Ron receptor-dependent gene regulation of Kupffer cells during endotoxemia

Abstract

Background: Ron receptor tyrosine kinase signaling in macrophages, including Kupffer cells and alveolar macrophages, suppresses endotoxin-induced proinflammatory cytokine/chemokine production. Further, we have also identified genes from Ron replete and Ron deplete livers that were differentially expressed during the progression of liver inflammation associated with acute liver failure in mice by microarray analyses. While important genes and signaling pathways have been identified downstream of Ron signaling during progression of inflammation by this approach, the precise role that Ron receptor plays in regulating the transcriptional landscape in macrophages, and particular in isolated Kupffer cells, has still not been investigated.

Methods: Kupffer cells were isolated from wild-type (TK+/+) and Ron tyrosine kinase deficient (TK-/-) mice. Ex vivo, the cells were treated with lipopolysaccharide (LPS) in the presence or absence of the Ron ligand, hepatocyte growth factor-like protein (HGFL). Microarray and qRT-PCR analyses were utilized to identify alterations in gene expression between genotypes.

Results: Microarray analyses identified genes expressed differentially in TK+/+ and TK-/- Kupffer cells basally as well as after HGFL and LPS treatment. Interestingly, our studies identified Mefv, a gene that codes for the anti-inflammatory protein pyrin, as an HGFL-stimulated Ron-dependent gene. Moreover, lipocalin 2, a proinflammatory gene, which is induced by LPS, was significantly suppressed by HGFL treatment. Microarray results were validated by qRT-PCR studies on Kupffer cells treated with LPS and HGFL.

Conclusion: The studies herein suggest a novel mechanism whereby HGFL-induced Ron receptor activation promotes the expression of anti-inflammatory genes while inhibiting genes involved in inflammation with a net effect of diminished inflammation in macrophages.

Figure 1. Global changes in gene expression in TK+/+ Kupffer cells compared to TK−/− cells prior to and after treatment with LPS and HGFL

TK+/+ and TK−/− Kupffer cells were untreated (-) or treated with LPS for 30 minutes, HGFL 60 minutes, or overnight HGFL followed by 30 minutes LPS in duplicate. RNA was isolated and subjected to microarray analysis. 935 genes were identified which changed significantly over untreated TK+/+ control RNA and are depicted in the heat map.

Figure 2. Gene expression changes in Kupffer cells after LPS and HGFL treatments

(A) Heat map of 24 genes differentially expressed between TK+/+ and TK−/− Kupffer cells in the absence of treatment (basal changes). (B) Heat map of 43 genes differentially expressed in TK+/+ Kupffer cells after LPS treatment for 30 minutes. (C) Heat map of 56 genes differentially expressed in TK−/− Kupffer cells after LPS treatment for 30 minutes. (D) Heat map of 13 genes differentially expressed in TK+/+ Kupffer cells after HGFL treatment for 1 hour. Gene ontology function of the web-based program, Panther, was used to classify the genes that were upregulated or down regulated basally or after each treatment into different biological processes categories (A–D).

Figure 3. Differential gene expression in Kupffer cells with LPS and HGFL treatment

(A) Venn diagram representing genes induced after LPS treatment in TK−/− compared to TK+/+ Kupffer cells. Ten unique genes were induced in TK−/− but suppressed in TK+/+ Kupffer cells after LPS treatment. (B) Venn diagram representing genes suppressed after LPS treatment in TK−/− compared to TK+/+ Kupffer cells. Analyses identified 13 unique genes that were suppressed in TK−/− Kupffer cells but were induced in TK+/+ cells after LPS treatment. (C) Venn diagram representing genes changed in TK+/+ Kupffer cells after LPS and/or HGFL treatment. Three unique genes were induced in Kupffer cells after dual treatment. Five genes induced by LPS were suppressed by HGFL and one gene, which was suppressed by LPS treatment, was induced after HGFL and LPS treatment. Seven unique genes were suppressed by HGFL and LPS treatment. The biological processes categories are noted for the differential treatment groups (A-B).

Figure 4. Validation of microarray data

qRT-PCR analyses on TK+/+ and TK−/− Kupffer cells showed EGR-1 mRNA was induced 1.8 fold more in the TK−/− compared to TK+/+ Kupffer cells after 30 minutes of LPS treatment (A). IL-6 mRNA was induced 2.1 fold more in the TK+/+ compared to TK−/− Kupffer cells after LPS treatment (B). qRT-PCR analyses showed that HGFL treatment for 1 hour modestly increased IL-6 (3 fold) and TNF-α (2.9 fold) mRNA levels (C). LPS induced IL-6 (6.67 fold) mRNA levels were suppressed by overnight HGFL treatment followed by LPS treatment (3.31 fold) (D). Cytokine array of conditioned media from TK+/+ and TK−/− Kupffer cells showing basal (E) and LPS-stimulated cytokine protein levels (F). *denotes P<0.05.

Figure 5. HGFL treatment induces Mefv (pyrin) and suppresses Lcn2 in TK+/+ Kupffer cells

RNA from Kupffer cells was obtained after HGFL and LPS treatment. qRT-PCR for Mefv (pyrin) (A) and Lcn2 (B) were performed under various treatment conditions. Western blot for Lcn2 protein expression in TK+/+ Kupffer cells after various treatment conditions (C). Secreted levels of IL-1β in conditioned media from TK+/+ Kupffer cells after various treatment conditions (D). *denotes P<0.05.

Figure 6. Working Model

Based on our data, we propose a role for pyrin as a downstream mediator of Ron signaling in inhibiting LPS-induced NF-κB mediated inflammation in Kupffer cells. Further, we propose that Ron signaling inhibits Lcn2 expression, which may act through multiple mechanisms to regulate inflammation.