LncRNA XR_001779380 Primes Epithelial Cells for IFN-γ-Mediated Gene Transcription and Facilitates Age-Dependent Intestinal Antimicrobial Defense.

Interferon (IFN) signaling is key to mucosal immunity in the gastrointestinal tract, but cellular regulatory elements that determine interferon gamma (IFN-γ)-mediated antimicrobial defense in intestinal epithelial cells are not fully understood. We report here that a long noncoding RNA (lncRNA), GenBank accession no. XR_001779380, was increased in abundance in murine intestinal epithelial cells following infection by Cryptosporidium, an important opportunistic pathogen in AIDS patients and a common cause of diarrhea in young children. Expression of XR_001779380 in infected intestinal epithelial cells was triggered by TLR4/NF-κB/Cdc42 signaling and epithelial-specific transcription factor Elf3. XR_001779380 primed epithelial cells for IFN-γ-mediated gene transcription through facilitating Stat1/Swi/Snf-associated chromatin remodeling. Interactions between XR_001779380 and Prdm1, which is expressed in neonatal but not adult intestinal epithelium, attenuated Stat1/Swi/Snf-associated chromatin remodeling induced by IFN-γ, contributing to suppression of IFN-γ-mediated epithelial defense in neonatal intestine. Our data demonstrate that XR_001779380 is an important regulator in IFN-γ-mediated gene transcription and age-associated intestinal epithelial antimicrobial defense. IMPORTANCE Epithelial cells along the mucosal surface provide the front line of defense against luminal pathogen infection in the gastrointestinal tract. These epithelial cells represent an integral component of a highly regulated communication network that can transmit essential signals to cells in the underlying intestinal mucosa that, in turn, serve as targets of mucosal immune mediators. LncRNAs are recently identified long noncoding transcripts that can regulate gene transcription through their interactions with other effect molecules. In this study, we demonstrated that lncRNA XR_001779380 was upregulated in murine intestinal epithelial cells following infection by a mucosal protozoan parasite Cryptosporidium. Expression of XR_001779380 in infected cells primed host epithelial cells for IFN-γ-mediated gene transcription, relevant to age-dependent intestinal antimicrobial defense. Our data provide new mechanistic insights into how intestinal epithelial cells orchestrate intestinal mucosal defense against microbial infection.

A host cell long noncoding RNA NR_033736 regulates type I interferon-mediated gene transcription and modulates intestinal epithelial anti-Cryptosporidium defense.

The gastrointestinal epithelium guides the immune system to differentiate between commensal and pathogenic microbiota, which relies on intimate links with the type I IFN signal pathway. Epithelial cells along the epithelium provide the front line of host defense against pathogen infection in the gastrointestinal tract. Increasing evidence supports the regulatory potential of long noncoding RNAs (lncRNAs) in immune defense but their role in regulating intestinal epithelial antimicrobial responses is still unclear. Cryptosporidium, a protozoan parasite that infects intestinal epithelial cells, is an important opportunistic pathogen in AIDS patients and a common cause of diarrhea in young children in developing countries. Recent advances in Cryptosporidium research have revealed a strong type I IFN response in infected intestinal epithelial cells. We previously identified a panel of host cell lncRNAs that are upregulated in murine intestinal epithelial cells following microbial challenge. One of these lncRNAs, NR_033736, is upregulated in intestinal epithelial cells following Cryptosporidium infection and displays a significant suppressive effect on type I IFN-controlled gene transcription in infected host cells. NR_033736 can be assembled into the ISGF3 complex and suppresses type I IFN-mediated gene transcription. Interestingly, upregulation of NR_033736 itself is triggered by the type I IFN signaling. Moreover, NR_033736 modulates epithelial anti-Cryptosporidium defense. Our data suggest that upregulation of NR_033736 provides negative feedback regulation of type I IFN signaling through suppression of type I IFN-controlled gene transcription, and consequently, contributing to fine-tuning of epithelial innate defense against microbial infection.

Microglia induce neurogenic protein expression in primary cortical cells by stimulating PI3K/AKT intracellular signaling in vitro.

Emerging evidence suggests that microglia can support neurogenesis. Little is known about the mechanisms by which microglia regulate the cortical environment and stimulate cortical neurogenesis. We used an in vitro co-culture model system to investigate the hypothesis that microglia respond to soluble signals from cortical cells, particularly following mechanical injury, to alter the cortical environment and promote cortical cell proliferation, differentiation, and survival. Analyses of cortical cell proliferation, cell death, neurogenic protein expression, and intracellular signaling were performed on uninjured and injured cortical cells in co-culture with microglial cell lines. Microglia soluble cues enhanced cortical cell viability and proliferation cortical cells. Co-culture of injured cortical cells with microglia significantly reduced cell death of cortical cells. Microglial co-culture significantly increased Nestin + and α-internexin + cortical cells. Multiplex ELISA and RT-PCR showed decreased pro-inflammatory cytokine production by microglia co-cultured with injured cortical cells. Inhibition of AKT phosphorylation in cortical cells blocked microglial-enhanced cortical cell viability and expression of neurogenic markers in vitro. This in vitro model system allows for assessment of the effect of microglial-derived soluble signals on cortical cell viability, proliferation, and stages of differentiation during homeostasis or following mechanical injury. These data suggest that microglia cells can downregulate inflammatory cytokine production following activation by mechanical injury to enhance proliferation of new cells capable of neurogenesis via activation of AKT intracellular signaling. Increasing our understanding of the mechanisms that drive microglial-enhanced cortical neurogenesis during homeostasis and following injury in vitro will provide useful information for future primary cell and in vivo studies.

m6A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation.

N6­methyladenosine (m6A) RNA modification maintained by N6­methyltransferases and demethylases is involved in multiple biological functions. Methyltransferase like 3 (METTL3) is a major N6­methyltransferase. However, the role of METTL3 and its installed m6A modification in colorectal tumorigenesis remains to be fully elucidated. METTL3 is highly expressed as indicated in colorectal cancer samples in the TCGA and Oncomine databases, implying its potential role in colon tumorigenesis. SW480 cell line with stable METTL3 knockout (METTL3­KO) was generated using CRISPR/Cas9 and were confirmed by the loss of METTL3 expression and suppression of m6A modification. The proliferation of METTL3­KO cells was significantly inhibited compared with that of control cells. METTL3­KO decreased the decay rate of suppressor of cytokine signaling 2 (SOCS2) RNA, resulting in elevated SOCS2 protein expression. m6A­RNA immunoprecipitation­qPCR (MeRIP­qPCR) revealed that SOCS2 mRNA was targeted by METTL3 for m6A modification. Similar to METTL3­KO SW480 cells, SW480 cells treated with 3­deazaadenosine, an RNA methylation inhibitor, exhibited elevated SOCS2 protein expression. Increased levels of SOCS2 in METTL3­KO SW480 cells were associated with decreased expression of leucine­rich repeat­containing G protein­coupled receptor 5 (LGR5), contributing to the inhibition of cell proliferation. The underlying associations among METTL3, SOCS2, and LGR5 were further confirmed in SW480 cells transfected with si­METTL3 and in tumor samples from patients with colorectal cancer. Taken together, our data demonstrate that an increased level of METTL3 may maintain the tumorigenicity of colon cancer cells by suppressing SOCS2.

Use of miRNAs to Study Host Cell-Parasite Interactions.

MicroRNAs (miRNAs) represent a subclass of endogenous small noncoding RNAs that have been identified in both mammalian and nonmammalian cells. miRNAs are an essential part of the complex regulatory networks that control numerous biological processes and may play an important role in host defense and/or microbial offense during host-parasite interactions. Here, several methodologies to explore the role for miRNAs in host-parasite interactions are briefly summarized, including the detection, quantification, and intracellular localization of miRNAs, identification and validation of miRNA targets, and functional manipulation of specific miRNAs.

Induction of Inflammatory Responses in Splenocytes by Exosomes Released from Intestinal Epithelial Cells following Cryptosporidium parvum Infection.

Cryptosporidium, a protozoan parasite that infects the gastrointestinal epithelium and other mucosal surfaces in humans and animals, is an important opportunistic pathogen in AIDS patients and one of the most common enteric pathogens affecting young children in developing regions. This parasite is referred to as a "minimally invasive" mucosal pathogen, and epithelial cells play a central role in activating and orchestrating host immune responses. We previously demonstrated that Cryptosporidium parvum infection stimulates host epithelial cells to release exosomes, and these released exosomes shuttle several antimicrobial peptides to carry out anti-C. parvum activity. In this study, we detected the upregulation of inflammatory genes in the liver and spleen following C. parvum intestinal infection in neonatal mice. Interestingly, exosomes released from intestinal epithelial cells following C. parvum infection could activate the nuclear factor kappa B signaling pathway and trigger inflammatory gene transcription in isolated primary splenocytes. Several epithelial cell-derived proteins and a subset of parasite RNAs were detected in the exosomes released from C. parvum-infected intestinal epithelial cells. Shuttling of these effector molecules, including the high mobility group box 1 protein, was involved in the induction of inflammatory responses in splenocytes induced by the exosomes released from infected cells. Our data indicate that exosomes released from intestinal epithelial cells upon C. parvum infection can activate immune cells by shuttling various effector molecules, a process that may be relevant to host systemic responses to Cryptosporidium infection.

Attenuation of Intestinal Epithelial Cell Migration During Cryptosporidium parvum Infection Involves Parasite Cdg7_FLc_1030 RNA-Mediated Induction and Release of Dickkopf-1.

Intestinal infection by Cryptosporidium is known to cause epithelial cell migration disorder but the underlying mechanisms are unclear. Previous studies demonstrated that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected epithelial cells. Using multiple models of intestinal cryptosporidiosis, we report here that C. parvum infection induces expression and release of the dickkopf protein 1 (Dkk1) from intestinal epithelial cells. Delivery of parasite Cdg7_FLc_1030 RNA to intestinal epithelial cells triggers transactivation of host Dkk1 gene during C. parvum infection. Release of Dkk1 is involved in C. parvum-induced inhibition of cell migration of epithelial cells, including noninfected bystander cells. Moreover, Dkk1-mediated suppression of host cell migration during C. parvum infection involves inhibition of Cdc42/Par6 signaling. Our data support the hypothesis that attenuation of intestinal epithelial cell migration during Cryptosporidium infection involves parasite Cdg7_FLc_1030 RNA-mediated induction and release of Dkk1 from infected cells.

The role of VEGF in the diagnosis and treatment of malignant pleural effusion in patients with non­small cell lung cancer (Review).

Malignant pleural effusion (MPE) is a severe medical condition, which can result in breathlessness, pain, cachexia and reduced physical activity. It can occur in almost all types of malignant tumors; however, lung cancer is the most common cause of MPE, accounting for ~1/3 of clinical cases. Although there are numerous therapeutic approaches currently available for the treatment of MPE, none are fully effective and the majority can only alleviate the symptoms of the patients. Vascular endothelial growth factor (VEGF) has now been recognized as one of the most important regulatory factors in tumor angiogenesis, which participates in the entire process of tumor growth through its function to stimulate tumor angiogenesis, activate host vascular endothelial cells and promote malignant proliferation. Novel drugs targeting VEGF, including endostar and bevacizumab, have been developed and approved for the treatment of various tumors. Data from recent clinical studies have demonstrated that drugs targeting VEGF are effective and safe for the clinical management of MPE. Therefore, VEGF­targeting represents a promising novel strategy for the diagnosis and treatment of MPE. The present review summarized recent advances in the role of VEGF in the pathogenesis, diagnosis and clinical management of MPE in patients with non­small cell lung cancer.

Nuclear delivery of parasite Cdg2_FLc_0220 RNA transcript to epithelial cells during Cryptosporidium parvum infection modulates host gene transcription.

Intestinal infection by the zoonotic protozoan, Cryptosporidium parvum, causes significant alterations in the gene expression profile in host epithelial cells. The molecular mechanisms of how C. parvum may modulate host cell gene transcription and the pathological significance of such alterations are largely unclear. Previous studies demonstrate that a panel of parasite RNA transcripts are delivered into infected host cells and may modulate host gene transcription. Using in vitro models of intestinal cryptosporidiosis, in this study, we analyzed the impact of host delivery of C. parvum Cdg2_FLc_0220 RNA transcript on host gene expression profile. We found that alterations in host gene expression profile following C. parvum infection were partially associated with the nuclear delivery of Cdg2_FLc_0220. Specifically, we identified a total of 46 overlapping upregulated genes and 8 overlapping downregulated genes in infected cells and cells transfected with Full-Cdg2_FLc_0220. Trans-suppression of the DAZ interacting zinc finger protein 1 like (DZIP1L) gene, the top overlapping downregulated gene in host cells following C. parvum infection and cells transfected with Full-Cdg2_FLc_0220, was mediated by G9a, independent of PRDM1. Cdg2_FLc_0220-mediated trans-suppression of the DZIP1L gene was independent of H3K9 and H3K27 methylation. Data from this study provide additional evidence that delivery of C. parvum Cdg2_FLc_0220 RNA transcript in infected epithelial cells modulates the transcription of host genes, contributing to the alterations in the gene expression profile in host epithelial cells during C. parvum infection.

Involvement of Cryptosporidium parvum Cdg7_FLc_1000 RNA in the Attenuation of Intestinal Epithelial Cell Migration via Trans-Suppression of Host Cell SMPD3.

Intestinal infection by Cryptosporidium parvum causes inhibition of epithelial turnover, but underlying mechanisms are unclear. Previous studies demonstrate that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected epithelial cells. Using in vitro and in vivo models of intestinal cryptosporidiosis, we report here that host delivery of parasite Cdg7_FLc_1000 RNA results in inhibition of epithelial cell migration through suppression of the gene encoding sphingomyelinase 3 (SMPD3). Delivery of Cdg7_FLc_1000 into infected cells promotes the histone methyltransferase G9a-mediated H3K9 methylation in the SMPD3 locus. The DNA-binding transcriptional repressor, PR domain zinc finger protein 1, is required for the assembly of Cdg7_FLc_1000 into the G9a complex and associated with the enrichment of H3K9 methylation at the gene locus. Pathologically, nuclear transfer of Cryptosporidium parvum Cdg7_FLc_1000 RNA is involved in the attenuation of intestinal epithelial cell migration via trans-suppression of host cell SMPD3.