Lesion-Specific Immune Response in Granulomas of Patients with Pulmonary Tuberculosis: A Pilot Study

The formation and maintenance of granulomas is central to the host response to Mycobacterium tuberculosis (Mtb) infection. It is widely accepted that the lungs of patients with tuberculosis (TB) usually contain multiple infection foci, and that the granulomas evolve and differentiate independently, resulting in considerable heterogeneity. Although gene expression profiles of human blood cells have been proposed as biomarkers of Mtb infection and/or active disease, the immune profiles of discrete lesion types has not been studied extensively. Using histology, immunopathology and genome-wide transcriptome analysis, we explored the immunological profile of human lung TB granulomas. We show that although the different granulomas share core similarities in their immunological/inflammatory characteristics, they also exhibit significant divergence. Despite similar numbers of CD68+ macrophages in the different lesions, the extent of immune reactivity, as determined by the density of CD3+ T cells in the macrophage rich areas, and the extent of fibrosis, shows considerable variation. Both quantitative and qualitative differences among significantly differentially expressed genes (SDEG) were noted in each of the lesion types studied. Further, network/pathway analysis of SDEG revealed differential regulation of inflammatory response, immune cell trafficking, and cell mediated immune response in the different lesions. Our data highlight the formidable challenges facing ongoing efforts to identify peripheral blood biomarkers due to the diversity of lesion types and complexity of local immune responses in the lung

Gene ontology (GO) analysis of SDEG in TB lung granulomas.

<p>Gene ontology (GO) analysis of SDEG in TB lung granulomas.</p

Expression of blood biomarkers of active TB in different TB granulomas.

<p>(A). Venn diagram showing comparison of blood TB biomarker profile (green circle) to SDEG from cavitary lung lesion with high AFB (AFB-h; grey circle). (B). Intensity plot of blood TB biomarker genes expressed in cavitary granulomas with scanty (AFB-s) or high (AFB-h) bacillary load, fibrotic nodule (FN) or common to all lesions versus pooled lung transcriptome (Combo). The up-regulated SDEG are in red and down-regulated SDEG are in blue and the intensity of the color is proportional to their expression level (i.e., stronger expression is represented as dark shades). Expression pattern in Combo is sorted in descending order (top to bottom). Scale bar ranges from +3 (red) to -3(blue). (C). Venn diagram showing comparison of blood TB biomarker profile (green circle) to SDEG from cavitary granuloma containing scarce bacillary load (AFB-s; orange circle) in the TB lungs. (D). Venn diagram showing comparison of blood TB biomarker profile (green circle) to SDEG from fibrotic nodule (FN; purple circle) in the TB lungs. For A, C and D, numbers in parenthesis indicate total number of SDEG.</p

Histopathology of granulomas in the lungs of TB patients.

<p>H&E and AFB stained lung sections from TB patients showing various types of granulomatous lesions. (A-D). H&E stained section of a closed fibrotic caseous nodule showing a necrotic center with a thickened fibrous wall (arrows in A), composed of fibroblasts (arrows in B) and scattered leukocytes. (C). The fibrotic area was surrounded by a rim of loosely accumulated macrophages, interspersed with T cells and few MNG. (D). No AFB was detected in these lesions. (E-H). H&E stained sections of an open cavitary lesion showing necrotic cellular debris (arrows in E) surrounded by a layer of activated epithelioid macrophages, MNG cells (thin arrows in F), and numerous scattered lymphocytes (F and G). (H). Abundant AFB were detected at the luminal surface of the cavity. (I-L). H&E stained section of a cavitary lesion showing necrotic debris (arrows in I) surrounded by layer (arrows in J) composed of macrophages, MNG (arrow in K) and lymphocytes (J and K). (L). Few if any AFB were detected at the luminal surface of the cavity. Magnification: A, E, I 4x; B-D, F-H, J-K, 40x; L, 100x.</p

Overview of host gene expression profile in the cavitary and fibrotic granulomas of human TB lungs.

<p>(A). Venn diagram showing the number of unique or shared SDEG between the cavitary granulomas (green circle) and fibrotic nodules (purple circle). Numbers in parenthesis shows total number of SDEG. (B). Intensity plot showing the expression pattern of shared SDEG between cavitary (Cav) and fibrotic (FN) granulomas. Up-regulated SDEG are in red and down-regulated are in blue. Expression patterns are sorted in descending order (left to right). Numbers in parenthesis shows the up- and down-regulated SDEG in each lesion type. The scale bar ranges from +3 (red) to -3 (blue).</p

Differential regulation of interferon signaling pathway in the cavitary granulomas with different bacterial loads.

<p>Expression pattern and interaction of SDEG involved in canonical IFN signaling pathway in the cavitary granulomas with numerous (A) or scanty (B) AFB. For both (A) and (B), the up-regulated SDEG are in red and down-regulated SDEG are in green and the intensity of the color is proportional to their expression level (i.e., stronger expression is represented as dark shades).</p

Distribution of immune cells in different type of TB lung granulomas^*.

<p>Average number of cells +/− SEM per x100 microscopic field; 3–7 fields per tissue sample;</p><p>* cell counts are independent of bacillary load for cavitary lesion;</p><p>**all CD68⁺ cells;</p><p>*** multinucleated giant cells;</p><p>n/d—not determined; n/f-not found</p><p>Distribution of immune cells in different type of TB lung granulomas^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132249#t002fn002" target="_blank">*</a>}.</p

Network analysis of SDEG in the lung cavitary (Cav) and fibrotic (FN) granulomas.

<p>(A). Intensity plot showing the expression pattern of up-regulated (red) and down-regulated (blue) SDEG in the immune cell movement network. Expression pattern in cavitary lesions is sorted in descending order (top to bottom). (B). Intensity plot showing the expression pattern of SDEG and their gene symbols in the STAT1-mediated T cell activation network. Expression pattern in cavitary lesions is sorted in descending order (top to bottom). (C). Intensity plot showing the expression pattern of SDEG and their gene symbols in the fibrosis and wound healing network. Expression pattern in fibrotic lesions is sorted in descending order (top to bottom). For A, B and C, red color indicates up-regulated and blue color represent down-regulated SDEG and the scale bar ranges from +5 (red) to -5 (blue).</p

Pathway/Network analysis of SDEG in TB lung granulomas with different bacillary load.

<p>* cavitary lesion with high bacillary load;</p><p>**cavitary lesion with scanty bacillary load.</p><p>Pathway/Network analysis of SDEG in TB lung granulomas with different bacillary load.</p