Commensal-epithelial signaling mediated via formyl peptide receptors

Abstract

Commensal bacteria and/or their products engender beneficial effects to the mammalian gut, including stimulating physiological cellular turnover and enhancing wound healing, without activating overt inflammation. In the present study, we observed commensal bacteria-mediated activation of the noninflammatory extracellular signal-regulated kinase[ERK]/mitogen-activated protein kinase and Akt signaling pathways in gut epithelial cells and delineated a mechanism for this bacterially activated signaling. All tested strains of commensal bacteria induced ERK phosphorylation without stimulating pro-inflammatory phospho-IκB or pro-apoptotic phospho-c-Jun NH(2)-terminal kinase, with Lactobacillus species being most potent. This pattern of signaling activation was recapitulated using the peptide N-formyl-Met-Leu-Phe, a bacterial product known to stimulate signaling events in mammalian phagocytes. Sensing of N-formyl-Met-Leu-Phe by gut epithelial cells occurs via recently characterized formyl peptide receptors located in the plasma membrane. Both commensal bacteria and N-formyl-Met-Leu-Phe application to the apical surface of polarized gut epithelial cells resulted in specific formyl peptide receptor activation. In addition, pretreatment of model epithelia and murine colon with Boc2 (a specific peptide antagonist) or pertussis toxin (a G(i)-protein inhibitor) abolished commensal-mediated ERK phosphorylation. Taken together, these data show that commensal bacteria specifically activate the ERK/mitogen-activated protein kinase pathway in an formyl peptide receptor-dependent manner, delineating a mechanism by which commensal bacteria contribute to cellular signaling in gut epithelia.

Figure 1

Apically applied enteric bacteria induce ERK phosphorylation in polarized T84 epithelial cells. All experiments were immunoblots of T84 lysates probed with a phospho-ERK specific antibody. A: Apical cell stimulation with 5 × 10⁷ cfu/ml of Lactobacillus rhamnosus GG, Lactobacillus casei, Lactobacillus acidophilus, Bacteriodes thetaiotaomicron, Streptococcus thermophilus, Escherichia coli, or Salmonella typhimurium strains for 30 minutes. B: Apical cell stimulation with L. rhamnosus GG titered as indicated for 30 minutes. C: Apical cell stimulation with L. rhamnosus GG [5 × 10⁷ cfu/ml] or L. rhamnosus GG cell wall preparation (described in methods) for 1 hour. All immunoblot experiments were repeated at least five times.

Figure 2

Apically applied L. rhamnosus GG or fMLF specifically activate ERK/Akt pathway signaling. A: Immunoblot analysis of cultured T84 cells apically stimulated with L. rhamnosus GG [5 × 10⁷ cfu/ml], L. rhamnosus GG cell wall preparation, fMLF [500 nmol/L] or S. typhimurium [1 × 10⁸ cfu/ml] or basolaterally with TNF-α [10 ng/ml] for 1 hour, lysed, and probed with the indicated antibodies. B: Murine bone marrow derived macrophages stimulated with L. rhamnosus GG [5 × 10⁷cfu/ml] or fMLF [500 nmol/L] over 30 minutes, lysed, and probed with antibodies indicated. C: Immunofluorescence analysis of fixed T84 cultured cells apically stimulated with L. rhamnosus GG [5 × 10⁷ cfu/ml] or fMLF [500 nmol/L] for 30 minutes. DNA (blue), Phospho-ERK (green). Confocal microscope magnification, ×63. D: ERK, JNK, or NF-κB pathway specific luciferase reporter gene assay of transfected SK-CO15 cells treated with L. rhamnosus GG [5 × 10⁷ cfu/ml], fMLF [500 nmol/L]. EGF [200 ng/ml] or TNF- α [10 ng/ml] served as activating controls for ERK and JNK/NF-κB, respectively. Data are shown as fold induction over unstimulated media control. *P < 0.05; **P < 0.001. All immunoblot experiments repeated at least five times, and immunofluorescence experiments were repeated at least three times. For the luciferase assay, n = 6 for each experimental treatment. E: EdU incorporation into cultured SK-CO15 cells incubated for 12 hours with L. rhamnosus GG [5 × 10⁷ cfu/ml] or fMLF [500 nmol/L]. DNA (blue), EdU (red). Confocal microscope magnification ×63 F: Quantitative representation of EdU positive cells in (E). Number of EdU positive cells per 10 fields of view for three replicates per treatment. *P < 0.05.

Figure 3

Inhibition of G-protein coupled receptors or formyl peptide receptors attenuates ERK phosphorylation in response to apical L. rhamnosus GG or fMLF stimulation. A: Immunoblot analysis of cultured T84 cells treated apically with PTx [1 μg/ml] or Boc2 [100 μg/ml] 30 minutes before apical stimulation with L. rhamnosus GG [5 × 10⁷ cfu/ml], S. typhimurium [1 × 10⁸ cfu/ml], or fMLF [500 nmol/L] for 1 hour. B: ERK pathway-specific luciferase reporter gene assay from transfected SK-CO15 cells treated with either PTx [1 μg/ml] or Boc2 [100 μg/ml] 30 minutes before L. rhamnosus GG [5 × 10⁷ cfu/ml], fMLF [500 nmol/L] or EGF [200 ng/ml] stimulation. Data are shown as fold induction over unstimulated media control.*P < 0.05; **P < 0.001 C: Immunoblot analysis of T84 cells treated basolaterally with either PTx [1 μg/ml] or Boc2 [100 μg/ml] 30 minutes before basolateral stimulation with flagellin [100 ng/ml]. D: Immunoblot analysis of T84 cells treated apically with Boc2 [100 μg/ml] 30 minutes before basolateral EGF [200 ng/ml] stimulation. All experiments repeated at least three times.

Figure 4

Apical L. rhamnosus GG and fMLF induce phosphorylation of FPR. Immunoblot analysis of immunoprecipitated FPR from cultured SK-CO15 cells apically pretreated with either PTx [1 μg/ml], Boc2 [100 μg/ml] or Dulbecco’s modified Eagle’s medium 30 minutes before stimulation with L. rhamnosus GG [5 × 10⁷ cfu/ml] or fMLF [500 nmol/L]. Immunoprecipitated proteins were immunoblotted against antibodies to pSer/Thr and FPR. Immunoblot experiments repeated at least three times. Densitometric analysis normalized to background using Scion Image β.

Figure 5

L. rhamnosus GG or fMLF treatment of murine colon stimulates ERK activation. A: Immunoblot analysis for phospho-ERK in T84 cells apically treated with cecal luminal preparations (as described in Materials and Methods) for 30 minutes. B: Immunoblot analysis of phospho-ERK in mouse colonic epithelial cell scrapings treated in vivo with 100 μl L. rhamnosus GG [1 × 10⁷ cfu/ml] or fMLF [500 nmol/L] for 7 minutes. C: Immunofluorescence of phospho-ERK of intestinal whole mount preparations (as described in Material and Methods) in either baseline intestinal mucosa or pretreated in vivo with either PTx [1 μg/ml] or Boc2 [100 μg/ml] 30 minutes before 100 μl L. rhamnosus GG [1 × 10⁷ cfu/ml] or fMLF [500 nmol/L] for 7 minutes. D: Experiment performed identically as in C on a MyD88 null background. n ≤3 for each experimental murine treatment.