Immune Dysregulation in Human Telomere Diseases

Germline variants in a diversity of genes involved in telomere maintenance result in premature telomere shortening and cause clinical conditions that are collectively called telomeropathies, which range from mucocutaneous findings (skin hypopigmentation, nail dystrophy and oral leukoplakia) to bone marrow failure, myeloid neoplasms, pulmonary fibrosis and cirrhosis. Even though pulmonary fibrosis and liver cirrhosis develop in up to a quarter of patients with telomeropathies, the exact mechanisms promoting the fibrotic pathology remain unclear. Considering that the hematopoietic compartment is particularly sensitive to telomere erosion, we aimed to investigate whether the immune response may play a role on the development of fibrotic lesions in a murine model, and to evaluate immunological biomarkers in patients diagnosed with telomere diseases.

To assess the effects of telomere attrition on the development of liver fibrosis and immune response, G3 Terc^-/- and Tert^-/- mice (background C57BL/6J) and wild-type (WT) mice were infected with S. mansoni cercariae and euthanized after 12 weeks. Telomere length (TL) was determined by qPCR in leukocytes and liver sections and confirmed to be shorter in both knockout (KO) models in comparison to WT animals (p≤0.01). Collagen deposition in liver tissue was quantified with Picro Sirius Red staining and showed larger fibrotic areas in WT mice (14 ± 5,2%) when compared to Terc^-/- (7,2 ± 4,3%; p≤0.05) and Tert^-/- (6,3 ± 2,4%; p≤0.05). Macrophage infiltration was evaluated by immunostaining using antibodies against F4/80, CD206 and iNOS, and revealed less positive cells within the liver of Terc^-/- and Tert^-/- mice in comparison to WT (p≤0.001). We studied the ability of bone marrow-derived macrophages (BMDMs) to be polarized towards M1 and M2-like phenotypes ex vivo, upon stimulation with either LPS or IL-4, respectively. Expression of TNF-alpha, IL-6 and MMP9 demonstrated that the classical activation of Terc^-/- and Tert^-/- BMDMs was impaired in a similar fashion, and these cells did not exhibit the M1-like phenotype as observed in the WT BMDMs. Conversely, CD206 and ARG1 were significantly upregulated in KOs stimulated with IL-4, suggesting that telomerase-null BMDMs are skewed towards an M2 polarization.

We also evaluated 18 patients with telomere diseases (median age, 33.9 years; range, 12.8-62.5 years; male/female, 9/9; marrow failure, 15; pulmonary manifestations, 5; hepatic manifestations, 5) and 10 healthy individuals (median age, 32.1; range, 13.9-56.3 years; male/female, 5/5) for their immune profile. Eight patients had very short TL (<1^st percentile) and ten had short TL (<10^th percentile). All healthy individuals had TL >10^th percentile. We measured serum levels of cytokines, chemokines and growth factors using a customized Luminex panel to assess 32 analytes in a single assay. Spearman's correlation coefficient was used to analyze associations between TL z-scores and the variables of interest. Analysis showed significant correlations (p≤0.05) with angiopoietin-1, IL-1α, IL-1β, IL-1RA, IL-3, IL-4, IL-7, IL-23, IL-27, MCP-1, M-CSF, MIP-3α and PDGF-BB and, in all cases, Spearman's rho was positive, indicating that shorter telomeres were correlated with decreased protein serum levels.

In order to study the maturation of T- and B-cells, T-cell receptor excision circles (TRECs) and kappa-deleting excision circles (KRECs) were quantified by qPCR in dried blood spot samples from ten patients with short telomeres and ten age-matched healthy individuals. Both biomarkers were significantly reduced in patients (p≤0.05), showing that production and/or maturation of T- and B-lymphocytes is impaired. TRECs were negatively correlated to age in the control group, (r_s = -0.66; p = 0.043), but not in patients (p>0.05), suggesting that TRECs do not follow the same pattern of age-dependent decrease in short telomere syndromes.

Altogether, the results demonstrate that telomere erosion contributes to impaired immune responses and T- and B-cell maturation, which may contribute to the pathophysiology of several clinical manifestations in telomeropathies. Further studies are necessary to deeply characterize innate and adaptative immune response in telomere syndromes and better understand how it correlates to fibrosis development.

Calado:Agios: Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria; Novartis: Honoraria.