The Importance of Hypoxia-Inducible Factors (HIF-1 and HIF-2) for the Pathophysiology of Inflammatory Bowel Disease
Abstract
:1. Introduction
2. Results
2.1. The Severity of Colon Inflammation is Reduced in Mice Lacking Functional HIF-1α, but Higher in Mice Lacking Functional HIF-2α in Myeloid Cells
2.2. Tissue Destruction after DSS Treatment in Mice without Functional HIF-1α is Reduced, but Higher in Mice without Functional HIF-2α in Myeloid Cells
2.3. Tissue Destruction Reduced the Mucin Production in the Colon
2.4. Macrophage Migration towards the Inflamed Colon is not Altered by Myeloid HIF-2α Knockout
2.5. Mice without Functional HIF-2α in Myeloid Cells Recruit more Neutrophils towards the Gut Lumen
2.6. Mice without Functional HIF-2α in Myeloid Cells Recruit more Neutrophils, CD4+, and CD8a+ T-Cells and Tregs towards the Inflamed Colon
2.7. Mice without Functional HIF-2α in Myeloid Cells Induce Migration of Lymphocytes towards the Gut
2.8. HIF-2α Loss in Myeloid Cells induced High Pro- and Anti-Inflammatory Gene Expressions in the Inflamed Gut
3. Discussion
4. Materials and Methods
4.1. Animal Model
4.2. Cell Culture
4.3. Histology
4.4. Real-Time PCR
4.5. Statistical Analyses
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
BMDM | Bone-marrow-derived macrophage |
CCR9 | C-C chemokine receptor type 9 |
C-TAD | C-terminal transactivation domain |
CXCL1 | chemokine (C-X-C motif) ligand 1 |
DAI | Disease Activity Index |
DC | Dendritic cell |
DNA | deoxyribonucleic acid |
DSS | dextran sodium sulfate |
FIH | Factor Inhibiting HIF |
HIF | Hypoxia-inducible factor |
HRE | Hypoxia-Response Element |
IBD | Inflammatory bowel disease |
IFNγ | Interferon-γ |
IL | Interleukin |
ODD | Oxygen-dependent degradation |
O2 | Oxygen |
PHD | Prolyl hydroxylase |
pVHL | von-Hippel Lindau protein |
TGFβ | Transforming growth factor beta |
TNFα | Tumor necrosis factor alpha |
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Score | Weight Loss | Stool Consistency | Hema Screen Test (Occult Blood) |
---|---|---|---|
0 | none | normal | negative |
1 | 1–5% | normal | negative |
2 | 5–10% | loose | positive |
3 | 10–20% | loose | positive |
4 | >20% | diarrhea | visible bleeding |
Score | Infiltrating Immune Cells | Extent of Injury | Crypt Damage |
---|---|---|---|
0 | rare | none | intact crypts |
1 | slightly dispersed | mucosal | basal 1/3 damaged |
2 | moderately increased | mucosal and submucosal | basal 2/3 damaged |
3 | severely large areas: loss of tissue structure | transmural | only surface epithelium intact |
4 | - | - | loss of entire crypt and epithelium |
Antibody | Concentration | Catalog Number and Company |
---|---|---|
rat anti-F4/80 | 0.180556 | #MCA497B, Bio-Rad AbD Serotec GmbH, Puchheim, Germany |
goat antirat secondary Ab | 0.388889 | #A11077, Invitrogen Thermo Fisher Scientific, Dreieich, Germany |
goat antimyeloperoxidase | 0.180556 | # AF3667, R&D systems |
rabbit antigoat secondary Ab | 0.388889 | # sc2774, Santa Cruz Biotechnology, Heidelberg, Germany |
goat anti-CD3-ε | 0.215278 | #sc-1127, Santa Cruz Biotechnology, Heidelberg, Germany |
rabbit antigoat Alexa Fluor 488 | 0.388889 | #A-11078, Thermo Fisher Scientific, Dreieich, Germany |
Rat anti-FoxP3 | 0.111111 | # FJK-16s, eBioscience Thermo Fisher Scientific, Dreieich, Germany |
goat antirat Alexa Fluor 568 | 0.388889 | #A-11077, Thermo Fisher Scientific, Dreieich, Germany |
rabbit anti-HIF-1alpha (C-Term) | 1:10,000 | #CAY-10006421, Cayman Chemical Biomol GmbH, Hamburg, Germany |
rabbit anti-HIF-2 alpha/EPAS1 | 1:10,000 | #NB100-122, Novus Biologicals, Wiesbaden, Germany |
Polyclonal goat-antirabbit Immunoglobulin/HRP | 0.388889 | #P044801-2, DAKO Agilent Technologies, Waldbronn, Germany |
Target Gene | Accession-Number | Sequence | Product Length (bp) |
---|---|---|---|
5′ Adgre1 3′ Adgre1 | NM_010130 | TCTGGGGAGCTTACGATGGA GAATCCCGCAATGATGGCAC | 237 |
5′ Arg1 3′ Arg1 | NM_007482 | AACACGGCAGTGGCTTTAACC GGTTTTCATGTGGCGCATTC | 117 |
5′ Ccr9 3′ Ccr9 | NM_001166625 | CCAAGGTGCCCACAATGAAC ACTCACAAGCCTTATTCCTGGC | 179 |
5′ Cd4 3′ Cd4 | NM_013488 | TGAAGGAAACGCTCCCACTC AGCAGTGCTGATGTCTTGCT | 136 |
5′ Cd8a 3′ Cd8a | NM_001081110 | ACCCTTGGCCGGAATCTGCG CTGTCTGACTAGCGGCCTGGGA | 112 |
5′ Cd11c 3′ Cd11c | NM_021334 | GGACGGTGCTGAGTTCGGACACAG CCACAAGCCAACAGCCAGGAAGG | 231 |
5′ Cxcl1 3′ Cxcl1 | NM_008176 | CAGGGTCAAGGCAAGCCTC CTGGGATTCACCTCAAGAACATC | 117 |
5′ Foxp3 3′ Foxp3 | NM_001199347 | CTGGCGAAGGGCTCGGTAGTCCT CTCCCAGAGCCCATGGCAGAAGT | 250 |
5′Hif-1a exon 2 3′ Hif-1a exon 2 | NM_001313919 | CATCCAGAAGTTTTCTCACACG GGCGAAGCAAAGAGTCTGAA | 138 |
5′Hif-2a exon 2 3′ Hif-2a exon 2 | NM_010137 | AGGAGACGGAGGTCTTCTATGA ACAGGAGCTTATGTGTCCGA | 126 |
5′ Ifng 3′ Ifng | NM_008337 | GGTCAACAACCCACAGGTCC CAGCGACTCCTTTTCCGCTT | 105 |
5′ Il6 3′ Il6 | NM_031168 | TCCTACCCCAATTTCCAATGC CATAACGCACTAGGTTTGCCG | 151 |
5′ Il10 3′ Il10 | NM_010548 | TGCCCCAGGCAGAGAAGCAT GGGAGAAATCGATGACAGCGCC | 109 |
5′ Il23a 3′ Il23a | NM_031252 | ACCAGCGGGACATATGAATCT AGACCTTGGCGGATCCTTTG | 147 |
5′ Il17a 3′ Il17a | NM_010552 | TCATCCCTCAAAGCTCAGCG TTCATTGCGGTGGAGAGTCC | 167 |
5′ Ly6g 3′ Ly6g | NM_023463 | GTACCTTGGGAAGATGTGGGT GTTCAGGCCCAGCTTATGGT | 103 |
5′ Tgfb1 3′ Tgfb1 | NM_011577 | TGGCCAGATCCTGTCCAAAC CATAGATGGCGTTGTTGCGG | 215 |
5′ Tnfa 3′ Tnfa | NM_011609 | TACCTCCTCCGCTTGCAAAT GAGTAGACTTCGGGCCTCCAC | 151 |
5′ Actb 3′ Actb | NM_007393 | TAGGCACCAGGGTGTGATGG CTCGGTGAGCAGCACAGG | 208 |
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Kerber, E.L.; Padberg, C.; Koll, N.; Schuetzhold, V.; Fandrey, J.; Winning, S. The Importance of Hypoxia-Inducible Factors (HIF-1 and HIF-2) for the Pathophysiology of Inflammatory Bowel Disease. Int. J. Mol. Sci. 2020, 21, 8551. https://doi.org/10.3390/ijms21228551
Kerber EL, Padberg C, Koll N, Schuetzhold V, Fandrey J, Winning S. The Importance of Hypoxia-Inducible Factors (HIF-1 and HIF-2) for the Pathophysiology of Inflammatory Bowel Disease. International Journal of Molecular Sciences. 2020; 21(22):8551. https://doi.org/10.3390/ijms21228551
Chicago/Turabian StyleKerber, Evelyn L., Claudia Padberg, Nora Koll, Vera Schuetzhold, Joachim Fandrey, and Sandra Winning. 2020. "The Importance of Hypoxia-Inducible Factors (HIF-1 and HIF-2) for the Pathophysiology of Inflammatory Bowel Disease" International Journal of Molecular Sciences 21, no. 22: 8551. https://doi.org/10.3390/ijms21228551