Calcineurin negatively regulates TLR-Mediated activation pathways

In innate immunity, microbial components stimulate macrophages to produce antimicrobial substances, cytokines, other proinflammatory mediators, and IFNs via TLRs, which trigger signaling pathways activating NF-kappa B, MAPKs, and IFN response factors. We show in this study that, in contrast to its activating role in T cells, in macrophages the protein phosphatase calcineurin negatively regulates NF-kappa B, MAPKs, and IFN response factor activation by inhibiting the TLR-mediated signaling pathways. Evidence for this novel role for calcineurin was provided by the findings that these signaling pathways are activated when calcineurin is inhibited either by the inhibitors cyclosporin A or FK506 or by small interfering RNA-targeting calcineurin, and that activation of these pathways by TLR ligands is inhibited by the overexpression of a constitutively active form of calcineurin. We further found that I kappa B-alpha degradation, MAPK activation, and TNF-alpha production by FK506 were reduced in macrophages from mice deficient in MyD88, Toll/IL-1R domain-containing adaptor-inducing IFN-beta (TRIF), TLR2, or TLR4, whereas macrophages from TLR3-deficient or TLR9 mutant mice showed the same responses to FK506 as those of wild-type cells. Biochemical studies indicate that calcineurin interacts with MyD88, TRIF, TLR2, and TLR4, but not with TLR3 or TLR9. Collectively, these results suggest that calcineurin negatively regulates TLR-mediated activation pathways in macrophages by inhibiting the adaptor proteins MyD88 and TRIF, and a subset of TLRs

Reversibility of Defective Hematopoiesis Caused by Telomere Shortening in Telomerase Knockout Mice.

Telomere shortening is common in bone marrow failure syndromes such as dyskeratosis congenita (DC), aplastic anemia (AA) and myelodysplastic syndromes (MDS). However, improved knowledge of the lineage-specific consequences of telomere erosion and restoration of telomere length in hematopoietic progenitors is required to advance therapeutic approaches. We have employed a reversible murine model of telomerase deficiency to compare the dependence of erythroid and myeloid lineage differentiation on telomerase activity. Fifth generation Tert-/- (G5 Tert-/-) mice with shortened telomeres have significant anemia, decreased erythroblasts and reduced hematopoietic stem cell (HSC) populations associated with neutrophilia and increased myelopoiesis. Intracellular multiparameter analysis by mass cytometry showed significantly reduced cell proliferation and increased sensitivity to activation of DNA damage checkpoints in erythroid progenitors and in erythroid-biased CD150hi HSC, but not in myeloid progenitors. Strikingly, Cre-inducible reactivation of telomerase activity restored hematopoietic stem and progenitor cell (HSPC) proliferation, normalized the DNA damage response, and improved red cell production and hemoglobin levels. These data establish a direct link between the loss of TERT activity, telomere shortening and defective erythropoiesis and suggest that novel strategies to restore telomerase function may have an important role in the treatment of the resulting anemia

Comparison of HSC and MPP numbers between G0 <i>Tert</i>+/- and G5 <i>Tert</i>-/- mice.

<p>(A) Representative FACS profiles of lineage-, c-Kit+ and Sca1+ cells separated based on CD34 and CD150 expression, showing absolute numbers of HSC (Lin-c-Kit+Sca1+CD34-CD150+), MPP-A (Lin-c-Kit+Sca1+CD34+CD150+) and MPP-B (Lin-c-Kit+Sca1+CD34+CD150-) populations in G0 <i>Tert</i>+/- and G5 <i>Tert</i>-/- BM. (B-D) Comparison of absolute numbers of (B) HSC, (C) MPP-A and (D) MPP-B in the femurs of G0 <i>Tert</i>+/- (n = 17) and G5 <i>Tert</i>-/- (n = 11) mice aged 11–20 months. The ends of the whiskers represent minimum and maximum values while the bar indicates the median value (50^th percentile). p values are based on a 2-tailed <i>t</i> test. (E) Representative FACS profile of CD34-LKS cells subdivided into CD150hi, CD150lo and CD150negative fractions in G0 <i>Tert</i>+/- and G5 <i>Tert</i>-/- BM cells. (F) Ratios of the three CD150 fractions within CD34-LKS cells from G0 <i>Tert</i>+/- (n = 6) and G5 <i>Tert</i>-/- (n = 9) mice aged 11–20 months, as calculated from the absolute numbers of total HSC population. p value < 0.01(two-way ANOVA) demonstrating that the differences between the three CD150 fractions in G0 <i>Tert</i>+/- and G5 <i>Tert</i>-/- mice are statistically significant.</p

Simultaneous Determinations of Cell Cycle and DNA Damage Response in MEP and GMP Populations as Determined by Mass Cytometry.

<p>Scatter plots showing (A and D) pATM, (B and E) p-γH2AX and (C and F) p53 expression in Ki-67 positive proliferating and Ki-67 negative non-proliferating cells in MEP and GMP populations. Bars indicate standard deviations and the p values are based on a 2-tailed <i>t</i> test. Statistically significant differences between WT and G5 <i>Tert</i>-/- mice are indicated by * (p value < 0.05) or ** (p value < 0.01). There were no significant differences between WT, G0 <i>Tert</i>+/- and TxG5 <i>Tert</i>-/- mice.</p

Telomerase Reactivation in G5 <i>Tert</i>-/- Mice.

<p>(A) PCR showing Cre mediated excision of LSL cassette within intron 2 (in2) of <i>Tert</i> gene using <i>Tert</i> in2 F/R primers in DNA extracted from G5 <i>Tert</i>-/- spleen: untreated (lanes 4, 9, 11, 13 and 15); vehicle treated (lanes 8 and 10); tamoxifen treated (lanes 12 and 14). WT (lanes 2, 3 and 6) and G0 <i>Tert</i>+/- spleen (lanes 5 and 7) were used as positive controls. (B) TRAP assay for telomerase activity in 1–2 μg ileum lysate from 11–14 months old WT or tamoxifen treated G5 <i>Tert</i>-/- (TxG5 Tert-/-) mice (lanes 1,2,3) or treated with vehicle (lanes 4,5,6) for 3 months. Negative (buffer only) and positive (293T lysate) controls are shown on the far right. (C) Percentage increase in body weight in tamoxifen and vehicle treated G5 <i>Tert</i>-/- mice. Bars indicate mean values. (D and E) Comparison of RBC numbers and hemoglobin levels in the peripheral blood in tamoxifen or vehicle treated G5 <i>Tert</i>-/- mice before and 3 months after treatment. (F and G) Effects of tamoxifen treatment on erythroblast (CD71+Ter119+) and MEP (Lin-c-Kit+ Sca1- CD34- FcγRII/III-) populations. Bars indicate standard deviation and p values are based on a 2-tailed <i>t</i> test. (H) Ratios of the CD34-LKS CD150 hi, CD150 lo and CD150 negative populations from WT, vehicle and tamoxifen G5 <i>Tert</i>-/- treated mice, as calculated from absolute numbers of HSC population. p value < 0.01 (two-way ANOVA). The results shown are combined data from two separate experiments.</p

Defective Erythropoiesis in G5 <i>Tert</i>-/- mice.

<p>(A-C) Murine BM cells were separated based on levels of CD71 and Ter119 expression into three stages (I-III) of erythroid cell maturation. Whisker plot represents the number of (A) CD71hi; (B) CD71lo; (C) CD71- erythroblasts in the femur of G0 <i>Tert</i>+/- (n = 17) and G5 <i>Tert</i>-/- (n = 11) mice aged 11–20 months. (D-F) CD34 and FcγRII/III surface expression in lineage negative BM cells in G0 <i>Tert</i>+/- and G5 <i>Tert</i>-/- mice to define CMP (Lin-c-Kit+ Sca1- CD34+ FcγRII/IIIlo), MEP (Lin-c-Kit+ Sca1- CD34- FcγRII/III-) and GMP (Lin-c-Kit+ Sca1- CD34+ FcγRII/IIIhi) populations. Whisker plots represent the number of (D) CMP, (E) MEP and (F) GMP in G0 <i>Tert</i>+/- (n = 17) and G5 <i>Tert</i>-/- (n = 11) mice aged 11–20 months. The ends of the whiskers represent minimum and maximum values and the bar indicates the median value (50^th percentile). (G and H) Q-FISH assay showing telomere length from 15–30 interphase nuclei comparing sorted MEP and GMP populations from G0 <i>Tert</i>+/- and G5 <i>Tert</i>-/- mice using Telometer software. Data shown is from two of the three experiments.</p

Cell Cycle Analysis of MEP and GMP Populations by Mass Cytometry.

<p>(A and B) Scatter plots comparing uridine incorporation and Ki-67 expression in MEPs and GMPs in WT, G0 <i>Tert</i>+/-, G5 <i>Tert</i>-/- and TxG5 <i>Tert</i>-/- mice. Percentages for Ki-67 + (proliferating) and Ki-67 negative (non-proliferating) cells were derived from the absolute numbers of MEPs and GMPs. Bars indicate standard deviations and the p values are based on a 2-tailed <i>t</i> test. Statistically significant differences between WT and G5 <i>Tert</i>-/- mice are indicated by ** (p value < 0.01). There were no significant differences between WT, G0 <i>Tert</i>+/- and TxG5 <i>Tert</i>-/- mice.</p

Effect of Telomerase Depletion on the Differentiation of Human CD34+ Progenitor Cells.

<p>(A-B) Scatter plots showing median values for (A) pRb and (B) pATM expression in CD34-LKS CD150hi, CD150lo and CD150neg populations from WT <i>Tert</i>+/+ (n = 4), G0 <i>Tert</i>+/- (n = 4), G5 <i>Tert</i>-/- (n = 4) and TxG5 <i>Tert</i>-/- (n = 4) mice. Bars indicate standard deviation and the p values are based on a 2-tailed <i>t</i> test. Statistically significant differences between WT and G5 Tert-/- mice are indicated by * (p value < 0.05) and ** (p value < 0.01). There were no significant differences between WT, G0 <i>Tert</i>+/- and TxG5 <i>Tert</i>-/- mice. (C) Proposed model for the consequences of reduced telomere length: erythropoiesis is decreased and myelopoiesis is increased due to differential susceptibility of erythroid and myeloid progenitors to the DDR induced by short telomeres. Reactivation of telomerase activity restores normal hematopoiesis, while cumulative DNA damage may lead to myeloid malignancies.</p