Peroxisome deficiency underlies failures in hepatic immune cell development and antigen presentation in a severe Zellweger disease model.

Peroxisome biogenesis disorders (PBDs) represent a group of metabolic conditions that cause severe developmental defects. Peroxisomes are essential metabolic organelles, present in virtually every eukaryotic cell and mediating key processes in immunometabolism. To date, the full spectrum of PBDs remains to be identified, and the impact PBDs have on immune function is unexplored. This study presents a characterization of the hepatic immune compartment of a neonatal PBD mouse model at single-cell resolution to establish the importance and function of peroxisomes in developmental hematopoiesis. We report that hematopoietic defects are a feature in a severe PBD murine model. Finally, we identify a role for peroxisomes in the regulation of the major histocompatibility class II expression and antigen presentation to CD4+ T cells in dendritic cells. This study adds to our understanding of the mechanisms of PBDs and expands our knowledge of the role of peroxisomes in immunometabolism.

The diverse roles of C-type lectin-like receptors in immunity.

Our understanding of the C-type lectin-like receptors (CTLRs) and their functions in immunity have continued to expand from their initial roles in pathogen recognition. There are now clear examples of CTLRs acting as scavenger receptors, sensors of cell death and cell transformation, and regulators of immune responses and homeostasis. This range of function reflects an extensive diversity in the expression and signaling activity between individual CTLR members of otherwise highly conserved families. Adding to this diversity is the constant discovery of new receptor binding capabilities and receptor-ligand interactions, distinct cellular expression profiles, and receptor structures and signaling mechanisms which have expanded the defining roles of CTLRs in immunity. The natural killer cell receptors exemplify this functional diversity with growing evidence of their activity in other immune populations and tissues. Here, we broadly review select families of CTLRs encoded in the natural killer cell gene complex (NKC) highlighting key receptors that demonstrate the complex multifunctional capabilities of these proteins. We focus on recent evidence from research on the NKRP1 family of CTLRs and their interaction with the related C-type lectin (CLEC) ligands which together exhibit essential immune functions beyond their defined activity in natural killer (NK) cells. The ever-expanding evidence for the requirement of CTLR in numerous biological processes emphasizes the need to better understand the functional potential of these receptor families in immune defense and pathological conditions.

Towards early detection of neurodegenerative diseases: A gut feeling.

The gastrointestinal tract communicates with the nervous system through a bidirectional network of signaling pathways called the gut-brain axis, which consists of multiple connections, including the enteric nervous system, the vagus nerve, the immune system, endocrine signals, the microbiota, and its metabolites. Alteration of communications in the gut-brain axis is emerging as an overlooked cause of neuroinflammation. Neuroinflammation is a common feature of the pathogenic mechanisms involved in various neurodegenerative diseases (NDs) that are incurable and debilitating conditions resulting in progressive degeneration and death of neurons, such as in Alzheimer and Parkinson diseases. NDs are a leading cause of global death and disability, and the incidences are expected to increase in the following decades if prevention strategies and successful treatment remain elusive. To date, the etiology of NDs is unclear due to the complexity of the mechanisms of diseases involving genetic and environmental factors, including diet and microbiota. Emerging evidence suggests that changes in diet, alteration of the microbiota, and deregulation of metabolism in the intestinal epithelium influence the inflammatory status of the neurons linked to disease insurgence and progression. This review will describe the leading players of the so-called diet-microbiota-gut-brain (DMGB) axis in the context of NDs. We will report recent findings from studies in model organisms such as rodents and fruit flies that support the role of diets, commensals, and intestinal epithelial functions as an overlooked primary regulator of brain health. We will finish discussing the pivotal role of metabolisms of cellular organelles such as mitochondria and peroxisomes in maintaining the DMGB axis and how alteration of the latter can be used as early disease makers and novel therapeutic targets.

Alveolar macrophage metabolic programming via a C-type lectin receptor protects against lipo-toxicity and cell death.

Alveolar macrophages (AM) hold lung homeostasis intact. In addition to the defense against inhaled pathogens and deleterious inflammation, AM also maintain pulmonary surfactant homeostasis, a vital lung function that prevents pulmonary alveolar proteinosis. Signals transmitted between AM and pneumocytes of the pulmonary niche coordinate these specialized functions. However, the mechanisms that guide the metabolic homeostasis of AM remain largely elusive. We show that the NK cell-associated receptor, NKR-P1B, is expressed by AM and is essential for metabolic programming. Nkrp1b-/- mice are vulnerable to pneumococcal infection due to an age-dependent collapse in the number of AM and the formation of lipid-laden AM. The AM of Nkrp1b-/- mice show increased uptake but defective metabolism of surfactant lipids. We identify a physical relay between AM and alveolar type-II pneumocytes that is dependent on pneumocyte Clr-g expression. These findings implicate the NKR-P1B:Clr-g signaling axis in AM-pneumocyte communication as being important for maintaining metabolism in AM.

Clr-f expression regulates kidney immune and metabolic homeostasis.

The C-type lectin-related protein, Clr-f, encoded by Clec2h in the mouse NK gene complex (NKC), is a member of a family of immune regulatory lectins that guide immune responses at distinct tissues of the body. Clr-f is highly expressed in the kidney; however, its activity in this organ is unknown. To assess the requirement for Clr-f in kidney health and function, we generated a Clr-f-deficient mouse (Clr-f-/-) by targeted deletions in the Clec2h gene. Mice lacking Clr-f exhibited glomerular and tubular lesions, immunoglobulin and C3 complement protein renal deposits, and significant abdominal and ectopic lipid accumulation. Whole kidney transcriptional profile analysis of Clr-f-/- mice at 7, 13, and 24 weeks of age revealed a dynamic dysregulation in lipid metabolic processes, stress responses, and inflammatory mediators. Examination of the immune contribution to the pathologies of Clr-f-/- mouse kidneys identified elevated IL-12 and IFNγ in cells of the tubulointerstitium, and an infiltrating population of neutrophils and T and B lymphocytes. The presence of these insults in a Rag1-/-Clr-f-/- background reveals that Clr-f-/- mice are susceptible to a T and B lymphocyte-independent renal pathogenesis. Our data reveal a role for Clr-f in the maintenance of kidney immune and metabolic homeostasis.

Modulation of the cell membrane lipid milieu by peroxisomal ß-oxidation induces Rho1 signaling to trigger inflammatory responses.

Phagocytosis, signal transduction, and inflammatory responses require changes in lipid metabolism. Peroxisomes have key roles in fatty acid homeostasis and in regulating immune function. We find that Drosophila macrophages lacking peroxisomes have perturbed lipid profiles, which reduce host survival after infection. Using lipidomic, transcriptomic, and genetic screens, we determine that peroxisomes contribute to the cell membrane glycerophospholipid composition necessary to induce Rho1-dependent signals, which drive cytoskeletal remodeling during macrophage activation. Loss of peroxisome function increases membrane phosphatidic acid (PA) and recruits RhoGAPp190 during infection, inhibiting Rho1-mediated responses. Peroxisome-glycerophospholipid-Rho1 signaling also controls cytoskeleton remodeling in mouse immune cells. While high levels of PA in cells without peroxisomes inhibit inflammatory phenotypes, large numbers of peroxisomes and low amounts of cell membrane PA are features of immune cells from patients with inflammatory Kawasaki disease and juvenile idiopathic arthritis. Our findings reveal potential metabolic markers and therapeutic targets for immune diseases and metabolic disorders.

Hematochezia in children with acute diarrhea seeking emergency department care - a prospective cohort study.

OBJECTIVES: Although the passage of blood in stools in children represents a medical emergency, children seeking emergency department (ED) care remain poorly characterized. Our primary objective was to compare clinical characteristics and etiologic pathogens in children with acute diarrhea with and without caregiver-reported hematochezia. Secondary objectives were to characterize interventions and resource utilization. METHODS: We conducted a secondary analysis of the Alberta Provincial Pediatric EnTeric Infection TEam (APPETITE) database. Children <18 years presenting to two pediatric EDs within a 24-hour period and <7 days of symptoms were consecutively recruited. RESULTS: Of 1,061 participants, 115 (10.8%) reported hematochezia at the enrollment visit at which time those with hematochezia, compared to those without, had more diarrheal episodes/24-hour period (9 vs. 6; difference: 2; 95% confidence interval [CI]: 2.0, 4.0; p < 0.001), and were less likely to have experienced vomiting (54.8% vs. 80.2%; difference: -25.4; 95% CI: -34.9, -16.0; p < 0.001). They were more likely to receive intravenous fluids (33.0% vs. 17.9%; difference: 15.2; 95% CI: 6.2, 24.1; p < 0.001) and require repeat health care visits (45.5% vs. 34.7%; difference: 10.7; 95% CI: 0.9, 20.6; p = 0.03). A bacterial pathogen was identified in 33.0% of children with hematochezia versus 7.9% without (difference: 25.1; 95% CI: 16.3, 33.9; p < 0.001); viruses were detected in 31.3% of children with hematochezia compared to 72.3% in those without (difference: -41.0%, 95% CI: -49.9, -32.1; p < 0.001). CONCLUSION: In children with acute diarrhea, caregiver report of hematochezia, compared to the absence of hematochezia, was associated with more diarrheal but fewer vomiting episodes, and greater resource consumption. The former group of children was also more likely to have bacteria detected in their stool.

Identification of Shiga-Toxin-Producing Shigella Infections in Travel and Non-Travel Related Cases in Alberta, Canada.

It has long been accepted that Shiga toxin (Stx) only exists in Shigella dysenteriae serotype 1. However, in recent decades, the presence of Shiga toxin genes (stx) in other Shigella spp. have been reported. We screened 366 Shigella flexneri strains from Alberta, Canada (2003 to 2016) for stx and 26 positive strains were identified. These isolates are highly related with the majority originating from the Dominican Republic and three isolates with Haiti origin. Both phylogenetic and spanning tree analysis of the 26 Alberta and 29 stx positive S. flexneri originating from the U.S., France, Canada (Quebec) and Haiti suggests that there are geographic specific distribution patterns (Haiti and Dominican Republic clades). This study provides the first comprehensive whole genome based phylogenetic analysis of stx positive S. flexneri strains as well as their global transmission, which signify the public health risks of global spreading of these strains.

Direct Clostridioides difficile ribotyping from stool using capillary electrophoresis.

Clostridioides difficile(C. difficile) genotyping is essential for surveillance of emerging strains, transmissions, and outbreak investigations, but culture is lengthy and may not be routinely performed, which necessitates culture-independent genotyping methods. We aimed to develop a direct from stool C. difficile PCR ribotyping algorithm using capillary electrophoresis. Ribotypes were generated directly from 66.8% of stools with 33.2% requiring broth enrichment. 16S and tcdB cycle thresholds (Ct) were significantly lower (P< 0.001) in directly ribotyped stools compared to enriched stools, and Ct correlated with direct ribotyping (area under the curve: 0.97 and 0.96, respectively). Direct and isolate ribotypes were 94.7% concordant. Mixed C. difficile ribotypes were presumptively identified in 14 (7.5%) samples with 12 (6.4%) mixtures confirmed. We have developed a rapid PCR ribotyping algorithm allowing for direct C. difficile genotyping from stool using capillary electrophoresis with occasional detection of mixed C. difficile populations in stool, which is a limitation of conventional isolate genotyping.

Association of Ct Values from Real-Time PCR with Culture in Microbiological Clearance Samples for Shiga Toxin-Producing Escherichia coli (STEC).

Shiga toxin-producing Escherichia coli (STEC) are associated with acute gastroenteritis worldwide, which induces a high economic burden on both healthcare and individuals. Culture-independent diagnostic tests (CIDT) in frontline microbiology laboratories have been implemented in Alberta since 2019. The objectives of this study were to determine the association between gene detection and culture positivity over time using STEC microbiological clearance samples and also to establish the frequency of specimen submission. Both stx genes' amplification by real-time PCR was performed with DNA extracted from stool samples using the easyMAG system. Stools were inoculated onto chromogenic agar for culture. An association between gene detection and culture positivity was found to be independent of which stx gene was present. CIDT can provide rapid reporting with less hands-on time and technical expertise. However, culture is still important for surveillance and early cluster detection. In addition, stool submissions could be reduced from daily to every 3-5 days until a sample is negative by culture.

A Central Role for Ly49 Receptors in NK Cell Memory.

In the past decade, the study of NK cells was transformed by the discovery of three ways these "innate" immune cells display adaptive immune behavior, including the ability to form long-lasting, Ag-specific memories of a wide variety of immunogens. In this review, we examine these types of NK cell memory, highlighting their unique features and underlying similarities. We explore those similarities in depth, focusing on the role that Ly49 receptors play in various types of NK cell memory. From this Ly49 dependency, we will build a model by which we understand the three types of NK cell memory as aspects of what is ultimately the same adaptive immune process, rather than separate facets of NK cell biology. We hope that a defined model for NK cell memory will empower collaboration between researchers of these three fields to further our understanding of this surprising and clinically promising immune response.

Defective Influenza A Virus RNA Products Mediate MAVS-Dependent Upregulation of Human Leukocyte Antigen Class I Proteins.

Influenza A virus (IAV) increases the presentation of class I human leukocyte antigen (HLA) proteins that limit antiviral responses mediated by natural killer (NK) cells, but molecular mechanisms for these processes have not yet been fully elucidated. We observed that infection with A/Fort Monmouth/1/1947(H1N1) IAV significantly increased the presentation of HLA-B, -C, and -E on lung epithelial cells. Virus entry was not sufficient to induce HLA upregulation because UV-inactivated virus had no effect. Aberrant internally deleted viral RNAs (vRNAs) known as mini viral RNAs (mvRNAs) and defective interfering RNAs (DI RNAs) expressed from an IAV minireplicon were sufficient for inducing HLA upregulation. These defective RNAs bind to retinoic acid-inducible gene I (RIG-I) and initiate mitochondrial antiviral signaling (MAVS) protein-dependent antiviral interferon (IFN) responses. Indeed, MAVS was required for HLA upregulation in response to IAV infection or ectopic mvRNA/DI RNA expression. The effect was partially due to paracrine signaling, as we observed that IAV infection or mvRNA/DI RNA-expression stimulated production of IFN-ß and IFN-λ1 and conditioned media from these cells elicited a modest increase in HLA surface levels in naive epithelial cells. HLA upregulation in response to aberrant viral RNAs could be prevented by the Janus kinase (JAK) inhibitor ruxolitinib. While HLA upregulation would seem to be advantageous to the virus, it is kept in check by the viral nonstructural 1 (NS1) protein; we determined that NS1 limits cell-intrinsic and paracrine mechanisms of HLA upregulation. Taken together, our findings indicate that aberrant IAV RNAs stimulate HLA presentation, which may aid viral evasion of innate immunity.IMPORTANCE Human leukocyte antigens (HLAs) are cell surface proteins that regulate innate and adaptive immune responses to viral infection by engaging with receptors on immune cells. Many viruses have evolved ways to evade host immune responses by modulating HLA expression and/or processing. Here, we provide evidence that aberrant RNA products of influenza virus genome replication can trigger retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral signaling (MAVS)-dependent remodeling of the cell surface, increasing surface presentation of HLA proteins known to inhibit the activation of an immune cell known as a natural killer (NK) cell. While this HLA upregulation would seem to be advantageous to the virus, it is kept in check by the viral nonstructural 1 (NS1) protein, which limits RIG-I activation and interferon production by the infected cell.

The Drosophila melanogaster as Genetic Model System to Dissect the Mechanisms of Disease that Lead to Neurodegeneration in Adrenoleukodystrophy.

Drosophila melanogaster is the most successful genetic model organism to study different human disease with a recent increased popularity to study neurological disorders. Drosophila melanogaster has a complex yet well-defined brain with defined anatomical regions with specific functions. The neuronal network in the adult brain has a structural organization highly similar to human neurons, but in a brain that is much more amenable for complex analyses. The availability of sophisticated genetic tools to study neurons permits to examine neuronal functions at the single cell level in the whole brain by confocal imaging, which does not require sections. Thus, Drosophila has been used to successfully study many neurological disorders such as Parkinson's disease and has been recently adopted to understand the complex networks leading to neurological disorders with metabolic origins such as Leigh disease and X-linked adrenoleukodystrophy (X-ALD).In this review, we will describe the genetic tools available to study neuronal structures and functions and also illustrate some limitations of the system. Finally, we will report the experimental efforts that in the past 10 years have established Drosophila melanogaster as an excellent model organism to study neurodegenerative disorders focusing on X-ALD.

Epidemiology of Shiga Toxin-Producing Escherichia coli O157 in the Province of Alberta, Canada, 2009-2016.

Shiga toxin-producing Escherichia coli (STEC) infections are the product of the interaction between bacteria, phages, animals, humans, and the environment. In the late 1980s, Alberta had one of the highest incidences of STEC infections in North America. Herein, we revisit and contextualize the epidemiology of STEC O157 human infections in Alberta for the period 2009-2016. STEC O157 infections were concentrated in large urban centers, but also in rural areas with high cattle density. Hospitalization was often required when the Shiga toxin genotype stx2a stx2c was involved, however, only those aged 60 years or older and infection during spring months (April to June) independently predicted that need. Since the late 1980s, the rate of STEC O157-associated hemolytic uremic syndrome (HUS) in Alberta has remained unchanged at 5.1%, despite a marked drop in the overall incidence of the infection. While Shiga toxin genotypes stx1a stx2c and stx2a stx2c seemed associated with HUS, only those aged under 10 years and infection during spring months were independently predictive of that complication. The complexity of the current epidemiology of STEC O157 in Alberta highlights the need for a One Health approach for further progress to be made in mitigating STEC morbidity.

Identification of Enteric Viruses in Oral Swabs from Children with Acute Gastroenteritis.

Stool is the diagnostic specimen of choice to identify enteropathogens in pediatric gastroenteritis. However, stool collection is challenging and its diagnostic characteristics in patients with isolated vomiting are unknown. Therefore, we evaluated if oral swabs are a suitable alternative specimen to stools. In total, 738 oral swabs and 577 stool specimens were collected from 738 children with vomiting and/or diarrhea. All specimens were tested by a laboratory-developed quantitative RT-PCR Gastroenteritis Virus Panel; 150 oral swabs and 577 stool specimens were tested by the commercial gastroenteritis pathogen panel. The Gastroenteritis Virus Panel identified adenovirus (n = 38), norovirus (n = 21), and rotavirus (n = 16) commonly in oral swabs. In stool specimens, rotavirus (n = 139), norovirus (n = 86), and adenovirus (n = 69) were detected commonly. Compared with stool specimens, the specificity of oral swabs was 99% (95% CI, 96%-100%); the sensitivity of oral swabs was 18% (95% CI, 14%-22%) for the detection of enteric viruses. The Gastrointestinal Pathogen Panel identified enteric bacteria and parasites in stool but not in oral swabs. Given the lower sensitivity of oral swabs, stool remains a preferable specimen to detect enteric viruses. However, with their high specificity, oral swabs can be considered as a suitable specimen if stool specimens are unavailable. Nevertheless, negative oral swabs require a confirmative test of stool specimens.

High genetic variability of norovirus leads to diagnostic test challenges.

BACKGROUND: It is important to understand the diagnostic accuracy of multiplex panels such as the Luminex xTAG® Gastrointestinal Pathogen Panel (GPP) as they are increasingly employed for routine diagnostics worldwide. Recent evaluations in our laboratory identified lower detection rates of norovirus genogroup II (NoV GII) using GPP compared to our laboratory-developed RT-qPCR, Gastroenteritis Virus Panel (GVP). OBJECTIVES: To characterize the cases of discordant NoV GII results between GPP and GVP and determine the sensitivity of the two assays for specific NoV GII genotypes. STUDY DESIGN: We genotyped discordant NoV GII strains identified in stool samples or rectal swabs collected prospectively from a cohort of children with acute gastroenteritis between December 2014 and July 2016. The sensitivities of GVP and GPP for NoV GII were compared by analyses of GVP threshold cycle (Ct) and ten-fold serial dilutions of positive samples of various NoV GII genotypes. RESULTS: All discordant samples (63/607) were NoV GII positive by GVP but negative by GPP. Twenty-two were successfully genotyped, fourteen of which were NoV GII genotype 2 (GII.2). The median Ct value of concordant positives was lower than that of discordant results (19.8 vs. 33.7; P<0.0001). GVP was 10 and at least 10,000-fold more sensitive than GPP in detecting NoV GII.3 and GII.2, respectively, but has similar sensitivity for NoV GII.4. Discordant GII.2 variant differed genetically from concordant GII.2 variants. CONCLUSIONS: GPP has lower sensitivity to detect NoV GII.2 than GVP and its use may lead to undetected cases clinically, and an underestimation of NoV disease burden at the population level.

Detection, Characterization, and Typing of Shiga Toxin-Producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) are responsible for gastrointestinal diseases reported in numerous outbreaks around the world. Given the public health importance of STEC, effective detection, characterization and typing is critical to any medical laboratory system. While non-O157 serotypes account for the majority of STEC infections, frontline microbiology laboratories may only screen for STEC using O157-specific agar-based methods. As a result, non-O157 STEC infections are significantly under-reported. This review discusses recent advances on the detection, characterization and typing of STEC with emphasis on work performed at the Alberta Provincial Laboratory for Public Health (ProvLab). Candidates for the detection of all STEC serotypes include chromogenic agars, enzyme immunoassays (EIA) and quantitative real time polymerase chain reaction (qPCR). Culture methods allow further characterization of isolates, whereas qPCR provides the greatest sensitivity and specificity, followed by EIA. The virulence gene profiles using PCR arrays and stx gene subtypes can subsequently be determined. Different non-O157 serotypes exhibit markedly different virulence gene profiles and a greater prevalence of stx1 than stx2 subtypes compared to O157:H7 isolates. Finally, recent innovations in whole genome sequencing (WGS) have allowed it to emerge as a candidate for the characterization and typing of STEC in diagnostic surveillance isolates. Methods of whole genome analysis such as single nucleotide polymorphisms and k-mer analysis are concordant with epidemiological data and standard typing methods, such as pulsed-field gel electrophoresis and multiple-locus variable number tandem repeat analysis while offering additional strain differentiation. Together these findings highlight improved strategies for STEC detection using currently available systems and the development of novel approaches for future surveillance.

A deregulated intestinal cell cycle program disrupts tissue homeostasis without affecting longevity in Drosophila.

Recent studies illuminate a complex relationship between the control of stem cell division and intestinal tissue organization in the model system Drosophila melanogaster. Host and microbial signals drive intestinal proliferation to maintain an effective epithelial barrier. Although it is widely assumed that proliferation induces dysplasia and shortens the life span of the host, the phenotypic consequences of deregulated intestinal proliferation for an otherwise healthy host remain unexplored. To address this question, we genetically isolated and manipulated the cell cycle programs of adult stem cells and enterocytes. Our studies revealed that cell cycle alterations led to extensive cell death and morphological disruptions. Despite the extensive tissue damage, we did not observe an impact on longevity, suggesting a remarkable degree of plasticity in intestinal function.

A direct phenotypic comparison of siRNA pools and multiple individual duplexes in a functional assay.

BACKGROUND: RNAi is a prominent tool for the identification of novel regulatory elements within complex cellular pathways. In invertebrates, RNAi is a relatively straightforward process, where large double-stranded RNA molecules initiate sequence-specific transcript destruction in target cells. In contrast, RNAi in mammalian cell culture assays requires the delivery of short interfering RNA duplexes to target cells. Due to concerns over off-target phenotypes and extreme variability in duplex efficiency, investigators typically deliver and analyze multiple duplexes per target. Currently, duplexes are delivered and analyzed either individually or as a pool of several independent duplexes. A choice between experiments based on siRNA pools or multiple individual duplexes has considerable implications for throughput, reagent requirements and data analysis in genome-wide surveys, yet there are relatively few data that directly compare the efficiency of the two approaches. METHODOLOGY/PRINCIPAL FINDINGS: To address this critical issue, we conducted a direct comparison of siRNA pools and multiple single siRNAs that target all human phosphatases in a robust functional assay. We determined the frequency with which both approaches uncover loss-of-function phenotypes and compared the phenotypic severity for siRNA pools and the constituent individual duplexes. CONCLUSIONS/SIGNIFICANCE: Our survey indicates that screens with siRNA pools have several significant advantages over identical screens with the corresponding individual siRNA duplexes. Of note, we frequently observed greater phenotypic penetrance for siRNA pools than for the parental individual duplexes. Thus, our data indicate that experiments with siRNA pools have a greater likelihood of generating loss-of-function phenotypes than individual siRNA duplexes.