Influenza A virus (IAV) is the etiological agent of a highly contagious acute respiratory disease that causes epidemics and considerable mortality annually. Recently, we demonstrated, using an in vitro approach, that the pattern recognition Toll-like receptor (TLR)3 plays a key role in the immune response of lung epithelial cells to IAV. In view of these data and the fact that the functional role of TLR3 in vivo is still debated, we designed an investigation to better understand the role of TLR3 in the mechanisms of IAV pathogenesis and host immune response using an experimental murine model. The time-course of several dynamic parameters, including animal survival, respiratory suffering, viral clearance, leukocyte recruitment into the airspaces and secretion of critical inflammatory mediators, was compared in infected wild-type and TLR3 −/− mice. First, we found that the pulmonary expression of TLR3 is constitutive and markedly upregulated following influenza infection in control mice. Notably, when compared to wild-type mice, infected TLR3 −/− animals displayed significantly reduced inflammatory mediators, including RANTES (regulated upon activation, normal T cell expressed and secreted), interleukin-6, and interleukin-12p40/p70 as well as a lower number of CD8+ T lymphocytes in the bronchoalveolar airspace. More important, despite a higher viral production in the lungs, mice deficient in TLR3 had an unexpected survival advantage. Hence, to our knowledge, our findings show for the first time that TLR3-IAV interaction critically contributes to the debilitating effects of a detrimental host inflammatory response.

Listeria monocytogenes is a human intracellular pathogen that is able to survive in the gastrointestinal environment and replicate in macrophages, thus bypassing the early innate immune defenses. Peptidoglycan (PG) is an essential component of the bacterial cell wall readily exposed to the host and, thus, an important target for the innate immune system. Characterization of the PG from L. monocytogenes demonstrated deacetylation of N-acetylglucosamine residues. We identified a PG N-deacetylase gene, pgdA, in L. monocytogenes genome sequence. Inactivation of pgdA revealed the key role of this PG modification in bacterial virulence because the mutant was extremely sensitive to the bacteriolytic activity of lysozyme, and growth was severely impaired after oral and i.v. inoculations. Within macrophage vacuoles, the mutant was rapidly destroyed and induced a massive IFN-␤ response in a TLR2 and Nod1-dependent manner. Together, these results reveal that PG N-deacetylation is a highly efficient mechanism used by Listeria to evade innate host defenses. The presence of deacetylase genes in other pathogenic bacteria indicates that PG N-deacetylation could be a general mechanism used by bacteria to evade the host innate immune system. cytokine ͉ macrophage ͉ pathogenesis ͉ virulence ͉ cell wall

Pulmonary epithelial cells are continuously exposed to microbial challenges as a result of breathing. It is recognized that immune myeloid cells express Toll-like receptors (TLRs), which play a major role in detecting microbes and initiating innate immune responses. In contrast, little is known concerning the expression of TLR in pulmonary epithelial cells per se, their distribution within the cell, their function, and the signaling pathways involved. In this work, we demonstrated by reverse transcription-PCR and/or immunoblot that TLR4 and the accessory molecule MD-2 are constitutively expressed in distinct human alveolar and bronchial epithelial cells. We further characterized by flow cytometry, biotinylation/precipitation, and confocal microscopy the intracellular localization of TLR4 in these cells. Despite this intracellular compartmentalization of TLR4, pulmonary epithelial cells were responsive to the TLR4 activator lipopolysaccharide (LPS), a potent Gram-negative bacteria-associated molecular pattern. Using respiratory epithelial cells isolated from TLR4 knock-out and wild type mice, we demonstrated that TLR4 is the actual activating receptor for LPS in these cells. Furthermore we showed that this cell response to LPS involves a signaling complex including the kinases interleukin-1 receptor-associated kinase (IRAK), p38, Jnk, and ERK1/2. Moreover, using vectors expressing dominant-negative forms of MyD88 and TRAF6, we established that LPS-induced activation of respiratory epithelial cells is largely dependent on TLR4 signaling intermediates. Altogether these data demonstrate that TLR4 is a key element in the response of pulmonary epithelial cells to molecules derived from Gram-negative bacteria. The intracellular localization of TLR4 in lung epithelia is expected to play an important role in the prevention of the development of chronic inflammatory disease.

Antibiotic-resistant bacteria threaten life worldwide. Although new antibiotics are scarce, the use of bacteriophages, viruses that infect bacteria, is rarely proposed as a means of offsetting this shortage. Doubt also remains widespread about the efficacy of phage therapy despite recent encouraging results. Using a bioluminescent Pseudomonas aeruginosa strain, we monitored and quantified the efficacy of a bacteriophage treatment in mice during acute lung infection. Bacteriophage treatment not only was effective in saving animals from lethal infection, but also was able to prevent lung infection when given 24 h before bacterial infection, thereby extending the potential use of bacteriophages as therapeutic agents to combat bacterial lung infection.

SummaryThe in vivo composition of the mycelial extracellular matrix (ECM) of Aspergillus fumigatus during host invasion is reported here for the first time. A new galactosaminogalactan and the galactomannan were the major polysaccharides of the in vivo ECM. The composition of the ECM in vivo varied with the aspergillosis pathologies.

Leptospira interrogans is a spirochete that is responsible for leptospirosis, a zoonotic disease. This bacterium possesses an unusual LPS that has been shown to use TLR2 instead of TLR4 for signaling in human cells. The structure of its lipid A was recently deciphered. Although its overall hexa-acylated disaccharide backbone is a classical feature of all lipid A forms, the lipid A of L. interrogans is peculiar. In this article, the functional characterization of this lipid A was studied in comparison to whole parental leptospiral LPS in terms of cell activation and use of TLR in murine and human cells. Lipid A from L. interrogans did not coagulate the Limulus hemolymph. Although leptospiral lipid A activated strongly murine RAW cells, it did not activate human monocytic cells. Results obtained from stimulation of peritoneal-elicited macrophages from genetically deficient mice for TLR2 or TLR4 clearly showed that lipid A stimulated the cells through TLR4 recognition, whereas highly purified leptospiral LPS utilized TLR2 as well as TLR4. In vitro experiments with transfected human HEK293 cells confirmed that activation by lipid A occurred only through murine TLR4-MD2 but not through human TLR4-MD2, nor murine or human TLR2. Similar studies with parental leptospiral LPS showed that TLR2/TLR1 were the predominant receptors in human cells, whereas TLR2 but also TLR4 contributed to activation in murine cells. Altogether these results highlight important differences between human and mouse specificity in terms of TLR4-MD2 recognition that may have important consequences for leptospiral LPS sensing and subsequent susceptibility to leptospirosis.

Aspergillus fumigatus causes invasive pulmonary aspergillosis (IPA). This disease is one of the most lifethreatening opportunistic infections in immunocompromised patients. The type of immunosuppressive regimen under which IPA occurs has rarely been investigated. In this study, we evaluated various parameters of the innate immune response during the progression of murine IPA induced by the intratracheal administration of A. fumigatus conidia as a function of two immunosuppressive treatments: a corticosteroid and a chemotherapeutic agent. We compared host responses various times after infection in terms of survival, pulmonary production of pro-and anti-inflammatory cytokines, cellular trafficking in the airways, lung injury, respiratory distress, and fungal development. We found that IPA pathogenesis involved predominantly fungal development in mice treated by chemotherapy and an adverse host response in mice treated with a corticosteroid. These previously unrecognized differences should be taken into account in evaluations of the pathogenesis of IPA in animal models.Aspergillus fumigatus belongs to a family of saprophytic filamentous fungi found in most environments. It sporulates abundantly, leading to the release into the atmosphere of large quantities of conidia. A. fumigatus spreads within the environment by means of airborne conidia, and the small diameter of these spores (2 to 3 m) enables them to reach the lung alveoli (19). Pulmonary host defenses against A. fumigatus are mediated by phagocytic cells of the innate immune system. Resident alveolar macrophages are thought to eliminate conidia by phagocytosis, whereas recruited polymorphonuclear neutrophils (PMN) destroy hyphae by producing reactive oxygen species (30,34).If the immune defense system of the lung is weakened, then conidia germinate and produce hyphae that invade the surrounding lung tissues (39), leading to the development of invasive pulmonary aspergillosis (IPA). This disease is one of the most life-threatening opportunistic infections in immunocompromised patients (19), and its incidence has increased in recent decades (14, 18). This increase in incidence has been associated with an increase in the number of people at risk due to solid organ transplantation, hematopoeitic stem cell transplantation, and intensive chemotherapeutic or immunosuppressive regimens (24). Furthermore, the difficulty of diagnosing IPA early and the low efficacy of treatments result in a high rate of mortality due to this disease in immunocompromised patients (10,23).Despite the increase in the number of IPA patients, the pathogenesis of this mycosis remains poorly understood. Experimental animal models of IPA have been developed for investigation of the role of innate immunity in the progression of the disease. Various infection protocols have been used and have generated conflicting data. More importantly, the type of immunosuppressive regimen in which IPA develops has received very little attention. In this study, we investigated various parameters of the innate...

The collectin surfactant protein-A (SP-A) is involved in the innate host defense and the regulation of inflammatory processes in the lung. In this work we investigated the molecular mechanisms related to the immunostimulatory activity of SP-A using macrophages from C3H/HeJ mice, which carry an inactivating mutation in the Toll-like receptor (TLR)4 gene, and TLR4-transfected Chinese hamster ovary cells. We demonstrate that SP-A-induced activation of the NF-κB signaling pathway and up-regulation of cytokine synthesis such as TNF-α and IL-10 are critically dependent on the TLR4 functional complex. These findings support the concept that TLR4 is a pattern recognition receptor that signals in response to both foreign pathogens and endogenous host mediators.