Replication of Human Sapovirus in Human-Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Cells.

Sapoviruses, like noroviruses, are single-stranded positive-sense RNA viruses classified in the family Caliciviridae and are recognized as a causative pathogen of diarrhea in infants and the elderly. Like human norovirus, human sapovirus (HuSaV) has long been difficult to replicate in vitro. Recently, it has been reported that HuSaV can be replicated in vitro by using intestinal epithelial cells (IECs) derived from human tissues and cell lines derived from testicular and duodenal cancers. In this study, we report that multiple genotypes of HuSaV can sufficiently infect and replicate in human-induced pluripotent stem cell-derived IECs. We also show that this HuSaV replication system can be used to investigate the conditions for inactivation of HuSaV by heat and alcohol, and the effects of virus neutralization of antisera obtained by immunization with vaccine antigens, under conditions closer to the living environment. The results of this study confirm that HuSaV can also infect and replicate in human normal IECs regardless of their origin and are expected to contribute to future virological studies.

Community based multi-disease health screening as an opportunity for early detection of HIV cases and linking them to care.

BACKGROUND: The 95-95-95 UNAIDS global strategy was adapted to end the AIDS epidemic by 2030. The target is based on the premise that early detection of HIV-infected persons and linking them to treatment regardless of their CD4 counts will lead to sustained viral suppression. HIV testing strategies to increase uptake of testing in Western and Central Africa remain inadequate. Hence, a high proportion of people living with HIV in this region do not know their status. This report describes the implementation of a community based multi-disease health screening (also known as "Know Your Status" -KYS), as part of basic science research, in a way that contributed to achieving public health goals. METHODS: A community based multi-disease health screening was conducted in 7 communities within the Eastern region of Ghana between November 2017 and April 2018, to recruit and match HIV seronegative persons to HIV seropositive persons in a case-control HIV gut microbiota study. Health assessments included blood pressure, body mass index, blood sugar, Hepatitis B virus, syphilis, and HIV testing for those who consented. HIV seronegative participants who consented were consecutively enrolled in an ongoing HIV gut microbiota case-control study. Descriptive statistics (percentages) were used to analyze data. RESULTS: Out of 738 people screened during the exercise, 700 consented to HIV testing and 23 (3%) were HIV positive. Hepatitis B virus infection was detected in 4% (33/738) and Syphilis in 2% (17/738). Co-infection of HIV and HBV was detected in 4 persons. The HIV prevalence of 3% found in these communities is higher than both the national prevalence of 1.7% and the Eastern Regional prevalence of 2.7 in 2018. CONCLUSION: Community based multi-disease health screening, such as the one undertaken in our study could be critical for identifying HIV infected persons from the community and linking them to care. In the case of HIV, it will greatly contribute to achieving the first two 95s and working towards ending AIDS by 2030.

Genotype Diversity of Enteric Viruses in Wastewater Amid the COVID-19 Pandemic.

Viruses remain the leading cause of acute gastroenteritis (AGE) worldwide. Recently, we reported the abundance of AGE viruses in raw sewage water (SW) during the COVID-19 pandemic, when viral AGE patients decreased dramatically in clinics. Since clinical samples were not reflecting the actual state, it remained important to determine the circulating strains in the SW for preparedness against impending outbreaks. Raw SW was collected from a sewage treatment plant in Japan from August 2018 to March 2022, concentrated by polyethylene-glycol-precipitation method, and investigated for major gastroenteritis viruses by RT-PCR. Genotypes and evolutionary relationships were evaluated through sequence-based analyses. Major AGE viruses like rotavirus A (RVA), norovirus (NoV) GI and GII, and astrovirus (AstV) increased sharply (10-20%) in SW during the COVID-19 pandemic, though some AGE viruses like sapovirus (SV), adenovirus (AdV), and enterovirus (EV) decreased slightly (3-10%). The prevalence remained top in the winter. Importantly, several strains, including G1 and G3 of RVA, GI.1 and GII.2 of NoV, GI.1 of SV, MLB1 of AstV, and F41 of AdV, either emerged or increased amid the pandemic, suggesting that the normal phenomenon of genotype changing remained active over this time. This study crucially presents the molecular characteristics of circulating AGE viruses, explaining the importance of SW investigation during the pandemic when a clinical investigation may not produce the complete scenario.

Organoid-derived intestinal epithelial cells are a suitable model for preclinical toxicology and pharmacokinetic studies.

Intestinal organoids are physiologically relevant tools used for cellular models. However, the suitability of organoids to examine biological functions over existing established cell lines lacks sufficient evidence. Cytochrome P450 3A4 (CYP3A4) induction by pregnane X receptor ligands, glucose uptake via sodium/glucose cotransporter 1, and microsomal triglyceride transfer protein-dependent ApoB-48 secretion, which are critical for human intestinal metabolism, were observed in organoid-derived two-dimensional cells but little in Caco-2 cells. CYP3A4 induction evaluation involved a simplified method of establishing organoids that constitutively expressed a reporter gene. Compound screening identified several anticancer drugs with selective activities toward Caco-2 cells, highlighting their characteristics as cancer cells. Another compound screening revealed a decline in N-(4-hydroxyphenyl)retinamide cytotoxicity upon rifampicin treatment in organoid-derived cells, under CYP3A4-induced conditions. This study shows that organoid-derived intestinal epithelial cells (IECs) possess similar physiological properties as intestinal epithelium and can serve as tools for enhancing the prediction of biological activity in humans.

Microbial-Driven Immunological Memory and Its Potential Role in Microbiome Editing for the Prevention of Colorectal Cancer.

Over the last several years, many advances have been made in understanding the role of bacteria in the pathogenesis of gastrointestinal cancers. Beginning with Helicobacter pylori being recognized as the first bacterial carcinogen and the causative agent of most gastric cancers, more recent studies have examined the role of enteric microbes in colorectal cancer. In the digestive tract, these communities are numerous and have a complex interrelationship with local immune/inflammatory responses that impact the health of the host. As modifying the microbiome in the stomach has decreased the risk of gastric cancer, modifying the distal microbiome may decrease the risk of colorectal cancers. To date, very few studies have considered the notion that mucosal lymphocyte-dependent immune memory may confound attempts to change the microbial components in these communities. The goal of this review is to consider some of the factors impacting host-microbial interactions that affect colorectal cancer and raise questions about how immune memory responses to the local microbial consortium affect any attempt to modify the composition of the intestinal microbiome.

Role of interleukin-6 in antigen-specific mucosal immunoglobulin A induction by cationic liposomes.

The COVID-19 pandemic, caused by a highly virulent and transmissible pathogen, has proven to be devastating to society. Mucosal vaccines that can induce antigen-specific immune responses in both the systemic and mucosal compartments are considered an effective measure to overcome infectious diseases caused by pathogenic microbes. We have recently developed a nasal vaccine system using cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium-propane and cholesteryl 3ß-N-(dimethylaminoethyl)carbamate in mice. However, the comprehensive molecular mechanism(s), especially the host soluble mediator involved in this process, by which cationic liposomes promote antigen-specific mucosal immune responses, remain to be elucidated. Herein, we show that intranasal administration of cationic liposomes elicited interleukin-6 (IL-6) expression at the site of administration. Additionally, both nasal passages and splenocytes from mice nasally immunized with cationic liposomes plus ovalbumin (OVA) were polarized to produce IL-6 when re-stimulated with OVA in vitro. Furthermore, pretreatment with anti-IL-6R antibody, which blocks the biological activities of IL-6, attenuated the production of OVA-specific nasal immunoglobulin A (IgA) but not OVA-specific serum immunoglobulin G (IgG) responses. In this study, we demonstrated that IL-6, exerted by nasally administered cationic liposomes, plays a crucial role in antigen-specific IgA induction.

Comparison of gene expression and activation of transcription factors in organoid-derived monolayer intestinal epithelial cells and organoids.

Intestinal organoids better represent in vivo intestinal properties than conventionally used established cell lines in vitro. However, they are maintained in three-dimensional culture conditions that may be accompanied by handling complexities. We characterized the properties of human organoid-derived two-dimensionally cultured intestinal epithelial cells (IECs) compared with those of their parental organoids. We found that the expression of several intestinal markers and functional genes were indistinguishable between monolayer IECs and organoids. We further confirmed that their specific ligands equally activate intestinal ligand-activated transcriptional regulators in a dose-dependent manner. The results suggest that culture conditions do not significantly influence the fundamental properties of monolayer IECs originating from organoids, at least from the perspective of gene expression regulation. This will enable their use as novel biological tools to investigate the physiological functions of the human intestine.

Distinct Age-Specific miRegulome Profiling of Isolated Small and Large Intestinal Epithelial Cells in Mice.

The intestinal epithelium serves as a dynamic barrier to protect the host tissue from exposure to a myriad of inflammatory stimuli in the luminal environment. Intestinal epithelial cells (IECs) encompass differentiated and specialized cell types that are equipped with regulatory genes, which allow for sensing of the luminal environment. Potential inflammatory cues can instruct IECs to undergo a diverse set of phenotypic alterations. Aging is a primary risk factor for a variety of diseases; it is now well-documented that aging itself reduces the barrier function and turnover of the intestinal epithelium, resulting in pathogen translocation and immune priming with increased systemic inflammation. In this study, we aimed to provide an effective epigenetic and regulatory outlook that examines age-associated alterations in the intestines through the profiling of microRNAs (miRNAs) on isolated mouse IECs. Our microarray analysis revealed that with aging, there is dysregulation of distinct clusters of miRNAs that was present to a greater degree in small IECs (22 miRNAs) compared to large IECs (three miRNAs). Further, miRNA-mRNA interaction network and pathway analyses indicated that aging differentially regulates key pathways between small IECs (e.g., toll-like receptor-related cascades) and large IECs (e.g., cell cycle, Notch signaling and small ubiquitin-related modifier pathway). Taken together, current findings suggest novel gene regulation pathways by epithelial miRNAs in aging within the gastrointestinal tissues.

Enzymatically polymerised polyphenols prepared from various precursors potentiate antigen-specific immune responses in both mucosal and systemic compartments in mice.

Despite significant modern medicine progress, having an infectious disease is a major risk factor for humans. Mucosal vaccination is now widely considered as the most promising strategy to defeat infectious diseases; however, only live-attenuated and inactivated mucosal vaccines are used in the clinical field. To date, no subunit mucosal vaccine was approved mainly because of the lack of safe and effective methodologies to either activate or initiate host mucosal immune responses. We have recently elucidated that intranasal administration of enzymatically polymerised caffeic acid potentiates antigen-specific mucosal and systemic antibody responses in mice. However, our earlier study has not confirmed whether these effects are specific to the polymer synthesised from caffeic acid. Here, we show that enzymatically polymerised polyphenols (EPPs) from various phenolic compounds possess mucosal adjuvant activities when administered nasally with an antigen to mice. Potentiation of antigen-specific immune responses by all EPPs tested in this study showed no clear difference among the precursors used. We found that intranasal administration of ovalbumin as the antigen, in combination with all enzymatically polymerised polyphenols used in this study, induced ovalbumin-specific mucosal IgA in the nasal cavity, bronchoalveolar lavage fluid, vaginal fluids, and systemic IgG, especially IgG1, in sera. Our results demonstrate that the mucosal adjuvant activities of polyphenols are not limited to polymerised caffeic acid but are broadly observable across the studied polyphenols. These properties of polyphenols may be advantageous for the development of safe and effective nasal vaccine systems to prevent and/or treat various infectious diseases.

Lipopolysaccharide from Gut-Associated Lymphoid-Tissue-Resident Alcaligenes faecalis: Complete Structure Determination and Chemical Synthesis of Its Lipid†A.

Alcaligenes faecalis is the predominant Gram-negative bacterium inhabiting gut-associated lymphoid tissues, Peyer's patches. We previously reported that an A. faecalis lipopolysaccharide (LPS) acted as a weak agonist for Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2) receptor as well as a potent inducer of IgA without excessive inflammation, thus suggesting that A. faecalis LPS might be used as a safe adjuvant. In this study, we characterized the structure of both the lipooligosaccharide (LOS) and LPS from A. faecalis. We synthesized three lipid A molecules with different degrees of acylation by an efficient route involving the simultaneous introduction of 1- and 4'-phosphates. Hexaacylated A. faecalis lipid A showed moderate agonistic activity towards TLR4-mediated signaling and the ability to elicit a discrete interleukin-6 release in human cell lines and mice. It was thus found to be the active principle of the LOS/LPS and a promising vaccine adjuvant candidate.

Stratified layer analysis reveals intrinsic leptin stimulates cryptal mesenchymal cells for controlling mucosal inflammation.

Mesenchymal cells in the crypt play indispensable roles in the maintenance of intestinal epithelial homeostasis through their contribution to the preservation of stem cells. However, the acquisition properties of the production of stem cell niche factors by the mesenchymal cells have not been well elucidated, due to technical limitations regarding the isolation and subsequent molecular and cellular analyses of cryptal mesenchymal cells. To evaluate the function of mesenchymal cells located at the large intestinal crypt, we established a novel method through which cells are harvested according to the histologic layers of mouse colon, and we compared cellular properties between microenvironmental niches, the luminal mucosa and crypts. The gene expression pattern in the cryptal mesenchymal cells showed that receptors of the hormone/cytokine leptin were highly expressed, and we found a decrease in Wnt2b expression under conditions of leptin receptor deficiency, which also induced a delay in cryptal epithelial proliferation. Our novel stratified layer isolation strategies thus revealed new microenvironmental characteristics of colonic mesenchymal cells, including the intrinsic involvement of leptin in the control of mucosal homeostasis.

Maternal ω3 docosapentaenoic acid inhibits infant allergic dermatitis through TRAIL-expressing plasmacytoid dendritic cells in mice.

BACKGROUND: Maternal dietary exposures are considered to influence the development of infant allergies through changes in the composition of breast milk. Cohort studies have shown that ω3 polyunsaturated fatty acids (PUFAs) in breast milk may have a beneficial effect on the preventing of allergies in infants; however, the underlying mechanisms remain to be investigated. We investigated how the maternal intake of dietary ω3 PUFAs affects fatty acid profiles in the breast milk and their pups and reduced the incidence of allergic diseases in the pups. METHODS: Contact hypersensitivity (CHS) induced by 2,4-dinitrofluorobenzene (DNFB) and fluorescein isothiocyanate was applied to the skin in pups reared by mother maintained with diets mainly containing ω3 or ω6 PUFAs. Skin inflammation, immune cell populations, and expression levels of immunomodulatory molecules in pups and/or human cell line were investigated by using flow cytometric, immunohistologic, and quantitative RT-PCR analyses. ω3 PUFA metabolites in breast milk and infant's serum were evaluated by lipidomics analysis using LC-MS/MS. RESULTS: We show that maternal intake of linseed oil, containing abundant ω3 α-linolenic acid, resulted in the increased levels of ω3 docosapentaenoic acid (DPA) and its 14-lipoxygenation products in the breast milk of mouse dams; these metabolites increased the expression of TNF-related apoptosis-inducing ligand (TRAIL) on plasmacytoid dendritic cells (pDCs) in their pups and thus inhibited infant CHS. Indeed, the administration of DPA-derived 14-lipoxygenation products to mouse pups ameliorated their DNFB CHS. CONCLUSION: These findings suggest that an inhibitory mechanism in infant skin allergy is induced through maternal metabolism of dietary ω3 PUFAs in mice.

Persistent colonization of non-lymphoid tissue-resident macrophages by Stenotrophomonas maltophilia.

Accumulating evidence has revealed that lymphoid tissue-resident commensal bacteria (e.g. Alcaligenes spp.) survive within dendritic cells. We extended our previous study by investigating microbes that persistently colonize colonic macrophages. 16S rRNA-based metagenome analysis using DNA purified from murine colonic macrophages revealed the presence of Stenotrophomonas maltophilia. The in situ intracellular colonization by S. maltophilia was recapitulated in vitro by using bone marrow-derived macrophages (BMDMs). Co-culture of BMDMs with clinically isolated S. maltophilia led to increased mitochondrial respiration and robust IL-10 production. We further identified a 25-kDa protein encoded by the gene assigned as smlt2713 (recently renamed as SMLT_RS12935) and secreted by S. maltophilia as the factor responsible for enhanced IL-10 production by BMDMs. IL-10 production is critical for maintenance of the symbiotic condition, because intracellular colonization by S. maltophilia was impaired in IL-10-deficient BMDMs, and smlt2713-deficient S. maltophilia failed to persistently colonize IL-10-competent BMDMs. These findings indicate a novel commensal network between colonic macrophages and S. maltophilia that is mediated by IL-10 and smlt2713.

Intrinsic Control of Surface Immune and Epithelial Homeostasis by Tissue-Resident Gut Stromal Cells.

The epithelial layer creates a chemical and physical barrier at the forefront of intestinal mucosa, and immune cells beneath the surface epithelium are poised to react to extrinsic factors, to maintain tissue homeostasis. Importantly, the nexus of epithelial-immune responses at mucosal surfaces is dexterously modulated by intrinsic stromal-mesenchymal cells. First, organogenesis of lymphoid tissues, including Peyer's patches, requires dynamic interplay between lymphoid cells and stromal cells, which have become known as "lymphoid organizers." Second, correct spatiotemporal interaction between these cell populations is essential to generate the infrastructure for gut immune responses. Moreover, immune cells at the intestinal barrier are functionally modulated by stromal cells; one such example is the stromal cell-mediated differentiation of innate immune cells, including innate lymphoid cells and mast cells. Ultimately, mucosal stromal cells orchestrate the destinations of epithelial and immune cells to maintain intestinal immune homeostasis.

Mast cells play role in wound healing through the ZnT2/GPR39/IL-6 axis.

Zinc (Zn) is an essential nutrient and its deficiency causes immunodeficiency and skin disorders. Various cells including mast cells release Zn-containing granules when activated; however, the biological role of the released Zn is currently unclear. Here we report our findings that Zn transporter ZnT2 is required for the release of Zn from mast cells. In addition, we found that Zn and mast cells induce IL-6 production from inflammatory cells such as skin fibroblasts and promote wound healing, a process that involves inflammation. Zn induces the production of a variety of pro-inflammatory cytokines including IL-6 through signaling pathways mediated by the Zn receptor GPR39. Consistent with these findings, wound healing was impaired in mice lacking IL-6 or GPR39. Thus, our results show that Zn and mast cells play a critical role in wound healing through activation of the GPR39/IL-6 signaling axis.

Fucosyltransferase 2 induces lung epithelial fucosylation and exacerbates house dust mite-induced airway inflammation.

BACKGROUND: One of the pathognomonic features of asthma is epithelial hyperproduction of mucus, which is composed of a series of glycoproteins; however, it remains unclear how glycosylation is induced in lung epithelial cells from asthmatic patients and how glycan residues play a role in the pathogenesis of asthma. OBJECTIVE: The objective of this study was to explore comprehensive epithelial glycosylation status induced by allergic inflammation and reveal its possible role in the pathogenesis of asthma. METHODS: We evaluated the glycosylation status of lung epithelium using a lectin microarray. We next searched for molecular mechanisms underlying epithelial glycosylation. We also examined whether epithelial glycosylation is involved in induction of allergic inflammation. RESULTS: On allergen inhalation, lung epithelial cells were heavily α(1,2)fucosylated by fucosyltransferase 2 (Fut2), which was induced by the IL-13-signal transducer and activator of transcription 6 pathway. Importantly, Fut2-deficient (Fut2-/-) mice, which lacked lung epithelial fucosylation, showed significantly attenuated eosinophilic inflammation and airway hyperresponsiveness in house dust mite (HDM)-induced asthma models. Proteome analyses and immunostaining of the HDM-challenged lung identified that complement C3 was accumulated in fucosylated areas. Indeed, Fut2-/- mice showed significantly reduced levels of C3a and impaired accumulation of C3a receptor-expressing monocyte-derived dendritic cells in the lung on HDM challenge. CONCLUSION: Fut2 induces epithelial fucosylation and exacerbates airway inflammation in asthmatic patients in part through C3a production and monocyte-derived dendritic cell accumulation in the lung.

Dietary coconut oil ameliorates skin contact hypersensitivity through mead acid production in mice.

Coconut oil is used as a dietary oil worldwide, and its healthy effects are recognized by the fact that coconut oil is easy to digest, helps in weight management, increases healthy cholesterol, and provides instant energy. Although topical application of coconut oil is known to reduce skin infection and inflammation, whether dietary coconut oil has any role in decreasing skin inflammation is unknown. In this study, we showed the impact of dietary coconut oil in allergic skin inflammation by using a mouse model of contact hypersensitivity (CHS). Mice maintained on coconut oil showed amelioration of skin inflammation and increased levels of cis-5, 8, 11-eicosatrienoic acid (mead acid) in serum. Intraperitoneal injection of mead acid inhibited CHS and reduced the number of neutrophils infiltrating to the skin. Detailed mechanistic studies unveiled that mead acid inhibited the directional migration of neutrophils by inhibiting the filamentous actin polymerization and leukotriene B4 production required for secondary recruitment of neutrophils. Our findings provide valuable insights into the preventive roles of coconut oil and mead acid against skin inflammation, thereby offering attractive therapeutic possibilities.

Polymeric Caffeic Acid Is a Safer Mucosal Adjuvant That Augments Antigen-Specific Mucosal and Systemic Immune Responses in Mice.

Infections remain a major threat to human lives. To overcome the threat caused by pathogens, mucosal vaccines are considered a promising strategy. However, no inactivated and/or subunit mucosal vaccine has been approved for human use, largely because of the lack of a safe and effective mucosal adjuvant. Here, we show that enzymatically synthesized polymeric caffeic acid (pCA) can act as a potent mucosal adjuvant in mice. Intranasal administration of ovalbumin (OVA) in combination with pCA resulted in the induction of OVA-specific mucosal IgA and serum IgG, especially IgG1. Importantly, pCA was synthesized from caffeic acid and horseradish peroxidase from coffee beans and horseradish, respectively, which are commonly consumed. Therefore, pCA is believed to be a highly safe material. In fact, administration of pCA did not show distinct toxicity in mice. These data indicate that pCA has merit for use as a mucosal adjuvant for nasal vaccine formulations.

ATP as a Pathophysiologic Mediator of Bacteria-Host Crosstalk in the Gastrointestinal Tract.

Extracellular nucleotides, such as adenosine triphosphate (ATP), are released from host cells including nerve termini, immune cells, injured or dead cells, and the commensal bacteria that reside in the gut lumen. Extracellular ATP interacts with the host through purinergic receptors, and promotes intercellular and bacteria-host communication to maintain the tissue homeostasis. However, the release of massive concentrations of ATP into extracellular compartments initiates acute and chronic inflammatory responses through the activation of immunocompetent cells (e.g., T cells, macrophages, and mast cells). In this review, we focus on the functions of ATP as a pathophysiologic mediator that is required for the induction and resolution of inflammation and inter-species communication.