Leptin signaling in intestinal epithelium mediates resistance to enteric infection by Entamoeba histolytica - PubMed (original) (raw)

Leptin signaling in intestinal epithelium mediates resistance to enteric infection by Entamoeba histolytica

X Guo et al. Mucosal Immunol. 2011 May.

Abstract

Leptin is an adipocytokine that links nutrition to immunity. Previous observation that a genetic polymorphism in the leptin receptor affected susceptibility to Entamoeba histolytica infection led to the hypothesis that leptin signaling has a protective role during intestinal amebic infection. In this study we show that mice lacking the functional leptin receptor developed devastating mucosal destruction after E. histolytica infection. Bone marrow chimera experiments demonstrated that leptin receptor expressed on hematopoietic cells was not sufficient to confer resistance. Similarly, peripheral knockout of the leptin receptor rendered animals susceptible, indicating that central expression of the leptin receptor was not sufficient to confer protection. The site of leptin action was localized to the gut via an intestinal epithelium-specific deletion of the leptin receptor, which rendered mice susceptible to infection and mucosal destruction by the parasite. Mutation of tyrosine 985 or 1138 in the intracellular domain of the leptin receptor, which mediates signaling through the SH2-containing tyrosine phosphatase/extracellular signal-regulated kinase (SHP2/ERK) and signal transducer and activator of transcription 3 (STAT3) pathways, respectively, demonstrated that both were important for mucosal protection. We conclude that leptin-mediated resistance to amebiasis is via its actions on intestinal epithelium rather than hematopoietic cells or the brain, and requires leptin receptor signaling through both the STAT3 and SHP2/ERK pathways.

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Conflict of interest statement

Disclosure

The authors have no financial conflict of interest.

Figures

Figure 1*

Figure 1*. Long form leptin receptor (LRb) signaling

Leptin binding to the long form LRb results in the activation and tyrosine phosphorylation of Janus kinase 2 (JAK2), which induces subsequent phosphorylation of downstream tyrosine residues (Tyr985, Tyr1077 and Tyr1138) in the intracellular tail of LRb. Phosphorylated Tyr1138 binds and mediates the phosphorylation-dependent activation of signal transducer and activator of transcription 3 (STAT3), which activates transcription of suppressor of cytokine signaling 3 (SOCS3) and other positive effectors of leptin action. Phosphorylated Tyr985 recruits the SH2-containing tyrosine phosphatase SHP-2, which activates the signaling pathway that culminates in extracellular signal-regulated kinase (ERK) activation. During prolonged stimulation, phosphorylated Tyr985 binds SOCS3 to mediate feedback inhibition of LRb signaling. Phosphorylated Tyr1077 recruits STAT5 to induce STAT5-dependent transcription activation; however, its functional effect is less clear. Signals mediated via phosphorylated Jak2 include insulin receptor substrate protein (IRS) and the PI3K/Akt pathway. The PI3K/Akt signaling is important for the LRb-mediated anti-apoptotic and pro-proliferative effects via down-regulating pro-apoptotic (eg. Bad, Bim, Bax and p53) while up-regulating anti-apoptotic/survival factors (eg. Bcl-2, Bcl-XL and Cyclin D). *Figure adapted from Febbraio MA, J Clin Invest. 2007; 117(4): 841–849.

Figure 2

Figure 2. Deficiency in leptin signaling renders the mice more susceptible to intestinal infection by E. histolytica

(a) Mice genetically deficient in leptin (ob/ob) and in functional leptin receptor (db/db) were more susceptible to intestinal infection with E. histolytica as determined by cecal amebic antigen and cultural positivity (presented as infection rate); (b) An unusual moribund phenotype was observed in db/db and ob/ob mice that started within 24 h post challenge. The Kaplan-Meier survival curves depict time to death for mice challenged with E. histolytica in hours. The experiment ended after 48 hours. Logrank test for B6 versus db/db: p = 0.005 (no B6 WT or sham-challenged db/db mice died, 50% of infected db/db mice died); db/db versus ob/ob: p = 0.09 (17% of ob/ob mice died). For each group n = 12. (c) Mucosal hyperplasia was observed in the wild type mice (B6) that were infected (left), while ob/ob (middle) and db/db (right) mice had extensive epithelial loss and trophozoite invasion (arrows). Images are 100× except insets (bottom row) are 200×.

Figure 3

Figure 3. Leptin-mediated resistance to E. histolytica was not restored by transplant of a wild type marrow into db/db mice

Recipient mice were reconstituted i.v. with bone marrow cells prepared from donor femur and tibia. Engraftment of donor hematopoietic cells were confirmed by FACS for Thy1.1 and Thy1.2 at 8 wk post-transfer on PBMCs (a) (n = 8/group, one representative mouse per group shown). At 9 wk post-transfer, mice were challenged intracecally with E. histolytica trophozoites and sacrificed after 48 hr for evaluation of infection according to cecal amebic antigen (b), culture positivity (c), and early mortality (d). Logrank test for difference in survival: (db/db recipients vs WT recipients: p <0.0001; db/db to db/db vs WT to db/db: p =0.11. N = 16/group.

Figure 4

Figure 4. Peripheral disruption of leptin receptor resulted in increased susceptibility to E. histolytica

Leprflox/flox/Cre (+) and Leprflox/flox/Cre (−) mice were treated with tamoxifen for 5 days. (a) After tamoxifen treatment, genomic DNA was extracted from the brain (Br) and the cecum (Cec), and PCR was performed using primers recognizing the flanking sequences of the LoxP to examine the deletion of leptin receptor exon 17. (b) Tam-Cre mice with a peripheral disruption of the leptin receptor and control mice (Tam-Non-cre) with intact leptin receptor were intracecally infected with E. histolytica, and susceptibility to amebiasis was assessed by cecal amebic antigen and cultural positivity (presented as infection rate). (c) Representative histopathology of infected ceca from Tam-Cre (top) mice showed more severe destruction of mucosal architecture with large ulcerations and extensive infiltration compared to the infected Tam-non cre controls (bottom). Amebic trophozoites are indicated by arrows. Original magnifications: left 40×, right 100×.

Figure 5

Figure 5. Intestine-specific deletion of leptin receptor rendered mice susceptible to amebiasis

Mice engineered with loxP sites flanking the first coding exon of the leptin receptor were mated with mice expressing the Vil-Cre recombinase. (a) Intestine-specific deletion of exon 1 was confirmed by PCR using primers flanking the LoxP sites (►). The primers amplified a single band from the _lepr_Δ allele, while no product was generated from the control WT or floxed lepr allele. Genomic DNA was extracted from the cecum (Cec), liver (Liv) or the adipose tissue (Adi) associated with the intestine to examine the intestine-specific deletion of lepr. (b) Deletion of leptin receptor was further confirmed by its absence as demonstrated by immunohistochemistry in the Vil-Cre KO (bottom), but not the flox/flox control (top) cecum. Abundant basolateral staining of leptin receptor was seen in the flox/flox cecum (insert). Original magnifications: 200×; insert: 400×. (c) Vil-Cre KO mice and the non-cre flox/flox controls were intracecally challenged with E. histolytica and sacrificed 4 days after challenge. Intestinal infection was determined by culture positivity and cecal amebic antigen. (d) Representative cecal histopathology demonstrated substantial mucosal erosion and epithelial loss in the infected Vil-Cre KO mice but none of the flox/flox controls. Original magnifications: 100 ×.

Figure 6

Figure 6. Signaling pathways through tyrosine 1138 and tyrosine 985 were both important in leptin-mediated protection

Mice with tyrosine residue substitutions, S1138 (a) or L985 (b) on the leptin receptor were more susceptible to intestinal infection with E. histolytica compared to wild type littermates, p = 0.001 and 0.02, respectively; Infection rate was determined by both cecal amebic antigen and cultural positivity. None of the variants had the rapid death phenotype as seen in the db/db mice. * p<0.05; *** p<0.001 compared to wild type littermates. Most of the infected S1138 mice showed tissue destruction including mucosal erosion, ulceration, and tissue necrosis (c); Infected L985 mice were mainly manifested with mucosal hyperplasia with very little mucosal destruction (d). Loss of epithelial integrity was seen in some S1138 mice with aggressively invading trophozoites (arrows) along the lamina propria and basement membrane (e, f). Original magnification: C: 40×, D: 100×, E: 200×, F: 400×.

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