[Show abstract][Hide abstract]ABSTRACT: Type 1 diabetes (T1D) occurs through a breakdown of self-tolerance resulting in the autoimmune destruction of the insulin producing β-islets of the pancreas. A numerical and functional waning of CD4+Foxp3+ regulatory T (Treg) cells, prompted by a pancreatic IL-2 deficiency, accompanies Th1 autoimmunity and T1D progression in non-obese diabetic (NOD) mice. Recently, we identified a dominant subset of intra-islet Treg cells that expresses the ICOS costimulatory receptor and promotes self-tolerance delaying the onset of T1D. ICOS co-stimulation potently enhances IL-2 induced survival and proliferation, and suppressive activity of Treg cells in situ. Here, we propose an ICOS-dependent mechanism of Treg cell homing to the β-islets during pre-diabetes in the NOD model via upregulation of the CXCR3 chemokine receptor. The islet-specific ICOS+ Treg cell subset preferentially expresses CXCR3 in the pancreatic lymph nodes (pLN) in response to Teff cell-mediated pancreatic inflammation, an expression correlating with the onset and magnitude of IFN-γ production by Teff cells in pancreatic sites. We also reveal that intra-pancreatic APC populations and insulin-producing β, but not α nor δ, islet cells secrete the CXCR3 chemokines, CXCL9, 10 and 11, and selectively promote ICOS+CXCR3+ Treg cell chemotaxis in vitro. Strikingly, islet-derived Treg cells also produce these chemokines suggesting an auto-regulation of homing by this subset. Unlike ICOS- cells, ICOS+ Treg cells adopt a Th1-like Treg phenotype while maintaining their suppressive capacity, characterized by expression of T-bet and CXCR3 and production of IFN-γ in the draining pLNs. Finally, in vivo neutralization of IFN-γ blocked Treg cell CXCR3 upregulation evincing its role in regulating expression of this chemokine receptor by Treg cells. Thus, CXCR3-mediated trafficking of Treg cells could represent a mechanism of homeostatic immunoregulation during diabetogeneesis.
[Show abstract][Hide abstract]ABSTRACT: Genetic and immunological analysis of host-pathogen interactions can reveal fundamental mechanisms of susceptibility and resistance to infection. Modeling human infectious diseases among inbred mouse strains is a proven approach but is limited by naturally occurring genetic diversity. Using N-ethyl-N-nitrosourea mutagenesis, we created a recessive loss-of-function point mutation in Unc93b1 (unc-93 homolog B1 (C. elegans)), a chaperone for endosomal Toll-like receptors (TLR)3, TLR7 and TLR9, which we termed Letr for 'loss of endosomal TLR response'. We used Unc93b1(Letr/Letr) mice to study the role of Unc93b1 in the immune response to influenza A/PR/8/34 (H1N1), an important global respiratory pathogen. During the early phase of infection, Unc93b1(Letr/Letr) mice had fewer activated exudate macrophages and decreased expression of CXCL10, interferon (IFN)-γ and type I IFN. Mutation of Unc93b1 also led to reduced expression of the CD69 activation marker and a concomitant increase in the CD62L naive marker on CD4(+) and CD8(+) T cells in infected lungs. Finally, loss of endosomal TLR signaling resulted in delayed viral clearance that coincided with increased tissue pathology during infection. Taken together, these findings establish a role for Unc93b1 and endosomal TLRs in the activation of both myeloid and lymphoid cells during the innate immune response to influenza.Genes and Immunity advance online publication, 22 May 2014; doi:10.1038/gene.2014.22.
[Show abstract][Hide abstract]ABSTRACT: Susceptibility to progressive infection with the fungus Cryptococcus neoformans is associated with an allergic pattern of lung inflammation, yet the factors that govern this host response are not clearly understood. Using a clinically relevant mouse model of inhalational infection with virulent C. neoformans H99, we demonstrate a role for IL-33-dependent signaling in host immune defense. Infection of BALB/c mice with 10(4) CFU of C. neoformans H99 caused a time-dependent induction of IL-33 with accumulation of type 2 pulmonary innate lymphoid cells and alternatively activated macrophages in the lungs as well as Th2-polarized CD4(+) T cells in draining lymph nodes. IL-33R subunit T1/ST2-deficient (T1/ST2(-/-)) mice infected with C. neoformans H99 had improved survival with a decreased fungal burden in the lungs, spleen, and brain, compared with wild-type mice. Signaling through T1/ST2 was required for the accumulation and early production of IL-5 and IL-13 by lung type 2 pulmonary innate lymphoid cells. Further analysis of T1/ST2(-/-) mice revealed increased fungicidal exudate macrophages in the lungs and decreased C. neoformans-specific Th2 cells in the mediastinal lymph nodes. T1/ST2 deficiency also diminished goblet cell hyperplasia, mucus hypersecretion, bronchoalveolar lavage eosinophilia, alternative activation of macrophages, and serum IgE. These observations demonstrate that IL-33-dependent signaling contributes to the expansion of innate type 2 immunity and subsequent Th2-biased lung immunopathology that facilitates C. neoformans growth and dissemination.
[Show abstract][Hide abstract]ABSTRACT: Quantification of VSV and Dengue virions. (A) Left panel: VSV virus titer from the supernatants from the experiment described in Fig. 6A was determined by standard plaque assay. Right panel: WCEs were analysed by western blotting for VSV protein expression. (B) Left panel: Dengue virus titer from the supernatants from the experiment described in Fig. 6A was determined by standard plaque assay. Right panel: RNA was extracted and analysed by real-time RT-PCR using primers specific for Dengue RNA (Forward 5′ CAA GGC GAG ATG AAG CTG TA 3′; Reverse 5′ GGT CTT TCC CAG CGT CAA TA 3′).
[Show abstract][Hide abstract]ABSTRACT: Stimulation of an immune response by 5′pppRNA in vivo. C57Bl/6 mice were injected intravenously with 25 ug of 5′pppRNA in complex with In vivo Jet-PEI or non-treated (NT, 0 h). (A) Serum (left panels) and lung homogenate (20% w/v in PBS; right panels) were collected at the indicated time point and ELISA for IFNβ (top panels) and IFNα (bottom panels) were performed. Error bars represent SEM from three different animals. (B) Spleen (left panels) and lungs (right panels) were collected at the indicated time point. RNA was extracted subjected to real-time RT-PCR. Error bars represent SEM from three different animals. (C) Lungs were collected at 6 h and 24 h post 5′pppRNA administration. Lungs were minced and digested with collagenase IV and DNase I for 30 minutes, with vigorous mixing after 15 minutes, and then filtered through a 70 µm nylon filter. Cells were analysed by flow cytometry and the proportion of cells relative to CD45+ leukocyte is reported. Error bars represent SEM from four different animals.
[Show abstract][Hide abstract]ABSTRACT: Transcriptional profiling following 5′pppRNA stimulation of A549 cells. (A) Left panel: Temporal analysis of up-regulated genes at 6 h (FC≥2 at 6 h). Right panel: Temporal analysis of genes that are exclusively induced significantly at 24 h (FC≤2 at 6 h and FC≥2 at 24 h). Genes with highest fold change under these conditions are listed. (B) Node analyses of transcriptome activation 6 h (left panel) and 24 h (right panel) post 10 ng/ml 5′pppRNA stimulation. Ingenuity Pathway Analysis software was used to generate transcriptome signalling networks centered around IRF1 and IRF7. Genes coloured in red were up-regulated, in green were down-regulated. (C) Node analyses of transcriptome activation 24 h post stimulation with either 10 ng/ml 5′pppRNA (left panel) or 1000 IU/ml IFNα-2b (right panel). Ingenuity Pathway Analysis Software was used to generate signaling networks surrounding IRF7 and NF-κB. Genes in red were up-regulated (FC≥2.0), in green were down-regulated (FC≤2.0).
[Show abstract][Hide abstract]ABSTRACT: Quantitative real-time RT-PCR validation of genes induced by 5′pppRNA treatment. Quantitative real-time RT-PCR was performed to validate the kinetic observed in the microarray for selected genes. A549 cells were transfected with 10 ng/ml of 5′pppRNA using Lipofectamine RNAiMax for designated periods of time. RNA was extracted from time-course samples and subjected to real-time RT-PCR analysis. Results are from a representative experiment; error bars represent SEM of three independent samples.
[Show abstract][Hide abstract]ABSTRACT: The RIG-I like receptor pathway is stimulated during RNA virus infection by interaction between cytosolic RIG-I and viral RNA structures that contain short hairpin dsRNA and 5' triphosphate (5'ppp) terminal structure. In the present study, an RNA agonist of RIG-I was synthesized in vitro and shown to stimulate RIG-I-dependent antiviral responses at concentrations in the picomolar range. In human lung epithelial A549 cells, 5'pppRNA specifically stimulated multiple parameters of the innate antiviral response, including IRF3, IRF7 and STAT1 activation, and induction of inflammatory and interferon stimulated genes - hallmarks of a fully functional antiviral response. Evaluation of the magnitude and duration of gene expression by transcriptional profiling identified a robust, sustained and diversified antiviral and inflammatory response characterized by enhanced pathogen recognition and interferon (IFN) signaling. Bioinformatics analysis further identified a transcriptional signature uniquely induced by 5'pppRNA, and not by IFNα-2b, that included a constellation of IRF7 and NF-kB target genes capable of mobilizing multiple arms of the innate and adaptive immune response. Treatment of primary PBMCs or lung epithelial A549 cells with 5'pppRNA provided significant protection against a spectrum of RNA and DNA viruses. In C57Bl/6 mice, intravenous administration of 5'pppRNA protected animals from a lethal challenge with H1N1 Influenza, reduced virus titers in mouse lungs and protected animals from virus-induced pneumonia. Strikingly, the RIG-I-specific transcriptional response afforded partial protection from influenza challenge, even in the absence of type I interferon signaling. This systems approach provides transcriptional, biochemical, and in vivo analysis of the antiviral efficacy of 5'pppRNA and highlights the therapeutic potential associated with the use of RIG-I agonists as broad spectrum antiviral agents.
[Show abstract][Hide abstract]ABSTRACT: Genetic factors that regulate the pathogenesis of pneumonia caused by the fungus Cryptococcus neoformans are poorly understood. Through a phenotypic strain survey we observed that inbred C3H/HeN mice develop a significantly greater
lung fungal burden than mice of the resistant CBA/J strain 4 weeks following intratracheal infection with C. neoformans ATCC 24067. The aim of the present study was to characterize the inflammatory response of C3H/HeN mice following C. neoformans pulmonary infection and to identify genetic loci that regulate host defense. Following cryptococcal infection, C3H/HeN mice
demonstrated a Th2 immune response with heightened airway and tissue eosinophilia, goblet cell metaplasia, and significantly
higher lung interleukin-5 (IL-5) and IL-13 protein expression relative to CBA/J mice. Conversely, CBA/J mice exhibited greater
airway and tissue neutrophilia that was associated with significantly higher pulmonary expression of gamma interferon, CXCL10,
and IL-17 proteins than C3H/HeN mice. Using the fungal burden at 4 weeks postinfection as a phenotype, genome-wide quantitative
trait locus (QTL) analysis among 435 segregating (C3H/HeN × CBA/J)F2 (C3HCBAF2) hybrids identified two significant QTLs on
chromosomes 1 (Cnes4) and 9 (Cnes5) that control susceptibility to cryptococcal pneumonia in an additive manner. Susceptible C3H/HeN mice carry a resistance
allele at Cnes4 and a susceptibility allele at Cnes5. These studies reveal additional genetic complexity of the host response to C. neoformans that is associated with divergent patterns of pulmonary inflammation.
Full-text Article · Sep 2012 · Infection and immunity
[Show abstract][Hide abstract]ABSTRACT: To determine the utility of angiopoietin-1 and angiopoietin-2 as potentially novel biomarkers of morbidity and mortality in patients with severe sepsis.
Multicenter longitudinal cohort study.
Three tertiary hospital intensive care units in Hamilton, Ontario, Canada.
A total of 70 patients with severe sepsis were enrolled within 24 hrs of meeting the inclusion criteria for severe sepsis and followed until day 28, hospital discharge, or death.
Clinical data and plasma samples were obtained at intensive care unit admission for all 70 patients and then daily for 1 wk and weekly thereafter for a subset of 43 patients. Levels of angiopoietin-1 and angiopoietin-2 in stored plasma samples were measured and compared with clinical characteristics, including the primary outcomes of 28-day mortality and morbidity measured by the Multiple Organ Dysfunction score.
Lower angiopoietin-1 plasma levels (≤ 5.5 ng/mL) at admission were associated with increased likelihood of death (relative risk 0.49 [95% confidence interval of 0.25-0.98], p = .046). Lower angiopoietin-1 levels remained a significant predictor of 28-day mortality in a multiple logistic regression model (adjusted odds ratio of 0.282 [95% confidence interval of 0.086-0.93], p = .037). Analysis of serial data using linear mixed models confirmed that sepsis survivors had higher levels of angiopoietin-1 (p = .012) and lower daily levels of angiopoietin-2 (p = .022) than nonsurvivors. Furthermore, survivors had higher peak angiopoietin-1 levels (median 13 vs. 10 ng/mL, p = .019) and lower nadir angiopoietin-2 levels (median 2.8 vs. 6.2 ng/mL, p = .013) than nonsurvivors. A score incorporating angiopoietin-1 and angiopoietin-2 and three other markers of endothelial activation discriminated with high accuracy between fatal and nonfatal cases (c-index of 0.80 [95% confidence interval of 0.69-0.90], p < .001). Plasma levels of angiopoietin-2 correlated with clinical markers of organ dysfunction and molecular markers of endothelial cell activation.
Angiopoietin-1 levels at admission and both angiopoietin-1 and angiopoietin-2 levels measured serially correlated with 28-day mortality in severe sepsis. Angiopoietin-2 levels also correlated with organ dysfunction/injury and a validated clinical sepsis score. These results suggest the use of angiopoietins as clinically informative biomarkers of disease severity and patient outcome in severe sepsis.
Full-text Article · Apr 2011 · Critical care medicine
[Show abstract][Hide abstract]ABSTRACT: By virtue of its direct contact with the environment, the lung is constantly challenged by infectious and non-infectious stimuli that necessitate a robust yet highly controlled host response coordinated by the innate and adaptive arms of the immune system. Mammalian Toll-like receptors (TLRs) function as crucial sentinels of microbial and non-infectious antigens throughout the respiratory tract and mediate host innate immunity. Selective induction of inflammatory responses to harmful environmental exposures and tolerance to innocuous antigens are required to maintain tissue homeostasis and integrity. Conversely, dysregulated innate immune responses manifest as sustained and self-perpetuating tissue damage rather than controlled tissue repair. In this article we review aspects of Toll-like receptor function that are relevant to the development of acute lung injury and chronic obstructive lung diseases as well as resistance to frequently associated microbial infections.
Full-text Article · Nov 2010 · Journal of Inflammation
[Show abstract][Hide abstract]ABSTRACT: Severe and cerebral malaria are associated with endothelial activation. Angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2) are major regulators of endothelial activation and integrity. The aim of this study was to investigate the clinical utility of whole blood angiopoietin (ANG) levels as biomarkers of disease severity in Plasmodium falciparum malaria.
The utility of whole blood ANG levels was examined in Thai patients to distinguish cerebral (CM; n = 87) and severe (non-cerebral) malaria (SM; n = 36) from uncomplicated malaria (UM; n = 70). Comparative statistics are reported using a non-parametric univariate analysis (Kruskal-Wallis test or Chi-squared test, as appropriate). Multivariate binary logistic regression was used to examine differences in whole blood protein levels between groups (UM, SM, CM), adjusting for differences due to ethnicity, age, parasitaemia and sex. Receiver operating characteristic curve analysis was used to assess the diagnostic accuracy of the ANGs in their ability to distinguish between UM, SM and CM. Cumulative organ injury scores were obtained for patients with severe disease based on the presence of acute renal failure, jaundice, severe anaemia, circulatory collapse or coma.
ANG-1 and ANG-2 were readily detectable in whole blood. Compared to UM there were significant decreases in ANG-1 (p < 0.001) and significant increases in ANG-2 (p < 0.001) levels and the ratio of ANG-2: ANG-1 (p < 0.001) observed in patients with SM and CM. This effect was independent of covariates (ethnicity, age, parasitaemia, sex). Further, there was a significant decrease in ANG-1 levels in patients with SM (non-cerebral) versus CM (p < 0.001). In participants with severe disease, ANG-2, but not ANG-1, levels correlated with cumulative organ injury scores; however, ANG-1 correlated with the presence of renal dysfunction and coma. Receiver operating characteristic curve analysis demonstrated that the level of ANG-1, the level of ANG-2 or the ratio of ANG-2: ANG-1 discriminated between individuals with UM and SM (area under the curve, p-value: ANG-2, 0.763, p < 0.001; ANG-1, 0.884, p < 0.001; Ratio, 0.857, p < 0.001) or UM and CM (area under the curve, p-value: ANG-2, 0.772, p < 0.001; ANG-1, 0.778, p < 0.001; Ratio, 0.820, p < 0.001).
These results suggest that whole blood ANG-1/2 levels are promising clinically informative biomarkers of disease severity in malarial syndromes.
[Show abstract][Hide abstract]ABSTRACT: Limited tools exist to identify which individuals infected with Plasmodium falciparum are at risk of developing serious complications such as cerebral malaria (CM). The objective of this study was to assess serum biomarkers that differentiate between CM and non-CM, with the long-term goal of developing a clinically informative prognostic test for severe malaria.
Based on the hypothesis that endothelial activation and blood-brain-barrier dysfunction contribute to CM pathogenesis, we examined the endothelial regulators, angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2), in serum samples from P. falciparum-infected patients with uncomplicated malaria (UM) or CM, from two diverse populations--Thai adults and Ugandan children. Angiopoietin levels were compared to tumour necrosis factor (TNF). In both populations, ANG-1 levels were significantly decreased and ANG-2 levels were significantly increased in CM versus UM and healthy controls (p<0.001). TNF was significantly elevated in CM in the Thai adult population (p<0.001), but did not discriminate well between CM and UM in African children. Receiver operating characteristic curve analysis showed that ANG-1 and the ratio of ANG-2:ANG-1 accurately discriminated CM patients from UM in both populations. Applied as a diagnostic test, ANG-1 had a sensitivity and specificity of 100% for distinguishing CM from UM in Thai adults and 70% and 75%, respectively, for Ugandan children. Across both populations the likelihood ratio of CM given a positive test (ANG-1<15 ng/mL) was 4.1 (2.7-6.5) and the likelihood ratio of CM given a negative test was 0.29 (0.20-0.42). Moreover, low ANG-1 levels at presentation predicted subsequent mortality in children with CM (p = 0.027).
ANG-1 and the ANG-2/1 ratio are promising clinically informative biomarkers for CM. Additional studies should address their utility as prognostic biomarkers and potential therapeutic targets in severe malaria.
[Show abstract][Hide abstract]ABSTRACT: Through the convergence of nano- and microtechnologies (quantum dots and microfluidics), we have created a diagnostic system capable of multiplexed, high-throughput analysis of infectious agents in human serum samples. We demonstrate, as a proof-of-concept, the ability to detect serum biomarkers of the most globally prevalent blood-borne infectious diseases (i.e., hepatitis B, hepatitis C, and HIV) with low sample volume (<100 microL), rapidity (<1 h), and 50 times greater sensitivity than that of currently available FDA-approved methods. We further show precision for detecting multiple biomarkers simultaneously in serum with minimal cross-reactivity. This device could be further developed into a portable handheld point-of-care diagnostic system, which would represent a major advance in detecting, monitoring, treating, and preventing infectious disease spread in the developed and developing worlds.