Biology subjects

Sirard, J.-C.

Publications and source records attributed to Sirard, J.-C..

9 recordsLinked to original sources

bioRxiv2025

Local tolerance and innate immune activation of primary human respiratory cells exposed to flagellin

Antibiotic-resistant respiratory infections can lead to treatment failure, highlighting the need for alternative strategies. FLAMOD, a recombinant flagellin, stimulates innate immunity via Toll-like receptor 5 when delivered intranasally. In mice, FLAMOD protects against bacterial pneumonia. The protection is associated with activation of airway epithelial cells. This study aimed to assess the tolerance of human primary respiratory epithelium to FLAMOD administered apically as liquid droplets or by nebulization, to measure the innate immune response, and the pharmacokinetics of FLAMOD. We used epithelia reconstituted from human nasal and bronchial (MucilAir), small airways (SmallAir) and alveolar (AlveolAir) primary epithelial cells, cultured at the air-liquid interface. We report that daily administration of escalating doses of FLAMOD for 5 days was well tolerated by epithelia as barrier integrity, cilia motion and cell viability were not affected. FLAMOD was rapidly degraded without leakage into the basal compartment. Each epithelial model exhibited responses involving pathways of innate defense and immune cell infiltration, which were dose-dependent, with an effective concentration of FLAMOD in the picomolar range. Similar tolerance profile and immune responses were obtained with airway epithelium from cystic fibrosis and chronic obstructive pulmonary disease patients. In conclusion, this study supports the stimulation of epithelial Toll-like receptor 5 signaling to fight against infections of vulnerable patients.

cell biology↗

bioRxiv2025

Flagellin-mediated TLR5 activation enhances innate immune responses in healthy and diseased human airway epithelium

Bacterial pneumonia poses a significant challenge to public health, often leading to antibiotic treatment failure. Enhancing innate immunity represents a promising adjunctive strategy to conventional antibiotic therapy. Bacterial flagellin, a Toll-like receptor 5 (TLR5) agonist, has been shown to stimulate innate immune defenses when delivered via the respiratory route, demonstrating efficacy in both preventing and treating bacterial pneumonia in murine models. This protective effect is primarily mediated through TLR5-driven activation of airway epithelial cells. This study aimed to characterize the immunomodulatory effects of flagellin on human primary respiratory epithelium. Using the MucilAir air-liquid interface model and RNA sequencing, we demonstrated that apical administration of flagellin induced robust immune responses in airway epithelium derived from healthy individuals, as well as patients with chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). TLR5-mediated epithelial signaling triggered key immune-related pathways, including cytokine production, leukocyte chemotaxis, neutrophil recruitment, and antimicrobial defense, with strong commonalities across healthy and diseased airway epithelia. Furthermore, we demonstrated that flagellin effectively activated epithelial immune responses even in the presence of the bacteria Pseudomonas aeruginosa or Streptococcus pneumoniae. However, epithelial activation alone was insufficient to directly limit bacterial colonization or replication, highlighting the potential role of epithelial-immune cell interactions in achieving effective bacterial clearance. These findings support TLR5 activation as a promising therapeutic strategy to enhance host defense mechanisms and improve treatment outcomes for bacterial pneumonia in both healthy individuals and patients with COPD or CF.

immunology↗

bioRxiv2025

Flagellin nebulization enhances respiratory immune responses in the porcine model

Respiratory delivery of the Toll-like receptor 5 agonist FLAMOD, a recombinant flagellin, offers a promising approach for treating bacterial pneumonia. FLAMOD stimulates the airway epithelium, mobilizing and activating immune cells and effectors to combat infections. While previous evidences were obtained in mouse models, this study represents the first comprehensive assessment of FLAMOD delivered by nebulization in pigs. Our results demonstrate that a single nebulization of FLAMOD did not cause any adverse effects on clinical parameters. Histological analysis supported that FLAMOD treatment led to immune cell infiltration in the lung tissue, indicative of an active immune response. Flow cytometry confirmed granulocyte recruitment in conducting airways. RNA sequencing established immune activation across the respiratory tract, from the nose, trachea, bronchi to the lungs, highlighting innate immunity, bacterial defense, cytokine and chemokine signaling, and granulocyte chemotaxis as key biological pathways. These findings demonstrated the capacity of FLAMOD to induce a robust and common immune response throughout the porcine respiratory system as well as specific compartmentalized immune signatures. This study establishes FLAMOD as a potent activator of innate immunity, providing a proof-of-concept for inhalation-based therapeutic strategies to combat bacterial pneumonia in the clinical setting.

immunology↗

bioRxiv2025

Flagellin aerosol administration improves the efficacy of antibiotic treatment in Actinobacillus pleuropneumoniae infected pigs

Swine infections with Actinobacillus pleuropneumoniae (App), the causative agent of porcine pleuropneumonia, lead to significant production losses. Surviving pigs may harbor antibiotic-resistant bacteria, highlighting the need for alternatives to reduce antimicrobial use. Stimulation of respiratory innate immunity via Toll-like receptor 5 (TLR5) agonists such as FLAMOD (a recombinant flagellin), has emerged as a promising strategy for combating bacterial lung infections. In a recent study, aerosolized administration of FLAMOD was found to elicit respiratory immune responses in pigs. This study assessed FLAMODs protective effects against App, either as a prophylactic intervention or as an adjunct to antibiotic therapy. Prophylactic administration of aerosolized FLAMOD via nebulization before App challenge failed to confer protection. However, in a therapeutic setting, where pigs were infected with App and treated with either a subtherapeutic dose of penicillin G (PenG) alone or a combination of PenG and aerosolized FLAMOD. Notably, the FLAMOD and PenG combination significantly improved bacterial clearance from the lungs. Moreover, FLAMOD-PenG treated pigs exhibited a trend toward increased weight gain and fewer lung lesions compared to those receiving PenG alone. These findings provide proof for the concept that FLAMOD enhances antibiotic efficacy, potentially lowering the required antibiotic dose and contributing to antimicrobial resistance mitigation in pigs.

immunology↗

bioRxiv2024

Targeted delivery of flagellin by nebulization offers optimized respiratory immunity and defense against pneumococcal pneumonia

Novel therapeutic strategies are urgently needed to combat pneumonia caused by Streptococcus pneumoniae strains resistant to standard-of-care antibiotics. Previous studies have shown that targeted stimulation of lung innate immune defenses through intranasal administration of the Toll-like receptor 5 agonist flagellin, improves the treatment of pneumonia when combined with antibiotics. To promote translation to the clinic application, this study assessed the direct delivery of flagellin to the airways through nebulization using a vibrating mesh nebulizer in mice. Intranasal delivery achieved approximately 40% lung deposition of the administered flagellin dose, whereas nebulization yielded less than 1%. Despite these differences, nebulized flagellin induced a transient activation of lung innate immunity characterized by cytokine/chemokine production and neutrophil infiltration into airways analogous to intranasal administration. Furthermore, inhalation by nebulization resulted in an accelerated resolution of systemic pro-inflammatory responses. Lastly, adjunct therapy combining nebulized flagellin and amoxicillin proved effective against antibiotic-resistant pneumococcal pneumonia in mice. We posit that flagellin aerosol therapy represents a safe and promising approach to address bacterial pneumonia within the context of antimicrobial resistance.

microbiology↗

bioRxiv2023

A conserved antigen induces respiratory Th17-mediated serotype-independent protection against pneumococcal superinfection

Several vaccines targeting bacterial pathogens show reduced efficacy in the context of intercurrent viral infection indicating a new vaccinology approach is required to protect against such superinfections. To find antigens for the human pathogen Streptococcus pneumoniae that are effective following influenza infection, we performed CRISPRi-seq in a murine model of superinfection and identified the highly conserved lafB gene as virulence factor. We show that LafB is a membrane-associated, intracellular protein that catalyzes the formation of galactosyl-glucosyl-diacylglycerol, a glycolipid we show is important for cell wall homeostasis. Respiratory vaccination with recombinant LafB, in contrast to subcutaneous vaccination, was highly protective against all serotypes in a murine model. In contrast to standard pneumococcal capsule-based conjugate vaccines, protection did not require LafB-specific antibodies but was dependent on airway CD4+ T helper 17 cells. Healthy human individuals can elicit LafB-specific immune responses, suggesting its merit as a universal pneumococcal vaccine antigen that remains effective following influenza infection. One-Sentence SummaryDiscovery of a universal pneumococcal vaccine protective during superinfection.

microbiology↗

bioRxiv2022

Competence remodels the pneumococcal cell wall providing resistance to fratricide and surface exposing key virulence factors

Competence development in the human pathogen Streptococcus pneumoniae controls several features such as genetic transformation, biofilm formation and virulence. Competent bacteria produce so called fratricins such as CbpD, that kill non-competent siblings by cleaving peptidoglycan (PGN). CbpD is a choline-binding protein (CBP) that binds to phosphorylcholine residues found on wall- and lipoteichoic acids (WTA and LTA) that together with PGN are major constituents of the pneumococcal cell wall. Competent pneumococci are protected against fratricide by producing the immunity protein ComM. How competence and fratricide contribute to virulence is unknown. Here, using a genome-wide CRISPRi-seq screen, we show that genes involved in teichoic acid biosynthesis are essential during competence. We demonstrate that LytR is the major enzyme mediating the final step in WTA formation, and that, together with ComM, is essential for immunity against CbpD. Importantly, we show that key virulence factors PspA and PspC become more surface-exposed at midcell during competence, in a CbpD-dependent manner. Together, our work supports a model in which activation of competence is crucial for host adherence by increased surface exposure of its various CBPs.

microbiology↗

bioRxiv2021

Amoxicillin-resistant Streptococcus pneumoniae can be resensitized by targeting the mevalonate pathway as identified by sCRilecs-seq

Antibiotic resistance in the important opportunistic human pathogen Streptococcus pneumoniae is on the rise. This is particularly problematic in the case of the {beta}-lactam antibiotic amoxicillin, which is the first-line therapy. It is therefore crucial to uncover targets that would kill or resensitize amoxicillin-resistant pneumococci. To do so, we developed a genome-wide, single-cell based, gene silencing screen using CRISPR interference called sCRilecs-seq (subsets of CRISPR interference libraries extracted by fluorescence activated cell sorting coupled to next generation sequencing). Since amoxicillin affects growth and division, sCRilecs-seq was used to identify targets that are responsible for maintaining proper cell size. Our screen revealed that downregulation of the mevalonate pathway leads to extensive cell elongation. Further investigation into this phenotype indicates that it is caused by insufficient transport of cell wall precursors across the cell membrane due to a limitation in the production of undecaprenyl phosphate (Und-P), the lipid carrier responsible for this process. The data suggest that whereas peptidoglycan synthesis continues even with reduced Und-P levels, cell constriction is specifically halted. We successfully exploited this knowledge to create a combination treatment strategy where the FDA-approved drug clomiphene, an inhibitor of Und-P synthesis, is paired up with amoxicillin. Our results show that clomiphene potentiates the antimicrobial activity of amoxicillin and that combination therapy resensitizes amoxicillin-resistant S. pneumoniae. These findings could provide a starting point to develop a solution for the increasing amount of hard-to-treat amoxicillin-resistant pneumococcal infections.

microbiology↗

bioRxiv2020

Boosting Toll-like receptor 4 signaling enhances the therapeutic outcome of antibiotic therapy in pneumococcal pneumonia

The emergence and spread of antibiotic resistance emphasize the need for alternative treatment strategies against bacterial infections. Boosting the host innate immunity is not only readily deployable in most individuals but can also mobilize many different antibacterial defenses. This study tested the hypothesis whereby stimulation of the innate immune receptor Toll-like receptor 4 (TLR4) can be combined with antibiotics in the treatment of invasive pneumonia. In a mouse model of Streptococcus pneumoniae infection, a single oral administration of low-dose amoxicillin (AMX) or the systemic delivery of monophosphoryl lipid A (MPLA, a clinically-approved TLR4 activator) decreased the bacterial load in lung and spleen, although this was not sufficient for long-term survival. In contrast, a single treatment with a combination of MPLA and AMX induced significant bacterial clearance with little to no regrowth over time, and was associated with longer survival. Upregulation of genes related to granulocyte infiltration in lung tissue and elevation of blood levels of pro-inflammatory cytokines was immediate and transient in MPLA-treated mice; this indicates activation of the innate immune system in a context of infection. Combination treatment was associated with a well-preserved lung tissue architecture and more rapid recovery from inflammation - suggesting that immune activation by MPLA does not exacerbate pneumonia-induced damage. After AMX administration, plasma AMX concentrations rapidly reached the maximum and declined, whereas the downstream effects of MPLA extended beyond AMX elimination; these findings suggested a two-step effect. Our results demonstrated that leveraging host innate immunity increases the efficacy of antibiotic therapy in bacterial pneumonia.

microbiology↗