Impaired IFN-α-mediated signal in dendritic cells differentiates active from latent tuberculosis

<div><p>Individuals exposed to <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) may be infected and remain for the entire life in this condition defined as latent tuberculosis infection (LTBI) or develop active tuberculosis (TB). Among the multiple factors governing the outcome of the infection, dendritic cells (DCs) play a major role in dictating antibacterial immunity. However, current knowledge on the role of the diverse components of human DCs in shaping specific T-cell response during <i>Mtb</i> infection is limited. In this study, we performed a comparative evaluation of peripheral blood circulating DC subsets as well as of monocyte-derived Interferon-α DCs (IFN-DCs) from patients with active TB, subjects with LTBI and healthy donors (HD). The proportion of circulating myeloid BDCA3⁺ DCs (mDC2) and plasmacytoid CD123⁺ DCs (pDCs) declined significantly in active TB patients compared to HD, whereas the same subsets displayed a remarkable activation in LTBI subjects. Simultaneously, the differentiation of IFN-DCs from active TB patients resulted profoundly impaired compared to those from LTBI and HD individuals. Importantly, the altered developmental trait of IFN-DCs from active TB patients was associated with down-modulation of IFN-linked genes, marked changes in molecular signaling conveying antigen (Ag) presentation and full inability to induce Ag-specific T cell response. Thus, these data reveal an important role of IFN-α in determining the induction of <i>Mtb-</i>specific immunity.</p></div

Downregulation of IFN-target genes in IFN-DCs derived from active TB patients.

<p><i>A</i>, Scatter plot of gene expression levels between active TB and HD groups showing downregulation of selected genes in active TB compared to HD. <i>B</i>, Heat map of selected genes downregulated in active TB compared to HD; median centered Log2 values are shown. Expression values of LTBI are also included. <i>C</i>, Hierarchical clustering of 25 genes belonging to IFN signal differentially expressed among active TB, LTBI and HD, built on mean corrected values. In both <i>B</i> and <i>C</i>, values are expressed on a blue-black-yellow gradient. Yellow, increased expression; blue, decreased expression. <i>D</i>, Mean centered expression levels of genes coding transcriptional factors regulating DC development. Statistical significance was analyzed by Student’s t-test (n = 5/group).</p

Analysis of circulating DC subsets in peripheral blood of active TB, LTBI or HD individuals.

<p><i>A</i>, Percentage of peripheral blood Lin^-/HLA-DR⁺ DCs, CD11c⁺ mDC1, BDCA3⁺ mDC2 and CD123⁺ pDCs were determined by flow cytometry. Median values are shown. <i>B</i>, CD86 and CD80 expression on total DCs and DC subsets are shown. Data are reported as mean fluorescence intensity (MFI) and median values are indicated. Statistical significance was analyzed by Mann-Whitney test using Bonferroni correction. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001; ****<i>P</i><0.0001. <i>C</i>, Expression of the type-1 and type-2 myeloid DC markers, BDCA1 and BDCA3, and the plasmacytoid marker CD123 in IFN-DCs derived from active TB, LTBI and HD individuals by flow cytometry. Active TB, LTBI and HD indicate IFN-DCs derived from active TB, LTBI and HD individuals, respectively. Percentages of positive cells are shown. SSC, side scatter. Data are representative of three independent experiments (N = 3).</p

IFN-DCs derived from active TB patients are impaired for DC-related response gene signatures.

<p><i>A</i>, GSEA results for comparison between active TB and HD. GeneSets relevant to DC biology are shown. Plot of the running sum for enrichment score (ES) in selected data set, the normalized enrichment score (NES) and location of genes (hits) in the list ranked according to expression differences (barcode) are shown as explained in the legend. <i>B</i>, Dot plot representation of GeneSet expression level in comparisons among active TB, LTBI and HD groups, as expressed by NES and q-value. As detailed in the legend, dot area is proportional to NES (<1 = no enrichment; >2 = maximum expression); dot color intensity is proportional to q-value significance, dot color indicates the group in which the GeneSet is overexpressed.</p

Analysis of differentially expressed genes across active TB, LTBI and HD individuals.

<p>Hereafter, active TB, LTBI and HD indicate TB-DCs, LTBI-DCs and HD-DCs, respectively. <i>A</i>, Venn’s diagram showing the number of transcripts modulated and their overlaps across IFN-DCs derived from active TB, LTBI and HD individuals. Genes selected on the threshold of <i>P</i> <0.005 by Student’s t-test from five biological replicates (n = 5) for each group were included in the analysis. <i>B</i>, PCA of differentially expressed genes across active TB, LTBI and HD; PC1 and PC2 were chosen as the axes explicating most of the data variance. <i>C</i>, Heat map showing differential expression across active TB, LTBI and HD, built on the selected 170 genes differentially expressed between active TB and HD individuals. The expression level average of five samples for each group is shown. Values are average corrected and shown on a blue-black-yellow gradient: yellow, increased expression; blue, decreased expression.</p

IPA analysis identifies specific impaired functional terms in IFN-DCs derived from active TB patients.

<p>Gene lists of 1.5-fold differentially expressed genes in TB-DCs <i>versus</i> HD-DCs were mapped by IPA software, revealing the modulation of several pathways and genes. <i>A</i>, Genes forming “activation dendritic cells” and “apoptosis dendritic cells” annotation found to be affected in TB-DCs. The direction of change is color coded with green and red indicating down-regulation and up-regulation, respectively. <i>B</i>, Regulator Effect Analysis of TB-DCs indicating upstream regulators (at the top) and downstream effects (at the bottom) hypothesized by IPA based on expression level of key genes (in the middle). Color legend as in <i>A</i>. <i>C</i>, Network of genes most significantly altered in TB-DCs. Genes in green showed a 1.5 fold down-regulation compared to HD-DCs.</p

Defective T cell response to <i>Mtb</i> Ag stimulation in active TB patients.

<p><i>A</i>, IFN-DCs generated from active TB, LTBI and HD individuals were pulsed for 24 hr with RD1 or PPD <i>Mtb</i> Ags and co-cultured with autologous T cells at indicated DC:T cell ratio. Data represent the mean cpm ± SEM of three independent experiments (N = 3; n = 3). <i>P</i> values refer to TB-DC and LTBI-DC comparison (Mann-Whitney test). <i>B</i>, Analysis of cytokine produced by T cells after stimulation for 24 or 72 hr with autologous RD1-loaded IFN-DCs. Cell culture supernatants were tested by flow cytometry using a cytokine bead array kit. <i>C</i>, CD1a and LAMP3 transcripts were quantified by qRT-PCR in IFN-DCs from active TB and LTBI subjects upon 24 hr RD1 stimulation and normalized to β-actin. Means ± SEM are shown (Mann-Whitney test). <i>P</i> values: *<0.05, **<0.01, ***<0.001.</p

Demographic characteristics of enrolled subjects.

<p>Demographic characteristics of enrolled subjects.</p

Background analysis of CE and controls sera.

Analysis of the background using a home-made ELISA by testing sera from the validation cohort comprising 29 patients with cystic echinococcosis and 14 controls with non-parasitic focal liver lesions. The optical density (OD) read for the wells where no peptides were adsorbed (no Ag) (“peptide-/serum+/secondary antibody+”) was considered as the background and compared with the OD read where peptides were adsorbed (Ag) (“peptide+/serum+/secondary antibody+”). Horizontal lines represent medians. P value was considered significant if (DOCX)</p

None of the peptides associate to specific CE cyst stage.

Validation of microarray results using a home-made ELISA by testing sera from the validation cohort comprising 29 patients with cystic echinococcosis and 14 controls with non-parasitic focal liver lesions. Peptides resulting from the manual examination of the microarray results promising for the diagnosis of CE infection (EGR_08002; EGR_03286; EGR_03099; EGR_10786) or for discriminating between active and inactive CE cysts are analyzed comparing CE patients with cysts at different stages. The following groups are compared: active cysts (CE1 and CE2/3b), transitional cysts (CE3a), inactive cysts (CE4/CE5 no therapy), and inactive cysts that received therapy in the last 5 years (t (DOCX)</p