Impact of thermogenesis induced by chronic ß3-adrenergic receptor agonist treatment on inflammatory and infectious response during bacteremia in mice.

White adipocytes store energy differently than brown and brite adipocytes which dissipate energy under the form of heat. Studies have shown that adipocytes are able to respond to bacteria thanks to the presence of Toll-like receptors at their surface. Despite this, little is known about the involvement of each class of adipocytes in the infectious response. We treated mice for one week with a ß3-adrenergic receptor agonist to induce activation of brown adipose tissue and brite adipocytes within white adipose tissue. Mice were then injected intraperitoneally with E. coli to generate acute infection. The metabolic, infectious and inflammatory parameters of the mice were analysed during 48 hours after infection. Our results shown that in response to bacteria, thermogenic activity promoted a discrete and local anti-inflammatory environment in white adipose tissue characterized by the increase of the IL-1RA secretion. More generally, activation of brown and brite adipocytes did not modify the host response to infection including no additive effect with fever and an equivalent bacteria clearance and inflammatory response. In conclusion, these results suggest an IL-1RA-mediated immunomodulatory activity of thermogenic adipocytes in response to acute bacterial infection and open a way to characterize their effect along more chronic infection as septicaemia.

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.

Modulation of the inflammatory response to LPS by the recruitment and activation of brown and brite adipocytes in mice.

Numerous studies have shown that the recruitment and activation of thermogenic adipocytes, which are brown and beige/brite, reduce the mass of adipose tissue and normalize abnormal glycemia and lipidemia. However, the impact of these adipocytes on the inflammatory state of adipose tissue is still not well understood, especially in response to endotoxemia, which is a major aspect of obesity and metabolic diseases. First, we analyzed the phenotype and metabolic function of white and brite primary adipocytes in response to lipopolysaccharide (LPS) treatment in vitro. Then, 8-wk-old male BALB/c mice were treated for 1 wk with a ß3-adrenergic receptor agonist (CL316,243, 1 mg/kg/day) to induce recruitment and activation of brown and brite adipocytes and were subsequently injected with LPS (Escherichia coli lipopolysaccharide, 100 µg/mouse ip) to generate acute endotoxemia. The metabolic and inflammatory parameters of the mice were analyzed 6 h later. Our results showed that in response to LPS, thermogenic activity promoted a local anti-inflammatory environment with high secretion of IL-1 receptor antagonist (IL-1RA) without affecting other anti- or proinflammatory cytokines. Interestingly, activation of brite adipocytes reduced the LPS-induced secretion of leptin. However, thermogenic activity and adipocyte function were not altered by LPS treatment in vitro or by acute endotoxemia in vivo. In conclusion, these results suggest an IL-1RA-mediated immunomodulatory activity of thermogenic adipocytes specifically in response to endotoxemia. This encourages potential therapy involving brown and brite adipocytes for the treatment of obesity and associated metabolic diseases.NEW & NOTEWORTHY Recruitment and activation of brown and brite adipocytes in the adipose tissue of mice lead to a local low-grade anti-inflammatory phenotype in response to acute endotoxemia without alteration of adipocyte phenotype and function.

Diet Supplementation in ω3 Polyunsaturated Fatty Acid Favors an Anti-Inflammatory Basal Environment in Mouse Adipose Tissue.

Oxylipins are metabolized from dietary ω3 and ω6 polyunsaturated fatty acids and are involved in an inflammatory response. Adipose tissue inflammatory background is a key factor of metabolic disorders and it is accepted that dietary fatty acids, in terms of quality and quantity, modulate oxylipin synthesis in this tissue. Moreover, it has been reported that diet supplementation in ω3 polyunsaturated fatty acids resolves some inflammatory situations. Thus, it is crucial to assess the influence of dietary polyunsaturated fatty acids on oxylipin synthesis and their impact on adipose tissue inflammation. To this end, mice fed an ω6- or ω3-enriched standard diet (ω6/ω3 ratio of 30 and 3.75, respectively) were analyzed for inflammatory phenotype and adipose tissue oxylipin content. Diet enrichment with an ω3 polyunsaturated fatty acid induced an increase in the oxylipins derived from ω6 linoleic acid, ω3 eicosapentaenoic, and ω3 docosahexaenoic acids in brown and white adipose tissues. Among these, the level of pro-resolving mediator intermediates, as well as anti-inflammatory metabolites, were augmented. Concomitantly, expressions of M2 macrophage markers were increased without affecting inflammatory cytokine contents. In vitro, these metabolites did not activate macrophages but participated in macrophage polarization by inflammatory stimuli. In conclusion, we demonstrated that an ω3-enriched diet, in non-obesogenic non-inflammatory conditions, induced synthesis of oxylipins which were involved in an anti-inflammatory response as well as enhancement of the M2 macrophage molecular signature, without affecting inflammatory cytokine secretion.

Pseudomonas aeruginosa Exolysin promotes bacterial growth in lungs, alveolar damage and bacterial dissemination.

Exolysin (ExlA) is a recently-identified pore-forming toxin secreted by a subset of Pseudomonas aeruginosa strains identified worldwide and devoid of Type III secretion system (T3SS), a major virulence factor. Here, we characterized at the ultrastructural level the lesions caused by an ExlA-secreting strain, CLJ1, in mouse infected lungs. CLJ1 induced necrotic lesions in pneumocytes and endothelial cells, resulting in alveolo-vascular barrier breakdown. Ectopic expression of ExlA in an exlA-negative strain induced similar tissue injuries. In addition, ExlA conferred on bacteria the capacity to proliferate in lungs and to disseminate in secondary organs, similar to bacteria possessing a functional T3SS. CLJ1 did not promote a strong neutrophil infiltration in the alveoli, owing to the weak pro-inflammatory cytokine reaction engendered by the strain. However, CLJ1 was rapidly eliminated from the blood in a bacteremia model, suggesting that it can be promptly phagocytosed by immune cells. Together, our study ascribes to ExlA-secreting bacteria the capacity to proliferate in the lung and to damage pulmonary tissues, thereby promoting metastatic infections, in absence of substantial immune response exacerbation.

Immunoadjuvant Properties of the Rho Activating Factor CNF1 in Prophylactic and Curative Vaccination against Leishmania infantum.

There is a need to develop new effective immunoadjuvants for prophylactic or therapeutic vaccines against intracellular pathogens. The activation of Rho GTPases by bacterial cytotoxic necrotizing factor 1 (CNF1) elicits humoral protective responses against protein antigens. Here, we set out to investigate whether CNF1 activity initiates humoral immunity against co-administered parasite antigens and anti-microbial immune signaling. We report that co-administration of wild-type (WT) CNF1 with Leishmania (L.) promastigote antigens at the nasal mucosa triggered prophylactic and curative vaccine responses against this parasite. Vaccination of the mucosa with promastigote lysate antigens combined with WT CNF1 conferred protection against high inoculum L. infantum infection, which reached 82% in the spleen. Immune parameter analysis by antigen recall indicated robust T-helper (Th)1 polarization of immune memory cells, with high IL-2 and IFN-γ production combined with decreased IL-4 production. Additionally, we explored the curative effect of WT CNF1 on previously infected animals. We observed that PL combined with WT CNF1, but not the inactive C866S mutant CNF1 (mCNF1), induced a 58% decrease in the parasite burden in the spleen.

The Saccharomyces boulardii CNCM I-745 strain shows protective effects against the B. anthracis LT toxin.

The probiotic yeast Saccharomyces boulardii (S. boulardii) has been prescribed for the prophylaxis and treatment of several infectious diarrheal diseases. Gastrointestinal anthrax causes fatal systemic disease. In the present study, we investigated the protective effects conferred by Saccharomyces boulardii CNCM I-745 strain on polarized T84 columnar epithelial cells intoxicated by the lethal toxin (LT) of Bacillus anthracis. Exposure of polarized T84 cells to LT affected cell monolayer integrity, modified the morphology of tight junctions and induced the formation of actin stress fibers. Overnight treatment of cells with S. boulardii before incubation with LT maintained the integrity of the monolayers, prevented morphological modification of tight junctions, restricted the effects of LT on actin remodeling and delayed LT-induced MEK-2 cleavage. Mechanistically, we demonstrated that in the presence of S. boulardii, the medium is depleted of both LF and PA sub-units of LT and the appearance of a cleaved form of PA. Our study highlights the potential of the S. boulardii CNCM I-745 strain as a prophylactic agent against the gastrointestinal form of anthrax.

EDIN-B Promotes the Translocation of Staphylococcus aureus to the Bloodstream in the Course of Pneumonia.

It is crucial to define risk factors that contribute to host invasion by Staphylococcus aureus. Here, we demonstrate that the chromosomally encoded EDIN-B isoform from S. aureus contributes to the onset of bacteremia during the course of pneumonia. Deletion of edinB in a European lineage community-acquired methicillin resistant S. aureus (CA-MRSA) strain (ST80-MRSA-IV) dramatically decreased the frequency and magnitude of bacteremia in mice suffering from pneumonia. This deletion had no effect on the bacterial burden in both blood circulation and lung tissues. Re-expression of wild-type EDIN-B, unlike the catalytically inactive mutant EDIN-R185E, restored the invasive characteristics of ST80-MRSA-IV.

Escherichia coli α-hemolysin counteracts the anti-virulence innate immune response triggered by the Rho GTPase activating toxin CNF1 during bacteremia.

The detection of the activities of pathogen-encoded virulence factors by the innate immune system has emerged as a new paradigm of pathogen recognition. Much remains to be determined with regard to the molecular and cellular components contributing to this defense mechanism in mammals and importance during infection. Here, we reveal the central role of the IL-1ß signaling axis and Gr1+ cells in controlling the Escherichia coli burden in the blood in response to the sensing of the Rho GTPase-activating toxin CNF1. Consistently, this innate immune response is abrogated in caspase-1/11-impaired mice or following the treatment of infected mice with an IL-1ß antagonist. In vitro experiments further revealed the synergistic effects of CNF1 and LPS in promoting the maturation/secretion of IL-1ß and establishing the roles of Rac, ASC and caspase-1 in this pathway. Furthermore, we found that the α-hemolysin toxin inhibits IL-1ß secretion without affecting the recruitment of Gr1+ cells. Here, we report the first example of anti-virulence-triggered immunity counteracted by a pore-forming toxin during bacteremia.

Cherubism allele heterozygosity amplifies microbe-induced inflammatory responses in murine macrophages.

Cherubism is a rare autoinflammatory bone disorder that is associated with point mutations in the SH3-domain binding protein 2 (SH3BP2) gene, which encodes the adapter protein 3BP2. Individuals with cherubism present with symmetrical fibro-osseous lesions of the jaw, which are attributed to exacerbated osteoclast activation and defective osteoblast differentiation. Although it is a dominant trait in humans, cherubism appears to be recessively transmitted in mice, suggesting the existence of additional factors in the pathogenesis of cherubism. Here, we report that macrophages from 3BP2-deficient mice exhibited dramatically reduced inflammatory responses to microbial challenge and reduced phagocytosis. 3BP2 was necessary for LPS-induced activation of signaling pathways involved in macrophage function, including SRC, VAV1, p38MAPK, IKKα/ß, RAC, and actin polymerization pathways. Conversely, we demonstrated that the presence of a single Sh3bp2 cherubic allele and pathogen-associated molecular pattern (PAMP) stimulation had a strong cooperative effect on macrophage activation and inflammatory responses in mice. Together, the results from our study in murine genetic models support the notion that infection may represent a driver event in the etiology of cherubism in humans and suggest limiting inflammation in affected individuals may reduce manifestation of cherubic lesions.

An Unusual Recurrence of Signet Ring Cell Gastric Adenocarcinoma Treated by Right Hemicolectomy, Pancreaticoduodenectomy, and IVC Resection: Controversies and Dilemmas of Following Standard Treatment Pathways.

We present the case of a 67-year-old male patient with a past history of previously resected T3 right adrenocortical carcinoma and T3N1 signet ring cell adenocarcinoma of the stomach who presented with recurrence of gastric cancer in the form of a large solitary mass in the right abdomen. He was treated with ECX (epirubicin, cisplatin and capecitabine) chemotherapy and multivisceral resection. This recurrence pattern is the first such description in the literature, and we discuss the controversies and arguments in favour of offering surgical resection.

Saccharomyces boulardii modifies Salmonella typhimurium traffic and host immune responses along the intestinal tract.

Salmonella enterica serovar Typhimurium (ST) is an enteropathogenic Gram-negative bacterium that causes infection following oral ingestion. ST spreads rapidly along the gastrointestinal tract (GIT) and invades the intestinal epithelium to ultimately reach internal body organs. The probiotic yeast Saccharomyces boulardii BIOCODEX (S.b-B) is prescribed for prophylaxis of diarrheal infectious diseases. We previously showed that S.b-B prevents weight loss in ST-infected mice and significantly decreases bacterial translocation to the spleen and liver. This study was designed to investigate the effect of S.b-B on ST migration along the GIT and the impact of the yeast on the host's early innate immune responses. Bioluminescent imaging (BLI) was used to evaluate the effect of S.b-B on the progression of luminescent Salmonella Typhimurium (ST-lux) in the GIT of mice pretreated with streptomycin. Photonic emission (PE) was measured in GIT extracts (stomach, small intestine, cecum and colon) at various time periods post-infection (PI). PE analysis revealed that, 45 min PI, ST-lux had migrated slightly faster in the mice treated with S.b-B than in the untreated infected animals. At 90 min PI, ST-lux had reached the cecum in both groups of mice. Adhesion of ST to S.b-B was visualized in the intestines of the mice and probably accounts for (1) the faster elimination of ST-lux in the feces, and (2) reduced translocation of ST to the spleen and liver. In the early phase of infection, S.b-B also modifies the host's immune responses by (1) increasing IFN-γ gene expression and decreasing IL-10 gene expression in the small intestine, and (2) elevating both IFN-γ, and IL-10 mRNA levels in the cecum. BLI revealed that S.b-B modifies ST migration and the host immune response along the GIT. Study findings shed new light on the protective mechanisms of S.b-B during the early phase of Salmonella pathogenesis.

Modification of Salmonella Typhimurium motility by the probiotic yeast strain Saccharomyces boulardii.

BACKGROUND: Motility is an important component of Salmonella enterica serovar Typhimurium (ST) pathogenesis allowing the bacteria to move into appropriate niches, across the mucus layer and invade the intestinal epithelium. In vitro, flagellum-associated motility is closely related to the invasive properties of ST. The probiotic yeast Saccharomyces boulardii BIOCODEX (S.b-B) is widely prescribed for the prophylaxis and treatment of diarrheal diseases caused by bacteria or antibiotics. In case of Salmonella infection, S.b-B has been shown to decrease ST invasion of T84 colon cell line. The present study was designed to investigate the impact of S.b-B on ST motility. METHODOLOGY/PRINCIPAL FINDINGS: Experiments were performed on human colonic T84 cells infected by the Salmonella strain 1344 alone or in the presence of S.b-B. The motility of Salmonella was recorded by time-lapse video microscopy. Next, a manual tracking was performed to analyze bacteria dynamics (MTrackJ plugin, NIH image J software). This revealed that the speed of bacterial movement was modified in the presence of S.b-B. The median curvilinear velocity (CLV) of Salmonella incubated alone with T84 decreased from 43.3 µm/sec to 31.2 µm/sec in the presence of S.b-B. Measurement of track linearity (TL) showed similar trends: S.b-B decreased by 15% the number of bacteria with linear tract (LT) and increased by 22% the number of bacteria with rotator tract (RT). Correlation between ST motility and invasion was further established by studying a non-motile flagella-deficient ST strain. Indeed this strain that moved with a CLV of 0.5 µm/sec, presented a majority of RT and a significant decrease in invasion properties. Importantly, we show that S.b-B modified the motility of the pathogenic strain SL1344 and significantly decreased invasion of T84 cells by this strain. CONCLUSIONS: This study reveals that S.b-B modifies Salmonella's motility and trajectory which may account for the modification of Salmonella's invasion.

The E3 ubiquitin-ligase HACE1 catalyzes the ubiquitylation of active Rac1.

Rac1 small GTPase controls essential aspects of cell biology and is a direct target of numerous bacterial virulence factors. The CNF1 toxin of pathogenic Escherichia coli addresses Rac1 to ubiquitin-proteasome system (UPS). We report the essential role of the tumor suppressor HACE1, a HECT-domain containing E3 ubiquitin-ligase, in the targeting of Rac1 to UPS. HACE1 binds preferentially GTP-bound Rac1 and catalyzes its polyubiquitylation. HACE1 expression increases the ubiquitylation of Rac1, when the GTPase is activated by point mutations or by the GEF-domain of Dbl. RNAi-mediated depletion of HACE1 blocks the ubiquitylation of active Rac1 and increases GTP-bound Rac1 cellular levels. HACE1 antagonizes cell isotropic spreading, a hallmark of Rac1 activation, and is required for endothelial cell monolayer invasion by bacteria. Together, these data establish the role of the HACE1 E3 ubiquitin-ligase in controlling Rac1 ubiquitylation and activity.

cAMP signaling by anthrax edema toxin induces transendothelial cell tunnels, which are resealed by MIM via Arp2/3-driven actin polymerization.

RhoA-inhibitory bacterial toxins, such as Staphylococcus aureus EDIN toxin, induce large transendothelial cell macroaperture (TEM) tunnels that rupture the host endothelium barrier and promote bacterial dissemination. Host cells repair these tunnels by extending actin-rich membrane waves from the TEM edges. We reveal that cyclic-AMP signaling produced by Bacillus anthracis edema toxin (ET) also induces TEM formation, which correlates with increased vascular permeability. We show that ET-induced TEM formation resembles liquid dewetting, a physical process of nucleation and growth of holes within a thin liquid film. We also identify the cellular mechanisms of tunnel closure and reveal that the I-BAR domain protein Missing in Metastasis (MIM) senses de novo membrane curvature generated by the TEM, accumulates at the TEM edge, and triggers Arp2/3-dependent actin polymerization, which induces actin-rich membrane waves that close the TEM. Thus, the balance between ET-induced TEM formation and resealing likely determines the integrity of the host endothelium barrier.

Luciferase-expressing Leishmania infantum allows the monitoring of amastigote population size, in vivo, ex vivo and in vitro.

Here we engineered transgenic Leishmania infantum that express luciferase, the objectives being to more easily monitor in real time their establishment either in BALB/c mice--the liver and spleen being mainly studied-or in vitro. Whatever stationary phase L. infantum promastigotes population--wild type or engineered to express luciferase-the parasite burden was similar in the liver and the spleen at day 30 post the intravenous inoculation of BALB/c mice. Imaging of L. infantum hosting BALB/C mice provided sensitivity in the range of 20,000 to 40,000 amastigotes/mg tissue, two tissues-liver and spleen-being monitored. Once sampled and processed ex vivo for their luciferin-dependent bioluminescence the threshold sensitivity was shown to range from 1,000 to 6,000 amastigotes/mg tissue. This model further proved to be valuable for in vivo measurement of the efficiency of drugs such as miltefosine and may, therefore, additionally be used to evaluate vaccine-induced protection.

The Staphylococcus aureus epidermal cell differentiation inhibitor toxin promotes formation of infection foci in a mouse model of bacteremia.

Inactivation of the host GTPase RhoA by staphylococcal epidermal cell differentiation inhibitor (EDIN) exotoxins triggers the formation of large transcellular tunnels, named macroapertures, in endothelial cells. We used bioluminescent strains of Staphylococcus aureus to monitor the formation of infection foci during the first 24 h of hematogenous bacterial dissemination. Clinically derived EDIN-expressing S. aureus strains S25 and Xen36 produced many disseminated foci. EDIN had no detectable impact on infection foci in terms of histopathology or the intensity of emitted light. Moreover, EDIN did not modify the course of bacterial clearance from the bloodstream. In contrast, we show that EDIN expression promotes a 5-fold increase in the number of infection foci produced by Xen36. This virulence activity of EDIN requires RhoA ADP-ribosyltranferase activity. These results suggest that EDIN is a risk factor for S. aureus dissemination through the vasculature by virtue of its ability to promote the formation of infection foci in deep-seated tissues.

Injection of Staphylococcus aureus EDIN by the Bacillus anthracis protective antigen machinery induces vascular permeability.

Systemic injection of Bacillus anthracis lethal toxin (LT) produces vascular leakage and animal death. Recent studies suggest that LT triggers direct endothelial cell cytotoxicity that is responsible for the vascular leakage. LT is composed of heptamers of protective antigen (PA), which drives the endocytosis and translocation into host cells of the lethal factor (LF), a mitogen-activated protein kinase kinase protease. Here we investigated the consequences of injection of an endothelium-permeabilizing factor using LT as a "molecular syringe." To this end, we generated the chimeric factor LE, corresponding to the PA-binding domain of LF (LF(1-254)) fused to EDIN exoenzyme. EDIN ADP ribosylates RhoA, leading to actin cable disruption and formation of transcellular tunnels in endothelial cells. We report that systemic injection of LET (LE plus PA) triggers a PA-dependent increase in the pulmonary endothelium permeability. We also report that native LT induces a progressive loss of endothelium barrier function. We established that there is a direct correlation between the extent of endothelium permeability induced by LT and the cytotoxic activity of LT. This suggests new ways to design therapeutic drugs against anthrax directed toward vascular permeability.