臺灣博碩士論文加值系統

根據美國腎臟病數據系統(USRDS)的資料顯示，2009年台灣地區因末期腎臟病進行透析人口的盛行率高居全球之冠。慢性腎臟病為一種慢性的發炎疾病，許多免疫細胞參與其中，包含巨噬細胞、淋巴球及嗜中性白血球等，這些免疫細胞浸潤到腎組織間質當中，引發過度的發炎反應而造成腎臟組織結構及功能上的損傷。在2000年所發現的髓細胞觸發受體(triggering receptor expressed on myeloid cells, TREMs)家族當中，TREM-2具有相當多元的功能，並表現在許多不同的骨髓來源細胞上，如巨噬細胞、樹突細胞、蝕骨細胞等。過去的研究發現TREM-2透過誘導巨噬細胞不同亞型(M1/M2)的轉換以減緩發炎反應，促進傷口的癒合。在本研究當中，我們利用單側輸尿管阻塞(unilateral ureteral obstruction, UUO)作為慢性腎臟病的動物模式。誘發阻塞性腎損傷後的7天和14天，TREM-2訊息核酸表現量顯著地增加，因此，我們利用TREM-2基因剔除小鼠來探討TREM-2在此動物模式中所扮演的角色。結果顯示，TREM-2缺失不會影響巨噬細胞浸潤的程度，但卻明顯增強了UUO對於腎小管的傷害及間質纖維化程度。因此，我們進一步的探討是否TREM-2調控了巨噬細胞亞型的轉換。M1巨噬細胞的重要指標，誘發型一氧化碳合成酶及介白素-6的表現在TREM-2剔除小鼠阻塞性腎炎組織中大量表現，但其他相關的指標如腫瘤壞死因子、介白素-1則在野生型及基因剔除小鼠的阻塞性腎炎組織中並沒有顯著的差異，另一方面，具有組織修護功能的M2巨噬細胞的相關指標，如甘露醣受器、幾丁質分解酶、精胺酸酶-1、介白素-10及轉化生長因子-表現在TREM-2基因剔除小鼠阻塞性腎炎組織中並未如預期的降低，顯然從M1/M2的轉換上不足以解釋TREM-2基因剔除小鼠在阻塞性腎炎後所造成較嚴重腎損傷的結果。有趣的是，我們發現基因剔除小鼠的阻塞性腎炎組織中可以觀察到存在許多的多核球，利用Ly6G免疫組織染色，也確認其為嗜中性白血球，浸潤程度在TREM-2基因剔除小鼠14天的阻塞性腎炎組織中顯著增加，我們進一步的將TREM-2小鼠利用靜脈注射專一性抗體將血液中嗜中性白血球清除，發現在誘發阻塞性腎炎14天後，腎小管傷害有顯著的減緩，表示嗜中性白血球在阻塞性腎損傷扮演了重要角色。過去的研究指出，嗜中性白血球的浸潤會受到促發炎細胞激素介白素-17的影響，而我們發現介白素-17的表現量在TREM-2基因剔除小鼠的阻塞性腎炎組織中顯著的增加。在細胞實驗當中，我們更進一步探討TREM-2在調控主要產生介白素-17的輔助T淋巴球17分化過程中所扮演的角色。給予阻塞性腎炎腎組織均質液後，剔除TREM-2之樹突細胞相較於野生型有較強的刺激T淋巴球分化成輔助T淋巴球17的能力，並顯著提升介白素-17的表現量，但輔助T淋巴球17相關的分化細胞激素，如介白素-6及介白素-23的生成在野生型及基因剔除組別之間沒有顯著的差異。根據以上的結果，我們推論：雖然TREM-2不參與在調控樹突細胞釋出有助於輔助T淋巴球17分化的細胞激素，但給予了阻塞性腎炎腎組織均質液後，TREM-2確實在輔助T淋巴球17的分化上扮演一定的角色，進而抑制介白素-17釋出並阻斷嗜中性白血球浸潤造成的腎損害。

According to the United States Renal Data System (USRDS), the prevalence of end stage renal disease (ESRD) is the highest in Taiwan in 2009. Accumulation of leukocytes, particularly macrophages, lymphocytes and neutrophils, in the tubulointerstitium plays a pivotal role in the development of chronic renal inflammatory disease and causes structural and functional injury. It has been reported that triggering receptor expressed on myeloid cells-2 (TREM-2) is a negative immune regulator of macrophage transition in wound healing. We found TREM-2 was upregulated in renal tissues of unilateral ureteral obstruction (UUO) mice. We further conducted a murine UUO model in TREM-2 deficiency (TREM-2-/-) mice to assess the pathologic role of TREM-2 in kidney injury. Our data showed that TREM-2-/- mice displayed significantly more tubular injury and interstitial fibrosis than wild-type (WT) mice did 14 days after UUO. F4/80+ cell infiltration was comparable between WT and TREM-2-/- UUO mice. The expression of inducible nitric oxide synthase and IL-6, makers of M1 macrophage, were significantly elevated in TREM2-/- UUO mice, but other markers of M1 and M2 macrophages showed inconsistent results. Intriguingly, a significant increase of neutrophil infiltration was observed in TREM2-/- mice 14 days after UUO. As our expectation, the tubular injury was significantly ameliorated after neutrophil depletion in TREM-2-/- in 14 days of UUO, indicating that neutrophils play an important role in late stage of obstruction nephropathy when lost TREM-2. Correspondingly, the mRNA expression of interleukin-17 (IL-17), a widely recognized inflammatory cytokine for neutrophil recruitment, was upregulated in TREM2-/- UUO mice. To investigate whether TREM-2 and UUO tissue lysate mediate the differentiation of naïve lymphocytes into IL-17-producing T lymphocyte, we co-cultured WT and TREM-2-/- bone marrow-derived dendritic cells (BMDCs) with CD4+ T cells respectively. Current result showed that IL-17 expression was upregulated in the TREM-2-/- group after stimulated with UUO tissue lysate. To further investigate the role of TREM-2 in Th17 activation, we cultured BMDCs alone with administration of UUO tissue lysate but the IL-17 related cytokine, IL-6 and IL-23 showed no significant difference between WT and TREM-2-/- group. Taken collectively, although TREM-2 doesn’t involved in mediating IL-17 related cytokines release from BMDCs, TREM-2 does play a role in downregulation of Th17 development and IL-17-mediated neutrophil infiltration, therefore attenuates the tubulointerstitium injury in UUO WT mice.

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Abbreviations Table V
致謝 Ⅶ
中文摘要 Ⅷ
Abstract Ⅹ
1. Introduction 1
1-1. Chronic kidney disease 1
1-2. The etiology of CKD 1
1-3. Renal inflammation on structural injury 2
1-4. Diversity of inflammatory cells in renal disease 4
1-5. UUO animal model 5
1-6. Tiggering receptor expressed on myeloid cell-2 (TREM-2): a negative regulator of inflammation 6
2. Hypothesis and specific aims of this study 8
3. Materials and Methods 9
3-1. Animals 9
3-2. UUO animal model 9
3-3. RNA isolation and real-time quantitative polymerase chain reaction 9
3-4. Histological stain and pathological assessment 10
3-5. Immunohistochemical stain (IHC) 11
3-6. Neutrophil depletion 11
3-7. Kidney cell dissociation 12
3-8. Intracellular cytokine analysis and flow cytometry 12
3-9. Preparation of UUO tissue lysate 13
3-10. Cell preparation and mixed lymphocyte reaction 13
3-11. Statistical analysis 14
4. Results 15
4-1. TREM-2 expression after UUO-induced renal tubulointerstitial injury 15
4-2. TREM-2-/- mice develop more sever renal tubular injury and fibrosis in the UUO model 16
4-3. The role of TREM-2 in infiltrated inflammatory cells after UUO 16
4-4. The role of TREM-2 in M1 vs. M2 cells polarization 17
4-5. Neutrophil participated in UUO model and neutrophil depletion ameliorates renal tubular injury in UUO model. 19
4-6. TREM-2 plays a role in neutrophil recruitment via releasing pro-inflammatory cytokine IL-17A 20
4-7. UUO-injured kidney contains an unidentified TREM-2 ligand that could trigger naïve CD4+ T lymphocyte differentiation into Th17 cell 21
5. Discussion 23
6. Reference 26

Figure 1 Study design of animal experiments 35
Figure 2 The mRNA expression of TREM-2 normalized to 18S in wild type and TREM-2-/- groups 7 and 14 days after UUO 36
Figure 3 More severe renal tubular injury in UUO kidneys of TREM-2-/- mice 37
Figure 4 TREM-2-/- mice develop more severe renal interstitial fibrosis 14 days after
UUO 38
Figure 5 Macrophage infiltrated in renal interstitium 7 and 14 days after UUO 39
Figure 6 Role of TREM-2 in M1 macrophage polarization 40
Figure 7 Role of TREM-2 in M1 macrophage polarization 41
Figure 8 Role of TREM-2 in M2 Macrophage transition 42
Figure 9 Increased neutrophil infiltration in the UUO kidneys of TREM-2-/- mice 44
Figure 10 Neutrophil depletion reduced tubular injury in TREM-2-/- mice 14 days after UUO 46
Figure 11 The expression of IL-17 was upregulated in the UUO kidneys of
TREM-2-/- mice 48
Figure 12 Allogenetic mix lymphocyte reaction (MLR) 50
Figure 13 The mRNA expression of IL-17 after mix lymphocyte reaction 52
Figure 14 BMDCs cultured alone with administration of PBS, UUO tissue lysate and
LPS 53
Figure 15 Proposed mechanism of TREM-2 reduced renal tubular injury and
interstitial fibrosis via downregulation of Th17 and inhibition of neutrophil
recruitment 55

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