Gowda Ranjita N, Redfern Rachel, Frikeche Jihane, Pinglay Sudarshan, Foster James William, Lema Carolina, Cope Leslie, Chakravarti Shukti
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, United States of America.
The Ocular Surface Institute, College of Optometry, University of Houston, Houston, TX, United States of America.
PLoS One. 2015 Sep 2;10(9):e0137129. doi: 10.1371/journal.pone.0137129. eCollection 2015.
Functions of antimicrobial peptidoglycan recognition proteins (Pglyrp1-4) at the ocular surface are poorly understood. Earlier, we reported an antibacterial role for Pglyrp-1 in Pseudomonas aeruginosa keratitis. Here we investigated functions of three other related genes Pglyrp-2, -3 and -4 in a mouse model of P. aeruginosa keratitis.
Wild type (WT) and each of the Pglyrp-null genotypes were challenged with P. aeruginosa keratitis. The eyes were scored in a blinded manner 24 and 48h post infection. Viable bacterial counts and inflammatory factors (IL-12, TNF-α, IFN-γ, CCL2, IL-6 and IL-10) were measured in whole eye homogenates using cytometric bead arrays. Expressions of Pglyrp-1-4, mouse beta defensins (mBD)-2,-3, cathelicidin-related antimicrobial peptide (CRAMP) were determined by qRTPCR in total RNA extracts of uninfected and infected eyes of WT and each of the Pglyrp-null mouse types.
The Pglyrp-2-/- mice showed reduced disease and lower induction of pro-inflammatory TNF-α (p = 0.02) than WT or the other Pglyrp null mice. Viable bacterial yield was significantly lower in the Pglyrp-2-/- (p = 0.0007) and the Pglyrp-4-/- (p = 0.098) mice. With regards to expression of these antimicrobial genes, Pglyrp-2 expression was induced after infection in WT mice. Pglyrp-3 expression was low before and after infection in WT mice, while Pglyrp-4 expression was slightly elevated after infection in WT, Pglyrp-2 and -3 null mice. Pglyrp-1 expression was slightly elevated after infection in all genotypes without statistical significance. Transcripts for antimicrobial peptides mBD2, mBD3 and CRAMP were elevated in infected Pglyrp-2-/- males without statistical significance.
Efficient resolution of keratitis in the Pglyrp-2-/- mice may be due to a reduced pro-inflammatory microenvironment and synergistic antibacterial activities of defensins, CRAMP and Pglyrp-1. Therefore, in ocular infections the pro-inflammatory functions of Pglyrp-2 must be regulated to benefit the host.
人们对眼表抗菌肽聚糖识别蛋白（Pglyrp1 - 4）的功能了解甚少。此前，我们报道了Pglyrp - 1在铜绿假单胞菌角膜炎中的抗菌作用。在此，我们在铜绿假单胞菌角膜炎小鼠模型中研究了其他三个相关基因Pglyrp - 2、- 3和- 4的功能。
野生型（WT）和每种Pglyrp基因敲除型小鼠均感染铜绿假单胞菌角膜炎。在感染后24小时和48小时以盲法对眼睛进行评分。使用细胞计数珠阵列测定全眼球匀浆中的活菌计数和炎症因子（IL - 12、TNF -α、IFN -γ、CCL2、IL - 6和IL - 10）。通过qRTPCR测定WT和每种Pglyrp基因敲除型小鼠未感染和感染眼睛的总RNA提取物中Pglyrp - 1 - 4、小鼠β - 防御素（mBD）- 2、- 3、cathelicidin相关抗菌肽（CRAMP）的表达。
与WT或其他Pglyrp基因敲除型小鼠相比，Pglyrp - 2 - / - 小鼠的病情减轻，促炎因子TNF -α的诱导水平降低（p = 0.02）。Pglyrp - 2 - / - 小鼠（p = 0.0007）和Pglyrp - 4 - / - 小鼠（p = 0.098）的活菌产量显著降低。关于这些抗菌基因的表达，WT小鼠感染后Pglyrp - 2表达被诱导。WT小鼠感染前后Pglyrp - 3表达均较低，而WT、Pglyrp - 2和- 3基因敲除型小鼠感染后Pglyrp - 4表达略有升高。所有基因型小鼠感染后Pglyrp - 1表达均略有升高，但无统计学意义。感染的Pglyrp - 2 - / - 雄性小鼠中抗菌肽mBD2、mBD3和CRAMP的转录本升高，但无统计学意义。
Pglyrp - 2 - / - 小鼠角膜炎的有效消退可能是由于促炎微环境的减少以及防御素、CRAMP和Pglyrp - 1的协同抗菌活性。因此，在眼部感染中，必须调节Pglyrp - 2的促炎功能以利于宿主。
