A newly established in vitro culture using transgenic Drosophila reveals functional coupling between the phospholipase A2-generated fatty acid cascade and lipopolysaccharide-dependent activation of the immune deficiency (imd) pathway in insect immunity

Innate immunity is the first line of defence against infectious micro-organisms, and the basic mechanisms of pathogen recognition and response activation are evolutionarily conserved. In mammals, the innate immune response in combination with antigen-specific recognition is required for the activation of adaptive immunity. Therefore, innate immunity is a pharmaceutical target for the development of immune regulators. Here, for the purpose of pharmaceutical screening, we established an in vitro culture based on the innate immune response of Drosophila. The in vitro system is capable of measuring lipopolysaccharide (LPS)-dependent activation of the immune deficiency (imd) pathway, which is similar to the tumour necrosis factor signalling pathway in mammals. Screening revealed that well-known inhibitors of phospholipase A2 (PLA2), dexamethasone (Dex) and p-bromophenacyl bromide (BPB) inhibit LPS-dependent activation of the imd pathway. The inhibitory effects of Dex and BPB were suppressed by the addition of an excess of three (arachidonic acid, eicosapentaenoic acid and γ-linolenic acid) of the fatty acids so far tested. Arachidonic acid, however, did not activate the imd pathway when used as the sole agonist. These findings indicate that PLA2 participates in LPS-dependent activation of the imd pathway via the generation of arachidonic acid and other mediators, but requires additional signalling from LPS stimulation. Moreover, PLA2 was activated in response to bacterial infection in Sarcophaga. These results suggest a functional link between the PLA2-generated fatty acid cascade and the LPS-stimulated imd pathway in insect immunity.

Download Full-text

Kdo Hydrolase Is Required for Francisella tularensis Virulence and Evasion of TLR2-Mediated Innate Immunity

mBio ◽

10.1128/mbio.00638-12 ◽

2013 ◽

Vol 4 (1) ◽

Cited By ~ 13

Author(s):

Nihal A. Okan ◽

Sabina Chalabaev ◽

Tae-Hyun Kim ◽

Avner Fink ◽

Robin A. Ross ◽

...

Keyword(s):

Immune Response ◽

Innate Immunity ◽

Innate Immune Response ◽

Francisella Tularensis ◽

Innate Immune ◽

Content Type ◽

Tnf Α ◽

Schu S4 ◽

Highly Virulent

ABSTRACT The highly virulent Francisella tularensis subsp. tularensis has been classified as a category A bioterrorism agent. A live vaccine strain (LVS) has been developed but remains unlicensed in the United States because of an incomplete understanding of its attenuation. Lipopolysaccharide (LPS) modification is a common strategy employed by bacterial pathogens to avoid innate immunity. A novel modification enzyme has recently been identified in F. tularensis and Helicobacter pylori. This enzyme, a two-component Kdo (3-deoxy-d-manno-octulosonic acid) hydrolase, catalyzes the removal of a side chain Kdo sugar from LPS precursors. The biological significance of this modification has not yet been studied. To address the role of the two-component Kdo hydrolase KdhAB in F. tularensis pathogenesis, a ΔkdhAB deletion mutant was constructed from the LVS strain. In intranasal infection of mice, the ΔkdhAB mutant strain had a 50% lethal dose (LD50) 2 log10 units higher than that of the parental LVS strain. The levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) in bronchoalveolar lavage fluid were significantly higher (2-fold) in mice infected with the ΔkdhAB mutant than in mice infected with LVS. In vitro stimulation of bone marrow-derived macrophages with the ΔkdhAB mutant induced higher levels of TNF-α and IL-1β in a TLR2-dependent manner. In addition, TLR2−/− mice were more susceptible than wild-type mice to ΔkdhAB bacterial infection. Finally, immunization of mice with ΔkdhAB bacteria elicited a high level of protection against the highly virulent F. tularensis subsp. tularensis strain Schu S4. These findings suggest an important role for the Francisella Kdo hydrolase system in virulence and offer a novel mutant as a candidate vaccine. IMPORTANCE The first line of defense against a bacterial pathogen is innate immunity, which slows the progress of infection and allows time for adaptive immunity to develop. Some bacterial pathogens, such as Francisella tularensis, suppress the early innate immune response, killing the host before adaptive immunity can mature. To avoid an innate immune response, F. tularensis enzymatically modifies its lipopolysaccharide (LPS). A novel LPS modification—Kdo (3-deoxy-d-manno-octulosonic acid) saccharide removal—has recently been reported in F. tularensis. We found that the ∆kdhAB mutant was significantly attenuated in mice. Additionally, the mutant strain induced an early innate immune response in mice both in vitro and in vivo. Immunization of mice with this mutant provided protection against the highly virulent F. tularensis strain Schu S4. Thus, our study has identified a novel LPS modification important for microbial virulence. A mutant lacking this modification may be used as a live attenuated vaccine against tularemia.

Download Full-text

Effects of Dietary Maltol on Innate Immunity, Gut Health, and Growth Performance of Broiler Chickens Challenged With Eimeria maxima

Frontiers in Veterinary Science ◽

10.3389/fvets.2021.667425 ◽

2021 ◽

Vol 8 ◽

Author(s):

Inkyung Park ◽

Doyun Goo ◽

Hyoyoun Nam ◽

Samiru S. Wickramasuriya ◽

Kichoon Lee ◽

...

Keyword(s):

Immune Response ◽

Innate Immunity ◽

Growth Performance ◽

Innate Immune Response ◽

Broiler Chickens ◽

Muscle Cells ◽

Innate Immune ◽

Gut Health ◽

Eimeria Maxima

Two studies were conducted to evaluate the effects of maltol as a postbiotic on innate immunity, gut health, and enteric infection. In the first study, an in vitro culture system was used to evaluate the effects of maltol on the innate immune response of chicken macrophage cells (CMC), gut integrity of chicken intestinal epithelial cells (IEC), anti-parasitic activity against Eimeria maxima, and differentiation of quail muscle cells (QMC) and primary chicken embryonic muscle cells (PMC). All cells seeded in the 24-well plates were treated with maltol at concentrations of 0.1, 1.0, and 10.0 μg. CMC and IEC were stimulated by lipopolysaccharide to induce an innate immune response, and QMC and PMC were treated with 0.5 and 2% fetal bovine serum, respectively. After 18 h of incubation, pro-inflammatory cytokines, tight junction proteins (TJPs), and muscle cell growth markers were measured. In the second study, the dietary effect of maltol was evaluated on disease parameters in broiler chickens infected with E. maxima. Eighty male 1-day-old broiler chickens were allocated into the following four treatment groups: (1) Control group without infection, (2) Basal diet with E. maxima, (3) High maltol (HI; 10.0 mg /kg feed) with E. maxima, and (4) Low maltol (LO; 1.0 mg/kg feed) with E. maxima. Body weights (BW) were measured on days 0, 7, 14, 20, and 22. All chickens except the CON group were orally infected with 104E. maxima per chicken on day 14. Jejunum samples were collected for gut lesion scoring, and the gene expression of cytokines and TJPs. Data was analyzed using PROC MIXED in SAS. In vitro, maltol not only increased TJPs in IEC and cytokines in the LPS-stimulated CMC but also showed direct cytotoxicity against sporozoites of E. maxima. In vivo, the HI group improved the BW, reduced the gut lesion scores and fecal oocyst shedding, and decreased jejunal TNFSF15 and IL-1β expression in E. maxima-infected chickens. In conclusion, these results demonstrate the beneficial effects of dietary maltol in the enhancement of growth performance, gut health, and coccidiosis resistance and the applicability of maltol as a postbiotic for the replacement of antibiotic growth promoters in commercial poultry production.

Download Full-text

High content screening and computational prediction reveal viral genes that suppress innate immune response

10.1101/2021.12.14.472572 ◽

2021 ◽

Author(s):

Tai L Ng ◽

Erika J Olson ◽

Tae Yeon Yoo ◽

H. Sloane Weiss ◽

Yukiye Koide ◽

...

Keyword(s):

Immune Response ◽

Innate Immune Response ◽

Nuclear Transport ◽

Computational Prediction ◽

Viral Proteins ◽

Innate Immune ◽

Type I ◽

Viral Genes ◽

Genes Encoding

Suppression of the host innate immune response is a critical aspect of viral replication. Upon infection, viruses may introduce one or more proteins that inhibit key immune pathways, such as the type I interferon pathway. However, the ability to predict and evaluate viral protein bioactivity on targeted pathways remains challenging and is typically done on a single virus/gene basis. Here, we present a medium-throughput high-content cell-based assay to reveal the immunosuppressive effects of viral proteins. To test the predictive power of our approach, we developed a library of 800 genes encoding known, predicted, and uncharacterized human viral genes. We find that previously known immune suppressors from numerous viral families such as Picornaviridae and Flaviviridae recorded positive responses. These include a number of viral proteases for which we further confirmed that innate immune suppression depends on protease activity. A class of predicted inhibitors encoded by Rhabdoviridae viruses was demonstrated to block nuclear transport, and several previously uncharacterized proteins from uncultivated viruses were shown to inhibit nuclear transport of the transcription factors NF-kB and IRF3. We propose that this pathway-based assay, together with early sequencing, gene synthesis, and viral infection studies, could partly serve as the basis for rapid in vitro characterization of novel viral proteins.

Download Full-text

The Hepatitis C Virus-Induced Membranous Web in Liver Tissue

Cells ◽

10.3390/cells7110191 ◽

2018 ◽

Vol 7 (11) ◽

pp. 191

Author(s):

Emmanuelle Blanchard ◽

Philippe Roingeard

Keyword(s):

Immune Response ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Innate Immune Response ◽

Liver Tissue ◽

Hepatoma Cell ◽

Membrane Vesicles ◽

Innate Immune ◽

Host Cell Membrane

Host cell membrane rearrangements induced by the hepatitis C virus (HCV) have been exclusively studied in vitro. These studies have shown that HCV induces double-membrane vesicles (DMVs), which probably serve to separate replication sites from the cytoplasmic sensors of the innate immune response. We report for the first time the observation of HCV-induced membrane rearrangements in liver biopsy specimens from patients chronically infected with HCV. Unlike observations performed in vitro, the membranous web detected in liver tissue seems essentially made of clusters of single-membrane vesicles derived from the endoplasmic reticulum and close to lipid droplets. This suggests that the DMVs could be a hallmark of laboratory-adapted HCV strains, possibly due to their ability to achieve a high level of replication. Alternatively, the concealment of viral RNA in DMVs may be part of innate immune response mechanisms particularly developed in hepatoma cell lines cultured in vitro. In any case, this constitutes the first report showing the differences in the membranous web established by HCV in vitro and in vivo.

Download Full-text

An In Vitro Assessment of Immunostimulatory Responses to Ten Model Innate Immune Response Modulating Impurities (IIRMIs) and Peptide Drug Product, Teriparatide

Molecules ◽

10.3390/molecules26247461 ◽

2021 ◽

Vol 26 (24) ◽

pp. 7461

Author(s):

Claire K. Holley ◽

Edward Cedrone ◽

Duncan Donohue ◽

Barry W. Neun ◽

Daniela Verthelyi ◽

...

Keyword(s):

Immune Response ◽

Immune Responses ◽

Innate Immune Response ◽

Innate Immune ◽

Cytokine Secretion ◽

Poly I:C ◽

In Vitro Assays ◽

Innate Immune Responses ◽

Vitro Assay

Understanding, predicting, and minimizing the immunogenicity of peptide-based therapeutics are of paramount importance for ensuring the safety and efficacy of these products. The so-called anti-drug antibodies (ADA) may have various clinical consequences, including but not limited to the alteration in the product’s distribution, biological activity, and clearance profiles. The immunogenicity of biotherapeutics can be influenced by immunostimulation triggered by the presence of innate immune response modulating impurities (IIRMIs) inadvertently introduced during the manufacturing process. Herein, we evaluate the applicability of several in vitro assays (i.e., complement activation, leukocyte proliferation, and cytokine secretion) for the screening of innate immune responses induced by ten common IIRMIs (Bacillus subtilis flagellin, FSL-1, zymosan, ODN2006, poly(I:C) HMW, poly(I:C) LMW, CLO75, MDP, ODN2216, and Escherichia coli O111:B4 LPS), and a model biotherapeutic Forteo™ (teriparatide). Our study identifies cytokine secretion from healthy human donor peripheral blood mononuclear cells (PBMC) as a sensitive method for the in vitro monitoring of innate immune responses to individual IIRMIs and teriparatide (TP). We identify signature cytokines, evaluate both broad and narrow multiplex cytokine panels, and discuss how the assay logistics influence the performance of this in vitro assay.

Download Full-text

RNA sensors as a mechanism of innate immune evasion among SARS-CoV2, HIV and Nipah viruses

Current Protein and Peptide Science ◽

10.2174/1389203722666210322142725 ◽

2021 ◽

Vol 22 ◽

Author(s):

Dalia Cicily Kattiparambil Dixon ◽

Chameli Ratan ◽

Bhagyalakshmi Nair ◽

Sabitha Mangalath ◽

Rachy Abraham ◽

...

Keyword(s):

Immune Response ◽

Innate Immunity ◽

Innate Immune Response ◽

Vaccine Development ◽

Innate Immune ◽

Interferon Response ◽

Host Immune System ◽

Type I ◽

Adaptive Responses ◽

Rna Sensors

: Innate immunity is the first line of defence elicited by the host immune system to fight against invading pathogens such as viruses and bacteria. From this elementary immune response, the more complex antigen-specific adaptive responses are recruited to provide a long-lasting memory against the pathogens. Innate immunity gets activated when the host cell utilizes a diverse set of receptors known as pattern recognition receptors (PRR) to recognize the viruses that have penetrated the host and respond with cellular processes like complement system, phagocytosis, cytokine release and inflammation and destruction of NK cells. Viral RNA or DNA or viral intermediate products are recognized by receptors like toll-like receptors(TLRs), nucleotide oligomerization domain(NOD)-like receptors (NLRs) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) thereby, inducing type I interferon response (IFN) and other proinflammatory cytokines in infected cells or other immune cells. But certain viruses can evade the host innate immune response to replicate efficiently, triggering the spread of the viral infection. The present review describes the similarity in the mechanism chosen by viruses from different families -HIV, SARS-CoV2 and Nipah viruses to evade the innate immune response and how efficiently they establish the infection in the host. The review also addresses the stages of developments of various vaccines against these viral diseases and the challenges encountered by the researchers during vaccine development.

Download Full-text

Ursodeoxycholic Acid (UDCA) Mitigates the Host Inflammatory Response during Clostridioides difficile Infection by Altering Gut Bile Acids

Infection and Immunity ◽

10.1128/iai.00045-20 ◽

2020 ◽

Vol 88 (6) ◽

Author(s):

Jenessa A. Winston ◽

Alissa J. Rivera ◽

Jingwei Cai ◽

Rajani Thanissery ◽

Stephanie A. Montgomery ◽

...

Keyword(s):

Immune Response ◽

Bile Acid ◽

Inflammatory Response ◽

Innate Immune Response ◽

Innate Immune ◽

Colonization Resistance ◽

Content Type ◽

Clostridioides Difficile

ABSTRACT Clostridioides difficile infection (CDI) is associated with increasing morbidity and mortality posing an urgent threat to public health. Recurrence of CDI after successful treatment with antibiotics is high, thus necessitating discovery of novel therapeutics against this enteric pathogen. Administration of the secondary bile acid ursodeoxycholic acid (UDCA; ursodiol) inhibits the life cycles of various strains of C. difficile in vitro, suggesting that the FDA-approved formulation of UDCA, known as ursodiol, may be able to restore colonization resistance against C. difficile in vivo. However, the mechanism(s) by which ursodiol is able to restore colonization resistance against C. difficile remains unknown. Here, we confirmed that ursodiol inhibits C. difficile R20291 spore germination and outgrowth, growth, and toxin activity in a dose-dependent manner in vitro. In a murine model of CDI, exogenous administration of ursodiol resulted in significant alterations in the bile acid metabolome with little to no changes in gut microbial community structure. Ursodiol pretreatment resulted in attenuation of CDI pathogenesis early in the course of disease, which coincided with alterations in the cecal and colonic inflammatory transcriptome, bile acid-activated receptors nuclear farnesoid X receptor (FXR) and transmembrane G-protein-coupled membrane receptor 5 (TGR5), which are able to modulate the innate immune response through signaling pathways such as NF-κB. Although ursodiol pretreatment did not result in a consistent decrease in the C. difficile life cycle in vivo, it was able to attenuate an overly robust inflammatory response that is detrimental to the host during CDI. Ursodiol remains a viable nonantibiotic treatment and/or prevention strategy against CDI. Likewise, modulation of the host innate immune response via bile acid-activated receptors FXR and TGR5 represents a new potential treatment strategy for patients with CDI.

Download Full-text

Human PSC-Derived Hepatocytes Express Low Levels of Viral Pathogen Recognition Receptors, but Are Capable of Mounting an Effective Innate Immune Response

International Journal of Molecular Sciences ◽

10.3390/ijms21113831 ◽

2020 ◽

Vol 21 (11) ◽

pp. 3831

Author(s):

Lena Fischer ◽

Baltasar Lucendo-Villarin ◽

David C. Hay ◽

Cliona O’Farrelly

Keyword(s):

Immune Response ◽

Innate Immune Response ◽

Innate Immune ◽

Receptor Expression ◽

Cytochrome P450 3A ◽

Metabolic Switch ◽

Viral Pathogen ◽

Pathogen Recognition Receptors ◽

Viral Responses

Hepatocytes are key players in the innate immune response to liver pathogens but are challenging to study because of inaccessibility and a short half-life. Recent advances in in vitro differentiation of hepatocyte-like cells (HLCs) facilitated studies of hepatocyte–pathogen interactions. Here, we aimed to define the anti-viral innate immune potential of human HLCs with a focus on pattern recognition receptor (PRR)-expression and the presence of a metabolic switch. We analysed cytoplasmic PRR and endosomal toll-like receptor (TLR)-expression, as well as activity and adaptation of HLCs to an inflammatory environment. We found that transcript levels of retinoic acid inducible gene I (RIG-I), melanoma differentiation antigen 5 (MDA5), and TLR3 became downregulated during differentiation, indicating the acquisition of a more tolerogenic phenotype, as expected in healthy hepatocytes. HLCs responded to activation of RIG-I by producing interferons (IFNs) and IFN-stimulated genes. Despite low-level levels of TLR3, receptor expression was upregulated in an inflammatory environment. TLR3 signalling induced expression of proinflammatory cytokines at the gene level, indicating that several PRRs need to interact for successful innate immune activation. The inflammatory responsiveness of HLCs was accompanied by the downregulation of cytochrome P450 3A and 1A2 activity and decreased serum protein production, showing that the metabolic switch seen in primary hepatocytes during anti-viral responses is also present in HLCs.

Download Full-text