Rapid Biodistribution of Fluorescent Outer-Membrane Vesicles from the Intestine to Distant Organs via the Blood in Mice.

A cell's ability to secrete extracellular vesicles (EVs) for communication is present in all three domains of life. Notably, Gram-negative bacteria produce a specific type of EVs called outer membrane vesicles (OMVs). We previously observed the presence of OMVs in human blood, which could represent a means of communication from the microbiota to the host. Here, in order to investigate the possible translocation of OMVs from the intestine to other organs, the mouse was used as an animal model after OMVs administration. To achieve this, we first optimized the signal of OMVs containing the fluorescent protein miRFP713 associated with the outer membrane anchoring peptide OmpA by adding biliverdin, a fluorescence cofactor, to the cultures. The miRFP713-expressing OMVs produced in E. coli REL606 strain were then characterized according to their diameter and protein composition. Native- and miRFP713-expressing OMVs were found to produce homogenous populations of vesicles. Finally, in vivo and ex vivo fluorescence imaging was used to monitor the distribution of miRFP713-OMVs in mice in various organs whether by intravenous injection or oral gavage. The relative stability of the fluorescence signals up to 3 days post-injection/gavage paves the way to future studies investigating the OMV-based communication established between the different microbiotas and their host.

Elicitation of potent SARS-CoV-2 neutralizing antibody responses through immunization with a versatile adenovirus-inspired multimerization platform.

Virus-like particles (VLPs) are highly suited platforms for protein-based vaccines. In the present work, we adapted a previously designed non-infectious adenovirus-inspired 60-mer dodecahedric VLP (ADDomer) to display a multimeric array of large antigens through a SpyTag/SpyCatcher system. To validate the platform as a potential COVID-19 vaccine approach, we decorated the newly designed VLP with the glycosylated receptor binding domain (RBD) of SARS-CoV-2. Cryoelectron microscopy structure revealed that up to 60 copies of this antigenic domain could be bound on a single ADDomer particle, with the symmetrical arrangements of a dodecahedron. Mouse immunization with the RBD decorated VLPs already showed a significant specific humoral response following prime vaccination, greatly reinforced by a single boost. Neutralization assays with SARS-CoV-2 spike pseudo-typed virus demonstrated the elicitation of strong neutralization titers, superior to those of COVID-19 convalescent patients. Notably, the presence of pre-existing immunity against the adenoviral-derived particles did not hamper the immune response against the antigen displayed on its surface. This plug and play vaccine platform represents a promising new highly versatile tool to combat emergent pathogens.

A New Plasmacytoid Dendritic Cell-Based Vaccine in Combination with Anti-PD-1 Expands the Tumor-Specific CD8+ T Cells of Lung Cancer Patients.

The purpose of immune checkpoint inhibitor (ICI)-based therapies is to help the patient's immune system to combat tumors by restoring the immune response mediated by CD8+ cytotoxic T cells. Despite impressive clinical responses, most patients do not respond to ICIs. Therapeutic vaccines with autologous professional antigen-presenting cells, including dendritic cells, do not show yet significant clinical benefit. To improve these approaches, we have developed a new therapeutic vaccine based on an allogeneic plasmacytoid dendritic cell line (PDC*line), which efficiently activates the CD8+ T-cell response in the context of melanoma. The goal of the study is to demonstrate the potential of this platform to activate circulating tumor-specific CD8+ T cells in patients with lung cancer, specifically non-small-cell lung cancer (NSCLC). PDC*line cells loaded with peptides derived from tumor antigens are used to stimulate the peripheral blood mononuclear cells of NSCLC patients. Very interestingly, we demonstrate an efficient activation of specific T cells for at least two tumor antigens in 69% of patients irrespective of tumor antigen mRNA overexpression and NSCLC subtype. We also show, for the first time, that the antitumor CD8+ T-cell expansion is considerably improved by clinical-grade anti-PD-1 antibodies. Using PDC*line cells as an antigen presentation platform, we show that circulating antitumor CD8+ T cells from lung cancer patients can be activated, and we demonstrate the synergistic effect of anti-PD-1 on this expansion. These results are encouraging for the development of a PDC*line-based vaccine in NSCLC patients, especially in combination with ICIs.

Anticancer chemotherapy-induced intratumoral recruitment and differentiation of antigen-presenting cells.

The therapeutic efficacy of anthracyclines relies on antitumor immune responses elicited by dying cancer cells. How chemotherapy-induced cell death leads to efficient antigen presentation to T cells, however, remains a conundrum. We found that intratumoral CD11c(+)CD11b(+)Ly6C(hi) cells, which displayed some characteristics of inflammatory dendritic cells and included granulomonocytic precursors, were crucial for anthracycline-induced anticancer immune responses. ATP released by dying cancer cells recruited myeloid cells into tumors and stimulated the local differentiation of CD11c(+)CD11b(+)Ly6C(hi) cells. Such cells efficiently engulfed tumor antigens in situ and presented them to T lymphocytes, thus vaccinating mice, upon adoptive transfer, against a challenge with cancer cells. Manipulations preventing tumor infiltration by CD11c(+)CD11b(+)Ly6C(hi) cells, such as the local overexpression of ectonucleotidases, the blockade of purinergic receptors, or the neutralization of CD11b, abolished the immune system-dependent antitumor activity of anthracyclines. Our results identify a subset of tumor-infiltrating leukocytes as therapy-relevant antigen-presenting cells.

Immune Checkpoint Inhibitor Rechallenge and Resumption: a Systematic Review.

PURPOSE OF THE REVIEW: The reintroduction of immune checkpoint inhibitors (ICIs) after disease progression (rechallenge) or immune-related adverse events (irAEs) recovering (resumption) raises questions in terms of efficacy and safety. RECENT FINDINGS: Here, we reviewed literature data about ICIs rechallenge/resumption in cancer patients along with their clinical characteristics to explore those factors associated with better outcomes. Heterogenous results were pointed out across rechallenge studies with an overall response rate between 0 and 54%, and a progression free survival ranged from 1.5 to 12.9 months and an overall survival between 6.5 and 23.8 months. Better outcomes have been recorded in patients with good ECOG PS, longer duration of initial ICI, discontinuation reason of initial ICI other than progression, and those who received ICI sequence other than the switch between anti-PD1 and anti-PDL1. Studies about ICI resumption highlighted that certain types of irAEs were more likely to relapse at retreatment. These results suggest that ICI rechallenge/resumption can be an interesting strategy for selected patients.

Microbiota-Derived Extracellular Vesicles Detected in Human Blood from Healthy Donors.

The microbiota constitutes an important part of the holobiont in which extracellular vesicles (EVs) are key players in health, especially regarding inter- and intra-kingdom communications. Analysis of EVs from the red blood cell concentrates of healthy donors revealed variable amounts of OmpA and LPS in 12 of the 14 analyzed samples, providing indirect experimental evidence of the presence of microbiota EVs in human circulating blood in the absence of barrier disruption. To investigate the role of these microbiota EVs, we tracked the fusion of fluorescent Escherichia coli EVs with blood mononuclear cells and showed that, in the circulating blood, these EVs interacted almost exclusively with monocytes. This study demonstrates that bacterial EVs constitute critical elements of the host-microbiota cellular communication. The analysis of bacterial EVs should thus be systematically included in any characterization of human EVs.

Photopheresis efficacy in the treatment of rheumatoid arthritis: a pre-clinical proof of concept.

BACKGROUND: Despite major advances in rheumatoid arthritis outcome, not all patients achieve remission, and there is still an unmet need for new therapeutic approaches. This study aimed at evaluating in a pre-clinical murine model the efficacy of extracorporeal photopheresis (ECP) in the treatment of rheumatoid arthritis, and to provide a relevant study model for dissecting ECP mechanism of action in autoimmune diseases. METHODS: DBA/1 mice were immunized by subcutaneous injection of bovine collagen type II, in order to initiate the development of collagen-induced arthritis (CIA). Arthritic mice received 3 ECP treatments every other day, with psoralen + UVA-treated (PUVA) spleen cells obtained from arthritic mice. Arthritis score was measured, and immune cell subsets were monitored. RESULTS: ECP-treated mice recovered from arthritis as evidenced by a decreasing arthritic score over time. Significant decrease in the frequency of Th17 cells in the spleen of treated mice was observed. Interestingly, while PUVA-treated spleen cells from healthy mouse had no effect, PUVA-treated arthritic mouse derived-spleen cells were able to induce control of arthritis development. CONCLUSIONS: Our results demonstrate that ECP can control arthritis in CIA-mice, and clarifies ECP mechanisms of action, showing ECP efficacy and Th17 decrease only when arthritogenic T cells are contained within the treated sample. These data represent a pre-clinical proof of concept supporting the use of ECP in the treatment of RA in Human.

In vitro PUVA treatment triggers calreticulin exposition and HMGB1 release by dying T lymphocytes in GVHD: New insights in extracorporeal photopheresis.

BACKGROUND: Extracorporeal photopheresis (ECP) is an effective therapy for graft vs host disease (GVHD), based on infusion of UVA-irradiated and 8 methoxy-psoralen (PUVA)-treated leukocytes. Reinfusion of these apoptosing cells affects the functionality of pathogenic T cells through poorly understood immunomodulatory mechanisms. Apoptosis is usually a silent, tolerance-associated process, but can also be immunogenic, depending on death-inducers and environmental context. METHODS: To understand ECP mechanisms of action, human alloreactive T cells generated in an in vitro model mimicking GVHD were used, as well as primary cells from GVHD patients. Cells were submitted to PUVA treatment and their phenotype and immunogenicity were analyzed, using cell culture and flow cytometry. RESULTS: In vitro PUVA treatment induced the expression of several damage-associated molecular patterns (DAMPs) by dying T cells (calreticulin, high-mobility group box-1, and to a lesser extent heat shock proteins 70 and 90), especially upon T cell activation, leading to their phagocytosis by macrophages and dendritic cells (DCs). Allogeneic DCs preincubated with PUVA treated T cells induced comparable naive T cell proliferation and polarization as control allogeneic DC. CONCLUSION: Altogether, in our experimental settings, in vitro PUVA-treatment induces a partially immunogenic phenotype allowing phagocytosis of apoptotic cells by macrophages and DC, however not sufficient to induce dendritic cell maturation and T cell activation. These data refine current models of ECP-mediated immune modulation and emphasize the need to further analyze PUVA-treated cell interactions with immune cells.

Harnessing γδ T cells in anticancer immunotherapy.

γδ T lymphocytes are involved in the stress response to injured epithelia and in tissue homeostasis by limiting the dissemination of malignant or infected cells and by regulating the nature of the subsequent adaptive immune response. γδ T cells have potent MHC-unrestricted cytotoxicity, a high potential for cytokine release and broad-spectrum recognition of cancer cells, and as such, are attractive effectors for cancer immunotherapy. Current expectations are going beyond ex vivo manipulation of the Vγ9Vδ2 T subset, and target novel γδ T cell subsets, properties or receptors, to harness these unconventional T lymphocytes against cancer. This Opinion article discusses novel aspects of γδ T cell function during the course of anticancer therapies, as well as new avenues for their clinical implementation.

An ethically guided preclinical device for phenotyping H2 production in laboratory rodents.

BACKGROUND: Dihydrogen (H2) is produced endogenously by the intestinal microbiota through the fermentation of diet carbohydrates. Over the past few years, numerous studies have demonstrated the significant therapeutic potential of H2 in various pathophysiological contexts, making the characterization of its production in laboratory species of major preclinical importance. METHODS: This study proposes an innovative solution to accurately monitor H2 production in free-moving rodents while respecting animal welfare standards. The developed device consisted of a wire rodent cage placed inside an airtight chamber in which the air quality was maintained, and the H2 concentration was continuously analyzed. After the airtightness and efficiency of the systems used to control and maintain air quality in the chamber were checked, tests were carried out on rats and mice with different metabolic phenotypes, over 12 min to 1-h experiments and repeatedly. H2 production rates (HPR) were obtained using an easy calculation algorithm based on a first-order moving average. RESULTS: HPR in hyperphagic Zucker rats was found to be twice as high as in control Wistar rats, respectively, 2.64 and 1.27 nmol.s-1 per animal. In addition, the ingestion of inulin, a dietary fiber, stimulated H2 production in mice. HPRs were 0.46 nmol.s-1 for animals under control diet and 1.99 nmol.s-1 for animals under inulin diet. CONCLUSIONS: The proposed device coupled with our algorithm enables fine analysis of the metabolic phenotype of laboratory rats or mice with regard to their endogenous H2 production.

Leveraging a powerful allogeneic dendritic cell line towards neoantigen-based cancer vaccines.

In recent years, immunotherapy has finally found its place in the anti-cancer therapeutic arsenal, even becoming standard of care as first line treatment for metastatic forms. The clinical benefit provided by checkpoint blockers such as anti-PD-1/PD-L1 in many cancers revolutionized the field. However, too many patients remain refractory to these treatments due to weak baseline anti-cancer immunity. There is therefore a need to boost the frequency and function of patients' cytotoxic CD8+ cellular effectors by targeting immunogenic and tumor-restricted antigens, such as neoantigens using an efficient vaccination platform. Dendritic cells (DC) are the most powerful immune cell subset for triggering cellular immune response. However, autologous DC-based vaccines display several limitations, such as the lack of reproducibility and the limited number of cells that can be manufactured. Here we discuss the advantages of a new therapeutic vaccine based on an allogeneic Plasmacytoid DC cell line, which is easy to produce and represents a powerful platform for priming and expanding anti-neoantigen cytotoxic CD8+ T-cells.

Inulin Prebiotic Protects against Lethal Pseudomonas aeruginosa Acute Infection via γδ T Cell Activation.

Pseudomonas aeruginosa (P. aeruginosa) causes harmful lung infections, especially in immunocompromised patients. The immune system and Interleukin (IL)-17-producing γδ T cells (γδ T) are critical in controlling these infections in mice. The gut microbiota modulates host immunity in both cancer and infection contexts. Nutritional intervention is a powerful means of modulating both microbiota composition and functions, and subsequently the host's immune status. We have recently shown that inulin prebiotic supplementation triggers systemic γδ T activation in a cancer context. We hypothesized that prophylactic supplementation with inulin might protect mice from lethal P. aeruginosa acute lung infection in a γδ T-dependent manner. C57Bl/6 mice were supplemented with inulin for 15 days before the lethal P. aeruginosa lung infection, administered intranasally. We demonstrate that prophylactic inulin supplementation triggers a higher proportion of γδ T in the blood, accompanied by a higher infiltration of IL-17-producing γδ T within the lungs, and protects 33% of infected mice from death. This observation relies on γδ T, as in vivo γδ TcR blocking using a monoclonal antibody completely abrogates inulin-mediated protection. Overall, our data indicate that inulin supplementation triggers systemic γδ T activation, and could help resolve lung P. aeruginosa infections. Moreover, our data suggest that nutritional intervention might be a powerful way to prevent/reduce infection-related mortality, by reinforcing the microbiota-dependent immune system.

Stimulation of the immune system by a tumor antigen-bearing adenovirus-inspired VLP allows control of melanoma growth.

Virus-like particles (VLPs) are versatile protein-based platforms that can be used as a vaccine platform mainly in infectiology. In the present work, we compared a previously designed, non-infectious, adenovirus-inspired 60-mer dodecahedric VLP to display short epitopes or a large tumor model antigen. To validate these two kinds of platforms as a potential immuno-stimulating approach, we evaluated their ability to control melanoma B16-ovalbumin (OVA) growth in mice. A set of adjuvants was screened, showing that polyinosinic-polycytidylic acid (poly(I:C)) was well suited to generate a homogeneous cellular and humoral response against the desired epitopes. In a prophylactic setting, vaccination with the VLP displaying these epitopes resulted in total inhibition of tumor growth 1 month after vaccination. A therapeutic vaccination strategy showed a delay in grafted tumor growth or its total rejection. If the "simple" epitope display on the VLP is sufficient to prevent tumor growth, then an improved engineered platform enabling display of a large antigen is a tool to overcome the barrier of immune allele restriction, broadening the immune response, and paving the way for its potential utilization in humans as an off-the-shelf vaccine.

Inulin prebiotic reinforces host cancer immunosurveillance via ɣδ T cell activation.

The gut microbiota is now recognized as a key parameter affecting the host's anti-cancer immunosurveillance and ability to respond to immunotherapy. Therefore, optimal modulation for preventive and therapeutic purposes is very appealing. Diet is one of the most potent modulators of microbiota, and thus nutritional intervention could be exploited to improve host anti-cancer immunity. Here, we show that an inulin-enriched diet, a prebiotic known to promote immunostimulatory bacteria, triggers an enhanced Th1-polarized CD4+ and CD8+ αß T cell-mediated anti-tumor response and attenuates tumor growth in three preclinical tumor-bearing mouse models. We highlighted that the inulin-mediated anti-tumor effect relies on the activation of both intestinal and tumor-infiltrating ɣδ T cells that are indispensable for αß T cell activation and subsequent tumor growth control, in a microbiota-dependent manner. Overall, our data identified these cells as a critical immune subset, mandatory for inulin-mediated anti-tumor immunity in vivo, further supporting and rationalizing the use of such prebiotic approaches, as well as the development of immunotherapies targeting ɣδ T cells in cancer prevention and immunotherapy.

[Antitumoral immunization during cancer chemotherapy]. / Immunisation par les chimiothérapies anti- cancéreuses: le point en 2012.

Most anticancer agents are thought to act through direct induction of tumoral, stromal and endothelial cell death by apoptosis or necrosis. In a 2008 issue of Bulletin de l'Académie Nationale de Médecine, we described an alternative (or complementary) theory whereby the immune system participates in the antitumoral effects of some chemotherapy or radiotherapy regimens by promoting an immunogenic cell death pathway. In particular, we showed the critical importance of two pre-mortem stressors that determine the immunogenicity of dying tumor cells. The first, an ER stress response culminating in calreticuline exposure at the tumor cell surface, is mandatory for the uptake and efficient phagocytosis of apoptotic bodies by dendritic cells. In the second, autophagy leads to the release of ATP by dying tumor cells, resulting in the recruitment of inflammatory phagocytes and antigen-presenting cells, and also triggering the inflammasome that causes IL-1beta release and CD8+ T cell polarization. The tumor microenvironment changes following chemotherapy, favoring sequential accumulation of a series of innate and cognate effectors that act in a coordinated fashion to promote tumor eradication. These findings will help to identify immune predictors of the response to conventional anticancer treatments and to design innovative combinatorial immunochemotherapy regimens.

Cryopreservation of mononuclear cells before extracorporeal photochemotherapy does not impair their anti-proliferative capabilities.

BACKGROUND AIMS: The clinical benefits of extracorporeal photochemotherapy (ECP) are well recognized, but its clinical use is limited by logistical difficulties, especially because of the need to perform repeated aphereses. The cryopreservation of mononuclear cells could allow maintenance of the ECP schedule while reducing the number of aphereses. The aim of this work was to assess whether previous cryopreservation impairs the immunomodulatory function of ECP-treated peripheral blood mononuclear cells (PBMC). METHODS: Fresh or previously cryopreserved PBMC were exposed to ECP and added on day 0 into a mixed leukocyte reaction. Proliferation of alloreactive lymphocytes was measured by carboxyfluorescein succinimidyl ester (CFSE) dye dilution. Apoptosis was quantified by annexin-7AAD staining. RESULTS: ECP-induced apoptosis was slightly increased in cryopreserved cells but the kinetics of apoptosis were similar to fresh cells. Lymphocytes stimulated in the presence of ECP-treated PBMC displayed a significant decrease in proliferation. The suppression was enforced when ECP-treated cells had been activated previously by allogeneic stimulation. Cryopreservation before ECP exposure did not impact apoptosis triggering or anti-proliferative properties of ECP-treated cells. CONCLUSIONS: Cryopreservation before ECP does not impair the immunomodulatory effects of treated cells. These data warrant investigation of the clinical use of cryopreserved PBMC for ECP.

Evaluation of the human type 3 adenoviral dodecahedron as a vector to target acute myeloid leukemia.

Intensive systemic chemotherapy is the gold standard of acute myeloid leukemia (AML) treatment and is associated with considerable off-target toxicities. Safer and targeted delivery systems are thus urgently needed. In this study, we evaluated a virus-like particle derived from the human type 3 adenovirus, called the adenoviral dodecahedron (Dd) to target AML cells. The vectorization of leukemic cells was proved very effective at nanomolar concentrations in a time- and dose-dependent manner, without vector toxicity. The internalization involved clathrin-mediated energy-dependent endocytosis and strongly correlated with the expression of αVß3 integrin. The treatment of healthy donor peripheral blood mononuclear cells showed a preferential targeting of monocytes compared to lymphocytes and granulocytes. Similarly, monocytes but also AML blasts were the best-vectorized populations in patients while acute lymphoid leukemia blasts were less efficiently targeted. Importantly, AML leukemic stem cells (LSCs) could be addressed. Finally, Dd reached peripheral monocytes and bone marrow hematopoietic stem and progenitor cells following intravenous injection in mice, without excessive spreading in other organs. These findings reveal Dd as a promising myeloid vector especially for therapeutic purposes in AML blasts, LSCs, and progenitor cells.

What We Know So Far about the Metabolite-Mediated Microbiota-Intestinal Immunity Dialogue and How to Hear the Sound of This Crosstalk.

Trillions of microorganisms, termed the "microbiota", reside in the mammalian gastrointestinal tract, and collectively participate in regulating the host phenotype. It is now clear that the gut microbiota, metabolites, and intestinal immune function are correlated, and that alterations of the complex and dynamic host-microbiota interactions can have deep consequences for host health. However, the mechanisms by which the immune system regulates the microbiota and by which the microbiota shapes host immunity are still not fully understood. This article discusses the contribution of metabolites in the crosstalk between gut microbiota and immune cells. The identification of key metabolites having a causal effect on immune responses and of the mechanisms involved can contribute to a deeper insight into host-microorganism relationships. This will allow a better understanding of the correlation between dysbiosis, microbial-based dysmetabolism, and pathogenesis, thus creating opportunities to develop microbiota-based therapeutics to improve human health. In particular, we systematically review the role of soluble and membrane-bound microbial metabolites in modulating host immunity in the gut, and of immune cells-derived metabolites affecting the microbiota, while discussing evidence of the bidirectional impact of this crosstalk. Furthermore, we discuss the potential strategies to hear the sound of such metabolite-mediated crosstalk.

Identification of a predictive metabolic signature of response to immune checkpoint inhibitors in non-small cell lung cancer: METABO-ICI clinical study protocol.

BACKGROUND: Immune checkpoints inhibitors (ICI) are becoming new standards of care for the treatment of non-small cell lung cancer (NSCLC), both as first (alone or in association with chemotherapy) and second line. However, no powerful predictive biomarker of therapeutic response to ICI has been found to date. It has been recently shown that microbiota composition could influence the ability of patients to respond to ICI. Indeed, the microbiota produces circulating metabolites that will subsequently act on immune system, the investigators hypothesized that plasma metabolic signature, reflecting a global microbiota function, could represent a predictive biomarker of response to ICI. METHODS: Monocentric prospective study. Primary objective is to identify baseline metabolic signature (metabolomics analysis by mass spectrometry) associated to ICI response. Secondary objectives are to link metabolic signature with microbiota composition (metagenomics analysis RNA 16S) and immune profile, and altogether with clinic response to ICI. The study will include 60 NSCLC patients treated by ICI in 1st, 2nd or 3rd line of treatment at the Grenoble Alpes University hospital (CHUGA) in 18 months. Patients that have received antibiotic or steroid treatment, 2 or 4 weeks before ICI initiation, respectively, will be excluded. Blood and feces will be collected prior to, at 2 months after ICI treatment initiation, and at 6 months or at progression. EXPECTED RESULTS: We expect to highlight a metabolic profile predictive of response to ICI. By identifying factors associated with early progression, we could avoid to treat potential non-responding patients. Moreover, by restoring a favorable microbiota, patients' ability to respond to these treatments might be restored.

Metabotypes of Pseudomonas aeruginosa Correlate with Antibiotic Resistance, Virulence and Clinical Outcome in Cystic Fibrosis Chronic Infections.

Pseudomonas aeruginosa (P.a) is one of the most critical antibiotic resistant bacteria in the world and is the most prevalent pathogen in cystic fibrosis (CF), causing chronic lung infections that are considered one of the major causes of mortality in CF patients. Although several studies have contributed to understanding P.a within-host adaptive evolution at a genomic level, it is still difficult to establish direct relationships between the observed mutations, expression of clinically relevant phenotypes, and clinical outcomes. Here, we performed a comparative untargeted LC/HRMS-based metabolomics analysis of sequential isolates from chronically infected CF patients to obtain a functional view of P.a adaptation. Metabolic profiles were integrated with expression of bacterial phenotypes and clinical measurements following multiscale analysis methods. Our results highlighted significant associations between P.a "metabotypes", expression of antibiotic resistance and virulence phenotypes, and frequency of clinical exacerbations, thus identifying promising biomarkers and therapeutic targets for difficult-to-treat P.a infections.