Lung Interstitial Macrophages Can Present Soluble Antigens and Induce Foxp3+ Regulatory T Cells.

Lung macrophages constitute a sophisticated surveillance and defense system that contributes to tissue homeostasis, host defense, and allows the host to cope with the myriad of insults and antigens to which the lung mucosa is exposed. As opposed to alveolar macrophages, lung interstitial macrophages express high levels of type 2 major histocompatibility complex (MHC-II), a hallmark of antigen-presenting cells. Here, we showed that lung IMs, like dendritic cells (DCs), possess the machinery to present soluble antigens in an MHC-II-restricted way. Using ex vivo ovalbumin (OVA)-specific T cell proliferation assays, we found that OVA-pulsed IMs could trigger OVA-specific CD4+ T cell proliferation and Foxp3 expression via MHC-II-, IL-10 and Tgfß-dependent mechanisms. Moreover, we showed that IMs efficiently captured locally instilled antigens in vivo, did not migrate to the draining lymph nodes and enhanced local interactions with CD4+ T cells in a model of OVA-induced allergic asthma. These results support that IMs can present antigens to CD4+ T cells and trigger regulatory T cells, which might attenuate lung immune responses and have functional consequences for lung immunity and T-cell-mediated disorders.

Tirap controls Mycobacterium tuberculosis phagosomal acidification.

Progression of tuberculosis is tightly linked to a disordered immune balance, resulting in inability of the host to restrict intracellular bacterial replication and its subsequent dissemination. The immune response is mainly characterized by an orchestrated recruitment of inflammatory cells secreting cytokines. This response results from the activation of innate immunity receptors that trigger downstream intracellular signaling pathways involving adaptor proteins such as the TIR-containing adaptor protein (Tirap). In humans, resistance to tuberculosis is associated with a loss-of-function in Tirap. Here, we explore how genetic deficiency in Tirap impacts resistance to Mycobacterium tuberculosis (Mtb) infection in a mouse model and ex vivo. Interestingly, compared to wild type littermates, Tirap heterozygous mice were more resistant to Mtb infection. Upon investigation at the cellular level, we observed that mycobacteria were not able to replicate in Tirap-deficient macrophages compared to wild type counterparts. We next showed that Mtb infection induced Tirap expression which prevented phagosomal acidification and rupture. We further demonstrate that the Tirap-mediated anti-tuberculosis effect occurs through a Cish-dependent signaling pathway. Our findings provide new molecular evidence about how Mtb manipulates innate immune signaling to enable intracellular replication and survival of the pathogen, thus paving the way for host-directed approaches to treat tuberculosis.

MafB-restricted local monocyte proliferation precedes lung interstitial macrophage differentiation.

Resident tissue macrophages (RTMs) are differentiated immune cells that populate distinct niches and exert important tissue-supportive functions. RTM maintenance is thought to rely either on differentiation from monocytes or on RTM self-renewal. Here, we used a mouse model of inducible lung interstitial macrophage (IM) niche depletion and refilling to investigate the development of IMs in vivo. Using time-course single-cell RNA-sequencing analyses, bone marrow chimeras and gene targeting, we found that engrafted Ly6C+ classical monocytes proliferated locally in a Csf1 receptor-dependent manner before differentiating into IMs. The transition from monocyte proliferation toward IM subset specification was controlled by the transcription factor MafB, while c-Maf specifically regulated the identity of the CD206+ IM subset. Our data provide evidence that, in the mononuclear phagocyte system, the ability to proliferate is not merely restricted to myeloid progenitor cells and mature RTMs but is also a tightly regulated capability of monocytes developing into RTMs in vivo.

Airway Macrophages Encompass Transcriptionally and Functionally Distinct Subsets Altered by Smoking.

Alveolar macrophages (AMs) are functionally important innate cells involved in lung homeostasis and immunity and whose diversity in health and disease is a subject of intense investigations. Yet, it remains unclear to what extent conditions like smoking or chronic obstructive pulmonary disease (COPD) trigger changes in the AM compartment. Here, we aimed to explore heterogeneity of human AMs isolated from healthy nonsmokers, smokers without COPD, and smokers with COPD by analyzing BAL fluid cells by flow cytometry and bulk and single-cell RNA sequencing. We found that subpopulations of BAL fluid CD206+ macrophages could be distinguished based on their degree of autofluorescence in each subject analyzed. CD206+ autofluorescenthigh AMs were identified as classical, self-proliferative AM, whereas autofluorescentlow AMs were expressing both monocyte and classical AM-related genes, supportive of a monocytic origin. Of note, monocyte-derived autofluorescentlow AMs exhibited a functionally distinct immunoregulatory profile, including the ability to secrete the immunosuppressive cytokine IL-10. Interestingly, single-cell RNA-sequencing analyses showed that transcriptionally distinct clusters of classical and monocyte-derived AM were uniquely enriched in smokers with and without COPD as compared with healthy nonsmokers. Of note, such smoking-associated clusters exhibited gene signatures enriched in detoxification, oxidative stress, and proinflammatory responses. Our study independently confirms previous reports supporting that monocyte-derived macrophages coexist with classical AM in the airways of healthy subjects and patients with COPD and identifies smoking-associated changes in the AM compartment that may favor COPD initiation or progression.

University population-based prospective cohort study of SARS-CoV-2 infection and immunity (SARSSURV-ULiège): a study protocol.

INTRODUCTION: For a safe and sustainable return to normal functioning of academic activities in higher education, objective-driven testing strategies that are flexible and rapidly adaptable are essential to effectively monitor and respond to new developments of the COVID-19 pandemic. To date, prospective longitudinal research on SARS-CoV-2 antibody testing in saliva and seroprevalence in higher education contexts is substantially lacking, limiting our understanding of COVID-19 prevalence, incidence and nature of the immune response to SARS-CoV-2 at various stages of the infection and vaccination. To address this lack of evidence, a prospective population-based cohort study (SARSSURV-ULiège) has recently been started. METHODS AND ANALYSIS: Students (n=1396) and staff members (n=1143) of the University of Liège are followed up over more than 1 year. All participants are required to complete anamnestic, clinical and vaccine hesitancy questionnaires for medical histories and undertaken treatments. Previous proven or suspected SARS-CoV-2 infection is also registered. In phase 1, weekly saliva samples to perform RT-qPCR to detect SARS-CoV-2 and monthly COVID-19 serological rapid test results are collected. Once being positive to either saliva RT-qPCR assay for SARS-CoV-2 presence or to serological test, the participant is invited to enter phase 2. If participants get vaccinated during the study period, they are invited to phase 2. In this second phase, besides weekly saliva self-test, depending on the participants' profiles, both gargle and blood samples are collected to obtain various biological data to measure the presence of neutralising antibodies against SARS-CoV-2, determine the magnitude and the duration of antibody responses over time. ETHICS AND DISSEMINATION: The study has received the approval from the University Hospital of Liège Ethics Committee (reference number 2021/96, dated 26 March 2021). Potential protocol amendments will be presented to the Research Ethics Committee. The findings of the present study will be presented at scientific conferences and the results published in peer-review publications. Weekly reports will be submitted to the risk assessment group and the risk management group against COVID-19 of the university to enable a timely public health action if necessary.

Lead Drives Complex Dynamics of a Conjugative Plasmid in a Bacterial Community.

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.

Characterization of the Bronchoalveolar Lavage Fluid by Single Cell Gene Expression Analysis in Healthy Dogs: A Promising Technique.

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.

Identification of Pro-Fibrotic Macrophage Populations by Single-Cell Transcriptomic Analysis in West Highland White Terriers Affected With Canine Idiopathic Pulmonary Fibrosis.

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.

The Roles of Spinochromes in Four Shallow Water Tropical Sea Urchins and Their Potential as Bioactive Pharmacological Agents.

Spinochromes are principally known to be involved in sea urchin pigmentation as well as for their potentially interesting pharmacological properties. To assess their biological role in sea urchin physiology, experiments are undertaken on crude extracts from four species and on four isolated spinochromes in order to test their antibacterial, antioxidant, inflammatory and cytotoxic activities. First, the antibacterial assays show that the use of crude extracts as representatives of antibacterial effects of spinochromes are inaccurate. The assays on purified spinochromes showed a decrease in the growth of four strains with an intensity depending on the spinochromes/bacteria system, revealing the participation of spinochromes in the defense system against microorganisms. Secondly, in the 2,2-diphenyl-1-picrylhydrazyl antioxidant assays, spinochromes show an enhanced activity compared to the positive control. This latter observation suggests their involvement in ultraviolet radiation protection. Third, spinochromes present a pro-inflammatory effect on lipopolysaccharide-stimulated macrophages, highlighting their possible implication in the sea urchin immune system. Finally, cytotoxicity assays based on Trypan blue exclusion, performed in view of their possible future applications as drugs, show a weak cytotoxicity of these compounds against human cells. In conclusion, all results confirm the implication of spinochromes in sea urchin defense mechanisms against their external environment and reveal their potential for pharmacological and agronomical industries.

Exposure to Bacterial CpG DNA Protects from Airway Allergic Inflammation by Expanding Regulatory Lung Interstitial Macrophages.

Living in a microbe-rich environment reduces the risk of developing asthma. Exposure of humans or mice to unmethylated CpG DNA (CpG) from bacteria reproduces these protective effects, suggesting a major contribution of CpG to microbe-induced asthma resistance. However, how CpG confers protection remains elusive. We found that exposure to CpG expanded regulatory lung interstitial macrophages (IMs) from monocytes infiltrating the lung or mobilized from the spleen. Trafficking of IM precursors to the lung was independent of CCR2, a chemokine receptor required for monocyte mobilization from the bone marrow. Using a mouse model of allergic airway inflammation, we found that adoptive transfer of IMs isolated from CpG-treated mice recapitulated the protective effects of CpG when administered before allergen sensitization or challenge. IM-mediated protection was dependent on IL-10, given that Il10-/- CpG-induced IMs lacked regulatory effects. Thus, the expansion of regulatory lung IMs upon exposure to CpG might underlie the reduced risk of asthma development associated with a microbe-rich environment.

Are Th17 Cells Playing a Role in Immunity to Dermatophytosis?

Despite their superficial localization in the skin, pathogenic dermatophytes can induce a complex but still misunderstood immune response in their hosts. The cell-mediated immunity (CMI) is correlated with both clinical recovery and protection against reinfection, and CD4+ T lymphocytes have been recognized as a crucial component of the immune defense against dermatophytes. Before the discovery of the Th17 pathway, CMI was considered to be only dependent of Th1 cells, and thus most studies on the immunology of dermatophytosis have focused on the Th1 pathway. Nevertheless, the fine comparative analysis of available scientific data on immunology of dermatophytosis in one hand and on the Th17 pathway mechanisms involved in opportunistic mucosal fungal infections in the other hand reveals that some key elements of the Th17 pathway can be activated by dermatophytes. Stimulation of the Th17 pathway could occur through the activation of some C-type lectin-like receptors and inflammasome in antigen-presenting cells. The Th17 cells could go back to the affected skin and by the production of signature cytokines could induce the effector mechanisms like the recruitment of polymorphonuclear neutrophils and the synthesis of antimicrobial peptides. In conclusion, besides the Th1 pathway, which is important to the immune response against dermatophytes, there are also growing evidences for the involvement of the Th17 pathway.

Generation of a soluble recombinant trimeric form of bovine CD40L and its potential use as a vaccine adjuvant in cows.

Vaccination is the most cost-effective way to control infectious diseases in cattle. However, many infectious diseases leading to severe economical losses worldwide still remain for which a really effective and safe vaccine is not available. These diseases are most often due to intracellular pathogens such as bacteria or viruses, which are, by their localization, protected from antibiotics and/or CD4(+) T cell-dependent humoral responses. We therefore postulated that strategies leading to induction of not only CD4(+) T cell responses but also CD8(+) cytotoxic T lymphocyte (CTL) responses against infected cells should be privileged in the development of new vaccines against problematic intracellular pathogens in bovines. CD40 signaling in antigen-presenting cells may lead to the induction of robust CD4-independent CTL responses and several studies, especially in mice, have used CD40 stimulation to promote CD8(+) T cell-mediated immunity. For example, we have recently shown that immunization of mice with heat-killed Staphylococcus aureus (HKSA) and agonistic anti-CD40 monoclonal antibodies elicits strong CTL responses capable of protecting mice from subsequent staphylococcal mastitis. Unfortunately, there is at present no tool available to efficiently stimulate CD40 in cattle. In this study, we therefore first produced a soluble recombinant trimeric form of the natural bovine CD40 ligand (sboCD40LT). We then observed that sboCD40LT was able to potently stimulate bovine cells in vitro. Finally, we provide evidence that immunization of cows with sboCD40LT combined with HKSA was able to significantly increase the number of both HKSA-specific CD4(+) and CD8(+) T cells in the draining lymph nodes. In conclusion, we suggest that this new molecular tool could help in the development of vaccine strategies against bovine diseases caused by intracellular pathogens.

Antigen presenting cell-derived IL-6 restricts Th2-cell differentiation.

The identification of DC-derived signals orchestrating activation of Th1 and Th17 immune responses has advanced our understanding on how these inflammatory responses develop. However, whether specific signals delivered by DCs also participate in the regulation of Th2 immune responses remains largely unknown. In this study, we show that administration of antigen-loaded, IL-6-deficient DCs to naïve mice induced an exacerbated Th2 response, characterized by the differentiation of GATA-3-expressing T lymphocytes secreting high levels of IL-4, IL-5, and IL-13. Coinjection of wild type and IL-6-deficient bone marrow-derived dendritic cells (BMDCs) confirmed that IL-6 exerted a dominant, negative influence on Th2-cell development. This finding was confirmed in vitro, where exogenously added IL-6 was found to limit IL-4-induced Th2-cell differentiation. iNKT cells were required for optimal Th2-cell differentiation in vivo although their activation occurred independently of IL-6 secretion by the BMDCs. Collectively, these observations identify IL-6 secretion as a major, unsuspected, mechanism whereby DCs control the magnitude of Th2 immunity.

Increased hypoxia-inducible factor 1α expression in lung cells of horses with recurrent airway obstruction.

BACKGROUND: Recurrent airway obstruction (RAO, also known as equine heaves) is an inflammatory condition caused by exposure of susceptible horses to organic dusts in hay. The immunological processes responsible for the development and the persistence of airway inflammation are still largely unknown. Hypoxia-inducible factor (Hif) is mainly known as a major regulator of energy homeostasis and cellular adaptation to hypoxia. More recently however, Hif also emerged as an essential regulator of innate immune responses. Here, we aimed at investigating the potential involvement of Hif1-α in myeloid cells in horse with recurrent airway obstruction. RESULTS: In vitro, we observed that Hif is expressed in equine myeloid cells after hay dust stimulation and regulates genes such as tumor necrosis factor alpha (TNF-α), interleukin-8 (IL-8) and vascular endothelial growth factor A (VEGF-A). We further showed in vivo that airway challenge with hay dust upregulated Hif1-α mRNA expression in myeloid cells from the bronchoalveolar lavage fluid (BALF) of healthy and RAO-affected horses, with a more pronounced effect in cells from RAO-affected horses. Finally, Hif1-α mRNA expression in BALF cells from challenged horses correlated positively with lung dysfunction. CONCLUSION: Taken together, our results suggest an important role for Hif1-α in myeloid cells during hay dust-induced inflammation in horses with RAO. We therefore propose that future research aiming at functional inactivation of Hif1 in lung myeloid cells could open new therapeutic perspectives for RAO.

Sirtuin inhibition attenuates the production of inflammatory cytokines in lipopolysaccharide-stimulated macrophages.

In several inflammatory conditions such as rheumatoid arthritis or sepsis, the regulatory mechanisms of inflammation are inefficient and the excessive inflammatory response leads to damage to the host. Sirtuins are class III histone deacetylases that modulate the activity of several transcription factors that are implicated in immune responses. In this study, we evaluated the impact of sirtuin inhibition on the activation of lipopolysaccharide (LPS)-stimulated J774 macrophages by assessing the production of inflammatory cytokines. The pharmacologic inhibition of sirtuins decreased the production of tumour necrosis factor-alpha (TNF-α) interleukin 6 (IL-6) and Rantes. The reduction of cytokine production was associated with decreased nuclear factor kappa B (NF-κB) activity and inhibitor kappa B alpha (IκBα) phosphorylation while no impact was observed on the phosphorylation status of p38 mitogen-activated kinase (p38 MAPK). This work shows that sirtuin pharmacologic inhibitors are a promising tool for the treatment of inflammatory conditions.

CD40 triggering induces strong cytotoxic T lymphocyte responses to heat-killed Staphylococcus aureus immunization in mice: a new vaccine strategy for staphylococcal mastitis.

Staphylococcus (S.) aureus is a major pathogen involved in chronic bovine mastitis. Staphylococcal mastitis is difficult to control due to the ability of S. aureus to invade and survive within host cells. We therefore postulated that induction of CD8(+) cytotoxic T lymphocyte (CTL) responses leading to destruction of infected cells could help in the control of S. aureus mastitis. We demonstrate that immunization of mice with heat-killed S. aureus together with agonistic anti-CD40 monoclonal antibodies elicits strong CTL responses capable of reducing the severity of subsequent staphylococcal mastitis. Our study shows promise for CTL-dependent vaccination against S. aureus mastitis.

Sirtuin 1 promotes Th2 responses and airway allergy by repressing peroxisome proliferator-activated receptor-γ activity in dendritic cells.

Sirtuins are a unique class of NAD(+)-dependent deacetylases that regulate diverse biological functions such as aging, metabolism, and stress resistance. Recently, it has been shown that sirtuins may have anti-inflammatory activities by inhibiting proinflammatory transcription factors such as NF-κB. In contrast, we report in this study that pharmacological inhibition of sirtuins dampens adaptive Th2 responses and subsequent allergic inflammation by interfering with lung dendritic cell (DC) function in a mouse model of airway allergy. Using genetic engineering, we demonstrate that sirtuin 1 represses the activity of the nuclear receptor peroxisome proliferator-activated receptor-γ in DCs, thereby favoring their maturation toward a pro-Th2 phenotype. This study reveals a previously unappreciated function of sirtuin 1 in the regulation of DC function and Th2 responses, thus shedding new light on our current knowledge on the regulation of inflammatory processes by sirtuins.

Nicotinamide enhances apoptosis of G(M)-CSF-treated neutrophils and attenuates endotoxin-induced airway inflammation in mice.

Neutrophils constitute the first line of host defense against invading microorganisms. Yet their removal from the inflammatory environment is fundamental for injury restraint and resolution of inflammation. Nicotinamide, a component of vitamin B(3), is known to modulate cell survival. In this study, we assessed the influence of nicotinamide on neutrophil apoptosis, both in vitro and in vivo in a mouse model of endotoxin-induced lung inflammation. In vitro, nicotinamide promoted apoptosis of human blood neutrophils in a dose-dependent manner in the presence of the apoptosis inhibitors granulocyte colony-stimulating factor and granulocyte/macrophage colony-stimulating factor. The highest concentration of nicotinamide completely neutralized the pro-survival effect of granulocyte (macrophage) colony-stimulating factor. Nicotinamide proapoptotic effect was associated with enhanced caspase-3 activity. In addition, nicotinamide slightly reduced neutrophil chemotaxis in vitro. In vivo, pulmonary nicotinamide delivery decreased the levels of cellular and biochemical inflammation markers and increased the percentage of apoptotic neutrophils in bronchoalveolar lavages. Our findings suggest that nicotinamide is an apoptotic stimulus for neutrophils, thereby contributing to the resolution of neutrophilic inflammation in the lungs.

Effects of formoterol and ipratropium bromide on repeated cadmium inhalation-induced pulmonary inflammation and emphysema in rats.

The anti-inflammatory properties of inhaled formoterol and ipratropium bromide, alone or in combination, were investigated in a rat model of chronic pulmonary inflammation with airspace enlargement induced by cadmium inhalation. At the end of the protocol, cadmium-induced increase of airway resistance was prevented by formoterol (4 mg/30 ml) or ipratropium (0.20 mg/20 ml). Formoterol elicited a significant decrease in total cell and neutrophil counts in bronchoalveolar lavage fluid as well as on the activity of gelatinase B (MMP-9), an enzyme strongly expressed in alveolar macrophages and epithelial cells. Additionally, a significant attenuation of the lung lesions characterized by inflammatory cell infiltration within the alveoli and the interstitium and a decrease in mean linear intercept were observed. Although ipratropium alone had no effects on the cadmium-induced pulmonary inflammation and emphysema, its combination with an inefficient concentration of formoterol (1 mg/30 ml) showed a synergistic inhibitory effect on neutrophil and total cell counts as well as on the mean linear intercept associated with a synergistic inhibition on the MMP-9 activity. Gelatinase A (MMP-2) activity was not influenced by drug pretreatments. Neither macrophage metalloelastase (MMP-12) activity nor levels of cytokines IL-1ß, TNF-α and GM-CSF in bronchoalveolar lavage fluid were modified in rats chronically exposed to cadmium. No desensitization of ß(2)-adrenoceptors or cholinergic receptors on airway smooth muscles and inflammatory cells during the protocol was observed. In conclusion, formoterol alone or combined with ipratropium bromide partially protects the lungs against the chronic inflammation and airspace enlargement by reducing neutrophilic infiltration possibly via the inhibition of MMP-9 activity.