Supplementary Material for: Murine Ribonuclease 6 Limits Bacterial Dissemination During Experimental Urinary Tract Infection

Introduction: The Ribonuclease (RNase) A superfamily encodes cationic antimicrobial proteins with potent microbicidal activity toward uropathogenic bacteria. Ribonuclease 6 (RNase6) is an evolutionarily conserved, leukocyte-derived antimicrobial peptide with potent microbicidal activity toward uropathogenic Escherichia coli (UPEC), the most common cause of bacterial urinary tract infections (UTI). In this study, we generated Rnase6 deficient mice to investigate the hypothesis that endogenous RNase 6 limits host susceptibility to UTI.

Methods: We generated a Rnase6EGFP knock-in allele to identify cellular sources of Rnase6 and determine the consequences of homozygous Rnase6 deletion on antimicrobial activity and UTI susceptibility.

Results: We identified monocytes and macrophages as the primary cellular sources of Rnase6 in bladders and kidneys of Rnase6EGFP/+ mice. Rnase6 deficiency (i.e., Rnase6EGFP/EGFP) resulted in increased upper urinary tract UPEC burden during experimental UTI, compared to Rnase6+/+ controls. UPEC displayed increased intracellular survival in Rnase6 deficient macrophages.

Conclusion: Our findings establish that RNase6 prevents pyelonephritis by promoting intracellular UPEC killing in monocytes and macrophages and reinforce the overarching contributions of endogenous antimicrobial RNase A proteins to host UTI defense.

引言：核糖核酸酶（RNase）A超家族编码一类对尿路致病菌具有强效杀菌活性的阳离子抗菌蛋白。核糖核酸酶6（RNase6）是一类进化保守、白细胞源性的抗菌肽，对尿路致病性大肠杆菌（UPEC）——细菌性尿路感染（UTI）最常见的致病菌——具有强效杀菌活性。本研究构建了Rnase6基因缺陷小鼠，以验证内源性RNase6可降低宿主对UTI易感性这一假说。
方法：我们构建了Rnase6EGFP敲入等位基因，用于定位Rnase6的细胞来源，并探究纯合子Rnase6缺失对抗菌活性以及UTI易感性的影响。
结果：在Rnase6EGFP/+小鼠的膀胱与肾脏组织中，单核细胞与巨噬细胞是Rnase6的主要细胞来源。与Rnase6+/+野生型对照组相比，Rnase6缺陷（即Rnase6EGFP/EGFP）小鼠在实验性UTI模型中出现上尿路UPEC载量升高的现象。UPEC在Rnase6缺陷巨噬细胞内的存活能力显著增强。
结论：本研究结果证实，RNase6可通过促进单核细胞与巨噬细胞内UPEC的杀伤作用，从而阻止肾盂肾炎的发生；同时进一步明确了内源性抗菌RNase A超家族蛋白在宿主UTI防御中的核心作用。