Hemoglobin alpha regulates T-lymphocyte activation and mitochondrial function

Abstract

Hemoglobinopathies affect approximately 7% of the global population and are associated with an increased risk of autoimmune disorders, though the underlying mechanisms remain unclear. Hemoglobin is traditionally recognized for its role in oxygen transport within erythrocytes, but its expression in other cell types has been documented. We have recently discovered hemoglobin alpha a1 (Hbα-a1 mRNA and Hbα protein) in T-lymphocytes and previously reported that its expression was sensitive to mitochondrial redox perturbations. However, outside of its incidence and basic characterization, the functional role of Hbα in T-lymphocytes remained unknown. Herein, we identify Hbα in both CD4⁺ and CD8⁺ T-lymphocyte subsets, and found its expression is highly dynamic, differs between the two subtypes, and is dependent upon activation stage. Further, the loss of Hbα impairs mitochondrial function, dysregulates cytokine production, and lowers the activation threshold primarily in CD4⁺ T-lymphocytes, indicating a critical role for Hbα within this subset. While these data suggested the loss of Hbα in T-lymphocytes may promote aberrant activation of autoreactive T-lymphocytes, surprisingly, we discovered that mice lacking Hbα in T-lymphocytes exhibited reduced severity of experimental autoimmune encephalomyelitis (EAE) compared to wild-type control animals. Interestingly, T-lymphocytes lacking Hbα in vivo appeared to function identically to wild-type controls, which did not explain the protection against EAE. In contrast, T-lymphocyte Hbα knock-out mice displayed significantly reduced levels of circulating immunoglobulins and CD40L expression compared to their wild-type counterparts during EAE, suggesting impaired intercellular communication. These data elucidate a previously unrecognized role for Hbα in T-lymphocyte function with implications for hemoglobinopathies.

Keywords: EAE; Hemoglobinopathy; immune; inflammation; redox.

Figure 1:. Activation state of T-lymphocytes determines intracellular Hbα levels and impact on mitochondria.

A-D: Hbα-a1 mRNA (N=7) and Hbα protein expression (N=7) over 24, 48, 72-hour time points after activation of CD4⁺ (A-B) and CD8⁺ (C-D) T-lymphocytes compared to naive T-lymphocytes (N=6). E-H: Splenic CD4⁺ and CD8⁺ T-lymphocytes were isolated and activated for 72 hours, then assessed by flow cytometric analysis of MitoSOX (E-F) and TMRE MFI (G-H). I-L: Splenic CD4⁺ and CD8⁺ T-lymphocytes were isolated and activated for 72 hours, then assessed using Seahorse mitochondrial stress test (N=5). R+A = rotenone + antimycin A. M-T: Splenic T-lymphocytes were activated for 72 hours, then puromycin MFI was measured via SCENITH protocol in CD4⁺ and CD8⁺ T-lymphocytes to obtain calculations graphed (reported in percentage). Statistics measured by a one-way ANOVA with Dunnett’s multiple comparisons test, unpaired Student’s t-test, or two-way ANOVA with Šídák’s multiple comparisons test where appropriate.

Figure 2:. Activated HbKO T-lymphocytes exhibit an accelerated and enhanced proinflammatory phenotype.

A-H: Extracellular cytokine protein measured from CD4⁺ (A-D) and CD8⁺ (E-H) T-lymphocytes at 24, 48, 72 hours post-activation (pg/mL per 10⁶ cells) (N=5). I-P: Extracellular cytokine protein measured from CD4⁺ (I-L) and CD8⁺ (M-P) T-lymphocytes treated for 24 hours with anti-CD28 and varying concentrations of anti-CD3 (N=3). Statistics measured using unpaired Student’s t-test, mixed-effects analysis, or two-way ANOVA with Šídák’s multiple comparisons test where appropriate.

Figure 3:. HbKO EAE animals exhibit better disease phenotypes but similar inflammatory profiles as WT EAE animals.

A: Schematic of 28-day EAE experimental design. B-C: EAE severity scores (0–5) and weights (g) over 28-day incubation period (WT N=14, HbKO N=10). D-G: Protein concentration (pg/mL) of cytokines in plasma. H-K: Percentage of CD4⁺ and CD8⁺ present in the spleen and inguinal lymph nodes at day 28. L-O: Percentage of splenic CD4⁺ polarized T-lymphocytes. P-S: Splenocytes restimulated with 10 μg/mL MOG_35–55 for 72 hours, then assessed for extracellular cytokine protein concentration (pg/mL per 10⁶ cells). Statistics measured by two-way ANOVA with Šídák’s multiple comparisons test or Student’s t-test where appropriate.

Figure 4:. Loss of Hbα impairs T-lymphocyte intercellular communication.

A-H: Splenic T-lymphocytes were isolated and activated with a 1:1 ratio of Dynabeads for 72 hours, replated into transwell inserts over control, RANTES, or CXCL12 media for 4 hours, then total cell counts (A-D) and ratio of cells migrated over control well migration (E-H) were assessed by flow cytometry. I-J: Relative IgM and lambda immunoglobulin concentrations in plasma from 14-day EAE animals (AU: arbitrary units). K-L: Splenic T-lymphocytes were isolated and assessed at 4-hour activated (L) and 24-hour activated (K) for receptor expression by flow cytometry. Statistics measured using Student’s t-test. Statistics (only significant shown) were measured by two-way ANOVA with Šídák’s multiple comparisons test or student’s t-test where appropriate.