A Landscape of Dendritic Cells Development, Activation and Migration in Mouse Model of Sickle Cell Anemia

Dendritic cells (DCs) are crucial players of innate immunity in initiating, polarizing, and regulating the adaptive immune response. They become activated upon encountering either pathogen- or damage-associated molecular patterns (PAMPs or DAMPs), which are often released due to tissue necrosis or intravascular hemolysis, common events in individuals with sickle cell disease (SCD), one of the most common hemoglobinophaties that affects millions of people worldwide. Upon activation, DCs migrate to peripheral lymph nodes, where they present antigens to T lymphocyte together with co-stimulatory or inhibitory molecules. In addition, they can polarize T cell response by releasing pro- or anti-inflammatory cytokines. We previously showed that SCD patients have higher circulating total and inflammatory DCs, whereas the ratio of type 1 conventional DCs (cDC1) is lower than control individuals. These changes correlated with number of reticulocytes and with the skewed T response towards Th17. Alterations in the development and activation of DC subpopulations, as well as in their function can impair the immune response or tolerance, which may account for the dysfunction in T lymphocyte responses observed in SCD patients.

In order to investigate the development, activation and migration of DCs during SCD, we characterized these cells by flow cytometry in lymphoid organs of Townes mice, which have human S hemoglobin (HbS). Our data show that Townes mice (SS) have lower number of total DCs in bone marrow (p=0.057; N=8 each), however, they have higher number of these cells in the spleen (p<0.0001; N=8 each) and lymph nodes (p<0.0021; N=8 each) when compared with the littermate WT mice, suggesting migration of DCs to these peripheral organs. Evaluation of the ratios of DCs subsets shows that the frequency of cDC1 (CD11c ⁺CD8a ⁺CD11b ^-) among total DCs is higher in the three organs of SS mice compared with WT mice (p=0.004 in spleen; p=0.0015 in bone marrow; and p=0.01 in lymph nodes; N=8 each group). In contrast, the percentage of type 2 conventional DCs, cDC2 (CD11c ⁺CD8a ^-CD11b ⁺) is similar or lower in SS mice than WT mice (p=0.0005 in spleen; N=8 each). In addition, the frequency of plasmacytoid dendritic cell, pDC (CD11c ⁺B220 ⁺SiglecH ⁺) is higher in lymph nodes (p=0.0006; N=8 each) and lower in bone marrow (p=0.0017; N=8 each) of SS mice when compared with WT mice, indicating that the migration of DCs to peripheral organs is due to cDC1 and pDC.

Regarding their maturation and activation phenotype, cDC1 and cDC2 from SS mice have a more immature or inhibitory phenotype than WT mice, as showed by MHC-II (cDC1: p=0.01 in spleen; p=0.0004 in lymph nodes; N=8 each) and CD86 expression (cDC1: p=0.013 in spleen; p=0.0003 in bone marrow; and p= 0.03 in lymph nodes; cDC2: p=0.0067 in bone marrow and p=0.038 in lymph nodes; N=8 each). On the other hand, pDCs from the three organs of SS mice showed a more mature profile than WT mice, as demonstrated by an increase in Sca-1 expression (p=0.0024 in spleen; p=0.037 in bone marrow; and p=0.04 in lymph nodes; N=8 each), indicating that cDCs are being inhibited whereas pDCs are activated during SCD even in steady state.

Aimed to understand whether at least part of the alterations on DCs is due to changes in their development, we quantified monocyte and DC progenitor, MDP (Lin ^-(CD3/CD19/CD56/CD14) CD135 ⁺CD11c ^-CD115 ⁺CD117 ⁺) and common DC progenitor, CDP (Lin ^-CD135 ⁺CD11c ^-CD115 ⁺CD117 ^-) in bone barrow. Our data show that the ratio of MDP, the earlier progenitor, is increased (p=0.0009; N=8 each), whereas CDP, the progenitor committed to DCs, is diminished (p=0.014; N=8 each) in SS mice when compared with WT mice, indicating a change in the DC compartment in SS mice since their developmental stage. When we performed an in vitro differentiation of DC from bone marrow cells in the presence of flt3l, we did not observe statistical difference in either development and apoptosis of DCs, or in the percentages of DC subsets between SS and WT mice, indicating that the alteration in DC development in SS mice may be due to environmental conditions instead of CDP or DC intrinsic factor.

Our findings so far improve our understanding of DCs dynamics in SCD, including the development, activation and migration of the different subsets that may have an important impact on adaptive immune response disfunction in SCD, which may account for the chronic inflammatory state and the susceptibility to infections.