Infection Of Intestinal Epithelial Cells Research Articles

Candida albicans translocation through the intestinal epithelial barrier is promoted by fungal zinc acquisition and limited by NFκB-mediated barrier protection.

The opportunistic fungal pathogen Candida albicans thrives on human mucosal surfaces as a harmless commensal, but frequently causes infections under certain predisposing conditions. Translocation across the intestinal barrier into the bloodstream by intestine-colonizing C. albicans cells serves as the main source of disseminated candidiasis. However, the host and microbial mechanisms behind this process remain unclear. In this study we identified fungal and host factors specifically involved in infection of intestinal epithelial cells (IECs) using dual-RNA sequencing. Our data suggest that host-cell damage mediated by the peptide toxin candidalysin-encoding gene ECE1 facilitates fungal zinc acquisition. This in turn is crucial for the full virulence potential of C. albicans during infection. IECs in turn exhibit a filamentation- and damage-specific response to C. albicans infection, including NFκB, MAPK, and TNF signaling. NFκB activation by IECs limits candidalysin-mediated host-cell damage and mediates maintenance of the intestinal barrier and cell-cell junctions to further restrict fungal translocation. This is the first study to show that candidalysin-mediated damage is necessary for C. albicans nutrient acquisition during infection and to explain how IECs counteract damage and limit fungal translocation via NFκB-mediated maintenance of the intestinal barrier.

Development and management of gastrointestinal symptoms in long-term COVID-19.

Emerging evidence reveals that SARS-CoV-2 possesses the capability to disrupt the gastrointestinal (GI) homeostasis, resulting in the long-term symptoms such as loss of appetite, diarrhea, gastroesophageal reflux, and nausea. In the current review, we summarized recent reports regarding the long-term effects of COVID-19 (long COVID) on the gastrointestine. To provide a narrative review of abundant clinical evidence regarding the development and management of long-term GI symptoms in COVID-19 patients. Long-term persistent digestive symptoms are exhibited in a majority of long-COVID patients. SARS-CoV-2 infection of intestinal epithelial cells, cytokine storm, gut dysbiosis, therapeutic drugs, psychological factors and exacerbation of primary underlying diseases lead to long-term GI symptoms in COVID-19 patients. Interventions like probiotics, prebiotics, fecal microbiota transplantation, and antibiotics are proved to be beneficial in preserving intestinal microecological homeostasis and alleviating GI symptoms. Timely diagnosis and treatment of GI symptoms in long-COVID patients hold great significance as they may contribute to the mitigation of severe conditions and ultimately lead to the improvement of outcomes of the patients.

Identification of differences in gene expression implicated in the Adherent-Invasive Escherichia coli phenotype during in vitro infection of intestinal epithelial cells.

Adherent-invasive Escherichia coli (AIEC) is strongly associated with the pathogenesis of Crohn's disease (CD). However, no molecular markers currently exist for AIEC identification. This study aimed to identify differentially expressed genes (DEGs) between AIEC and non-AIEC strains that may contribute to AIEC pathogenicity and to evaluate their utility as molecular markers. Comparative transcriptomics was performed on two closely related AIEC/non-AIEC strain pairs during Intestine-407 cell infection. DEGs were quantified by RT-qPCR in the same RNA extracts, as well as in 14 AIEC and 23 non-AIEC strains to validate the results across a diverse strain collection. Binary logistical regression was performed to identify DEGs whose quantification could be used as AIEC biomarkers. Comparative transcriptomics revealed 67 differences in expression between the two phenotypes in the strain pairs, 50 of which (81.97%) were corroborated by RT-qPCR. When explored in the whole strain collection, 29 DEGs were differentially expressed between AIEC and non-AIEC phenotypes (p-value < 0.042), and 42 genes between the supernatant fraction of infected cell cultures and the cellular fraction containing adhered and intracellular bacteria (p-value < 0.049). Notably, six DEGs detected in the strain collection were implicated in arginine biosynthesis and five in colanic acid synthesis. Furthermore, two biomarkers based on wzb and cueR gene expression were proposed with an accuracy of ≥ 85% in our strain collection. This is the first transcriptomic study conducted using AIEC-infected cell cultures. We have identified several genes that may be involved in AIEC pathogenicity, two of which are putative biomarkers for identification.

STAT1/MUC4 activation promotes antimicrobial peptide production to reduce intestinal epithelium barrier injury caused by enteropathogenic Escherichia coli infection

IntroductionAntimicrobial peptides (AMPs) are endogenous peptides that have been identified to alleviate intestinal epithelial barrier inflammation and dysfunction caused by enteropathogenic Escherichia coli (EPEC) infection; nonetheless, the upstream molecular mechanism of the production of AMPs is poorly understood.Material and methodsThe binding of signal transducer and activator of transcription (STAT) 1 (STAT1) to mucin 4 (MUC4) was examined by Co-Immunoprecipitation assay. To detect the influence of STAT1 and MUC4 expression, C57BL/6 mice model of EPEC infection in vivo and EPEC infected intestinal epithelial cells (IEC) in vitro model was established. Expressions of STAT1, MUC4, phosphorylated (p)-STAT1, proinflammatory cytokines, zonula occludens-1 (ZO-1) and AMP-related genes in mouse ileum and/or IEC were analyzed by immunohistochemical test, immunofluorescence assay, Western blot, and/or qRT-PCR. Meanwhile, IEC viability and apoptosis were measured using CCK-8 assay and flow cytometry.Resultsp-STAT1, MUC4, ZO-1 and AMP-related gene were lowly expressed in the ileum of EPEC-infected mice. p-STAT1 and MUC4 bound to each other. The expressions of STAT1 and MUC4 were decreased in EPEC-infected IEC. Overexpression of STAT1 resisted EPEC-induced viability decrease, apoptosis promotion, ZO-1 activity inhibition, release of proinflammatory cytokines, and downregulation of MUC4 and AMP-related genes in IEC. MUC4 knockdown partly counteracted the effect of STAT1 overexpression, but did not affect the forced STAT1 overexpression in EPEC-infected IEC.ConclusionsSTAT1/MUC4 pathway activation promotes AMP production to mitigate intestinal epithelium barrier injury caused by EPEC infection.

Giardia duodenalis-induced G0/G1 intestinal epithelial cell cycle arrest and apoptosis involve activation of endoplasmic reticulum stress in vitro.

Giardia duodenalis is a zoonotic intestinal protozoan parasite that may cause host diarrhea and chronic gastroenteritis, resulting in great economic losses annually and representing a significant public health burden across the world. However, thus far, our knowledge on the pathogenesis of Giardia and the related host cell responses is still extensively limited. The aim of this study is to assess the role of endoplasmic reticulum (ER) stress in regulating G0/G1 cell cycle arrest and apoptosis during in vitro infection of intestinal epithelial cells (IECs) with Giardia. The results showed that the mRNA levels of ER chaperone proteins and ER-associated degradation genes were increased and the expression levels of the main unfolded protein response (UPR)-related proteins (GRP78, p-PERK, ATF4, CHOP, p-IRE1, XBP1s and ATF6) were increased upon Giardia exposure. In addition, cell cycle arrest was determined to be induced by UPR signaling pathways (IRE1, PERK and ATF6) through upregulation of p21 and p27 levels and promotion of E2F1-RB complex formation. Upregulation of p21 and p27 expression was shown to be related to Ufd1-Skp2 signaling. Therefore, the cell cycle arrest was induced by ER stress when infected with Giardia. Furthermore, the apoptosis of the host cell was also assessed after exposure to Giardia. The results indicated that apoptosis would be promoted by UPR signaling (PERK and ATF6), but would be suppressed by the hyperphosphorylation of AKT and hypophosphorylation of JNK that were modulated by IRE1 pathway. Taken together, both of the cell cycle arrest and apoptosis of IECs induced by Giardia exposure involved the activation of the UPR signaling. The findings of this study will deepen our understanding of the pathogenesis of Giardia and the associated regulatory network.

Cytosolic galectin-4 enchains bacteria, restricts their motility, and promotes inflammasome activation in intestinal epithelial cells

Galectin-4, a member of the galectin family of animal glycan-binding proteins (GBPs), is specifically expressed in gastrointestinal epithelial cells and is known to be able to bind microbes. However, its function in host-gut microbe interactions remains unknown. Here, we show that intracellular galectin-4 in intestinal epithelial cells (IECs) coats cytosolic Salmonella enterica serovar Worthington and induces the formation of bacterial chains and aggregates. Galectin-4 enchains bacteria during their growth by binding to the O-antigen of lipopolysaccharides. Furthermore, the binding of galectin-4 to bacterial surfaces restricts intracellular bacterial motility. Galectin-4 enhances caspase-1 activation and mature IL-18 production in infected IECs especially when autophagy is inhibited. Finally, orally administered S. enterica serovar Worthington, which is recognized by human galectin-4 but not mouse galectin-4, translocated from the intestines to mesenteric lymph nodes less effectively in human galectin-4-transgenic mice than in littermate controls. Our results suggest that galectin-4 plays an important role in host-gut microbe interactions and prevents the dissemination of pathogens. The results of the study revealed a novel mechanism of host-microbe interactions that involves the direct binding of cytosolic lectins to glycans on intracellular microbes.

High yield production of norovirus GII.4 virus-like particles using silkworm pupae and evaluation of their protective immunogenicity.

Noroviruses (NoVs) are one of the major causes of acute viral gastroenteritis in humans. Virus-like particles (VLPs) without genomes that mimic the capsid structure of viruses are promising vaccine candidates for the prevention of NoVs infection. To produce large amounts of recombinant protein, including VLPs, the silkworm-expression vector system (silkworm-BEVS) is an efficient and powerful tool. In this study, we constructed a recombinant baculovirus that expresses VP1 protein, the major structural protein of NoV GII.4. Expression analysis showed that the baculovirus-infected silkworm pupae expressed NoV VP1 protein more efficiently than silkworm larval fat bodies. We obtained about 4.9mg of purified NoV VP1 protein from only five silkworm pupae. The purified VP1 protein was confirmed by dynamic light scattering and electron microscopy to form VLPs of approximately 40nm in diameter. Antisera from mice immunized with the antigen blocked NoV VLPs binding to histo-blood group antigens of pig gastric mucin and also blocked NoV infection in intestinal epithelial cells derived from human induced pluripotent stem (iPS) cells. Our findings demonstrated that NoV VLP eliciting protective antibodies could be obtained in milligram quantities from a few silkworm pupae using the silkworm-BEVS.

MiR-615 facilitates porcine epidemic diarrhea virus replication by targeting IRAK1 to inhibit type III interferon expression.

Porcine epidemic diarrhea virus (PEDV) in the Coronavirus family is a highly contagious enteric pathogen in the swine industry, which has evolved mechanisms to evade host innate immune responses. The PEDV-mediated inhibition of interferons (IFNs) has been linked to the nuclear factor-kappa B (NF-κB) pathway. MicroRNAs (miRNAs) are involved in virus-host interactions and IFN-I regulation. However, the mechanism by which the PEDV regulates IFN during PEDV infection has not yet been investigated in its natural target cells. We here report a novel mechanism of viral immune escape involving miR-615, which was screened from a high-throughput sequencing library of porcine intestinal epithelial cells (IECs) infected with PEDV. PEDV infection altered the profiles of miRNAs and the activities of several pathways involved in innate immunity. Overexpression of miR-615 increased PEDV replication, inhibited IFN expression, downregulated the NF-κB pathway, and blocked p65 nuclear translocation. In contrast, knockdown of miR-615 enhanced IFN expression, suppressed PEDV replication, and activated the NF-κB pathway. We further determined that IRAK1 is the target gene of miR-615 in IECs. Our findings show that miR-615 suppresses activation of the NF-κB pathway by suppressing the IRAK1 protein and reducing the generation of IFN-IIIs, which in turn facilitates PEDV infection in IECs. Moreover, miR-615 inhibited PEDV replication and NF-κB pathway activation in both IECs and MARC-145 cells. These findings support an important role for miR-615 in the innate immune regulation of PEDV infections and provide a novel perspective for developing new treatments.

Development of Toxoplasma gondii Chinese I genotype Wh6 Strain in Cat Intestinal Epithelial Cells.

Felids are the unique definitive host of Toxoplasma gondii. The intestine of felid is the only site for initiating Toxoplasma gondii sexual reproduction. T. gondii excretes millions of infectious oocysts from the intestine, which are the primary source of infection. There are many difficulties in developing vaccines and drugs to control oocyst excretion due to the lack of an appropriate experimental model. Here, we established an in vitro feline intestinal epithelial cell (IEC) infection system and an efficient animal model of T. gondii Chinese 1 genotype, Wh6 strain (TgCtwh6). The Kunming mice brain tissues containing TgCtwh6 cysts were harvested 42-day post-infection. The bradyzoites were co-cultured with cat IECs in vitro at a ratio of 1:10. Five 3-month-old domestic cats were orally inoculated with 600 cysts each. The oocysts were detected by daily observation of cat feces by microscopy and polymerase chain reaction. We found that the parasite adhered and invaded cat IECs in vitro, transformed into tachyzoites, and then divided to form rose-like structures. These parasites eventually destroyed host cells, escaped, and finished the asexual reproduction process. Schizonts associated with sexual reproduction have not been observed during development in vitro cultured cells. However, schizonts were detected in all infected cat intestinal epithelial cells, and oocysts were presented in all cat feces. Our study provides a feasible cell model and an efficient infection system for the following studies of T. gondii sexual reproduction, and also lays a foundation to develop drugs and vaccines for blocking excretion and transmission of oocysts.

The miseducation of T cells in autoimmunity

Abstract Celiac disease (CeD) is an immune-mediated enteropathy characterized by an inflammatory T-helper type 1 (Th1) immune response against dietary gluten. Although 40% of individuals in the U.S. have the genetic factors required for the development of CeD, less than 1% will be diagnosed with CeD. This suggests that additional environmental factors are necessary for the development of CeD. Our previous work demonstrates that viral infections can disrupt tolerance to oral antigens, leading to immune activation as seen in CeD. However, the mechanisms by which viral infections trigger inflammatory responses to dietary antigen remains unknown. Here we show that upon infection with reovirus, a subset of migratory dendritic cells, cDC1, sense and uptake virally infected and necroptotic intestinal epithelial cells and secrete type I interferon (IFN), resulting in a dietary antigen specific Th1 immune response. Our results identify a novel mechanism by which certain viruses can trigger inflammatory immune responses to dietary antigen. We also demonstrate that only a subset of enteric viruses can disrupt homeostatic response to dietary antigens. Only those that both induce type I IFN, and cause necroptosis will result in such pathology. This data identifies specific viral targets for clinical intervention via vaccine that could potentially reduce the incidence of CeD and other dietary-triggered immune enteropathies. More generally, knowledge from our work enhance our understanding of how viral-host interactions drive autoimmune disorders at the mucosal interface. Supported by grant from NIH (RO1 5R01DK098435-06)

The Long Non-Coding RNA Nostrill Regulates Transcription of Irf7 Through Interaction With NF-κB p65 to Enhance Intestinal Epithelial Defense Against Cryptosporidium parvum.

The cells of the intestinal epithelium establish the frontline for host defense against pathogens in the gastrointestinal tract and play a vital role in the initiation of the immune response. Increasing evidence supports the role of long non-coding RNAs (lncRNAs) as critical regulators of diverse cellular processes, however, their role in antimicrobial host defense is incompletely understood. In this study, we provide evidence that the lncRNA Nostrill is upregulated in the intestinal epithelium following infection by Cryptosporidium parvum, a globally prevalent apicomplexan parasite that causes significant diarrheal disease and an important opportunistic pathogen in the immunocompromised and AIDS patients. Induction of Nostrill in infected intestinal epithelial cells was triggered by NF-κB signaling and was observed to enhance epithelial defense by decreasing parasitic infection burden. Nostrill participates in the transcriptional regulation of C. parvum-induced Irf7 expression through interactions with NF-κB p65, and induction of Nostrill promotes epigenetic histone modifications and occupancy of RNA polymerase II at the Irf7 promoter. Our data suggest that the induction of Nostrill promotes antiparasitic defense against C. parvum and enhances intestinal epithelial antimicrobial defense through contributions to transcriptional regulation of immune-related genes, such as Irf7.

Lactobacillus crispatus Strain KT-11 S-Layer Protein Inhibits Rotavirus Infection.

S-layer proteins (SLPs), which are present in the external layer of certain strains of lactic acid bacteria isolated from the intestinal tract, are known to recognize and bind to specific proteins and glycan structures and contribute to adsorption to the host intestinal mucosa. The binding properties of certain SLPs are considered to exert a competitive inhibitory effect on infection because similar properties are involved in the infection mechanisms of several viruses. However, little is known regarding whether SLPs directly inhibit viral infection. In the present study, we investigated the effect of an SLP of the Lactobacillus crispatus KT-11 strain, a probiotic strain isolated from a healthy human infant, on human rotavirus infection. The impact of KT-11 lithium chloride extract (KT-11 LE), which contains SLP, on the infection of the P[4] genotype human rotavirus strain DS-1 was evaluated by monitoring the amplification of viral protein 6 (VP6) expression in human intestinal epithelial Caco-2 cells by quantitative reverse transcription-polymerase chain reaction assay after infection. KT-11 LE showed a significant suppressive effect on DS-1 infection in a dose-dependent manner with pre-infection treatment, whereas post-infection treatment was not effective. A 45 KDa protein isolated from KT-11 LE was investigated for homology using the BLAST database and was found to be a novel SLP. KT-11 SLP concentrate (KT-11 SLP) significantly inhibited the proliferative process of the DS-1 strain but not that of the P[8] genotype human rotavirus strain Wa. KT-11 SLP exerted significant inhibitory effect on DS-1 infection by pre-infection treatment even after digestion with gastric juice up to 2 h. Our results provided crucial evidence that SLPs from certain Lactobacillus strains can inhibit human rotavirus infection of intestinal epithelial cells.

Attachment of Enterohemorrhagic Escherichia coli to Host Cells Reduces O Antigen Chain Length at the Infection Site That Promotes Infection.

ABSTRACTMany enteropathogenic bacteria express a needle-like type III secretion system (T3SS) that translocates effectors into host cells promoting infection. O antigen (OAg) constitutes the outer layer of Gram-negative bacteria protecting bacteria from host immune responses. Shigella constitutively shortens the OAg molecule in its three-dimensional conformation by glucosylation, leading to enhanced T3SS function. However, whether and how other enteropathogenic bacteria shorten the OAg molecule that probably facilitates infection remain unknown. For the first time, we report a smart mechanism by which enterohemorrhagic Escherichia coli specifically reduces the size of the OAg molecule at the infection site upon sensing mechanical signals of intestinal epithelial cell attachment via the membrane protein YgjI. YgjI represses expression of the OAg chain length regulator gene fepE via the global regulator H-NS, leading to shortened OAg chains and injection of more T3SS effectors into host cells. However, bacteria express long-chain OAg in the intestinal lumen benefiting their survival. Animal experiments show that blocking this regulatory pathway significantly attenuates bacterial virulence. This finding enhances our understanding of interactions between the surfaces of bacterial and host cells and the way this interaction enhances bacterial pathogenesis.

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

ABSTRACTInterferon (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.

Human small intestinal infection by SARS-CoV-2 is characterized by a mucosal infiltration with activated CD8+ T cells

The SARS-CoV-2 pandemic has so far claimed over three and a half million lives worldwide. Though the SARS-CoV-2 mediated disease COVID-19 has first been characterized by an infection of the upper airways and the lung, recent evidence suggests a complex disease including gastrointestinal symptoms. Even if a direct viral tropism of intestinal cells has recently been demonstrated, it remains unclear, whether gastrointestinal symptoms are caused by direct infection of the gastrointestinal tract by SARS-CoV-2 or whether they are a consequence of a systemic immune activation and subsequent modulation of the mucosal immune system. To better understand the cause of intestinal symptoms we analyzed biopsies of the small intestine from SARS-CoV-2 infected individuals. Applying qRT-PCR and immunohistochemistry, we detected SARS-CoV-2 RNA and nucleocapsid protein in duodenal mucosa. In addition, applying imaging mass cytometry and immunohistochemistry, we identified histomorphological changes of the epithelium, which were characterized by an accumulation of activated intraepithelial CD8+ T cells as well as epithelial apoptosis and subsequent regenerative proliferation in the small intestine of COVID-19 patients. In summary, our findings indicate that intraepithelial CD8+ T cells are activated upon infection of intestinal epithelial cells with SARS-CoV-2, providing one possible explanation for gastrointestinal symptoms associated with COVID-19.

Gastrointestinal Perspective of Coronavirus Disease 2019 in Children-An Updated Review.

ABSTRACTGastrointestinal symptoms are common findings in children with severe acute respiratory syndrome coronavirus 2 infection, including vomiting, diarrhoea, abdominal pain, and difficulty in feeding, although these symptoms tend to be mild. The hepato-biliary system and the pancreas may also be involved, usually with a mild elevation of transaminases and, rarely, pancreatitis. In contrast, a late hyper-inflammatory phenomenon, termed multisystem inflammatory syndrome (MIS-C), is characterized by more frequent gastrointestinal manifestations with greater severity, sometimes presenting as peritonitis. Gastrointestinal and hepato-biliary manifestations are probably related to a loss in enterocyte absorption capability and microscopic mucosal damage caused by a viral infection of intestinal epithelial cells, hepatocytes and other cells through the angiotensin conversion enzyme 2 receptor resulting in immune cells activation with subsequent release of inflammatory cytokines. Specific conditions such as inflammatory bowel disease (IBD) and liver transplantation may pose a risk for the more severe presentation of coronavirus disease 2019 (COVID-19) but as adult data accumulate, paediatric data is still limited. The aim of this review is to summarize the current evidence about the effect of COVID-19 on the gastrointestinal system in children, with emphasis on the emerging MIS-C and specific considerations such as patients with IBD and liver transplant recipients.

Eimeria acervulina Microneme Protein 3 Inhibits Apoptosis of the Chicken Duodenal Epithelial Cell by Targeting the Casitas B-Lineage Lymphoma Protein.

Eimeria acervulina (E. acervulina) causes coccidiosis in poultry which persists as economic pain worldwide. Most damage to the intestinal mucosa results from apoptosis of the infected intestinal epithelial cells. The Microneme protein 3 (MIC3) protein is a key virulence factor in some parasites involved in host cell apoptosis inhibition. Here, we studied whether and how MIC3 affects the apoptosis in E. acervulina infected chicken duodenal epithelial cells. Through flow cytometry (FCM), we found that the presence of merozoites and the overexpression of MIC3 significantly decreased apoptosis and the activity of caspase-3 in chicken duodenal epithelial cells at 4, 6, and 8 h post merozoite infection (P < 0.01). Silencing the Casitas B-lineage lymphoma (CBL) protein, a host receptor for MIC3 with shRNA was shown to promote apoptosis in the chicken duodenal epithelial cells. The early apoptotic rate of host cells in the lentiviral-MIC3 group was significantly lower than that in the lentiviral-MIC3 + shRNA CBL group at 4 h after MIC3 expression (P < 0.01), and it was moderately decreased in the lentiviral-MIC3 + shRNA CBL group compared with that in the shRNA CBL group. Our data indicated that MIC3 inhibited early apoptosis of E. acervulina infected chicken duodenal epithelial cells by targeting host receptor-CBL protein. These findings unveiled one of the mechanisms of how intracellular parasites affect the apoptosis of infected host cells, which provided a deeper understanding of their pathogenesis.

LPS modifications and AvrA activity of Salmonella enterica serovar Typhimurium are required to prevent Perforin-2 expression by infected fibroblasts and intestinal epithelial cells

Cellular Perforin-2 (MPEG1) is a pore-forming MACPF family protein that plays a critical role in the defense against bacterial pathogens. Macrophages, neutrophils, and several other cell types that are part of the front line of innate defenses constitutively express high levels of Perforin-2; whereas, most other cell types must be induced to express Perforin-2 by interferons (α, β and γ) and/or PAMPs such as LPS. In this study, we demonstrate that many bacterial pathogens can limit the expression of Perforin-2 in cells normally inducible for Perforin-2 expression, while ordinarily commensal or non-pathogenic bacteria triggered high levels of Perforin-2 expression in these same cell types. The mechanisms by which pathogens suppress Perforin-2 expression was explored further using Salmonella enterica serovar Typhimurium and cultured MEFs as well as intestinal epithelial cell lines. These studies identified multiple factors required to minimize the expression of Perforin-2 in cell types inducible for Perforin-2 expression. These included the PmrAB and PhoPQ two-component systems, select LPS modification enzymes and the Type III secretion effector protein AvrA.

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.

CD300lf Conditional Knockout Mouse Reveals Strain-Specific Cellular Tropism of Murine Norovirus.

Noroviruses are a leading cause of gastrointestinal infection in humans and mice. Understanding human norovirus (HuNoV) cell tropism has important implications for our understanding of viral pathogenesis. Murine norovirus (MNoV) is extensively used as a surrogate model for HuNoV. We previously identified CD300lf as the receptor for MNoV. Here, we generated a Cd300lf conditional knockout (CD300lfF/F ) mouse to elucidate the cell tropism of persistent and nonpersistent strains of murine norovirus. Using this mouse model, we demonstrated that CD300lf expression on intestinal epithelial cells (IECs), and on tuft cells in particular, is essential for transmission of the persistent MNoV strain CR6 (MNoVCR6) in vivo In contrast, the nonpersistent MNoV strain CW3 (MNoVCW3) does not require CD300lf expression on IECs for infection. However, deletion of CD300lf in myelomonocytic cells (LysM Cre+) partially reduces CW3 viral load in lymphoid and intestinal tissues. Disruption of CD300lf expression on B cells (CD19 Cre), neutrophils (Mrp8 Cre), and dendritic cells (CD11c Cre) did not affect MNoVCW3 viral RNA levels. Finally, we show that the transcription factor STAT1, which is critical for the innate immune response, partially restricts the cell tropism of MNoVCW3 to LysM+ cells. Taken together, these data demonstrate that CD300lf expression on tuft cells is essential for MNoVCR6; that myelomonocytic cells are a major, but not exclusive, target cell of MNoVCW3; and that STAT1 signaling restricts the cellular tropism of MNoVCW3 This study provides the first genetic system for studying the cell type-specific role of CD300lf in norovirus pathogenesis.IMPORTANCE Human noroviruses (HuNoVs) are a leading cause of gastroenteritis resulting in up to 200,000 deaths each year. The receptor and cell tropism of HuNoV in immunocompetent humans are unclear. We use murine norovirus (MNoV) as a model for HuNoV. We recently identified CD300lf as the sole physiologic receptor for MNoV. Here, we leverage this finding to generate a Cd300lf conditional knockout mouse to decipher the contributions of specific cell types to MNoV infection. We demonstrate that persistent MNoVCR6 requires CD300lf expression on tuft cells. In contrast, multiple CD300lf+ cell types, dominated by myelomonocytic cells, are sufficient for nonpersistent MNoVCW3 infection. CD300lf expression on epithelial cells, B cells, neutrophils, and dendritic cells is not critical for MNoVCW3 infection. Mortality associated with the MNoVCW3 strain in Stat1-/- mice does not require CD300lf expression on LysM+ cells, highlighting that both CD300lf receptor expression and innate immunity regulate MNoV cell tropism in vivo.