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Abstract
Objective
We have previously shown that DICAM inhibits LPS-mediated macrophage differentiation. However, less is known about the exact action mechanisms of DICAM on the macrophage function and differentiation.
Methods
To induce differentiation into a resting M0 macrophage, THP-1 cells were cultured with 100 nM PMA for 24 h, and then rested for 3 days. THP-1 cells were infected with 50 moi of control LacZ- or DICAM-containing adenovirus. The RNA expression profile associated with DICAM during THP-1 differentiation was analyzed with a microarray chip and in silico analysis with Ingenuity Pathway Analysis (IPA) program.
Results
A disease and function analysis of the microarray data in DICAM-overexpressed THP-1 cells revealed a suppression in the expression of multiple genes involved in the response of myeloid cells and phagocytes, and an increase of genes associated with apoptosis of fibroblast cell-line, and viral infection and replication. The canonical pathway analysis also showed the most prominent changes of signaling pathways that involve inflammation responses. An upstream regulator analysis identifyingmolecules upstream of the genes that potentially explain the observed expression changes revealed that IRF7 and the genes in type 1 interferon system, such as IFNA2 and IFNAR, was significantly attenuated by DICAM. A mechanistic network analysis confirmed a direct causal association between IRF7 and type 1 interferon system. A real-time RT-PCR analysis validating the microarray data verified the significant suppression of IRFs, IFNA2, and IFNB1.
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Figure 1.
Top hits for canonical pathways of differentially expressed proteins by DICAM and their overlap connections. (A) The most highly represented canonical pathways of genes differentially expressed during the macrophage differentiation of THP-1 cells. The columns represent the -log of the p-value calculated based on Fisher's exact test. The dot points represents the ratio of the number of genes in a given pathway that meet cut off criteria and total number of genes that make up that pathway. (B) The network of canonical pathways highlights the relationships between pathways and classify the functional domains.
Figure 2.
IRF7-associated mechanistic networks of upstream regulators. IRF7, mostly suppressed by DICAM overexpression, is postu-lated to affect the expression of type I interferon and their downstream signaling molecules such as IRFs, STAT1/2/3 and RelA (p65). The set of total 15 regulators in mechanistic network has a connection with 187 dataset genes (not shown) among which 40 genes directly connect to IRF7. The blue color indicates a more confident predicted inhibition and the pale blue indicates a less confident predicted inhibition.
Figure 3.
The predicted DICAM-mediated inhibition of IRF canonical pathway. The canonical pathway of IRF activation by cytosolic pattern recognition receptor is analyzed in silico using Molecule Activity Predictor analysis of IPA.
Figure 4.
DICAM-mediated inhibition of IRFs and type I interferon system. The microarray data is validated with real-time RT-PCR analysis. In the presence of PMA 100 nM, THP-1 cells infected with adenovirus encoding DICAM or LacZ for 24 h, and then left unstimulated for 3 days. Real-time RT-PCR analysis of (A) IRFs mRNA and (B) type I interferon (IFNA1, IFNA2 and IFNB1) and type 2 interferon (IFNG).
Table 1.
The 8 mostly affected category of disease and biologic functions by DICAM overexpression in THP-1 cells that was analyzed with Downstream Effects Analysis using IPA
| Category | Disease or functions annotation | p-value* | Predicted activation state | Activation z-score† | #Molecules‡ |
|---|---|---|---|---|---|
| Cell-To-Cell Signaling and Interaction | Response of myeloid cells | 2.84E-05 | Decreased | −3.049 | 25 |
| Inflammatory Response | Response of phagocytes | 6.18E-06 | Decreased | −2.990 | 28 |
| Cell-To-Cell Signaling and Interaction | Response of phagocytes | 6.18E-06 | Decreased | −2.990 | 28 |
| Lipid Metabolism | Biosynthesis of polyunsaturated fatty acids | 1.30E-08 | Decreased | −2.951 | 35 |
| Cell Death and Survival | Apoptosis of fibroblast cell lines | 7.84E-06 | Increased | 3.126 | 40 |
| Infectious Disease | Viral Infection | 1.12E-09 | Increased | 3.095 | 168 |
| Infectious Disease | Replication of RNA virus | 1.33E-07 | Increased | 3.056 | 62 |
| Infectious Disease | Replication of virus | 3.98E-08 | Increased | 2.769 | 68 |
* The p-value was calculated with the Fischer's exact test, and reflects the likelyhood that the association between a set of genes in dataset and a related biological function is significant.
Table 2.
The 12 mostly activated and inhibited upstream regulators by DICAM overexpression in THP-1 cells predicted by the Upstream Regulator Analysis in IPA
| Upstream regulator | Fold change | Molecule type | Predicted activation state | Activation z-score* | p-value of overlap† |
|---|---|---|---|---|---|
| IRF7 | −38.745 | Tanscription regulator | Inhibited | −5.247 | 2.70E-18 |
| IFNA2 | Cytokine | Inhibited | −4.914 | 1.18E-15 | |
| Ifnar | Group | Inhibited | −4.636 | 1.58E-18 | |
| Tretinoin | Chemical – endogenous mammalian | Inhibited | −4.599 | 2.01E-19 | |
| IFNL1 | Cytokine | Inhibited | −4.463 | 4.80E-16 | |
| Lipopolysaccharide | Chemical drug | Inhibited | −4.345 | 4.02E-45 | |
| MYC | 2.052 | Transcription regulator | Activated | 5.330 | 4.19E-14 |
| TRIM24 | Transcription regulator | Activated | 4.208 | 7.73E-15 | |
| E2F1 | Transcription regulator | Activated | 4.060 | 6.09E-13 | |
| INSIG1 | −6.816 | Other | Activated | 3.957 | 3.90E-13 |
| TBX2 | Transcription regulator | Activated | 3.900 | 4.44E-10 | |
| IL1RN | −5.747 | Cytokine | Activated | 3.816 | 1.99E-11 |
IRF7: interferon regulatory factor7, IFNA2: interferon alpha-2, Ifnar: interferon-α/β receptor, IFNL1: interferon lambda1, MYC: v-myc avian myelocytomatosis viral oncogene homolog, TRIM24: Tripartite Motif Containing 24, E2F1: E2F transcription factor 1, INSIG1: insulin induced gene 1, TBX2: T-box transcription factor2, IL1RN: interleukin-1 receptor antagonist.
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