Role of LPS/TLR4 and complement signaling in liver regeneration after partial hepatectomy

Abstract

Liver regeneration after partial hepatectomy (PH) is a complex process involving an inflammatory response that is followed by the proliferation of both parenchymal and nonparenchymal liver cells to restore the lost liver mass. The production of the proinflammatory cytokines TNF- and IL-6 is an essential component of hepatocyte priming. There is evidence that LPS plays a role in the initiation of liver regeneration, but the mechanism remains to be elucidated. The LPS hyporesponsive C3H/HeJ mouse, which has a mutation in the LPS receptor, TLR4, shows delayed liver regeneration. In addition to signaling via TLR4, LPS also activates the Complement pathway. There is evidence that modulation of the Complement pathway can also affect liver regeneration. LPS signaling via either TLR4 or activation of the Complement pathway results in the production of IL-6 and TNF-. The aim of this study was to clarify the mechanism of LPS signaling in liver regeneration after PH through the use of mice lacking functional TLR4 (C3H/HeJ), inhibition of the Complement pathway via the blockade of receptors for key Complement components (C3a and C5a), the blockade of TLR4 and via the depletion of LPS in the gut using antibiotics. Furthermore, examined the liver transcriptome before and after PH to identify expression patterns that may contribute to the delayed regeneration seen in C3H/HeJ mice. We measured liver weight/body weight ratio (LW/BW), hepatocyte proliferation (mitotic index and Ki67 immunohistochemistry), plasma cytokines (TNF- and IL-6), Complement activation products (C3a and C5a) and expression of downstream signaling components (STAT3 and Cyclin D1) following PH in wild-type mice (C3H/HeN), LPS hyporesponsive mice (C3H/HeJ mice) and mice of both strains treated with C3a and/or C5a receptor blockers. Liver regeneration was delayed in C3H/HeJ mice compared to mice with functional TLR4 (C3H/HeN), indicated by a reduced LW/BW at all time-points until 28 days, and reduced proliferation, this delay was accompanied by reduced TNF- and IL-6 levels and reduced STAT3 and Cyclin D1 expression. The use of C3a and/or C5a receptor antagonists delayed liver regeneration in both C3H/HeJ and C3H/HeN mice, and reduced cytokine levels compared to their untreated counterparts accompanied this delay. There was almost complete suppression of regeneration in the C3H/HeJ mice treated with both C3a and C5a receptor antagonists, and this group of mice had no detectable expression of STAT3 or Cyclin D1 at 3 days post-PH. However, we demonstrated Complement activation, assessed by plasma levels of C3a and C5a for the first time the, was unchanged by either a lack of a functional TLR4 or by C3a and/or C5a receptor blockade. We then further investigated the role of the LPS/TLR4 pathway in liver regeneration through the use of a monoclonal anti-TLR4 mAb (MTS-510) or the depletion of gut-derived LPS via antibiotic treatment in C3H/HeN (wild-type) mice. We found that markers of hepatocyte proliferation (PCNA and mitotic figures) were reduced in the MTS-510 treated mice to levels similar to those seen in C3H/HeJ mice, and plasma levels of IL-6 and TNF- were also significantly lower than in the untreated C3H/HeN mice. Similarly, liver regeneration was reduced in antibiotic-treated C3H/HeN mice, with significantly reduced LW/BW and proliferation markers, indicating that gut-derived LPS is required for normal liver regeneration after PH. Finally, we used RNA-seq for the first time to assess differences in the transcriptome between C3H/HeN and C3H/HeJ mice at baseline, and also in both the early phase (6-hour) and late phase (48-hour) after PH in an attempt to identify possible contributors to the delayed proliferative response to PH seen in C3H/HeJ mice. In this analysis, we found that C3H/HeJ mice livers have distinct gene expression signature when compared to C3H/HeN mice. However post-PH, significantly more number of genes in liver transcriptome were active in were active at 6- hours in C3H/HeN while more number of genes were active at 48-hours in C3H/HeJ mice. This shows the delayed onset of gene expression in C3H/HeJ mice. In addition to this, many genes in functional groups implicated in liver regeneration like TNF, TGF, IL6/STAT3, cell cycle, wound healing, etc. were highly upregulated at 48-hours in C3H/HeJ, but not in C3H/HeN. This pattern explains the delayed regenerative response in C3H/HeJ mice. In conclusion, this study has demonstrated that both the Complement pathway and the LPS/TLR4 pathways contribute to liver regeneration following PH. In the absence of either pathway, liver regeneration is delayed but the lost mass is eventually restored. However, if both pathways are impaired, and therefore LPS cannot activate either signaling mechanism, then the regenerative response is almost completely suppressed.