Sialylated IVIg promotes clinical improvements in a rabbit dry eye model by regulating inflammatory cytokines.

Dry eye disease (DED) is caused by a loss of homeostasis of the tear film, which results in visual disturbance, ocular surface inflammation and damage, and neurosensory abnormalities. Although it is prevalent in 5-50% of the global population, there are limited clinical options for its treatment. This study explored the potential use of human intravenous immunoglobulin (IVIg) and its enriched fractions of sialylation, sialylated IVIg (sIVIg), as a treatment for DED. Fifteen female New Zealand white rabbits were topically instilled with 0.2% benzalkonium chloride (BAC) twice daily for five consecutive days to induce experimental dry eye. Saline, 0.4% IVIg, or 0.04% sIVIg eye drops were instilled twice daily for 20 consecutive days. Clinical evaluations, such as non-invasive tear break-up time (NIBUT) and corneal fluorescein staining (CFS), were conducted. mRNA levels of mucin 4, mucin 16, TNF-α, IL-1ß, MMP9, IL-10, TGF-ß, and CD209 in rabbit conjunctival tissues were examined using reverse transcription polymerase chain reaction (RT-PCR) or quantitative RT-PCR (qRT-PCR). The relationships between CD209 family members in rabbits and various mammalian species were analyzed using a phylogenetic tree. IVIg or sIVIg treatment resulted in clinical improvements in the rabbit DED model. The inflammatory cytokines, TNF-α and IL-1ß, were increased and mucin 4 and mucin 16, cell surface-associated mucins, were decreased in BAC-induced dry eye. Following IVIg or sIVIg treatment, inflammatory cytokines decreased, whereas the anti-inflammatory cytokine, IL-10, increased substantially. Moreover, a 10-fold lower sIVIg treatment dose resulted in prolonged IL-10 production, representing a significantly improved DED compared to IVIg. Furthermore, the expression of rabbit CD209 mRNA in the rabbit conjunctiva and its close relationship with primate homologs suggest that it may interact with IVIg or sIVIg to promote IL-10 expression, as previously described in humans. At a lower dosage, sIVIg showed a more efficient improvement in DED, making it a promising new candidate medication for DED.

Sialylated IVIg binding to DC-SIGN+ Hofbauer cells induces immune tolerance through the caveolin-1/NF-kB pathway and IL-10 secretion.

Although the use of IVIg has increased in various immune-driven diseases and even in pregnancy, the exact action mechanisms of IVIg are not fully understood. Dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) is a known receptor for α-2,6-sialylated IgG (sIVIg), which is responsible for the anti-inflammatory effect of IVIg. DC-SIGN is expressed on Hofbauer cells (HBCs) of the fetal villi of the placenta which act as an innate immune modulator at the maternal-fetal interface. Preeclampsia is a major complication in pregnancy and is related to IL-10, a cytokine with an important role in immune tolerance. DC-SIGN interaction with sIVIg in HBCs promoted IL-10 secretion through the activation of the caveolin-1/NF-κB pathway, especially in plasma lipid rafts. Consistent results were obtained for HBCs from patients with preeclampsia. Collectively, the stimulation of DC-SIGN+ HBCs with sIVIg enhanced immune tolerance in the feto-maternal environment, suggesting the therapeutic application of sIVIg to prevent preeclampsia.

A Dual Role of DDX3X in dsRNA-Derived Innate Immune Signaling.

DEAD-Box Helicase 3 X-Linked (DDX3X) is essential for RNA metabolism and participates in various cellular processes involving RNA. DDX3X has been implicated in cancer growth and metastasis. DDX3X is involved in antiviral responses for viral RNAs and contributes to pro- or anti-microbial responses. A better understanding of how human cells regulate innate immune response against the viral "non-self" double-stranded RNAs (dsRNAs) and endogenous viral-like "self" dsRNAs is critical to understanding innate immune sensing, anti-microbial immunity, inflammation, immune cell homeostasis, and developing novel therapeutics for infectious, immune-mediated diseases, and cancer. DDX3X has known for activating the viral dsRNA-sensing pathway and innate immunity. However, accumulating research reveals a more complex role of DDX3X in regulating dsRNA-mediated signaling in cells. Here, we discuss the role of DDX3X in viral dsRNA- or endogenous dsRNA-mediated immune signaling pathways.

USP13 promotes development and metastasis of high-grade serous ovarian carcinoma in a novel mouse model.

Epithelial ovarian cancer is the most lethal gynecologic malignancy and one of the most common causes of cancer mortality among women worldwide. Ubiquitin-Specific Peptidase 13 (USP13) gene copy is strongly amplified in human epithelial ovarian cancer, and high USP13 expression is correlated with poor survival outcomes. Yet, its pathological contribution to ovarian tumorigenesis remains unknown. We crossed a conditional Usp13 overexpressing knock-in mouse with a conditional knockout of Trp53 and Pten mouse and generated a novel ovarian cancer genetically engineered mouse model (GEMM), which closely recapitulates the genetic changes driving ovarian cancer in humans. Overexpression of USP13 with deletion of Trp53 and Pten in murine ovarian surface epithelium accelerated ovarian tumorigenesis and led to decreased survival in mice. Notably, USP13 greatly enhanced peritoneal metastasis of ovarian tumors with frequent development of hemorrhagic ascites. The primary and metastatic tumors exhibited morphology and clinical behavior similar to human high-grade serous ovarian cancer. Co-inhibition of USP13 and AKT significantly decreased the viability of the primary murine ovarian cancer cells isolated from the GEMM. USP13 also increased the tumorigenic and metastatic abilities of primary murine ovarian cancer cells in a syngeneic mouse study. These findings suggest a critical role of USP13 in ovarian cancer development and reveal USP13 as a potential therapeutic target for ovarian cancer.

Targeting DDX3X Triggers Antitumor Immunity via a dsRNA-Mediated Tumor-Intrinsic Type I Interferon Response.

Induction of nucleic acid sensing-mediated type I interferon (IFN) has emerged as a novel approach to activate the immune system against cancer. Here we show that the depletion of DEAD-box RNA helicase 3X (DDX3X) triggers a tumor-intrinsic type I IFN response in breast cancer cells. Depletion or inhibition of DDX3X activity led to aberrant cytoplasmic accumulation of cellular endogenous double-stranded RNAs (dsRNA), which triggered type I IFN production through the melanoma differentiation-associated gene 5 (MDA5)-mediated dsRNA-sensing pathway. Furthermore, DDX3X interacted with dsRNA-editing ADAR1 and dual depletion of DDX3X and ADAR1 synergistically activated the cytosolic dsRNA pathway in breast cancer cells. Loss of DDX3X in mouse mammary tumors enhanced antitumor activity by increasing the tumor-intrinsic type I IFN response, antigen presentation, and tumor infiltration of cytotoxic T and dendritic cells. These findings may lead to the development of a novel therapeutic approach for breast cancer by targeting DDX3X in combination with immune-checkpoint blockade. SIGNIFICANCE: This study elucidates the novel role of DDX3X in regulating endogenous cellular dsRNA homeostasis and type I IFN signaling in breast cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/13/3607/F1.large.jpg.

Dominant role of splenic marginal zone lipid rafts in the classical complement pathway against S. pneumoniae.

Lipid rafts (LRs) play crucial roles in complex physiological processes, modulating innate and acquired immune responses to pathogens. The transmembrane C-type lectins human dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and its mouse homolog SIGN-R1 are distributed in LRs and expressed on splenic marginal zone (MZ) macrophages. The DC-SIGN-C1q or SIGN-R1-C1q complex could mediate the immunoglobulin (Ig)-independent classical complement pathway against Streptococcus pneumoniae. Precise roles of LRs during this complement pathway are unknown. Here we show that LRs are indispensable for accelerating the DC-SIGN- or SIGN-R1-mediated classical complement pathway against S. pneumoniae, thus facilitating rapid clearance of the pathogen. The trimolecular complex of SIGN-R1-C1q-C4 was exclusively enriched in LRs of splenic MZ macrophages and their localization was essential for activating C3 catabolism and enhancing pneumococcal clearance, which were abolished in SIGN-R1-knockout mice. However, DC-SIGN replacement on splenic MZ macrophage's LRs of SIGN-R1-depleted mice reversed these defects. Disruption of LRs dramatically reduced pneumococcal uptake and decomposition. Additionally, DC- SIGN, C1q, C4, and C3 were obviously distributed in splenic LRs of cadavers. Therefore, LRs on splenic SIGN-R1+ or DC-SIGN+ macrophages could provide spatially confined and optimal bidirectional platforms, not only for usual intracellular events, for example recognition and phagocytosis of pathogens, but also an unusual extracellular event such as the complement system. These findings improve our understanding of the orchestrated roles of the spleen, unraveling a new innate immune system initiated from splenic MZ LRs, and yielding answers to several long-standing problems, including the need to understand the profound role of LRs in innate immunity, the need to identify how such a small portion of splenic SIGN-R1+ macrophages (<0.05% of splenic macrophages) effectively resist S. pneumoniae, and the need to understand how LRs can promote the protective function of DC-SIGN against S. pneumoniae in the human spleen.

CXCR2 is a negative regulator of p21 in p53-dependent and independent manner via Akt-mediated Mdm2 in ovarian cancer.

Ovarian cancer (OC) has the highest rate of mortality among gynecological malignancy. Chemokine receptor CXCR2 in OC is associated with poor outcomes. However, the mechanisms by which CXCR2 regulates OC proliferation remain poorly understood. We generated CXCR2-positive cells from parental p53 wild-type (WT), mutant and null OC cells, and assessed the roles of CXCR2 on proliferation of OC cells in p53-dependent and independent manner. CXCR2 promoted cell growth rate: p53WT > mutant = null cells. Nutlin-3, a p53 stabilizer, inhibited cell proliferation in p53WT cells, but had little effect in p53-mutant or null cells, indicating p53-dependence of CXCR2-mediated proliferation. CXCR2 decreased p53 protein, a regulator of p21, and downregulated p21 promoter activity only in p53WT cells. The p53 responsive element (RE) of p21 promoter played a critical role in this CXCR2-mediated p21 downregulation. Moreover, CXCR2-positive cells activated more Akt than CXCR2-negative cells followed by enhanced murine double minute (Mdm2). Silencing Mdm2 or Akt1 upregulated p21 expression, whereas Akt1 overexpression downregulated p21 at the promoter and protein levels in p53WT cells. Cell cycle analysis revealed that CXCR2 decreased p21 gene in p53-null cells. Interestingly, romidepsin (histone deacetylase inhibitor)-induced p21 upregulation did not involve the p53 RE in the p21 promoter in p53-null cells. Romidepsin decreased the protein levels of Akt1 and Mdm2, leading to induction of p21 in p53-null cells. CXCR2 reduced romidepsin-induced p21 upregulation by activating Akt-induced Mdm2. Taken together, CXCR2 enhances cell proliferation by suppressing p21 through Akt-Mdm2 signaling in p53-dependent and independent manner.

Localization of Serum Amyloid A3 in the Mouse Ovary.

Tumor necrosis factor-α (TNF-α) induces serum amyloid A (SAA) 3 among acute-phase proteins in mouse granulosa cells by activating NF-κB signaling via p55 TNF-α receptor type 1. However, the localization of SAA3 within the ovary is unknown. Here we investigated ovarian localization of SAA3 in a mouse ovulation model and in response to IL-1ß, a proinflammatory mediator. For the ovulation model, equine chorionic gonadotropin (eCG; 2.5 IU) was administered to mice subcutaneously (sc) to stimulate follicular development on day 25 of age and then 50 h after eCG, human chorionic gonadotropin (hCG; 2.5 IU) was administered sc to induce ovulation. The mouse ovulation model was characterized by the localization of CYP19 mRNA expression to granulosa layers of larger follicles. SAA3 mRNA, determined by in situ hybridization, was broadly expressed throughout the whole ovary. Granulosa layers and small follicles expressed higher SAA3 mRNA compared to thecal-interstitial layers and large follicles, respectively. Interestingly, atretic follicles contained cells expressing intense SAA3 mRNA. After ovulation, SAA3 mRNA expression was intensely evident in ruptured follicles and corpora lutea (CL). The intraperitoneal administration of IL-1ß revealed the intense and extensive appearance of specific cells expressing SAA3 mRNA around follicles and in CL. In addition, Gene Expression Omnibus (GEO) database analysis supported expression pattern of SAA3 mRNA observed in mouse ovulation model. Taken together, SAA3 was broadly distributed through the whole ovary, but intensely expressed in atretic follicles and CL. Furthermore, proinflammatory mediators could trigger the intense appearance of SAA3 around follicles and in CL.

Augmented Serum Amyloid A1/2 Mediated by TNF-induced NF-κB in Human Serous Ovarian Epithelial Tumors.

Tumor necrosis factor-α (TNF) is well known to be involved in the immune system and ovarian inflammation. Ovarian cancer is an inflammation-related malignancy that lacks early screening strategies, resulting in late diagnosis followed by high mortality. Based on our previous data, TNF induced abundant serum amyloid A (SAA), an acute phase protein linked to inflammation, in ovarian granulosal cells. To date, the regulation and expression of SAA in ovarian cancer is not fully elucidated. Here, we investigated the relationship between TNF and SAA by comparing human normal ovarian tissues and serous ovarian tumors. We found that SAA1/2 was significantly expressed in tumor tissues, but no or trace expression levels in normal tissues. TNF was also significantly upregulated in ovarian tumor tissues compared to normal tissues. Moreover, TNF significantly increased SAA1/2 levels in human ovarian cancer cell lines, OVCAR-3 and SKOV-3, in a time-dependent manner. Since the SAA1 promoter contains two nuclear factor (NF)-κB sites, we examined whether TNF regulates SAA1 promoter activity. Deletion analysis revealed that the proximal NF-κB site (-95/-85) played a critical role in regulating TNF-induced SAA1 promoter activity. Within 2 h after intraperitoneal injection of lipopolysaccharide, a product known to stimulate release of TNF, SAA preferably localized to ovarian epithelial cells and the thecal-interstitial layers compared to granulosal cell layers. Based on Gene Expression Omnibus (GEO) database, SAA1/2 and TNF were dominantly expressed in advanced grade ovarian cancer. Taken together, the accumulation of SAA1/2 in ovarian cancer could be mediated by TNF-induced NF-κB activation.

SIGN-R1 and complement factors are involved in the systemic clearance of radiation-induced apoptotic cells in whole-body irradiated mice.

Although SIGN-R1-mediated complement activation pathway has been shown to enhance the systemic clearance of apoptotic cells, the role of SIGN-R1 in the clearance of radiation-induced apoptotic cells has not been characterized and was investigated in this study. Our data indicated that whole-body γ-irradiation of mice increased caspase-3(+) apoptotic lymphocyte numbers in secondary lymphoid organs. Following γ-irradiation, SIGN-R1 and complements (C4 and C3) were simultaneously increased only in the mice spleen tissue among the assessed tissues. In particular, C3 was exclusively activated in the spleen. The delayed clearance of apoptotic cells was markedly prevalent in the spleen and liver of SIGN-R1 KO mice, followed by a significant increase of CD11b(+) cells. These results indicate that SIGN-R1 and complement factors play an important role in the systemic clearance of radiation-induced apoptotic innate immune cells to maintain tissue homeostasis after γ-irradiation.

Intravenous immunoglobulin-mediated immunosuppression and the development of an IVIG substitute.

Immunoglobulins are glycoproteins produced by the cells of the immune system. Their primary function is to protect the body from pathogenic infection. Moreover, a concentrated polyclonal mixture of immunoglobulin G (IgG), the so-called intravenous IgG (IVIG), has been used to treat various chronic and systemic disorders of the immune system. Studies on the effects of IVIG in autoimmune disease models have revealed that IgG Fc fragments confer protection against various autoimmune diseases. The identification of this IgG Fc immunomodulatory component is important for the development of IVIG substitutes. The focus of this review is to introduce one of the Fc regulatory entities and to provide a summary of the current knowledge of the putative general mechanisms underlying IVIG activity in vivo on the basis of these Fc fragments. We also address the recent insights into several approaches for the development of IVIG substitutes.

Enhanced immune response of T-cell independent or dependent antigens in SIGN-R1 knock-out mice.

Dextran was used to explore a novel method of enhancing an immune response against T-cell independent type 2 (TI-2) polysaccharide antigens, because of its suitability as a model for the immunogenecity of many TI-2 polysaccharide antigens and its high affinity to SIGN-R1. Here we showed that the primary immune response of IgM, IgG3, and IgG2b was enhanced by dextran in SIGN-R1 knock-out (KO) mice, further evoking the induction of a secondary immune response to IgG2b in parallel. On the other hand, an immune response of IgG1 and IgG2b against T-cell dependent (TD) antigen was strongly enhanced by the administration of ovalbumin (OVA) in SIGN-R1 KO mice. These results indicate that SIGN-R1 is critical in the regulation of immune responses. Therefore, our study suggests that inhibition of TI-2 polysaccharide antigen uptake in SIGN-R1(+) macrophages contributes to the development of novel vaccination strategies against TI-2 polysaccharide antigens.

SIGN-R1, a C-type lectin, binds to Bip/GRP78 and this interaction mediates the regurgitation of T-cell-independent type 2 antigen dextran through the endoplasmic reticulum.

Capsular polysaccharides of Streptococcus pneumoniae are representative T-cell-independent type 2 (TI-2) antigens, frequently causing serious infections in children, the elderly, and immunocompromised patients. However, the detailed mechanism of this immune escape by CPSs is poorly understood. To pursue this question, polysaccharide dextran, ligand of SIGN-R1 as well as an appropriate model of the immunogenicity of many TI-2 polysaccharide antigens was used. SIGN-R1 bound to binding immunoglobulin protein (BiP), a well-characterized endoplasmic reticulum (ER) chaperone, primarily in non-ER compartments. Interestingly, SIGN-R1(+) macrophages in the MZ showed high expression of BiP, implying an important role of SIGN-R1 binding to BiP in vivo. To our surprise, dextran is rapidly transported into the ER and subsequently regurgitated out of cells in vitro or in vivo. BiP down-regulation in SIGN-R1 transfectant reduced the regurgitation of dextran, causing the accumulation of dextran in the ER. Therefore, these results demonstrated the first example to describe the intracellular trafficking and the regurgitation of TI-2 antigen dextran, suggesting the novel pathway of TI-2 antigen presentation to immune cells.