Assessment of the pulmonary adaptive immune response to Cladosporium cladosporioides infection using an experimental mouse model

Abstract

Cladosporium cladosporioides causes asthma and superficial and deep infections, mostly in immunodeficient individuals and animals. This study aimed to investigate whether C. cladosporioides spores can enter the lungs through pulmonary circulation and influence pulmonary immune response. We intravenously injected mice with C. cladosporioides spore suspension and conducted several assays on the lungs. Pulmonary hemorrhage symptoms and congestion were most severe on days 1, 2, and 3 post-inoculation (PI). Extensive inflammatory cell infiltration occurred throughout the period of infection. More spores and hyphae colonizing the lungs were detected on days 1, 2, and 3 PI, and fewer spores and hyphae were observed within 21 d of infection. Numerous macrophages, dendritic cells, and neutrophils were observed on day 5 PI, along with upregulation of CD54, an intercellular adhesion molecule. Th1 and Th2 cells increased after infection; specifically, Th2 cells increased considerably on day 5 PI. These results suggest that days 2 and 5 PI represent the inflammatory peak in the lungs and that the Th2 and Th1 signaling pathways are potentially involved in pulmonary immune responses. In conclusion, the further adaptive immune responses played important roles in establishing effective pulmonary immunity against C. cladosporioides systemic infections based on innate immune responses.

Figure 1

Histological assessment of lung lesions in mice intravenously infected with Cladosporium cladosporioides. Severe pulmonary hemorrhage and congestion were observed on days 1, 2, and 3 post-inoculation (PI), and extensive infiltration of inflammatory cells was observed throughout the infection period, especially around veins. Data are representative of three independent experiments (n = 3 mice per group). Scale bars, left-200 μm, right-50 μm.

Figure 2

Assessment of the colonization of fungal spores and mycelia in pulmonary structures in mice intravenously infected with Cladosporium cladosporioides. A larger number of spores and mycelia colonized the lung tissue on days 1, 2, and 3 post-inoculation (PI) compared to days 5, 9, 14, and 21 PI. The spores and mycelia are indicated with arrows. Data are representative of three independent experiments (n = 3 mice per group). Scale bars, left-200 μm, right-50 μm.

Figure 3

Assessment of the fungal clearance in lungs infected with Cladosporium cladosporioides. The fungal burden in lung homogenates were determined on days 2, 5, 9, and 14 post-inoculation. Data are representative of three independent experiments (n = 8 mice per group). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4

The recruitment of immune cells in the lungs in mice intravenously infected with Cladosporium cladosporioides. Numerous polymorphonuclear neutrophils (neutrophil⁺), macrophages, and dendritic cells (CD11C⁺) were recruited in the lung tissue in mice intravenously infected with Cladosporium cladosporioides, along with marked CD54 expression (CD54⁺) (red fluorescence increased). Data are representative of three independent experiments (n = 4 mice per group). Scale bars, 200 μm.

Figure 5

Expression levels of pattern recognition receptor (PRR) and cytokine mRNAs in the lung tissue in the mice intravenously infected with Cladosporium cladosporioides on days 0, 2, 5, 9 and 14 post-inoculation (PI). The mRNA expression levels of PRRs and cytokines in the lung tissue of mice in the test groups (days 2, 5, 9, and 14 PI) were measured relative to those of the control group (day 0 PI). Cytokines: PRRs (Dectin-1, TLR-2, TLR-4); Th1 (IFN-γ); Th2 (IL-4, IL-13); Th17 (IL-17A, IL-17F, IL-22); anti-inflammatory cytokines (IL-10, TGF-β); pro-inflammatory cytokines (IL-12, IL-23, IL-1β, TNF-α). TLR-2, TLR-4, IL-1β, IL-10, IL-23, TGF-β, IL-4, and IL-22 mRNAs were significantly upregulated on day 5 PI. IFN-γ, IL-13, and IL-17A were significantly upregulated on day 2 PI. TLR-2, TLR-4, IL-12, TNF-α, and IL-17F were significantly upregulated on day 14 PI. Data are representative of three independent experiments and are expressed as means ± SEMs (n = 8 mice per group).*p < 0.05; **p < 0.01; ***p < 0.001.

Figure 6

Cladosporium cladosporioides induced more Th1 and Th2 cells after infection. We detected Th cells (Th1, Th2, and Th17) with the antibodies IFN-γ, IL-4, and IL-17A, respectively. More Th1 and Th2 cells differentiated after infection. Data are expressed as mean ± SEM (n = 6 mice per group).*p < 0.05; **p < 0.01; ***p < 0.001.