Lactobacillus rhamnosus GG (LGG) is the most widely used probiotic, but the mechanisms underlying its beneficial effects remain unresolved. Previous studies typically inoculated LGG in hosts with ...established gut microbiota, limiting the understanding of specific impacts of LGG on host due to numerous interactions among LGG, commensal microbes, and the host. There has been a scarcity of studies that used gnotobiotic animals to elucidate LGG-host interaction, in particular for gaining specific insights about how it modifies the metabolome. To evaluate whether LGG affects the metabolite output of pathobionts, we inoculated with LGG gnotobiotic mice containing Propionibacterium acnes, Turicibacter sanguinis, and Staphylococcus aureus (PTS).
16S rRNA sequencing of fecal samples by Ion Torrent and MinION platforms showed colonization of germ-free mice by PTS or by PTS plus LGG (LTS). Although the body weights and feeding rates of mice remained similar between PTS and LTS groups, co-associating LGG with PTS led to a pronounced reduction in abundance of P. acnes in the gut. Addition of LGG or its secretome inhibited P. acnes growth in culture. After optimizing procedures for fecal metabolite extraction and metabolomic liquid chromatography-mass spectrometry analysis, unsupervised and supervised multivariate analyses revealed a distinct separation among fecal metabolites of PTS, LTS, and germ-free groups. Variables-important-in-projection scores showed that LGG colonization robustly diminished guanine, ornitihine, and sorbitol while significantly elevating acetylated amino acids, ribitol, indolelactic acid, and histamine. In addition, carnitine, betaine, and glutamate increased while thymidine, quinic acid and biotin were reduced in both PTS and LTS groups. Furthermore, LGG association reduced intestinal mucosal expression levels of inflammatory cytokines, such as IL-1α, IL-1β and TNF-α.
LGG co-association had a negative impact on colonization of P. acnes, and markedly altered the metabolic output and inflammatory response elicited by pathobionts.
The naked mole rat (NMR), Heterocephalus glaber, the longest-living rodent, provides a unique opportunity to explore how evolution has shaped adult stem cell (ASC) activity and tissue function with ...increasing lifespan. Using cumulative BrdU labelling and a quantitative imaging approach to track intestinal ASCs (Lgr5
) in their native in vivo state, we find an expanded pool of Lgr5
cells in NMRs, and these cells specifically at the crypt base (Lgr5
) exhibit slower division rates compared to those in short-lived mice but have a similar turnover as human LGR5
cells. Instead of entering quiescence (G0), NMR Lgr5
cells reduce their division rates by prolonging arrest in the G1 and/or G2 phases of the cell cycle. Moreover, we also observe a higher proportion of differentiated cells in NMRs that confer enhanced protection and function to the intestinal mucosa which is able to detect any chemical imbalance in the luminal environment efficiently, triggering a robust pro-apoptotic, anti-proliferative response within the stem/progenitor cell zone.
The effects of polarized membrane trafficking in mature epithelial tissue on cell growth and cancer progression have not been fully explored
. A majority of colorectal cancers have reduced and ...mislocalized Rab11, a small GTPase dedicated to trafficking of recycling endosomes. Patients with low Rab11 protein expression have poor survival rates. Using genetic models across species, we show that intact recycling endosome function restrains aberrant epithelial growth elicited by APC or RAS mutations. Loss of Rab11 protein led to epithelial dysplasia in early animal development and synergized with oncogenic pathways to accelerate tumor progression initiated by carcinogen, genetic mutation, or aging. Transcriptomic analysis uncovered an immediate expansion of the intestinal stem cell pool along with cell-autonomous Yki/Yap activation following disruption of Rab11a-mediated recycling endosomes. Intestinal tumors lacking Rab11a traffic exhibited marked elevation of nuclear Yap, upd3/IL6-Stat3, and amphiregulin-MAPK signaling, whereas suppression of Yki/Yap or upd3/IL6 reduced gut epithelial dysplasia and hyperplasia. Examination of Rab11a function in enteroids or cultured cell lines suggested that this endosome unit is required for suppression of the Yap pathway by Hippo kinases. Thus, recycling endosomes in mature epithelia constitute key tumor suppressors, loss of which accelerates carcinogenesis. SIGNIFICANCE: Recycling endosome traffic in mature epithelia constitutes a novel tumor suppressing mechanism.
Like nucleated cells, erythrocytes (red blood cells, RBCs) are capable of executing programmed cell death pathways. RBCs undergo necroptosis in response to CD59-specific pore-forming toxins (PFTs). ...The relationship between blood bank storage and RBC necroptosis was explored in this study.
Human RBCs were stored in standard blood bank additive solutions (AS-1, AS-3, or AS-5) for 1 week and hemolysis was evaluated in the context of necroptosis inhibitors and reactive oxygen species (ROS) scavengers. Activation of key factors including RIP1, RIP3, and MLKL was determined using immunoprecipitations and western blot. RBC vesiculation and formation of echinocytes was determined using phase-contrast microscopy. The effect of necroptosis and storage on RBC clearance was determined using a murine transfusion model.
Necroptosis is associated with increased RBC clearance post-transfusion. Moreover, storage in AS-1, AS-3, or AS-5 sensitizes RBCs for necroptosis. Importantly, storage-sensitized RBCs undergo necroptosis in response to multiple PFTs, regardless of specificity for CD59. Storage-sensitized RBCs undergo necroptosis via NADPH oxidase-generated ROS. RBC storage led to RIP1 phosphorylation and necrosome formation in an NADPH oxidase-dependent manner suggesting the basis for this sensitization. In addition, storage led to increased RBC clearance post-transfusion. Clearance of these RBCs was due to Syk-dependent echinocyte formation.
Storage-induced sensitization to RBC necroptosis and clearance is important as it may be relevant to hemolytic transfusion reactions.
The regulatory mechanisms enabling the intestinal epithelium to maintain a high degree of regenerative capacity during mucosal injury remain unclear. Ex vivo survival and clonogenicity of intestinal ...stem cells (ISCs) strictly required growth response mediated by cell division control 42 (Cdc42) and Cdc42-deficient enteroids to undergo rapid apoptosis. Mechanistically, Cdc42 engaging with EGFR was required for EGF-stimulated, receptor-mediated endocytosis and sufficient to promote MAPK signaling. Proteomics and kinase analysis revealed that a physiologically, but nonconventionally, spliced Cdc42 variant 2 (V2) exhibited stronger MAPK-activating capability. Human CDC42-V2 is transcriptionally elevated in some colon tumor tissues. Accordingly, mice engineered to overexpress Cdc42-V2 in intestinal epithelium showed elevated MAPK signaling, enhanced regeneration, and reduced mucosal damage in response to irradiation. Overproducing Cdc42-V2 specifically in mouse ISCs enhanced intestinal regeneration following injury. Thus, the intrinsic Cdc42-MAPK program is required for intestinal epithelial regeneration, and elevating this signaling cascade is capable of initiating protection from genotoxic injury.
Although the influence of the gut microbiota on structure and proliferation of intestinal epithelial cells has been established, there is disconcordance on information related to cell type ...differentiation, because most studies on probiotics reflected an outcome from interactions among Lactobacillus, numerous existing microbes, and the host, with limited insight on the specific impact of Lactobacilli spp. For example, L. reuteri enhanced goblet and Paneth cell differentiation in intestine of conventionally‐raised chickens with an established commensal gut microbiota, confounding results. Here we fed gnotobiotic mice with tryptophan (t+) or trp‐free (t–) diets, followed by monocolonization with LGG (L+, ATCC 53103) or without (control, L–) for a total of 4 groups (L+t+, L+t–, L–t+, L–,t–), then ileum transcriptome was analyzed 3 wk later. L– mice remained germ‐free throughout while only LGG was associated with L+. Principal component analysis shows L+ or t+ by itself to modestly cluster away from L–t–, but the cluster depicting the combination of L+t+ was far removed from the others. The heatmap of whole ileum transcriptome shows several distinct groups of genes affected by t+ or by L+ alone. However, the simultaneous impact of L+t+ dramatically upregulates numerous gene clusters while downregulating a few. Volcano plots and Venn diagrams comparing L–t– with L–t+, or L+t– with L–t– indicate ~2300 unique genes affected by L+ (LGG) or t+ (tryptophan) alone. Comparisons between L+t+ and L+t– (effect of t with L) or between L+t+ and L–t+ (effect of L with t) each impact >3800 genes, suggesting that synergistic interactions between L+ and t+ primarily underlie the probiotic impact ascribed to LGG and dietary t. Many of the genes upregulated by L+t+ have functions typical of mature enterocytes: tight junctions, brush border membrane transporters and enzymes, intestinal bile acid metabolism, lipid synthesis and fatty acid b‐oxidation. Many downregulated genes are antimicrobial and secreted peptides typical of differentiated Paneth and goblet cells. This latter result was recapitulated when reexamined in conventional mice perfused for 4 h with LGG, as expression of many defensins decreased by >10‐fold, suggesting not only that this probiotic’s effects can arise from both acute ex vivo and chronic in vivo interactions with the host, but also in hosts housing a complex microbiota. Remarkably, Notch ligands Dll1 and Dll4 typically expressed in Paneth cells as well as Yap1, Axin2 and Ctnnb1 are upregulated in L+t+ ileum, suggesting downstream upregulation of Hes1 and repression of Atoh1, leading to suppression of the secretory lineage. In conclusion, LGG monocolonization may lead to synthesis of t+‐derived metabolites by both host and bacteria, leading to an enhancement of absorptive enterocyte functions and suppression of those by goblet and Paneth cells.
Human enterocytes are primary targets of infection by invasive bacterium Salmonella Typhimurium, and studies using nonintestinal epithelial cells established that S. Typhimurium activates Rho family ...GTPases, primarily CDC42, to modulate the actin cytoskeletal network for invasion. The host intracellular protein network that engages CDC42 and influences the pathogen's invasive capacity are relatively unclear. Here, proteomic analyses of canonical and variant CDC42 interactomes identified a poorly characterized CDC42 interacting protein, CDC42EP1, whose intracellular localization is rapidly redistributed and aggregated around the invading bacteria. CDC42EP1 associates with SEPTIN‐7 and Villin, and its relocalization and bacterial engagement depend on host CDC42 and S. Typhimurium's capability of activating CDC42. Unlike CDC42, CDC42EP1 is not required for S. Typhimurium's initial cellular entry but is found to associate with Salmonella‐containing vacuoles after long‐term infections, indicating a contribution to the pathogen's intracellular growth and replication. These results uncover a new host regulator of enteric Salmonella infections, which may be targeted to restrict bacterial load at the primary site of infection to prevent systemic spread.
Lysozyme is a β-1,4-glycosidase that hydrolyzes the polysaccharide backbone of bacterial cell walls. With an additional bactericidal function mediated by a separate protein domain, lysozyme is ...considered a uniquely important antimicrobial molecule contributing to the host's innate immune response to infection. Elevated lysozyme production is found in various inflammatory conditions while patients with genetic risks for inflammatory bowel diseases demonstrate abnormal lysozyme expression, granule packaging, and secretion in Paneth cells. However, it remains unclear how a gain- or loss-of-function in host lysozyme may impact the host inflammatory responses to pathogenic infection. We challenged Lyz1−/− and ectopic Lyz1-expressing (Villin-Lyz1TG) mice with S. Typhimurium and then comprehensively assessed the inflammatory disease progression. We conducted proteomics analysis to identify molecules derived from human lysozyme-mediated processing of live Salmonella. We examined the barrier-impairing effects of these identified molecules in human intestinal epithelial cell monolayer and enteroids. Lyz1−/− mice are protected from infection in terms of morbidity, mortality, and barrier integrity, whereas Villin-Lyz1TG mice demonstrate exacerbated infection and inflammation. The growth and invasion of Salmonella in vitro are not affected by human or chicken lysozyme, whereas lysozyme encountering of live Salmonella stimulates the release of barrier-disrupting factors, InvE-sipC and Lpp1, which directly or indirectly impair the tight junctions. The direct engagement of host intestinal lysozyme with an enteric pathogen such as Salmonella promotes the release of virulence factors that are barrier-impairing and pro-inflammatory. Controlling lysozyme function may help alleviate the inflammatory progression.
Paneth cells are the primary source of C-type lysozyme, a β-1,4-N-acetylmuramoylhydrolase that enzymatically processes bacterial cell walls. Paneth cells are normally present in human cecum and ...ascending colon, but are rarely found in descending colon and rectum; Paneth cell metaplasia in this region and aberrant lysozyme production are hallmarks of inflammatory bowel disease (IBD) pathology. Here, we examined the impact of aberrant lysozyme production in colonic inflammation. Targeted disruption of Paneth cell lysozyme (Lyz1) protected mice from experimental colitis. Lyz1-deficiency diminished intestinal immune responses to bacterial molecular patterns and resulted in the expansion of lysozyme-sensitive mucolytic bacteria, including Ruminococcus gnavus, a Crohn’s disease-associated pathobiont. Ectopic lysozyme production in colonic epithelium suppressed lysozyme-sensitive bacteria and exacerbated colitis. Transfer of R. gnavus into Lyz1−/− hosts elicited a type 2 immune response, causing epithelial reprograming and enhanced anti-colitogenic capacity. In contrast, in lysozyme-intact hosts, processed R. gnavus drove pro-inflammatory responses. Thus, Paneth cell lysozyme balances intestinal anti- and pro-inflammatory responses, with implications for IBD.
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•Abnormal production of Paneth cell lysozyme is a feature of human IBD•Intestinal luminal lysozyme determines the abundance of mucolytic commensal bacteria•Processing lysozyme-sensitive bacterial cell wall drives colitis in Lyz1+/+ hosts•Lyz1−/− mice have reduced NLR signaling but an anti-colitogenic type 2 immune response
Paneth cell metaplasia in the colon and rectum and aberrant lysosome production are hallmarks of inflammatory bowel disease in humans. Using mouse models where Lyz1 is deleted or ectopically expressed, Yu et al. show that Paneth cell lysozyme regulates the abundance of mucolytic commensal bacteria and thereby the intestinal inflammatory response.
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