scholarly journals Imd pathway-specific immune assays reveal NF-κB stimulation by viral RNA PAMPs in Aedes aegypti Aag2 cells

2020 ◽  
Author(s):  
Tiffany A. Russell ◽  
Andalus Ayaz ◽  
Andrew D. Davidson ◽  
Ana Fernandez-Sesma ◽  
Kevin Maringer
Keyword(s):  
Immune System ◽  
Aedes Aegypti ◽  
Immune Responses ◽  
Health Impact ◽  
Global Public Health ◽  
Toll Pathway ◽  
Experimental Conditions ◽  
Imd Pathway ◽  
Arboviral Diseases ◽  
Transgenic Insects

ABSTRACTBackgroundThe mosquito Aedes aegypti is a major vector for the arthropod-borne viruses (arboviruses) chikungunya, dengue, yellow fever and Zika viruses. Vector immune responses pose a major barrier to arboviral transmission, and transgenic insects with altered immunity have been proposed as tools for reducing the global public health impact of arboviral diseases. However, a better understanding of virus-immune interactions is needed to progress the development of such transgenic insects. Although the NF-κB-regulated Toll and ‘immunodeficiency’ (Imd) pathways are increasingly thought to be antiviral, relevant pattern recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs) remain poorly characterised in A. aegypti.Methodology/Principle FindingsWe developed novel RT-qPCR and luciferase reporter assays to measure induction of the Toll and Imd pathways in the commonly used A. aegypti-derived Aag2 cell line. We thus determined that the Toll pathway is not inducible by exogenous stimulation with bacterial, viral or fungal stimuli in Aag2 cells under our experimental conditions. We used our Imd pathway-specific assays to demonstrate that the viral dsRNA mimic poly(I:C) is sensed by the Imd pathway, likely through intracellular and extracellular PRRs. The Imd pathway was also induced during infection with the model insect-specific virus cricket paralysis virus (CrPV).Conclusions/SignificanceOur demonstration that a general PAMP shared by many arboviruses is sensed by the Imd pathway paves the way for future studies to determine how viral RNA is sensed by mosquito PRRs at a molecular level. Our data also suggest that studies measuring inducible immune pathway activation through antimicrobial peptide (AMP) expression in Aag2 cells should be interpreted cautiously given that the Toll pathway is not responsive under all experimental conditions. With no antiviral therapies and few effective vaccines available to treat arboviral diseases, our findings provide new insights relevant to the development of transgenic mosquitoes as a means of reducing arbovirus transmission.AUTHOR SUMMARYThe mosquito Aedes aegypti, found globally across the tropics and subtropics, transmits viral diseases with a significant global public health impact, including chikungunya, dengue, yellow fever and Zika viruses. There are no antiviral drugs to treat these diseases and few effective vaccines. One way of reducing the global burden of mosquito-borne diseases would be to develop genetically modified mosquitoes unable to transmit viruses. One approach would be to alter the mosquitoes’ immune system to allow them to better fight viral infections. To do so, we first need to understand how viruses are detected by the mosquito immune system. We developed new methods of measuring immune responses in laboratory-cultured mosquito cells and used them to show that one specific arm of the immune system, called the ‘Imd pathway’, can detect the RNA that constitutes the genome of mosquito-borne viruses. These findings pave the way for future immune studies that could inform the development of transmission-incompetent mosquitoes. We also found that another arm of the immune system, called the ‘Toll pathway’, is not functional under any experimental conditions used in this study. This finding has implications for how different laboratories interpret data from these particular cultured cells.

scholarly journals Imd pathway-specific immune assays reveal NF-κB stimulation by viral RNA PAMPs in Aedes aegypti Aag2 cells

2021 ◽  
Vol 15 (2) ◽  
pp. e0008524
Author(s):  
Tiffany A. Russell ◽  
Andalus Ayaz ◽  
Andrew D. Davidson ◽  
Ana Fernandez-Sesma ◽  
Kevin Maringer
Keyword(s):  
Aedes Aegypti ◽  
Viral Rna ◽  
Poly I:C ◽  
Luciferase Reporter ◽  
Toll Pathway ◽  
Experimental Conditions ◽  
Major Barrier ◽  
Imd Pathway ◽  
Arboviral Diseases ◽  
Transgenic Insects

Background The mosquito Aedes aegypti is a major vector for the arthropod-borne viruses (arboviruses) chikungunya, dengue, yellow fever and Zika viruses. Vector immune responses pose a major barrier to arboviral transmission, and transgenic insects with altered immunity have been proposed as tools for reducing the global public health impact of arboviral diseases. However, a better understanding of virus-immune interactions is needed to progress the development of such transgenic insects. Although the NF-κB-regulated Toll and ‘immunodeficiency’ (Imd) pathways are increasingly thought to be antiviral, relevant pattern recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs) remain poorly characterised in A. aegypti. Methodology/Principle findings We developed novel RT-qPCR and luciferase reporter assays to measure induction of the Toll and Imd pathways in the commonly used A. aegypti-derived Aag2 cell line. We thus determined that the Toll pathway is not inducible by exogenous stimulation with bacterial, viral or fungal stimuli in Aag2 cells under our experimental conditions. We used our Imd pathway-specific assays to demonstrate that the viral dsRNA mimic poly(I:C) is sensed by the Imd pathway, likely through intracellular and extracellular PRRs. The Imd pathway was also induced during infection with the model insect-specific virus cricket paralysis virus (CrPV). Conclusions/Significance Our demonstration that a general PAMP shared by many arboviruses is sensed by the Imd pathway paves the way for future studies to determine how viral RNA is sensed by mosquito PRRs at a molecular level. Our data also suggest that studies measuring inducible immune pathway activation through antimicrobial peptide (AMP) expression in Aag2 cells should be interpreted cautiously given that the Toll pathway is not responsive under all experimental conditions. With no antiviral therapies and few effective vaccines available to treat arboviral diseases, our findings provide new insights relevant to the development of transgenic mosquitoes as a means of reducing arbovirus transmission.

scholarly journals Aedes aegypti (Diptera: Culicidae) Immune Responses with Different Feeding Regimes Following Infection by the Entomopathogenic Fungus Metarhizium anisopliae

Insects ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 95 ◽  
Author(s):  
Sara Cabral ◽  
Adriano de Paula ◽  
Richard Samuels ◽  
Rodrigo da Fonseca ◽  
Simone Gomes ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Immune Responses ◽  
Antimicrobial Peptides ◽  
Fungal Infection ◽  
Fat Body ◽  
Negative Regulator ◽  
Toll Pathway ◽  
Promising Alternative ◽  
Imd Pathway ◽  
Feeding Regimes

The mosquito Aedes aegypti is the most notorious vector of illness-causing viruses. The use of entomopathogenic fungi as bioinsecticides is a promising alternative for the development of novel mosquito control strategies. We investigate whether differences in immune responses could be responsible for modifications in survival rates of insects following different feeding regimes. Sucrose and blood-fed adult A. aegypti females were sprayed with M. anisopliae 1 × 106 conidia mL−1, and after 48 h, the midgut and fat body were dissected. We used RT-qPCR to monitor the expression of Cactus and REL1 (Toll pathway), IMD, REL2, and Caspar (IMD pathway), STAT and PIAS (JAK-STAT pathway), as well as the expression of antimicrobial peptides (Defensin A, Attacin and Cecropin G). REL1 and REL2 expression in both the midgut and fat body were higher in blood-fed fungus-challenged A. aegypti than in sucrose-fed counterparts. Interestingly, infection of sucrose-fed insects induced Cactus expression in the fat body, a negative regulator of the Toll pathway. The IMD gene was upregulated in the fat body in response to fungal infection after a blood meal. Additionally, we observed the induction of antimicrobial peptides in the blood-fed fungus-challenged insects. This study suggests that blood-fed A. aegypti are less susceptible to fungal infection due to the rapid induction of Toll and IMD immune pathways.

scholarly journals Serum bactericidal activity as indicator of innate immunity in pacu Piaractus mesopotamicus (Holmberg, 1887)

2013 ◽  
Vol 65 (6) ◽  
pp. 1745-1751 ◽  
Author(s):  
J.D. Biller-Takahashi ◽  
L.S. Takahashi ◽  
F. Pilarski ◽  
F.A. Sebastião ◽  
E.C. Urbinati
Keyword(s):  
Immune System ◽  
Immune Responses ◽  
Bactericidal Activity ◽  
Aeromonas Hydrophila ◽  
Activity Assay ◽  
Experimental Conditions ◽  
Piaractus Mesopotamicus ◽  
Colony Forming Unit ◽  
Serum Bactericidal Activity ◽  
Protective Proteins

The immune system of teleost fish has mechanisms responsible for the defense against bacteria through protective proteins in several tissues. The protein action can be evaluated by serum bactericidal activity and this is an important tool to analyze the immune system. Pacu, Piaractus mesopotamicus, is one of the most important fish in national aquaculture. However there is a lack of studies on its immune responses. In order to standardize and assess the accuracy of the serum bactericidal activity assay, fish were briefly challenged with Aeromonas hydrophila and sampled one week after the challenge. The bacterial infection increased the concentration of protective proteins, resulting in a decrease of colony-forming unit values expressed as well as an enhanced serum bactericidal activity. The protocol showed a reliable assay, appropriate to determine the serum bactericidal activity of pacu in the present experimental conditions.

scholarly journals Functional characterization of two clip domain serine proteases in innate immune responses of Aedes aegypti

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Hao-Cheng Wang ◽  
Qiu-Hui Wang ◽  
Biswajit Bhowmick ◽  
Yi-Xun Li ◽  
Qian Han
Keyword(s):  
Innate Immunity ◽  
Transcription Factors ◽  
Immune Responses ◽  
Serine Proteases ◽  
Fungal Infections ◽  
Tissue Injury ◽  
Functional Characterization ◽  
Innate Immune Responses ◽  
Toll Pathway ◽  
Imd Pathway

Abstract Background Clip domain serine proteases (CLIPs), a very diverse group of proteolytic enzymes, play a crucial role in the innate immunity of insects. Innate immune responses are the first line of defense in mosquitoes against the invasion of pathogenic microorganisms. The Toll pathway, immunodeficiency (IMD) pathway and melanization are the main processes of innate immunity in Aedes aegypti. CLIPS are classified into five subfamilies—CLIPA, CLIPB, CLIPC, CLIPD, and CLIPE—based on their sequence specificity and phylogenetic relationships. We report the functional characterization of the genes that code for two CLIPs in Ae. aegypti (Ae): Ae-CLIPB15 and Ae-CLIPB22. Methods Clustal Omega was used for multiple amino acid sequence alignment of Ae-CLIPB15 and Ae-CLIPB22 with different CLIP genes from other insect species. The spatiotemporal expression profiles of Ae-CLIPB15 and Ae-CLIPB22 were examined. We determined whether Ae-CLIPB15 and Ae-CLIPB22 respond to microbial challenge and tissue injury. RNA interference (RNAi) was used to explore the function of Ae-CLIPB15 and Ae-CLIPB22 in the defense of Ae. aegypti against bacterial and fungal infections. The expression levels of nuclear factor kappa B (NF-κB) transcription factors REL1 and REL2 in the Toll pathway and IMD pathway after bacterial infection were investigated. Finally, the change in phenoloxidase (PO) activity in Ae-CLIPB15 and Ae-CLIPB22 knockdown adults was investigated. Results We performed spatiotemporal gene expression profiling of Ae-CLIPB15 and Ae-CLIPB22 genes in Ae. aegypti using quantitative real-time polymerase chain reaction. These genes were expressed in different stages and tissues. The messenger RNA (mRNA) levels for both genes were also up-regulated by Gram-negative bacteria Escherichia coli, Gram-positive bacteria Staphylococcus aureus and fungal Beauveria bassiana infections, as well as in the tissue injury experiments. RNAi-mediated knockdown of Ae-CLIPB15 led to a significant decrease of PO activity in the hemolymph of Ae. aegypti, while other RNAi experiments revealed that both Ae-CLIPB15 and Ae-CLIPB22 were involved in immune defense against bacterial and fungal infections. The mRNA expression of NF-κB transcription factors REL1 and REL2 in the Toll pathway and IMD pathway differed between Ae-CLIPB15 and Ae-CLIPB22 knockdown mosquitoes infected with bacteria and wild type mosquitoes infected with bacteria. Conclusions Our findings suggest that Ae-CLIPB15 and Ae-CLIPB22 play a critical role in mosquito innate immunity, and that they are involved in immune responses to injury and infection. Their regulation of transcription factors and PO activity indicates that they also play a specific role in the regulation of innate immunity. Graphical Abstract

scholarly journals Natural variation in the contribution of microbial density to inducible immune dynamics

2019 ◽  
Author(s):  
Derrick Jent ◽  
Abby Perry ◽  
Justin Critchlow ◽  
Ann T. Tate
Keyword(s):  
Immune System ◽  
Immune Responses ◽  
Natural Variation ◽  
Temporal Dynamics ◽  
Acute Infection ◽  
Theoretical Dependence ◽  
System Evolution ◽  
Immune System Evolution ◽  
Imd Pathway ◽  
Microbial Density

AbstractImmune responses evolve to balance the benefits of microbial killing against the costs of autoimmunity and energetic resource use. Models that explore the evolution of optimal immune responses generally include a term for constitutive immunity, or the level of immunological investment prior to microbial exposure, and for inducible immunity, or investment in immune function after microbial challenge. However, studies rarely consider the functional form of inducible immune responses with respect to microbial density, despite the theoretical dependence of immune system evolution on microbe-versus immune-mediated damage to the host. In this study, we analyze antimicrobial peptide (AMP) gene expression from seven wild-caught flour beetle populations (Tribolium spp.) during acute infection with the virulent bacteria Bacillus thuringiensis (Bt) and Photorhabdus luminescens (P.lum) to demonstrate that inducible immune responses mediated by the humoral IMD pathway exhibit natural variation in both microbe density-dependent and independent temporal dynamics. Beetle populations that exhibited greater AMP expression sensitivity to Bt density were also more likely to die from infection, while populations that exhibited higher microbe density-independent AMP expression were more likely to survive P. luminescens infection. Reduction in pathway signaling efficiency through RNAi-mediated knockdown of the imd gene reduced the magnitude of both microbe-independent and dependent responses and reduced host resistance to Bt growth, but had no net effect on host survival. This study provides a framework for understanding natural variation in the flexibility of investment in inducible immune responses and should inform theory on the contribution of non-equilibrium host-microbe dynamics to immune system evolution.

scholarly journals Immunological responses to the relapsing fever spirochete Borrelia turicatae in infected Rhesus macaques: implications for pathogenesis and diagnosis.

2018 ◽  
Author(s):  
Monica E Embers ◽  
Aparna Krishnavjhala ◽  
Brittany Armstrong ◽  
Michael W. Curtis ◽  
Bapi Pahar ◽  
...  
Keyword(s):  
Immune Responses ◽  
B Cell ◽  
Rhesus Macaques ◽  
Health Impact ◽  
Surface Proteins ◽  
Vaccine Antigen ◽  
Global Public Health ◽  
Relapsing Fever ◽  
Primate Model ◽  
Immunological Responses

The global public health impact of relapsing fever (RF) spirochetosis is significant, as the pathogens exist on five of seven continents. The hallmark sign of infection is episodic fever and the greatest threat is to the unborn. With the goal of better understanding the specificity of B cell responses and the role of immune responses in pathogenicity, we infected Rhesus macaques with Borrelia turicatae (a new world RF spirochete species) by tick bite and monitored the immune responses generated in response to the pathogen. Specifically, we evaluated inflammatory mediator induction by the pathogen, host antibody responses to specific antigens, and peripheral lymphocyte population dynamics. Our results indicate that B. turicatae elicits from peripheral blood cells key inflammatory response mediators (IL-1β and TNF-α) which are associated with pre-term abortion. Moreover, a global decline in peripheral B cell populations was observed in all animals at 14 days post-infection. Serological responses were also evaluated to assess the antigenicity of three surface proteins, BipA, BrpA and Bta112. Interestingly, a distinction was observed between antibodies generated in non-human primates (NHPs) and mice. Our results provide support for the nonhuman primate model not only in studies of prenatal pathogenesis, but for diagnostic and vaccine antigen identification and testing.

scholarly journals Identification of dengue virus immunodeficiency (IMD) pathway antagonists in Aedes Aegypti cells

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Tiffany Russell ◽  
Louisa Wallace ◽  
Kevin Maringer
Keyword(s):  
Aedes Aegypti ◽  
Immune Responses ◽  
Dengue Virus ◽  
Mosquito Vector ◽  
Dengue Disease ◽  
Host Interactions ◽  
Host Determinants ◽  
Imd Pathway ◽  
Insight Into ◽  
Potential Antagonist

The flavivirus dengue virus (DENV) is the most significant arthropod borne virus (arbovirus) of humans, causing serious morbidity and mortality, with nearly half of the world’s population at risk of infection. Due to a lack of antivirals and limited vaccine options, vector control remains a vital defence against dengue disease. The mosquito Aedes aegypti is the major vector for DENV, and understanding mosquito immune responses and how DENV may evade them is critical. We have shown that DENV-2 can inhibit the exogenous induction of immune deficiency (IMD) signalling by classical immune stimuli. Therefore, we aimed to identify DENV antagonists of the IMD pathway, and define the molecular virus and host determinants of IMD antagonism in a well characterised Ae. aegypti derived cell line, Aag2. Each DENV protein was expressed individually in Aag2 cells and tested for their ability to block IMD signalling induced by exogenous stimuli. This screen identified NS4A as a potential antagonist of the IMD pathway. Further, we have found that the N-terminus of NS4A is responsible for this inhibition. The antagonism of IMD signalling is specific to flaviviruses transmitted by a mosquito vector, illustrating the importance of both the IMD pathway for mosquito immunity and the antagonism of this pathway by DENV. By enhancing our understanding of how DENV evades the mosquito immune response at a molecular level, we will gain insight into virus-host interactions constraining arbovirus transmission and emergence, which may be exploited for developing transmission-incompetent vectors to reduce the burden of dengue disease.

CNS–immune system interactions: Conditioning phenomena

1985 ◽  
Vol 8 (3) ◽  
pp. 379-395 ◽  
Author(s):  
Robert Ader ◽  
Nicholas Cohen
Keyword(s):  
Immune System ◽  
Autoimmune Disease ◽  
Immune Responses ◽  
Self Regulation ◽  
Immunosuppressive Drug ◽  
Conditioned Stimuli ◽  
Biological Impact ◽  
Experimental Conditions ◽  
Progression Of Disease ◽  
The Brain

AbstractConverging data from different disciplines indicate that central nervous system processes are capable of influencing immune responses. This paper concentrates on recent studies documenting behaviorally conditioned suppression and enhancement of immunity. Exposing rats or mice to a conditioned stimulus previously paired with an immunomodulating agent results in alterations in humoral and cell-mediated immune responses to antigenic stimuli, and unreinforced reexposures to the conditioned stimuli result in extinction of the conditioned response. Although the magnitude of such conditioning effects has not been large, the phenomenon has been independently verified under a variety of experimental conditions. The biological impact of conditioned alterations in immune function is illustrated by studies in which conditioning operations were applied in the pharmacotherapy of autoimmune disease in New Zealand mice. In conditioned animals, substituting conditioned stimuli for active drugs delays the onset of autoimmune disease relative to nonconditioned animals using a dose of immunosuppressive drug that, by itself, is ineffective in modifying the progression of disease. The hypothesis that such conditioning effects are mediated by elevations in adrenocortical steroid levels receives no support from available data. Despite its capacity for self-regulation, it appears that the immune system is integrated with other psychophysiological processes and subject to modulation by the brain.

A Simple PCR Assay for Discrimination of Dengue Vectors from Nsukka LGA, Nigeria

2020 ◽  
Vol 10 (1) ◽  
pp. 67-77
Author(s):  
Amos Watentena ◽  
Ikem Chris Okoye ◽  
Ikechukwu Eugene Onah ◽  
Onwude Cosmas Ogbonnaya ◽  
Emmanuel Ogudu
Keyword(s):  
Aedes Aegypti ◽  
Aedes Albopictus ◽  
Mosquito Species ◽  
Public Health Problem ◽  
Major Public Health Problem ◽  
Mosquito Vector ◽  
Aedes Mosquitoes ◽  
Allele Specific ◽  
Arboviral Diseases ◽  
Dengue Vectors

Mosquitoes of Aedes species are vectors of several arboviral diseases which continue to be a major public health problem in Nigeria. This study among other things, morphologically identified Aedes mosquitoes collected from Nsukka LGA and used an allele specific PCR amplification for discrimination of dengue vectors. Larval sampling, BG-sentinel traps and modified human landing catches were used for mosquito sampling in two selected autonomous communities of Nsukka LGA (Nsukka and Obimo). A total of 124 Aedes mosquitoes consisting of five (5) different species were collected from April to June, 2019 in a cross-sectional study that covered 126 households, under 76 distinct geographical coordinates. Larvae was mainly collected from plastic containers 73% (n=224), metallic containers 14% (n=43), earthen pots 9% (n=29) and used car tyres 3% (n=9), reared to adult stage 69.35% (n=86), and all mosquitoes were identified using standard morphological keys. Five (5) Aedes mosquito species were captured; Aedes aegypti 83(66.94%), Aedes albopictus 33(26.61%), Aedes simpsoni (4.48%), Aedes luteocephalus (≤1%) and Aedes vittatus (≤1%). Nsukka autonomous community had higher species diversity than Obimo. Allele specific amplification confirmed dengue vectors, Aedes aegypti and Aedes albopictus species on a 2% agarose gel. Since the most recent re-emergence of arboviral diseases is closely associated with Aedes species, findings of this study, therefore, give further evidence about the presence of potential arboviral vectors in Nigeria and describe the role of a simple PCR in discriminating some. Further entomological studies should integrate PCR assays in mosquito vector surveillance.

scholarly journals The immune response of T cells and therapeutic targets related to regulating the levels of T helper cells after ischaemic stroke

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Tian-Yu Lei ◽  
Ying-Ze Ye ◽  
Xi-Qun Zhu ◽  
Daniel Smerin ◽  
Li-Juan Gu ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Immune System ◽  
Immune Responses ◽  
Ischaemic Stroke ◽  
Immune Cells ◽  
Tissue Injury ◽  
Recovery Period ◽  
Vital Functions ◽  
Anti Inflammatory

AbstractThrough considerable effort in research and clinical studies, the immune system has been identified as a participant in the onset and progression of brain injury after ischaemic stroke. Due to the involvement of all types of immune cells, the roles of the immune system in stroke pathology and associated effects are complicated. Past research concentrated on the functions of monocytes and neutrophils in the pathogenesis of ischaemic stroke and tried to demonstrate the mechanisms of tissue injury and protection involving these immune cells. Within the past several years, an increasing number of studies have elucidated the vital functions of T cells in the innate and adaptive immune responses in both the acute and chronic phases of ischaemic stroke. Recently, the phenotypes of T cells with proinflammatory or anti-inflammatory function have been demonstrated in detail. T cells with distinctive phenotypes can also influence cerebral inflammation through various pathways, such as regulating the immune response, interacting with brain-resident immune cells and modulating neurogenesis and angiogenesis during different phases following stroke. In view of the limited treatment options available following stroke other than tissue plasminogen activator therapy, understanding the function of immune responses, especially T cell responses, in the post-stroke recovery period can provide a new therapeutic direction. Here, we discuss the different functions and temporal evolution of T cells with different phenotypes during the acute and chronic phases of ischaemic stroke. We suggest that modulating the balance between the proinflammatory and anti-inflammatory functions of T cells with distinct phenotypes may become a potential therapeutic approach that reduces the mortality and improves the functional outcomes and prognosis of patients suffering from ischaemic stroke.

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