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Long-lived multipotent stem cells (ISCs) at the base of intestinal crypts adjust their phenotypes to accommodate normal maintenance and post-injury regeneration of the epithelium. Their long life,lineage plasticity,and proliferative potential underlie the necessity for tight homeostatic regulation of the ISC compartment. In that context,the guanylate cyclase C (GUCY2C) receptor and its paracrine ligands regulate intestinal epithelial homeostasis,including proliferation,lineage commitment,and DNA damage repair. However,a role for this axis in maintaining ISCs remains unknown. Transgenic mice enabling analysis of ISCs (Lgr5-GFP) in the context of GUCY2C elimination (Gucy2c -/- ) were combined with immunodetection techniques and pharmacological treatments to define the role of the GUCY2C signaling axis in supporting ISCs. ISCs were reduced inGucy2c -/- mice,associated with loss of active Lgr5+cells but a reciprocal increase in reserve Bmi1+cells. GUCY2C was expressed in crypt base Lgr5+cells in which it mediates canonical cyclic (c) GMP-dependent signaling. Endoplasmic reticulum (ER) stress,typically absent from ISCs,was elevated throughout the crypt base inGucy2c -/- mice. The chemical chaperone tauroursodeoxycholic acid resolved this ER stress and restored the balance of ISCs,an effect mimicked by the GUCY2C effector 8Br-cGMP. Reduced ISCs inGucy2c -/- mice was associated with greater epithelial injury and impaired regeneration following sub-lethal doses of irradiation. These observations suggest that GUCY2C provides homeostatic signals that modulate ER stress and cell vulnerability as part of the machinery contributing to the integrity of ISCs. View Publication

Background and Aims Disturbance of intestinal homeostasis is associated with the development of inflammatory bowel disease (IBD),and TGF-beta$ signaling impairment in mononuclear phagocytes (MPs) causes murine colitis with goblet cell depletion. Here,we examined an organoid-MP co-culture system to study the role of MPs in intestinal epithelial differentiation and homeostasis. Methods Intestinal organoids were co-cultured with lamina propria leukocytes and bone marrow-derived dendritic cells (BMDCs) from CD11c-cre Tgfbr2fl/fl mice. Organoid-MP adhesive interactions were evaluated by microscopy,RT-PCR,and flow cytometry. Murine colitis models (dextran sodium sulphate (DSS),CD11c-cre Tgfbr2fl/fl,T-cell-transfer) were used for histological and immunohistochemical analysis. Anti-E-cadherin antibody treatment or CD11c+-cell-specific CDH1 gene deletion were performed for E-cadherin neutralization or knockout. Colonic biopsies from patients with ulcerative colitis were analyzed by flow cytometry. Results Intestinal organoids co-cultured with CD11c+ lamina propria leukocytes or BMDCs from CD11c-cre Tgfbr2fl/fl mice showed morphological changes and goblet cell depletion with Notch signal activation,analogous to CD11c-cre Tgfbr2fl/fl colitis. E-cadherin was upregulated in CD11c+ MPs,especially CX3CR1+CCR2+ monocytes,of CD11c-cre Tgfbr2fl/fl mice. E-cadherin-mediated BMDC adhesion promoted Notch activation and cystic changes in organoids. Anti-E-cadherin antibody treatment attenuated colitis in CD11c-cre Tgfbr2fl/fl and T-cell-transferred mice. In addition,E-cadherin deletion in CD11c+ cells attenuated colitis in both CD11c-cre Tgfbr2fl/fl and DSS-treated mice. In patients with ulcerative colitis,E-cadherin expressed by intestinal CD11c+ leukocytes was enhanced compared with that in healthy controls. Conclusions E-cadherin-mediated MP-epithelium adhesion is associated with the development of colitis,and blocking these adhesions may have therapeutic potential for IBD. View Publication

BackgroundThere is substantial evidence that signaling through Toll-like receptor 4 (TLR4) contributes to the pathogenesis of necrotizing enterocolitis (NEC). Pregnane X receptor (PXR),a xenobiotic sensor and signaling intermediate for certain host-bacterial metabolites,has been shown to negatively regulate TLR4 signaling. Here we investigated the relationship between PXR and TLR4 in the developing murine intestine and explored the capacity of PXR to modulate inflammatory pathways involved in experimental NEC.MethodsWild-type and PXR-/- mice were studied at various time points of development in an experimental model of NEC. In addition,we studied the ability of the secondary bile acid lithocholic acid (LCA),a known PXR agonist in liver,to activate intestinal PXR and reduce NEC-related intestinal inflammation.ResultsWe found a reciprocal relationship between the developmental expression of PXR and TLR4 in wild-type murine intestine,with PXR acting to reduce TLR4 expression by decreasing TLR4 mRNA stability. In addition,PXR-/- mice exhibited a remarkably heightened severity of disease in experimental NEC. Moreover,LCA attenuated intestinal proinflammatory responses in the early stages of experimental NEC.ConclusionThese findings provide proactive insights into the regulation of TLR4 in the developing intestine. Targeting PXR may be a novel approach for NEC prevention. View Publication

Intraepithelial lymphocytes (IELs) expressing the gamma$delta$ TCR (gamma$delta$ IELs) provide continuous surveillance of the intestinal epithelium. However,the mechanisms regulating the basal motility of these cells within the epithelial compartment have not been well defined. We investigated whether IL-15 contributes to gamma$delta$ IEL localization and migratory behavior in addition to its role in IEL differentiation and survival. Using advanced live cell imaging techniques in mice,we find that compartmentalized overexpression of IL-15 in the lamina propria shifts the distribution of gamma$delta$ T cells from the epithelial compartment to the lamina propria. This mislocalization could be rescued by epithelial IL-15 overexpression,indicating that epithelial IL-15 is essential for gamma$delta$ IEL migration into the epithelium. Furthermore,in vitro analyses demonstrated that exogenous IL-15 stimulates gamma$delta$ IEL migration into cultured epithelial monolayers,and inhibition of IL-2Rbeta$ significantly attenuates the basal motility of these cells. Intravital microscopy showed that impaired IL-2Rbeta$ signaling induced gamma$delta$ IEL idling within the lateral intercellular space,which resulted in increased early pathogen invasion. Similarly,the redistribution of gamma$delta$ T cells to the lamina propria due to local IL-15 overproduction also enhanced bacterial translocation. These findings thus reveal a novel role for IL-15 in mediating gamma$delta$ T cell localization within the intestinal mucosa and regulating gamma$delta$ IEL motility and patrolling behavior as a critical component of host defense. View Publication

Whipworms are parasitic nematodes that live in the gut of more than 500 million people worldwide. Owing to the difficulty in obtaining parasite material,the mouse whipwormTrichuris murishas been extensively used as a model to study human whipworm infections. These nematodes secrete a multitude of compounds that interact with host tissues where they orchestrate a parasitic existence. Herein we provide the first comprehensive characterization of the excretory/secretory products ofT. muris. We identify 148 proteins secreted byT. murisand show for the first time that the mouse whipworm secretes exosome-like extracellular vesicles (EVs) that can interact with host cells. We use an Optiprep{\textregistered} gradient to purify the EVs,highlighting the suitability of this method for purifying EVs secreted by a parasitic nematode. We also characterize the proteomic and genomic content of the EVs,identifying {\textgreater}350 proteins,56 miRNAs (22 novel) and 475 full-length mRNA transcripts mapping toT. murisgene models. Many of the miRNAs putatively mapped to mouse genes are involved in regulation of inflammation,implying a role in parasite-driven immunomodulation. In addition,for the first time to our knowledge,colonic organoids have been used to demonstrate the internalization of parasite EVs by host cells. Understanding how parasites interact with their host is crucial to develop new control measures. This first characterization of the proteins and EVs secreted byT. murisprovides important information on whipworm-host communication and forms the basis for future studies. View Publication

The sympathetic (SNS) and parasympathetic (PNS) branches of the autonomic nervous system have been implicated in the modulation of the renewal of many tissues,including the intestinal epithelium. However,it is not known whether these mechanisms are direct,requiring an interaction between autonomic neurotransmitters and receptors on proliferating epithelial cells. To evaluate the existence of a molecular framework for a direct effect of the SNS or PNS on intestinal epithelial renewal,we measured gene expression for the main autonomic neurotransmitter receptors in this tissue. We separately evaluated intestinal epithelial regions comprised of the stem,progenitor,and mature cells,which allowed us to investigate the distinct contributions of each cell population to this proposed autonomic effect. Notably,we found that the stem cells expressed the receptors for the SNS-associated alpha2A adrenoreceptor and the PNS-associated muscarinic acetylcholine receptors (M1 and M3). In a separate experiment,we found that the application of norepinephrine or acetylcholine decreases the expression of cyclin D1,a gene necessary for cell cycle progression,in intestinal epithelial organoids compared with controls (P {\textless} 0.05). Together,these results provide evidence of a direct mechanism for the autonomic nervous system influence on intestinal epithelial stem cell proliferation. View Publication

Tryptamine,a tryptophan-derived monoamine similar to 5-hydroxytryptamine (5-HT),is produced by gut bacteria and is abundant in human and rodent feces. However,the physiologic effect of tryptamine in the gastrointestinal (GI) tract remains unknown. Here,we show that the biological effects of tryptamine are mediated through the 5-HT4 receptor (5-HT4R),a G-protein-coupled receptor (GPCR) uniquely expressed in the colonic epithelium. Tryptamine increases both ionic flux across the colonic epithelium and fluid secretion in colonoids from germ-free (GF) and humanized (ex-GF colonized with human stool) mice,consistent with increased intestinal secretion. The secretory effect of tryptamine is dependent on 5-HT4R activation and is blocked by 5-HT4R antagonist and absent in 5-HT4R-/- mice. GF mice colonized by Bacteroides thetaiotaomicron engineered to produce tryptamine exhibit accelerated GI transit. Our study demonstrates an aspect of host physiology under control of a bacterial metabolite that can be exploited as a therapeutic modality. VIDEO ABSTRACT. View Publication

Multiciliated cells (MCCs) promote fluid flow through coordinated ciliary beating,which requires properly organized basal bodies (BBs). Airway MCCs have large numbers of BBs,which are uniformly oriented and,as we show here,align linearly. The mechanism for BB alignment is unexplored. To study this mechanism,we developed a long-term and high-resolution live-imaging system and used it to observe green fluorescent protein"centrin2"labeled BBs in cultured mouse tracheal MCCs. During MCC differentiation,the BB array adopted four stereotypical patterns,from a clustering floret? pattern to the linear alignment.? This alignment process was correlated with BB orientations,revealed by double immunostaining for BBs and their asymmetrically associated basal feet (BF). The BB alignment was disrupted by disturbing apical microtubules with nocodazole and by a BF-depleting Odf2 mutation. We constructed a theoretical model,which indicated that the apical cytoskeleton,acting like a viscoelastic fluid,provides a self-organizing mechanism in tracheal MCCs to align BBs linearly for mucociliary transport. View Publication

BACKGROUND The Rhinovirus C (RV-C),first identified in 2006,produce high symptom burdens in children and asthmatics,however,their primary target host cell in the airways remains unknown. Our primary hypotheses were that RV-C target ciliated airway epithelial cells (AECs),and that cell specificity is determined by restricted and high expression of the only known RV-C cell-entry factor,cadherin related family member 3 (CDHR3). METHODS RV-C15 (C15) infection in differentiated human bronchial epithelial cell (HBEC) cultures was assessed using immunofluorescent and time-lapse epifluorescent imaging. Morphology of C15-infected differentiated AECs was assessed by immunohistochemistry. RESULTS C15 produced a scattered pattern of infection,and infected cells were shed from the epithelium. The percentage of cells infected with C15 varied from 1.4 to 14.7% depending on cell culture conditions. Infected cells had increased staining for markers of ciliated cells (acetylated-alpha-tubulin [aat],p < 0.001) but not markers of goblet cells (wheat germ agglutinin or Muc5AC,p = ns). CDHR3 expression was increased on ciliated epithelial cells,but not other epithelial cells (p < 0.01). C15 infection caused a 27.4% reduction of ciliated cells expressing CDHR3 (p < 0.01). During differentiation of AECs,CDHR3 expression progressively increased and correlated with both RV-C binding and replication. CONCLUSIONS The RV-C only replicate in ciliated AECs in vitro,leading to infected cell shedding. CDHR3 expression positively correlates with RV-C binding and replication,and is largely confined to ciliated AECs. Our data imply that factors regulating differentiation and CDHR3 production may be important determinants of RV-C illness severity. View Publication

Bio-engineered organs for transplantation may ultimately provide a personalized solution for end-stage organ failure,without the risk of rejection. Building upon the process of whole organ perfusion decellularization,we aimed to develop novel,translational methods for the recellularization and regeneration of transplantable lung constructs. We first isolated a proliferative KRT5+TP63+ basal epithelial stem cell population from human lung tissue and demonstrated expansion capacity in conventional 2D culture. We then repopulated acellular rat scaffolds in ex vivo whole organ culture and observed continued cell proliferation,in combination with primary pulmonary endothelial cells. To show clinical scalability,and to test the regenerative capacity of the basal cell population in a human context,we then recellularized and cultured isolated human lung scaffolds under biomimetic conditions. Analysis of the regenerated tissue constructs confirmed cell viability and sustained metabolic activity over 7 days of culture. Tissue analysis revealed extensive recellularization with organized tissue architecture and morphology,and preserved basal epithelial cell phenotype. The recellularized lung constructs displayed dynamic compliance and rudimentary gas exchange capacity. Our results underline the regenerative potential of patient-derived human airway stem cells in lung tissue engineering. We anticipate these advances to have clinically relevant implications for whole lung bioengineering and ex vivo organ repair. View Publication

Little is known about monocyte differentiation in the lung mucosal environment and about how the epithelium shapes monocyte function. We studied the role of the soluble component of bronchial epithelial cells (BECs) obtained under basal culture conditions in innate and adaptive monocyte responses. Monocytes cultured in bronchial epithelial cell-conditioned media (BEC-CM) specifically upregulate CD141,CD123,and DC-SIGN surface levels andFLT3expression,as well as the release of IL-1β,IL-6,and IL-10. BEC-conditioned monocytes stimulate naive T cells to produce IL-17 through IL-1β mechanism and also trigger IL-10 production by memory T cells. Furthermore,monocytes cultured in an inflammatory environment induced by the cytokines IL-6,IL-8,IL-1β,IL-15,TNF-α,and GM-CSF also upregulate CD123 and DC-SIGN expression. However,only inflammatory cytokines in the epithelial environment boost the expression of CD141. Interestingly,we identified a CD141/CD123/DC-SIGN triple positive population in the bronchoalveolar lavage fluid (BALF) from patients with different inflammatory conditions,demonstrating that this monocyte population existsin vivo. The frequency of this monocyte population was significantly increased in patients with sarcoidosis,suggesting a role in inflammatory mechanisms. Overall,these data highlight the specific role that the epithelium plays in shaping monocyte responses. Therefore,the unraveling of these mechanisms contributes to the understanding of the function that the epithelium may playin vivo. View Publication

Human bocavirus 1 (HBoV1) is a human parvovirus that causes acute respiratory tract infections in young children. In this study,we confirmed that,when polarized/well-differentiated human airway epithelia are infected with HBoV1in vitro,they develop damage characterized by barrier function disruption and cell hypotrophy. Cell death mechanism analyses indicated that the infection induced pyroptotic cell death characterized by caspase-1 activation. Unlike infections with other parvoviruses,HBoV1 infection did not activate the apoptotic or necroptotic cell death pathway. When the NLRP3-ASC-caspase-1 inflammasome-induced pathway was inhibited by short hairpin RNA (shRNA),HBoV1-induced cell death dropped significantly; thus,NLRP3 mediated by ASC appears to be the pattern recognition receptor driving HBoV1 infection-induced pyroptosis. HBoV1 infection induced steady increases in the expression of interleukin 1α (IL-1α) and IL-18. HBoV1 infection was also associated with the marked expression of the antiapoptotic genesBIRC5andIFI6When the expression ofBIRC5and/orIFI6was inhibited by shRNA,the infected cells underwent apoptosis rather than pyroptosis,as indicated by increased cleaved caspase-3 levels and the absence of caspase-1.BIRC5and/orIFI6gene inhibition also significantly reduced HBoV1 replication. Thus,HBoV1 infection of human airway epithelial cells activates antiapoptotic proteins that suppress apoptosis and promote pyroptosis. This response may have evolved to confer a replicative advantage,thus allowing HBoV1 to establish a persistent airway epithelial infection. This is the first report of pyroptosis in airway epithelia infected by a respiratory virus.IMPORTANCEMicrobial infection of immune cells often induces pyroptosis,which is mediated by a cytosolic protein complex called the inflammasome that senses microbial pathogens and then activates the proinflammatory cytokines IL-1 and IL-18. While virus-infected airway epithelia often activate NLRP3 inflammasomes,studies to date suggest that these viruses kill the airway epithelial cells via the apoptotic or necrotic pathway; involvement of the pyroptosis pathway has not been reported previously. Here,we show for the first time that virus infection of human airway epithelia can also induce pyroptosis. Human bocavirus 1 (HBoV1),a human parvovirus,causes lower respiratory tract infections in young children. This study indicates that HBoV1 kills airway epithelial cells by activating genes that suppress apoptosis and thereby promote pyroptosis. This strategy appears to promote HBoV1 replication and may have evolved to allow HBoV1 to establish persistent infection of human airway epithelia. View Publication