Etanercept blocks inflammatory responses orchestrated by TNF-α to promote transplanted cell engraftment and proliferation in rat liver

Abstract

Engraftment of transplanted cells is critical for liver-directed cell therapy, but most transplanted cells are rapidly cleared from liver sinusoids by proinflammatory cytokines/chemokines/receptors after activation of neutrophils or Kupffer cells (KCs). To define whether tumor necrosis factor alpha (TNF-α) served roles in cell-transplantation-induced hepatic inflammation, we used the TNF-α antagonist, etanercept (ETN), for studies in syngeneic rat hepatocyte transplantation systems. After cell transplantation, multiple cytokines/chemokines/receptors were overexpressed, whereas ETN before cell transplantation essentially normalized these responses. Moreover, ETN down-regulated cell-transplantation-induced intrahepatic release of secretory cytokines, such as high-mobility group box 1. These effects of ETN decreased cell-transplantation-induced activation of neutrophils, but not of KCs. Transplanted cell engraftment improved by several-fold in ETN-treated animals. These gains in cell engraftment were repeatedly realized after pretreatment of animals with ETN before multiple cell transplantation sessions. Transplanted cell numbers did not change over time, indicating absence of cell proliferation after ETN alone. By contrast, in animals preconditioned with retrorsine and partial hepatectomy, cell transplantation after ETN pretreatment significantly accelerated liver repopulation, compared to control rats.

Conclusion: TNF-α plays a major role in orchestrating cell-transplantation-induced inflammation through regulation of multiple cytokines/chemokines/receptor expression. Because TNF-α antagonism by ETN decreased transplanted cell clearance, improved cell engraftment, and accelerated liver repopulation, this pharmacological approach to control hepatic inflammation will help optimize clinical strategies for liver cell therapy.

Figure 1. Effect of ETN on transplanted cell engraftment

(A) Protocol for analyzing gene expression, cell engraftment and tissue changes in rats after cell transplantation alone or cell transplantation after treatment of rats with ETN or vehicle. Studies used 3–6 rats per time-point. (B) RT-PCR showing cell transplantation rapidly and persistently induced hepatic expression of TNF. Lane 1, untreated control liver sample and lanes 2–4, liver samples 6, 24 and 48 h after cell transplantation. (C) Representative liver sections showing DPPIV+ transplanted hepatocytes (red color) 1 d or 7 d after cell transplantation in controls and ETN-treated rats. Orig. mag., ×100. (D) Morphometric data showed cell engraftment was superior in ETN-treated rats at all times.

Figure 2. Differences in hepatic expression of chemokines/cytokines/receptors after cell transplantation

(A) Graphic depiction indicating 25 of 84 genes were upregulated after cell transplantation versus 3 of 84 genes in animals after ETN and cell transplantation. (B) Distribution of upregulated genes in ontology groups. (C) Differences in normalized expression against housekeeping genes of cytokines/chemokines/receptors after cell transplantation with or without prior ETN. Genes indicated in bold letters were upregulated only in ETN-treated rats after cell transplantation.

Figure 3. Cell transplantation-induced activation of inflammatory cells and soluble cytokine expression

(A) Cell transplantation increased number of MPO+ PMN compared with untreated control rats but this increase in MPO+ PMN was less in ETN-treated rats. (B) KC showed greater carbon uptake after cell transplantation. This was unchanged in ETN-treated rats. (C) Western blot for serum HMGB1 showed increased expression after GalN and also cell transplantation. In ETN-treated rats, serum HMGB1 was not detected. Lane 1, HMGB1 protein; lanes 2–4, animals 5 d after GalN as positive controls; lanes 5–10, replicate animals 6, 24 and 48 h after cell transplantation; lanes 11–12, replicate ETN-treated animals 24 h after cell transplantation with no HMGB1. (D) Morphometric data showing cell engraftment did not increase in nicotine-treated animals (n=3 ea).

Figure 4. ETN and repeated cell transplantation

(A) Protocol for administering ETN each time before 3 cell transplantation thrice at intervals followed by cell engraftment analysis in 3–6 rats after 38 d. (B) Representative tissue examples showing cell engraftment after 3 sessions of cell transplantation in control rat and ETN-treated rat with the latter showing more DPPIV+ transplanted cells (red color). (C) Morphometric analysis showing significant differences in number of transplanted cells in animals after only one session of cell transplantation versus after 3 sessions of cell transplantation with or without prior ETN treatments.

Figure 5. Effect of ETN on kinetics of liver repopulation

(A) Protocol indicating use of retrorsine and PH preconditioning for liver repopulation analysis. Animals were given cells followed by analysis of liver repopulation after 1 mo and 3 mo. (B) Tissue staining for DPPIV showing extent of liver repopulation in control rat and ETN-treated rat after 3 mo. (C) Morphometric data indicating liver repopulation was significantly greater after 1 mo as well as 3 mo in ETN-treated rats.

Figure 6. Antagonism of TNF in cultured primary rat hepatocytes

(A) Shows protocols for studies of cells including cytotoxicity assays in the short-term in vitro as well as transplantation of ETN-treated cells for engraftment analysis after 1 week. (B) Chart indicates abrogation of TNF cytotoxicity when cells were cultured after incubation for 1 h with ETN. (C) Engraftment of control DPPIV+ transplanted cells and those cells treated with ETN in vitro. (D) Morphometric analysis of cell engraftment indicating no benefit of ETN treatment of cells in vitro.