Figure 3.

Reduced expression of pulmonary XCL1 results in changes in the granulomatous structure and increased fibrosis. (A) Images from lung tissue sections (10×) obtained from each mouse either at 3 or 5 days after treatment, stained by hematoxylin and eosin (H&E), and evaluated by a pathologist. The histopathological analysis of these sections demonstrated less lymphocyte infiltration and disruption of the tuberculoid granuloma structure in XCL1-targeting siRNA–treated mice after 3 and 5 days of treatment when compared with control mice (PBS or nontargeting siRNA). (B) Representative images (40×) from lung tissue sections obtained from each group stained by Masson's trichromic staining showing higher fibrosis in mice treated with XCL1-targeting siRNA at either 3 or 5 days after treatment. Immunohistochemistry (IHC) using antibodies to detect (C) transforming growth factor (TGF)-β and (D) IL-4 on tissue sections obtained from lungs of each mouse correlating with areas of fibrosis. The IHC demonstrated that there was no association between fibrosis and TGF-β or IL-4 expression (red staining). Expression of these two cytokines changed between groups; whereas TGF-β and IL-4 expression were clustered in the core of the granuloma in tissue sections from control mice, positive staining for these cytokines in sections from XCL1-targeting siRNA–treated mice were diffused throughout the lung tissue. Many cell types, such as lymphocytes, fibroblasts, macrophages, and dendritic cells, were positive for TGF-β and IL-4. (E) Quantitative image analysis of fibrosis using Masson's trichrome staining. The extent of fibrosis was quantified by Masson's trichrome staining and computer-assisted image analysis, as previously reported (18, 19). Positive blue signal, indicating collagen, was selected based on its color ranges, and the proportional area in each image was quantified using the histogram tool in Adobe Photoshop CS and expressed as a percentage of the image. Fibrosis in mice treated with XCL1-targeting siRNA (open bars) was two- to fourfold higher than in control mice (gray and closed bars). (F) Fold change of TGF-β mRNA expression compared with the housekeeping gene, GADPH, between lung samples obtained from chronically infected mice after 5 days of treatment with PBS (closed bars), nontargeting siRNA (gray bars), or XCL1-targeting siRNA (open bars). The relative levels of expression of TGF-β mRNA expression in the lungs at 5 days after treatment were significantly higher than in control mice.