Local Necrotic Cells Trigger Systemic Immune Activation via Gut Microbiome Dysbiosis in Drosophila.

Necrotic cells elicit an inflammatory response through their endogenous factors with damage-associated molecular patterns. Blocking apoptosis in Drosophila wings leads to the necrosis-driven systemic immune response by unknown mechanisms. Here, we demonstrate that immune activation in response to necrotic cells is mediated by commensal gut microbiota. Removing the microbiome attenuates hyperactivation of the innate immune signaling IMD pathway in necrosis-induced flies. Necrotic cells in wings trigger Gluconobacter expansion in the gut. An isolated Gluconobacter sp. strain is sufficient for pathological IMD activation in necrosis-induced flies, while it is not inflammatory for control animals. In addition, bacterial colonization shifts the host metabolome and shortens the lifespan of necrosis-induced flies. This study shows that local necrosis triggers a pathological systemic inflammatory response through interaction between the host and the dysbiotic gut microbiome.

The growing feather as a dermal test site: Comparison of leukocyte profiles during the response to Mycobacterium butyricum in growing feathers, wattles, and wing webs.

Using the response to Mycobacterium butyricum as the test-immune response, the main goal of this study was to demonstrate the suitability of the growing feather (GF) as a dermal test site and window into in vivo cellular/tissue responses (US-Patent 8,216,551). Using M. butyricum immunized chickens, the specific objectives were to: 1) compare the leukocyte infiltration response to intra-dermally injected M. butyricum in GF, wattles, and wing webs; 2) use GF as the test site to monitor leukocyte response profiles to recall antigen in the same individuals; and 3) gain new knowledge regarding the local response to M. butyricum in chickens. For objective 1, chickens were euthanized for tissue collection at 4 to 6, 24, 48, and 72 h after intra-dermal antigen injection. Leukocyte infiltration profiles were determined using immunochemical and conventional histology. Data from this study established the similarities between the cellular response in GF, wattles, and wing webs and uncovered many advantages of working with GF. For objective 2, antigen was injected into multiple GF per individual. GF were collected before and at 0.25, 1, 2, 3, and 7 d post injection and processed for cell population analysis by flow cytometry. Advantages of the approach used in objective 2 included a technically easier, more comprehensive, and more objective leukocyte profile analysis; same-day data acquisition; and, most importantly, easy, minimally invasive sample collection from the same individual throughout the study. Both studies contributed new knowledge regarding the local cutaneous response to M. butyricum in M. butyricum immunized chickens and confirmed the cell-mediated nature of the immune response to M. butyricum (e.g., elevated levels [P < 0.05] of T cells [CD4+ and CD8+], macrophages and MHC class II+-cells on days one to 3 post injection in M. butyricum- compared to PBS-injected tissues). The use of GF as an "in vivo test tube" to monitor local innate and adaptive immune activities will find direct application in vaccine development, as well as in the assessment and optimization of immune system development and function in poultry.

The White-Nose Syndrome Transcriptome: Activation of Anti-fungal Host Responses in Wing Tissue of Hibernating Little Brown Myotis.

White-nose syndrome (WNS) in North American bats is caused by an invasive cutaneous infection by the psychrophilic fungus Pseudogymnoascus destructans (Pd). We compared transcriptome-wide changes in gene expression using RNA-Seq on wing skin tissue from hibernating little brown myotis (Myotis lucifugus) with WNS to bats without Pd exposure. We found that WNS caused significant changes in gene expression in hibernating bats including pathways involved in inflammation, wound healing, and metabolism. Local acute inflammatory responses were initiated by fungal invasion. Gene expression was increased for inflammatory cytokines, including interleukins (IL) IL-1ß, IL-6, IL-17C, IL-20, IL-23A, IL-24, and G-CSF and chemokines, such as Ccl2 and Ccl20. This pattern of gene expression changes demonstrates that WNS is accompanied by an innate anti-fungal host response similar to that caused by cutaneous Candida albicans infections. However, despite the apparent production of appropriate chemokines, immune cells such as neutrophils and T cells do not appear to be recruited. We observed upregulation of acute inflammatory genes, including prostaglandin G/H synthase 2 (cyclooxygenase-2), that generate eicosanoids and other nociception mediators. We also observed differences in Pd gene expression that suggest host-pathogen interactions that might determine WNS progression. We identified several classes of potential virulence factors that are expressed in Pd during WNS, including secreted proteases that may mediate tissue invasion. These results demonstrate that hibernation does not prevent a local inflammatory response to Pd infection but that recruitment of leukocytes to the site of infection does not occur. The putative virulence factors may provide novel targets for treatment or prevention of WNS. These observations support a dual role for inflammation during WNS; inflammatory responses provide protection but excessive inflammation may contribute to mortality, either by affecting torpor behavior or causing damage upon emergence in the spring.

The expression of a sexually selected trait correlates with different immune defense components and survival in males of the American rubyspot.

Recent studies have suggested that courtship trait expression indicates immune strength. However, most studies have measured only one immune parameter, have not assessed individual differences in immune ability according to time and have not controlled for ecological differences among individuals after an immune challenge. In this work, we tested this hypothesis and controlled for these factors using males of the American rubyspot damselfly which bear a wing red spot whose size is evolutionarily maintained via male-male territorial competition. Our general hypothesis was that territorial, large-spotted males, had a better immune ability compared to nonterritorial, small-spotted males. We expected that the following variables were greater in territorial males compared to nonterritorial males: spot size, phenoloxidase (PO) and hydrolytic enzymatic (HE) activity in males challenged and nonchallenged with a nylon implant, PO and HE activity rate; PO activity after a Serratia marcescens challenge, and survival after a nylon challenge controlling for activity and feeding differences. We found that territorial males showed larger spot areas, greater PO and HE activity (independently of whether they were challenged or not), a higher rate of PO and HE activity (but only expressed at 8h), greater PO production after the bacterial challenge, and a higher survival after the challenge. These results corroborate that males with more pronounced sexual traits have a superior immune function.

Toxicity of cadmium in Japanese quail: evaluation of body weight, hepatic and renal function, and cellular immune response.

Cadmium (Cd) is an environmental pollutant that is able to alter the immune function. Previous studies have shown that, in mammals, chronic exposure to Cd decreases the release of macrophagic cytokines such as IL1 and TN alpha and decreases phagocytosis activity. On the other hand contradictory results showed an increase in the humoral response. The cellular response could be decreased by exposure to Cd. These alterations were observed in mammals. The present study aimed to investigate some of the toxic effects of Cd exposure in birds. In particular, the main objective of this work was to elucidate the effects of exposure to this pollutant on the cellular immune function of the Japanese quail as a model for the study of toxicity in animals exposed in nature. The animals were exposed to the metal (100 ppm, per os) during development, i.e., from 1 to 28 days old. Body weight, biochemical parameters, and cellular immune response were measured during and at the end of treatment. The results showed that the exposure to Cd for 28 days significantly reduced the body weight and induced hepatic toxicity. The kidney function and cellular immune response were not affected by the Cd exposure.

Experimental removal of the male parent negatively affects growth and immunocompetence in nestling great tits.

Given the available empirical evidence on the benefits and costs associated with immune defence, a role for the immune system in the trade-off between current and future reproduction has been predicted. This hypothesis was studied in a free-living population of great tits (Parus major) by examining the effects of male removal on the immunocompetence, body condition, and recapture probability in the widowed females and their nestlings. Furthermore, we investigated whether growth and its relation to immunocompetence were affected in the nestlings. For a short-lived species such as the great tit, one could predict that widowed females will compensate for the lack of any male assistance in feeding of their chicks and that they consequently might jeopardize their own health. However, we did not find any negative effects of male removal on body mass or condition, nor on humoral immunocompetence against sheep red blood cells in the widowed females by the end of the feeding period. In contrast, we observed significantly reduced body mass and size as well as a reduced T-lymphocyte cell-mediated immune response (expressed as the thickness of the swelling to a subcutaneous injection with phytohemagglutinin) in the experimental nestlings compared to the control nestlings. In addition, the experimental nestlings showed a tendency for a reduced chance to be found breeding the following year. Furthermore, our results showed that in the experimental nestlings, which suffered from unfavourable growth conditions, tarsus length was inversely related to cell-mediated immunocompetence, whereas in control nestlings this relationship was significantly positive. The relationship between cell-mediated immunity and body condition was found to be significantly positive in the experimental nestlings while in control nestlings there was no relationship between both variables. The latter finding suggests different priorities of investment in body condition between different growth conditions.

Occupational allergy in laboratory workers caused by the African migratory grasshopper Locusta migratoria.

BACKGROUND: Recent reports of fatal asthma cases associated with swarms of locusts affecting African countries have highlighted the importance of this insect in causing asthma morbidity and mortality. However, only limited information is available about the allergic health outcomes such as asthma and its determinants in exposed individuals. In this study, workers exposed to the African migratory locust Locusta migratoria were evaluated for allergic health outcomes as well as the nature of the offending allergens. METHODS: Ten scientists and technicians exposed to locusts in a laboratory were investigated for locust-related allergy using questionnaires and immunological tests. The presence of allergy was determined by quantification of specific IgE and IgG to L. migratoria using the UniCAP system and via skin-prick testing (SPT). The allergens were characterized by Western blot and ImmunoCAP inhibition assays. RESULTS: Six of the 10 workers experienced symptoms ranging from urticaria and rhinoconjuctivitis to asthma. Seven individuals demonstrated sensitivity on SPT and five had specific IgE antibodies to L. migratoria. Significant cross-reactivity was demonstrated for allergens in the locust faeces, body and wings but not to cockroach allergens. Novel allergens with molecular weights of approximately 70 kDa were identified in locust wings, which are distinctly different from other known allergen sources from locusts. CONCLUSION: Exposure to L. migratoria allergens is a potential sensitizer in exposed individuals. Raised levels of locust-specific IgE can be readily quantified. The wings of this insect species have been identified as a novel allergen source.

Nerve-muscle interactions regulate motor terminal growth and myoblast distribution during muscle development.

Interactions between motoneurons and muscles influence many aspects of neuromuscular development in all animals. These interactions can be readily investigated during adult muscle development in holometabolous insects. In this study, the development of the dorsolongitudinal flight muscle (DLM) and its innervation is investigated in the moth, Manduca sexta, to address the specificity of neuromuscular interactions. The DLM develops from an anlage containing both regressed larval template fibers and imaginal myoblasts. In the adult, each fiber bundle (DLM1-5) is innervated by a single motoneuron (MN1-MN5), with the dorsal-most fiber bundle (DLM5) innervated by a mesothoracic motoneuron (MN5). The DLM failed to develop following complete denervation because myoblasts failed to accumulate in the DLM anlage. After lesioning MN1-4, MN5 retained its specificity for the DLM5 region of the anlage and failed to rescue DLM1-4. Thus specific innervation of the DLM fiber bundles does not depend on interactions among motoneurons. Myoblast accumulation, but not myonuclear proliferation, increased around the MN5 terminals, producing a hypertrophied adult DLM5. Therefore, motoneurons compete for uncommitted myoblasts. MN5 terminals subsequently grew more rapidly over the hypertrophied DLM5 anlage, indicating that motoneuron terminal expansion is regulated by the size of the target muscle anlage.

Influence of dose and route of inoculation on responses of chickens to recombinant fowlpox virus vaccines.

The influence of dose and route of inoculation on responses of chickens to vaccination with recombinant fowlpox viruses (rFPVs) expressing an influenza haemagglutinin (HA) (FPV-HA) and the infectious bursal disease virus (IBDV) VP2 antigen (FPV-VP2) has been evaluated. Antibody responses to influenza and fowlpox virus were generated following vaccination via the wing web by subcutaneous inoculation or skin scarification. Intranasal and conjunctival inoculation failed to induce antibodies to FPV or influenza. Following direct intratracheal inoculation antibodies developed to influenza but not FPV. Dose response studies with the FPV-HA and FPV-VP2 recombinants showed that good responses to FPV and the vaccine antigen could be generated over a wide (10000 fold) dose range following wing web inoculation. The responses generated by the FPV-VP2 recombinant over this vaccine dose range protected against IBDV infection of the bursae following challenge with the Australian IBDV 002/73 isolate. These data suggest that effective application of rFPVs for poultry vaccination may be restricted to wing web and parenteral routes of inoculation.

Developmental analysis of Drosophila position-specific antigens.

The distributions of three position-specific (PS) antigens have been examined in different Drosophila tissues and at various developmental times, using both immunofluorescence and affinity purification procedures. In the imaginal discs the PS antigens show nonuniform and nonhomologous distributions, and the expression of the antigens in a particular disc region can vary during development. In general, PS antigen expression appears to correlate with morphogenetic events in the disc epithelia, suggesting that the antigens are involved in cell-cell recognition and/or adhesion processes. PS antigens are also found in many other tissues, and in embryos as early as the cellular blastoderm stage. Affinity-purified PS antigens from different tissues or stages appear to be similar, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results are discussed in relation to Drosophila developmental events, with particular regard to the dorsoventral cell lineage restriction in the wing disc.