Identification of trypsin-degrading commensals in the large intestine.

Increased levels of proteases, such as trypsin, in the distal intestine have been implicated in intestinal pathological conditions1-3. However, the players and mechanisms that underlie protease regulation in the intestinal lumen have remained unclear. Here we show that Paraprevotella strains isolated from the faecal microbiome of healthy human donors are potent trypsin-degrading commensals. Mechanistically, Paraprevotella recruit trypsin to the bacterial surface through type IX secretion system-dependent polysaccharide-anchoring proteins to promote trypsin autolysis. Paraprevotella colonization protects IgA from trypsin degradation and enhances the effectiveness of oral vaccines against Citrobacter rodentium. Moreover, Paraprevotella colonization inhibits lethal infection with murine hepatitis virus-2, a mouse coronavirus that is dependent on trypsin and trypsin-like proteases for entry into host cells4,5. Consistently, carriage of putative genes involved in trypsin degradation in the gut microbiome was associated with reduced severity of diarrhoea in patients with SARS-CoV-2 infection. Thus, trypsin-degrading commensal colonization may contribute to the maintenance of intestinal homeostasis and protection from pathogen infection.

Novel bile acid biosynthetic pathways are enriched in the microbiome of centenarians.

Centenarians have a decreased susceptibility to ageing-associated illnesses, chronic inflammation and infectious diseases1-3. Here we show that centenarians have a distinct gut microbiome that is enriched in microorganisms that are capable of generating unique secondary bile acids, including various isoforms of lithocholic acid (LCA): iso-, 3-oxo-, allo-, 3-oxoallo- and isoallolithocholic acid. Among these bile acids, the biosynthetic pathway for isoalloLCA had not been described previously. By screening 68 bacterial isolates from the faecal microbiota of a centenarian, we identified Odoribacteraceae strains as effective producers of isoalloLCA both in vitro and in vivo. Furthermore, we found that the enzymes 5α-reductase (5AR) and 3ß-hydroxysteroid dehydrogenase (3ß-HSDH) were responsible for the production of isoalloLCA. IsoalloLCA exerted potent antimicrobial effects against Gram-positive (but not Gram-negative) multidrug-resistant pathogens, including Clostridioides difficile and Enterococcus faecium. These findings suggest that the metabolism of specific bile acids may be involved in reducing the risk of infection with pathobionts, thereby potentially contributing to the maintenance of intestinal homeostasis.

Sessile Trichomes Play Major Roles in Prey Digestion and Absorption, While Stalked Trichomes Function in Prey Predation in Byblis guehoi.

Carnivorous plants in the genus Byblis obtain nutrients by secreting viscous glue drops and enzymes that trap and digest small organisms. Here, we used B. guehoi to test the long-held theory that different trichomes play different roles in carnivorous plants. In the leaves of B. guehoi, we observed a 1:2.5:14 ratio of long-stalked, short-stalked, and sessile trichomes. We demonstrated that the stalked trichomes play major roles in the production of glue droplets, while the sessile trichomes secrete digestive enzymes, namely proteases and phosphatases. In addition to absorbing digested small molecules via channels/transporters, several carnivorous plants employ a more efficient system: endocytosis of large protein molecules. By feeding B. guehoi fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) to monitor protein transport, we found that sessile trichomes exhibited more endocytosis than long- and short-stalked trichomes. The uptaken FITC-BSA was delivered to the neighboring short epidermal cells in the same row as the sessile trichomes, then to the underlying mesophyll cells; however, no signals were detected in the parallel rows of long epidermis cells. The FITC control could be taken up by sessile trichomes but not transported out. Our study shows that B. guehoi has developed a well-organized system to maximize its food supply, consisting of stalked trichomes for prey predation and sessile trichomes for prey digestion. Moreover, the finding that sessile trichomes transfer large, endocytosed protein molecules to the underlying mesophyll, and putatively to the vascular tissues, but not laterally to the terminally differentiated epidermis, indicates that the nutrient transport system has evolved to maximize efficiency.

Toward the development of defined microbial therapeutics.

The collection of micro-organisms living in the mammalian gastrointestinal tract, termed the gut microbiota, has been shown to have profound impacts on host health and increasingly is regarded as a viable therapeutic target. Clinical studies of fecal microbiota transplantation have demonstrated potential efficacy of microbiota-based therapies for diseases including Clostridioides difficile infections, inflammatory bowel disease, graft-versus-host disease and cancer. However, the lack of understanding of the active ingredients and potential risks of such therapies pose challenges for clinical application. Meanwhile, efforts are being made to identify effector microbes directly associated with a given phenotype, to establish causality and to devise well-characterized microbial therapeutics for clinical use. Strategies based on defined microbial components will likely enhance the potential of microbiota-targeted therapies.

Cell-Type-Specific Transcription of Innate Immune Regulators in response to HMPV Infection.

Human metapneumovirus (HMPV) may cause severe respiratory disease. The early innate immune response to viruses like HMPV is characterized by induction of antiviral interferons (IFNs) and proinflammatory immune mediators that are essential in shaping adaptive immune responses. Although innate immune responses to HMPV have been comprehensively studied in mice and murine immune cells, there is less information on these responses in human cells, comparing different cell types infected with the same HMPV strain. The aim of this study was to characterize the HMPV-induced mRNA expression of critical innate immune mediators in human primary cells relevant for airway disease. In particular, we determined type I versus type III IFN expression in human epithelial cells and monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs). In epithelial cells, HMPV induced only low levels of IFN-ß mRNA, while a robust mRNA expression of IFN-λs was found in epithelial cells, MDMs, and MDDCs. In addition, we determined induction of the interferon regulatory factors (IRFs) IRF1, IRF3, and IRF7 and critical inflammatory cytokines (IL-6, IP-10, and IL-1ß). Interestingly, IRF1 mRNA was predominantly induced in MDMs and MDDCs. Overall, our results suggest that for HMPV infection of MDDCs, MDMs, NECs, and A549 cells (the cell types examined), cell type is a strong determinator of the ability of HMPV to induce different innate immune mediators. HMPV induces the transcription of IFN-ß and IRF1 to higher extents in MDMs and MDDCs than in A549s and NECs, whereas the induction of type III IFN-λ and IRF7 is considerable in MDMs, MDDCs, and A549 epithelial cells.

Dual roles of PGE2-EP4 signaling in mouse experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS). Although prostaglandin (PG) concentrations are increased in cerebrospinal fluid of MS patients, the role of PGs in MS is unknown. We examined this issue by subjecting mice deficient in each PG receptor type or subtype to EAE induction and using agonists or antagonists selective for each of the four PGE receptor (EP) subtypes. Among PG receptor-deficient mice, only EP4(-/-) mice manifested significant suppression of EAE, which was mimicked in wild-type mice and to a greater extent, in EP2(-/-) mice by administration of the EP4 antagonist ONO-AE3-208 during the immunization phase. EP4 antagonism during immunization also suppressed the generation of antigen-specific T helper (Th) 1 and Th17 cells in wild-type mice and to a greater extent, in EP2(-/-) mice. ONO-AE3-208 administration at EAE onset had little effect on disease severity, and its administration throughout the experimental period did not cause significant reduction of the peak of disease, suggesting that, in addition to its facilitative action during the immunization phase, EP4 exerts a preventive action in the elicitation phase. Administration of the EP4 agonist ONO-AE1-329 at EAE onset delayed and suppressed disease progression as well as inhibited the associated increase in permeability of the blood-brain barrier. Thus, PGE(2) exerts dual functions in EAE, facilitating Th1 and Th17 cell generation redundantly through EP4 and EP2 during immunization and attenuating invasion of these cells into the brain by protecting the blood-brain barrier through EP4.

Profiling of mismatch discrimination in RNAi enabled rational design of allele-specific siRNAs.

Silencing specificity is a critical issue in the therapeutic applications of siRNA, particularly in the treatment of single nucleotide polymorphism (SNP) diseases where discrimination against single nucleotide variation is demanded. However, no generally applicable guidelines are available for the design of such allele-specific siRNAs. In this paper, the issue was approached by using a reporter-based assay. With a panel of 20 siRNAs and 240 variously mismatched target reporters, we first demonstrated that the mismatches were discriminated in a position-dependent order, which was however independent of their sequence contexts using position 4th, 12th and 17th as examples. A general model was further built for mismatch discrimination at all positions using 230 additional reporter constructs specifically designed to contain mismatches distributed evenly along the target regions of different siRNAs. This model was successfully employed to design allele-specific siRNAs targeting disease-causing mutations of PIK3CA gene at two SNP sites. Furthermore, conformational distortion of siRNA-target duplex was observed to correlate with the compromise of gene silencing. In summary, these findings could dramatically simplify the design of allele-specific siRNAs and might also provide guide to increase the specificity of therapeutic siRNAs.

Staphylococcus cohnii is a potentially biotherapeutic skin commensal alleviating skin inflammation.

Host-microbe interactions orchestrate skin homeostasis, the dysregulation of which has been implicated in chronic inflammatory conditions such as atopic dermatitis and psoriasis. Here, we show that Staphylococcus cohnii is a skin commensal capable of beneficially inhibiting skin inflammation. We find that Tmem79-/- mice spontaneously develop interleukin-17 (IL-17)-producing T-cell-driven skin inflammation. Comparative skin microbiome analysis reveals that the disease activity index is negatively associated with S. cohnii. Inoculation with S. cohnii strains isolated from either mouse or human skin microbiota significantly prevents and ameliorates dermatitis in Tmem79-/- mice without affecting pathobiont burden. S. cohnii colonization is accompanied by activation of host glucocorticoid-related pathways and induction of anti-inflammatory genes in the skin and is therefore effective at suppressing inflammation in diverse pathobiont-independent dermatitis models, including chemically induced, type 17, and type 2 immune-driven models. As such, S. cohnii strains have great potential as effective live biotherapeutics for skin inflammation.

Inhibition of arsenite methylation induces synergistic genotoxicity of arsenite and benzo(a)pyrene diol epoxide in SCC-7 cells.

As is well-known, arsenite (As(iii)) is a human carcinogen associated with many human cancers. As(iii) can act as a co-carcinogen to induce DNA damage with other carcinogens. Benzo(a)pyrene diol epoxide (BPDE) is one of the most-studied environmental carcinogens, which exists ubiquitously in our daily life. The elucidation of the mechanism of As(iii) as a co-carcinogen with BDPE in cells causing genotoxicity is beneficial for the evaluation of its bioeffect. In this study, a comprehensive analytical system is used for DNA damage evaluation, BPDE-DNA adduct detection, arsenic speciation and gene expression analysis. Based on the experimental results, it can be inferred that BPDE and As(iii) synergistically cause genotoxicity, and the possible mechanism is that BPDE inhibits arsenic methylation, leading to cellular As(iii) enrichment. As(iii) inhibits nucleotide excision repair (NER) of the DNA adduct damage caused by BPDE. The synergistic effect of BPDE and As(iii) causes DNA strand break damage, which further results in carcinogenesis.

Human Metapneumovirus Infection Inhibits Cathelicidin Antimicrobial Peptide Expression in Human Macrophages.

Human cathelicidin antimicriobial peptide (CAMP) is a critical component of host innate immunity with both antimicrobial and immunomodulatory functions. Several pathogens have been shown to downregulate CAMP expression, yet it is unclear if such modulation occurs during a viral infection. In this study, we showed that infection with human metapneumovirus (hMPV), one of the leading causes of respiratory tract infections in young children, strongly suppressed basal and vitamin-D induced CAMP expression in human macrophages. hMPV-mediated suppression of CAMP did not correlate with reduced transcriptional expression of key vitamin D signaling components, such as CYP27B1 or vitamin D receptor, suggesting a vitamin D-independent mechanism. Blocking interferon-signaling pathways did not reverse hMVP-mediated suppression of CAMP, indicating that the suppressive effect is largely interferon-independent. Instead, we identified C/EBPα as the key modulator of hMPV-mediated suppression of CAMP. hMPV infection strongly repressed the expression of C/EBPα, and a knockdown study confirmed that C/EBPα is critical for CAMP expression in human macrophages. Such modulation of CAMP (and C/EBPα) could be reproduced by TLR1/2 ligand treatment in human macrophages, suggesting a common mechanism underlying pathogen-mediated downregulation of CAMP through C/EBPα. This study opens up a new understanding of altered human antimicrobial responses following infections.

Biomethylation metabolism study of arsenite in SCC-7 cells by reversed phase ion pair high performance liquid chromatography-inductively coupled plasma-mass spectrometry.

Arsenite (As(III)) has been considered as a human carcinogen associated with many human cancers especially skin cancer. Elucidation of the transformed species of As(III) during its metabolism in cells is beneficial for evaluation of its bioeffect. In this work, a hyphenated method of reversed phase ion pair high performance liquid chromatography - inductively coupled plasma mass spectrometry (RP-IP-HPLC-ICP-MS) equipped with collision/reaction cell technology (CCT) was developed for speciation of As(III) and its metabolites (arsenate [As(V)], monomethylarsonic acid [MMA(V)], and dimethylarsinic acid [DMA(V)]) in SCC-7 cells. The developed analytical method exhibits low limits of detection for interest arsenic species in the range of 14-27 ng/L and wide linear range up to four orders of magnitude, providing a sensitive tool for arsenic metabolites analysis and further understanding the metabolism of As(III) in SCC-7 cells. The effect of exposure time, exposure concentrations and elimination time on the arsenic species and total arsenic in SCC-7 cells incubated by As(III) were systematically studied. At low exposure concentrations (< 5 µM), large proportion of intracellular As(III) transformed to methylated metabolites, and the final methylated metabolite DMA(V), which could not be completely removed from the cells in the elimination process, is considered to play as the primary carcinogen. While at high exposure concentrations (> 5 µM), most of intracellular As(III) probably bound to biomacromolecules rather than followed biomethylation process, exhibiting different metabolism.

Characterization of signaling pathways regulating the expression of pro-inflammatory long form thymic stromal lymphopoietin upon human metapneumovirus infection.

Thymic stromal lymphopoietin (TSLP) is associated with several allergic diseases including asthma. Two isoforms of TSLP exist in humans, a long form (lfTSLP) and a short form (sfTSLP), displaying distinct immunological functions. Recently, TSLP was found to be upregulated in human airway cells upon human metapneumovirus (hMPV) infection, yet it remains unclear if the two isoforms are regulated differently during hMPV infection. Importantly, the molecular mechanisms underlying hMPV-mediated TSLP induction remain undescribed. In this study, we characterized the expression and regulation of TSLP in hMPV-infected human airway cells. We demonstrated that hMPV strongly induced the expression of pro-inflammatory lfTSLP in human airway epithelial cells and lung fibroblasts. Further, knockdown of pattern recognition receptors retinoic acid-inducible gene I (RIG-I) or Toll-like receptor 3 (TLR3), as well as downstream signal transducers, abrogated hMPV-mediated lfTSLP induction. Importantly, silencing of TANK-binding kinase 1 (TBK1) also impaired hMPV-mediated lfTSLP induction, which could be attributed to compromised NF-κB activation. Overall, these results suggest that TBK1 may be instrumental for hMPV-mediated activation of NF-κB downstream RIG-I and TLR3, leading to a specific induction of lfTSLP in hMPV-infected human airway cells.

Prostaglandin E2-EP4 signaling promotes immune inflammation through Th1 cell differentiation and Th17 cell expansion.

Two distinct helper T (TH) subsets, TH1 and TH17, mediate tissue damage and inflammation in animal models of various immune diseases such as multiple sclerosis, rheumatoid arthritis, inflammatory bowel diseases and allergic skin disorders. These experimental findings, and the implication of these TH subsets in human diseases, suggest the need for pharmacological measures to manipulate these TH subsets. Here we show that prostaglandin E2 (PGE2) acting on its receptor EP4 on T cells and dendritic cells not only facilitates TH1 cell differentiation but also amplifies interleukin-23-mediated TH17 cell expansion in vitro. Administration of an EP4-selective antagonist in vivo decreases accumulation of both TH1 and TH17 cells in regional lymph nodes and suppresses the disease progression in mice subjected to experimental autoimmune encephalomyelitis or contact hypersensitivity. Thus, PGE2-EP4 signaling promotes immune inflammation through TH1 differentiation and TH17 expansion, and EP4 antagonism may be therapeutically useful for various immune diseases.