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Cystic fibrosis is associated with chronic Pseudomonas aeruginosa colonization and inflammation. The role of MyD88, the shared adapter protein of the proinflammatory TLR and IL-1R families, in chronic P. aeruginosa biofilm lung infection is unknown. We report that chronic lung infection with the clinical P. aeruginosa RP73 strain is associated with uncontrolled lung infection in complete MyD88-deficient mice with epithelial damage, inflammation, and rapid death. Then, we investigated whether alveolar or myeloid cells contribute to heightened sensitivity to infection. Using cell-specific, MyD88-deficient mice, we uncover that the MyD88 pathway in myeloid or alveolar epithelial cells is dispensable, suggesting that other cell types may control the high sensitivity of MyD88-deficient mice. By contrast, IL-1R1–deficient mice control chronic P. aeruginosa RP73 infection and IL-1β Ab blockade did not reduce host resistance. Therefore, the IL-1R1/MyD88 pathway is not involved, but other IL-1R or TLR family members need to be investigated. Our data strongly suggest that IL-1 targeted neutralizing therapies used to treat inflammatory diseases in patients unlikely reduce host resistance to chronic P. aeruginosa infection.

Idiopathic pulmonary fibrosis (IPF) is the most common and severe type of interstitial lung disease for which current treatments display limited efficacy. IPF is largely driven by host-derived danger signals released upon recurrent local tissue damage. Here we explored the roles of self-DNA and stimulator of interferon genes (STING), a protein belonging to an intracellular DNA sensing pathway that leads to type I and/or type III interferon (IFN) production upon activation. Using a mouse model of IPF, we report that STING deficiency leads to exacerbated pulmonary fibrosis with increased collagen deposition in the lungs and excessive remodeling factors expression. We further show that STING-mediated protection does not rely on type I IFN signaling nor on IL-17A or TGF-β modulation but is associated with dysregulated neutrophils. Together, our data support an unprecedented immunoregulatory function of STING in lung fibrosis.

Cigarette smoke (CS) is the major cause of chronic lung injuries, such as chronic obstructive pulmonary disease (COPD). In patients with severe COPD, tertiary lymphoid follicles containing B lymphocytes and B cell-activating factor (BAFF) overexpression are associated with disease severity. In addition, BAFF promotes adaptive immunity in smokers and mice chronically exposed to CS. However, the role of BAFF in the early phase of innate immunity has never been investigated. We acutely exposed C57BL/6J mice to CS and show early BAFF expression in the bronchoalveolar space and lung tissue that correlates to airway neutrophil and macrophage influx. Immunostaining analysis revealed that neutrophils are the major source of BAFF. We confirmed in vitro that neutrophils secrete BAFF in response to cigarette smoke extract (CSE) stimulation. Antibody-mediated neutrophil depletion significantly dampens lung inflammation to CS exposure but only partially decreases BAFF expression in lung tissue and bronchoalveolar space suggesting additional sources of BAFF. Importantly, BAFF deficient mice displayed decreased airway neutrophil recruiting chemokines and neutrophil influx while the addition of exogenous BAFF significantly enhanced this CS-induced neutrophilic inflammation. This demonstrates that BAFF is a key proinflammatory cytokine and that innate immune cells in particular neutrophils, are an unconsidered source of BAFF in early stages of CS-induced innate immunity.

Cigarette smoke exposure is a leading cause of chronic obstructive pulmonary disease (COPD), a major health issue characterized by airway inflammation with fibrosis and emphysema. Here we demonstrate that acute exposure to cigarette smoke causes respiratory barrier damage with the release of self-dsDNA in mice. This triggers the DNA sensor cGAS (cyclic GMP-AMP synthase) and stimulator of interferon genes (STING), driving type I interferon (IFN I) dependent lung inflammation, which are attenuated in cGAS, STING or type I interferon receptor (IFNAR) deficient mice. Therefore, we demonstrate a critical role of self-dsDNA release and of the cGAS-STING-type I interferon pathway upon cigarette smoke-induced damage, which may lead to therapeutic targets in COPD.

Introduction B cell activating factor (BAFF) is a TNF family cytokine mainly produced by adaptive immune cells in particular B and T lymphocytes. Depending on the context, innate immune cells can also produce BAFF. First of all, BAFF was known to play an important role in the maturation and survival of B lymphocytes in humoral immune response. Recent data indicate that BAFF may also participate in the regulation of innate immune responses and in the pathophysiology of pulmonary diseases. In models of mice chronically exposed to cigarette smoke (CS), BAFF plays a major role in the generation of pulmonary antinuclear antibodies and tertiary lymphoid follicles. However, the implication of BAFF in the development of innate immunity to cigarette smoking remains unknown. Methods Wild-type or BAFF deficient mice were exposed to 3R4F cigarette smoke 3 times a day for 4 days. Mice were treated or not with mouse anti-GR1 or recombinant mouse BAFF during CS exposure. Broncho-alveolar lavage fluids (BALF) were done and cell infiltration was determined. Different cytokines and chemokines were measured in BALF and lung homogenates. Immunostaining on cytospin were done. Bone marrow derived neutrophils (BMDN) and Murine tracheal epithelial cells (MTEC) were stimulated in vitro by CSE (Cigarette Smoke Extract). Cell viability and BAFF levels were determined. Results Acute CS exposure promotes BAFF expression in airway recruited neutrophils. Immunostaining analysis revealed that neutrophils are the major source of BAFF. We confirmed in vitro that neutrophils secrete BAFF in response to CSE stimulation. Neutrophil depletion partially decreases BAFF expression in lung tissue and bronchoalveolar space suggesting additional sources of BAFF. Importantly, BAFF deficient mice displayed decreased airway neutrophil recruiting chemokines and neutrophil influx while the addition of exogenous BAFF significantly enhanced this CS-induced neutrophilic inflammation. Conclusion These results demonstrate that BAFF is a key pro-inflammatory cytokine and that innate immune cells in particular neutrophils, are an unconsidered source of BAFF in early stages of CS-induced innate immunity.

Introduction Chronic obstructive pulmonary disease is a major health problem and has been predicted to become the third cause of death in the world by 2020. Using mice model of cigarette smoke and elastase-induced injury, we previously showed that interleukin (IL-) 1beta is essential to inflammation, remodelling and emphysema. In addition, secretion of mature IL-1beta, inflammation and emphysema were reduced in mice deficient for the inflammasome adaptor molecule ASC but not in mice deficient for the receptor NLRP3, suggesting that another NLRP can be involved in inflammasome scaffolding and IL-1beta maturation. Recently, it was shown that genetic ablation of NLRP6 in mice profoundly reduces inflammasome activation in the intestine, and increases the animals’ susceptibility to colitis. However, the role of NLRP6 in lung inflammation and repair upon injury remains completely undiscovered. Methods Wild type mice (B6) or Nlpr6 deficient mice (Nlrp6 ko) were exposed to the smoke of 4 cigarettes (3R4F), 3 times a day during 4 days. Mice were sacrificed 16 hours after the last exposure and inflammatory and remodeling parameters were evaluated. Broncho-alveolar lavage fluids (BALF) were done and cell infiltration was determined. Myeloperoxidase (MPO) activities and content of pro-inflammatory cytokines and remodeling factors were measured in BALF and lung homogenates. Results In comparison to wild type mice, Nlrp6 deficient mice presented greatly reduced cell recruitment and in particular reduced macrophage and neutrophil influx into the BALF. MPO activities and levels of the proinflammatorty cytokine IL-1beta and of the B cell activating factor (BAFF) into BALF and lung homogenates were also diminished. Moreover the production of the remodeling factors a matrix metalloproteinase, member 9 (MMP-9) and tissue inhibitor of metalloproteinase member 1 (TIMP-1) were attenuated in Nlrp6 KO mice. Conclusion Our results demonstrate for the first time that NLRP6 is a critical player in acute inflammation and remodeling induced by cigarette smoke exposure. Better understanding of the role of NLRP6 may allow identifying new therapeutic targets for potential curative treatments in very severe and irreversible disease such as COPD or emphysema.

The NLR pyrin domain containing 3 (NLRP3) inflammasome is a major component of the innate immune system, but its mechanism of activation by a wide range of molecules remains largely unknown. Widely used nano-sized inorganic metal oxides such as silica dioxide (nano-SiO2) and titanium dioxide (nano-TiO2) activate the NLRP3 inflammasome in macrophages similarly to silica or asbestos micro-sized particles. By investigating towards the molecular mechanisms of inflammasome activation in response to nanoparticles, we show here that active adenosine triphosphate (ATP) release and subsequent ATP, adenosine diphosphate (ADP) and adenosine receptor signalling are required for inflammasome activation. Nano-SiO2 or nano-TiO2 caused a significant increase in P2Y1, P2Y2, A2A and/or A2B receptor expression, whereas the P2X7 receptor was downregulated. Interestingly, IL-1β secretion in response to nanoparticles is increased by enhanced ATP and ADP hydrolysis, whereas it is decreased by adenosine degradation or selective A2A or A2B receptor inhibition. Downstream of these receptors, our results show that nanoparticles activate the NLRP3 inflammasome via activation of PLC-InsP3 and/or inhibition of adenylate cyclase (ADCY)-cAMP pathways. Finally, a high dose of adenosine triggers inflammasome activation and IL-1β secretion through adenosine cellular uptake by nucleotide transporters and by its subsequent transformation in ATP by adenosine kinase. In summary, we show for the first time that extracellular adenosine activates the NLRP3 inflammasome by two ways: by interacting with adenosine receptors at nanomolar/micromolar concentrations and through cellular uptake by equilibrative nucleoside transporters at millimolar concentrations. These findings provide new molecular insights on the mechanisms of NLRP3 inflammasome activation and new therapeutic strategies to control inflammation.

International audience; Deposition of uric acid crystals in joints causes the acute and chronic inflammatory disease known as gout and prolonged airway exposure to silica crystals leads to the development of silicosis, an irreversible fibrotic pulmonary disease. Aluminum salt (Alum) crystals are frequently used as vaccine adjuvant. The mechanisms by which crystals activate innate immunity through the Nlrp3 inflammasome are not well understood. Here, we show that uric acid, silica and Alum crystals trigger the extracellular delivery of endogenous ATP, which just precedes the secretion of mature interleukin-1β (IL-1β) by macrophages, both events depending on purinergic receptors and connexin/pannexin channels. Interestingly, not only ATP but also ADP and UTP are involved in IL-1β production upon these Nlrp3 inflammasome activators through multiple purinergic receptor signaling. These findings support a pivotal role for nucleotides as danger signals and provide a new molecular mechanism to explain how chemically and structurally diverse stimuli can activate the Nlrp3 inflammasome.