LIGHT/TNFSR14 can regulate hepatic lipase expression by hepatocytes independent of T cells and Kupffer cells

Abstract

LIGHT/TNFSF14 is a costimulatory molecule expressed on activated T cells for activation and maintenance of T cell homeostasis. LIGHT over expressed in T cells also down regulates hepatic lipase levels in mice through lymphotoxin beta receptor (LTβR) signaling. It is unclear whether LIGHT regulates hepatic lipase directly by interacting with LTβR expressing cells in the liver or indirectly by activation of T cells, and whether Kupffer cells, a major cell populations in the liver that expresses the LTβR, are required. Here we report that LIGHT expression via an adenoviral vector (Ad-LIGHT) is sufficient to down regulate hepatic lipase expression in mice. Depletion of Kupffer cells using clodronate liposomes had no effect on LIGHT-mediated down regulation of hepatic lipase. LIGHT-mediated regulation of hepatic lipase is also independent of LIGHT expression by T cells or activation of T cells. This is demonstrated by the decreased hepatic lipase expression in the liver of Ad-LIGHT infected recombination activating gene deficient mice that lack mature T cells and by the Ad-LIGHT infection of primary hepatocytes. Hepatic lipase expression was not responsive to LIGHT when mice lacking LTβR globally or only on hepatocytes were infected with Ad-LIGHT. Therefore, our data argues that interaction of LIGHT with LTβR on hepatocytes, but not Kupffer cells, is sufficient to down regulate hepatic lipase expression and that this effect can be independent of LIGHT's costimulatory function.

Figure 1. LIGHT-mediated HL regulation in mice is independent of the presence of Kupffer cells.

To deplete Kupffer cells, liposomes containing clodronate were injected through the tail vein into wild type C57BL/6 mice or Tg-LIGHT mice every 5^th day for 14 days. Control liposomes did not contain clodronate. (A) F4/80 staining for liver Kupffer cells in control liposome and clodronate liposome injected Tg-LIGHT mice. (B) Real-time PCR data for HL mRNA expression in the liver of control (C) and clodronate (CL) liposome injected wild type (WT) and Tg-LIGHT mice. (n = 3; *p<0.01 WT vs. Tg-LIGHT).

Figure 2. Decreased HL expression in Ad-LIGHT infected mice is independent of T cells and IL-1β.

Mice were injected with PBS or adenoviral vectors (1.25×10⁹ pfu/mouse) and sacrificed on the 7^th day. (A) Liver HL mRNA expression in LIGHT adenovirus (Ad-L), human apoA-I adenovirus (Ad-AI) or PBS (P) injected LDLR^−/− mice was analyzed by real time PCR. (B) Morphology of liver from PBS and adenoviral infected mice (H & E staining, 20x objective). (C) Liver HL mRNA expression in Ad-L and P injected RAG^−/−LDLR^−/− and LIGHT^−/−LDLR^−/− mice. (D) Liver HL and IL-1β mRNA expression in Ad-L injected LDLR^−/− mice treated with control (C) or clodronate (CL) liposomes. The virus was injected 2 days after clodronate injection. (n = 3; *p<0.05, **p<0.01; for panels A and C: vs. PBS treated mice; for Panel D: vs. control liposome treated mice.).

Figure 3. LTβR expression on hepatocytes is sufficient for HL regulation by Ad-LIGHT.

(A) Liver LTβR and HVEM expression in LDLR^−/−, HVEM^−/−LDLR^−/− and hepatocyte-specific knockout of LTβR (H-LTβR^−/−) LDLR^−/− mice. (B and C) Mice were injected with PBS or adenovirus and sacrificed on the 7^th day. Liver HL mRNA expression in Ad-LIGHT (Ad-L) and PBS (P) injected LTβR^−/−LDLR^−/− mice (B) and (H-LTβR^−/−LDLR^−/− and HVEM^−/−LDLR^−/− mice C). (n = 3; **p<0.01 vs. PBS injected mice).

Figure 4. LIGHT and HL expression in Ad-LIGHT transduced primary hepatocytes in vitro.

(A) HL mRNA expression in Ad-LIGHT (Ad-L) and Null adenovirus (Ad-N) infected primary hepatocytes from wild type mice 18, 24 and 36 hours post-infection. The numbers indicate the % decrease in expression in the Ad-LIGHT infected cells relative to the Ad-N infected cells. (B) Total protein from 1×10⁶ Ad-LIGHT infected hepatocytes (in triplicate) at various times post-infection were immunoblotted with anti-mouse LIGHT antibody. Lane 12 is from non-infected hepatocytes and lane 13 is from Tg-LIGHT mouse spleen. (C) Liver HL mRNA expression in Ad-N and Ad-L infected hepatocytes from LDLR^−/− and LTβR^−/−LDLR^−/− mice. (n = 3; *p<0.05, **p<0.01 vs. Ad-N).

Figure 5. Evidence for trans-regulation by LIGHT and lack of a role of liver non-parenchymal cells.

(A) HL mRNA expression in hepatocytes from LDLR^−/− (WT) and LTβR^−/−LDLR^−/− mice cocultured with Ad-LIGHT (Ad-L) or Ad-Null (Ad-N) infected FL83B cells. (B) HL mRNA expression in hepatocytes from LTβR^−/−LDLR^−/− mice cocultured with Ad-N or Ad-L infected FL83B cells and Tg-LIGHT mouse liver non-parenchymal cells (NPC). (n = 3; *<0.05 vs. Ad-N infected FL83B coculture).