Effector-Triggered Trained Immunity: An Innate Immune Memory to Microbial Virulence Factors?

In the last decade, a major dogma in the field of immunology has been called into question by the identification of a cell autonomous innate immune memory. This innate immune memory (also named trained immunity) was found to be mostly carried by innate immune cells and to be characterized by an exacerbated inflammatory response with a heightened expression of proinflammatory cytokines, including TNF-α, IL-6 and IL-1ß. Unlike the vast majority of cytokines, IL-1ß is produced as a proform (pro-IL-1ß) and requires a proteolytic cleavage to exert its biological action. This cleavage takes place mainly within complex molecular platforms named inflammasomes. These platforms are assembled upon both the infectious or sterile activation of NOD-like receptors (NLRs), thereby allowing for the recruitment and activation of caspases and the subsequent maturation of pro-IL-1ß into IL-1ß. The NLRP3 inflammasome has recently been implicated both in western diet-induced trained immunity, and in the detection of microbial virulence factors (effector-triggered immunity (ETI)). Here, we will attempt to link these two immune processes and provide arguments to hypothesize the existence of trained immunity triggered by microbial virulence factors (effector-triggered trained immunity (ETTI)).

Heterogeneous NLRP3 inflammasome signature in circulating myeloid cells as a biomarker of COVID-19 severity.

Dysregulated immune response is the key factor leading to unfavorable coronavirus disease 2019 (COVID-19) outcome. Depending on the pathogen-associated molecular pattern, the NLRP3 inflammasome can play a crucial role during innate immunity activation. To date, studies describing the NLRP3 response during severe acute respiratory syndrome coronavirus 2 infection in patients are lacking. We prospectively monitored caspase-1 activation levels in peripheral myeloid cells from healthy donors and patients with mild to critical COVID-19. The caspase-1 activation potential in response to NLRP3 inflammasome stimulation was opposed between nonclassical monocytes and CD66b+CD16dim granulocytes in severe and critical COVID-19 patients. Unexpectedly, the CD66b+CD16dim granulocytes had decreased nigericin-triggered caspase-1 activation potential associated with an increased percentage of NLRP3 inflammasome impaired immature neutrophils and a loss of eosinophils in the blood. In patients who recovered from COVID-19, nigericin-triggered caspase-1 activation potential in CD66b+CD16dim cells was restored and the proportion of immature neutrophils was similar to control. Here, we reveal that NLRP3 inflammasome activation potential differs among myeloid cells and could be used as a biomarker of a COVID-19 patient's evolution. This assay could be a useful tool to predict patient outcome. This trial was registered at www.clinicaltrials.gov as #NCT04385017.

Escherichia coli Rho GTPase-activating toxin CNF1 mediates NLRP3 inflammasome activation via p21-activated kinases-1/2 during bacteraemia in mice.

Inflammasomes are signalling platforms that are assembled in response to infection or sterile inflammation by cytosolic pattern recognition receptors. The consequent inflammasome-triggered caspase-1 activation is critical for the host defence against pathogens. During infection, NLRP3, which is a pattern recognition receptor that is also known as cryopyrin, triggers the assembly of the inflammasome-activating caspase-1 through the recruitment of ASC and Nek7. The activation of the NLRP3 inflammasome is tightly controlled both transcriptionally and post-translationally. Despite the importance of the NLRP3 inflammasome regulation in autoinflammatory and infectious diseases, little is known about the mechanism controlling the activation of NLRP3 and the upstream signalling that regulates the NLRP3 inflammasome assembly. We have previously shown that the Rho-GTPase-activating toxin from Escherichia coli cytotoxic necrotizing factor-1 (CNF1) activates caspase-1, but the upstream mechanism is unclear. Here, we provide evidence of the role of the NLRP3 inflammasome in sensing the activity of bacterial toxins and virulence factors that activate host Rho GTPases. We demonstrate that this activation relies on the monitoring of the toxin's activity on the Rho GTPase Rac2. We also show that the NLRP3 inflammasome is activated by a signalling cascade that involves the p21-activated kinases 1 and 2 (Pak1/2) and the Pak1-mediated phosphorylation of Thr 659 of NLRP3, which is necessary for the NLRP3-Nek7 interaction, inflammasome activation and IL-1ß cytokine maturation. Furthermore, inhibition of the Pak-NLRP3 axis decreases the bacterial clearance of CNF1-expressing UTI89 E. coli during bacteraemia in mice. Taken together, our results establish that Pak1 and Pak2 are critical regulators of the NLRP3 inflammasome and reveal the role of the Pak-NLRP3 signalling axis in vivo during bacteraemia in mice.

Temperature affects the biology of Schmidtea mediterranea.

Studies of tissue regeneration and host-pathogen interactions using the model planarian Schmidtea mediterranea have been performed at an experimental temperature of 19 °C. S. mediterranea planarians exposed to 19 °C-32 °C were observed for survival, mobility, feeding and regeneration for three months and elimination of the Staphylococcus aureus pathogen over six days. S. mediterranea planarians died at 30 °C-32 °C after 18 days of observation but tolerated temperatures of 19 °C up to 28 °C with non-significant differences in mobility and feeding behavior. Genetic malleability tested by RNAi feeding was still efficient at 26 °C and 28 °C. Concerning the immune capacity of planarians, we reported an exacerbation of the immune response in worms infected by S. aureus at 26 °C and 28 °C. These observations suggest a temperature modulation of planarian stem cells and illustrate the importance of modulating experimental temperature when using planarians as model organisms to study regeneration and immune response.

In silico analysis of Schmidtea mediterranea TIR domain-containing proteins.

While genetic evidence points towards an absence of Toll-Like Receptors (TLRs) in Platyhelminthes, the Toll/IL-1 Receptor (TIR)-domains that drive the assembly of signalling complexes downstream TLR are present in these organisms. Here, we undertook the characterisation of the repertoire of TIR-domain containing proteins in Schmidtea mediterranea in order to gain valuable information on TLR evolution in metazoan. We report the presence of twenty proteins containing between one and two TIR domains. In addition, our phylogenetic-based reconstruction approach identified Smed-SARM and Smed-MyD88 as conserved TLR adaptors.

Trained Immunity Carried by Non-immune Cells.

"Trained immunity" is a term proposed by Netea to describe the ability of an organism to develop an exacerbated immunological response to protect against a second infection independent of the adaptative immunity. This immunological memory can last from 1 week to several months and is only described in innate immune cells such as monocytes, macrophages, and natural killer cells. Paradoxically, the lifespan of these cells in the blood is shorter than the duration of trained immunity. This observation suggested that trained immunity could be carried by long lifespan cells such as stem cells and non-immune cells like fibroblasts. It is now evident that in addition to performing their putative function in the development and maintenance of tissue homeostasis, non-immune cells also play an important role in the response to pathogens by producing anti-microbial factors, with long-term inflammation suggesting that non-immune cells can be trained to confer long-lasting immunological memory. This review provides a summary of the current relevant knowledge about the cells which possess immunological memory and discusses the possibility that non-immune cells may carry immunological memory and mechanisms that might be involved.

[Trained immunity in invertebrates: what do we know?] / La mémoire immunitaire entraînée chez les invertébrés - Que sait-on ?

One of the defense mechanisms of the host is the trained immunity, an immune component of the innate immunity, also known as innate immune memory. The trained immunity is defined as an exacerbated protection of an organism to a foreign body, such as a pathogenic microorganism, upon a second contact with it. This kind of immunity does not involve the components of acquired immunity, such as the B lymphocytes or T lymphocytes. It relies heavily on the cellular and molecular machinery of the innate response. Trained immunity is described as being present in both invertebrates and vertebrates, but its mechanisms have only recently begun to be elucidated, particularly in the vertebrates. Today, most of the research about innate immune memory deals with vertebrate models, forgetting that this mechanism exists in invertebrates. Investigating trained immunity in invertebrates gives the advantage to work with models naturally lacking acquired immunity. The aim of this review is to take stock of the knowledge concerning the trained immunity entailed in invertebrates.

Enzymatic degradation of organophosphorus insecticides decreases toxicity in planarians and enhances survival.

Organophosphorus insecticides (OPs) are toxic compounds used for agricultural purposes and responsible for severe types of contamination worldwide. OPs may also induce chronic deleterious effects and developmental disruption. Finding remediation strategies is a major concern to diminish their impact on environment and human health. Enzymes have emerged as a promising eco-friendly route for decontaminating OPs. The enzyme SsoPox from the archaea Sulfolobus solfataricus has been particularly studied, considering both its tremendous stability and phosphotriesterase activity. However, the toxicity of the degradation products generated through enzyme hydrolysis has been poorly investigated. To address both neurotoxicity and developmental perturbation, freshwater planarians from Platyhelminthes were considered to evaluate the impact of OP and degradation product exposure. Planarians have a large proportion of stem cells that give them an unconventional capacity for regeneration. OPs were found to be highly toxic to planarians and enzyme decontamination drastically enhanced survival rate. Although not completely innocuous, the degradation products were found to be less toxic than insecticides and reduced poisoning effects by increasing NOEC values by up to eight-fold. SsoPox also limited detrimental consequences on planarian mobility and enabled them to recover a non-exposed type regeneration process suggesting that enzymatic decontamination is a promising alternative to bioremediation.

Staphylococcus aureus Promotes Smed-PGRP-2/Smed-setd8-1 Methyltransferase Signalling in Planarian Neoblasts to Sensitize Anti-bacterial Gene Responses During Re-infection.

Little is known about how organisms exposed to recurrent infections adapt their innate immune responses. Here, we report that planarians display a form of instructed immunity to primo-infection by Staphylococcus aureus that consists of a transient state of heightened resistance to re-infection that persists for approximately 30days after primo-infection. We established the involvement of stem cell-like neoblasts in this instructed immunity using the complementary approaches of RNA-interference-mediated cell depletion and tissue grafting-mediated gain of function. Mechanistically, primo-infection leads to expression of the peptidoglycan receptor Smed-PGRP-2, which in turn promotes Smed-setd8-1 histone methyltransferase expression and increases levels of lysine methylation in neoblasts. Depletion of neoblasts did not affect S. aureus clearance in primo-infection but, in re-infection, abrogated the heightened elimination of bacteria and reduced Smed-PGRP-2 and Smed-setd8-1 expression. Smed-PGRP-2 and Smed-setd8-1 sensitize animals to heightened expression of Smed-p38 MAPK and Smed-morn2, which are downstream components of anti-bacterial responses. Our study reveals a central role of neoblasts in innate immunity against S. aureus to establish a resistance state facilitating Smed-sted8-1-dependent expression of anti-bacterial genes during re-infection.

Antimicrobial capacity of the freshwater planarians against S. aureus is under the control of Timeless.

Planarians, which are non-parasitic flatworms, are highly resistant to bacterial infections. To better understand the mechanisms underlying this resistance, we investigated the role of the circadian machinery in the anti-bacterial response of the freshwater planarian Schmidtea mediterranea. We identified Smed-Tim from S. mediterranea as a homolog of the mammalian clock gene Tim. We showed via RNA interference that Smed-Tim is required for the anti-microbial activities of Schmidtea mediterranea against Staphylococcus aureus infection during the light/dark cycle. Indeed, S. aureus infection leads to the expression of Smed-Tim, which in turn promotes Smed-Traf6 and Smed-morn2, but not Smed-p38 MAPK expression, 2 master regulators of planarian anti-microbial responses.

Circadian Control of Antibacterial Immunity: Findings from Animal Models.

Most of the biological functions, including the immune system, are linked to circadian rhythms in living organisms. Changes occurring to biological parameters as the result of these circadian rhythms can therefore affect the outcome of a disease. For decades, model organisms have proven to be a great tool to understanding biological mechanisms such as circadian cycle and immunity. In this review, we created an inventory of the use of model organisms in order to decipher the relation between circadian rhythms and antibacterial immunity.

Screening in planarians identifies MORN2 as a key component in LC3-associated phagocytosis and resistance to bacterial infection.

Dugesia japonica planarian flatworms are naturally exposed to various microbes but typically survive this challenge. We show that planarians eliminate bacteria pathogenic to Homo sapiens, Caenorhabditis elegans, and/or Drosophila melanogaster and thus represent a model to identify innate resistance mechanisms. Whole-transcriptome analysis coupled with RNAi screening of worms infected with Staphylococcus aureus or Legionella pneumophila identified 18 resistance genes with nine human orthologs, of which we examined the function of MORN2. Human MORN2 facilitates phagocytosis-mediated restriction of Mycobacterium tuberculosis, L. pneumophila, and S. aureus in macrophages. MORN2 promotes the recruitment of LC3, an autophagy protein also involved in phagocytosis, to M. tuberculosis-containing phagosomes and subsequent maturation to degradative phagolysosomes. MORN2-driven trafficking of M. tuberculosis to single-membrane, LC3-positive compartments requires autophagy-related proteins Atg5 and Beclin-1, but not Ulk-1 and Atg13, highlighting the importance of MORN2 in LC3-associated phagocytosis. These findings underscore the value of studying planarian defenses to identify immune factors.

What RNAi screens in model organisms revealed about microbicidal response in mammals?

The strategies evolved by pathogens to infect hosts and the mechanisms used by the host to eliminate intruders are highly complex. Because several biological pathways and processes are conserved across model organisms, these organisms have been used for many years to elucidate and understand the mechanisms of the host-pathogen relationship and particularly to unravel the molecular processes enacted by the host to kill pathogens. The emergence of RNA interference (RNAi) and the ability to apply it toward studies in model organisms have allowed a breakthrough in the elucidation of host-pathogen interactions. The aim of this mini-review is to highlight and describe recent breakthroughs in the field of host-pathogen interactions using RNAi screens of model organisms. We will focus specifically on the model organisms Drosophila melanogaster, Caenorhabditis elegans, and Danio rerio. Moreover, a recent study examining the immune system of planarian will be discussed.