Seminal plasma promotes decidualization of endometrial stromal fibroblasts in vitro from women with and without inflammatory disorders in a manner dependent on interleukin-11 signaling.

STUDY QUESTION: Do seminal plasma (SP) and its constituents affect the decidualization capacity and transcriptome of human primary endometrial stromal fibroblasts (eSFs)? SUMMARY ANSWER: SP promotes decidualization of eSFs from women with and without inflammatory disorders (polycystic ovary syndrome (PCOS), endometriosis) in a manner that is not mediated through semen amyloids and that is associated with a potent transcriptional response, including the induction of interleukin (IL)-11, a cytokine important for SP-induced decidualization. WHAT IS KNOWN ALREADY: Clinical studies have suggested that SP can promote implantation, and studies in vitro have demonstrated that SP can promote decidualization, a steroid hormone-driven program of eSF differentiation that is essential for embryo implantation and that is compromised in women with the inflammatory disorders PCOS and endometriosis. STUDY DESIGN, SIZE, DURATION: This is a cross-sectional study involving samples treated with vehicle alone versus treatment with SP or SP constituents. SP was tested for the ability to promote decidualization in vitro in eSFs from women with or without PCOS or endometriosis (n = 9). The role of semen amyloids and fractionated SP in mediating this effect and in eliciting transcriptional changes in eSFs was then studied. Finally, the role of IL-11, a cytokine with a key role in implantation and decidualization, was assessed as a mediator of the SP-facilitated decidualization. PARTICIPANTS/MATERIALS, SETTING, METHODS: eSFs and endometrial epithelial cells (eECs) were isolated from endometrial biopsies from women of reproductive age undergoing benign gynecologic procedures and maintained in vitro. Assays were conducted to assess whether the treatment of eSFs with SP or SP constituents affects the rate and extent of decidualization in women with and without inflammatory disorders. To characterize the response of the endometrium to SP and SP constituents, RNA was isolated from treated eSFs or eECs and analyzed by RNA sequencing (RNAseq). Secreted factors in conditioned media from treated cells were analyzed by Luminex and ELISA. The role of IL-11 in SP-induced decidualization was assessed through Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-9-mediated knockout experiments in primary eSFs. MAIN RESULTS AND THE ROLE OF CHANCE: SP promoted decidualization both in the absence and presence of steroid hormones (P < 0.05 versus vehicle) in a manner that required seminal proteins. Semen amyloids did not promote decidualization and induced weak transcriptomic and secretomic responses in eSFs. In contrast, fractionated SP enriched for seminal microvesicles (MVs) promoted decidualization. IL-11 was one of the most potently SP-induced genes in eSFs and was important for SP-facilitated decidualization. LARGE SCALE DATA: RNAseq data were deposited in the Gene Expression Omnibus repository under series accession number GSE135640. LIMITATIONS, REASONS FOR CAUTION: This study is limited to in vitro analyses. WIDER IMPLICATIONS OF THE FINDINGS: Our results support the notion that SP promotes decidualization, including within eSFs from women with inflammatory disorders. Despite the general ability of amyloids to induce cytokines known to be important for implantation, semen amyloids poorly signaled to eSFs and did not promote their decidualization. In contrast, fractionated SP enriched for MVs promoted decidualization and induced a transcriptional response in eSFs that overlapped with that of SP. Our results suggest that SP constituents, possibly those associated with MVs, can promote decidualization of eSFs in an IL-11-dependent manner in preparation for implantation. STUDY FUNDING/COMPETING INTEREST(S): This project was supported by NIH (R21AI116252, R21AI122821 and R01AI127219) to N.R.R. and (P50HD055764) to L.C.G. The authors declare no conflict of interest.

Peli1 facilitates virus replication and promotes neuroinflammation during West Nile virus infection.

The E3 ubiquitin ligase Pellino 1 (Peli1) is a microglia-specific mediator of autoimmune encephalomyelitis. Its role in neurotropic flavivirus infection is largely unknown. Here, we report that mice deficient in Peli1 (Peli1-/-) were more resistant to lethal West Nile virus (WNV) infection and exhibited reduced viral loads in tissues and attenuated brain inflammation. Peli1 mediates chemokine and proinflammatory cytokine production in microglia and promotes T cell and macrophage infiltration into the CNS. Unexpectedly, Peli1 was required for WNV entry and replication in mouse macrophages and mouse and human neurons and microglia. It was also highly expressed on WNV-infected neurons and adjacent inflammatory cells from postmortem patients who died of acute WNV encephalitis. WNV passaged in Peli1-/- macrophages or neurons induced a lower viral load and impaired activation in WT microglia and thereby reduced lethality in mice. Smaducin-6, which blocks interactions between Peli1 and IRAK1, RIP1, and IKKε, did not inhibit WNV-triggered microglia activation. Collectively, our findings suggest a nonimmune regulatory role for Peli1 in promoting microglia activation during WNV infection and identify a potentially novel host factor for flavivirus cell entry and replication.

Oncogenic mTOR signalling recruits myeloid-derived suppressor cells to promote tumour initiation.

Myeloid-derived suppressor cells (MDSCs) play critical roles in primary and metastatic cancer progression. MDSC regulation is widely variable even among patients harbouring the same type of malignancy, and the mechanisms governing such heterogeneity are largely unknown. Here, integrating human tumour genomics and syngeneic mammary tumour models, we demonstrate that mTOR signalling in cancer cells dictates a mammary tumour's ability to stimulate MDSC accumulation through regulating G-CSF. Inhibiting this pathway or its activators (for example, FGFR) impairs tumour progression, which is partially rescued by restoring MDSCs or G-CSF. Tumour-initiating cells (TICs) exhibit elevated G-CSF. MDSCs reciprocally increase TIC frequency through activating Notch in tumour cells, forming a feedforward loop. Analyses of primary breast cancers and patient-derived xenografts corroborate these mechanisms in patients. These findings establish a non-canonical oncogenic role of mTOR signalling in recruiting pro-tumorigenic MDSCs and show how defined cancer subsets may evolve to promote and depend on a distinct immune microenvironment.

A West Nile virus NS4B-P38G mutant strain induces cell intrinsic innate cytokine responses in human monocytic and macrophage cells.

Previous studies have shown that an attenuated West Nile virus (WNV) nonstructural (NS) 4B-P38G mutant induces stronger innate and adaptive immune responses than wild-type WNV in mice, which has important applications to vaccine development. To investigate the mechanism of immunogenicity, we characterized WNV NS4B-P38G mutant infection in two human cell lines-THP-1 cells and THP-1 macrophages. Although the NS4B-P38G mutant produced more viral RNA than the parental WNV NY99 in both cell types, there was no detectable infectious virus in the supernatant of either cell type. Nonetheless, the attenuated mutant boosted higher innate cytokine responses than virulent parental WNV NY99 in these cells. The NS4B-P38G mutant infection of THP-1 cells led to more diverse and robust innate cytokine responses than that seen in THP-1 macrophages, which were mediated by toll-like receptor (TLR)7 and retinoic acid-inducible gene 1(RIG-I) signaling pathways. Overall, these results suggest that a defective viral life cycle during NS4B-P38G mutant infection in human monocytic and macrophage cells leads to more potent cell intrinsic innate cytokine responses.

A hamster-derived West Nile virus strain is highly attenuated and induces a differential proinflammatory cytokine response in two murine cell lines.

Increasing evidence suggests that West Nile virus (WNV) induces a persistent infection in some humans and animals. Here, we characterized infection of mouse macrophage and kidney epithelial cell lines with a strain of WNV (H8912), cultured from urine of a persistently infected hamster. WNV H8912 had a reduced replication rate, concurrent with a lower interferon (IFN)-ß gene expression in both cell types compared to its parent strain - WNV NY99. In WNV H8912-infected macrophages, we observed higher interleukin (IL)-6 and tumor necrosis factor (TNF)-α expression and more nuclear factor kappa B (NF-κB) activation than in cells infected with WNV NY99. In contrast, there were reduced levels of TNF-α and IL-6 expression, as well as less NF-κB activation following WNV H8912 infection in the kidney epithelial cells compared to WNV NY99. Overall, our results demonstrate that the WNV isolate obtained from hamster urine is an attenuated virus and induces a differential proinflammatory cytokine response in mouse macrophage and kidney epithelial cell lines.

Rab23 negatively regulates Gli1 transcriptional factor in a Su(Fu)-dependent manner.

Hedgehog (Hh) signaling, via the key signal transducer Smoothened (SMO) and Gli transcription factors, is essential for embryonic development and carcinogenesis. While the biological relevance of hedgehog signaling to cancer is well established, very little is known about the molecular mechanisms by which signaling transduction of this pathway occurs. Rab23 was discovered as a negative regulator of the Hh pathway through a mouse genetic study. Here we report that Rab23 directly associates with Su(Fu) and inhibits Gli1 function in a Su(Fu)-dependent manner. By confocal microscope and immunoprecipitation, we detected interaction between Rab23 and Su(Fu). Using Gli1-mediated reporter gene analysis, we found that Rab23 can suppress Gli1 transcriptional activity in wild type but not Su(Fu) null fibroblasts. Similarly, Rab23 expression reduced the nuclear localization of Gli1 in wild type but not Su(Fu) null fibroblast cells. Consistent with the GTPase motif in the protein, we showed that Rab23 has GTPase activity. The dominant negative form of Rab23 was unable to suppress Gli1-mediated transcriptional activity. Taken together, these data provide evidence to support that Rab23 negatively regulates Gli1 activity in a Su(Fu)-dependent manner.

Tumor shrinkage by cyclopamine tartrate through inhibiting hedgehog signaling.

The link of hedgehog (Hh) signaling activation to human cancer and synthesis of a variety of Hh signaling inhibitors raise great expectation that inhibiting Hh signaling may be effective in human cancer treatment. Cyclopamine (Cyc), an alkaloid from the Veratrum plant, is a specific natural product inhibitor of the Hh pathway that acts by targeting smoothened (SMO) protein. However, its poor solubility, acid sensitivity, and weak potency relative to other Hh antagonists prevent the clinical development of Cyc as a therapeutic agent. Here, we report properties of cyclopamine tartrate salt (CycT) and its activities in Hh signaling-mediated cancer in vitro and in vivo. Unlike Cyc, CycT is water soluble (5-10 mg/mL). The median lethal dose (LD50) of CycT was 62.5 mg/kg body weight compared to 43.5 mg/kg for Cyc, and the plasma half-life (T1/2) of CycT was not significantly different from that of Cyc. We showed that CycT had a higher inhibitory activity for Hh signaling-dependent motor neuron differentiation than did Cyc (IC50 = 50 nmol/L for CycT vs. 300 nmol/L for Cyc). We also tested the antitumor effectiveness of these Hh inhibitors using two mouse models of basal cell carcinomas (K14cre:Ptch1(neo/neo) and K14cre:SmoM2(YFP)). After topical application of CycT or Cyc daily for 21 days, we found that all CycT-treated mice had tumor shrinkage and decreased expression of Hh target genes. Taken together, we found that CycT is an effective inhibitor of Hh signaling-mediated carcinogenesis.

Expression of hedgehog signaling molecules in lung cancer.

Abnormal hyperactivation of the hedgehog (Hh) pathway has been reported in many types of human cancers, including lung cancer. However, most reports are based on studies of fewer than three Hh target genes and the data vary between different studies. In the present report, we have determined the expression levels of several important components of the Hh pathway in lung cancers by using RT-PCR, in situ hybridization and immunohistochemistry. These molecules include Smoothened (SMO), Rab23, the downstream target platelet-derived growth factor receptor alpha (PDGFRα), hedgehog interacting protein (HIP) and hepatocyte nuclear factor 3-beta (HNF3ß). Our data show that some components of the hedgehog pathway, such as SMO, Rab23 and PDGFRα are expressed in many lung cancer specimens, although other hedgehog target genes are infrequently detected in lung cancer. Loss of HIP expression was found in several cases of lung cancers. Our study indicates that there might be some additional mechanisms involved in the hyperactivation of the Hh pathway. Thus, we suggest that lung cancer with heterogeneous tumor type harboring Hh signaling activation may have some novel and different regulatory mechanisms.

Activation of the hedgehog pathway in human hepatocellular carcinomas.

Liver cancers, the majority of which are hepatocellular carcinomas (HCCs), rank as the fourth in cancer mortality worldwide and are the most rapidly increasing type of cancer in the United States. However, the molecular mechanisms underlying HCC development are not well understood. Activation of the hedgehog pathway is shown to be involved in several types of gastrointestinal cancers. Here, we provide evidence to indicate that hedgehog signaling activation occurs frequently in HCC. We detect expression of Shh, PTCH1 and Gli1 in 115 cases of HCC and in 44 liver tissues adjacent to the tumor. Expression of Shh is detectable in about 60% of HCCs examined. Consistent with this, hedgehog target genes PTCH1 and Gli1 are expressed in over 50% of the tumors, suggesting that the hedgehog pathway is frequently activated in HCCs. Of five cell lines screened, we found Hep3B, Huh7 and PLC/PRF/5 cells with detectable hedgehog target genes. Specific inhibition of hedgehog signaling in these three cell lines by smoothened (SMO) antagonist, KAAD-cyclopamine, or with Shh neutralizing antibodies decreases expression of hedgehog target genes, inhibits cell growth and results in apoptosis. In contrast, no effects are observed after these treatments in HCC36 and HepG2 cells, which do not have detectable hedgehog signaling. Thus, our data indicate that hedgehog signaling activation is an important event for development of human HCCs.