Local radiotherapy for murine breast cancer increases risk of metastasis by promoting the recruitment of M-MDSCs in lung.

BACKGROUND: Radiotherapy is one of the effective methods for treatment of breast cancer; however, controversies still exist with respect to radiotherapy for patients with TNBC. Here, we intend to explore the mechanism by which local radiotherapy promotes the recruitment of M-MDSCs in the lung and increases the risk of lung metastasis in TNBC tumor-bearing mice. METHODS: A single dose of 20 Gy X-ray was used to locally irradiate the primary tumor of 4T1 tumor-bearing mice. Tumor growth, the number of pulmonary metastatic nodules, and the frequency of MDSCs were monitored in the mice. Antibody microarray and ELISA methods were used to analyze the cytokines in exosomes released by irradiated (IR) or non-IR 4T1 cells. The effects of the exosomes on recruitment of MDSCs and colonization of 4T1 cells in the lung of normal BALB/c mice were observed with the methods of FCM and pathological section staining. T lymphocytes or 4T1 cells co-cultured with MDSCs were performed to demonstrate the inhibitory effect on T lymphocytes or accelerative migration effect on 4T1 cells. Finally, a series of in vitro experiments demonstrated how the exosomes promote the recruitment of M-MDSCs in lung of mice. RESULTS: Even though radiotherapy reduced the burden of primary tumors and larger lung metastatic nodules (≥ 0.4 mm2), the number of smaller metastases (< 0.4 mm2) significantly increased. Consistently, radiotherapy markedly potentiated M-MDSCs and decreased PMN-MDSCs recruitment to lung of tumor-bearing mice. Moreover, the frequency of M-MDSCs of lung was positively correlated with the number of lung metastatic nodules. Further, M-MDSCs markedly inhibited T cell function, while there was no difference between M-MDSCs and PMN-MDSCs in promoting 4T1 cell migration. X-ray irradiation promoted the release of G-CSF, GM-CSF and CXCl1-rich exosomes, and facilitated the migration of M-MDSCs and PMN-MDSCs into the lung through CXCL1/CXCR2 signaling. While irradiated mouse lung extracts or ir/4T1-exo treated macrophage culture medium showed obvious selective chemotaxis to M-MDSCs. Mechanistically, ir/4T1-exo induce macrophage to produce GM-CSF, which further promoted CCL2 release in an autocrine manner to recruit M-MDSCs via CCL2/CCR2 axis. CONCLUSIONS: Our work has identified an undesired effect of radiotherapy that may promote immunosuppressive premetastatic niches formation by recruiting M-MDSCs to lung. Further studies on radiotherapy combined CXCR2 or CCR2 signals inhibitors were necessary.

HCV Core Protein Induces Chemokine CCL2 and CXCL10 Expression Through NF-κB Signaling Pathway in Macrophages.

HCV core protein is the first structural protein synthesized during hepatitis C virus (HCV) infection and replication. It is released from virus infected liver cells and mediates multiple functions to affect host cell response. The innate immune response is the first line of defense against viral infection. After HCV infection, Kupffer cells (KCs) which are liver macrophages play an important role in host innate immune response. Kupffer cells act as phagocytes and release different cytokines and chemokines to counter viral infection and regulate inflammation and fibrosis in liver. Earlier, we have demonstrated that HCV core protein interacts with gC1qR and activates MAPK, NF-κB and PI3K/AKT pathways in macrophages. In this study, we explored the effect of HCV core protein on CCL2 and CXCL10 expression in macrophages and the signaling pathways involved. Upon silencing of gC1qR, we observed a significant decrease expression of CCL2 and CXCL10 in macrophages in the presence of HCV core protein. Inhibiting NF-κB pathway, but not P38, JNK, ERK and AKT pathways greatly reduced the expression of CCL2 and CXCL10. Therefore, our results indicate that interaction of HCV core protein with gC1qR could induce CCL2 and CXCL10 secretion in macrophages via NF-κB signaling pathway. These findings may shed light on the understanding of how leukocytes migrate into the liver and exaggerate host-derived immune responses and may provide novel therapeutic targets in HCV chronic inflammation.

HSP90-dependent PUS7 overexpression facilitates the metastasis of colorectal cancer cells by regulating LASP1 abundance.

BACKGROUND: Pseudouridine synthase (PUS) 7 is a member of the PUS family that catalyses pseudouridine formation. It has been shown to be involved in intellectual development and haematological malignancies. Nevertheless, the role and the underlying molecular mechanisms of PUS7 in solid tumours, such as colorectal cancer (CRC), remain unexplored. This study elucidated, for the first time, the role of PUS7 in CRC cell metastasis and the underlying mechanisms. METHODS: We conducted immunohistochemistry, qPCR, and western blotting to quantify the expression of PUS7 in CRC tissues as well as cell lines. Besides, diverse in vivo and in vitro functional tests were employed to establish the function of PUS7 in CRC. RNA-seq and proteome profiling analysis were also applied to identify the targets of PUS7. PUS7-interacting proteins were further uncovered using immunoprecipitation and mass spectrometry. RESULTS: Overexpression of PUS7 was observed in CRC tissues and was linked to advanced clinical stages and shorter overall survival. PUS7 silencing effectively repressed CRC cell metastasis, while its upregulation promoted metastasis, independently of the PUS7 catalytic activity. LASP1 was identified as a downstream effector of PUS7. Forced LASP1 expression abolished the metastasis suppression triggered by PUS7 silencing. Furthermore, HSP90 was identified as a client protein of PUS7, associated with the increased PUS7 abundance in CRC. NMS-E973, a specific HSP90 inhibitor, also showed higher anti-metastatic activity when combined with PUS7 repression. Importantly, in line with these results, in human CRC tissues, the expression of PUS7 was positively linked to the expression of HSP90 and LASP1, and patients co-expressing HSP90/PUS7/LASP1 showed a worse prognosis. CONCLUSIONS: The HSP90-dependent PUS7 upregulation promotes CRC cell metastasis via the regulation of LASP1. Thus, targeting the HSP90/PUS7/LASP1 axis may be a novel approach for the treatment of CRC.

Impact of Laparoscopic Converted to Open Gastrectomy on Short- and Long-Term Outcomes of Patients with Locally Advanced Gastric Cancer: A Propensity Score-Matched Analysis.

BACKGROUND: It remains unclear whether laparoscopic conversation to open gastrectomy causes higher morbidity and has an adverse effect on the long-term survival outcomes of patients with gastric cancer. This study was designed to evaluate the impact of the conversion on short and long-term outcomes of patients with locally advanced gastric cancer (AGC). METHODS: We retrospectively investigated 871 patients who initially underwent laparoscopic gastrectomy (LG) for pathologically confirmed diagnosis of AGC between February 2009 and April 2018. The patients were grouped as the conversion (CONV) group and completed laparoscopic (LAP) group. The 1:2 propensity score matching was performed to reduce the effect of bias due to the imbalanced baseline features between the two groups. Multivariate analyses were performed to identify risk factors for conversion and poor survival. RESULTS: After propensity-score matching, 168 patients (56 in the CONV group and 112 in the LAP group) were studied. The CONV group was associated with significantly longer operation time (252.4 vs. 216.7 min, P < 0.001) and greater estimated blood loss (234.8 vs. 171.2 ml, P < 0.001) as compared with the LAP group. The time to first flatus (3.8 vs. 3.3 days, P = 0.043), time to start a liquid diet (4.1 vs. 3.5 days, P = 0.021), and postoperative hospital stay (8.7 vs. 7.6 days, P = 0.020) were significantly longer in the CONV group than that in the LAP group. The overall complication rate did not differ significantly between the CONV group and the LAP group (16.1% vs. 12.5%, P = 0.692). Both 5-year overall survival (OS) and 5-year disease-free survival (DFS) did not differ significantly between the CONV group and the LAP group (P = 0.805, P = 0.945, respectively). Multivariate analysis showed that lymphovascular invasion and stage III were independent prognostic factors for poor OS and DFS, whereas conversion was not. CONCLUSIONS: The conversion from laparoscopic to open gastrectomy had no negative impact on morbidity and long-term survival outcomes for patients with locally AGC.

Indoleamine 2,3-Dioxygenase 1: A Promising Therapeutic Target in Malignant Tumor.

Tumorigenesis is a complex multifactorial and multistep process in which tumors can utilize a diverse repertoire of immunosuppressive mechanisms to evade host immune attacks. The degradation of tryptophan into immunosuppressive kynurenine is considered an important immunosuppressive mechanism in the tumor microenvironment. There are three enzymes, namely, tryptophan 2,3-dioxygenase (TDO), indoleamine 2,3-dioxygenase 1 (IDO1), and indoleamine 2,3-dioxygenase 2 (IDO2), involved in the metabolism of tryptophan. IDO1 has a wider distribution and higher activity in catalyzing tryptophan than the other two; therefore, it has been studied most extensively. IDO1 is a cytosolic monomeric, heme-containing enzyme, which is now considered an authentic immune regulator and represents one of the promising drug targets for tumor immunotherapy. Collectively, this review highlights the regulation of IDO1 gene expression and the ambivalent mechanisms of IDO1 on the antitumoral immune response. Further, new therapeutic targets via the regulation of IDO1 are discussed. A comprehensive analysis of the expression and biological function of IDO1 can help us to understand the therapeutic strategies of the inhibitors targeting IDO1 in malignant tumors.

Metabolomic characterization of semen from asthenozoospermic patients using ultra-high-performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry.

Asthenozoospermia (AS) is a common factor of male infertility, and its pathogenesis remains unclear. The purpose of this study was to investigate the differential seminal plasma metabolic pattern in asthenozoospermic men and to identify potential biomarkers in relation to spermatogenic dysfunction using sensitive ultra-high-performance liquid chromatography-tandem quadruple time-of-flight MS (UHPLC-Q-TOF/MS). The samples of seminal plasma from patients with AS (n = 20) and healthy controls (n = 20) were checked and differentiated by UHPLC-Q-TOF/MS. Compared with the control group, the AS group showed a total of nine significantly different metabolites, including increases in creatinine, uric acid, N6 -methyladenosine (m6 A), uridine, and taurine and decreases in carnitine, nicotinamide, N-acetylputrescine and l-palmitoylcarnitine. By analyzing the correlation among these metabolites and clinical computer-assisted semen analysis reports, we found that m6 A is significantly correlated with not only the four decreased metabolites but also with sperm count, motility, and curvilinear velocity. Furthermore, nicotinamide was shown to correlate with other identified metabolites, indicating its important role in the metabolic pathway of AS. Current results implied that sensitive untargeted seminal plasma metabolomics could identify distinct metabolic patterns of AS and would help clinicians by offering novel cues for discovering the pathogenesis of male infertility.

Nudix Hydrolase NUDT16 Regulates 53BP1 Protein by Reversing 53BP1 ADP-Ribosylation.

53BP1 controls two downstream subpathways, one mediated by PTIP and Artemis and the other by RIF1 and MAD2L2/Shieldin, to coordinate DNA repair pathway choices. However, the upstream regulator(s) of 53BP1 function in DNA repair remain unknown. We and others recently reported that TIRR associates with 53BP1 to stabilize it and prevents 53BP1 localization to DNA damage sites by blocking 53BP1 Tudor domain binding to H4K20me2 sites. Here, we report that the Nudix hydrolase NUDT16, a TIRR homolog, regulates 53BP1 stability. We identified a novel posttranslational modification of 53BP1 by ADP-ribosylation that is targeted by a PAR-binding E3 ubiquitin ligase, RNF146, leading to 53BP1 polyubiquitination and degradation. In response to DNA damage, ADP-ribosylated 53BP1 increased significantly, resulting in its ubiquitination and degradation. These data suggest that NUDT16 plays a major role in controlling 53BP1 levels under both normal growth conditions and during DNA damage. Notably, overexpression of a NUDT16 catalytically inactive mutant blocked 53BP1 localization to double-strand breaks because (i) the mutant binding to TIRR increased after IR; (ii) the mutant enhanced 53BP1 Tudor domain binding to TIRR, and (iii) the mutant impaired the interaction of 53BP1 Tudor domain with H4K20me2. Moreover, NUDT16's catalytic hydrolase activity was required for 53BP1 de-ADP-ribosylation, 53BP1 protein stability, and its function in cell survival. In summary, we demonstrate that NUDT16 regulates 53BP1 stability and 53BP1 recruitment at double-strand breaks, providing yet another mechanism of 53BP1 regulation.Significance: This study provides a novel mechanism of 53BP1 regulation by demonstrating that NUDT16 has hydrolase activities that remove ADP-ribosylation of 53BP1 to regulate 53BP1 stability and 53BP1 localization at DSBs.

M Protein of Group a Streptococcus Plays an Essential Role in Inducing High Expression of A20 in Macrophages Resulting in the Downregulation of Inflammatory Response in Lung Tissue.

Group A streptococcus (GAS), a common pathogen, is able to escape host immune attack and thus survive for longer periods of time. One of the mechanisms used by GAS is the upregulated expression of immunosuppressive molecules, which leads to a reduction in the production of inflammatory cytokines in immune cells. In the present study, we found that macrophages produced lower levels of proinflammatory cytokines (IL-1ß, TNF-α, IL-6) when challenged with GAS than they did when challenged with Escherichia coli (E. coli). Simultaneously, in a mouse model of lung infection, GAS appeared to induce a weaker inflammatory response compared to E. coli. Our data also indicated that the expression of the A20 transcriptional regulator was higher in GAS-infected macrophages than that in macrophages infected with E. coli, and that high expression of A20 correlated with a reduction in the production of TRAF6. SiRNA targeting of A20 led to the increased production of TRAF6, IL-1ß, TNF-α, and IL-6, suggesting that A20 inhibits synthesis of these key proinflammatory cytokines. We also investigated the pathway underlying A20 production and found that the synthesis of A20 depends on My88, and to a lower extent on TNFR1. Finally, we showed a significant reduction in the expression of A20 in macrophages stimulated by M protein-mutant GAS, however, a speB-GAS mutant, which is unable to degrade M protein, induced a greater level of A20 production than wild type GAS. Collectively, our data suggested that M protein of GAS was responsible for inducing A20 expression in macrophages, which in turn down-regulates the inflammatory cytokine response in order to facilitate GAS in evading immune surveillance and thus prolong survival in the host.

Respiratory Syncytial Virus Replication Is Promoted by Autophagy-Mediated Inhibition of Apoptosis.

Respiratory syncytial virus (RSV) is the main cause of acute lower respiratory tract infection (ALRI) in children worldwide. Virus-host interactions affect the progression and prognosis of the infection. Autophagy plays important roles in virus-host interactions. Respiratory epithelial cells serve as the front line of host defense during RSV infection, However, it is still unclear how they interact with RSV. In this study, we found that RSV induced autophagy that favored RSV replication and exacerbated lung pathology in vivo Mechanistically, RSV induced complete autophagy flux through reactive oxygen species (ROS) generation and activation of the AMP-activated protein kinase/mammalian target of rapamycin (AMPK-MTOR) signaling pathway in HEp-2 cells. Furthermore, we evaluated the functions of autophagy in RSV replication and found that RSV replication was increased in HEp-2 cells treated with rapamycin but decreased remarkably in cells treated with 3-methylademine (3-MA) or wortmannin. Knockdown key molecules in the autophagy pathway with short hairpinp RNA (shRNA) against autophagy-related gene 5 (ATG5), autophagy-related gene 7 (ATG7), or BECN1/Beclin 1 or treatment with ROS scavenger N-acetyl-l-cysteine (NAC) and AMPK inhibitor (compound C) suppressed RSV replication. 3-MA or shATG5/BECN1 significantly decreased cell viability and increased cell apoptosis at 48 hours postinfection (hpi). Blocking apoptosis with Z-VAD-FMK partially restored virus replication at 48 hpi. Those results provide strong evidence that autophagy may function as a proviral mechanism in a cell-intrinsic manner during RSV infection.IMPORTANCE An understanding of the mechanisms that respiratory syncytial virus utilizes to interact with respiratory epithelial cells is critical to the development of novel antiviral strategies. In this study, we found that RSV induces autophagy through a ROS-AMPK signaling axis, which in turn promotes viral infection. Autophagy favors RSV replication through blocking cell apoptosis at 48 hpi. Mechanistically, RSV induces mitophagy, which maintains mitochondrial homeostasis and therefore decreases cytochrome c release and apoptosis induction. This study provides a novel insight into this virus-host interaction, which may help to exploit new antiviral treatments targeting autophagy processes.

HCV core protein binds to gC1qR to induce A20 expression and inhibit cytokine production through MAPKs and NF-κB signaling pathways.

Hepatitis C virus (HCV) infection is characterized by a strong propensity toward chronicity. During chronic HCV infection, HCV core protein is implicated in deregulating cytokine expression that associates with chronic inflammation. A20 is known as a powerful suppressor in cytokine signaling, in this study, we explored the A20 expression in macrophages induced by HCV core protein and the involved signaling pathways. Results demonstrated that HCV core protein induced A20 expression in macrophages. Silencing A20 significantly enhanced the secretion of IL-6, IL-1ß and TGF-ß1, but not IL-8 and TNF. Additionally, HCV core protein interacted with gC1qR, but not TLR2, TLR3 and TLR4 in pull-down assay. Silencing gC1qR abrogated core-induced A20 expression. Furthermore, HCV core protein activated MAPK, NF-κB and PI3K/AKT pathways in macrophages. Inhibition of P38, JNK and NF-κB but not ERK and AKT activities greatly reduced the A20 expression. In conclusion, the study suggests that HCV core protein ligates gC1qR to induce A20 expression in macrophages via P38, JNK and NF-κB signaling pathways, which leads to a low-grade chronic inflammation during HCV infection. It represents a novel mechanism by which HCV usurps the host for persistence.

Group A Streptococcus induces less p65 nuclear translocation and non-classical nuclear factor kappa B activation in macrophages, which possibly leads to a weaker inflammatory response.

OBJECTIVES: The aim of this study was to explore the pathogenic mechanism of group A Streptococcus (GAS) and to investigate how GAS evades phagocytosis by immune cells. METHODS: The classical inflammatory signaling pathway of macrophages infected with GAS was investigated by protein microarray, real-time PCR, Western blot, immunoprecipitation, and flow cytometry. RESULTS: GAS induced a lower level of inflammatory mediators in macrophages than either the Gram-positive Staphylococcus aureus or the Gram-negative Escherichia coli. Therefore, the conventional inflammatory signal pathway was investigated. It was found that GAS and S. aureus induced both toll-like receptor (TLR)2 and TLR4 expression, while Gram-negative E. coli only activated TLR4 in RAW264.7 cells. Although MyD88, the main adaptor protein, was activated by the three pathogens, there was no difference in MyD88 expression in macrophages. Nuclear factor kappa B (NF-κB) is the classical transcription factor of inflammatory signals, and the results of the present study showed that GAS, similar to E. coli, induced a weaker p65 nuclear translocation compared to S. aureus. Interestingly, GAS activated NF-κB by inducing p65-p52 heterodimer, but not the classical heterodimer of NF-κB (p65-p50), while E. coli activated NF-κB by inducing both p65-p50 and p65-p52 heterodimers. CONCLUSIONS: Compared to S. aureus and E. coli infection, GAS induced a weaker nuclear translocation and distinct combination of NF-κB subunits in macrophages, which probably leads to a weak inflammatory response.

Role of the exogenous HCV core protein in the interaction of human hepatocyte proliferation and macrophage sub-populations.

BACKGROUND: The core protein of hepatitis C virus (HCV) is found in the cytoplasm and nuclei of infected cells, including hepatocytes and other cells in the liver. The core protein could be secreted as well. Resident liver macrophages are dependent on the tissue micro-environment and external stimuli to differentiate M1 and M2 hypotypes with distinct functions, and increased expression of the nuclear transcription factor STAT3 was seen in M2-polarized macrophages. In contrast to proinflammatory M1 macrophages, M2 macrophages serve beneficial roles in chronic inflammation, immunosuppression, and tumorigenesis. METHODS: Monocyte-derived human macrophage line (mTHP-1) was treated with the exogenous HCV core protein. Next, the mTHP-1 culture supernatant or cell pellets were added to culture media of normal human liver cell line (L02). RESULTS: Only the culture supernatant stimulated L02 cells proliferation, which was associated with phosphorylated ERK expression. Core protein activated mTHP-1 cells showed enhanced pro- and anti-inflammatory cytokines secretion, which was accompanied by high expression of phosphorylated NF-κB105 and NF-κB65. However, phosphorylated STAT1, and STAT3, which are normally associated with M1 and M2 macrophage polarization, and cell surface expression of CD206, CD14, CD16, and CD86, were unaltered. A transwell co-culture system showed that only in mTHP-1 co-cultured with L02 in the presence of exogenous core protein, were higher levels of phosphorylated STAT3 and CD206 seen. CONCLUSIONS: We showed L02 cells proliferation was accelerated by the culture supernatant of mTHP-1 cells treated with the exogenous HCV core protein. The exogenous core protein mediated the interaction between macrophages and hepatocytes in co-culture, which enhanced the expression of phosphorylated STAT3 and CD206 in macrophages.

[Construction of the eukaryotic expression vector of human syk and its effect on MHC-I expression of human breast cancer cells].

AIM: To construct the eukaryotic expression vector of human spleen tyrosine kinase (Syk) and study the effect of Syk on MHC-I expression of human breast cancer cells. METHODS: A CDS fragment of human syk was obtained by RT-PCR from human breast cancer cells MDA-MB-468 and then the fragment was cloned into the expression vector pcDNA3.1D/V5-His-TOPO. After restriction endonuclease digestion, PCR and DNA sequencing confirmation, the recombinant hsyk expression plasmid was transfected into human breast cancer cells MDA-MB-231 by using lipofectamine protocols. After G418 selection, the cells stably expressed Syk. The expression of Syk, MHC-I and ICAM-I in the transfected cells was detected by Western blot, RT-PCR and flow cytometry (FCM). RESULTS: The eukaryotic expression plamid pcDNA3.1D/V5-His-TOPO/hsyk was constructed and it was expressed in the human breast cancer cells MDA-MB-231. The MDA-MB-231/Syk cells highly expressed MHC-I and ICAM-I. CONCLUSION: The successful construction of eukaryotic expression plamid pcDNA3.1D/V5-His-TOPO/hsyk and its expression in eukaryotic cells will be useful for further study of tumor immunity.