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Respiratory mucosal surfaces are continuously exposed to not only innocuous non-self antigens but also pathogen-associated molecular patterns (PAMPs) originating from environmental or symbiotic microbes. According to either “self/non-self” or “danger” models, this should systematically result in homeostasis breakdown and the development of immune responses directed to inhaled harmless antigens, such as T helper type (Th)2-mediated asthmatic reactions, which is fortunately not the case in most people. This discrepancy implies the existence, in the lung, of regulatory mechanisms that tightly control immune homeostasis. Although such mechanisms have been poorly investigated in comparison to the ones that trigger immune responses, a better understanding of them could be useful in the development of new therapeutic strategies against lung diseases (e.g., asthma). Here, we review current knowledge on innate immune cells that prevent the development of aberrant immune responses in the lung, thereby contributing to mucosal homeostasis.

BackgroundUnderstanding and measuring the individual level of immune protection and its persistence at both humoral and cellular levels after SARS-CoV-2 vaccination is mandatory for the management of the vaccination booster campaign. Our prospective study was designed to assess the immunogenicity of the BNT162b2 mRNA vaccine in triggering the cellular and humoral immune response in healthcare workers up to 12 months after the initial vaccination, with one additional boosting dose between 6 and 12 months.MethodsThis prospective study enrolled 208 healthcare workers (HCWs) from the Liège University Hospital (CHU) of Liège in Belgium. Participants received two doses of BioNTech/Pfizer COVID-19 vaccine (BNT162b2) and a booster dose 6-12 months later. Fifty participants were SARS-CoV-2 experienced and 158 were naïve before the vaccination. Blood sampling was performed at the day of the first (T0) and second (T1) vaccine doses administration, then at 2 weeks (T2), 4 weeks (T3), 6 months (T4) and 12 months (T5) after the second dose. Between T4 and T5, participants also got the third boosting vaccine dose. A total of 1145 blood samples were collected. All samples were tested for the presence of anti-Spike antibodies, using the DiaSorin LIAISON SARS-CoV-2 Trimeric S IgG assay, and for anti-Nucleocapsid antibodies, using Elecsys anti-SARS-CoV-2 assay​​. Neutralizing antibodies against the SARS-CoV-2 Wuhan-like variant strain were quantified in all samples using a Vero E6 cell-based neutralization assay. Cell-mediated immune response was evaluated at T4 and T5 on 80 and 55 participants, respectively, by measuring the secretion of IFN-γ on peripheral blood lymphocytes using the QuantiFERON Human IFN-γ SARS-CoV-2, from Qiagen. We analyzed separately the naïve and experienced participants.FindingsWe found that anti-spike antibodies and neutralization capacity levels were significantly higher in SARS-CoV-2 experienced HCWs compared to naïve HCWs at all time points analyzed except the one after boosting dose. Cellular immune response was also higher in experienced HCWs six months following vaccination. Besides the impact of SARS-CoV-2 infection history on immune response to BNT162b2 mRNA vaccine, we observed a significant negative association between age and persistence of humoral response. The booster dose induced an increase in humoral and cellular immune responses, particularly in naive individuals. Breakthrough infections resulted in higher cellular and humoral responses after the booster dose.ConclusionsOur data strengthen previous findings demonstrating that immunization through vaccination combined with natural infection is better than 2 vaccine doses immunization or natural infection alone. The benefit of the booster dose was greater in naive individuals. It may have implications for personalizing mRNA vaccination regimens used to prevent severe COVID-19 and reduce the impact of the pandemic on the healthcare system. More specifically, it may help prioritizing vaccination, including for the deployment of booster doses.

Plasmids carrying metal resistance genes (MRGs) have been suggested to be key ecological players in the adaptation of metal-impacted microbial communities, making them promising drivers of bio-remediation processes. However, the impact of metals on plasmid-mediated spread of MRGs through selection, plasmid loss, and transfer is far from being fully understood. In the present study, we used two-member bacterial communities to test the impact of lead on the dispersal of the IncP plasmid pKJK5 from a Pseudomonas putida KT2440 plasmid donor and two distinct recipients, Variovorax paradoxus B4 or Delftia acidovorans SPH-1 after 4 and 10 days of mating. Two versions of the plasmid were used, carrying or not carrying the lead resistance pbrTRABCD operon, to assess the importance of fitness benefit and conjugative potential for the dispersal of the plasmid. The spread dynamics of metal resistance conveyed by the conjugative plasmid were dependent on the recipient and the lead concentration: For V. paradoxus, the pbr operon did not facilitate neither lead resistance nor variation in plasmid spread. The growth gain brought by the pbr operon to D. acidovorans SPH-1 and P. putida KT2440 at 1 mM Pb enhanced the spread of the plasmid. At 1.5 mM Pb after 4 days, the proteomics results revealed an oxidative stress response and an increased abundance of pKJK5-encoded conjugation and partitioning proteins, which most likely increased the transfer of the control plasmid to D. acidovorans SPH-1 and ensured plasmid maintenance. As a consequence, we observed an increased spread of pKJK5-gfp. Conversely, the pbr operon reduced the oxidative stress response and impeded the rise of conjugation- and partitioning-associated proteins, which slowed down the spread of the pbr carrying plasmid. Ultimately, when a fitness gain was recorded in the recipient strain, the spread of MRG-carrying plasmids was facilitated through positive selection at an intermediate metal concentration, while a high lead concentration induced oxidative stress with positive impacts on proteins encoding plasmid conjugation and partitioning.

Canine idiopathic pulmonary fibrosis (CIPF) affects old dogs from the West Highland white terrier (WHWT) breed and mimics idiopathic pulmonary fibrosis (IPF) in human. The disease results from deposition of fibrotic tissue in the lung parenchyma causing respiratory failure. Recent studies in IPF using single-cell RNA sequencing (scRNA-seq) revealed the presence of profibrotic macrophage populations in the lung, which could be targeted for therapeutic purpose. In dogs, scRNA-seq was recently validated for the detection of cell populations in bronchoalveolar lavage fluid (BALF) from healthy dogs. Here we used the scRNA-seq to characterize disease-related heterogeneity within cell populations of macrophages/monocytes (Ma/Mo) in the BALF from five WHWTs affected with CIPF in comparison with three healthy WHWTs. Gene set enrichment analysis was also used to assess pro-fibrotic capacities of Ma/Mo populations. Five clusters of Ma/Mo were identified. Gene set enrichment analyses revealed the presence of pro-fibrotic monocytes in higher proportion in CIPF WHWTs than in healthy WHWTs. In addition, monocyte-derived macrophages enriched in pro-fibrotic genes in CIPF compared with healthy WHWTs were also identified. These results suggest the implication of Ma/Mo clusters in CIPF processes, although, further research is needed to understand their role in disease pathogenesis. Overexpressed molecules associated with pulmonary fibrosis processes were also identified that could be used as biomarkers and/or therapeutic targets in the future.

Single-cell mRNA-sequencing (scRNA-seq) is a technique which enables unbiased, high throughput and high-resolution transcriptomic analysis of the heterogeneity of cells within a population. This recent technique has been described in humans, mice and other species in various conditions to cluster cells in populations and identify new subpopulations, as well as to study the gene expression of cells in various tissues, conditions and origins. In dogs, a species for which markers of cell populations are often limiting, scRNA-seq presents with elevated yet untested potential for the study of tissue composition. As a proof of principle, we used scRNA-seq to identify cellular populations of the bronchoalveolar lavage fluid (BALF) in healthy dogs (n = 4). A total of 5,710 cells were obtained and analyzed by scRNA-seq. Fourteen distinct clusters of cells were identified, further identified as macrophages/monocytes (4 clusters), T cells (2 clusters) and B cells (1 cluster), neutrophils (1 cluster), mast cells (1 cluster), mature or immature dendritic cells (1 cluster each), ciliated or non-ciliated epithelial cells (1 cluster each) and cycling cells (1 cluster). We used for the first time in dogs the scRNA-seq to investigate cellular subpopulations of the BALF of dog. This study hence expands our knowledge on dog lung immune cell populations, paves the way for the investigation at single-cell level of lower respiratory diseases in dogs, and establishes that scRNA-seq is a powerful tool for the study of dog tissue composition.

Allergic asthma is a chronic Th2 inflammatory disease of the lower airways affecting a growing number of people worldwide. The impact of infections and microbiota composition on allergic asthma has been investigated frequently. Until now, however, there have been few attempts to investigate the impact of asthma on the control of infectious microorganisms and the underlying mechanisms. In this work, we characterize the consequences of allergic asthma on intranasal (i.n.) infection by Brucella bacteria in mice. We observed that i.n. sensitization with extracts of the house dust mite Dermatophagoides farinae or the mold Alternaria alternata (Alt) significantly increased the number of Brucella melitensis, Brucella suis, and Brucella abortus in the lungs of infected mice. Microscopic analysis showed dense aggregates of infected cells composed mainly of alveolar macrophages (CD11c+ F4/80+ MHCII+) surrounded by neutrophils (Ly-6G+). Asthma-induced Brucella susceptibility appears to be dependent on CD4+ T cells, the IL-4/STAT6 signaling pathway and IL-10, and is maintained in IL-12- and IFN-γR-deficient mice. The effects of the Alt sensitization protocol were also tested on Streptococcus pneumoniae and Mycobacterium tuberculosis pulmonary infections. Surprisingly, we observed that Alt sensitization strongly increases the survival of S. pneumoniae infected mice by a T cell and STAT6 independent signaling pathway. In contrast, the course of M. tuberculosis infection is not affected in the lungs of sensitized mice. Our work demonstrates that the impact of the same allergic sensitization protocol can be neutral, negative, or positive with regard to the resistance of mice to bacterial infection, depending on the bacterial species.

Eosinophils are typically considered to be specialized effector cells that are recruited to the tissues as a result of T helper type 2 (Th2) cell responses associated with helminth infections or allergic diseases such as asthma. Once at the site of injury, eosinophils release their cytotoxic granule proteins as well as preformed cytokines and lipid mediators, contributing to parasite destruction but also to exacerbation of inflammation and tissue damage. Accumulating evidence indicates that, besides their roles in Th2 responses, eosinophils also regulate homeostatic processes at steady state, thereby challenging the exclusive paradigm of the eosinophil as a destructive and inflammatory cell. Indeed, under baseline conditions, eosinophils rapidly leave the bloodstream to enter tissues, mainly the gastrointestinal tract, lungs, adipose tissue, thymus, uterus, and mammary glands, where they regulate a variety of important biological functions, such as immunoregulation, control of glucose homeostasis, protection against obesity, regulation of mammary gland development, and preparation of the uterus for pregnancy. This article provides an overview of the characteristics and functions of these homeostatic eosinophils.