Induction of Stress-Induced Renal Cellular Senescence In Vitro: Impact of Mouse Strain Genetic Diversity.

Cellular senescence, a stress-induced state of irreversible cell cycle arrest, is associated with organ dysfunction and age-related disease. While immortalized cell lines bypass key pathways of senescence, important mechanisms of cellular senescence can be studied in primary cells. Primary tubular epithelial cells (PTEC) derived from mouse kidney are highly susceptible to develop cellular senescence, providing a valuable tool for studying such mechanisms. Here, we tested whether genetic differences between mouse inbred strains have an impact on the development of stress-induced cellular senescence in cultured PTEC. Kidneys from 129S1, B6, NOD, NZO, CAST, and WSB mice were used to isolate PTEC. Cells were monitored for expression of typical senescence markers (SA-ß-galactosidase, γ-H2AX+/Ki67-, expression levels of CDKN2A, lamin B1, IL-1a/b, IL-6, G/M-CSF, IFN-g, and KC) at 3 and 10 days after pro-senescent gamma irradiation. Clear differences were found between PTEC from different strains with the highest senescence values for PTEC from WSB mice and the lowest for PTEC from 129S1 mice. PTEC from B6 mice, the most commonly used inbred strain in senescence research, had a senescence score lower than PTEC from WSB and CAST mice but higher than PTEC from NZO and 129S1 mice. These data provide new information regarding the influence of genetic diversity and help explain heterogeneity in existing data. The observed differences should be considered when designing new experiments and will be the basis for further investigation with the goal of identifying candidate loci driving pro- or anti-senescent pathways.

TLR7 Controls VSV Replication in CD169+ SCS Macrophages and Associated Viral Neuroinvasion.

Vesicular stomatitis virus (VSV) is an insect-transmitted rhabdovirus that is neurovirulent in mice. Upon peripheral VSV infection, CD169+ subcapsular sinus (SCS) macrophages capture VSV in the lymph, support viral replication, and prevent CNS neuroinvasion. To date, the precise mechanisms controlling VSV infection in SCS macrophages remain incompletely understood. Here, we show that Toll-like receptor-7 (TLR7), the main sensing receptor for VSV, is central in controlling lymph-borne VSV infection. Following VSV skin infection, TLR7-/- mice display significantly less VSV titers in the draining lymph nodes (dLN) and viral replication is attenuated in SCS macrophages. In contrast to effects of TLR7 in impeding VSV replication in the dLN, TLR7-/- mice present elevated viral load in the brain and spinal cord highlighting their susceptibility to VSV neuroinvasion. By generating novel TLR7 floxed mice, we interrogate the impact of cell-specific TLR7 function in anti-viral immunity after VSV skin infection. Our data suggests that TLR7 signaling in SCS macrophages supports VSV replication in these cells, increasing LN infection and may account for the delayed onset of VSV-induced neurovirulence observed in TLR7-/- mice. Overall, we identify TLR7 as a novel and essential host factor that critically controls anti-viral immunity to VSV. Furthermore, the novel mouse model generated in our study will be of valuable importance to shed light on cell-intrinsic TLR7 biology in future studies.

De Novo Fatty Acid Synthesis During Mycobacterial Infection Is a Prerequisite for the Function of Highly Proliferative T Cells, But Not for Dendritic Cells or Macrophages.

Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, is able to efficiently manipulate the host immune system establishing chronic infection, yet the underlying mechanisms of immune evasion are not fully understood. Evidence suggests that this pathogen interferes with host cell lipid metabolism to ensure its persistence. Fatty acid metabolism is regulated by acetyl-CoA carboxylase (ACC) 1 and 2; both isoforms catalyze the conversion of acetyl-CoA into malonyl-CoA, but have distinct roles. ACC1 is located in the cytosol, where it regulates de novo fatty acid synthesis (FAS), while ACC2 is associated with the outer mitochondrial membrane, regulating fatty acid oxidation (FAO). In macrophages, mycobacteria induce metabolic changes that lead to the cytosolic accumulation of lipids. This reprogramming impairs macrophage activation and contributes to chronic infection. In dendritic cells (DCs), FAS has been suggested to underlie optimal cytokine production and antigen presentation, but little is known about the metabolic changes occurring in DCs upon mycobacterial infection and how they affect the outcome of the immune response. We therefore determined the role of fatty acid metabolism in myeloid cells and T cells during Mycobacterium bovis BCG or Mtb infection, using novel genetic mouse models that allow cell-specific deletion of ACC1 and ACC2 in DCs, macrophages, or T cells. Our results demonstrate that de novo FAS is induced in DCs and macrophages upon M. bovis BCG infection. However, ACC1 expression in DCs and macrophages is not required to control mycobacteria. Similarly, absence of ACC2 did not influence the ability of DCs and macrophages to cope with infection. Furthermore, deletion of ACC1 in DCs or macrophages had no effect on systemic pro-inflammatory cytokine production or T cell priming, suggesting that FAS is dispensable for an intact innate response against mycobacteria. In contrast, mice with a deletion of ACC1 specifically in T cells fail to generate efficient T helper 1 responses and succumb early to Mtb infection. In summary, our results reveal ACC1-dependent FAS as a crucial mechanism in T cells, but not DCs or macrophages, to fight against mycobacterial infection.

Conventional Dendritic Cells Confer Protection against Mouse Cytomegalovirus Infection via TLR9 and MyD88 Signaling.

Cytomegalovirus (CMV) is an opportunistic virus severely infecting immunocompromised individuals. In mice, endosomal Toll-like receptor 9 (TLR9) and downstream myeloid differentiation factor 88 (MyD88) are central to activating innate immune responses against mouse CMV (MCMV). In this respect, the cell-specific contribution of these pathways in initiating anti-MCMV immunity remains unclear. Using transgenic mice, we demonstrate that TLR9/MyD88 signaling selectively in CD11c+ dendritic cells (DCs) strongly enhances MCMV clearance by boosting natural killer (NK) cell CD69 expression and IFN-γ production. In addition, we show that in the absence of plasmacytoid DCs (pDCs), conventional DCs (cDCs) promote robust NK cell effector function and MCMV clearance in a TLR9/MyD88-dependent manner. Simultaneously, cDC-derived IL-15 regulates NK cell degranulation by TLR9/MyD88-independent mechanisms. Overall, we compartmentalize the cellular contribution of TLR9 and MyD88 signaling in individual DC subsets and evaluate the mechanism by which cDCs control MCMV immunity.

MyD88 signalling in myeloid cells is sufficient to prevent chronic mycobacterial infection.

Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis that is responsible for almost 1.5 million deaths per year. Sensing of mycobacteria by the host's immune system relies on different families of receptors present on innate immune cells. Amongst them, several members of the TLR family are involved in the activation of immune cells by mycobacteria, yet the in vivo contribution of individual TLRs to the protective immune response remains controversial. On the contrary, MyD88, the adaptor molecule for most TLRs, plays a non-redundant role in the protection against tuberculosis and mice with a complete germline deletion of MyD88 succumb very early to infection. MyD88 is expressed in both immune and non-immune cells, but it is not clear whether control of mycobacteria requires ubiquitous or cell-type specific MyD88 expression. Therefore, using novel conditional switch-on mouse models, we aimed to investigate the importance of MyD88 signalling in DCs and macrophages for the induction of protective effector mechanisms against mycobacterial infection. We conclude that specific reactivation of MyD88 signalling in CD11c- or lysozyme M-expressing myeloid cells during Mycobacterium bovis Bacille Calmette-Guerin infection is sufficient to restore systemic and local inflammatory cytokine production and to control pathogen burden.