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.

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.

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.

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.

CR3 Engaged by PGL-I Triggers Syk-Calcineurin-NFATc to Rewire the Innate Immune Response in Leprosy.

Mycobacterium leprae, the causative agent of leprosy, is unique amongst human pathogens in its capacity to produce the virulence factor phenolic glycolipid (PGL)-I. In addition to mediating bacterial tropism for neurons, PGL-I interacts with Complement Receptor (CR)3 on macrophages (MPs) to promote infection. We demonstrate here that PGL-I binding to CR3 also enhances bacterial invasion of both polymorphonuclear neutrophils (PMNs) and dendritic cells (DCs). Moreover, in all cell types CR3 engagement by PGL-I activates the Syk tyrosine kinase, inducing calcineurin-dependent nuclear translocation of the transcription factor NFATc. This selectively augments the production of IL-2 by DCs, IL-10 by PMNs and IL-1ß by MPs. In intranasally-infected mice PGL-I binding to CR3 heightens mycobacterial phagocytosis by lung PMNs and MPs, and stimulates NFATc-controlled production of Syk-dependent cytokines. Our study thus identifies the CR3-Syk-NFATc axis as a novel signaling pathway activated by PGL-I in innate immune cells, rewiring host cytokine responses to M. leprae.

Experimental infection of cattle with Mycobacterium tuberculosis isolates shows the attenuation of the human tubercle bacillus for cattle.

The Mycobacterium tuberculosis complex (MTBC) is the collective term given to the group of bacteria that cause tuberculosis (TB) in mammals. It has been reported that M. tuberculosis H37Rv, a standard reference MTBC strain, is attenuated in cattle compared to Mycobacterium bovis. However, as M. tuberculosis H37Rv was isolated in the early 1930s, and genetic variants are known to exist, we sought to revisit this question of attenuation of M. tuberculosis for cattle by performing a bovine experimental infection with a recent M. tuberculosis isolate. Here we report infection of cattle using M. bovis AF2122/97, M. tuberculosis H37Rv, and M. tuberculosis BTB1558, the latter isolated in 2008 during a TB surveillance project in Ethiopian cattle. We show that both M. tuberculosis strains caused reduced gross pathology and histopathology in cattle compared to M. bovis. Using M. tuberculosis H37Rv and M. bovis AF2122/97 as the extremes in terms of infection outcome, we used RNA-Seq analysis to explore differences in the peripheral response to infection as a route to identify biomarkers of progressive disease in contrast to a more quiescent, latent infection. Our work shows the attenuation of M. tuberculosis strains for cattle, and emphasizes the potential of the bovine model as a 'One Health' approach to inform human TB biomarker development and post-exposure vaccine development.

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.

IL-17RA in Non-Hematopoietic Cells Controls CXCL-1 and 5 Critical to Recruit Neutrophils to the Lung of Mycobacteria-Infected Mice during the Adaptive Immune Response.

During chronic infection with Mycobacterium tuberculosis (Mtb), bacilli multiplication is constrained within lung granulomas until excessive inflammation destroys the lung. Neutrophils are recruited early and participate in granuloma formation, but excessive neutrophilia exacerbates the tuberculosis disease. Neutrophils thus appear as potential targets for therapeutic interventions, especially in patients for whom no antibiotic treatment is possible. Signals that regulate neutrophil recruitment to the lung during mycobacterial infection need to be better understood. We demonstrated here, in the mouse model, that neutrophils were recruited to the lung in two waves after intranasal infection with virulent Mtb or the live attenuated vaccine strain Bacillus Calmette Guérin (BCG). A first wave of neutrophils was swiftly recruited, followed by a subsequent adaptive wave that reached the lung together with IFN-γ- and IL-17A-producing T cells. Interestingly, the second neutrophil wave did not participate to mycobacteria control in the lung and established contacts with T cells. The adaptive wave was critically dependent on the expression of IL-17RA, the receptor for IL-17A, expressed in non-hematopoietic cells. In absence of this receptor, curtailed CXCL-1 and 5 production in the lung restrained neutrophil recruitment. CXCL-1 and 5 instillation reconstituted lung neutrophil recruitment in BCG-infected IL17RA-/- mice.

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.

Immunological responses and protective efficacy against Brucella melitensis induced by bp26 and omp31 B. melitensis Rev.1 deletion mutants in sheep.

The commonly used live attenuated vaccine in ovine brucellosis prophylaxis is Brucella melitensis Rev.1. This vaccine is known to induce antibody responses in vaccinated animals indistinguishable by the current conventional serological tests from those observed in challenged animals. Brucella BP26 and Omp31 proteins have shown an interesting potential as diagnostic antigens for ovine brucellosis. Accordingly, the bp26 gene and both bp26 and omp31 genes have been deleted from the vaccine strain Rev.1. Immunogenicity and vaccine efficacy of the parental Rev.1 strain and of both mutants in protecting sheep against B. melitensis strain H38 challenge was evaluated by clinical and bacteriological examination of ewes. They were conjunctivally or subcutaneously vaccinated when 4 months old and then challenged with B. melitensis H38 at the middle of the first pregnancy following vaccination. Deletion of bp26 and omp31 genes did not significantly affect the well recognised capacity of Rev.1 to protect sheep against B. melitensis challenge. However, the protection conferred by the CGV2631 mutant was significantly lower than that conferred by the CGV26 mutant or the Rev.1 strain. Vaccinated and challenged animals were detected positive in classical serological tests and in the IFN-gamma assay. A BP26-based ELISA was investigated to discriminate between ewes vaccinated by the mutants and ewes challenged with B. melitensis H38. The cut-off which was chosen in order to have 100% specificity resulted in a moderate sensitivity for the detection of challenged ewes. The use in the field of one of the mutants as vaccine against a B. melitensis infection, combined with classic diagnostic tests and a BP26 ELISA, could thus give an improvement in the differentiation between vaccinated and infected animals and contribute to the objective of eradication of brucellosis in small ruminants.