LncRNA Nostrill promotes interferon-γ-stimulated gene transcription and facilitates intestinal epithelial cell-intrinsic anti-Cryptosporidium defense.

Intestinal epithelial cells possess the requisite molecular machinery to initiate cell-intrinsic defensive responses against intracellular pathogens, including intracellular parasites. Interferons(IFNs) have been identified as cornerstones of epithelial cell-intrinsic defense against such pathogens in the gastrointestinal tract. Long non-coding RNAs (lncRNAs) are RNA transcripts (>200 nt) not translated into protein and represent a critical regulatory component of mucosal defense. We report here that lncRNA Nostrill facilitates IFN-γ-stimulated intestinal epithelial cell-intrinsic defense against infection by Cryptosporidium, an important opportunistic pathogen in AIDS patients and a common cause of diarrhea in young children. Nostrill promotes transcription of a panel of genes controlled by IFN-γ through facilitating Stat1 chromatin recruitment and thus, enhances expression of several genes associated with cell-intrinsic defense in intestinal epithelial cells in response to IFN-γ stimulation, including Igtp, iNos, and Gadd45g. Induction of Nostrill enhances IFN-γ-stimulated intestinal epithelial defense against Cryptosporidium infection, which is associated with an enhanced autophagy in intestinal epithelial cells. Our findings reveal that Nostrill enhances the transcription of a set of genes regulated by IFN-γ in intestinal epithelial cells. Moreover, induction of Nostrill facilitates the IFN-γ-mediated epithelial cell-intrinsic defense against cryptosporidial infections.

Cryptosporidium parvum hijacks a host's long noncoding RNA U90926 to evade intestinal epithelial cell-autonomous antiparasitic defense.

Cryptosporidium is a zoonotic apicomplexan parasite that infects the gastrointestinal epithelium and other mucosal surfaces in humans. It is an important opportunistic pathogen in AIDS patients and a leading cause of infectious diarrhea and diarrheal-related death in children worldwide. The intestinal epithelial cells provide the first line of defense against Cryptosporidium infection and play a central role in activating and regulating the host's antiparasitic response. Increasing evidence suggests that long noncoding RNAs (lncRNAs) participate in host-pathogen interactions and play a regulatory role in the pathogenesis of diseases but the underlying molecular mechanisms are not fully understood. We previously identified a panel of host lncRNAs that are upregulated in murine intestinal epithelial cells following Cryptosporidium infection, including U90926. We demonstrate here that U90926 is acting in a pro-parasitic manner in regulating intestinal epithelial cell-autonomous antiparasitic defense. Inhibition of U90926 resulted in a decreased infection burden of the parasite while overexpression of U90926 showed an increase in infection burden in cultured murine intestinal epithelial cells. Induction of U90926 suppressed transcription of epithelial defense genes involved in controlling Cryptosporidium infection through epigenetic mechanisms. Specifically, transcription of Aebp1, which encodes the Aebp1 protein, a potent modulator of inflammation and NF-κB signaling, was suppressed by U90926. Gain- or loss-of-function of Aebp1 in the host's epithelial cells caused reciprocal alterations in the infection burden of the parasite. Interestingly, Cryptosporidium carries the Cryptosporidium virus 1 (CSpV1), a double-stranded (ds) RNA virus coding two dsRNA fragments, CSpV1-dsRdRp and CSpV1-dsCA. Both CSpV1-dsRdRp and CSpV1-dsCA can be delivered into infected cells as previously reported. We found that cells transfected with in vitro transcribed CSpV1-dsCA or CSpV1-dsRdRp displayed an increased level of U90926, suggesting that CSpV1 is involved in the upregulation of U90926 during Cryptosporidium infection. Our study highlights a new strategy by Cryptosporidium to hijack a host lncRNA to suppress epithelial cell-autonomous antiparasitic defense and allow for a robust infection.

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.

m6A mRNA Methylation Regulates Epithelial Innate Antimicrobial Defense Against Cryptosporidial Infection.

Increasing evidence supports that N6-methyladenosine (m6A) mRNA modification may play an important role in regulating immune responses. Intestinal epithelial cells orchestrate gastrointestinal mucosal innate defense to microbial infection, but underlying mechanisms are still not fully understood. In this study, we present data demonstrating significant alterations in the topology of host m6A mRNA methylome in intestinal epithelial cells following infection by Cryptosporidium parvum, a coccidian parasite that infects the gastrointestinal epithelium and causes a self-limited disease in immunocompetent individuals but a life-threatening diarrheal disease in AIDS patients. Altered m6A methylation in mRNAs in intestinal epithelial cells following C. parvum infection is associated with downregulation of alpha-ketoglutarate-dependent dioxygenase alkB homolog 5 and the fat mass and obesity-associated protein with the involvement of NF-кB signaling. Functionally, m6A methylation statuses influence intestinal epithelial innate defense against C. parvum infection. Specifically, expression levels of immune-related genes, such as the immunity-related GTPase family M member 2 and interferon gamma induced GTPase, are increased in infected cells with a decreased m6A mRNA methylation. Our data support that intestinal epithelial cells display significant alterations in the topology of their m6A mRNA methylome in response to C. parvum infection with the involvement of activation of the NF-кB signaling pathway, a process that modulates expression of specific immune-related genes and contributes to fine regulation of epithelial antimicrobial defense.

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.

Knockdown of m6A methyltransferase METTL3 in gastric cancer cells results in suppression of cell proliferation.

N6-methyladenosine (m6A) RNA modification regulates multiple biological functions. Methyltransferase like 3 (METTL3), one of the major N6-methyltransferases, is highly expressed in gastric cancer, but its potential role in disease is unclear. The current study knocked out METTL3 (METTL3-KO) in human gastric cancer AGS cells using CRISPR/Cas9. METTL3-KO AGS cells exhibited decreased m6A methylation levels. A significant inhibition of cell proliferation was observed in METTL3-KO AGS cells. Silencing METTL3 in AGS cells altered the expression profile of many effector molecules that were previously demonstrated to serve key roles in AGS cell proliferation, including the suppressor of cytokine signaling (SOCS) family of proteins. The results further demonstrated that SOCS2 upregulation in METTL3-KO AGS cells was associated with a decreased RNA decay rate. Furthermore, SOCS2 KO or SOCS2 overexpression caused a significant increase and decrease in AGS cell proliferation, respectively. The current data suggested that METTL3-KO in gastric cancer cells resulted in the suppression of cell proliferation by inducing SOCS2, suggesting a potential role of elevated METTL3 expression in gastric cancer progression.

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.

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.

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.

Trans-suppression of host CDH3 and LOXL4 genes during Cryptosporidium parvum infection involves nuclear delivery of parasite Cdg7_FLc_1000 RNA.

Intestinal infection by Cryptosporidium parvum causes significant alterations in the gene expression profile in host epithelial cells. Previous studies demonstrate that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected host cells and may modulate host gene transcription. Using in vitro models of human intestinal cryptosporidiosis, we report here that trans-suppression of the cadherin 3 (CDH3) and lysyl oxidase like 4 (LOXL4) genes in human intestinal epithelial cells following C. parvum infection involves host delivery of the Cdg7_FLc_1000 RNA, a C. parvum RNA that has been previously demonstrated to be delivered into the nuclei of infected host cells. Downregulation of CDH3 and LOXL4 genes was detected in host epithelial cells following C. parvum infection or in cells expressing the parasite Cdg7_FLc_1000 RNA. Knockdown of Cdg7_FLc_1000 attenuated the trans-suppression of CDH3 and LOXL4 genes in host cells induced by infection. Interestingly, Cdg7_FLc_1000 was detected to be recruited to the promoter regions of both CDH3 and LOXL4 gene loci in host cells following C. parvum infection. Host delivery of Cdg7_FLc_1000 promoted the PH domain zinc finger protein 1 (PRDM1)-mediated H3K9 methylation associated with trans-suppression in the CDH3 gene locus, but not the LOXL4 gene. Therefore, our data suggest that host delivery of Cdg7_FLc_1000 causes CDH3 trans-suppression in human intestinal epithelial cells following C. parvum infection through PRDM1-mediated H3K9 methylation in the CDH3 gene locus, whereas Cdg7_FLc_1000 induces trans-suppression of the host LOXL4 gene through H3K9/H3K27 methylation-independent mechanisms.

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.

Delivery of parasite Cdg7_Flc_0990 RNA transcript into intestinal epithelial cells during Cryptosporidium parvum infection suppresses host cell gene transcription through epigenetic mechanisms.

Cryptosporidial infection causes dysregulated transcription of host genes key to intestinal epithelial homeostasis, but the underlying mechanisms remain obscure. Previous studies demonstrate that several Cryptosporidium parvum (C. parvum) RNA transcripts are selectively delivered into epithelial cells during host cell invasion and may modulate gene transcription in infected cells. We report here that C. parvum infection suppresses the transcription of LRP5, SLC7A8, and IL33 genes in infected intestinal epithelium. Trans-suppression of these genes in infected host cells is associated with promoter enrichment of suppressive epigenetic markers (i.e., H3K9me3). Cdg7_FLc_0990, a C. parvum RNA that has previously demonstrated to be delivered into the nuclei of infected epithelial cells, is recruited to the promoter regions of LRP5, SLC7A8, and IL33 genes. Cdg7_FLc_0990 appears to be recruited to their promoter regions together with G9a, a histone methyltransferase for H3K9 methylation. The PR domain zinc finger protein 1, a G9a-interacting protein, is required for the assembly of Cdg7_FLc_0990 to the G9a complex and gene-specific enrichment of H3K9 methylation. Our data demonstrate that cryptosporidial infection induces epigenetic histone methylations in infected cells through nuclear transfer of parasite Cdg7_Flc_0990 RNA transcript, resulting in transcriptional suppression of the LRP5, SLC7A8, and IL33 genes.

A long noncoding RNA, lincRNA-Tnfaip3, acts as a coregulator of NF-κB to modulate inflammatory gene transcription in mouse macrophages.

Long intergenic noncoding RNAs (lincRNAs) are long noncoding transcripts (>200 nt) from the intergenic regions of annotated protein-coding genes. We report here that the lincRNA gene lincRNA-Tnfaip3, located at mouse chromosome 10 proximal to the tumor necrosis factor α-induced protein 3 (Tnfaip3) gene, is an early-primary response gene controlled by nuclear factor-κB (NF-κB) signaling in murine macrophages. Functionally, lincRNA- Tnfaip3 appears to mediate both the activation and repression of distinct classes of inflammatory genes in macrophages. Specifically, induction of lincRNA-Tnfaip3 is required for the transactivation of NF-κB-regulated inflammatory genes in response to bacterial LPSs stimulation. LincRNA-Tnfaip3 physically interacts with the high-mobility group box 1 (Hmgb1), assembling a NF-κB/Hmgb1/lincRNA-Tnfaip3 complex in macrophages after LPS stimulation. This resultant NF-κB/Hmgb1/lincRNA-Tnfaip3 complex can modulate Hmgb1-associated histone modifications and, ultimately, transactivation of inflammatory genes in mouse macrophages in response to microbial challenge. Therefore, our data indicate a new regulatory role of NF-κB-induced lincRNA-Tnfaip3 to act as a coactivator of NF-κB for the transcription of inflammatory genes in innate immune cells through modulation of epigenetic chromatin remodeling.-Ma, S., Ming, Z., Gong, A.-Y., Wang, Y., Chen, X., Hu, G., Zhou, R., Shibata, A., Swanson, P. C., Chen, X.-M. A long noncoding RNA, LincRNA-Tnfaip3, acts as a coregulator of NF-κB to modulate inflammatory gene transcription in mouse macrophages.

Delivery of Parasite RNA Transcripts Into Infected Epithelial Cells During Cryptosporidium Infection and Its Potential Impact on Host Gene Transcription.

Cryptosporidium parvum is an important opportunistic parasite pathogen for immunocompromised individuals and a common cause of diarrhea in young children. Previous studies have identified a panel of RNA transcripts of very low protein-coding potential in C. parvum. Using an in vitro model of human intestinal cryptosporidiosis, we report here that some of these C. parvum RNA transcripts were selectively delivered into the nuclei of host epithelial cells during C. parvum infection. Nuclear delivery of several such parasitic RNAs, including Cdg7_FLc_0990, involved heat-shock protein 70-mediated nuclear importing mechanism. Overexpression of Cdg7_FLc_0990 in intestinal epithelial cells resulted in significant changes in expression levels of specific genes, with significant overlapping with alterations in gene expression profile detected in host cells after C. parvum infection. Our data demonstrate that C. parvum transcripts of low protein-coding potential are selectively delivered into epithelial cells during infection and may modulate gene transcription in infected host cells.

LincRNA-Cox2 modulates TNF-α-induced transcription of Il12b gene in intestinal epithelial cells through regulation of Mi-2/NuRD-mediated epigenetic histone modifications.

Long intergenic noncoding RNAs (lincRNAs) can regulate the transcription of inflammatory genes and thus may represent a new group of inflammatory mediators with a potential pathogenic role in inflammatory diseases. Here, our genome-wide transcriptomic data show that TNF-α stimulation caused up-regulation of 171 lincRNAs and down-regulation of 196 lincRNAs in murine intestinal epithelial cells in culture. One of the up-regulated lincRNAs, lincRNA-Cox2, is an early-responsive lincRNA induced by TNF-α through activation of the NF-ĸB signaling pathway. Knockdown of lincRNA-Cox2 resulted in reprogramming of the gene expression profile in intestinal epithelial cells in response to TNF-α stimulation. Specifically, lincRNA-Cox2 silencing significantly (P < 0.05) enhanced the transcription of Il12b, a secondary late-responsive gene induced by TNF-α. Mechanistically, lincRNA-Cox2 promoted the recruitment of the Mi-2/nucleosome remodeling and deacetylase (Mi-2/NuRD) repressor complex to the Il12b promoter region. Recruitment of the Mi-2/NuRD complex was associated with decreased H3K27 acetylation and increased H3K27 dimethylation at the Il12b promoter region, which might contribute to Il12b trans-suppression by lincRNA-Cox2. Thus, our data demonstrate a novel mechanism of epigenetic modulation by lincRNA-Cox2 on Il12b transcription, supporting an important role for lincRNAs in the regulation of intestinal epithelial inflammatory responses.

Cryptosporidium parvum induces SIRT1 expression in host epithelial cells through downregulating let-7i.

Epithelial cells along human gastrointestinal mucosal surface express pathogen-recognizing receptors and actively participate in the regulation of inflammatory reactions in response to microbial infection. The NAD-dependent deacetylase sirtuin-1 (SIRT1), one member of the sirtuin family of proteins and an NAD-dependent deacetylase has been implicated in the regulation of multiple cellular processes, including inflammation, longevity, and metabolism. In this study, we demonstrated that infection of cultured human biliary epithelial cells (H69 cholangiocytes) with a parasitic protozoan, Cryptosporidium parvum, induced SIRT1 expression at the protein level without a change in SIRT1 mRNA content. Using real-time PCR and Northern blot analyses, we found that C. parvum infection decreased the expression of let-7i in infected H69 cells. Down-regulation of let-7i caused relief of miRNA-mediated translational suppression of SIRT1 and consequently, resulted in an increased SIRT1 protein level in infected H69 cell cultures. Moreover, gain- and loss-of-function studies revealed that let-7i could modulate NF-κB activation through modification of SIRT1 protein expression. Thus, our data suggest that let-7i regulates SIRT1 expression in human biliary epithelial cells in response to microbial challenge, suggesting a new role of let-7i in the regulation of NF-κB-mediated epithelial innate immune response.

Histone deacetylases and NF-kB signaling coordinate expression of CX3CL1 in epithelial cells in response to microbial challenge by suppressing miR-424 and miR-503.

The NF-kB pathway is key to epithelial immune defense and has been implicated in secretion of antimicrobial peptides, release of cytokines/chemokines to mobilize immune effector cells, and activation of adaptive immunity. The expression of many inflammatory genes following infection involves the remodeling of the chromatin structure. We reported here that histone deacetylases (HDACs) and NF-kB signaling coordinate expression of CX3CL1 in epithelial cells following Cryptosporidium parvum infection. Upregulation of CX3CL1 was detected in cultured human biliary epithelial cells following infection. Expression of miR-424 and miR-503 was downregulated, and was involved in the induction of CX3CL1 in infected cells. C. parvum infection suppressed transcription of the mir-424-503 gene in a NF-kB- and HDAC-dependent manner. Increased promoter recruitment of NF-kB p50 and HDACs, and decreased promoter H3 acetylation associated with the mir-424-503 gene were observed in infected cells. Upregulation of CX3CL1 in biliary epithelial cells and increased infiltration of CX3CR1(+) cells were detected during C. parvum infection in vivo. Induction of CX3CL1 and downregulation of miR-424 and miR-503 were also detected in epithelial cells in response to LPS stimulation. The above results indicate that HDACs and NF-kB signaling coordinate epithelial expression of CX3CL1 to promote mucosal antimicrobial defense through suppression of the mir-424-503 gene.

Phenethyl isothiocyanate inhibits androgen receptor-regulated transcriptional activity in prostate cancer cells through suppressing PCAF.

SCOPE: Androgen receptor (AR) signaling is critical for all aspects of prostate growth and tumorigenesis. The glucosinolate-derived phenethyl isothiocyanate (PEITC) has recently been demonstrated to reduce the risk of prostate cancer (PCa) and inhibit PCa cell growth. We previously reported that p300/CBP-associated factor (PCAF), a co-regulator for AR, is upregulated in PCa cells through suppression of the mir-17 gene. Here, we assessed the effects of PEITC on PCAF expression and AR-regulated transcriptional activity in PCa cells. METHODS AND RESULTS: Using AR-responsive LNCaP cells, we observed the inhibitory effects of PEITC on the dihydrotestosterone-stimulated AR transcriptional activity and cell growth of PCa cells. Interestingly, overexpression of PCAF attenuated the inhibitory effects of PEITC on dihydrotestosterone-stimulated AR transcriptional activity. Expression of PCAF was upregulated in PCa cells through suppression of miR-17. PEITC treatment significantly decreased PCAF expression and promoted transcription of miR-17 in LNCaP cells. Functional inhibition of miR-17 attenuated the suppression of PCAF in cells treated by PEITC. CONCLUSION: Our results indicate that PEITC inhibits AR-regulated transcriptional activity and cell growth of PCa cells through miR-17-mediated suppression of PCAF, suggesting a new mechanism by which PEITC modulates PCa cell growth.

Release of luminal exosomes contributes to TLR4-mediated epithelial antimicrobial defense.

Exosomes are membranous nanovesicles released by most cell types from multi-vesicular endosomes. They are speculated to transfer molecules to neighboring or distant cells and modulate many physiological and pathological procedures. Exosomes released from the gastrointestinal epithelium to the basolateral side have been implicated in antigen presentation. Here, we report that luminal release of exosomes from the biliary and intestinal epithelium is increased following infection by the protozoan parasite Cryptosporidium parvum. Release of exosomes involves activation of TLR4/IKK2 signaling through promoting the SNAP23-associated vesicular exocytotic process. Downregulation of let-7 family miRNAs by activation of TLR4 signaling increases SNAP23 expression, coordinating exosome release in response to C. parvum infection. Intriguingly, exosomes carry antimicrobial peptides of epithelial cell origin, including cathelicidin-37 and beta-defensin 2. Activation of TLR4 signaling enhances exosomal shuttle of epithelial antimicrobial peptides. Exposure of C. parvum sporozoites to released exosomes decreases their viability and infectivity both in vitro and ex vivo. Direct binding to the C. parvum sporozoite surface is required for the anti-C. parvum activity of released exosomes. Biliary epithelial cells also increase exosomal release and display exosome-associated anti-C. parvum activity following LPS stimulation. Our data indicate that TLR4 signaling regulates luminal exosome release and shuttling of antimicrobial peptides from the gastrointestinal epithelium, revealing a new arm of mucosal immunity relevant to antimicrobial defense.