Lifespan-Extending Caloric Restriction or mTOR Inhibition Impair Adaptive Immunity of Old Mice By Distinct Mechanisms

Aging of the world population and a concomitant increase in age-related diseases and disabilities mandates the search for strategies to increase healthspan, the length of time an individual lives healthy and productively. Due to the age-related decline of the immune system, infectious diseases remain among the top 5–10 causes of mortality and morbidity in the elderly, and improving immune function during aging remains an important aspect of healthspan extension. Calorie restriction (CR) and more recently rapamycin (rapa) feeding have both been used to extend lifespan in mice. Preciously few studies have actually investigated the impact of each of these interventions upon in vivo immune defense against relevant microbial challenge in old organisms. We tested how rapa and CR each impacted the immune system in adult and old mice. We report that each intervention differentially altered T-cell development in the thymus, peripheral T-cell maintenance, T-cell function and host survival after West Nile virus infection, inducing distinct but deleterious consequences to the aging immune system. We conclude that neither rapa feeding nor CR, in the current form/administration regimen, may be optimal strategies for extending healthy immune function and, with it, lifespan

Neisseria gonorrhoeae Suppresses Dendritic Cell-Induced, Antigen-Dependent CD4 T Cell Proliferation

Neisseria gonorrhoeae is the second most common sexually transmitted bacterial pathogen worldwide. Diseases associated with N. gonorrhoeae cause localized inflammation of the urethra and cervix. Despite this inflammatory response, infected individuals do not develop protective adaptive immune responses to N. gonorrhoeae. N. gonorrhoeae is a highly adapted pathogen that has acquired multiple mechanisms to evade its host's immune system, including the ability to manipulate multiple immune signaling pathways. N. gonorrhoeae has previously been shown to engage immunosuppressive signaling pathways in B and T lymphocytes. We have now found that N. gonorrhoeae also suppresses adaptive immune responses through effects on antigen presenting cells. Using primary, murine bone marrow-derived dendritic cells and lymphocytes, we show that N. gonorrhoeae-exposed dendritic cells fail to elicit antigen-induced CD4+ T lymphocyte proliferation. N. gonorrhoeae exposure leads to upregulation of a number of secreted and dendritic cell surface proteins with immunosuppressive properties, particularly Interleukin 10 (IL-10) and Programmed Death Ligand 1 (PD-L1). We also show that N. gonorrhoeae is able to inhibit dendritic cell- induced proliferation of human T-cells and that human dendritic cells upregulate similar immunosuppressive molecules. Our data suggest that, in addition to being able to directly influence host lymphocytes, N. gonorrhoeae also suppresses development of adaptive immune responses through interactions with host antigen presenting cells. These findings suggest that gonococcal factors involved in host immune suppression may be useful targets in developing vaccines that induce protective adaptive immune responses to this pathogen

<em>Neisseria gonorrhoeae</em> Suppresses Dendritic Cell-Induced, Antigen-Dependent CD4 T Cell Proliferation

<div><p><em>Neisseria gonorrhoeae</em> is the second most common sexually transmitted bacterial pathogen worldwide. Diseases associated with <em>N. gonorrhoeae</em> cause localized inflammation of the urethra and cervix. Despite this inflammatory response, infected individuals do not develop protective adaptive immune responses to <em>N. gonorrhoeae</em>. <em>N. gonorrhoeae</em> is a highly adapted pathogen that has acquired multiple mechanisms to evade its host's immune system, including the ability to manipulate multiple immune signaling pathways. <em>N. gonorrhoeae</em> has previously been shown to engage immunosuppressive signaling pathways in B and T lymphocytes. We have now found that <em>N. gonorrhoeae</em> also suppresses adaptive immune responses through effects on antigen presenting cells. Using primary, murine bone marrow-derived dendritic cells and lymphocytes, we show that <em>N. gonorrhoeae</em>-exposed dendritic cells fail to elicit antigen-induced CD4+ T lymphocyte proliferation. <em>N. gonorrhoeae</em> exposure leads to upregulation of a number of secreted and dendritic cell surface proteins with immunosuppressive properties, particularly Interleukin 10 (IL-10) and Programmed Death Ligand 1 (PD-L1). We also show that <em>N. gonorrhoeae</em> is able to inhibit dendritic cell- induced proliferation of human T-cells and that human dendritic cells upregulate similar immunosuppressive molecules. Our data suggest that, in addition to being able to directly influence host lymphocytes, <em>N. gonorrhoeae</em> also suppresses development of adaptive immune responses through interactions with host antigen presenting cells. These findings suggest that gonococcal factors involved in host immune suppression may be useful targets in developing vaccines that induce protective adaptive immune responses to this pathogen.</p> </div

IL-10 inhibits OVA-DC-induced T cell proliferation.

<p>OVA-pulsed dendritic cells were co-cultured with CFSE-loaded OT-II T cells with or without IL-10 for seven days. <b>A</b>) Representative histogram overlay and bar graph show T cell proliferation profiles following culture with OVA-pulsed DCs (black) or OVA-pulsed DCs+IL-10 (red). The bar graph shows the proliferation of OT-II T cell in the presence of OVA-pulsed DCs with and without exogenous IL-10 from three independent experiments. Data are mean ± standard deviation (N = 3). <b>B</b>) Transwell experiment scheme. WT OVA-DC with OT-II T cell co-culture was placed in all transwell plates. In the insert medium treated-DCs or <i>N. gonorrhoeae</i>-treated DCs from wild type or <i>Il10^−/−</i> were co-cultured with OT-II T cells as indicated. T cell proliferation from the transwell plate is shown in the histogram overlays. OVA-induced T cell proliferation in the plate was inhibited by <i>N. gonorrhoeae</i>-treated wild type DCs in the insert (red) but not by wild type medium treated-DCs in the insert (blue). OVA-induced T cell proliferation in the plate was the same for <i>N. gonorrhoeae</i>-treated <i>Il10^−/−</i> DCs in the insert (green) and medium treated <i>Il10^−/−</i> DCs in the insert (purple). <b>C</b>) Ratio of proliferated T cells from transwell plates with inserts supplying <i>N. gonorrhoeae</i>-OVA-DCs or medium-DCs. Ratio of T cell proliferation in the plate was obtained by dividing the <i>N. gonorrhoeae</i>-OVA-DCs insert by medium-DCs insert. The black bars represent proliferation ratio from transwell plate supplied with wild type BMDCs in insert (N = 8), the open bars represent proliferation ratio from transwell plate supplied with <i>Il10^−/−</i> BMDCs in insert (N = 4).</p

Fold change in genes over-expressed in BMDCs with <i>N. gonorrhoeae</i> (MOI = 1) plus OVA versus OVA only.

<p>Fold change in genes over-expressed in BMDCs with <i>N. gonorrhoeae</i> (MOI = 1) plus OVA versus OVA only.</p

<i>N. gonorrhoeae</i> inhibits dendritic cell-induced T cell proliferation in human primary immune cells.

<p><b>A</b>) Representative histograms from 2 donors showing unregulated expression of CD11c, HLA-DR, CD274 and CD273 at 24 hours post stimulation with <i>N. gonorrhoeae</i> (MOI = 1, 10). <b>B</b>) IL-10 protein production by human DCs treated with <i>N. gonorrhoeae</i> (MOI = 1,10). <b>C</b>) MFI of PD-L1 expression on human DCs treated with <i>N. gonorrhoeae</i> (MOI = 1,10). <b>D</b>) <i>N. gonorrhoeae</i> inhibits human DCs induced allogeneic T cell proliferation in the Mixed Lymphocyte Reaction (MLR). CFSE proliferation profiles of CD4+ cells after non-adherent cells (NAD) co-cultured with human DCs treated with medium or <i>N. gonorrhoeae</i> (MOI = 10) for 7 days at the ratio of 10∶1.</p

PD-L1 and PD-L2 are induced on <i>N. gonorrhoeae</i> exposed BMDCs.

<p>BMDCs treated for 24 hours with medium only, OVA, <i>N. gonorrhoeae</i> (MOI = 1,10) with OVA were immunostained for flow cytometric analysis of CD273 and CD274 on DCs (B220−, CD11c+). Representative overlay histograms of: <b>A</b>) CD274 (PD-L1) and <b>B</b>) CD273 (PD-L2). <b>C</b>) Median fluorescence intensity (MFI) of PD-L1 and PD-L2 expression on BMDCs treated as indicated. <b>D</b>) Histogram of PD1 (CD279) expression on CD4+ Vβ5+ OT-II T cells prior to co-culture with BMDCs. <b>E–F</b>) Caspase 3&7 activity (FLICA) form CD4+ Vβ5+ OT-II T cells following co-culture with OVA or <i>N. gonorrhoeae</i> (MOI = 1) plus OVA (100 µg/mL) pulsed BMDCs. <b>E</b>) Representative overlay histograms of Caspase 3&7 activity (FLICA) from CD4+ Vβ5+ OT-II T cells following co-cultured with BMDCs for 24 hours. <b>F</b>) Percentage of apoptotic CD4+ Vβ5+ OT-II T cells following co-cultured with BMDCs for 24 hours. Data are mean ± standard deviation (N = 4 replicates). T cells treated with 1 µM staurosporine (ST) for 3 hours was used as positive control. <b>G</b>) Representative overlay histograms of OT-II T cell proliferation induced by BMDCs treated with OVA (green) versus <i>N. gonorrhoeae</i> (MOI = 0.1) with OVA plus anti-PD-L1 (1∶10 dilute, light blue), <i>N. gonorrhoeae</i> (MOI = 0.1) with OVA plus isotype control (1∶10 dilute, dark blue). <b>H</b>) Mean % ± SD of OT-II T cells proliferated through generations 0–1, 2–4, 5–7 following indicated culture conditions, N = 5–7.</p

Soluble factors in BMDC/T cell co-culture partially inhibit OVA-induced T cell proliferation.

<p><b>A–C</b>) CFSE proliferation profiles for OT-II T cells co-cultured with BMDCs under indicated conditions. Representative CFSE profiles for T cells from transwell insert (gray) and transwell itself (open) are shown (from three independent experiments). <b>D</b>) IL-10 protein production by BMDCs cultured with Medium, OVA, <i>N. gonorrhoeae</i> (MOI = 1, 1). Mean pg/mL ± SD, N = 3. <b>E</b>) <i>Il12a</i>, <i>Il12b</i>, <i>Il23a</i> and <i>Ebi3</i> mRNA steady-state expression in BMDCs cultured with OVA, <i>N. gonorrhoeae</i> (MOI = 1), or <i>N. gonorrhoeae</i> (MOI = 1) with OVA. Mean fold regulation ± SD, N = 3. <b>F</b>) Steady-state expression of mRNA encoding TGF-β 1, 2 and 3 in BMDCs cultured with OVA, <i>N. gonorrhoeae</i> (MOI = 1), or <i>N. gonorrhoeae</i> (MOI = 1) plus OVA. Mean fold regulation ± SD, N = 3. <b>G</b>) <i>Aldh1a2</i> mRNA steady-state expression in BMDCs cultured with OVA, <i>N. gonorrhoeae</i> (MOI = 1), or <i>N. gonorrhoeae</i> (MOI = 1) with OVA. Mean fold regulation (decrease) ± SD, N = 3.</p