Caspase-1-driven neutrophil pyroptosis and its role in host susceptibility to Pseudomonas aeruginosa.

Multiple regulated neutrophil cell death programs contribute to host defense against infections. However, despite expressing all necessary inflammasome components, neutrophils are thought to be generally defective in Caspase-1-dependent pyroptosis. By screening different bacterial species, we found that several Pseudomonas aeruginosa (P. aeruginosa) strains trigger Caspase-1-dependent pyroptosis in human and murine neutrophils. Notably, deletion of Exotoxins U or S in P. aeruginosa enhanced neutrophil death to Caspase-1-dependent pyroptosis, suggesting that these exotoxins interfere with this pathway. Mechanistically, P. aeruginosa Flagellin activates the NLRC4 inflammasome, which supports Caspase-1-driven interleukin (IL)-1ß secretion and Gasdermin D (GSDMD)-dependent neutrophil pyroptosis. Furthermore, P. aeruginosa-induced GSDMD activation triggers Calcium-dependent and Peptidyl Arginine Deaminase-4-driven histone citrullination and translocation of neutrophil DNA into the cell cytosol without inducing extracellular Neutrophil Extracellular Traps. Finally, we show that neutrophil Caspase-1 contributes to IL-1ß production and susceptibility to pyroptosis-inducing P. aeruginosa strains in vivo. Overall, we demonstrate that neutrophils are not universally resistant for Caspase-1-dependent pyroptosis.

Neutrophils expressing major histocompatibility complex class II molecules circulate in blood and milk during mastitis and show high microbicidal activity.

Bovine mastitis is mainly caused by bacterial infection and is responsible for important economic losses as well as alterations of the health and welfare of animals. The increase in somatic cell count (SCC) in milk during mastitis is mainly due to the influx of neutrophils, which have a crucial role in the elimination of pathogens. For a long time, these first-line defenders have been viewed as microbe killers, with a limited role in the orchestration of the immune response. However, their role is more complex: we recently characterized a bovine neutrophil subset expressing major histocompatibility complex class II (MHC-II) molecules (MHC-IIpos), usually distributed on antigen-presenting cells, as having regulatory capacities in cattle. In this study, our objective was to evaluate the implication of different neutrophils subsets in the mammary gland immunity during clinical and subclinical mastitis. Using flow cytometry, we analyzed the presence of MHC-IIpos neutrophils in blood and in milk during clinical mastitis at different time points of inflammation (n = 10 infected quarters) and during subclinical mastitis, defined as the presence of bacteria and an SCC >150,000 cells/mL (n = 27 infected quarters). Our results show, for the first time, that in blood and milk, neutrophils are a heterogeneous population and encompass at least 2 subsets distinguishable by their expression of MHC-II. In milk without mastitis, we observed higher production of reactive oxygen species and higher phagocytosis capacity of MHC-IIpos neutrophils compared with their MHC-IIneg counterparts, indicating the high bactericidal capacities of MHC-IIpos neutrophils. MHC-IIpos neutrophils are enriched in milk compared with blood during subclinical mastitis but not during clinical mastitis. Moreover, we observed a positive and highly significant correlation between MHC-IIpos neutrophils and T lymphocytes present in milk during subclinical mastitis. Our experiments involved a total of 47 cows (40 Holstein and 7 Normande cows). To conclude, our study opens the way to the discovery of new biomarkers of mastitis inflammation.

IL-17A Is an Important Effector of the Immune Response of the Mammary Gland to Escherichia coli Infection.

The cytokine IL-17A has been shown to play critical roles in host defense against bacterial and fungal infections at different epithelial sites, but its role in the defense of the mammary gland (MG) has seldom been investigated, although infections of the MG constitute the main pathology afflicting dairy cows. In this study, we showed that IL-17A contributes to the defense of the MG against Escherichia coli infection by using a mouse mastitis model. After inoculation of the MG with a mastitis-causing E. coli strain, the bacterial load increased rapidly, triggering an intense influx of leukocytes into mammary tissue and increased concentrations of IL-6, IL-22, TNF-α, and IL-10. Neutrophils were the first cells that migrated intensely to the mammary tissue, in line with an early production of CXCL2. Depletion of neutrophils induced an increased mammary bacterial load. There was a significant increase of IL-17-containing CD4(+) αß T lymphocyte numbers in infected glands. Depletion of IL-17A correlated with an increased bacterial colonization and IL-10 production. Intramammary infusion of IL-17A at the onset of infection was associated with markedly decreased bacterial numbers, decreased IL-10 production, and increased neutrophil recruitment. Depletion of CD25(+) regulatory T cells correlated with a decreased production of IL-10 and a reduced bacterial load. These results indicate that IL-17A is an important effector of MG immunity to E. coli and suggest that an early increased local production of IL-17A would improve the outcome of infection. These findings point to a new lead to the development of vaccines against mastitis.

The C3HeB/FeJ mouse model recapitulates the hallmark of bovine tuberculosis lung lesions following Mycobacterium bovis aerogenous infection.

Achieving the control of bovine tuberculosis (bTB) would require the discovery of an efficient combined immunodiagnostic and vaccine strategy. Since in vivo experiments on cattle are not ethically and economically acceptable there is a need for a cost-effective animal model capable of reproducing, as closely as possible, the physiopathology of bTB to (i) better characterize the cellular and molecular features of bTB immunopathogenesis and (ii) screen preclinical vaccine candidates. To develop such a model, we focused on the C3HeB/FeJ Kramnik's mouse forming hypoxic, encapsulated granulomas with a caseous necrotic center following Mycobacterium tuberculosis infection. Our work represents the first investigation on C3HeB/FeJ interaction with M. bovis, the main agent of bTB. Detailed histopathological analysis of C3HeB/FeJ lung lesions development following aerogenous M. bovis infection unraveled a bimodal evolution of the pathology. The C3HeB/FeJ recapitulated all the hallmarks of classical bovine lung granulomas but also developed, to some extend, lethal necrotic large lesions characterized by high mycobacterial and neutrophil load, and an inefficient collagen-driven lesion encapsulation. Interestingly these rapidly invasive pneumonia lesions, occurring in a constant percentage of the mice, shared all features with some exacerbated lung lesions that we and others have observed in lungs of cattle naturally or experimentally infected with M. bovis. Together, our findings demonstrate the relevance of the C3HeB/FeJ mouse as a comprehensive model to study bTB immunopathology that could be used for further vaccine therapies in the future.

Mycobacteria-infected dendritic cells attract neutrophils that produce IL-10 and specifically shut down Th17 CD4 T cells through their IL-10 receptor.

Neutrophils participate in the control of mycobacterial infection both by directly eliminating bacilli and by interacting with macrophages and dendritic cells (DCs). Despite host defenses, slow-growing mycobacteria can persist in the host for decades, mostly inside macrophages and DCs, and eventually destroy tissues after exacerbated inflammation. IL-17A-driven neutrophil recruitment may participate in this process. We report that mouse bone marrow-derived DCs infected with live Mycobacterium bovis Bacillus Calmette-Guérin (BCG) produced large amounts of CXCL1 and CXCL2, and attracted neutrophils. After physical contact with DCs infected with live BCG, the neutrophils produced large quantities of the immunosuppressive cytokine IL-10 via the MyD88 and spleen tyrosine kinase pathways. The CD11b integrin was involved in this neutrophil-DC interaction and allowed IL-10 production. TCR OVA transgenic mice immunized with a BCG strain producing OVA mounted an OVA-specific Th17 and Th1 CD4 response. Interestingly, IL-10-producing neutrophils specifically shut down IL-17A production by Th17 CD4 cells, but not IFN-γ production by Th1 cells. This was due to Th17 CD4 cell-restricted expression of the receptor for IL-10. After neutrophil depletion, total mouse lung cells produced less IL-10 but more IL-17A; IFN-γ production was not affected. Therefore, we suggest that during mycobacterial infection, regulatory neutrophils are instructed by infected reservoir DCs to produce IL-10 that specifically targets IL-10Rα-expressing Th17 CD4 T cells. This could be important to control the otherwise exuberant Th17 response.

Dual neutrophil subsets exacerbate or suppress inflammation in tuberculosis via IL-1ß or PD-L1.

Neutrophils can be beneficial or deleterious during tuberculosis (TB). Based on the expression of MHC-II and programmed death ligand 1 (PD-L1), we distinguished two functionally and transcriptionally distinct neutrophil subsets in the lungs of mice infected with mycobacteria. Inflammatory [MHC-II-, PD-L1lo] neutrophils produced inflammasome-dependent IL-1ß in the lungs in response to virulent mycobacteria and "accelerated" deleterious inflammation, which was highly exacerbated in IFN-γR-/- mice. Regulatory [MHC-II+, PD-L1hi] neutrophils "brake" inflammation by suppressing T-cell proliferation and IFN-γ production. Such beneficial regulation, which depends on PD-L1, is controlled by IFN-γR signaling in neutrophils. The hypervirulent HN878 strain from the Beijing genotype curbed PD-L1 expression by regulatory neutrophils, abolishing the braking function and driving deleterious hyperinflammation in the lungs. These findings add a layer of complexity to the roles played by neutrophils in TB and may explain the reactivation of this disease observed in cancer patients treated with anti-PD-L1.

Selective suppression of dendritic cell functions by Mycobacterium ulcerans toxin mycolactone.

Mycolactone is a polyketide toxin produced by Mycobacterium ulcerans (Mu), the causative agent of the skin disease Buruli ulcer (BU). Surprisingly, infected tissues lack inflammatory infiltrates. Structural similarities between mycolactone and immunosuppressive agents led us to investigate the immunomodulatory properties of mycolactone on dendritic cells (DCs), the key initiators and regulators of immune responses. At noncytotoxic concentrations, phenotypic and functional maturation of both mouse and human DCs was inhibited by mycolactone. Notably, mycolactone blocked the emigration of mouse-skin DCs to draining lymph nodes, as well as their maturation in vivo. In human peripheral blood-derived DCs, mycolactone inhibited the ability to activate allogeneic T cell priming and to produce inflammatory molecules. Interestingly, production of the cytokines interleukin (IL) 12, tumor necrosis factor alpha, and IL-6 was only marginally affected, whereas production of the chemokines macrophage inflammatory protein (MIP) 1alpha, MIP-1beta, regulated on activation, normal T cell expressed and secreted, interferon gamma-inducible protein 10, and monocyte chemoattractant protein 1 was abolished at nanomolar concentrations. Importantly, mycolactone endogenously expressed by Mu mediated similar inhibitory effects on beta-chemokine production by DCs. In accordance with the histopathological features of BUs, our results suggest that bacterial production of mycolactone may limit both the initiation of primary immune responses and the recruitment of inflammatory cells to the infection site. Moreover, they highlight a potential interest in mycolactone as a novel immunosuppressive agent.

The pig as a model for investigating the role of neutrophil serine proteases in human inflammatory lung diseases.

The serine proteases released by activated polymorphonuclear neutrophils [NSPs (neutrophil serine proteases)] contribute to a variety of inflammatory lung diseases, including CF (cystic fibrosis). They are therefore key targets for the development of efficient inhibitors. Although rodent models have contributed to our understanding of several diseases, we have previously shown that they are not appropriate for testing anti-NSP therapeutic strategies [Kalupov, Brillard-Bourdet, Dade, Serrano, Wartelle, Guyot, Juliano, Moreau, Belaaouaj and Gauthier (2009) J. Biol. Chem. 284, 34084-34091). Thus NSPs must be characterized in an animal model that is much more likely to predict how therapies will act in humans in order to develop protease inhibitors as drugs. The recently developed CFTR-/- (CFTR is CF transmembrane conductance regulator) pig model is a promising alternative to the mouse model of CF [Rogers, Stoltz, Meyerholz, Ostedgaard, Rokhlina, Taft, Rogan, Pezzulo, Karp, Itani et al. (2008) Science 321, 1837-1841]. We have isolated blood neutrophils from healthy pigs and determined their responses to the bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus, and the biochemical properties of their NSPs. We used confocal microscopy and antibodies directed against their human homologues to show that the three NSPs (elastase, protease 3 and cathepsin G) are enzymatically active and present on the surface of triggered neutrophils and NETs (neutrophil extracellular traps). All of the porcine NSPs are effectively inhibited by human NSP inhibitors. We conclude that there is a close functional resemblance between porcine and human NSPs. The pig is therefore a suitable animal model for testing new NSP inhibitors as anti-inflammatory agents in neutrophil-associated diseases such as CF.

Vitamin D induced microbicidal activity against Mycobacterium bovis BCG is dependent on the synergistic activity of bovine peripheral blood cell populations.

A growing appreciation is emerging of the beneficial role of vitamin D for health and resistance against infectious diseases, including tuberculosis. However, research has predominantly focused on murine and human species and functional data in bovines is limited. Therefore, the objective of this study was to assess the microbicidal activity and immunoregulatory effect of the vitamin D metabolite 1,25(OH)2D3 on bovine peripheral blood leukocytes (PBL) in response to Mycobacterium bovis BCG (BCG) infection using a combination of functional assays and gene expression profiling. Blood from Holstein-Friesian bull calves with low circulating levels of 25(OH)D was stimulated with 1,25(OH)2D3 for 2 h, and then infected with M. bovis BCG. Results showed that 1,25(OH)2D3 supplementation significantly increased BCG killing by on average 16 %, although responses varied between 1 % and 38 % killing. Serial cell subset depletion was then performed on PBL prior to 1,25(OH)2D3 incubation and BCG infected as before to analyse the contribution of major cell types to mycobacterial growth control. Specific antibodies and either magnetic cell separation or density gradient centrifugation of monocytes, granulocytes, CD3+, CD4+, and CD8+ T lymphocytes were used to capture each cell subset. Results showed that depletion of granulocytes had the greatest impact on BCG growth, leading to a significant enhancement of bacterial colonies. In contrast, depletion of CD4+ or CD8+ T cells individually, or in combination (CD3+), had no impact on mycobacterial growth control. In agreement with our previous data, 1,25(OH)2D3 significantly increased bacterial killing in PBL, in monocyte depleted samples, and a similar trend was observed in the granulocyte depleted subset. In addition, specific analysis of sorted neutrophils treated with 1,25(OH)2D3 showed an enhanced microbicidal activity against both BCG and a virulent strain of M. bovis. Lastly, data showed that 1,25(OH)2D3 stimulation increased reactive oxygen species (ROS) production and the expression of genes encoding host defence peptides (HDP) and pathogen recognition receptors (PRRs), factors that play an important role in the microbicidal activity against mycobacteria. In conclusion, the vitamin D metabolite 1,25(OH)2D3 improves antimycobacterial killing in bovine PBLs via the synergistic activity of monocytes and granulocytes and enhanced activation of innate immunity.

An isoniazid analogue promotes Mycobacterium tuberculosis-nanoparticle interactions and enhances bacterial killing by macrophages.

Nanoenabled drug delivery systems against tuberculosis (TB) are thought to control pathogen replication by targeting antibiotics to infected tissues and phagocytes. However, whether nanoparticle (NP)-based carriers directly interact with Mycobacterium tuberculosis and how such drug delivery systems induce intracellular bacterial killing by macrophages is not defined. In the present study, we demonstrated that a highly hydrophobic citral-derived isoniazid analogue, termed JVA, significantly increases nanoencapsulation and inhibits M. tuberculosis growth by enhancing intracellular drug bioavailability. Importantly, confocal and atomic force microscopy analyses revealed that JVA-NPs associate with both intracellular M. tuberculosis and cell-free bacteria, indicating that NPs directly interact with the bacterium. Taken together, these data reveal a nanotechnology-based strategy that promotes antibiotic targeting into replicating extra- and intracellular mycobacteria, which could actively enhance chemotherapy during active TB.

Mycobacterium bovis bacillus Calmette-Guérin vaccination mobilizes innate myeloid-derived suppressor cells restraining in vivo T cell priming via IL-1R-dependent nitric oxide production.

Early immune response to the largely used Mycobacterium bovis bacillus Calmette-Guérin (BCG) intradermal vaccine remains ill defined. Three days after BCG inoculation into the mouse ear, in addition to neutrophils infiltrating skin, we observed CD11b(+)Ly-6C(int)Ly-6G(-) myeloid cells. Neutrophil depletion markedly enhanced their recruitment. These cells differed from inflammatory monocytes and required MyD88-dependent BCG-specific signals to invade skin, whereas neutrophil influx was MyD88 independent. Upon BCG phagocytosis, CD11b(+)Ly-6C(int)Ly-6G(-) cells produced NO, which required the IL-1 receptor. Despite NO production, they were unable to kill BCG or the nonpathogenic Mycobacterium smegmatis. However, they markedly impaired T cell priming in the draining lymph node. Their elimination by all-trans retinoid acid treatment increased the number of IFN-gamma-producing CD4 T cells. Thus, BCG vaccination recruits innate myeloid-derived suppressor cells, akin to mouse tumor-infiltrating cells. These propathogenic cells dampen the early T cell response and might facilitate BCG persistence.

Specific NLRP3 Inflammasome Assembling and Regulation in Neutrophils: Relevance in Inflammatory and Infectious Diseases.

The NLRP3 inflammasome is a cytosolic multimeric protein platform that leads to the activation of the protease zymogen, caspase-1 (CASP1). Inflammasome activation mediates the proteolytic activation of pro-inflammatory cytokines (IL-1ß and IL-18) and program cell death called pyroptosis. The pyroptosis is mediated by the protein executioner Gasdermin D (GSDMD), which forms pores at the plasma membrane to facilitate IL-1ß/IL-18 secretion and causes pyroptosis. The NLRP3 inflammasome is activated in response to a large number of pathogenic and sterile insults. However, an uncontrolled inflammasome activation may drive inflammation-associated diseases. Initially, inflammasome-competent cells were believed to be limited to macrophages, dendritic cells (DC), and monocytes. However, emerging evidence indicates that neutrophils can assemble inflammasomes in response to various stimuli with functional relevance. Interestingly, the regulation of inflammasome in neutrophils appears to be unconventional. This review provides a broad overview of the role and regulation of inflammasomes-and more specifically NLRP3-in neutrophils.

In situ IL-12/23p40 production during mycobacterial infection is sustained by CD11bhigh dendritic cells localized in tissue sites distinct from those harboring bacilli.

Although IL-12/23p40 is known to play a major role in host resistance to Mycobacterium spp, the cellular source, tissue localization, and regulation of p40 production during mycobacterial infection in vivo has been unclear. In this study, we used IL-12/23p40eYFP (yet40) reporter mice to track expression of the cytokine following Mycobacterium bovis bacillus Calmette-Guérin (BCG) infection. We found that in spleens of these mice, p40 production is initiated by a transient burst from CD11b(low)CD11c(+) dendritic cells (DC) which are later replaced at the onset of granuloma formation by CD11b(high)CD11c(+) DC as the major source of the cytokine. The latter subset was also found to be the key producer of DC-derived p40 in nonlymphoid tissue and in both spleen and liver optimal production of the cytokine was regulated by endogenous TNF-alpha. Although BCG and p40-expressing DC were both observed in splenic white pulp, p40(+) DC rarely colocalized with bacilli. Indeed, in vitro flow cytometry and confocal microscopy indicated that the presence of intracellular bacteria is not required for p40 production by DC and Transwell experiments confirmed that soluble mycobacterial components are sufficient for inducing cytokine expression by these cells. Moreover, when stimulated with LPS, DC directly infected with BCG showed impaired IL-12p40 production in vitro. Together, our findings establish CD11b(high) DC as a major source of IL-12/23p40 during mycobacterial infection in situ and implicate both soluble mycobacterial products and TNF-alpha in stimulating sustained production of p40 by these cells.

TLR4- and TLR2-mediated B cell responses control the clearance of the bacterial pathogen, Leptospira interrogans.

Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira interrogans that are transmitted by asymptomatic infected rodents. Leptospiral lipoproteins and LPS have been shown to stimulate murine cells via TLRs 2 and 4. Host defense mechanisms remain obscure, although TLR4 has been shown to be involved in clearing Leptospira. In this study, we show that double (TLR2 and TLR4) knockout (DKO) mice rapidly died from severe hepatic and renal failure following Leptospira inoculation. Strikingly, the severe proinflammatory response detected in the liver and kidney from Leptospira-infected DKO mice appears to be independent of MyD88, the main adaptor of TLRs. Infection of chimeric mice constructed with wild-type and DKO mice, and infection of several lines of transgenic mice devoid of T and/or B lymphocytes, identified B cells as the crucial lymphocyte subset responsible for the clearance of Leptospira, through the early production of specific TLR4-dependent anti-Leptospira IgMs elicited against the leptospiral LPS. We also found a protective tissue compartmentalized TLR2/TLR4-mediated production of IFN-gamma by B and T lymphocytes, in the liver and kidney, respectively. In contrast, the tissue inflammation observed in Leptospira-infected DKO mice was further characterized to be mostly due to B lymphocytes in the liver and T cells in the kidney. Altogether these findings demonstrate that TLR2 and TLR4 play a key role in the early control of leptospirosis, but do not directly trigger the inflammation induced by pathogenic Leptospira.

Isolation of Bovine Neutrophils by Fluorescence- and Magnetic-Activated Cell Sorting.

Flow cytometry and magnetic bead technology enable the separation of cell populations with the highest degree of purity. Here, we describe protocols to sort bovine neutrophils from blood, the labeling and sorting, including gating strategies. We also provide advice to preserve neutrophil viability and detail a protocol to measure phagocytosis and oxidative species production.

Neutrophils Encompass a Regulatory Subset Suppressing T Cells in Apparently Healthy Cattle and Mice.

Neutrophils that reside in the bone marrow are swiftly recruited from circulating blood to fight infections. For a long time, these first line defenders were considered as microbe killers. However their role is far more complex as cross talk with T cells or dendritic cells have been described for human or mouse neutrophils. In cattle, these new roles are not documented yet. We identified a new subset of regulatory neutrophils that is present in the mouse bone marrow or circulate in cattle blood under steady state conditions. These regulatory neutrophils that display MHC-II on the surface are morphologically indistinguishable from classical MHC-IIneg neutrophils. However MHC-IIpos and MHC-IIneg neutrophils display distinct transcriptomic profiles. While MHC-IIneg and MHC-IIpos neutrophils display similar bacterial phagocytosis or killing activity, MHC-IIpos only are able to suppress T cell proliferation under contact-dependent mechanisms. Regulatory neutrophils are highly enriched in lymphoid organs as compared to their MHC-IIneg counterparts and in the mouse they express PDL-1, an immune checkpoint involved in T-cell blockade. Our results emphasize neutrophils as true partners of the adaptive immune response, including in domestic species. They open the way for discovery of new biomarkers and therapeutic interventions to better control cattle diseases.

Mycobacterial Infection of Precision-Cut Lung Slices Reveals Type 1 Interferon Pathway Is Locally Induced by Mycobacterium bovis but Not M. tuberculosis in a Cattle Breed.

Tuberculosis exacts a terrible toll on human and animal health. While Mycobacterium tuberculosis (Mtb) is restricted to humans, Mycobacterium bovis (Mb) is present in a large range of mammalian hosts. In cattle, bovine TB (bTB) is a noticeable disease responsible for important economic losses in developed countries and underestimated zoonosis in the developing world. Early interactions that take place between mycobacteria and the lung tissue early after aerosol infection govern the outcome of the disease. In cattle, these early steps remain poorly characterized. The precision-cut lung slice (PCLS) model preserves the structure and cell diversity of the lung. We developed this model in cattle in order to study the early lung response to mycobacterial infection. In situ imaging of PCLS infected with fluorescent Mb revealed bacilli in the alveolar compartment, in adjacent or inside alveolar macrophages, and in close contact with pneumocytes. We analyzed the global transcriptional lung inflammation signature following infection of PCLS with Mb and Mtb in two French beef breeds: Blonde d'Aquitaine and Charolaise. Whereas, lungs from the Blonde d'Aquitaine produced high levels of mediators of neutrophil and monocyte recruitment in response to infection, such signatures were not observed in the Charolaise in our study. In the Blonde d'Aquitaine lung, whereas the inflammatory response was highly induced by two Mb strains, AF2122 isolated from cattle in the UK and Mb3601 circulating in France, the response against two Mtb strains, H37Rv, the reference laboratory strain, and BTB1558, isolated from zebu in Ethiopia, was very low. Strikingly, the type I interferon pathway was only induced by Mb but not Mtb strains, indicating that this pathway may be involved in mycobacterial virulence and host tropism. Hence, the PCLS model in cattle is a valuable tool to deepen our understanding of early interactions between lung host cells and mycobacteria. It revealed striking differences between cattle breeds and mycobacterial strains. This model could help in deciphering biomarkers of resistance vs. susceptibility to bTB in cattle as such information is still critically needed for bovine genetic selection programs and would greatly help the global effort to eradicate bTB.

CFTR-deficient pigs display alterations of bone microarchitecture and composition at birth.

BACKGROUND: The lack of cystic fibrosis transmembrane conductance regulator (CFTR) function causes cystic fibrosis (CF), predisposing to severe lung disease, reduced growth and osteopenia. Both reduced bone content and strength are increasingly recognized in infants with CF before the onset of significant lung disease, suggesting a developmental origin and a possible role in bone disease pathogenesis. The role of CFTR in bone metabolism is unclear and studies on humans are not feasible. Deletion of CFTR in pigs (CFTR -/- pigs) displays at birth severe malformations similar to humans in the intestine, respiratory tract, pancreas, liver, and male reproductive tract. METHODS: We compared bone parameters of CFTR -/- male and female pigs with those of their wild-type (WT) littermates at birth. Morphological and microstructural properties of femoral cortical and trabecular bone were evaluated using micro-computed tomography (µCT), and their chemical compositions were examined using Raman microspectroscopy. RESULTS: The integrity of the CFTR -/- bone was altered due to changes in its microstructure and chemical composition in both sexes. Low cortical thickness and high cortical porosity were found in CFTR -/- pigs compared to sex-matched WT littermates. Moreover, an increased chemical composition heterogeneity associated with higher carbonate/phosphate ratio and higher mineral crystallinity was found in CFTR -/- trabecular bone, but not in CFTR -/- cortical bone. CONCLUSIONS: The loss of CFTR directly alters the bone composition and metabolism of newborn pigs. Based on these findings, we speculate that bone defects in patients with CF could be a primary, rather than a secondary consequence of inflammation and infection.

The cell surface-exposed glycopeptidolipids confer a selective advantage to the smooth variants of Mycobacterium smegmatis in vitro.

The cell surface of mycobacteria is quite rich in lipids. Glycopeptidolipids, surface-exposed lipids that typify some mycobacterial species, have been associated with a phenotypic switch between rough and smooth colony morphotypes. This conversion in Mycobacterium smegmatis is correlated with the absence/presence of glycopeptidolipids on the cell surface and is due to insertion sequence mobility. Here, we show that the occurrence of a high amount of glycopeptidolipids in the smooth variant leads to lower invasion abilities and lower internalization by macrophages. We further show that the high production of glycopeptidolipids on the cell surface can confer a selective advantage to the smooth variant when grown in vitro. This higher fitness under the laboratory condition might explain the selection of smooth variants in several independent laboratories. The implications of these findings are discussed.

Neutrophils and Close Relatives in the Hypoxic Environment of the Tuberculous Granuloma: New Avenues for Host-Directed Therapies?

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is one of the most prevalent lung infections of humans and kills ~1.7 million people each year. TB pathophysiology is complex with a central role played by granuloma where a delicate balance takes place to both constrain bacilli and prevent excessive inflammation that may destroy lung functions. Neutrophils reach the lung in waves following first encounter with bacilli and contribute both to early Mtb elimination and late deleterious inflammation. The hypoxic milieu where cells and bacilli cohabit inside the granuloma favors metabolism changes and the impact on TB infection needs to be more thoroughly understood. At the cellular level while the key role of the alveolar macrophage has long been established, behavior of neutrophils in the hypoxic granuloma remains poorly explored. This review will bring to the front new questions that are now emerging regarding neutrophils activity in TB. Are different neutrophil subsets involved in Mtb infection and how? How do neutrophils and close relatives contribute to shaping the granuloma immune environment? What is the role of hypoxia and hypoxia induced factors inside granuloma on neutrophil fate and functions and TB pathophysiology? Addressing these questions is key to the development of innovative host-directed therapies to fight TB.