used. MATERIALS AND METHODS: C57BL/6 (wild-type, WT) or inducible nitric oxide synthase-deficient (iNOS-/- ) mice were injected with PBS or 15 mg/kg ConA via tail vein. Detection of liver injury by histological examination and apoptosis, and flow cytometry to detect the effect of immune cells on liver injury. RESULTS: iNOS-/-  mice had lower levels of the liver enzymes aspartate aminotransferase and alanine aminotransferase, suggesting that they were protected against ConA-induced pathological liver injury and that iNOS participated in the regulation of hepatitis. Furthermore, iNOS deficiency was found to lower CD86 expression and suppressed the messenger RNA levels of inflammatory factors in the liver. In vitro experiments also demonstrated that iNOS deficiency suppressed the sequential phosphorylation of the mitogen-activated protein kinase pathway cascade, thereby inhibiting the M1 polarization of macrophages and consequently suppressing the transcription of inflammation factors. CONCLUSION: iNOS may contribute to ConA-induced inflammation by promoting the activation of proinflammatory macrophages.

[CD11b agonist leukadherin-1 inhibits activation of TLR7 and TLR9 in mouse bone marrow-derived dendritic cells by blocking NF-κB p65 pathway].

Objective To study the effect of CD11b agonist leukadherin-1 (LA1) on Toll-like receptor 7 (TLR7)- and TLR9-induced activation of mouse bone marrow-derived dendritic cells (BMDCs) and its specific mechanism. Methods BMDCs were successfully induced and the concentrations of LA1 used in the study were determined by CCK-8 assay and annexin V-FITC/PI double staining. BMDCs were treated with LA1 for 2 hours followed by stimulation of TLR7 agonist R837 and TLR9 agonist CpG1826. The expression of BMDCs surface markers CD40, CD86 and MHC-II were detected by flow cytometry; IL-6, IL-12p40 and tumor necrosis factor α (TNF-α) in the cell culture supernatant were detected by ELISA; the phosphorylation of NF-κB p65 in BMDCs was detected by Western blotting. Results LA1 concentration below 20 µmol/L had no effect on the viability and apoptosis of BMDCs. LA1 pretreatment significantly inhibited R837- and CpG 1826-induced expression of CD40, CD86 and MHC-II , and the secretion of IL-6, IL-12p40 and TNF-α in BMDCs. Moreover, LA1 pretreatment significantly inhibited the phosphorylation of NF-κB p65 activated by R837 and CpG1826 in BMDCs. Conclusion CD11b agonist LA1 can significantly inhibit the activation of TLR7 and TLR9 in BMDCs by blocking the NF-κB p65 signaling pathway.

Correction: Shi, H., et al. Arctigenin Attenuates Breast Cancer Progression through Decreasing GM-CSF/TSLP/STAT3/ß-Catenin Signaling. Int. J. Mol. Sci. 2020, 21, 6357.

The authors wish to make the following correction to this paper [...].

Arctigenin Attenuates Breast Cancer Progression through Decreasing GM-CSF/TSLP/STAT3/ß-Catenin Signaling.

Invasive breast cancer is highly regulated by tumor-derived cytokines in tumor microenvironment. The development of drugs that specifically target cytokines are promising in breast cancer treatment. In this study, we reported that arctigenin, a bioactive compound from Arctium lappa L., could decrease tumor-promoting cytokines GM-CSF, MMP-3, MMP-9 and TSLP in breast cancer cells. Arctigenin not only inhibited the proliferation, but also the invasion and stemness of breast cancer cells via decreasing GM-CSF and TSLP. Mechanistically, arctigenin decreased the promoter activities of GM-CSF and TSLP via reducing the nuclear translocation of NF-κB p65 which is crucial for the transcription of GM-CSF and TSLP. Furthermore, arctigenin-induced depletion of GM-CSF and TSLP inhibited STAT3 phosphorylation and ß-catenin signaling resulting in decreased proliferation, invasion and stemness of breast cancer cells in vitro and in vivo. Our findings provide new insights into the mechanism by which tumor-promoting cytokines regulate breast cancer progression and suggest that arctigenin is a promising candidate for cytokine-targeted breast cancer therapy.

Erratum: Enrichment and characterization of cancer stem-like cells in ultra-low concentration of serum and non-adhesive culture system.

[This corrects the article on p. 1552 in vol. 10, PMID: 29887968.].

Establishment of a Human Gastric Cancer Xenograft Model in Immunocompetent Mice Using the Microcarrier-6.

Gastric cancer is among the most common malignant tumors of the digestive tract. Establishing a robust and reliable animal model is the foundation for studying the pathogenesis of cancer. The present study established a mouse model of gastric carcinoma by inoculating immunocompetent mice with MKN45 cells using microcarrier. Sixty male C57BL/6 mice were randomly divided into three groups: a 2D group, an empty carrier group, and a 3D group, according to the coculture system of MKN45 and the microcarrier. The mouse models were established by hypodermic injection. Time to develop tumor, rate of tumor formation, and pathological features were observed in each group. In the 3D group, the tumorigenesis time was short, while the rate of tumor formation was high (75%). There was no detectable tumor formation in either the 2D or the empty carrier group. Both H&E and immunohistochemical staining of the tumor xenograft showed characteristic evidence of human gastric neoplasms. The present study successfully established a human gastric carcinoma model in immunocompetent mice, which provides a novel and valuable animal model for the cancer research and development of anticancer drugs.

The inhibitor of autophagy SBI-0206965 aggravates atherosclerosis through decreasing myeloid-derived suppressor cells.

Atherosclerosis (AS) is currently the leading cause of mortality worldwide, with the development of new strategies to prevent the formation and rupture of atherosclerotic plaques being a paramount area of research. Amounting evidence suggests autophagy has an important role in the pathogenesis of AS and may be a potential therapeutic target. In this study, the effect of SBI-0206965(6965), a novel inhibitor of autophagy, was tested on the development of AS in apolipoprotein E deficient (ApoE-/-) mice. Systemic application of 6965 was found to aggravate AS, with increased plaque size and decreased plaque stability in comparison with the control. Of note, it was observed that 6965 decreased the proportion of myeloid-derived suppressor cells (MDSCs). Further investigation demonstrated MDSCs markedly alleviated AS in ApoE-/- mice; while 6965 reduced the viability and promoted apoptosis of MDSCs in vitro. This is the first study describing an association between autophagy and MDSCs in AS models, providing a novel mechanism to potentially target in the management of this condition.

Granulocytic myeloid-derived suppressor cells contribute to IFN-I signaling activation of B cells and disease progression through the lncRNA NEAT1-BAFF axis in systemic lupus erythematosus.

Activation of interferon (IFN)-I signaling in B cells contributes to the pathogenesis of systemic lupus erythematosus (SLE). Recent studies have shown that myeloid-derived suppressor cells (MDSCs) significantly expand in SLE patients and lupus-prone MRL/lpr mice and contribute to the pathogenesis of SLE. However, the role of SLE-derived MDSCs in regulating IFN-I signaling activation of B cells remains unknown. Here, we demonstrate that expansions of MDSCs, including granulocyte (G)-MDSCs and monocytic (M)-MDSCs, during the progression of SLE were correlated with the IFN-I signature of B cells. Interestingly, G-MDSCs from MRL/lpr mice, but not M-MDSCs, could significantly promote IFN-I signaling activation of B cells and contribute to the pathogenesis of SLE. Mechanistically, we identified that the long non-coding RNA NEAT1 was over-expressed in G-MDSCs from MRL/lpr mice and could induce the promotion of G-MDSCs on IFN-I signaling activation of B cells through B cell-activating factor (BAFF) secretion. Importantly, NEAT1 deficiency significantly attenuated the lupus symptoms in pristane-induced lupus mice. In addition, there was a positive correlation between NEAT1 and BAFF with the IFN signature in SLE patients. In conclusion, G-MDSCs may contribute to the IFN signature in SLE B cells through the NEAT1-BAFF axis, highlighting G-MDSCs as a potential therapeutic target to treat SLE.

Hepatoprotective effect of capsaicin against concanavalin A-induced hepatic injury via inhibiting oxidative stress and inflammation.

Immune-mediated liver injury plays a crucial role in the pathogenesis of liver diseases, which can result from viral infections, autoimmunity, alcohol intake, and drug use. Concanavalin A (Con A)-induced hepatitis is a well-characterized murine model with similar pathophysiology to that of human viral and autoimmune hepatitis. Capsaicin, a selective agonist of the transient potential vanilloid subfamily member 1 (TRPV1) receptor, exhibits anti-inflammatory effects on various causes of inflammation. In the present study, we investigated the effect of capsaicin on Con A-induced hepatitis. Capsaicin (1 mg/kg body weight) was administered by intraperitoneal injection, after which (30 minutes), the mice were challenged intravenously with Con A (20 µg/g body weight). We collected serum for plasma transaminase analysis. Pro-inflammatory cytokine levels and hepatocyte apoptosis were assayed by ELISA and TUNEL, respectively. Liver samples were collected for real-time PCR, hematoxylin and eosin staining, and measuring oxidative stress and myeloperoxidase levels. Activation of splenocytes and hepatic mononuclear cells was analyzed by flow cytometry. Compared with control, the capsaicin-treated group showed significantly decreased aminotransferase levels and markedly prolonged mouse survival. Capsaicin pretreatment also attenuated hepatocyte apoptosis and oxidative stress. Furthermore, tumor necrosis factor-α and interferon-γ levels in serum and liver were significantly suppressed, while the percentage of myeloid-derived suppressor cells increased after capsaicin pretreatment. Our findings indicate that capsaicin pretreatment protects mice from Con A-induced hepatic damage and is partially involved in inhibiting hepatocyte apoptosis, oxidative stress, and inflammatory mediators as well as regulating activation and recruitment of intrahepatic leukocytes.

Interleukin-16 aggravates ovalbumin-induced allergic inflammation by enhancing Th2 and Th17 cytokine production in a mouse model.

Asthma is a chronic inflammatory disease that involves a variety of cytokines and cells. Interleukin-16 (IL-16) is highly expressed during allergic airway inflammation and is involved in its development. However, its specific mechanism of action remains unclear. In the present study, we used an animal model of ovalbumin (OVA)-induced allergic asthma with mice harboring an IL-16 gene deletion to investigate the role of this cytokine in asthma, in addition to its underlying mechanism. Increased IL-16 expression was observed during OVA-induced asthma in C57BL/6J mice. However, when OVA was used to induce asthma in IL-16-/- mice, a diminished inflammatory reaction, decreased bronchoalveolar lavage fluid (BALF) eosinophil numbers, and the suppression of OVA-specific IgE levels in the serum and BALF were observed. The results also demonstrated decreased levels of T helper type 2 (Th2) and Th17 cytokines upon OVA-induced asthma in IL-16-/- mice. Hence, we confirmed that IL-16 enhances the lung allergic inflammatory response and suggest a mechanism possibly associated with the up-regulation of IgE and the promotion of Th2 and Th17 cytokine production. This work explored the mechanism underlying the regulation of IL-16 in asthma and provides a new target for the clinical treatment of asthma.

Transplanted adult human hepatic stem/progenitor cells prevent histogenesis of advanced hepatic fibrosis in mice induced by carbon tetrachloride.

Transplantation of adult human hepatic stem/progenitor cells (hHSPCs) has been considered as an alternative therapy, replacing donor liver transplantation to treat liver cirrhosis. This study assessed the antifibrotic effects of hHSPCs in mice with fibrosis induced by carbon tetrachloride (CCl4) and examined the actions of hHSPCs on the fibrogenic activity of human hepatic stellate cells (HSCs) in a coculture system. Isolated hHSPCs expressed stem/progenitor cell phenotypic markers. Mice were given CCl4 (twice weekly for 7 weeks) and hHSPC transplantation weekly. CCl4 induced advanced fibrosis (bridging fibrosis and cirrhosis) in mice, which was prevented by hHSPC transplantation. The liver of hHSPC-transplanted mice showed only occasional short septa and focal parenchymal fibrosis, and a 50% reduction in hepatic collagen, assessed by Sirius red stain histomorphometry. Moreover, the proteins for α-smooth muscle actin (α-SMA) and collagen I were decreased. While α-SMA, collagen α1(I), and tissue inhibitor of metalloproproteinase-1 mRNAs were decreased, matrix metalloproteinase (MMP)-1 mRNA was increased, consistent with decreased fibrogenesis. MMP-2 and transforming growth factor-ß were not affected. Alanine aminotransferase and aspartate aminotransferase were lower, suggesting improvement of liver function/damage. In coculture, hHSPCs elicited changes of α-SMA and fibrogenic molecules in HSCs similar to those observed in vivo, providing evidence for a functional link between hHSPCs and HSCs. A decreased HSC proliferation was noted. Thus, transplantation of hHSPCs prevents histogenesis of advanced liver fibrosis caused by CCl4. hHSPCs mediate downregulation of HSC activation coincident with modulation of fibrogenic molecule expression, leading to suppression of fibrogenesis both in vivo and in vitro.

Leukadherin-1-Mediated Activation of CD11b Inhibits LPS-Induced Pro-inflammatory Response in Macrophages and Protects Mice Against Endotoxic Shock by Blocking LPS-TLR4 Interaction.

Dysregulation of macrophage has been demonstrated to contribute to aberrant immune responses and inflammatory diseases. CD11b, expressed on macrophages, plays a critical role in regulating pathogen recognition, phagocytosis, and cell survival. In the present study, we explored the effect of leukadherin-1 (LA1), an agonist of CD11b, on regulating LPS-induced pro-inflammatory response in macrophages and endotoxic shock. Intriguingly, we found that LA1 could significantly reduce mortalities of mice and alleviated pathological injury of liver and lung in endotoxic shock. In vivo studies showed that LA1-induced activation of CD11b significantly inhibited the LPS-induced pro-inflammatory response in macrophages of mice. Moreover, LA1-induced activation of CD11b significantly inhibited LPS/IFN-γ-induced pro-inflammatory response in macrophages by inhibiting MAPKs and NF-κB signaling pathways in vitro. Furthermore, the mice injected with LA1-treated BMDMs showed fewer pathological lesions than those injected with vehicle-treated BMDMs in endotoxic shock. In addition, we found that activation of TLR4 by LPS could endocytose CD11b and activation of CD11b by LA1 could endocytose TLR4 in vitro and in vivo, subsequently blocking the binding of LPS with TLR4. Based on these findings, we concluded that LA1-induced activation of CD11b negatively regulates LPS-induced pro-inflammatory response in macrophages and subsequently protects mice from endotoxin shock by partially blocking LPS-TLR4 interaction. Our study provides a new insight into the role of CD11b in the pathogenesis of inflammatory diseases.

Interleukin-35 in immune-related diseases: protection or destruction.

Interleukin-35 (IL-35) is a recently identified heterodimeric cytokine in the IL-12 family. It consists of an IL-12 subunit α chain (P35) and IL-27 subunit Epstein-Barr virus-induced gene 3 (EBI3) ß chain. Unlike the other IL-12 family members, it signals through four unconventional receptors: IL-12Rß2-IL-27Rα, IL-12Rß2-IL-12Rß2, IL-12Rß2-GP130, and GP130-GP130. Interleukin-35 signaling is mainly carried out through the signal transducer and activator of transcription family of proteins. It is secreted not only by regulatory T (Treg) cells, but also by CD8+ Treg cells, activated dendritic cells and regulatory B cells. It exhibits immunosuppressive functions distinct from those of other members of the IL-12 family; these are mediated primarily by the inhibition of T helper type 17 cell differentiation and promotion of Treg cell proliferation. Interleukin-35 plays a critical role in several immune-associated diseases, such as autoimmune diseases and viral and bacterial infections, as well as in tumors. In this review, we summarize the structure and function of IL-35, describe its role in immune-related disorders, and discuss the mechanisms by which it regulates the development and progression of diseases, including inflammatory bowel disease, collagen-induced arthritis, allergic airway disease, hepatitis, and tumors. The recent research on IL-35, combined with improved techniques of studying receptors and signal transduction pathways, allows for consideration of IL-35 as a novel immunotherapy target.

Ligation of CD180 contributes to endotoxic shock by regulating the accumulation and immunosuppressive activity of myeloid-derived suppressor cells through STAT3.

Myeloid-derived suppressor cells (MDSCs) play an immunosuppressive role in the pathogenesis of inflammatory diseases. CD180, a TLR-like protein, can regulate the proliferation and activation of immune cells. However, the roles of CD180 in regulating the accumulation and function of MDSCs have not been investigated. Here, we found that, compared with non-treated controls, the expression of CD180 was significantly elevated in MDSCs, especially granulocytic MDSCs (G-MDSCs), from mice challenged with lipopolysaccharide (LPS). Ligation of CD180 by the anti-CD180 antibody not only blocked the expansion of MDSCs by preventing the phosphorylation of signal transducer and activator of transcription 3 (STAT3), but also reduced the immunosuppressive activity of MDSCs on M1 macrophage polarization through inhibition of Arg-1 expression in vitro. In vivo studies showed that injection of anti-CD180 antibody significantly aggravated pathological lesions in mice challenged with LPS. Furthermore, injection of anti-CD180 antibody inhibited the accumulation of G-MDSCs in mice challenged with LPS and reduced the immunosuppressive activity of G-MDSCs on M1 macrophage polarization. Based on these findings, we conclude that ligation of CD180 contributes to the pathogenesis of endotoxic shock by inhibiting the accumulation and immunosuppressive activity of G-MDSCs, thus providing insight into the function of CD180 in inflammatory diseases.

[The activation of CD11b by leukadherin-1 alleviates endotoxic shock through promoting the aggregation and activation of myeloid-derived suppressor cells].

Objective To study the effect of CD11b agonist leukadherin-1 (LA1) on the aggregation and immunosuppressive function of myeloid-derived suppressor cells (MDSCs) and its therapeutic effect on the condition of endotoxic shock mice. Methods The percentages of MDSCs , granulocytic myeloid-derived suppressor cells(G-MDSCs)and monocytic myeloid-derived suppressor cells(M-MDSCs)in spleen were detected by flow cytometry, after C57BL/6 female mice were injected of LA1 to activate through abdominal cavity for 12 hours and 48 hours. MDSCs were induced from the femur and tibia of C57BL/6 female mice in vitro. The expression levels of immunosuppressive related factors, such as interleukin 10 (IL-10), NADPH oxidase 1 (NOX1) and inducible nitric oxide synthase (iNOS) , were detected by real time quantitative PCR. C57BL/6 female mice were randomly divided into PBS group, LA1 group, PBS combined LPS group and LA1 combined LPS group. Flow cytometry was utilized to detect the ratio changes of MDSCs, G-MDSCs and M-MDSCs as well as the expression of CD86 and CD40 in macrophage, hematoxylin-eosin staining of lung and liver was utilized to detect the pathological injury, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling(TUNEL)was used to detect the apoptosis of pneumonocyte and hepatocyte and mortality analysis was reflected the severity of the disease. Based on the above indicators, we analyzed the effects of LA1 on the aggregation of MDSCs and the condition of mice in endotoxic shock. Results The ratio of MDSCs was increased by LA1 treatment for 12 and 48 hours. Further analysis of the proportions of G-MDSCs showed that LA1 treatment for 12 hours increased the proportions of G-MDSCs compared with the control group. In vitro, mRNA levels of IL-10, NOX1 and iNOS were increased after LA1 treatment in MDSCs. In vivo experiments, compared with the PBS combined LPS group, the proportions of MDSCs and G-MDSCs in LA1 combined LPS group were increased, the injuries of liver and lung were alleviated, the mortalities were reduced, and the activations of macrophage were decreased. Conclusion The activation of CD11b by LA1 alleviates endotoxin shock by promoting the aggregation of MDSCs and the expression of immunosuppressive related factors.

[IL-16 aggravates dextran sulfate sodium (DSS)-induced mouse inflammatory bowel disease by promoting M1 polarization of macrophages].

Objective To investigate the role of interleukin-16 (IL-16) in the development of inflammatory bowel disease (IBD) and clarify its regulatory mechanism involved in the pathogenesis of IBD. Methods Seven-week-old wild-type C57BL/6 (WT) and IL-16 knockout (IL-16-/-) female mice were divided into WT control group, WT dextran sulfate sodium (DSS) treatment group, IL-16-/- control group and IL-16-/- DSS treatment group. The DSS model groups were given the water with 25 g/L DSS for 7 days to establish the IBD models, while the control groups were given the normal water. During the modeling period, the body mass of mice was recorded to calculate the body mass curve. After 7 days, the whole colon of the mice was dissected and the level of IL-16 mRNA in the colon tissue was detected by real-time PCR. The level of IL-16 protein in the colon tissue was detected by ELISA. The expression and localization of IL-16 in the colon tissue were observed by immunofluorescence technique. HE staining was used to detect colonic pathological injury in mice. TUNEL assay was used to detect cell apoptosis of the colon tissue. Flow cytometry was used to detect the number and polarization of macrophages in peritoneal cells (F4/80, CD86). Immunohistochemical staining was used to detect the distribution of macrophages in the colon tissues. Real-time PCR was used to detect IL-6 and IL-12 mRNA levels in the colon tissue, and IL-6 and IL-12 protein levels were detected by ELISA. Results DSS induced high expression of IL-16 in the colon tissue. Compared with WT DSS treatment group, IL-16-/- DSS treatment group showed less changes in body mass, less colon tissue damage, and markedly lower percents of apoptotic cells in the peritoneal or colonic tissues of IL-16-/- mice. What's more, the number of macrophages, the polarization level of M1 macrophages, and the levels of the iconic inflammatory factors IL-6 and IL-12 significantly decreased in IL-16-/- DSS treatment group compared with WT DSS treatment group. Conclusion IL-16 can aggravate DSS-induced IBD by promoting the polarization of M1 macrophages.

Arctigenin Ameliorates Inflammation by Regulating Accumulation and Functional Activity of MDSCs in Endotoxin Shock.

Endotoxin shock is a life-threatening response caused by a disordered immune response to an infection. MDSCs are accumulated and play a protective role in the pathogenesis of endotoxin shock. However, the regulation of MDSCs by small molecule remains unrevealed. Here, we report that arctigenin, a small molecule extracted from Arctium lappa, induces accumulation of functional MDSCs. Arctigenin was able to ameliorate LPS-induced inflammation through accumulating MDSCs, especially granulocytic MDSCs (G-MDSCs), and enhancing the immunosuppressive function of MDSCs in vivo and in vitro. Mechanistically, arctigenin promoted the accumulation of MDSCs through upregulating miR-127-5p which targets the 3'UTR of interferon regulatory factor-8 (IRF8) mRNA. In addition, arctigenin enhanced the immunosuppressive activity of MDSCs on M1 macrophage polarization by elevating the expression of arginase 1 (Arg-1) and inducible nitric oxide synthase (iNOS). Our study provides new insights into the regulation of functional MDSCs by arctigenin in exerting immune responses and pathogenesis of inflammatory diseases.

Enrichment and characterization of cancer stem-like cells in ultra-low concentration of serum and non-adhesive culture system.

Cancer stem cells (CSCs) play important roles in tumor initiation, metastasis, and progression. They are also mainly responsible for high treatment failure rates. Identification and characterization of CSCs are crucial for facilitating the detection, prevention, or therapy of cancer. Great efforts have been paid to develop an effective method and the ideal method for CSCs research is still in the going. In our study, we created an ultra-low concentration of serum and non-adhesive (ULCSN) culture system to enrich CSCs from murine lewis lung cancer cell line LL/2 with cell spheres structure and characterize the LL/2 CSCs properties. Their characteristics were investigated through colony formation, spheres formation, chemoresistance, flow cytometry for putative stem cell markers, such as CD133, CD34 and CD45, immunofluorescence staining and tumor initiation capacity in vivo. Tumor spheres were formed within 7-10 days under the condition of ULCSN culture system. Compared with adherent parental LL/2 cells, the colony capacity, chemo-resistance, and expression of stem cell markers increased significantly in addition to tumor-initiating capability in the tumor sphere cells. Using the ULCSN culture system, an available isolation method of lewis lung CSCs was established, which is simple, effective, and inexpensive compared with the cytokines attachment serum free culture method. The stem cell properties of the tumor sphere LL/2 cells reflected the CSCs phenotypes. We developed a useful CSCs model for basic and pre-clinical studies for lung cancer and other types of cancer.

A paralogous pair of mammalian host restriction factors form a critical host barrier against poxvirus infection.

Host restriction factors constitute a formidable barrier for viral replication to which many viruses have evolved counter-measures. Human SAMD9, a tumor suppressor and a restriction factor for poxviruses in cell lines, is antagonized by two classes of poxvirus proteins, represented by vaccinia virus (VACV) K1 and C7. A paralog of SAMD9, SAMD9L, is also encoded by some mammals, while only one of two paralogs is retained by others. Here, we show that SAMD9L functions similarly to SAMD9 as a restriction factor and that the two paralogs form a critical host barrier that poxviruses must overcome to establish infection. In mice, which naturally lack SAMD9, overcoming SAMD9L restriction with viral inhibitors is essential for poxvirus replication and pathogenesis. While a VACV deleted of both K1 and C7 (vK1L-C7L-) was restricted by mouse cells and highly attenuated in mice, its replication and virulence were completely restored in SAMD9L-/- mice. In humans, both SAMD9 and SAMD9L are poxvirus restriction factors, although the latter requires interferon induction in many cell types. While knockout of SAMD9 with Crispr-Cas9 was sufficient for abolishing the restriction for vK1L-C7L- in many human cells, knockout of both paralogs was required for abolishing the restriction in interferon-treated cells. Both paralogs are antagonized by VACV K1, C7 and C7 homologs from diverse mammalian poxviruses, but mouse SAMD9L is resistant to the C7 homolog encoded by a group of poxviruses with a narrow host range in ruminants, indicating that host species-specific difference in SAMD9/SAMD9L genes serves as a barrier for cross-species poxvirus transmission.

Autophagy regulates accumulation and functional activity of granulocytic myeloid-derived suppressor cells via STAT3 signaling in endotoxin shock.

Autophagy extensively participates in immune responses and inflammatory diseases. Myeloid-derived suppressor cells (MDSCs) are derived from CD11b+Gr1+ cells under pathological conditions and play an immunosuppressive role in the pathogenesis of cancer and inflammatory diseases. However, the role of autophagy in regulating the accumulation and activity of MDSCs remains unknown. In the present study, we evaluated the effects and mechanisms of autophagy on regulating accumulation and activity of MDSCs. We first found that granulocytic MDSCs (G-MDSCs), but not monocytic MDSCs (M-MDSCs), were accumulated in mice challenged by lipopolysaccharide (LPS) and showed an elevated autophagy activity. Pharmacological inhibition of autophagy significantly enhanced accumulation of G-MDSCs in vivo and in vitro. Notably, inhibition of autophagy enhanced the immunosuppressive activity of G-MDSCs on M1 macrophage polarization by promoting reactive oxygen species (ROS) production. Inhibition of autophagy promotes the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in G-MDSCs, which is required for the accumulation and activity of MDSCs. In addition, in vivo pharmacological inhibition of autophagy significantly attenuated the condition of mice challenged by LPS. Thus, we conclude that inhibition of autophagy contributes to accumulation and immunosuppressive function of G-MDSCs by promoting the activation of STAT3 signaling, suggesting that autophagy may play a critical role in regulating accumulation and activity of MDSCs. Our study provides new insights into understanding the mechanisms of autophagy in regulating immune responses and pathogenesis of inflammatory diseases.