Edited by: Francesca Granucci, University of Milano-Bicocca, Italy
Reviewed by: Min Wu, University of North Dakota, United States; William Durante, University of Missouri, United States
*Correspondence: Xiaofeng Yang,
This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
The mechanisms that underlie various inflammation paradoxes, metabolically healthy obesity, and increased inflammations after inflammatory cytokine blockades and deficiencies remain poorly determined. We performed an extensive –omics database mining, determined the expressions of 1367 innate immune regulators in 18 microarrays after deficiencies of 15 proinflammatory cytokines/regulators and eight microarray datasets of patients receiving Mab therapies, and made a set of significant findings: 1) proinflammatory cytokines/regulators suppress the expressions of innate immune regulators; 2) upregulations of innate immune regulators in the deficiencies of IFNγ/IFNγR1, IL-17A, STAT3 and miR155 are more than that after deficiencies of TNFα, IL-1β, IL-6, IL-18, STAT1, NF-kB, and miR221; 3) IFNγ, IFNγR and IL-17RA inhibit 10, 59 and 39 proinflammatory cytokine/regulator pathways, respectively; in contrast, TNFα, IL-6 and IL-18 each inhibits only four to five pathways; 4) The IFNγ-promoted and -suppressed innate immune regulators have four shared pathways; the IFNγR1-promoted and -suppressed innate immune regulators have 11 shared pathways; and the miR155-promoted and -suppressed innate immune regulators have 13 shared pathways, suggesting negative-feedback mechanisms in their conserved regulatory pathways for innate immune regulators; 5) Deficiencies of proinflammatory cytokine/regulator-suppressed, promoted programs share signaling pathways and increase the likelihood of developing 11 diseases including cardiovascular disease; 6) There are the shared innate immune regulators and pathways between deficiency of TNFα in mice and anti-TNF therapy in clinical patients; 7) Mechanistically, up-regulated reactive oxygen species regulators such as myeloperoxidase caused by suppression of proinflammatory cytokines/regulators can drive the upregulation of suppressed innate immune regulators. Our findings have provided novel insights on various inflammation paradoxes and proinflammatory cytokines regulation of innate immune regulators; and may re-shape new therapeutic strategies for cardiovascular disease and other inflammatory diseases.
Cardiovascular diseases (CVDs), which include coronary heart disease, hypertension, stroke, and peripheral artery disease, collectively comprise the number one cause of death globally (
Proinflammatory cytokines (PCs) are key regulators of inflammation, participating in acute (
Proinflammatory cytokine-blocking therapies paradoxically lead to increased inflammation.
Proinflammatory cytokines | Related findings | Changes of inflammation related genes | The related pathway | PMID | |
---|---|---|---|---|---|
TNF | Human | The incidences of infections and worsening RA were about 27.7% and 0.5% in Adalimumab treatment. | N/A | N/A | 27856432 |
The risk of serious infections increased 2 folds in patients with RA treated with anti-TNF antibody. | N/A | N/A | 16705109 | ||
Paradoxical inflammation occurred involving the skin, joints and lungs under anti-TNF treatment in patients with inflammatory bowel disease. | IFN-α production; IL12B and IL23R increased. | type I IFN signaling; the differentiation of naïve T cells towards TH1 (via IL-12) or TH17 (via IL-23) cells | 22751454 | ||
The incidences of pneumonia were 2.2% and 1.4% using TNF antibodies Infliximab and Etanercept for RA. | N/A | N/A | 20877307 | ||
Anti-TNFα therapy up-regulated IL6 and IL23p19, in patients with Crohn’s disease; IL-1B and IL17A remained up-regulated in patients refractory to anti-TNF α. | IL-1B and IL17A are up-regulated in nonresponders. | IL17A pathway | 24700437 | ||
During anti-TNF therapy, there are upregulations of IL-23p19, IL23R, and |
IL23R, IL17A, IL17F and TNFR2 are up-regulated in nonrespinders. | IL23R signalling | 29848778 | ||
Mouse | TNF overexpression was cardioprotective | N/A | canonical NF-κB pathway signaling | 26280121 | |
In macrophages, TNF produced less cytokines after challenged with LPS. | suppress IL6, TNF, IL-1β production | LPS-induced signaling | 21602809 | ||
In Tnf KO tumor tissues, tumor-promoting cytokines induced | the expression levels of IL-1b, IL-6, CXCL1 and CXCL2 increased. | COX-2/PGE2, IL-1b, IL-6 and CXCL1/2 pathways | 23975421 | ||
IL1B | Human | For atherosclerotic therapy, incidence rates of deaths attributed to infection or sepsis in Canakinumab groups were higher. | N/A | N/A | 28845751 |
Severe infections were more frequent in Canakinumab group in patients with JIA. | N/A | N/A | 23252526 | ||
IL6 | Human | Incidences of infections were about 28.8% when Sarilumab monotherapy treat patients with RA. | N/A | N/A | 27856432 |
In multiple myeloma patients, anti-IL6 antibodies did not prevent IL6 production. | IL6 | N/A | 8823310 | ||
Treating patients with RA with Tocilizumab increased infections. | N/A | N/A | 21884601 | ||
Mouse | IL-6 provided protection against influenza A infection. | Mcl-1 and Bcl-X L were down-regulated. | IL-6 or IL-6R signals | 22294047 | |
IL17A | Human | In patients with Crohn’s disease for treatment with Secukinumab, 51.3% infections were observed | CRP, and/or faecal calprotectin elevated | N/A | 22595313 |
Incidences of severe infection were 1% in Ixekizumab in the treatment of AS or RAS. | N/A | N/A | 30360964 | ||
IL18 | Human | Inhibition of IL-18 using GSK1070806 did not improve glucose control | N/A | N/A | 26930607 |
Mouse | Decrease in IL-18 in mice that were deficient in NLRP6 inflammasome was involved in enhanced colitogenic microbiota | NLRP6, ASC, caspase-1 | NLRP6 flammasome pathway | 21565393 | |
Il18 or Il18 receptor KO mice led to hyperphagia, obesity and insulin resistance | activation of STAT3 phosphorylation | STAT3 pathway | 16732281 |
TNF, tumor necrosis factor; LPS, Lipopolysaccharide; IL, Interleukin; IFN, interferon; NF-κB, nuclear factor kappa B; CXCL, chemokine (C-X-C motif) ligand; COX-2/PGE2, prostaglandin-endoperoxide 2; Mcl-1, MCL1 apoptosis regulator; CRP, C-reactive protein; NLRP6, NLR family pyrin domain containing 6; ASC, apoptosis-associated speck-like protein; STAT3, signal transducer and activator of transcription 3; RA, Rheumatoid arthritis; JIA, juvenile idiopathic arthritis; AS, ankylosing spondylitis; RAS, radiographic axial spondyloarthritis; N/A, Not applicable.
The increased incidences of infections occurred when antibodies blocking proinflammatory cytokines were used to treat patients with inflammatory diseases. Experimental animal studies showed proinflammatory cytokine knockout or blocking can induce other cytokines production and activate some inflammation related pathways.
In addition, various inflammation paradoxes have been reported including new inflammations occur when:
Similar to single cytokine targeting Mab therapies discussed above, one of the current research strategies is to use gene-deficient mouse models and transgenic mouse models to determine the dominant effects of these inflammatory regulators in disease models such as atherogenesis (
InnateDB (
The 18 murine microarray datasets of proinflammatory cytokine gene deficiencies and eight microarray datasets of patients receiving Mab therapies were collected from National Institutes of Health (NIH)-National Center for Biotechnology Information (NCBI)-Gene Expression Omnibus (GEO) databases (
18 microarray datasets were collected to analyze the changes of innate immunity molecules (innatomic genes, IGs) in deficiencies of proinflammatory regulators (
No. | Factors | GEO NO. | Method | Innatomic genes (total n=1376) | Background | Cell type/tissue | PMID | |||
---|---|---|---|---|---|---|---|---|---|---|
Up-regulatedN% | Down-regulatedN% | |||||||||
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1 | TNF | GSE43145 | Tnfa KOa |
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C57BL/6 | Glandular stomach | 23975421 |
2 | GSE33253 | Tnfr1,2 KOb | 32 | 2.33 | 171 | 12.43 | C57BL/6 | Tumor endothelial | 23056240 | |
3 | IFNG | GSE9892 | Ifng KOc | 81 | 5.89 | 138 | 10.03 | BALB/c | Liver | 19490417 |
4 | GSE39592 | Ifngr1 KO |
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C57BL/6 | CD4+ T | 23575689 | |
5 | IL1B | GSE15750 | Traf6 KO |
10 | 0.73 | 22 | 1.60 | C57BL/6 | CD8 T | 19494812 |
6 | GSE73875 | Irak1 KO |
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C57BL/6 | CD4+ CD26L+ T | 26561545 | |
7 | IL6 | GSE63761 | Il6 KO |
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C57BL/6 | adipose | 25738456 |
8 | IL17 | GSE88800 | Il17ra KO |
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N/A | kidney | 27814401 |
9 | IL18 | GSE64308 | Il18 KO |
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C57BL/6 | brown adipose | 30453990 |
10 | GSE64309 | Il18 KO | 40 | 2.91 | 51 | 3.71 | C57BL/6 | Liver | 27063959 | |
11 | GSE64310 | Il18 KO |
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C57BL/6 | Kidney | 29514661 | |
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12 | STAT | GSE40666 | Stat1 KO |
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C57BL/6 | CD8 T | 22968462 |
13 | GSE6846 | Stat3 KO |
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N/A | pulmonary type II epithelia | 18070348 | |
14 | NFKB | GSE45755 | Rela KO |
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C57BL/6 | lineage-Flk2-c-kit+Sca-1+ | 23670180 |
15 | GSE30049 | Ikk2 KO | 47 | 3.42 | 83 | 6.03 | N/A | tumor-derived cell line | 22327365 | |
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16 | MIR155 | GSE45122 | mir155 KO |
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C57BL/6 | CD4+ IL-17F RFP+ T | 23686497 |
17 | GSE66815 | mir155 KO |
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C57BL/6 | spleen | 25911753 | |
18 | MIR221 | GSE19777 | MIR221 KD | 18 | 1.31 | 39 | 2.83 | N/A* | breast cancer | 21057537 |
Up, up-regulated IGs; Down, down-regulated IGs; KO, Knockout; KD, Knockdown; N/A, Not applicable; The significant differential expressed IGs were the comparison results between the major inflammatory KO and the parallel control. Please note: aTnfa KO Gan mice vs. WT Gan mice; bTnfr1,2 KO B16F1 melanoma tumors vs. WT B16F1 melanoma tumors; cbased on the Tgfb KO model mice of autoimmune hepatitis; #the combined data of Traf6 KO 6 days and 10 days because of the small number of the regulated innate immune genes; *Homo sapiens.
Tnfr, tumor necrosis factor receptor superfamily; Ifngr1, interferon gamma receptor 1; Traf6, TNF receptor associated factor 6. It mediates signaling from members of the TNF receptor superfamily as well as the Toll/IL-1 family; Irak1, interleukin-1 receptor-associated kinase 1; Rela, v-rel reticuloendotheliosis viral oncogene homolog A; Ikk2, inhibitor of kappaB kinase beta.
In gene KO experiments, for the pro-inflammatory cytokines, we focus on the up-regulated innate immune genes, those maybe inhibited by the proinflammatory cytokines during inflammation.
Table showing the number and the ratio of the up-regulated and down-regulated IGs in proinflammatory molecules KO or KD microarrays. From the data, in majority microarrays, the ratio of up-regulated IGs is higher than that of down-regulated IGs (marked in bold).
The innatomic genes (IGs) were analyzed in cytokine-monoclonal antibodies therapy microarrays.
GEO# | Disease | Target | Drug | Tissue | Comparation | IGs | PMID | |
---|---|---|---|---|---|---|---|---|
up | down | |||||||
GSE15602 | rheumatoid arthritis | TNF | Adalimumab | synovium | poor responder vs. good responder |
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|
19389237 |
GSE111761 | Crohn’s disease | TNF | Infliximab or Adalimumab | intestine | none-responder vs. responder |
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29848778 |
GSE92415 | ulcerative colitis | TNF | Golimumab | colonic mucosa | none-responder vs. responder (6 weeks) |
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29981298 |
GSE14580 | ulcerative colitis | TNF | Infliximab | colonic mucosa | none-responder vs. responder |
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19700435 |
GSE24742 | rheumatoid arthritis | TNF | Rituximab | synovium | good-responder 12 weeks vs.0 week | 29 | 42 | 21337318 |
rheumatoid arthritis | TNF | Rituximab | synovium | moderate-responder 12 weeks vs.0 week | 21 | 73 | 21337318 | |
rheumatoid arthritis | TNF | Rituximab | synovium | poor-responder 12 weeks vs.0 week |
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|
21337318 | |
GSE45867 | rheumatoid arthrtis | IL6R | Tocilizumab | synovium | after therapy 12 weeks vs.before | 2 | 23 | 24449571 |
GSE31652 | psoriasis vulgaris | IL17A | LY2439821 | skin | LY2439821 2 weeks vs.0 weeks | 47 | 87 | 22677045 |
The results showed that the IGs were more down-regulated than up-regulated after therapy compared with before therapy or with placebo; the IGs were more up-regulated than down-regulated when poor responder or non-responder compared with responder or good responder (marked in bold).
Six house-keeping genes including ACTB, CHMP2A, RPL27, SRP14, RPL22 and OAZ1 (
We utilized Ingenuity Pathway Analysis (IPA, Qiagen,
The studies using the compound gene-deficient mice established with specific cytokine deficiency crossing to two atherogenic mouse models such as apolipoprotein E deficient (ApoE-/-), and low-density lipoprotein receptor-deficient (LDLR-/-) background have significantly improved our understanding on the roles of these PCs on atherosclerotic progression. Deficiencies of TNFα, IL-1β, IL-18 and interferon-γ (IFNγ) lead to a significant reduction of atherosclerotic lesions. However, the parts of atherosclerotic lesions remain in those proinflammatory cytokine deficient and ApoE-/- double gene KO mice. The atherosclerotic lesions remain 66% for TNF-/-/ApoE-/-, 66% for IL-1β-/-/ApoE-/-, 65% for IL-18-/-/ApoE-/- and 25% for IFNγ-/-/LDLR-/- mice, respectively (
Proinflammatory cytokine-suppressed proinflammatory/proatherogenic mechanisms contribute to atherosclerotic lesions remained in mouse models with cytokine deficiencies.
Since the pathways underlying inflammations and
In microarray datasets of patients receiving Mab therapy (
We hypothesized that PCs are cross-talked and share their regulation on the expression of IGs. To test this hypothesis, we performed a Venn Diagram analysis on the upregulated IGs from proinflammatory cytokine KO datasets. Among 15 IGs lists shared by two proinflammatory cytokine pathways, the five shared cytokine pathways including TNFα/IFNγ, IFNγ/IL-18, IL-6/IL-17, IFNγ/IL-17, and IL-17/IL6 pathways have the higher numbers of shared IGs (
Deficiencies of proinflammatory cytokines shared the up-regulated innatomic genes.
We then examined the signaling pathways in upregulated IGs in the deficiencies of five PCs such as TNFα, IFNγ, IL-6, IL-17 and IL-18. We used IPA to perform this analysis, which is a web-based bioinformatics application that allows the uploading of microarray and RNA-Seq data for functional pathway analysis and integration.
Ingenuity Pathway Analysis (IPA) results showed the significant pathways (∣Z score∣> 2) of up-regulated innatomic genes (IGs) in proinflammatory cytokine KO microarray datasets.
No. | Significant signaling pathways | Tnf-/- (GSE43145) | Tnfr1,2-/- (GSE33253) | Ifng-/- (GSE9892) | Ifngr1-/- (GSE39592) | Il6-/- (GSE63761) | Il17ra-/- (GSE88800) | Il18-/- (GSE64308) |
---|---|---|---|---|---|---|---|---|
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↑ | ↑ | ↑ | ↑ | |||
2 | Cardiac Hypertrophy Signaling (Enhanced) | ↑ | ↑ | ↑ | ↑ | |||
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↑ | ↑ | ↑ | ↑ | |||
4 | Colorectal Cancer Metastasis Signaling | ↑ | ↑ | ↑ | ↑ | |||
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↑ | ↑ | ↑ | ||||
6 | B Cell Receptor Signaling | ↑ | ↑ | ↑ | ||||
7 | Tec Kinase Signaling | ↑ | ↑ | ↑ | ||||
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↑ | ↑ | ↑ | ||||
9 | Integrin Signaling | ↑ | ↑ | ↑ | ||||
10 | FGF Signaling | ↑ | ↑ | |||||
11 | ILK Signaling | ↑ | ↑ | ↑ | ||||
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↑ | ↑ | ↑ | ||||
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↓ | ↓ | |||||
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↑ | ↑ | |||||
15 | Role of NFAT in Regulation of the Immune Response | ↑ | ↑ | |||||
16 | Acute Phase Response Signaling | ↑ | ↑ | |||||
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↑ | ↑ | |||||
18 | RANK Signaling in Osteoclasts | ↑ | ↑ | |||||
19 | Type II Diabetes Mellitus Signaling | ↑ | ↑ | |||||
20 | Adrenomedullin signaling pathway | ↑ | ↑ | |||||
21 | LPS-stimulated MAPK Signaling | ↑ | ↑ | |||||
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↑ | ↑ | |||||
23 | Signaling by Rho Family GTPases | ↑ | ↑ | |||||
24 | Cholecystokinin/Gastrin-mediated Signaling | ↑ | ↑ | |||||
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↑ | ↑ | |||||
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↑ | ↑ | |||||
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↑ | ↑ | |||||
28 | IL-1 Signaling | ↑ | ↑ | |||||
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↑ | ||||||
30 | ERK/MAPK Signaling | ↑ | ||||||
31 | Mouse Embryonic Stem Cell Pluripotency | ↑ | ||||||
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↑ | ||||||
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↑ | ||||||
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↑ | ||||||
35 | Endocannabinoid Cancer Inhibition Pathway | ↑ | ||||||
36 | NGF Signaling | ↑ | ||||||
37 | Cardiac Hypertrophy Signaling | ↑ | ||||||
38 | Synaptogenesis Signaling Pathway | ↑ | ||||||
39 | Fc Epsilon RI Signaling | ↑ | ||||||
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↑ | ||||||
41 | EGF Signaling | ↑ | ||||||
42 | Rac Signaling | ↑ | ||||||
43 | PDGF Signaling | ↑ | ||||||
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↑ | ||||||
45 | Pancreatic Adenocarcinoma Signaling | ↑ | ||||||
46 | Ephrin Receptor Signaling | ↑ | ||||||
47 | Cdc42 Signaling | ↑ | ||||||
48 | p70S6K Signaling | ↑ | ||||||
49 | Phospholipase C Signaling | ↑ | ||||||
50 | Neurotrophin/TRK Signaling | ↑ | ||||||
51 | ErbB Signaling | ↑ | ||||||
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↑ | ||||||
53 | Neuregulin Signaling | ↑ | ||||||
54 | Endocannabinoid Developing Neuron Pathway | ↑ | ||||||
55 | PFKFB4 Signaling Pathway | ↑ | ||||||
56 | GNRH Signaling | ↑ | ||||||
57 | Glioma Signaling | ↑ | ||||||
58 | Melatonin Signaling | ↑ | ||||||
59 | Role of NFAT in Cardiac Hypertrophy | ↑ | ||||||
60 | Acute Myeloid Leukemia Signaling | ↑ | ||||||
61 | Opioid Signaling Pathway | ↑ | ||||||
62 | Sphingosine-1-phosphate Signaling | ↑ | ||||||
63 | G Beta Gamma Signaling | ↑ | ||||||
64 | Gα12/13 Signaling | ↑ | ||||||
65 | Gαq Signaling | ↑ | ||||||
66 | Wnt/β-catenin Signaling | ↑ | ||||||
67 | Thrombin Signaling | ↑ | ||||||
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↑ | ||||||
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↑ | ||||||
70 | LXR/RXR Activation | ↓ | ||||||
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↑ | ||||||
72 | PPAR Signaling | ↑ | ||||||
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↑ | ||||||
74 | Type I Diabetes Mellitus Signaling | ↑ | ||||||
75 | p38 MAPK Signaling | ↑ | ||||||
76 | Antioxidant Action of Vitamin C | ↓ | ||||||
77 | MIF Regulation of Innate Immunity | ↑ | ||||||
78 | Inflammasome pathway | ↑ | ||||||
79 | Osteoarthritis Pathway | ↑ | ||||||
80 | STAT3 Pathway | ↑ | ||||||
81 | VDR/RXR Activation | ↑ | ||||||
82 | Role of IL-17F in Allergic Inflammatory Airway Diseases | ↑ | ||||||
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↑ | ||||||
84 | Angiopoietin Signaling | ↓ | ||||||
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↑ | ||||||
86 | Endothelin-1 Signaling | ↑ |
A total of 11 microarrays about proinflammatory cytokines in
The data showed a total of 28 shared significant signaling pathways of the up-regulated innate immune genes in PCs KO microarrays. 12 cellular immune response signals were activated and PD-1, PD-L1 cancer immunotherapy pathway was suppressed by the up-regulated IGs (marked in bold). And in 58 unique significant signaling pathways, 12 cellular immune response signals (marked in bold) are activated when one proinflammatory cytokine was KO. The detailed IPA results were showed in
IPA results showed the significant pathways (∣Z score∣> 2) of down-regulated IGs in pro-inflammatory cytokine KO microarray datasets.
No. | Significant signaling pathways | Tnfr1,2-/- (GSE33253) | Ifng-/- (GSE9892) | Ifngr1-/- (GSE39592) | Il18-/- (GSE64309) |
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↓ | ↓ | ↓ | ↓ |
2 | Cardiac Hypertrophy Signaling (Enhanced) | ↓ | ↓ | ↓ | |
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↓ | ↓ | ↓ | |
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↓ | ↓ | ↓ | |
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↓ | ↓ | ↓ | |
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↓ | ↓ | ↓ | |
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↓ | ↓ | ↓ | |
8 | iNOS Signaling | ↓ | ↓ | ↓ | |
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↓ | ↓ | ↓ | |
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↓ | ↓ | ↓ | |
11 | Colorectal Cancer Metastasis Signaling | ↓ | ↓ | ↓ | |
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↓ | ↓ | ↓ | |
13 | IL-1 Signaling | ↓ | ↓ | ↓ | |
14 | Adrenomedullin signaling pathway | ↓ | ↓ | ↓ | |
15 | Cholecystokinin/Gastrin-mediated Signaling | ↓ | ↓ | ↓ | |
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↓ | ↓ | ↓ | |
17 | HGF Signaling | ↓ | ↓ | ||
18 | Tec Kinase Signaling | ↓ | ↓ | ||
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↓ | ↓ | ||
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↓ | ↓ | ||
21 | Signaling by Rho Family GTPases | ↓ | ↓ | ||
22 | ERK/MAPK Signaling | ↓ | ↓ | ||
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↓ | ↓ | ||
24 | PI3K/AKT Signaling | ↓ | ↓ | ||
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↓ | ↓ | ||
26 | Endothelin-1 Signaling | ↓ | ↓ | ||
27 | Synaptogenesis Signaling Pathway | ↓ | ↓ | ||
28 | ILK Signaling | ↓ | ↓ | ||
29 | PDGF Signaling | ↓ | ↓ | ||
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↓ | ↓ | ||
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↓ | ↓ | ||
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↓ | ↓ | ||
33 | JAK/Stat Signaling | ↓ | ↓ | ||
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↓ | ↓ | ||
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↓ | ↓ | ||
36 | Cardiac Hypertrophy Signaling | ↓ | ↓ | ||
37 | Acute Phase Response Signaling | ↓ | ↓ | ||
38 | Rac Signaling | ↓ | |||
39 | B Cell Receptor Signaling | ↓ | |||
40 | Ephrin Receptor Signaling | ↓ | |||
41 | Role of IL-17F in Allergic Inflammatory Airway Diseases | ↓ | |||
42 | PPAR Signaling | ↑ | |||
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↓ | |||
44 | TNFR1 Signaling | ↓ | |||
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↓ | |||
46 | Integrin Signaling | ↓ | |||
47 | RhoA Signaling | ↓ | |||
48 | Type II Diabetes Mellitus Signaling | ↓ | |||
49 | Cdc42 Signaling | ↓ | |||
50 | Antioxidant Action of Vitamin C | ↑ | |||
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↓ | |||
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↓ | |||
53 | PAK Signaling | ↓ | |||
54 | Type I Diabetes Mellitus Signaling | ↓ | |||
55 | Renin-Angiotensin Signaling | ↓ | |||
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↓ | |||
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↓ | |||
58 | LPS-stimulated MAPK Signaling | ↓ | |||
59 | Fc Epsilon RI Signaling | ↓ | |||
60 | cAMP-mediated signaling | ↓ | |||
61 | Osteoarthritis Pathway | ↓ | |||
62 | Hepatic Fibrosis Signaling Pathway | ↓ | |||
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↓ | |||
64 | Oncostatin M Signaling | ↓ | |||
65 | Unfolded protein response | ↓ | |||
66 | Retinoic acid Mediated Apoptosis Signaling | ↓ | |||
67 | LXR/RXR Activation | ↑ | |||
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↓ | |||
69 | PPARα/RXRα Activation | ↑ | |||
70 | RhoGDI Signaling | ↑ |
A total of 10 microarrays about proinflammatory cytokines in
We also examined the signaling pathways in downregulated IGs in the deficiencies of four PCs such as TNFαR, IFNγ, IFNγR1, and IL-18. As shown in
It has been well documented that IFNγ secreted from Th1 cells and natural killer cells (NK) play proinflammatory roles (
Venn diagram showing the overlapping significant pathways of up-regulated and down-regulated IGs in Ifng KO microarray dataset (GSE9892). The shared pathways by up-regulated and down-regulated IGs were called “2nd and 1st inflammatory wave pathways”. The data showed in 4 common significant “2nd and 1st inflammatory wave pathways”, Role of NFAT in Regulation of the Immune Response is immune response related pathway. The data suggested that the up-regulated IGs have the same immune function to the down-regulated IGs by Ifng KO. Fcγ Receptor-mediated Phagocytosis in Macrophages and Monocytes, Th2 Pathway, PD-1, PD-L1 cancer immunotherapy pathway, Leukocyte Extravasation Signaling and Systemic Lupus Erythematosus In T Cell Signaling Pathway were unique cellular immune response pathways of “2nd inflammatory wave pathways”. The suppression of Interferon Signaling, Systemic Lupus Erythematosus In B Cell Signaling Pathway, and Role of Pattern Recognition Receptors in Recognition of Bacteria and Viruses, etc. 16 pathways were unique cellular immune response pathways of “1st inflammatory molecules”. The cellular immune response pathways were marked in bold.
Venn diagram showing 11 overlapping significant pathways of up-regulated and down-regulated IGs in Ifngr KO microarray dataset (GSE39592). The data showed that in 11 common significant “2nd and 1st inflammatory wave pathways”, there were six cellular immune response related pathways. The data suggested that the up-regulated IGs have the same immune function to the down-regulated IGs by Ifngr KO. The suppression of PD-1, PD-L1 cancer immunotherapy pathway, and the activation of CD28 Signaling in T Helper Cells, CD27 Signaling in Lymphocytes, Leukocyte Extravasation Signaling, etc. ten pathways were unique cellular immune response pathways of “2nd inflammatory molecules”. Interferon Signaling, iNOS Signaling, Systemic Lupus Erythematosus etc. eight pathways were unique cellular immune response pathways of “1st inflammatory wave pathways”. The cellular immune response pathways were marked in bold.
Venn diagram showing 13 overlapping significant pathways of up-regulated and down-regulated genes in mir155 KO microarray dataset (GSE66815). The data showed in 13 common significant “2nd and 1st inflammatory wave pathways”, there were nine cellular immune response related pathways. The data suggested that the up-regulated IGs have the same immune function to the down-regulated IGs by inflammatory miRNA155 KO. Six pathways were unique pathways of “2nd inflammatory molecules” and three pathways were cellular immune response related pathways. 89 pathways were unique pathways of “1st inflammatory molecules” and 15 pathways were cellular immune response related pathways. That is, the main significant pathways of miR155 are “1st inflammatory wave pathways”. And in “1st inflammatory wave pathways”, Leptin Signaling in Obesity was inhibited, which can explain the pathogenesis of MHO partly. The cellular immune response pathways were marked in bold.
These results have demonstrated that:
We hypothesized that the common signaling pathways shared in PCs-promoted-, and suppressed programs play significant roles in the initiation and development of inflammation. To examine this hypothesis, we compiled all the significantly downregulated and upregulated pathways in the deficiencies of all the proinflammatory molecules examined (
Venn diagram showing 51 overlapping significant pathways of up-regulated and down-regulated innatomic genes in proinflammatory cytokine KO microarray datasets. The significant pathways from down-regulated innate immune genes have the same expression pattern to proinflammatory cytokines. The data showed in 51 common significant pathways, there were about 22 cellular immune response related pathways (account for 43.14%). The data suggested that the up-regulated innate immune genes have the same immune function to the down-regulated innate immune genes by proinflammatory cytokines KO. Additionally, the suppression of PD-1, PD-L1 cancer immunotherapy pathway, Fcγ Receptor-mediated Phagocytosis in Macrophages and Monocytes, Th2 Pathway, Systemic Lupus Erythematosus In T Cell Signaling Pathway, CD27 Signaling in Lymphocytes, PFKFB4 Signaling Pathway and Inflammasome pathway were unique cellular immune response pathways of the up-regulated innate immunity genes after cytokines KO. Activation of IRF by Cytosolic Pattern Recognition Receptors, Th17 Activation Pathway, MIF-mediated Glucocorticoid Regulation, IL-7 Signaling Pathway were unique cellular immune response pathways of the down-regulated innate immunity genes after cytokines KO. The detailed IPA results were showed in
We then hypothesized that the common signaling pathways shared in proinflammatory transcription factors (TFs) KO, including STAT1, STAT3, NF-KB Rela and IKK2-promoted-, and suppressed programs play significant roles on initiation and development of inflammation. To examine this hypothesis, we compiled all the significantly downregulated and upregulated pathways in the deficiencies of all the proinflammatory TFs (
Venn diagram showing eight overlapping significant pathways of up-regulated and down-regulated innatomic genes in pro-inflammatory related transcription factor KO microarray datasets. The significant pathways from down-regulated innate immune genes have the same expression pattern to proinflammatory related transcription factors. The data showed that in 8 common significant pathways, there were 3 cellular immune response related pathways. The data suggested that the up-regulated innate immune genes have the same immune function to the down-regulated innate immune genes by proinflammatory related transcription factors KO. There are 67 significant pathways in up-regulated innate immune genes including TREM1 Signaling, Leukocyte Extravasation Signaling, CD28 Signaling in T Helper Cells etc. cellular immune response pathways. The cellular immune response pathways were marked in bold. The detailed IPA results were showed in
As shown in
The top 5 disease and disorders of the up-regulated and down-regulated IGs in cytokines KO microarrays.
PCs (upN/downN) | Up-regulated IGs | Down-regulated IGs | ||||
---|---|---|---|---|---|---|
Top Diseases and disorders | Top Diseases and disorders | |||||
Name | p-value range | # Molecules | Name | p-value range | # Molecules | |
|
||||||
(52/24) | Endocrine System Disorders | 5.47E-04 - 1.07E-15 | 25 | Infectious Diseases | 3.34E-02 - 2.12E-05 | 4 |
Gastrointestinal Disease | 5.77E-04 - 1.07E-15 | 29 | Organismal Injury and Abnormalities | 3.60E-02 - 2.12E-05 | 22 | |
Immunological Disease | 7.06E-04 - 1.07E-15 | 36 | Respiratory Disease | 3.34E-02 - 2.12E-05 | 4 | |
Metabolic Disease | 3.18E-04 - 1.07E-15 | 21 | Cardiovascular Disease | 3.04E-02 - 7.84E-05 | 7 | |
Organismal Injury and Abnormalities | 7.69E-04 - 1.07E-15 | 44 | Hematological Disease | 2.54E-02 - 7.84E-05 | 5 | |
|
Immunological Disease | 4.24E-03 - 1.49E-07 | 18 | Immunological Disease | 1.04E-08 - 1.62E-39 | 113 |
(32/171) | Cancer | 4.24E-03 - 5.41E-07 | 28 | Infectious Diseases | 1.07E-08 - 2.19E-33 | 87 |
Hematological Disease | 4.24E-03 - 5.41E-07 | 19 | Connective Tissue Disorders | 8.87E-09 - 1.04E-29 | 76 | |
Organismal Injury and Abnormalities | 4.24E-03 - 5.41E-07 | 28 | Inflammatory Disease | 5.62E-09 - 1.04E-29 | 92 | |
Tumor Morphology | 4.24E-03 - 9.23E-07 | 4 | Organismal Injury and Abnormalities | 1.18E-08 - 1.04E-29 | 152 | |
|
Cancer | 5.81E-04 - 8.88E-09 | 79 | Immunological Disease | 3.22E-08 - 2.68E-38 | 97 |
(81/138) | Organismal Injury and Abnormalities | 5.81E-04 - 8.88E-09 | 79 | Infectious Diseases | 2.26E-08 - 1.47E-34 | 82 |
Immunological Disease | 5.81E-04 - 1.00E-08 | 43 | Connective Tissue Disorders | 4.62E-08 - 5.68E-33 | 69 | |
Connective Tissue Disorders | 5.81E-04 - 1.47E-08 | 26 | Inflammatory Disease | 3.11E-08 - 5.68E-33 | 78 | |
Inflammatory Disease | 4.24E-04 - 1.47E-08 | 32 | Organismal Injury and Abnormalities | 4.62E-08 - 5.68E-33 | 131 | |
|
Organismal Injury and Abnormalities | 1.49E-04 - 1.51E-14 | 105 | Infectious Diseases | 2.01E-05 - 1.37E-21 | 46 |
(111/82) | Hematological Disease | 1.18E-04 - 9.22E-11 | 50 | Immunological Disease | 1.35E-05 - 9.81E-21 | 51 |
Immunological Disease | 1.31E-04 - 9.22E-11 | 44 | Inflammatory Response | 1.92E-05 - 2.13E-19 | 54 | |
Cardiovascular Disease | 1.46E-04 - 1.04E-10 | 29 | Connective Tissue Disorders | 1.48E-05 - 2.82E-19 | 40 | |
Cancer | 1.49E-04 - 1.19E-10 | 103 | Inflammatory Disease | 1.65E-05 - 2.82E-19 | 50 | |
|
Inflammatory Response | 1.39E-03 - 3.58E-17 | 34 | Endocrine System Disorders | 6.76E-03 - 1.87E-06 | 5 |
(44/13) | Immunological Disease | 1.23E-03 - 2.76E-12 | 35 | Gastrointestinal Disease | 6.38E-03 - 1.87E-06 | 13 |
Organismal Injury and Abnormalities | 1.40E-03 - 1.16E-10 | 40 | Metabolic Disease | 7.38E-03 - 1.87E-06 | 7 | |
Connective Tissue Disorders | 1.23E-03 - 2.07E-10 | 22 | Nutritional Disease | 5.22E-03 - 1.87E-06 | 4 | |
Inflammatory Disease | 1.23E-03 - 2.07E-10 | 27 | Organismal Injury and Abnormalities | 7.54E-03 - 1.87E-06 | 13 | |
|
Inflammatory Response | 7.74E-12 - 1.20E-39 | 100 | Inflammatory Response | 4.47E-03 - 3.68E-06 | 21 |
(141/50) | Connective Tissue Disorders | 5.90E-12 - 1.96E-37 | 72 | Neurological Disease | 4.47E-03 - 1.07E-05 | 21 |
Inflammatory Disease | 5.33E-12 - 1.96E-37 | 83 | Organismal Injury and Abnormalities | 4.47E-03 - 1.07E-05 | 49 | |
Organismal Injury and Abnormalities | 1.52E-11 - 1.96E-37 | 115 | Cancer | 4.37E-03 - 1.21E-05 | 48 | |
Skeletal and Muscular Disorders | 2.96E-12 - 1.96E-37 | 78 | Gastrointestinal Disease | 4.47E-03 - 1.21E-05 | 46 | |
|
Connective Tissue Disorders | 6.58E-04 - 5.71E-10 | 22 | Gastrointestinal Disease | 1.12E-02 - 1.32E-06 | 6 |
|
Inflammatory Disease | 1.80E-04 - 5.71E-10 | 22 | Hepatic System Disease | 3.22E-03 - 1.32E-06 | 4 |
(48/12) | Inflammatory Response | 6.55E-04 - 5.71E-10 | 28 | Organismal Injury and Abnormalities | 1.12E-02 - 1.32E-06 | 11 |
Organismal Injury and Abnormalities | 7.20E-04 - 5.71E-10 | 46 | Connective Tissue Disorders | 9.09E-03 - 7.38E-06 | 7 | |
Skeletal and Muscular Disorders | 6.45E-04 - 5.71E-10 | 24 | Immunological Disease | 1.02E-02 - 7.38E-06 | 6 |
upN/down: number of up-regulated IGs/number of down-regulated IGs.
The up- and down-regulated innatomic genes (IGs) share inflammatory diseases in deficiencies of proinflammatory cytokines (PCs).
Of note, as shown in
We made an interesting finding that, the IGs were more upregulated than downregulated when poor responder or non-responder compared with the group responders or good responders in several microarray datasets of patients receiving monoclonal antibody (Mab) therapy (
Up-regulated innatomic genes (IGs) in Tnf KO (GSE43145, Tnf KO Gan mice versus (vs.) Gan mice) and anti-TNF therapy (GSE111761, non-responder vs. responder) microarrays shared pathways.
Mitochondrial ROS (mtROS) are signaling molecules, which drive inflammatory cytokine production (
165 ROS regulators were analyzed in inflammatory molecules KO microarrays.
No. | PCs and regulators | GEO NO. | Method | ROS regulators | |||||
---|---|---|---|---|---|---|---|---|---|
Up-regulated | Down-regulated | ||||||||
N | % | Genes | N | % | Genes | ||||
|
|||||||||
1 | TNF | GSE43145 | Tnfa KO | 3 | 2.42 | Bst1,Cryab,Foxm1 | 4 | 2.42 | Ncf1,Noxo1,Sod1,Tlr2 |
2 | GSE33253 | Tnfr1,2 KO | 4 | 2.42 | Cyp1b1,Nfe2l2,Sirt5,Tgfbr2 | 13 | 7.88 | Acod1,Cd36,Cps1,Crp,Cybb,Ddit4, |
|
3 | IFNG | GSE9892 | Ifng KO | 7 | 4.24 | Ace2,Bmp7,Egfr,Fbln5,Mapt, |
14 | 8.48 | Acod1,Apoa4,Bnip3,Bst1,Cybb, |
4 | GSE39592 | Ifngr1 KO | 10 | 6.06 | Cd177,Cdkn1a,Cps1,Cyp1b1, |
11 | 6.67 | Acod1,Bco2,Bmp7,Bst1,Cryab, |
|
5 | IL1B | GSE15750 | Traf6 KO | 0 | 0.00 | N/A | 2 | 1.21 | Coq7,Syk |
6 | GSE73875 | Irak1 KO | 3 | 1.82 | Cybb,Fpr2,Tyrobp | 0 | 0.00 | N/A | |
7 | IL6 | GSE63761 | Il6 KO | 11 | 6.67 | Cyba,Gch1,Itgam,Itgb2,Ncf1, |
3 | 1.82 | Agt,Ddit4,Nos3 |
8 | IL17 | GSE88800 | Il17ra KO | 23 | 13.94 | Acod1,Cd177,Cdkn1a,Ddit4, |
3 | 1.82 | Bco2,Mapt,Tigar |
9 | IL18 | GSE64308 | Il18 KO | 6 | 3.64 | Cdkn1a,Duox1,Edn1,F2, |
1 | 0.61 | Alox12 |
10 | GSE64309 | Il18 KO | 7 | 4.24 | Apoa4,Cdkn1a,Ddit4,Gstp1, |
8 | 4.85 | Bco2,Bnip3,Cd36,Duox1,Gadd45a, |
|
11 | GSE64310 | Il18 KO | 1 | 0.61 | Apoa4 | 2 | 1.21 | Bco2,Sh3pxd2a | |
|
|||||||||
12 | STAT | GSE40666 | Stat1 KO | 6 | 3.64 | Cd36,Cdkn1a,Mt3,Pmaip1, |
0 | 0.00 | N/A |
13 | GSE6846 | Stat3 KO | 8 | 4.85 | Cybb,Hif1a,Itgam,Mapk14, |
3 | 1.82 | Brca1,Plin5,Sod1 | |
14 | NFKB | GSE45755 | Rela KO | 6 | 3.64 | Cybb,Mpo,Ncf1,Ncf4,Tspo, |
0 | 0.00 | N/A |
15 | GSE30049 | Ikk2 KO | 9 | 5.45 | Bcl2,Bmp7,Cryab,Cyp1b1, |
12 | 7.27 | Brca1,Cybb,Dhfr,Edn1,Ephx2,F2rl1, |
|
|
|||||||||
16 | MIR155 | GSE45122 | mir155 KO | 3 | 1.82 | Bmp7,Crp,Nqo2 | 3 | 1.82 | Cyp1b1,Lrrk2,Pax2 |
17 | GSE66815 | mir155 KO | 13 | 7.88 | Alox12,Bst1,Cyp1b1,Lrrk2, |
10 | 6.06 | Agt,Cd177,Cdkn1a,Egfr,Foxm1, |
|
18 | MIR221 | GSE19777 | MIR221 KD | 1 | 0.61 | CPS1 | 4 | 2.42 | ACOD1,CYBB,PLA2R1,SFTPD |
Mechanism: up-regulated reactive oxygen species (ROS) regulators can drive the upregulation of “suppressed innatomic genes”.
Our detailed results showed that deficiencies of IL17RA, IL18 and NF-kB Rela result in upregulated heme peroxidase myeloperoxidase (MPO) expression (
These progresses lead to the development of many cytokine blockage-based therapies for inflammatory diseases and CVDs. The CANTOS trial with the Mab Canakinumab to block proinflammatory cytokine IL-1β was a recent success in treating coronary artery disease (
The original microarray experiments used different cells, which prevented us from comparing the effects of proinflammatory regulators in regulating the expressions of IGs in the same cell types. Although our database mining approach was not ideal, however, as the first step to fill in the important knowledge gap this approach was justified. Actually, this was a common practice that we (
Indeed, in addition to PC regulation of IGs transcription examined in our study, PCs could also regulate innate immune regulators in several other modes: 1) mRNA stability (
To summarize our findings presented here, we propose a novel working model (
A new working model is proposed: Blocking proinflammatory regulators induces the proinflammatory regulators-suppressed “second waves of inflammation”. Single cytokine blockade therapies result in significant upregulation of innate immune regulators and signaling pathways, presumably “second wave of inflammation” as we proposed.
One limitation of the current study is that due to the low throughput nature of verification techniques, we could not verify every result we identified with the analyses of high throughput data. We acknowledge that carefully designed
All the datasets used in this study are publicly available. The analyzed results in this study are included within the article and the
ML carried out the data gathering, data analysis and prepared tables and figures. JS, RZ, YSh, YSu, WY, JW, LuL, CD, CJ, FS, YL, KX, LiL, XW, XJ, and HW aided with analysis of the data. XY supervised the experimental design, data analysis, and manuscript writing. All authors contributed to the article and approved the submitted version.
This work was partially supported by NIH grants to HW and XY.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
ML was supported by a fellowship from the School of Basic Medical Science, Shanxi Medical University.
The Supplementary Material for this article can be found online at:
The heatmap of diseases related to down-regulated innatomic genes from IPA analysis comparison.
Nine proinflammatory cytokine blocking monoclonal antibody therapies and clinical uses.
Ten additional inflammation paradoxes related to this study which have been reported.
The IGs and ROS regulators gene-lists used in this study.
The expression changes of housekeeping genes selected in this study.
The expression changes of up-regulated and down-regulated innatomic genes in deficiencies of proinflammatory cytokines and regulators. (
The expression changes of up-regulated and down-regulated innatomic genes in Mab therapy microarrays. (
IPA results of up-regulated innatomic genes in deficiencies of proinflammatory cytokines and regulators. (∣Z score∣> 2)
IPA results of down-regulated innatomic genes in deficiencies of proinflammatory cytokines and regulators. (∣Z score∣> 2)
Pathways from IPA results of up- and down-regulated innatomic genes in deficiencies of TFs. (∣Z score∣> 2)
IPA results of up- and down-regulated innatomic genes in anti-TNF therapy microarray.
The expression changes of ROS regulators in deficiencies of proinflammatory cytokines and regulators. (
CVDs, cardiovascular diseases; IGs, innatomic genes (innate immune genes/regulators); PCs, proinflammatory cytokines/regulators; ROS, reactive oxygen species; Treg, regulatory T cells; IL-1β interleukin-1β; miRs: microRNAs; miR155, microRNA-155; KO, knock-out; MHO, metabolically healthy obesity; RA, rheumatoid arthritis; TNFα, tumor necrosis factor-α; Mab, monoclonal antibody; IL-6R, IL-6 receptor; DAMPs, danger associated molecular patterns; PAMPs, pathogen associated molecular patterns; NCDs, non-communicable diseases; NIH, National Institutes of Health; NCBI, National Center for Biotechnology Information; GEO, Gene Expression Omnibus; IPA: Ingenuity Pathway Analysis; GSEA, Gene Set Enrichment Analysis; IFNγ, interferon-γ; STAT1, signal transducer and activator of transcription protein 1; IKK2, Inhibitor of NFKB kinase subunit-β; TRAF6, TNF receptor associated factor 6; IRAK1, interleukin-1 receptor-associated kinase 1; PBMCs, peripheral blood mononuclear cell populations; Th1, type 1 T helper cells; HMGB1, high mobility group box 1; STAT4, signal transducer and activator of transcription 4; Erk1/2, extracellular signal-regulated protein kinases 1 and 2; Pyk2, proline-rich tyrosine kinase 2; CrkL, CRK like proto-oncogene; iCOS, inducible T-cell co-stimulator; NF-AT, nuclear factor of activated T cells; IRF, interferon regulatory factor; HGF, hepatocyte growth factor; JAK, Janus kinases; MIF: macrophage migration inhibitory factor; PAK, p21-activated kinase; PI3K, phosphatidylinositol-3-kinase; AKT, protein kinase B; cAMP, cyclic adenosine monophosphate; TFs, transcription factors; PD-1, programmed cell death protein-1; DC, dendritic cells; mtROS, mitochondrial ROS; MPO, myeloperoxidase; NES, normalized enrichment score; FDR, false discovery rate; NK, natural killer.