結腸上皮細胞剔除類鐸受體4對腸道菌與生理的影響

Abstract

人類腸道內具有數萬億種微生物，其中包括細菌、病毒、寄生蟲等，而大部分的微生物分布於大腸中，與人體消化吸收、代謝、免疫息息相關，也能透過腸-腦軸(gut-brain axis)調控神經傳導。許多研究指出腸道菌群的生態失衡(dysbiosis)與人體種種疾病的發生有關，包括發炎、退化性與全身性疾病等，因此維持良好的腸道菌群相當重要。而脂多醣/類鐸受體4 (LPS/TLR4)訊號的活化被認為是導致腸道發炎與腸漏症的致病機制之一，可能增加腸道菌群失衡患者罹患遠端器官疾病的風險。 先前有研究透過將絨毛蛋白(villin)做為啟動子，進一步剔除腸上皮細胞的類鐸受體4(Tlr4)，實驗結果發現腸上皮細胞缺乏類鐸受體4的小鼠(TLR4△IEC)與對照組(Wild type, WT)小鼠腸道中主導的三大菌門雖然相同，但兩者的叢集(cluster)不同，且TLR4△IEC的α-diversity較WT低，表示將腸上皮細胞類鐸受體4剔除後腸道微生物群的豐富度會降低，也發現腸道菌功能類別顯著改變，進一步影響脂質、胺基酸、核苷酸等代謝。除此之外，腸上皮細胞缺乏類鐸受體4會導致小鼠罹患代謝症候群，TLR4△IEC小鼠與WT小鼠相比體重顯著增加，脂肪較多，且有葡萄糖不耐的情形。由於人類腸道菌群主要位於大腸，且未來預計進行腸-腎軸相關研究，因此本研究目的是要建立結腸特異性類鐸受體4剔除小鼠，研究其腸道微生物群的變化，並觀察是否造成病生理的影響。 首先，利用Tg(Car1-cre);Rosa26fstdTomato/+小鼠來確認使用的Car1-cre基因轉殖系統是否順利運作，由於製造出Tg(Car1-cre)小鼠的作者研究指出Car1除了表現於結腸外，在肝臟也有些微表現，且後續我們預計進行腎臟方面的研究，因此利用免疫螢光(immunofluorescence, IF)染色確認Car1在肝臟與腎臟中的表現。實驗結果發現肝細胞與腎臟中的內皮細胞和周細胞皆會表現Car1。接著利用Tg(Car1-cre);Tlr4F/F小鼠進行實驗，由於肝臟與腎臟中皆有細胞會表現Car1，因此測定了血清中肝腎功能相關生化指標，只有實驗組(KO)母鼠血清中白蛋白顯著高於對照組(WT)，其餘血清生化指標則皆無顯著差異。由於TLR4△IEC小鼠體重顯著高於WT小鼠，我們好奇在結腸上皮細胞剔除類鐸受體4是否會有相同的情形，因此在小鼠8周齡時開始記錄體重至24周齡，不論公鼠或母鼠，KO小鼠與WT小鼠體重皆沒有顯著差異，由於過程中看到KO小鼠相較於WT有體重較輕的趨勢，因此在24週齡時進行體組成分析，實驗結果發現公鼠與母鼠在脂肪組織重(Fat)、瘦體組織重(Lean body mass; LBM)、游離水含量(Free fluid)及身體總水分含量(Total water)，以及各組織重與體重之百分比在KO小鼠與WT間皆無顯著差異。也利用口服葡萄糖耐受性試驗(Oral glucose tolerance test, OGTT) 測量小鼠代謝葡萄糖的能力，觀察是否有葡萄糖不耐的情形，實驗結果發現管餵葡萄糖後所有小鼠有相似的血糖變化，KO小鼠並不像TLR4△IEC小鼠有葡萄糖不耐的情形。為了觀察將結腸上皮細胞類鐸受體4剔除是否會影響小鼠腸道菌群，在小鼠8、12、16、20、24周齡時收取糞便檢體進行腸道菌分析，主座標分析(Principal Coordinates Analysis; PCoA)的結果顯示KO小鼠與WT小鼠腸道菌分布並沒有明顯分離，若將公鼠與母鼠分週數各別分析的話，公鼠各週數兩組間皆沒有顯著差異，母鼠則在24周齡時KO小鼠與WT小鼠的腸道菌顯著分離。接著進行物種豐富度的分析，發現只有12周齡的WT母鼠物種豐富度顯著高於20周齡，其餘各組皆無顯著差異。物種均勻度則只有8周齡的KO公鼠顯著高於24周齡，其餘組別沒有顯著差異。在物種多樣性方面各組皆無顯著差異。最後利用LEfSe (Linear discriminant analysis (LDA) Effect Size)發現不論是公鼠還是母鼠，KO小鼠與WT小鼠間還是具有幾種有差異的細菌，表示結腸上皮細胞缺乏類鐸受體4 雖然對於小鼠腸道菌的物種豐富度、均勻度與多樣性沒有太大的影響，但仍然可以使部分菌種增加或下降。 我們的實驗結果與先前將整段腸道上皮細胞中Tlr4剔除的實驗結果相差甚遠，可能是因為Tg(Car1-Cre)小鼠的剔除效率不佳，如文獻所示Car1在結腸組織呈馬賽克狀分布，僅表現於部分的結腸上皮細胞，而非所有上皮細胞，從而影響Tlr4的剔除效率，導致僅少數結腸上皮細胞中的Tlr4被剔除。儘管剔除Tlr4未對腸道菌群產生顯著影響，仍觀察到部分菌種表現差異，若是進一步給予其他刺激(如高脂飲食)或是誘導疾病模型，可能會造成腸道菌群有更顯著的變化，並影響其他病生理表現，此部分值得未來深入探討。

The human gut hosts trillions of microorganisms, including bacteria, viruses, and parasites, with the majority residing in the colon. These microorganisms play a crucial role in digestion, absorption, metabolism, and immune function. They can also regulate neural transmission through the gut-brain axis. Numerous studies have shown that imbalances in gut microbiota, known as dysbiosis, are closely associated with various health conditions, ranging from inflammation and degenerative disorders to systemic diseases. Therefore, maintaining a healthy gut microbiota is essential. The activation of the lipopolysaccharide (LPS)/Toll-like receptor 4 (TLR4) signaling pathway has been identified as a key pathogenic mechanism underlying gut inflammation and leaky gut, potentially increasing the risk of distant organ diseases in patients with gut microbiota imbalances. Previous research utilized villin as a promoter to specifically delete Toll-like receptor 4 (Tlr4) in intestinal epithelial cells. Experimental results revealed that their microbial clusters differed. Additionally, mice lacking Tlr4 in intestinal epithelial cells (TLR4△IEC) exhibited lower α-diversity compared to control mice (Wild Type, WT), indicating a reduction in microbial richness upon deletion of Tlr4 in intestinal epithelial cells. Significant functional alterations in the gut microbiota were also observed, particularly affecting lipid, amino acid, and nucleotide metabolism. Furthermore, the absence of Tlr4 in intestinal epithelial cells led to the development of metabolic syndrome in mice. Compared to WT mice, TLR4△IEC mice showed significant weight gain, increased fat deposition, and glucose intolerance. Given that most human gut microbiota resides in the colon and that future research aims to investigate the gut-kidney axis, this study aims to establish a colon-specific Tlr4 knockout mouse. This model will enable the examination of changes in gut microbiota and the evaluation of their potential pathophysiological impacts. First, the Tg(Car1-cre); Rosa26fstdTomato/+ mouse model was used to confirm the functional activity of the Car1-cre transgenic system. According to the original study that developed the Tg(Car1-cre) mouse, Car1 expression is predominantly localized in the colon but also slightly expressed in the liver. Given our future research focus on renal studies, we utilized immunofluorescence (IF) staining to confirm Car1 expression in the liver and kidneys. The results showed that Car1 is expressed in hepatocytes, as well as in endothelial cells and pericytes within the kidney. The experiment was then conducted using Tg(Car1-cre); Tlr4F/F mice. Since Car1 is expressed in cells within the liver and kidney, plasma biochemical markers associated with liver and kidney function were analyzed. Among these markers, only the plasma albumin levels in female mice in the experimental group (KO) were significantly higher than those in the control group (WT), while all other plasma biochemical parameters showed no significant differences. Given that TLR4△IEC mice exhibited significantly higher body weights compared to WT mice, we explored whether KO mice would display a similar trend. The body weights of male and female mice were recorded from 6-week-old to 24-week-old. No significant differences in body weight were observed between KO and WT mice for either sex. Interestingly, KO mice showed a lower tendency for body weight compared to WT mice. To investigate this, body composition analysis was conducted at 24 weeks old. The results indicated no significant differences between KO and WT mice in fat mass, lean body mass (LBM), free fluid content, total water, or the percentages of these components relative to body weight in both sexes. The oral glucose tolerance test (OGTT) was performed to evaluate glucose metabolism. All mice exhibited similar blood glucose responses following glucose administration. Next, to assess the impact of colonic epithelial Tlr4 deletion on gut microbiota, fecal samples were collected at 8, 12, 16, 20,and 24 weeks of age for analysis. Principal Coordinates Analysis (PCoA) demonstrated no distinct separation in microbiota composition between KO and WT mice overall. However, when analyzed by sex and age, male mice showed no significant differences between groups at any age, whereas female mice displayed a significant separation in microbiota composition at 24 weeks of age between KO and WT mice. Community richness analysis revealed that only 12-week-old WT females had significantly higher richness than 20-week-old WT females. No significant differences in richness were observed in other groups. For community evenness, only 8-week-old KO males exhibited significantly higher evenness compared to 24-week-old KO males, with no other significant differences. Regarding community diversity, no significant differences were found across any group or age. Finally, LEfSe identified several bacterial taxa with differential abundances between KO and WT mice, in both sexes. These findings suggest that while colonic epithelial Tlr4 deletion has minimal effects on overall community richness, evenness, and diversity, it can alter the abundance of specific bacterial taxa, resulting in upregulation or downregulation in certain species. Our results differ from previous studies that deleted Tlr4 in the entire intestinal epithelium, likely due to the low efficiency of Tg(Car1-Cre) mice. Car1 exhibits a mosaic expression pattern in colonic epithelial cells, limiting Tlr4 deletion to a subset of cells. While Tlr4 deletion showed no significant impact on gut microbiota, some bacterial taxa exhibited differences. Maybe using additional stimulation, such as high-fat diets or disease models, may reveal more pronounced microbial and physiological changes.