Department of Surgery, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI, USA.
Surgery. 2013 Jan;153(1):17-24. doi: 10.1016/j.surg.2012.04.001. Epub 2012 Jun 13.
Parenteral nutrition (PN) increases infectious risk in critically ill patients compared with enteral feeding. Previously, we demonstrated that PN feeding suppresses the concentration of the Paneth cell antimicrobial protein secretory phospholipase A2 (sPLA2) in the gut lumen. sPLA2 and other Paneth cell proteins are released in response to bacterial components, such as lipopolysaccharide (LPS), and they modulate the intestinal microbiome. Because the Paneth cell protein sPLA2 was suppressed with PN feeding, we hypothesized PN would diminish the responsiveness of the small bowel to LPS through reduced secretions and as a result exhibit less bactericidal activity.
The distal ileum was harvested from Institute of Cancer Research mice, washed, and randomized for incubation with LPS (0, 1, or 10 μg/mL). Culture supernatant was collected and sPLA2 activity was measured. Bactericidal activity of the ileum segment secretions was assessed against Pseudomonas aeruginosa with and without an sPLA2 inhibitor at 2 concentrations, 100 nmol/L and 1 μmol/L. Institute of Cancer Research mice were randomized to chow or PN for 5 days. Tissue was collected for immunohistochemistry (IHC) and ileal segments were incubated with LPS (0 or 10 μg/mL). sPLA2 activity and bactericidal activity were measured in secretions from ileal segments.
Ileal segments responded to 10 μg/mL LPS with significantly greater sPLA2 activity and bactericidal activity. The bactericidal activity of secretions from LPS stimulated tissue was suppressed 50% and 70%, respectively, with the addition of the sPLA2-inhibitor. Chow displayed greater sPLA2 in the Paneth cell granules and secreted higher levels of sPLA2 than PN before and after LPS. Accordingly, media collected from chow was more bactericidal than PN. IHC confirmed a reduction in Paneth cell granules after PN.
This work demonstrates that ileal segments secrete bactericidal secretions after LPS exposure and the inhibition of the Paneth cell antimicrobial protein sPLA2 significantly diminishes this. PN feeding resulted in suppressed secretion of the sPLA2 and resulted in increased bacterial survival. This demonstrates that PN significantly impairs the innate immune response by suppressing Paneth cell function.
与肠内喂养相比，肠外营养（PN）会增加危重症患者的感染风险。此前，我们已经证明 PN 喂养会抑制肠道腔中潘氏细胞抗菌蛋白分泌型磷脂酶 A2（sPLA2）的浓度。sPLA2 和其他潘氏细胞蛋白会响应细菌成分（如脂多糖[LPS]）而释放，并调节肠道微生物组。由于 PN 喂养会抑制潘氏细胞蛋白 sPLA2 的分泌，我们假设 PN 喂养会通过减少分泌来降低小肠对 LPS 的反应性，因此表现出较弱的杀菌活性。
从研究所癌症研究小鼠的回肠末端采集组织，清洗并随机孵育 LPS（0、1 或 10μg/ml）。收集培养上清液并测量 sPLA2 活性。使用两种浓度（100nmol/L 和 1μmol/L）的 sPLA2 抑制剂评估回肠段分泌物对铜绿假单胞菌的杀菌活性。将研究所癌症研究小鼠随机分为标准饮食组或 PN 组，进行 5 天喂养。收集组织进行免疫组织化学（IHC）分析，并孵育 LPS（0 或 10μg/ml）。测量回肠段分泌物中的 sPLA2 活性和杀菌活性。
10μg/ml LPS 刺激回肠段产生明显更高的 sPLA2 活性和杀菌活性。添加 sPLA2 抑制剂后，LPS 刺激组织的杀菌活性分别抑制了 50%和 70%。标准饮食组的潘氏细胞颗粒中 sPLA2 含量更高，且在 LPS 刺激前后分泌的 sPLA2 水平均高于 PN 组。因此，标准饮食组的培养基比 PN 组具有更强的杀菌活性。IHC 证实 PN 喂养后潘氏细胞颗粒减少。
这项工作表明，回肠段在 LPS 暴露后会分泌杀菌分泌物，而潘氏细胞抗菌蛋白 sPLA2 的抑制会显著降低这种分泌。PN 喂养会导致 sPLA2 分泌减少，从而导致细菌存活率增加。这表明 PN 喂养通过抑制潘氏细胞功能显著损害了固有免疫反应。
