Figure 1.
Induction of the DosR-regulon upon culturing Mtb in hypoxia.
The devR transcripts and DevR protein dependent ‘devR regulon’ transcripts (e.g. Rv1738, hspX and tgs1 transcripts, ref. 41) in Mtb cultures grown under hypoxic (30 days) versus aerobic conditions is shown. Fold induction of dosR regulon genes in Mtb-H vs. Mtb-A bacterial cultures obtained by Real-time RT-qPCR performed in triplicates is expressed as Mean ± standard deviation (SD); for dosR 2.90±0.13, Rv1738 90.25±3.96, hspX 60.81±3.43 and tgs1 are 313.32±67.84. Data were normalized using 16S ribosomal RNA as an invariant transcript and calculated as fold induction using delta-delta Ct method (ΔΔCt) (ref. 21).
Figure 2.
Analysis of the replication of Mtb-A and Mtb-H in Rh-BMDMs.
(A) CFU assay. Growth progression of Mtb-A and reduction of Mtb-H during 72 hrs of Rh-BMDM infection measured by CFU analysis is shown. The figure shows the consistent decrease in CFU numbers of Mtb-H (***, p<0.005) at 4, 24 and 72 hrs from two biological replicate experiments, with each replicate data comprising of a summarized value from different relevant dilutions and multiple plating. For Statistics, unpaired Student’s t-test (parametric test) was performed using SAS 9.2 (SA Institute, Cary, NC). (B) Confocal Microscopy. Visual comparison of Mtb (green), Rh-BMDMs (red) at 0 hr, 24 hr and 72 hr post infection. For quantitation, bacilli were counted within Rh-BMDMs (more than 250 Rh-BMDMs in all cases) under a fixed magnification using a TCS-SP2 confocal microscope (Leica Microsystems). Total number of bacilli counted in Rh-BMDMs infected with Mtb-H, Mean±SD were 750±10 at 0 hr; 136±8 at 24 hr; 34±2 at 72 hr and with Mtb-A were 715±20 at 0 hr; 1394±40 at 24 hr; 650±20 at 72 hr. The average relative numbers and SDs determined by CFU assay and Confocal microscopy are shown from at least 3 independent experiments in duplicate or triplicate for each test group.
Figure 3.
Immune response analysis using microarrays, RT-qPCR, cytokine assay and confocal microscopy.
(A). Microarray. Venn diagram shows the total number of genes perturbed in Mtb-H or Mtb-A infected- relative to uninfected-Rh-BMDMs. Total 226 genes (164 genes induced (UP), 62 genes repressed (DN) upon infection with Mtb-A; 2996 genes (1474 genes induced (UP), 1522 genes repressed (DN) upon infection with Mtb-H). Common genes (from up- or down-regulated gene dataset in both Mtb-H and -A group) are shown with overlap. For a description of the common genes e.g. RIPK4 [42], see Supplement S2. Heat-map clusters; green, lower expression; red, higher expression. The data are shown from independent experiments with Rh-BMDMs isolated from two Rhesus macaques. (B) RT-qPCR. The relative fold change in transcripts (Mtb-H infected Rh-BMDM to Mtb-A infected Rh-BMDM) in microarray (grey bars) and RT-qPCR (white bars) is shown. The relative fold change values (Mtb-H to Mtb-A) microarray and RT-qPCR (within bracket) are shown below; TNF, 5.3 (14.42); IL5, 3.49 (1.38); CASP8AP2, 2.93 (2.71); CXCL10, −5.7 (−8.7). (C) Cytokine Assay and Multilabel confocal microscopy. Measurements of TNF in supernatants, Mtb-H (red) or Mtb-A (green). Experiment was performed in triplicate and values were plotted using GraphPad Prism version 6.0b. The data is statistically significant; Student’s t-test, **P = 0.0027. Confocal microscopy shows secretion of TNF (green signal) detected only in the Rh-BMDMs (blue signal) infected with Mtb-H (red signal) (top panels) at 24 hr and 72 hr. The results are shown from Rh-BMDMs derived from two rhesus macaques.
Table 1.
Functional category of upregulated genes in Rh-BMDMs infected with Mtb-H or Mtb-A.
Figure 4.
Signaling pathways involving growth arrest and DNA damage inducible genes.
Various genes (e.g. GADD45B 64.156 fold; GADD45G, 30.299 fold; MAP3K8 20.242 fold etc.), up-regulated in Rh-BMDMs in response to Mtb-H (A) are down-regulated in Rh-BMDM infected with Mtb-A (B). Grey lines represent multiple steps. ATF3-Activating transcription factor 3; CREB-cAMP response element-binding protein; CREM-cAMP response element modulator; KLF4-Kruppel like factor 4 that interact with CREB-binding protein [43]; CFLAR- CASP8AP2 and FADD like receptor. Figures were made using Ingenuity IPA software (Ingenuity Systems, Inc. USA).
Figure 5.
Functional validation of differential apoptosis in Rh-BMDMs infected with Mtb-H or Mtb-A by Tunel assay.
Apoptotic cells show Tunel-positive nuclei (green) and Rh-BMDMs (red). Representative results are shown for Rh-BMDMs infected with Mtb-H (A) and the Mtb-A (B) at the 24-hr post-infection. (C) Graph (from Figure 1A and B) shows percentage of Rh-BMDMs undergoing apoptosis after 24 hours of exposure to Mtb-H or Mtb-A quantified by in situ Tunel assay. ** Student’s t test, p<0.005.
Figure 6.
Rh-BMDMs infected with Mtb-H in the absence or presence of small interfering RNA (siRNA) for TNF gene is shown by A) CFU assay. B) RT-qPCR, Cytokine assay. C) Tunel assay. Percentages of Rh-BMDMs undergoing apoptosis (left panel) after 24 hours of exposure to Mtb-H alone (no Si RNA) or Mtb-H with SiRNA quantified (right panel) by in situ Tunel assay are shown from 3 independent experiments. Statistics; one way analysis of variance (Bonferroni’s multiple comparison test), **, P<0.005, ***, P = 0.008; unpaired student’s t-test *, p<0.02.