Introduction: In response to chronic peritonitis from Klebsiella pneumoniae, we have observed peritoneal macrophage reprogramming that is dependent on intraperitoneal (IP) concentration of bacteria over time. Such tolerance may lead to resolution of the early innate response, and may be responsible for incomplete bacterial clearance and a chronic inflammatory state. The hypothesis is that LPS pre-treatment of chronic peritonitis will induce early macrophage tolerance at the transcriptional level.

Methods: 24 hours after IP LPS (.01mg/g) or saline, C57BL/6 mice were given 103 colony forming units of K. pneumoniae IP. Gentamicin was then given BID. Peritoneal exudate cells were obtained through peritoneal lavage and RNA was isolated (n=3) at 4, 24, and 48 hours following infection. SA Bioscience RT2 Profiler PCR array mouse Toll-like receptor signaling pathway (PAMM_018A) data were analyzed by Ingenuity Pathway Inc. analysis (IPA). Student’s t-tests were used to calculate fold change and IPA for pathway significance.

Results: Of the 84 genes studied, 26 were significantly dysregulated (fold change>2.0 and p-value<0.05) in the saline-pretreated group at 4, 24, and 48 hours. All of the dysregulated genes were up regulated. In the LPS pretreated group, 35 genes were significantly up regulated (fold change>2.0 and p-value <0.05). Of the 35 significantly regulated genes in LPS pretreated mice, 13 (Cd86, Clec4e, Fos, Ikbkb, Il1a, Nfkb1, Nfkbia, Nr2c2, Pglyrp1, Rel, Rela, Ripk2, and Tlr2) were not increased the saline group. This left 22 genes dysregulated in common. At 4 Hours, of these 22 genes 6, (Chuck, Hmgb1, Hspd1, Irak2, Ly96, and Tlr4) were further 2 fold increased in the LPS pretreatment compared with saline pretreatment. Only Irak2 is 2 fold increased at 24 hours. By 48 hours no LPS effect was seen. The 22 common regulated genes are: Btk, Cd80, Chuck, Hmgb1, Hras1, Hrb, Hspd1, Irak2, Jun, Ly96, Map3k7, Peli, Eif2ak2, Ticam, Tirap, Tlr4, Tlr6, Tlr7, Tlr9, Tnfrsf1a, Traff6, and Ube2v1. When applying IPA analysis to the above data, 6 main canonical pathways were constantly dysregulated, and in the same significance order, in both the saline and LPS group at 4, 24, and 48 hours. These are: Toll-like receptor and NF-KB signaling, hepatic cholestasis, interleukin-6 and LPS mediated MAPK signaling pathways, and pattern recognition receptors of bacteria pathway.

Conclusion: Peritonitis increased PEC gene expression associated with sepsis and a pro-inflammatory response, which was further augmented by LPS pretreatment over 24 hours only. Prior exposure to LPS did not induce PEC gene tolerance to subsequent infection with Klebsiella. Up regulated pathways were common to the early innate response, and macrophage reprogramming occurs at the genetic level. Lack of bacterial clearance in this model is due to an inadequate innate response which resolves despite incomplete clearance of intraperitoneal bacteria.