Leptin protects host cells from Entamoeba histolytica cytotoxicity by a STAT3-dependent mechanism

Abstract

The adipocytokine leptin links nutritional status to immune function. Leptin signaling protects from amebiasis, but the molecular mechanism is not understood. We developed an in vitro model of ameba-host cell interaction to test the hypothesis that leptin prevents ameba-induced apoptosis in host epithelial cells. We demonstrated that activation of mammalian leptin signaling increased cellular resistance to amebic cytotoxicity, including caspase-3 activation. Exogenous expression of the leptin receptor conferred resistance in susceptible cells, and leptin stimulation enhanced protection. A series of leptin receptor signaling mutants showed that resistance to amebic cytotoxicity was dependent on activation of STAT3 but not the Src homology-2 domain-containing tyrosine phosphatase (SHP-2) or STAT5. A common polymorphism in the leptin receptor (Q223R) that increases susceptibility to amebiasis in humans and mice was found to increase susceptibility to amebic cytotoxicity in single cells. The Q223R polymorphism also decreased leptin-dependent STAT3 activation by 21% relative to that of the wild-type (WT) receptor (P = 0.035), consistent with a central role of STAT3 signaling in protection. A subset of genes uniquely regulated by STAT3 in response to leptin was identified. Most notable were the TRIB1 and suppressor of cytokine signaling 3 (SOCS3) genes, which have opposing roles in the regulation of apoptosis. Overall apoptotic genes were highly enriched in this gene set (P < 1E-05), supporting the hypothesis that leptin regulation of host apoptotic genes via STAT3 is responsible for protection. This is the first demonstration of a mammalian signaling pathway that restricts amebic pathogenesis and represents an important advance in our mechanistic understanding of how leptin links nutrition and susceptibility to infection.

Fig 1

Leptin decreased amebic cytotoxicity in cells endogenously expressing the long form of the leptin receptor. (A) Caspase-3 activation by E. histolytica in Caco2 cells, Jurkat T cells, and HEK293T cells after leptin treatment. Caco2 and Jurkat cells endogenously express LepR, while HEK293T cells do not. Cells were treated with 0 to 500 ng/ml leptin 3 h prior to challenge with E. histolytica trophozoites at a ratio of 1 parasite to 5 host cells for 1 h. A total of 50 and 500 ng of leptin significantly decreased amebic cytotoxicity for Caco2 and Jurkat T cells, respectively, but had no effect on HEK293T cells. (B) Host cell lysis by E. histolytica in Caco2 and HEK293T cells after leptin treatment was measured concurrently with caspase-3 activation (A). Amebic lysis of host cells was measured by LDH release assay. *, P < 0.05 compared to 0 ng/ml leptin.

Fig 2

Exogenous expression of LepR in HEK293T cells decreased amebic cytotoxicity in a leptin-dependent manner. (A) Immunoblot of cells transfected with LepR (WT), Q223R LepR (Q223R), Y985L, Y1077L, Y1138L, and vector-transfected (pcDNA) cells. (B) The effect of LepR expression on amebic cytotoxicity was measured by E. histolytica-mediated caspase-3 activation. HEK293T cells transfected with wild-type LepR (WT LepR), an intracellular truncation of LepR that abrogates the majority of downstream leptin signaling (Δ65 LepR), or an empty-vector control (pcDNA) were treated with 0 or 100 ng/ml leptin for 3 h prior to amebic challenge. E. histolytica was added at a ratio of 1 parasite to 5 host cells. Caspase-3 activation was measured after 1 h. (C) Host cell lysis by E. histolytica was measured by LDH release assay concurrently with caspase-3 activation. Leptin treatment significantly increased protection from cytotoxicity in cells expressing WT LepR (P = 0.04 for caspase-3 activation and cell lysis) but not Δ65 LepR or pcDNA. Additionally, the WT LepR conferred significant protection relative to both Δ65 LepR and pcDNA in the presence and absence of leptin for both caspase-3 activation (B) and cell lysis (C) (*, P < 0.05; **, P < 0.005.) (D) STAT3 induction was measured in HEK293T cells transfected with WT LepR and Δ65 LepR by cotransfection with a STAT3-driven luciferase reporter. Cells were treated with 0 to 1,000 ng/ml of leptin for 3 h, and STAT3 induction in response to leptin was measured by luciferase reporter activity. WT LepR significantly induced STAT3 in response to leptin relative to Δ65 LepR and pcDNA (*, P < 0.05).

Fig 3

Mutation of intracellular signaling tyrosines revealed that STAT3 was critical for leptin-mediated protection. (A) Amebic cytotoxicity was compared in HEK293T cells transfected with WT LepR and LepR with mutations of intracellular tyrosines critical for downstream leptin signaling (Y985L, Y1077L, Y1183L). Cells were treated with 0 or 100 ng/ml of leptin for 3 h prior to amebic challenge. Amebic cytotoxicity was measured by caspase-3 activation. (B) Host cell lysis by E. histolytica was measured by LDH release assay concurrently with caspase-3 activation. *, P < 0.05; **, P < 0.005.

Fig 4

STATTIC, a small-molecule inhibitor of STAT3, reversed leptin-mediated protection. (A) STATTIC dose response was assayed by measuring STAT3 activation in HEK293T cells coexpressing WT LepR and a STAT3 luciferase reporter. Cells were treated with 0 to 40 μm STATTIC and/or 100 ng/ml leptin. STAT3 induction was measured by luciferase assay after 3 h. (B) STATTIC reversed the protective effect of LepR expression and leptin treatment in HEK293T cells transfected with WT LepR. Cells expressing WT LepR that are treated with leptin but not STATTIC are significantly protected from amebic caspase-3 activation relative to cells expressing pcDNA. **, P = 0.002. Amebic caspase-3 activation was measured in HEK293T cells transfected with WT LepR in the presence or absence of leptin (100 ng/ml), STATTIC (40 μm), and the caspase-3 inhibitor ZVAD-FMK (20 μm). (C) STATTIC dose response in Caco2 cells expressing a STAT3 luciferase reporter. Cells were treated with 0, 10, or 100 ng/ml leptin and 0 or 40 μm STATTIC. STAT3 induction was measured by luciferase assay after 3 h. *, P < 0.005. (D) STATTIC reversed leptin protection (P = 0.058) in Caco2 cells but did not significantly increase susceptibility to caspase-3 activation by ameba in the absence of leptin stimulation. Caco2 cells were treated with leptin (100 ng/ml) and/or STATTIC (40 μm) for 1 h prior to challenge with E. histolytica at a ratio of 1 parasite to 5 Caco2 cells. Caspase-3 activation by E. histolytica was measured after 1 h.

Fig 5

The Q223R polymorphism decreases leptin-mediated protection from amebic cytotoxicity and STAT3 induction. (A) HEK293T cells transfected with the Q223R LepR were more susceptible to amebic cytotoxicity. HEK293T cells expressing WT or Q223R LepR were treated with 0 or 100 ng/ml of leptin for 3 h prior to challenge with E. histolytica (1 parasite to 5 host cells). Caspase-3 activation by E. histolytica was measured after 1 h. (B) Host cell lysis by E. histolytica was measured by LDH release assay concurrently with caspase-3 activation. (C) Q223R diminished STAT3 induction in response to leptin. STAT3 induction by leptin measured in cells expressing WT and Q223R LepR cotransfected with a STAT3-luciferase reporter. Cells were treated with 0 to 1,000 ng/ml leptin for 3 h, and STAT3 activity was measured by luciferase assay. *, P < 0.05 for 223R versus 223Q.

Fig 6

The Q223R polymorphism does not significantly alter expression of known STAT3-regulated antiapoptotic genes. Gene expression of STAT3, SOCS3, BIRC5, and Bcl-xL was measured in HEK293T cells expressing the WT, Q223R or Y1138L (STAT3 mutant) mutant, or vector alone (pcDNA). Expression levels were compared between cells treated with 100 ng/ml of leptin for 3 h and cells maintained in the absence of leptin.

Fig 7

Genes associated with apoptosis and cell proliferation are differentially upregulated in response to leptin, depending upon LepR STAT3. Normalized, clustered microarray-based gene expression profiles are shown on a blue-red (low- to high-expression) gradient for wild-type (WT) or Y1138L mutant (MU) cells, either treated with leptin or maintained in the absence of leptin. Genes exhibiting significant leptin- and/or mutant-associated effects were selected by FDR of <5%. Genes having gene ontology (GO) term associations for apoptosis or cell proliferation are marked along the left axis of the heat map. The dendrogram shows hierarchical clustering based on pairwise correlations.