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-β-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

Dendritic Cells Coordinate Innate Immunity via MyD88 Signaling to Control Listeria monocytogenes Infection

Listeria monocytogenes (LM), a facultative intracellular Gram-positive pathogen, can cause life-threatening infections in humans. In mice, the signaling cascade downstream of the myeloid differentiation factor 88 (MyD88) is essential for proper innate immune activation against LM, as MyD88-deficient mice succumb early to infection. Here, we show that MyD88 signaling in dendritic cells (DCs) is sufficient to mediate the protective innate response, including the production of proinflammatory cytokines, neutrophil infiltration, bacterial clearance, and full protection from lethal infection. We also demonstrate that MyD88 signaling by DCs controls the infection rates of CD8α+ cDCs and thus limits the spread of LM to the T cell areas. Furthermore, in mice expressing MyD88 in DCs, inflammatory monocytes, which are required for bacterial clearance, are activated independently of intrinsic MyD88 signaling. In conclusion, CD11c+ conventional DCs critically integrate pathogen-derived signals via MyD88 signaling during early infection with LM in vivo

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

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

Limited impact of Treg depletion on inflammatory cytokine production after <i>M. bovis</i> BCG infection.

<p>Intracellular cytokine production by FoxP3⁻CD4⁺ T cells was analysed in the spleen of day 7/8 and 14/15 double-depleted (black bars) or untreated DEREG mice (white bars) on day 20 after i.v. infection with 2×10⁶ CFU <i>M. bovis</i> BCG. Percentages of (A) IFN-γ, (B) IL-17A and (C) IL-10 production within live FoxP3⁻CD4⁺ T cells after PMA/ionomycin restimulation are shown. Bar graphs represent mean ± SD of 3–5 mice per group. N = 3. Statistical analysis: Mann-Whitney-U-Test. <i>*p<0.05; **p<0.01; and ***p<0.001</i>.</p

Preferential expansion of Teff cells over Tregs during acute BCG infection.

<p>WT mice were infected i.v. with 2×10⁶ CFU <i>M. bovis</i> BCG (black squares), or not (black dots), and the (A) frequency and (B) total cell number of FoxP3⁺ Tregs within the live CD4⁺ T cell gate was determined in spleen (left) and lungs (right) at day 20 p.i.. Data are pooled from three independent experiments and represent the mean ± SD of 11–12 mice per group. Each symbol represents an individual mouse. N = 3. Statistical analysis: Mann-Whitney-U-Test. <i>*p<0.05; **p<0.01; and ***p<0.001</i>.</p

Removal of Tregs after <i>Mtb</i> aerosol infection in DEREG mice does not influence pathogen control.

<p>DEREG mice were infected with approx. 100<i>Mtb</i> via the aerosol route. Tregs were depleted by DT administration on days 11/12, 18/19 and 25/26 (black dots/bars) or not (white dots/bars). (A) Experimental scheme. (B) Mycobacterial colony enumeration assays were performed in lungs (left), spleen (middle) and liver (right) on day 20, 28 and 42 p.i.. Data represent mean ± SD of 5 mice per group. (C) The frequency of ESAT6_1–20-specific IFN-γ- and IL-17A-producing CD4⁺ cells per 10⁵ total lung cells was determined by ELISPOT assay at different time points after infection. Bar graphs represent the mean ± SD of 5 mice per group. N = 3 (day 20), N = 1(day 28 and 42).</p

eGFP⁻ diTregs rapidly replenish the pool of Tregs in <i>M. bovis</i> BCG infected DEREG mice.

<p>DEREG mice were infected i.v. with 2×10⁶ CFU <i>M. bovis</i> BCG and treated, or not, with DT on days 7/8 and 14/15 p.i. and (A) the percentage of eGFP⁺ (grey)- and eGFP⁻ (white) Foxp3⁺CD4⁺ Tregs cells in the spleen (left) and lungs (right) or (B) the expression of the thymic Treg markers Helios (left) and Nrp-1 (right) in the eGFP⁺ (grey) and eGFP⁻ (white) Treg population were analysed in the spleen at day 20 p.i. in DT-treated mice. Bar graphs represent mean ± SD of 3–5 mice per group. N = 3. Statistical analysis: Mann-Whitney-U-Test. <i>*p<0.05; **p<0.01; and ***p<0.001</i>.</p

Presentation_1.PDF

<p>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.</p