Transcriptomic Analysis of the Host Response and Innate Resilience to Enterotoxigenic Escherichia coli Infection in Humans.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a globally prevalent cause of diarrhea. Though usually self-limited, it can be severe and debilitating. Little is known about the host transcriptional response to infection. We report the first gene expression analysis of the human host response to experimental challenge with ETEC. METHODS: We challenged 30 healthy adults with an unattenuated ETEC strain, and collected serial blood samples shortly after inoculation and daily for 8 days. We performed gene expression analysis on whole peripheral blood RNA samples from subjects in whom severe symptoms developed (n = 6) and a subset of those who remained asymptomatic (n = 6) despite shedding. RESULTS: Compared with baseline, symptomatic subjects demonstrated significantly different expression of 406 genes highlighting increased immune response and decreased protein synthesis. Compared with asymptomatic subjects, symptomatic subjects differentially expressed 254 genes primarily associated with immune response. This comparison also revealed 29 genes differentially expressed between groups at baseline, suggesting innate resilience to infection. Drug repositioning analysis identified several drug classes with potential utility in augmenting immune response or mitigating symptoms. CONCLUSIONS: There are statistically significant and biologically plausible differences in host gene expression induced by ETEC infection. Differential baseline expression of some genes may indicate resilience to infection.

Immunostimulatory effects of different aspects of aquaculture on the host response in the edible sea urchin, paracentrotus lividus

Aquaculture is a fast-growing industry contributing to global food security and sustainable aquaculture, which may reduce pressures on capture fisheries. The overall objective of this thesis was to look at the immunostimulatory effects of different aspects of aquaculture on the host response of the edible sea urchin, Paracentrotus lividus, which are a prized delicacy (roe) in many Asian and Mediterranean countries. In Chapter 1, the importance of understanding the biology, ecology, and physiology of P. lividus, as well as the current status in the culture of this organism for mass production and introducing the thesis objectives for following chapters is discussed. As the research commenced, the difficulties of identifying individuals for repeat sampling became clear; therefore, Chapter 2 was a tagging experiment that indicated PIT tagging was a successful way of identifying individual sea urchins over time with a high tag retention rate. However, it was also found that repeat sampling via syringe to measure host response of an individual caused stress which masked results and thus animals would be sampled and sacrificed going forward. Additionally, from personal observations and discussion with peers, it was suggested to look at the effect that diet has on sea urchin immune function and the parameters I measured which led to Chapter 3. In this chapter, both Laminaria digitata and Mytilus edulis were shown to influence measured immune parameters of differential cell counts, nitric oxide production, and lysozyme activity. Therefore, trials commencing after Trial 5 in Chapter 4, were modified to include starvation in order to remove any effect of diet. Another important aspect of culturing any organism is the study of their immune function and its response to several immunostimulatory agents (Chapter 4). Zymosan A was shown to be an effective immunostimulatory agent in P. lividus. Further work on handled/stored animals (Chapter 5) showed Zymosan A reduced the measured levels of some immune parameters measured relative to the control, which may reduce the amount of stress in the animals. In Chapter 6, animals were infected with Vibrio anguillarum and, although V. anguillarum, impacted immune parameters of P. lividus, it did not cause mortality as predicted. Lastly, throughout this thesis work, it was noted that the immune parameters measured produced different values at different times of the year (Chapter 7); therefore, using collated baseline (control) data, results were compiled to observe seasonal effects. It was determined that both seasonality and sourcing sites influenced immune parameter measurements taken at different times throughout the year. In conclusion, this thesis work fits into the framework of development of aquaculture practices that affect immune function of the host and future research focusing on the edible sea urchin, P. lividus.

Tipping the balance : sclerotinia sclerotiorum secreted oxalic acid suppresses host defenses by manipulating the host redox environment

Sclerotinia sclerotiorum is a necrotrophic ascomycete fungus with an extremely broad host range. This pathogen produces the non-specific phytotoxin and key pathogenicity factor, oxalic acid (OA). Our recent work indicated that this fungus and more specifically OA, can induce apoptotic-like programmed cell death (PCD) in plant hosts, this induction of PCD and disease requires generation of reactive oxygen species (ROS) in the host, a process triggered by fungal secreted OA. Conversely, during the initial stages of infection, OA also dampens the plant oxidative burst, an early host response generally associated with plant defense. This scenario presents a challenge regarding the mechanistic details of OA function; as OA both suppresses and induces host ROS during the compatible interaction. In the present study we generated transgenic plants expressing a redox-regulated GFP reporter. Results show that initially, Sclerotinia (via OA) generates a reducing environment in host cells that suppress host defense responses including the oxidative burst and callose deposition, akin to compatible biotrophic pathogens. Once infection is established however, this necrotroph induces the generation of plant ROS leading to PCD of host tissue, the result of which is of direct benefit to the pathogen. In contrast, a non-pathogenic OA-deficient mutant failed to alter host redox status. The mutant produced hypersensitive response-like features following host inoculation, including ROS induction, callose formation, restricted growth and cell death. These results indicate active recognition of the mutant and further point to suppression of defenses by the wild type necrotrophic fungus. Chemical reduction of host cells with dithiothreitol (DTT) or potassium oxalate (KOA) restored the ability of this mutant to cause disease. Thus, Sclerotinia uses a novel strategy involving regulation of host redox status to establish infection. These results address a long-standing issue involving the ability of OA to both inhibit and promote ROS to achieve pathogenic success.

Transcriptome analyses of amoebic gill disease-affected atlantic salmon (Salmo salar) tissues reveal localized host gene suppression

The transcriptome response of Atlantic salmon (Salmo salar) displaying advanced stages of amoebic gill disease (AGD) was investigated. Naïve smolt were challenged with AGD for 19 days, at which time all fish were euthanized and their severity of infection quantified through histopathological scoring. Gene expression profiles were compared between heavily infected and naïve individuals using a 17 K Atlantic salmon cDNA microarray with real-time quantitative RT-PCR (qPCR) verification. Expression profiles were examined in the gill, anterior kidney, and liver. Twenty-seven transcripts were significantly differentially expressed within the gill; 20 of these transcripts were down-regulated in the AGD-affected individuals compared with naïve individuals. In contrast, only nine transcripts were significantly differentially expressed within the anterior kidney and five within the liver. Again the majority of these transcripts were down-regulated within the diseased individuals. A down-regulation of transcripts involved in apoptosis (procathepsin L, cathepsin H precursor, and cystatin B) was observed in AGD-affected Atlantic salmon. Four transcripts encoding genes with antioxidant properties also were down-regulated in AGD-affected gill tissue according to qPCR analysis. The most up-regulated transcript within the gill was an unknown expressed sequence tag (EST) whose expression was 218-fold (± SE 66) higher within the AGD affected gill tissue. Our results suggest that Atlantic salmon experiencing advanced stages of AGD demonstrate general down-regulation of gene expression, which is most pronounced within the gill. We propose that this general gene suppression is parasite-mediated, thus allowing the parasite to withstand or ameliorate the host response. © 2008 Springer Science+Business Media, LLC.

The koala immunological toolkit: Sequence identification and comparison of key markers of the koala (Phascolarctos cinereus) immune response

The koala (Phascolarctos cinereus) is an Australian marsupial that continues to experience significant population declines. Infectious diseases caused by pathogens such as Chlamydia are proposed to have a major role. Very few species-specific immunological reagents are available, severely hindering our ability to respond to the threat of infectious diseases in the koala. In this study, we utilise data from the sequencing of the koala transcriptome to identify key immunological markers of the koala adaptive immune response and cytokines known to be important in the host response to chlamydial infection in other species. This report describes the identification and preliminary sequence analysis of (1) T lymphocyte glycoprotein markers (CD4, CD8); (2) IL-4, a marker for the Th2 response; (3) cytokines such as IL-6, IL-12 and IL-1β, that have been shown to have a role in chlamydial clearance and pathology in other hosts; and (4) the sequences for the koala immunoglobulins, IgA, IgG, IgE and IgM. These sequences will enable the development of a range of immunological reagents for understanding the koala’s innate and adaptive immune responses, while also providing a resource that will enable continued investigations into the origin and evolution of the marsupial immune system.

Temporal dynamics of host molecular responses differentiate symptomatic and asymptomatic influenza a infection.

Exposure to influenza viruses is necessary, but not sufficient, for healthy human hosts to develop symptomatic illness. The host response is an important determinant of disease progression. In order to delineate host molecular responses that differentiate symptomatic and asymptomatic Influenza A infection, we inoculated 17 healthy adults with live influenza (H3N2/Wisconsin) and examined changes in host peripheral blood gene expression at 16 timepoints over 132 hours. Here we present distinct transcriptional dynamics of host responses unique to asymptomatic and symptomatic infections. We show that symptomatic hosts invoke, simultaneously, multiple pattern recognition receptors-mediated antiviral and inflammatory responses that may relate to virus-induced oxidative stress. In contrast, asymptomatic subjects tightly regulate these responses and exhibit elevated expression of genes that function in antioxidant responses and cell-mediated responses. We reveal an ab initio molecular signature that strongly correlates to symptomatic clinical disease and biomarkers whose expression patterns best discriminate early from late phases of infection. Our results establish a temporal pattern of host molecular responses that differentiates symptomatic from asymptomatic infections and reveals an asymptomatic host-unique non-passive response signature, suggesting novel putative molecular targets for both prognostic assessment and ameliorative therapeutic intervention in seasonal and pandemic influenza.

Function of host defence peptide LL-37 in human periodontal disease.

Objectives: Fibroblasts play a significant role as regulators of the host response in periodontal disease, responding to bacterial stimulation by producing an array of inflammatory cytokines and chemokines. LL-37, a host defence peptide, inhibits LPS-induced cytokine signalling in macrophages, suggesting an immunomodulatory role. The objective was to investigate the interaction between LL-37 and gingival fibroblasts – both its direct regulation of fibroblast activity and its effect on fibroblast response to LPS activation. Methods: Human gingival fibroblasts (HGFs) were incubated for 24 hours in the presence of either P. gingivalis LPS (10µg/ml) or E. coli LPS (10ng/ml) along with LL-37 (0-50 µg/ml). IL-6 and IL-8 production by HGFs in the conditioned medium was determined by ELISA. Western blot was performed to determine the effect of LL-37 on LPS -induced IκBα degradation in HGFs following LPS stimulation over 2 hours. DNA microarray analysis was performed on cell populations incubated for 6 hr in the presence or absence of the peptide. Confirmation of LL-37 effects on specific gene expression was obtained by QPCR. Results: At low concentrations (≤ 5 µg/ml) LL-37 significantly inhibited LPS-induced cytokine production by HGFs. At higher concentrations LL-37 induced IL-8 production independent of LPS. Addition of LL-37 blocked LPS-induced IκBα degradation in HGFs. Microarray analysis revealed that LL-37 (50µg/ml) upregulated a significant number of cytokines and chemokines by > 5 fold. Upregulation of five of these, CXCL1, CXCL2, CXCL3, IL-24 and IL-8 was confirmed by Q-PCR. Conclusion: The host defence peptide LL-37, the only known human cathelicidin, appears to have pleiotrophic effects in innate immunity. At least some of these are mediated through cytokine and chemokine signalling networks. The ability of LL-37 to reduce bacterial LPS-induced cytokine production in gingival fibroblasts, at low concentrations, suggests a potential therapeutic role in the management of periodontal disease.

Parasitized snails take the heat : a case of host manipulation?

Infection-induced changes in a host’s thermal physiology can represent (1) a generalized host response to infection, (2) a pathological side-effect of infection, or (3), provided the parasite’s development is temperature-dependent, a subtle case of host manipulation. This study investigates parasite-induced changes in the thermal biology of a first intermediate host infected by two castrating trematodes (genera Maritrema and Philophthalmus) using laboratory experiments and Weld surveys. The heat tolerance and temperatures selected by the snail, Zeacumantus subcarinatus, displayed alterations upon infection that differed between the two trematodes. Upon heating, snails infected by Maritrema sustained activity for longer durations than uninfected snails, followed by a more rapid recovery, and selected higher temperatures in a thermal gradient. These snails were also relatively abundant in high shore localities in the summer only, corresponding with seasonal elevated microhabitat temperatures. By contrast, Philophthalmus infected snails fell rapidly into a coma upon heating and did not display altered thermal preferences. The respective heat tolerance of each trematode corresponded with the thermal responses induced in the snail: Maritrema survived exposure to 40°C, while Philophthalmus was less heat tolerant. Although both trematodes infect the same tissues, Philophthalmus leads to a reduction in the host’s thermal tolerance, a response consistent with a pathological side effect. By contrast, Maritrema induces heat tolerance in the snail and withstood exposure to high heat. As the developmental rate and infectivity of Maritrema increase with temperature up to 25°C, one adaptive explanation for our findings is that Maritrema manipulates the snail’s thermal responses to exploit warm microhabitats.

Response of macrophage Toll-like receptor 4 to a Sporothrix schenckii lipid extract during experimental sporotrichosis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Modulation of host cell signaling pathways as a therapeutic approach in periodontal disease

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Salivary epithelial cells as model to study immune response against cutaneous pathogens

The human skin not only provides passive protection as a physical barrier against external injury, but also mediates active surveillance via epidermal cell surface receptors that recognize and respond to potential invaders. Primary keratinocytes and immortalized cell lines, the commonly used sources to investigate immune responses of cutaneous epithelium are often difficult to obtain and/or potentially exhibit changes in cellular genetic make-up. Here we investigated the possibility of using salivary epithelial cells (SEC) to evaluate the host response to cutaneous microbes. Elevated secretion of IFN-γ and IL-12 was observed in the SEC stimulated with Staphylococcus aureus, a transient pathogen of the skin, as mono species biofilm as compared to SEC stimulated with a commensal microbe, the Staphylococcus epidermidis. Co-culture of the SEC with both microbes as dual species biofilm elicited maximum cytokine response. Stimulation with S. aureus alone but not with S. epidermidis alone induced maximum toll-like receptor-2 (TLR-2) expression in the SEC. Exposure to dual species biofilm induced a sustained upregulation of TLR-2 in the SEC for up to an hour. The data support novel application of the SEC as efficient biospecimen that may be used to investigate personalized response to cutaneous microflora. © 2013 Wiley Periodicals, Inc.

Modulation of host cell signaling pathways as a therapeutic approach in periodontal disease

Recently, new treatment approaches have been developed to target the host component of periodontal disease. This review aims at providing updated information on host-modulating therapies, focusing on treatment strategies for inhibiting signal transduction pathways involved in inflammation. Pharmacological inhibitors of MAPK, NFκB and JAK/STAT pathways are being developed to manage rheumatoid arthritis, periodontal disease and other inflammatory diseases. Through these agents, inflammatory mediators can be inhibited at cell signaling level, interfering on transcription factors activation and inflammatory gene expression. Although these drugs offer great potential to modulate host response, their main limitations are lack of specificity and developments of side effects. After overcoming these limitations, adjunctive host modulating drugs will provide new therapeutic strategies for periodontal treatment.

Host condition and host immunity affect parasite fitness in a bird-ectoparasite system

1. Parasites might preferentially feed on hosts in good nutritional condition as such hosts provide better resources for the parasites' own growth, survival and reproduction. However, hosts in prime condition are also better able to develop costly immunological or physiological defence mechanisms, which in turn reduce the parasites' reproductive success. The interplay between host condition, host defence and parasite fitness will thus play an important part in the dynamics of host-parasite systems.;2. In a 2 x 2 design, we manipulated both the access to food in great tit Parus major broods and the exposure of the nestlings to hen fleas Ceratophyllus gallinae, a common ectoparasite of hole-breeding birds. We subsequently investigated the role of manipulated host condition, host immunocompetence, and experimentally induced host defence in nestlings on the reproductive success of individual hen flea females.;3. The food supplementation of the nestlings significantly influenced the parasites' reproductive success. Female fleas laid significantly more eggs when feeding on food-supplemented hosts.;4. Previous parasite exposure of the birds affected the reproductive success of fleas. However, the impact of this induced host response on flea reproduction depended on the birds' natural level of immunocompetence, assessed by the phytohaemagglutinin (PHA) skin test. Flea fecundity significantly decreased with increasing PHA response of the nestlings in previously parasite-exposed broods. No relationship between flea fitness and host immunocompetence was, however, found in previously unexposed broods. The PHA response thus correlates with the nestlings' ability to mount immunological or physiological defence mechanisms against hen fleas. No significant interaction effect between early flea exposure and food supplementation on the parasites' reproductive success was found.;5. Our study shows that the reproductive success of hen fleas is linked to the hosts' food supply early in life and their ability to mount induced immunological or physiological defence mechanisms. These interactions between host quality and parasite fitness are likely to influence host preference, host choice and parasite virulence and thus the evolutionary dynamics in host-parasite systems.

Host responses to intestinal microbial antigens in gluten-sensitive mice

BACKGROUND AND AIMS: Excessive uptake of commensal bacterial antigens through a permeable intestinal barrier may influence host responses to specific antigen in a genetically predisposed host. The aim of this study was to investigate whether intestinal barrier dysfunction induced by indomethacin treatment affects the host response to intestinal microbiota in gluten-sensitized HLA-DQ8/HCD4 mice. METHODOLOGY/PRINCIPAL FINDINGS: HLA-DQ8/HCD4 mice were sensitized with gluten, and gavaged with indomethacin plus gluten. Intestinal permeability was assessed by Ussing chamber; epithelial cell (EC) ultra-structure by electron microscopy; RNA expression of genes coding for junctional proteins by Q-real-time PCR; immune response by in-vitro antigen-specific T-cell proliferation and cytokine analysis by cytometric bead array; intestinal microbiota by fluorescence in situ hybridization and analysis of systemic antibodies against intestinal microbiota by surface staining of live bacteria with serum followed by FACS analysis. Indomethacin led to a more pronounced increase in intestinal permeability in gluten-sensitized mice. These changes were accompanied by severe EC damage, decreased E-cadherin RNA level, elevated IFN-gamma in splenocyte culture supernatant, and production of significant IgM antibody against intestinal microbiota. CONCLUSION: Indomethacin potentiates barrier dysfunction and EC injury induced by gluten, affects systemic IFN-gamma production and the host response to intestinal microbiota antigens in HLA-DQ8/HCD4 mice. The results suggest that environmental factors that alter the intestinal barrier may predispose individuals to an increased susceptibility to gluten through a bystander immune activation to intestinal microbiota.