Severe persistent mycobacteria antigen stimulation causes lymphopenia through impairing hematopoiesis

Abstract

Miliary tubersculosis (TB), an acute systemic blood disseminated tuberculosis mainly caused by Mycobacterium tuberculosis (M. tuberculosis), can cause signs of lymphopenia in clinical patients. To investigate whether/how persistent mycobacteria antigen stimulation impairs hematopoiesis and the therapeutic effect of interleukin-7 (IL-7), a mouse model of Mycobacterium Bovis Bacillus Calmette-Guérin (BCG) intravenous infection with/without an additional stimulation with M. tuberculosis multi-antigen cocktail containing ESAT6-CFP10 (EC) and Mtb10.4-HspX (MH) was established. Consistent with what happened in miliary TB, high dose of BCG intravenous infection with/without additional antigen stimulation caused lymphopenia in peripheral blood. In which, the levels of cytokines IFN-γ and TNF-α in serum increased, and consequently the expression levels of transcription factors Batf2 and IRF8 involved in myeloid differentiation were up-regulated, while the expression levels of transcription factors GATA2 and NOTCH1 involved in lymphoid commitment were down-regulated, and the proliferating activity of bone marrow (BM) lineage^- c-Kit⁺ (LK) cells decreased. Furthermore, recombinant Adeno-Associated Virus 2-mediated IL-7 (rAAV2-IL-7) treatment could significantly promote the elevation of BM lymphoid progenitors. It suggests that persistent mycobacteria antigen stimulation impaired lymphopoiesis of BM hematopoiesis, which could be restored by complement of IL-7.

Keywords: BCG; IL-7; hematopoiesis; lymphopenia; miliary tuberculosis; mycobacterium tuberculosis.

Figure 1

The change in cytokine profile induced by high-dose BCG bloodstream infection. Mice were infected with BCG via intravenous injection, at 1, 2, 3, and 4 weeks after infection, the variation of serum cytokine profile was detected by LEGEND plex™ Multi-Analyte Flow Assay Kit. (A) Heatmap showing highly secreted cytokines (red) and low secreted cytokines (green). (B) The levels of cytokines (including IFN-γ, TNF-α, GM-CSF, and IL-22) that has changed significantly. P.I., post-infection. n = 3-4, * p < 0.05, ** p < 0.01.

Figure 2

High-dose BCG bloodstream infection reduced the proliferating activity of LK cells. Mice were infected with BCG via intravenous injection or intranasal route. At 3 weeks, 5 weeks, and 8 weeks after infection, BM cells were isolated and the proliferating activity of LK cells was determined by flow cytometry. (A) Gating strategy for detecting EdU incorporation into LK cells in BM. (B) Number of EdU-positive cells in LK cells. ^a p < 0.05, compared with PBS group; ^b p < 0.05, compared with Low BCG i.n. group; n = 5.

Figure 3

The expression of transcription factors involved in the differentiation of LK cells during high-dose BCG bloodstream infection. Mice were infected with BCG via intravenous injection or intranasal route. At 5 weeks after infection, LK cells were enriched with Lineage and c-Kit positive microbeads. Then, the relative quantities of transcription factors Batf2, IRF8, GATA2, and NOTCH1 mRNA in LK cells were determined by qRT-PCR. n = 5, * p < 0.05, ** p < 0.01.

Figure 4

rAAV2-IL-7 treatment increased the IL-7 levels and decreased IFN-γ and TNF-α levels. The C57BL/6 mice from high BCG plus Ag group were treated with rAAV2-IL-7 or rAAV2-EGFP. At 5 weeks after infection, the IL-7, IFN-γ, and TNF-α levels in serum were detected. (A) The levels of IL-7. (B) The levels of IFN-γ. (C) The levels of TNF-α. n = 4-6, ** p < 0.01.

Figure 5

rAAV2-IL-7 treatment restored the reduced lymphoid progenitors caused by high-dose BCG plus M. tuberculosis antigen stimulation. At 5 weeks after infection, BM cells were isolated and the percentage of Lineage^- c-Kit⁺ CD127⁺ cells was determined by flow cytometry. (A) Gating strategy for detecting lymphoid progenitors. (B) The percentage of lymphoid progenitors. n = 5-6, * p < 0.05.