Tuft cell-derived acetylcholine promotes epithelial chloride secretion and intestinal helminth clearance.

Epithelial cells secrete chloride to regulate water release at mucosal barriers, supporting both homeostatic hydration and the "weep" response that is critical for type 2 immune defense against parasitic worms (helminths). Epithelial tuft cells in the small intestine sense helminths and release cytokines and lipids to activate type 2 immune cells, but whether they regulate epithelial secretion is unknown. Here, we found that tuft cell activation rapidly induced epithelial chloride secretion in the small intestine. This response required tuft cell sensory functions and tuft cell-derived acetylcholine (ACh), which acted directly on neighboring epithelial cells to stimulate chloride secretion, independent of neurons. Maximal tuft cell-induced chloride secretion coincided with immune restriction of helminths, and clearance was delayed in mice lacking tuft cell-derived ACh, despite normal type 2 inflammation. Thus, we have uncovered an epithelium-intrinsic response unit that uses ACh to couple tuft cell sensing to the secretory defenses of neighboring epithelial cells.

A Tuft Act to Follow: Leukotrienes Take the Stage in Anti-worm Immunity.

Tuft cells are specialized taste-chemosensory cells that detect the presence of intestinal parasites and orchestrate type 2 immunity. In this issue of Immunity, McGinty et al. discover that parasitic worms, but not commensal protists, stimulate tuft cells to release cysteinyl leukotrienes to amplify anti-helminth immunity in the small intestine.

Tuft cells mediate commensal remodeling of the small intestinal antimicrobial landscape.

Succinate produced by the commensal protist Tritrichomonas musculis (T. mu) stimulates chemosensory tuft cells, resulting in intestinal type 2 immunity. Tuft cells express the succinate receptor SUCNR1, yet this receptor does not mediate antihelminth immunity nor alter protist colonization. Here, we report that microbial-derived succinate increases Paneth cell numbers and profoundly alters the antimicrobial peptide (AMP) landscape in the small intestine. Succinate was sufficient to drive this epithelial remodeling, but not in mice lacking tuft cell chemosensory components required to detect this metabolite. Tuft cells respond to succinate by stimulating type 2 immunity, leading to interleukin-13-mediated epithelial and AMP expression changes. Moreover, type 2 immunity decreases the total number of mucosa-associated bacteria and alters the small intestinal microbiota composition. Finally, tuft cells can detect short-term bacterial dysbiosis that leads to a spike in luminal succinate levels and modulate AMP production in response. These findings demonstrate that a single metabolite produced by commensals can markedly shift the intestinal AMP profile and suggest that tuft cells utilize SUCNR1 and succinate sensing to modulate bacterial homeostasis.

An infection-induced oxidation site regulates legumain processing and tumor growth.

Oxidative stress is a defining feature of most cancers, including those that stem from carcinogenic infections. Reactive oxygen species can drive tumor formation, yet the molecular oxidation events that contribute to tumorigenesis are largely unknown. Here we show that inactivation of a single, redox-sensitive cysteine in the host protease legumain, which is oxidized during infection with the gastric cancer-causing bacterium Helicobacter pylori, accelerates tumor growth. By using chemical proteomics to map cysteine reactivity in human gastric cells, we determined that H. pylori infection induces oxidation of legumain at Cys219. Legumain oxidation dysregulates intracellular legumain processing and decreases the activity of the enzyme in H. pylori-infected cells. We further show that the site-specific loss of Cys219 reactivity increases tumor growth and mortality in a xenograft model. Our findings establish a link between an infection-induced oxidation site and tumorigenesis while underscoring the importance of cysteine reactivity in tumor growth.

Expression of the Z Variant of α1-Antitrypsin Suppresses Hepatic Cholesterol Biosynthesis in Transgenic Zebrafish.

Individuals homozygous for the Pi*Z allele of SERPINA1 (ZAAT) are susceptible to lung disease due to insufficient α1-antitrypsin secretion into the circulation and may develop liver disease due to compromised protein folding that leads to inclusion body formation in the endoplasmic reticulum (ER) of hepatocytes. Transgenic zebrafish expressing human ZAAT show no signs of hepatic accumulation despite displaying serum insufficiency, suggesting the defect in ZAAT secretion occurs independently of its tendency to form inclusion bodies. In this study, proteomic, transcriptomic, and biochemical analysis provided evidence of suppressed Srebp2-mediated cholesterol biosynthesis in the liver of ZAAT-expressing zebrafish. To investigate the basis for this perturbation, CRISPR/Cas9 gene editing was used to manipulate ER protein quality control factors. Mutation of erlec1 resulted in a further suppression in the cholesterol biosynthesis pathway, confirming a role for this ER lectin in targeting misfolded ZAAT for ER-associated degradation (ERAD). Mutation of the two ER mannosidase homologs enhanced ZAAT secretion without inducing hepatic accumulation. These insights into hepatic ZAAT processing suggest potential therapeutic targets to improve secretion and alleviate serum insufficiency in this form of the α1-antitrypsin disease.

High-resolution mapping reveals that microniches in the gastric glands control Helicobacter pylori colonization of the stomach.

Lifelong infection of the gastric mucosa by Helicobacter pylori can lead to peptic ulcers and gastric cancer. However, how the bacteria maintain chronic colonization in the face of constant mucus and epithelial cell turnover in the stomach is unclear. Here, we present a new model of how H. pylori establish and persist in stomach, which involves the colonization of a specialized microenvironment, or microniche, deep in the gastric glands. Using quantitative three-dimensional (3D) confocal microscopy and passive CLARITY technique (PACT), which renders tissues optically transparent, we analyzed intact stomachs from mice infected with a mixture of isogenic, fluorescent H. pylori strains with unprecedented spatial resolution. We discovered that a small number of bacterial founders initially establish colonies deep in the gastric glands and then expand to colonize adjacent glands, forming clonal population islands that persist over time. Gland-associated populations do not intermix with free-swimming bacteria in the surface mucus, and they compete for space and prevent newcomers from establishing in the stomach. Furthermore, bacterial mutants deficient in gland colonization are outcompeted by wild-type (WT) bacteria. Finally, we found that host factors such as the age at infection and T-cell responses control bacterial density within the glands. Collectively, our results demonstrate that microniches in the gastric glands house a persistent H. pylori reservoir, which we propose replenishes the more transient bacterial populations in the superficial mucosa.

A transgenic zebrafish model of hepatocyte function in human Z α1-antitrypsin deficiency.

In human α1-antitrypsin deficiency, homozygous carriers of the Z (E324K) mutation in the gene SERPINA1 have insufficient circulating α1-antitrypsin and are predisposed to emphysema. Misfolding and accumulation of the mutant protein in hepatocytes also causes endoplasmic reticulum stress and underpins long-term liver damage. Here, we describe transgenic zebrafish (Danio rerio) expressing the wildtype or the Z mutant form of human α1-antitrypsin in hepatocytes. As observed in afflicted humans, and in rodent models, about 80% less α1-antitrypsin is evident in the circulation of zebrafish expressing the Z mutant. Although these zebrafish also show signs of liver stress, they do not accumulate α1-antitrypsin in hepatocytes. This new zebrafish model will provide useful insights into understanding and treatment of α1-antitrypsin deficiency.

The effect of horticultural therapy on the quality of life of palliative care patients.

Palliative care patients experience a variety of needs and perceive their quality of life as being only fair. This study adopted a single-group repeated-measure design to investigate the effect of horticultural therapy on the quality of life of palliative care patients using the Quality of Life Concern in End of Life Questionnaire. Significant differences in the domains of "existential distress" and "health care concern" were observed immediately postintervention and at 4 weeks postintervention, respectively. No other significant differences were seen in the other domains or in the total mean score of the outcome measure.

A Toxoplasma palmitoyl acyl transferase and the palmitoylated armadillo repeat protein TgARO govern apical rhoptry tethering and reveal a critical role for the rhoptries in host cell invasion but not egress.

Apicomplexans are obligate intracellular parasites that actively penetrate their host cells to create an intracellular niche for replication. Commitment to invasion is thought to be mediated by the rhoptries, specialized apical secretory organelles that inject a protein complex into the host cell to form a tight-junction for parasite entry. Little is known about the molecular factors that govern rhoptry biogenesis, their subcellular organization at the apical end of the parasite and subsequent release of this organelle during invasion. We have identified a Toxoplasma palmitoyl acyltransferase, TgDHHC7, which localizes to the rhoptries. Strikingly, conditional knockdown of TgDHHC7 results in dispersed rhoptries that fail to organize at the apical end of the parasite and are instead scattered throughout the cell. While the morphology and content of these rhoptries appears normal, failure to tether at the apex results in a complete block in host cell invasion. In contrast, attachment and egress are unaffected in the knockdown, demonstrating that the rhoptries are not required for these processes. We show that rhoptry targeting of TgDHHC7 requires a short, highly conserved C-terminal region while a large, divergent N-terminal domain is dispensable for both targeting and function. Additionally, a point mutant lacking a key residue predicted to be critical for enzyme activity fails to rescue apical rhoptry tethering, strongly suggesting that tethering of the organelle is dependent upon TgDHHC7 palmitoylation activity. We tie the importance of this activity to the palmitoylated Armadillo Repeats-Only (TgARO) rhoptry protein by showing that conditional knockdown of TgARO recapitulates the dispersed rhoptry phenotype of TgDHHC7 knockdown. The unexpected finding that apicomplexans have exploited protein palmitoylation for apical organelle tethering yields new insight into the biogenesis and function of rhoptries and may provide new avenues for therapeutic intervention against Toxoplasma and related apicomplexan parasites.

Manipulation of Proteostasis Networks in Transgenic ZAAT Zebrafish via CRISPR-Cas9 Gene Editing.

The CRISPR-Cas9 genome editing system is used to induce mutations in genes of interest resulting in the loss of functional protein. A transgenic zebrafish α1-antitrypsin deficiency (AATD) model displays an unusual phenotype, in that it lacks the hepatic accumulation of the misfolding Z α1-antitrypsin (ZAAT) evident in human and mouse models. Here we describe the application of the CRISPR-Cas9 system to generate mutant zebrafish with defects in key proteostasis networks likely to be involved in the hepatic processing of ZAAT in this model. We describe the targeting of the atf6a and man1b1 genes as examples.

Tuft cell-derived acetylcholine regulates epithelial fluid secretion.

Tuft cells are solitary chemosensory epithelial cells that can sense lumenal stimuli at mucosal barriers and secrete effector molecules to regulate the physiology and immune state of their surrounding tissue. In the small intestine, tuft cells detect parasitic worms (helminths) and microbe-derived succinate, and signal to immune cells to trigger a Type 2 immune response that leads to extensive epithelial remodeling spanning several days. Acetylcholine (ACh) from airway tuft cells has been shown to stimulate acute changes in breathing and mucocilliary clearance, but its function in the intestine is unknown. Here we show that tuft cell chemosensing in the intestine leads to release of ACh, but that this does not contribute to immune cell activation or associated tissue remodeling. Instead, tuft cell-derived ACh triggers immediate fluid secretion from neighboring epithelial cells into the intestinal lumen. This tuft cell-regulated fluid secretion is amplified during Type 2 inflammation, and helminth clearance is delayed in mice lacking tuft cell ACh. The coupling of the chemosensory function of tuft cells with fluid secretion creates an epithelium-intrinsic response unit that effects a physiological change within seconds of activation. This response mechanism is shared by tuft cells across tissues, and serves to regulate the epithelial secretion that is both a hallmark of Type 2 immunity and an essential component of homeostatic maintenance at mucosal barriers.

Validation of the Chinese Non-pharmacological Therapy Experience Scale in persons with intellectual disability.

Aim: To validate the 4- and 7-point Chinese Non-pharmacological Therapy Experience Scales and test the psychometric properties of the scales on persons with intellectual disability. Design: A validation study. Methods: Sixty-seven persons with intellectual disability were recruited from six hostels or centres for persons with intellectual disability in Hong Kong. A total of 1,163 and 1,161 observations were collected by the trained observers with the 4-point and 7-point scales, respectively. The floor and ceiling effects, inter-rater reliability, internal consistency, responsiveness of both scales and the scale equivalence were examined. Results: The Cronbach's α of the 4- and 7-point scales was .762 and .797, respectively. The correlation between the two scales was 0.906. The inter-rater reliability of the 4- and 7-point scales was 0.774 and 0.835, respectively. Neither scale had the floor or ceiling effects. The effect size of the 7-point scale was consistently higher than that of the 4-point scale.

Effects of Horticulture on Frail and Prefrail Nursing Home Residents: A Randomized Controlled Trial.

OBJECTIVE: Frail nursing home residents face multiple health challenges as a result of their frail status. The aim of this study was to examine the effects of HT on the psychosocial well-being of frail and prefrail nursing home residents. DESIGN: Randomized controlled trial. SETTING: Nursing homes. PARTICIPANTS: One hundred eleven participants were randomly allocated into the intervention [horticultural therapy (HT)] and control (social activities) conditions. INTERVENTION: HT group participants attended a weekly 60-minute session for 8 consecutive weeks. Control group activities were social in nature, without any horticulture components. MEASUREMENTS: The outcome measures include happiness, depressive symptoms, self-efficacy, well-being, social network, and social engagement. The time points of measurement were at baseline (T0), immediately postintervention (T1), and 12 weeks postintervention (T2). A modified intention-to-treat approach was adopted. A multivariate general estimating equation was used to analyze the data. RESULTS: Forty-six and 50 participants received at least 1 session of the intervention and control condition protocol, respectively. A significant interaction effect between group and time was observed only on the happiness scale (ß = 1.457, P = .036), but not on other outcome variables. In a follow-up cluster analysis of those who received HT, a greater effect on subjective happiness (mean difference = 6.23, P < .001) was observed for participants who were happier at baseline. CONCLUSION: HT was found to be effective in promoting subjective happiness for frail and prefrail nursing home residents. Its favorable effect suggests that HT should be used to promote the psychosocial well-being of those who are frail.

Toxoplasma ISP4 is a central IMC sub-compartment protein whose localization depends on palmitoylation but not myristoylation.

Apicomplexan parasites utilize a peripheral membrane system called the inner membrane complex (IMC) to facilitate host cell invasion and parasite replication. We recently identified a novel family of Toxoplasma IMC Sub-compartment Proteins (ISP1/2/3) that localize to sub-domains of the IMC using a targeting mechanism that is dependent on coordinated myristoylation and palmitoylation of a series of residues in the N-terminus of the protein. While the precise functions of the ISPs are unknown, deletion of ISP2 results in replication defects, suggesting that this family of proteins plays a role in daughter cell formation. Here we have characterized a fourth ISP family member (ISP4) and discovered that this protein localizes to the central IMC sub-compartment, similar to ISP2. Like ISP1/3, ISP4 is dispensable for the tachyzoite lytic cycle as the disruption of ISP4 does not produce any gross replication or growth defects. Surprisingly, targeting of ISP4 to the IMC membranes is dependent on residues predicted for palmitoylation but not myristoylation, setting its trafficking apart from the other ISP proteins and demonstrating distinct mechanisms of protein localization to the IMC membranes, even within a family of highly related proteins.

Estimation of the basic reproduction number of enterovirus 71 and coxsackievirus A16 in hand, foot, and mouth disease outbreaks.

BACKGROUND: Coxsackievirus A16 (Cox A16) and enterovirus 71 (EV71) are common pathogens causing hand, foot, and mouth disease (HFMD) in pediatric population. Little is known about the basic reproductive number (R0) for these enteroviruses. METHODS: We estimated the R0 of EV71 and of Cox A16 from laboratory-confirmed HFMD outbreaks reported to the Department of Health, from 2004 to 2009. We derived a mathematical model and calculated R0 based on the cumulative number of cases at the initial growth phase of the outbreaks, as determined by the epidemic curves. We tested the association of R0 with settings and sizes of the institution and total number of persons affected. RESULTS: We analyzed 34 outbreaks, 27 caused by Cox A16 and 7 caused by EV71. Assuming the incubation period to be 5 days, the median R0 of EV71 was 5.48 with an interquartile range of 4.20 to 6.51, whereas the median R0 of Cox A16 was 2.50 with an interquartile range of 1.96 to 3.67. The R0 of EV71 was significantly higher than that of CoxA16, P = 0.002; and sensitivity analysis showed the same results. The R0 was not associated with outbreak settings, sizes of the institutions, or number of persons affected. CONCLUSIONS: The R0 for EV71 and for Cox A16 was determined using a model which showed that the R0 for EV71 was higher than that of Cox A16. This finding helps better understand the transmission dynamics of HFMD outbreaks and formulate public health measures for controlling the disease.