Thymopoietic and bone marrow response to murine Pneumocystis pneumonia

Abstract

CD4(+) T cells play a key role in host defense against Pneumocystis infection. To define the role of naïve CD4(+) T cell production through the thymopoietic response in host defense against Pneumocystis infection, Pneumocystis murina infection in the lung was induced in adult male C57BL/6 mice with and without prior thymectomy. Pneumocystis infection caused a significant increase in the number of CCR9(+) multipotent progenitor (MPP) cells in the bone marrow and peripheral circulation, an increase in populations of earliest thymic progenitors (ETPs) and double negative (DN) thymocytes in the thymus, and recruitment of naïve and total CD4(+) T cells into the alveolar space. The level of murine signal joint T cell receptor excision circles (msjTRECs) in spleen CD4(+) cells was increased at 5 weeks post-Pneumocystis infection. In thymectomized mice, the numbers of naïve, central memory, and total CD4(+) T cells in all tissues examined were markedly reduced following Pneumocystis infection. This deficiency of naïve and central memory CD4(+) T cells was associated with delayed pulmonary clearance of Pneumocystis. Extracts of Pneumocystis resulted in an increase in the number of CCR9(+) MPPs in the cultured bone marrow cells. Stimulation of cultured bone marrow cells with ligands to Toll-like receptor 2 ([TLR-2] zymosan) and TLR-9 (ODN M362) each caused a similar increase in CCR9(+) MPP cells via activation of the Jun N-terminal protein kinase (JNK) pathway. These results demonstrate that enhanced production of naïve CD4(+) T lymphocytes through the thymopoietic response and enhanced delivery of lymphopoietic precursors from the bone marrow play an important role in host defense against Pneumocystis infection.

Fig. 1.

Copies of Pneumocystis rRNA in the whole lung. *, P < 0.05 versus week 1 sham-operated mice. ▴, P < 0.05 versus sham operated mice at the same time point (n = 5 samples/week). W, week.

Fig. 2.

CD4⁺ cell types recovered from BAL fluid. * and ‡, P < 0.05 versus the corresponding control group (uninfected mice); ▴, P < 0.05 versus sham-operated mice at the same time point (n = 5 samples/group).

Fig. 3.

CD4⁺ cell types isolated from lung-associated lymph nodes (LN) (A) and spleen (B). * and ‡, P < 0.05 verus the corresponding control group (uninfected mice); ▴, P < 0.05 versus Sham-operated mice at the same time point (n = 5 samples/group).

Fig. 4.

The number of earliest thymic progenitors (ETPs) (A) and double negative (DN) cells (B) in the thymus.*, ‡, †, and ^, P < 0.05 versus the corresponding cell type in the control group (uninfected mice) (n = 5). (C) The level of msjTREC DNA in CD4⁺ splenocytes. *, P < 0.05 verus the corresponding control group (uninfected mice); ▴, P < 0.05 versus Sham-operated mice at the same time point (n = 5 samples/group). D1-D4, DN1 to DN4 cells (Table 1).

Fig. 5.

Hematopoietic precursor cell types in the bone marrow (A) and circulation (B). * and ‡, P < 0.05 verus the corresponding control group (uninfected mice); ▴, P < 0.05 versus sham operated mice at the same time point (n = 5 samples/group). BMC, bone marrow cell; WBC, white blood cell.

Fig. 6.

Changes in bone marrow cell types following culture with different stimuli. *, P < 0.05 versus the corresponding control group (n = 5 samples/group). PC, Pneumocystis; ZYM, zymosan.

Fig. 7.

Changes in the number of CCR9⁺ MPPs and LKS cells in bone marrow cells following culture with different doses of TLR-2 and TLR-9 ligands. *, P < 0.05 versus the corresponding control group (n = 5 samples/group).

Fig. 8.

Phospho-JNK level in nucleated bone marrow cells following culture with TLR ligands (A). The image is a representative of four sets of cultures. *, P < 0.05 versus the control group. The effects of JNK inhibitor SP600125 (20 μM) on alteration of bone marrow cell types following culture with different stimuli (B). *, P < 0.05 versus the control group; ‡, P < 0.05 versus cells cultured without SP600125 (n = 5 samples/group).