mTOR Modulates Lymphocyte Differentiation through T-bet and Eomesodermin in Response to Invasive Pulmonary Aspergillosis in Rats

Abstract

Background: Aspergillosis infection is common in the patients with insufficient immunity. The role of mammalian target of rapamycin (mTOR), T-box expressed in T-cells (T-bet), and eomesodermin (EOMES) in mediating T lymphocytes differentiation in response to Aspergillus fumigatus infection in immunocompromised rats was investigated in this study.

Methods: Invasive pulmonary aspergillosis (IPA) of immunosuppressive twenty male rats were established and sacrificed at 24 h (n = 5), 48 h (n = 5), 72 h (n = 5), and 96 h (n = 5) after A. fumigatus infection. In addition, control (n = 5), cyclophosphamide (CTX) (n = 5), and aspergillosis (n = 5) group were also established the tissues and pathology of lung tissue was examined by hematoxylin and eosin staining. CD8+ T-cells was sorted by flow cytometry. Serum mTOR, S6K, T-bet, and EOMES were quantified by enzyme-linked immunosorbent assay.

Results: Histology of lung tissue indicated severe lung tissue injury including infiltration of inflammatory cells, alveolar wall damage or degradation, blood congestion, and hemorrhage in the CTX, IPA, and CTX + IPA rats. Hyphae were seen in the IPA, and CTX + IPA groups. The proportion of CD8+ T-cells was significantly increased in the animals of CTX + IPA. Memory CD8+ T-cells was significantly increased in early stage (24 h and 48 h, P < 0.001), but decreased in the late phase of fungal infection (72 h and 96 h) in the animals of CTX + IPA. In addition, at early stage of fungal infection (24 h and 48 h), serum mTOR (P < 0.001), S6K (P < 0.001), and T-bet (P < 0.05) was significantly higher, while EOMES was significantly lower (P < 0.001), in CTX + IPA group than that in control, CTX alone or IPA alone group. Conversely, serum mTOR, S6K, T-bet, and EOMES showed opposite changed in the late stage (72 h and 96 h). Pearson's correlation analysis indicated that mTOR and S6K were significantly correlated with T-bet (r = 0.901 and 0.91, respectively, P < 0.001), but negatively and significantly correlated with EOMES (r = -0.758 and -0.751, respectively, P < 0.001).

Conclusions: mTOR may regulate transcription factors of EOMES and T-bet, and by which mechanism, it may modulate lymphocytes differentiation in animals with immune suppression and fungal infection.

Figure 1

White blood cell, neutrophil, and lymphocytes in the blood of animal models (n = 5 each group). (a) Total white blood cell count. (b) Neutrophil count. (c) Lymphocytes count. Vertical axes: cell number (×10⁹/L). Horizontal axes: Groups *P < 0.05.

Figure 2

Histology of lung tissue stained with H and E. (a) Control animal. (b) Animals treated with CTX. (c) Animals with IPA. (d) Animals with CTX + IPA for 24 h. (e and h) Animals with CTX + IPA for 48 h. (f) Animals with CTX + IPA for 72 h. (g) Animals with CTX + IPA for 96 h. (h) The mycelium of pulmonary aspergillosis. (a-g: original magnification ×20); (h: original magnification ×40). CTX: Cyclophosphamide; IPA: Invasive pulmonary aspergillosis.

Figure 3

Comparison of CD8⁺ T-cells (a) and memory CD8⁺ T-cells (b). CD8⁺ T-cells and memory CD8⁺ T-cells were sorted by flow cytometry as described in the methods. Vertical axes: percent of CD8⁺ T-cells (%) or memory CD8⁺ T-cells (%). Horizontal axes: Groups *P < 0.05.

Figure 4

Serum levels of mTOR (a), S6K (b), EOMES (c), and T-bet (d). Serum was harvested, and protein levels of mTOR, S6K, EOMES, and T-bet were quantified by enzyme-linked immunosorbent assay as described in the methods. Vertical axes: Amount of mTOR (ng/ml) or S6K (U/L) or EOMES (ng/ml) or T-bet (pg/ml). Horizontal axes: Groups *P < 0.05. EOMES: Eomesodermin.

Figure 5

Correlation between mTOR, S6K, EOMES, and T-bet. (a) mTOR and EOMES. (b) mTOR and T-bet. (c) S6K and EOMES. (d) S6K and T-bet. EOMES: Eomesodermin.