Intestinal Alkaline Phosphatase Exerts Anti-Inflammatory Effects Against Lipopolysaccharide by Inducing Autophagy

Abstract

Intestinal alkaline phosphatase (IAP) regulates bicarbonate secretion, detoxifies lipopolysaccharide (LPS), regulates gut microbes, and dephosphorylates proinflammatory nucleotides. IAP also exhibits anti-inflammatory effects in a Toll-like Receptor-4 (TLR-4) dependent manner. However, it is not known whether IAP induces autophagy. We tested the hypothesis that IAP may induce autophagy which may mediate the anti-inflammatory effects of IAP. We found that exogenous IAP induced autophagy in intestinal epithelial cells and in macrophages. TLR4INC34 (C34), a TLR4 signaling inhibitor, suppressed IAP-induced autophagy. IAP also inhibited LPS-induced IL-1β mRNA expression and activation of NF-κB. When autophagy was blocked by 3-methyladenine (3MA) or by Atg5 siRNA, IAP failed to block LPS-mediated effects. IAP also upregulated autophagy-related gene expression in small intestine in mice. We administered either vehicle or IAP (100 U/ml) in drinking water for 14 days in C57BL/6 mice. Mice were sacrificed and ileal tissues collected. Increased expression of Atg5, Atg16, Irgm1, Tlr4, and Lyz genes was observed in the IAP treated group compared to the vehicle treated group. Increase in Atg16 protein expression and fluorescence intensity of LC3 was also observed in IAP-treated tissues compared to the vehicle-treated tissues. Thus, our study lays the framework for investigating how IAP and autophagy may act together to control inflammatory conditions.

Figure 1

IAP induces autophagy in HCT116 epithelial cells. (A) HCT116 cells were treated with 2.5, 10, or 25 U/ml of bovine IAP (A2356) for 24 hrs. Vehicle-treated cells were used as controls. Fifty µg of protein lysate was separated on SDS page and analyzed for LC3-II induction by Western blotting. Actin was used as a loading control. Representative image indicates the gel that was cut at 25 kDa and lower portion was probed for LC3 while upper portion was probed for actin. (B) Blots were quantified with ImageJ by analyzing the ratio of LC3-II/actin (mean ± SEM from three independent experiments). (C) Cells treated with IAP same as in A and probed for p62. The membranes were cut at 100 kDa and 25 kDa. Following p62 detection, the membranes were stripped and probed for actin. (D) Blots were quantified with ImageJ by analyzing the ratio of p62/actin (mean ± SEM from three independent experiments). (E) Cells treated with IAP same as in A, but in the presence of bafilomycin A1 (100 nM for 3 h towards the end of 24 hr period). LC3-II induction was analyzed by Western blotting and (F) quantified with ImageJ (mean ± SEM from three independent experiments). Blot represents a gel that was cut between 75 kDa (probed for actin ~37 kDa) and at 25 kDa (probed for LC3 at ~16 &14 kDa). LC3-I was detected only after higher exposure. (G) Cells were transfected with mCherry-EGFP-LC3B expressing plasmid for 24 hours using lipofectamine 2000. Cells were then treated with IAP for 24 hours and fixed with 4% paraformaldehyde. After washing with PBS, cells were mounted with permafluor mounting medium (ThermoFisher), observed, and imaged with the Olympus FluoView FV1200 confocal microscope. Scale bar = 10 µm. (H&I)Total number of LC3 puncta per transfected cell and mCherry⁺ EGFP⁺ (yellow) and mCherry⁺ EGFP⁻ (red) puncta per transfected cell, representing autophagosomes and autolysosome respectively, were counted and plotted. One way ANOVA and Dunnett’s Multiple Comparison Test was used to determine statistical significance. *P < 0.05 and **P < 0.01, compared to vehicle.

Figure 2

IAP induces autophagy in RAW264.7 macrophage cells. (A) RAW cells were treated with 2.5, 5, or 10 U/ml of bovine IAP (A2356) for 24 hrs. Vehicle-treated cells were used as controls. Fifty µg of protein lysate was separated on SDS page and analyzed for LC3-II induction by Western blotting. Actin was used as a loading control. Blot represents a gel that was cut between 75 kDa (probed for actin ~37 kDa) and at 25 kDa (probed for LC3-I at ~16 and LC3-II at ~14 kDa) (B) Blots were quantified with ImageJ by analyzing the ratio of LC3-II/actin (mean ± SEM from three independent experiments). (C) Cells were treated same as in A, but in the presence of Baf (100 nM for 3 hours towards the end). Protein samples were probed for LC3. Blot represents a gel that was cut between 75 kDa (probed for actin ~37 kDa) and at 25 kDa (probed for LC3-I at ~16 and LC3-II at ~14 kDa) (D) Blots were quantified with ImageJ by analyzing the ratio of LC3-II/actin (mean ± SEM from three independent experiments). (E) Cells were transfected with mCherry-EGFP-LC3B expressing plasmid using nucleofactor V kit. After 24 hrs, cells were then treated with IAP for 24 hours and fixed with 4% paraformaldehyde. After washing with PBS, cells were mounted with permaflour mounting medium (ThermoFisher), observed, and imaged with the Olympus FluoView FV1200 confocal microscope. Scale bar = 10 µm. (F&G)Total number of LC3 puncta per transfected cell and mCherry⁺ EGFP⁺ (yellow) and mCherry⁺ EGFP⁻ (red) puncta per transfected cell, representing autophagosomes and autolysosome respectively, were counted and plotted. One way ANOVA and Dunnett’s Multiple Comparison Test was used to determine statistical significance. *P < 0.05 and **P < 0.01, compared to vehicle.

Figure 3

IAP-induced autophagy is inhibited by L-Phe and by Atg5 knockdown. (A) RAW cells were treated with L-Phe (10 mM) or IAP alone or together for 24 hours and 100 nM Baf was added for 3 hrs towards the end. Fifty µg of protein samples were ran on SDS-PAGE and probed with LC3 and actin antibodies by Western blot. Blot represents a gel that was cut between 75 kDa (probed for actin ~37 kDa) and at 25 kDa (LC3-II at ~14 kDa) (B) Blots were quantified with ImageJ by analyzing the ratio of LC3-II/actin (mean ± SEM from three independent experiments). One way ANOVA and Dunnett’s Multiple Comparison Test was used to determine statistical significance. *P < 0.05 and Ϯ P > 0.05, compared to vehicle. (C) RAW cells were transfected with either Scr or siAtg5 siRNA for 48 hrs. Cells were then treated with either vehicle or IAP (25U/ml) for 24 hrs and Baf was added for 3 hrs towards the end of the experiment. Cells were harvested and protein samples were subjected to Western Blotting and probed for LC3 and Actin. Blot represents two gels that were cut between 75 kDa (probed for actin ~37 kDa) and at 25 kDa (LC3-I at ~16 kDa and LC3-II at ~14 kDa) (D) Quantification was carried out using ImageJ and data was plotted as LC3-II/Actin ratio (relative expression, arbitrary units). Students two-tailed T-test was used to compare significance between the vehicle and IAP treatment within a group. *P < 0.05 and Ϯ P > 0.05.

Figure 4

IAP-induced autophagy is dependent on TLR4 pathway. RAW cells were pre-treated without (vehicle-C34, IAP-C34) or with TLR4 INC34 (IAP25 + C34) at 15 µM for 30 mins followed by addition of vehicle or IAP (A2356) (25U/ml) for 24 hours in the absence (A,B) or presence of Baf added for 3 hrs towards the end (C,D). (A) Western blotting showing the expression of p62. Actin was used a loading control. The gel was cut between 100 kDa and 25 kDa (probed for p62 ~62 kDa). Following detection of p62, the blots were stripped and re-probed for Actin. (B) Quantification of the blots using image J (mean ± SEM from three independent experiments). (C) Western blotting showing the expression of LC3-II in the samples treated with Baf. Actin was used a loading control. The gel was cut between 75 kDa (probed for actin ~37 kDa) and at 25 kDa (LC3-II at ~14 kDa) (D) Quantification of the blots using image J (mean ± SEM from three independent experiments). One way ANOVA and Dunnett’s Multiple Comparison Test was used to determine statistical significance. *P < 0.05 and Ϯ > 0.05, compared to vehicle control.

Figure 5

IAP inhibits LPS-mediated proinflammatory effects in autophagy-dependent manner. (A) RAW cells were pretreated with or without 3MA (2 mM) for 1 hour before treatment with vehicle or bovine IAP (10 and 25 U/ml) for 24 hours followed by a challenge with LPS (25 ng/ml) for 24hrs. Cells were harvested, RNA was isolated and cDNA synthesized. qPCR was carried out to determine the gene expression of IL-1β with a 2^ddCt method using 18 s as a housekeeping gene. Values were compared to the LPS treatment in the respective group. (B) Cells were transfected for 24 hrs with either scrambled siRNA or siRNA against Atg5 and treated with vehicle or IAP (A2356) at 10 or 25 U/ml for 24 hours followed by addition of LPS (25 ng/ml) for the next 24 hours. qPCR was carried out to determine the gene expression of IL-1β. Values were compared to the LPS treatment in the respective group (C) NF-κB activation was assessed by the phosphorylation of RelA/p65 subunit. Cells were treated with or without 3MA (2 mM) for 1 hr before adding IAP (A2356, 10 or 25 U/ml) for 24 hours followed by addition of LPS (25 ng/ml) for 15 mins. Cells were harvested and protein lysates were obtained. Western blotting was performed to detect phospho-p65. Total p65 was used as a loading control. Blot represents a gel that was cut between 100 kDa and 25 kDa probed for phospho p65 at ~65 kDa. The membrane was stripped and re-probed for total p65. (D) Graph represents mean ± SEM from three independent experiments). Values were compared to the LPS treatment in the respective group. (E) NF-κB activation was assessed in cells transfected with either scrambled siRNA or siRNA against Atg5 and treated with vehicle or IAP (A2356) at 25 U/ml for 24 hours followed by addition of LPS(25 ng/ml) for the 15 mins. Cells were harvested and protein lysates were obtained. Western blotting was performed to detect phospho-p65. Total p65 was used as a loading control. Blot represents a gel that was cut between 100 kDa and 25 kDa probed for phospho p65 at ~65 kDa. The membrane was stripped and re-probed for total p65. (F) Graph represents mean ± SEM from three independent experiments. Values were compared to the LPS treatment in the respective group. One way ANOVA and Dunnett’s Multiple Comparison Test was used to determine statistical significance. *P < 0.05, ***P < 0.001 and Ϯ > 0.05.

Figure 6

IAP induces autophagy-related gene and protein expression in the small intestine. Small intestinal ileal tissues were collected from vehicle or IAP treated animals. RNA was isolated from tissue samples and cDNA was synthesized. qPCR was carried out using primers specific for Atg16 (A), Atg5 (B), Irgm1 (C), Tlr4 (D), and Lyz lysozyme (E) gene expression. Fold change values were calculated using 2^−ΔΔCt method and plotted as log base 2. Data are presented as median value with the 25^th and 75^th quartiles in each box plot. The whiskers represent highest and lowest data points. *P < 0.05. (F) Atg16 protein expression was analyzed in vehicle or IAP treated ileal tissues (N = 4/group) by Western blotting using anti-Atg16 antibody. Blots represent a gel that was cut at 100 kDa and at 25 kDa and probed for Atg16 ~68 kDa, followed by stripping and probing with actin antibody. (G) Quantification of Atg16 protein as relative expression of Atg16L1/Actin ratio. (H) Immunohistochemistry was performed on paraformaldehyde fixed and paraffin embedded segments of distal ileum from vehicle or IAP treated mice. Five micron sections were labeled for LC3B (Alexa Fluor 488 secondary), and imaged using a 40x oil immersion lens with wide field microscopy on an LSM 800 (Zeiss).Scale bar = 50 µm.