Identification of immune system modulators and generation of induced pluripotent stem cells for their therapeutic potential
- 주제(키워드) Flagelling , TLR5 , TNF , CRISPR/Cas9 , iPSCs
- 주제(DDC) 547
- 발행기관 아주대학교
- 지도교수 Sangdun Choi
- 발행년도 2022
- 학위수여년월 2022. 8
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000032202
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
The immune system of an organism constitutes multiple structures, receptors and biological processes that protect it from pathogens including bacteria, parasites, fungi, and viruses. This system is broadly classified into two main types i.e., innate immune system and adaptive immune system. Innate immune system is the first line of defense that elicits a quick response against invaders. At molecular level, it constitutes certain structures which recognize certain patterns of exogenous and endogenous stimuli, known as pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). These structures or receptors are known as pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) and NOD-like receptors (NLRs). TLRs are present on cell-surface or embedded in endosomes while NLRs are cytoplasmic structures; they both recognize specific PAMPs and/or DAMPs. For instance, TLR5 is present on the surface of cells where it recognizes flagellin protein, a major component of flagella. This interaction mediates intracellular Toll/interleukin-1 receptor (TIR) domain to bind with other cytosolic adaptor molecules containing TIR domain. This activates a cascade of signaling pathways which leads to the activation of various transcription factors such as activating protein 1 (AP-1), nuclear factor kappa light-chain-enhancer of activated B cells (NF-κB), and interferon-regulatory factors (IRFs). Cells primed with PAMPs also activate NOD-like receptor (NLR) family CARD domain-containing protein 4 (NLRC4) via detecting flagellin in cytosol during infection. NLRC4 protein cleaves pro-caspase 1 to mature caspase 1 that in turn activates the release of cytokines such as IL-18 and IL-1β along with execution of pyroptosis. The cytokines generated by TLR5 and NLRC4 prepare host to defend itself against pathogens through the cooperation of other immune components. Acidovorax avenae is a pathogenic bacterium to economically important crops. Given a huge devastation to plants, there is not much information about its ability to cause opportunistic infection in species like humans. The presence of A. avenae is confirmed in human patients of fever, cancer, sepsis; but, its definite mechanism of infection is not known for humans. The activation of PRRs also elicits the secretion of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α, also called as TNF). TNF-α is a homotrimer protein which is held together through noncovalent interactions among its monomeric units. It is mainly produced by dendritic cells, macrophages, natural killer cells, or T lymphocytes. It contributes in host protection such as tumor suppression, cell survival, differentiation, and proliferation. However, dysregulated production of TNF can lead to the development of diseases such as rheumatoid diseases, inflammatory bowel disease (IBD), and psoriatic arthritis. Along with that, it also contributes in cancer-progression, carcinogenesis, and metastasis. Due to involvement in various diseases, TNF-α is being targeted to develop therapeutics. Usually drug-like molecules contain aromatic cores which provide them high polarizability and conformational flexibility. It also allows them to react with various polar solvents; however, their solubility in aqueous solutions is a main obstacle in the drug discovery process. These kind of candidate molecules are usually dissolved in a most powerful organic solvent, dimethyl sulfoxide (DMSO). However, presence of DMSO can compromise the screening of TNF-α targeting therapeutics. The small-molecule compounds or short peptides are being developed to treat TNF-mediated inflammatory diseases. Crystal structure of the TNF homotrimer in complex with one of its receptors has facilitated identification and development of chemical or peptide-based probes through rational design. Induced pluripotent stem cells (iPSCs) has ability of indefinite can self-renewal in culture and to differentiate into any specialized cell types. They do not present naturally and instead are generated from somatic cells via ectopic expression of certain pluripotency factors. Owing to their generation from any patient or healthy person, iPSCs are reflected as a treasured reserve for regenerative medicine to substitute damaged or diseased tissues. Moreover, reprogramming knowledge has delivered an influential means to understand mechanisms of cell fate and model human diseases, thus significantly enhancing the likelihood to (i) discover new drugs for screening purposes and (ii) treat serious diseases via cell therapy-based strategies. Broad applications of iPSCs urge to design multiple approaches to facilitate their generation. The iPSCs can be generated by expressing reprogramming factors from a transgene integrated into the genome of specialized cells. However, existing non-viral and viral integrative approaches are not much safe. First, Acidovorax avenae is a flagella possessing pathogenic bacterium to multiple plant crops. It is also found in human patients of sepsis, fever, and haematological malignancy; but, its definite mechanism of infection is not known for humans. We hypothesized that flagellin protein purified from A. avenae (FLA-AA) may provoke immune response in humans by interacting with innate immune receptors. We found release of inflammatory cytokines including TNF-α, interleukin (IL)-6, and IL-8 on treating FLA-AA to human macrophages as well as human dermal fibroblasts. This response was mediated by the activation of TLR5 on these cells. We also found release of inflammatory cytokine, IL-1β, through recognition of FLA-AA by cytoplasmic NLRC4 protein. This data provides molecular basis for the activation of human innate immune response by opportunistic pathogen, A. avenae. Second, recognition of TNF-α by TNFR1 and TNFR2 activates downstream signaling pathways which lead to the release of cytokines as well as to the cell death. The functional, trimeric structure of TNF-α can be distorted by certain drug-dissolving solvents such as DMSO. Hence, we evaluated the effect of DMSO on the efficacy of TNF-α at various concentrations of DMSO. The solutions of DMSO in water were made at various concentrations of DMSO; for instance, 0.1%, 1%, 10%, 50%, and 100%. These solutions were incubated with rhTNF-α for 1 h followed by treatment on human dermal fibroblasts (HDFs) for 24 h. We found reduction in the release of IL-6 and IL-8 as well as cell-proliferation with an increasing concentration of DMSO. We also found reduction in TNF-mediated cell death of mouse and human fibroblasts with increasing concentration of DMSO. We confirmed by cross-linking experiment that this inhibition is due to the disruption in the homotrimeric state of TNF-α. Third, TNF-α activates various deleterious signaling pathways in cells and can be targeted to develop therapeutics. Here, we employed Rosetta PeptiDerive protocol to make 12-mer TNF-inhibiting decoys (TIDs). These TIDs were observed to suppress TNF signaling by distorting functional trimeric form of TNF. Among effective TIDs, TID3 and its derivative, TID3c, strongly distorted the trimeric form of TNF in order to make it inactive. The biophysical interactions by surface plasmon resonance confirmed binding of TID3 as well as TID3c with both mouse and human TNF. These results pave the way for a new class of TIDs with functional specificity. Fourth, dysregulated signaling by TNF is involved in multiple autoimmune diseases such as rheumatoid arthritis (RA), Crohn’s disease, and psoriatic arthritis. Although direct TNF inhibition by small molecule compounds is an alternative to currently approved biologics, available small molecule-based inhibitors exhibit low potency or are cytotoxic. Here, we describe a TNF-inhibitory molecule (TIM) that has potential to treat rheumatoid arthritis. The initial lead, TIM1, suppressed TNF-mediated necroptosis in mouse and human cells by inhibiting the activation of downstream signaling pathways. TIM1 also suppressed the release of prominflammatory cytokines i.e., IL-6 and IL-8 through distorting TNF homotrimeri assembly. An analog of TIM1, TIM1c, showed better in vitro activity and reversed the symptoms of RA in mouse model through oral administration. The biophysical interactions confirm binding of TIM1 and TIM1c to both human and mouse TNF. Fifth, induced pluripotent stem cells (iPSCs) play a role in biomedicine, pharmacology, cell therapy, and toxicology. Various approaches are being developed to make iPSCs by expressing reprogramming factors. Here, we made iPSCs after integrating a reprogramming cassette into genomic safe harbor, CASH-1, via precise genome editing tool, CRISPR/Cas9. We did not find any difference between parental and transgene-containing cells in terms of proliferation and IL-6 secretion after stimulating with ligands of various signaling pathways such as TNF-α receptor, IL-1 receptor, and TLRs. Furthermore, regulated expression of OCT4, SOX2, and KLF4 successfully reprogrammed transgene-containing human dermal fibroblasts and human embryonic kidney cells into iPSCs. The iPSCs generated by this technique showed ability to make embryoid bodies followed by differentiation into derivatives of each germ layer. Collectively, this data emphasizes usage of CASH-1 by CRISPR/Cas9 tool to reprogram engineered cells into iPSCs.
more목차
CHAPTER 1 1
1. Introduction 2
2. Methods 4
2.1 Sequence alignment 4
2.2 Cell lines and reagents 4
2.3 Cell viability assay 5
2.4 Western blot analysis 5
2.5 Immunofluorescence 6
2.6 Cytokine detection assays 6
2.7 Statistical analysis 7
3. Results and discussion 8
3.1. Results 8
3.1.1. Activation of immune signaling in human macrophages 8
3.1.2. Activation of immune signalling in primary human dermal fibroblasts (HDFs) 10
3.1.3. FLA-AA–mediated signaling is specific to TLR5 11
3.2. Discussion 13
3.3. Conclusion 16
CHAPTER 2 17
4. Introduction 18
5. Methods 21
5.1. Cell lines and reagents 21
5.2. Cell viability assay 21
5.3. IL-8 and IL-6 cytokine assays 22
5.4. Cell-death recovery assay 22
5.5. Western blot analysis 22
5.6. Dissociation of TNF-α trimerization assembly 23
5.7. Surface plasmon resonance (SPR) spectroscopy 23
5.8. Murine CIA model and grouping 24
5.9. Behavioral and histological analysis of animals 24
5.10. Statistical analysis 25
6. Results and discussion (1): The effect of DMSO on the biological activity of TNF-α 26
6.1. Results 26
6.1.1. DMSO Inhibits TNF-mediated Cytokine Release and Cell Proliferation 26
6.1.2. DMSO inhibits TNF-mediated cell death 27
6.1.3. DMSO deactivates TNF-mediated pathways 28
6.1.4. DMSO prevents TNF oligomerization 29
6.2. Discussion 29
6.3. Conclusion 31
7. Results and discussion (2): A series of decoy peptides that disrupt TNF-alpha oligomeric configuration 32
7.1. Results 32
7.1.1. Identification of TNF-inhibitory peptides 32
7.1.2. Designing derivatives of shortlisted peptides 36
7.1.3. TID3c inhibits TNF-mediated downstream signaling pathways 39
7.1.4. TID3 and TID3c distort trimeric form of TNF 40
7.1.5. Biophysical interaction between TNF and peptides 41
7.2. Discussion 43
7.3. Conclusion 45
8. Results and discussion (3): Designing an orally active small molecule inhibitor of TNF-alpha to alleviate rheumatoid arthritis 45
8.1. Results 45
8.1.1. Identification of TIM1 as a potential TNF inhibitor 45
8.1.2. TIM1 attenuates TNF–mediated death of cells 46
8.1.3. TIM1 deactivates TNF-dependent pathways 48
8.1.4. Derivatives of TIM1 showed improved activity against TNF signaling 49
8.1.5. TIM1 and TIM1c disrupt TNF trimer by binding to TNF 53
8.1.6. TIM1/TIM1c reduces arthritis symptoms in murine model through oral- administration 53
8.2. Discussion 54
8.3. Conclusion 58
CHAPTER 3 59
9. Introduction 60
10. Methods 63
10.1. Design and cloning of gRNAs 63
10.2. Cell culture and reagents 66
10.3. Off-target prediction 67
10.4. T7E1 endonuclease assay 67
10.5. Reprogramming-plasmid construction 68
10.6. Optimizing knock-in of donor cassette 68
10.7. Junction PCR 69
10.8. Flow cytometry 69
10.9. Cell viability assay 69
10.10. Cytokine detection assay 70
10.11. Generation and maintenance of iPSCs 70
10.12. In vitro differentiation of iPSCs 71
10.13. Immunocytochemistry 71
10.14. RT-PCR and quantitative RT-PCR 72
10.15. Western blotting 72
10.16. Statistical analysis 73
11. Results and Discussion 74
11.1. Results 74
11.1.1. Design and validation of guide RNAs 74
11.1.2. Knock-in of reprogramming-cassette 78
11.1.3. Proliferative and immunological validation 82
11.1.4. Generation and characterization of iPSCs 83
11.2. Discussion 86
11.3. Conclusion 88
12. References 89
