Intrapulmonary delivery of aerosolized small interfering RNA (siRNA) is confined to the lungs. Mice (C57BL/6 strain) were infected by low-dose aerosol challenge with M. tuberculosis. At 60 days of the infection, mice were treated via the intrapulmonary route with 15 μg of fluorescent-tagged siRNA (Alexa 488–XCL1-targeting siRNA). (Upper panel) Representative dot plots from lung samples. Numbers within the plots indicate the percentage of positive cells. Bar graph corresponds to the fold change in mean fluorescence channel (MFC), determined as the ratio of MFC in FL1 for samples obtained from mice treated with XCL1-targeting siRNA–Alexa 488 (closed bars), with the MFC in the same channel of cell suspensions obtained from the same organs of untreated mice (open bars). (Lower panel) Representative dot plots from lung samples. Numbers within the plots indicate the percentage of positive cells. The graph shows fold change in MFC determined as the ratio of MFC in FL1 for samples obtained from mice treated with XCL1-targeting siRNA–Alexa 488 (closed bars), with the MFC in the same channel of cell suspensions obtained from the same organs of untreated mice (open bars) after gating forward scatter/side scatter plot in the total lymphocyte gate followed by secondary gating in the CD4 or CD8 T–positive fluorescence.

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.

Lower levels of expression of pulmonary XCL1 results in reduced IFN-γ production. (A) Mice treated with XCL1-targeting siRNA (open bars), both at 3 or 5 days after treatment, had a one-third reduction in the total amount of IFN-γ protein when compared with similar samples obtained from control mice (closed and gray bars). (B) Fold change of inducible nitric oxide synthase (iNOS) mRNA compared with the housekeeping gene, GADPH, between lung samples obtained from chronically infected mice after treatment with PBS (closed bars), nontargeting siRNA (gray bars), or XCL1-targeting siRNA (open bars). The relative levels of expression of iNOS in the lungs at 3 days were lower, but recovered at 5 days after treatment. (C) Bacterial loads obtained from lung homogenates of chronically infected mice after treatment with PBS (closed bars), nontargeting siRNA (gray bars), or XCL1-targeting siRNA (open bars). Graphs are representative of three independent experiments, and represent the average (±SEM) of IFN-γ production, iNOS expression, and bacterial load from PBS, nontargeting siRNA, or XCL1-targeting siRNA.

XCL1 gene silencing and protein suppression. (A) siRNA targeting XCL1 induced transient gene silencing and suppression of protein expression. After one single dose (15 μg/mouse) of XCL1-targeting siRNA (open bars), nontargeting siRNA (gray bars), or PBS (closed bars), the levels of xcl1 transcripts in each mouse at 3 or 5 days after treatment were determined using quantitative RT-PCR. Mice at 3 days after treatment with XCL1-targeting siRNA had a 50% decreases in xcl1 transcripts, but, 5 days after treatment, expression levels of xcl1 transcripts were similar to those of control mice. (B) Levels of XCL1 protein. Mice receiving XCL1-targeting siRNA (open bars) at 3 and 5 days after treatment had a 40–50% reduction in the levels of XCL1 protein, respectively, when compared with similar samples obtained from control groups (closed and gray bars). Data presented represent the average (±SEM) of XCL1 mRNA (%) or XCL1 production from PBS, nontargeting siRNA, or XCL1-targeting siRNA.

Lower levels of expression of pulmonary XCL1 are associated with decreased numbers of CD4 and CD8 T cells. (A) Numbers of total CD4 and IFN-γ expressing CD4 T cells were reduced in mice receiving XCL1-targeting siRNA (open bars) either at 3 or 5 days after treatment when compared with control mice (closed and gray bars). (B) The same mice showed a reduction of 18–50% in IFN-γ CD4 T cells, total number of CD8 T cells, and CD8 expressing IFN-γ T cells. Mice receiving XCL1-targeting siRNA (open bars) had a 20–40% and a 28–42% reduction in CD8 T cells and CD8 T cells expressing IFN-γ, respectively, when compared with control mice (closed and gray bars). Data are representative of three independent experiments and represent the average (±SEM) of CD4 and CD8, and CD4 and CD8 expressing IFN-γ from PBS (closed bars), nontargeting siRNA (gray bars), or XCL1-targeting siRNA (open bars).

Lower levels of expression of pulmonary XCL1 had a long-term effect in the lung immunopathology. (A) Percentage of xcl1 transcripts at 180 d after therapy in PBS (closed bars), non targeting siRNA (gray bars) or XCL1 targeting siRNA (open bars) treated mice. Mice that received XCL1-targeting siRNA had increased by 45% the levels of xcl1 gene transcripts by 180 d after treatment. (B) Representative images of lymphocytic foci from each group. Bar indicates the size (μm) of lymphocytes foci in each sample. Images were taken using an Olympus BX41 microscope and DP70 camera. All images were obtained using a 10× objective. (C) For each group (n = 5), 25 randomly selected images (upper panels) of lymphocyte foci in H&E stained lung sections were measured in each group. Data represent the average (±SEM) of lymphocyte foci diameter for mice treated with PBS, nontargeting siRNA, or XCL1-targeting siRNA. The lower panels in (C) correspond to fibrosis determined by Masson's trichrome staining. (D) 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 in 25 images from each group, indicating collagen, was selected based on its color range, and the proportional area in each image was quantified using the histogram tool and expressed as a percentage of the image. Fibrosis in mice treated with XCL1-targeting siRNA was slightly higher than in control mice. Data are expressed as the mean (±SEM) values (n = 25). The data have been analyzed using Student's two-tailed t test for paired samples. P values less than 0.05 were considered significant.