| Inflammatory bowel diseases(IBD) are chronic debilitating inflammatory condition made up of Crohn’s disease(CD) and ulcerative colitis(UC). To date, over 200 susceptibility loci have been identified, with some genetic loci that are unique but others that are shared between the 2 conditions, indicating complexity in IBD pathophysiology. One of the genetic variants at position 300 in the autophagy gene ATG16L1 results in a threonine to alanine substitution(T300A) in the carboxy terminal domain. Macroautophagy(herein referred to as autophagy) is a bulk degradation system where cytosolic constituents is engulfed in a double-membrane vesicle and targeted for degradation by lysosomal fusion. Xenophagy is where intracellular pathogens are sequestered autophagosomes for lysosomal degradation and is recognized as an important mechanism for antimicrobial defense.In vitro functional analysis showed that the IBD associated ATG16L1 T300 A single-nucleotide polymorphism(SNP) is a loss-of-function SNP through increased caspase mediated cleavage of ATG16L1 protein, resulting in reduced autophagy. To better elucidate the functional consequences of ATG16L1 T300 A, mice with knock-in of the human ATG16L1 T300 A gene was generated and shown to have defective and abnormal appearing Paneth cells and goblet cells, reduced antibacterial autophagy(xenophagy), and increased Il1β production, and worsened cecal inflammation with salmonella infection as compared to WT mice. Another group independently showed reduced xenophagy and increased expression of Il1β in primary human ATG16L1 T300 A dentritic cells and mouse ATG16L1 T316A(corresponding to human T300A) dentritic cells.To better understand the cell-specific role of autophagy, conditional Atg16l1 knockout mice in which Atg16l1 was deleted specifically in intestinal epithelial cells or CD11c+ dendritic cells(DC). Compared to WT mice, epithelial Atg16l1 deficiency in mice exhibited Paneth cell abnormalities and are more susceptible to S. typhimurium infection whereas the phenotype of Atg16l1 deficiency in CD11c+ DC is similar to control WT mice. This illustrated that Atg16l1 in intestinal epithelial cells is important for xenophagy and maintain gut homeostasis but may be dispensible in CD11c+ DC. However, given the importance of dentritic cells in innate immune responses and the previous study showing the importance of another component of autophagy(Atg5) in protection against active tuberculosis and pulmonary inflammation, we aim to elucidate the role of Atg16l1 in in myeloid cells, in particular dentritic cells in mice. Here, we independently generated mice with Atg16l1 deficiency in dentritic cells showed worsened colonic inflammation in one acute and one chronic models of murine colitis. Additionally, mice with Atg16l1 deficiency in dentritic cells exhibit increased colitogenic Ig A coated bacteria. Atg16l1 deficiency impaired the function of primary murine dentritic cells and led to increased ROS production, reduced intracellular killing of salmonella, and shift of salmonella to phagocytosis vesicles. Together, these results indicate the importance of autophagy in dentritic cells in maintaining intestinal homeostasis.Part one: Mice and generation of Atg16l1f/f mice and Atg16l1f/f Cre-DC miceObjective: To confirm the generation of Atg16l1f/f mice and Atg16l1f/f Cre-DC mice.Methods: 1) The generation of Atg16l1f/f mice and Atg16l1f/f Cre-DC mice using Cre/flox P. Cloning of Atg16l1 targeting vector and generation of Atg16l1f/f mice were performed in collaboration with gen Oway(gen Oway). Briefly, Atg16l1 endogenous locus containing 5.6 kb upstream and 2.1 kb downstream of exon 3 were generated by PCR amplification using proprietary C57BL/6J library developed at gen Oway. Subsequently, two lox P sites were inserted flanking Atg16l1 exons 3 Positive selection neomycin gene flanked by FRT sites was inserted to the intron between exons 3 and 4 to generate the targeting vector. Every step of the cloning process was validated through restriction enzyme analysis and sequencing. The Atg16l1 gene targeting construct was linearized and electroporated into gen Oway proprietary embryonic stem(ES) cells with C57BL/6J background. Homologous recombinants were selected by G418 and confirmed by PCR and Southern blot analysis. ES clones with correct 5’ and 3’ recombination were microinjected into C57BL/6J blastocysts and introduced into pseudopregnant C57BL/6J mice. Male chimeric offspring were bred to obtain germ line mutant mice which were then bred to Flpe delete mouse strain to remove the neomycin cassette and confirmed by Southern blot. To generate mice with conditional targeting of Atg16l1, Atg16l1f/f mice were bred with mice expressing cre recombinase under the control of CD11c(CD11c-cre, Jax mice stock 007567). 2) The expression levels of Atg16l1 were detected by Q-PCR from tails of Atg16l1f/f mice and Atg16l1f/f Cre-DC mice.Results: 1) We generated Atg16l1f/f mice and Atg16l1f/f Cre-DC mice successfully. 2) Deletion of Atg16l1 in DC was confirmed by the lack of Atg16l1 m RNA.Part two: The regulation effects of ATG16L1 on inflammation of the intestinal mucosa in experimental acute and chronic colitisObjective: To intvestigate regulation effects of ATG16L1 on mucosal inflammation in experimental acute and chronic colitis.Methods: 1) Atg16l1f/f mice and Atg16l1f/f Cre-DC mice were used for in vivo studies. The salmonella acute colitis model was established by oral gavage 3x10^6cfu/mouse salmonella.typhimurium(salmonella) after pretreatment with 20 mg strepmycin/mouse. DAI was measured on Day1, day3 and Day5 after infection and we did mcie sacrifice on Day5. 2) 3% DSS actue model was established by administration of DSS(Dextran sodium sulfate,DSS) for 7 days. DAI was measured on Day2, day4 and Day6 and we did mcie sacrifice on Day7. 3) Chronic colitis was induced by 2.5% DSS drinking from 1 to 5 days, 8 to 12 days, 15 to 19 days and 22 to 26 days,and distilled water was given during the remaining time. Animals were sacrificed at 29 th day. 4) Severity of colitis was evaluated by body weight(BW) changes, disease activity index(DAI),colon length,colon histology changes and pathology score. 5) Expressions of TNF-a,IL-6 and IL-1b were detcted by Enzyme-linked immunosorbent assay(ELISA). 6) CD4+T cells activation markers of CD44 and CD69 were detected by Fluorescence Activated Cell Sorter(FACS). 7) Ig A+ bacteria and total Ig A were detected by FACS and ELISA in differernt models. 8) Total bacteria in the stool, ileun, colon and MLN were detected in the different models.Results: 1) The results of salmonella acute colitis showed that BW of Atg16L1f/f CD11c-Cre mice had significantly greater loss compared with that in Atg16L1f/f mice. DAI(6.9±1.21 vs 6.3±0.41,P>0.05) and pathology scores(2.13±0.14 vs 1.85±0.15,P>0.05) in Atg16L1f/f CD11c-Cre mice were a little higher than that in Atg16L1f/f mice; congestion and edema of the colon wall and infiltration into the mucosa superficial layers of granulocytes were much severity in Atg16L1f/f CD11c-Cre mice(5.88±0.03 vs 5.50±0.72,P>0.05). 2) The results of 3% DSS actue colitis showed that mice with Atg16l1 deficiency in myeloid showed worsened colonic inflammation. DAI(8.6±0.41 vs 6.25±0.75,P<0.01) significantly increased in the Atg16L1f/f CD11c-Cre mice, and congestion and edema of the colon wall and infiltration into the mucosa superficial layers of granulocytes were significantly worsened(10.67±1.17 vs 5.25±0.25, P<0.01). 3) The results of 2.5% DSS chronic colitis showed that significant increased DAI(7.33±0.33 vs 4.17±0.48,P<0.001) was observed in Atg16L1f/f CD11c-Cre mice as compared to control Atg16L1f/f mice. Histologic examination of the colon revealed worsened inflammation characterized by increased cellular infiltrate, mucin depletion, crypt abscess, and architectural changes in Atg16L1f/f CD11c-Cre mice as compared to Atg16L1f/f mice(10.37 ± 1.00 vs 7.08 ± 0.88, P<0.01). 4) Expressions of TNF-a, IL-6 and IL-1b were detcted by ELISA. In the salmonella actue model, expressions of TNF-a,IL-6 and IL-1b in Atg16L1f/f CD11c-Cre mice(51.54 pg/m L±5.22 pg/m L, 290.44 pg/m L±48.88 pg/m L, 922.55 pg/m L±147.70 pg/m L, P>0.05) were a little higher than that in Atg16L1f/f mice(24.26 pg/m L±6.57 pg/m L, 248.22 pg/m L±16.88 pg/m L, 619.72 pg/m L ± 149.89 pg/m L) in the supernatant in LPMC. Also the consistent results were got in MLN. Expressions of TNF-a,IL-6 and IL-1b in Atg16L1f/f CD11c-Cre mice(19.42 pg/m L±2.29 pg/m L, 429.02 pg/m L±133.91 pg/m L, 497.40 pg/m L±129.90 pg/m L, P>0.05) were higher than that in Atg16L1f/f mice(13.63 pg/m L±1.85 pg/m L, 244.58 pg/m L±27.30 pg/m L, 320.90 pg/m L±233.68 pg/m L), but there is no significance between Atg16l1f/f mice and Atg16l1f/f Cre-DC mice; The results of 3% DSS actue colitis showed that expressions of TNF-a,IL-6 and IL-1b in Atg16L1f/f CD11c-Cre mice(72.89 pg/m L±5.19 pg/m L, 363.93 pg/m L±14.80 pg/m L, 1783.53 pg/m L±268.42 pg/m L, P<0.05) were higher than that in Atg16L1f/f mice(22.19 pg/m L ± 2.12 pg/m L, 273.06 pg/m L ± 15.46 pg/m L, 1153.9 pg/m L±102.84 pg/m L) in the supernatant in LPMC. Also the consistent results were got in MLN. Expressions of TNF-a,IL-6 and IL-1b in Atg16L1f/f CD11c-Cre mice(2.48 pg/m L±0.134 pg/m L, 1126.60 pg/m L±19.07 pg/m L, 1307.92 pg/m L±74.74 pg/m L, P<0.05) were higher than that in Atg16L1f/f mice(0.78 pg/m L±0.28 pg/m L, 229.24 pg/m L±50.82 pg/m L, 728.00 pg/m L±64.10 pg/m L); In 2.5% DSS chronic colitis the results of ELISA showed that expressions of TNF-a,IL-6 and IL-1b in Atg16L1f/f CD11c-Cre mice(59.58 pg/m L±7.41 pg/m L, 441.40 pg/m L±19.43 pg/m L, 2942.92 pg/m L±438.95 pg/m L, P<0.05) were higher than that in Atg16L1f/f mice(24.76 pg/m L±2.03 pg/m L, 137.75 pg/m L±15.08 pg/m L, 1601.06 pg/m L±202.12 pg/m L) in the supernatant in LPMC. Also the consistent results were got in MLN. Expressions of TNF-a,IL-6 and IL-1b in Atg16L1f/f CD11c-Cre mice(19.46 pg/m L±3.34 pg/m L, 324.14 pg/m L±41.41 pg/m L and 1017. 62 pg/m L±90.92 pg/m L) were higher than that in Atg16L1f/f mice(6.61 pg/m L±1.60 pg/m L, 229.02 pg/m L±29.22 pg/m L, 403.54 pg/m L±122.88 pg/m L). 5) There are no differences of CD44 and CD69 between Atg16L1f/f CD11c-Cre mice and Atg16L1f/f mice in different models. 6) There are much more Ig A+ bacteria in Atg16L1f/f CD11c-Cre mice(44.00%±9.51%, P<0.01) compared to Atg16L1f/f mice(11.53%±2.42%) in salmonella actue model. Also we found higer percentage of Ig A+ bacteria in Atg16L1f/f CD11c-Cre mice (19.43%±0.77%, P<0.05) compared to Atg16L1f/f mice(12.98%±0.62%) in 3% DSS actue model. In 2.5% DSS chronic model, Ig A+ bacteria were gradually increased from week0, week2 to week4. And the result is consistent. In the different timepiont, there are much more Ig A+ bacteria in Atg16L1f/f CD11c-Cre mice compared to Atg16L1f/f mice. 7) There are much more total Ig A in Atg16L1f/f CD11c-Cre mice(2 850 μg/g±33 μg/g) compared to Atg16L1f/f mice(1 100 μg/g±24.2 μg/g) in salmonella actue model. Also we found on different in total Ig A in Atg16L1f/f CD11c-Cre mice(81 μg/g±9.2 μg/g, P>0.05) compared to Atg16L1f/f mice(70 μg/g±5.6 μg/g) in 3% DSS actue model. In 2.5% DSS chronic model, there are on different in total Ig A in Atg16L1f/f CD11c-Cre mice compared to Atg16L1f/f mice(50 μg/g±8.3 μg/g vs 48 μg/g±3.2 μg/g, P>0.05).8) There are no differernt bacteria in Atg16L1f/f mice in the stools(198 cfu/g±28.20 cfu/g), ileum(170.80x10^3 cfu/g±33 x10^3 cfu/g),colon(1 674 x10^6 cfu/g±169 x10^6 cfu/g) and MLN(201.50 x10^3 cfu/g±137 x10^3 cfu/g) compared to Atg16L1f/f CD11c-Cre mice in salmonella actue model in the stools(2 584 x10^6 cfu/g±590 x10^6 cfu/g), ileum(529.3 x10^3 cfu/g±354.6 x10^3 cfu/g), colon(246.9x10^3 cfu/g±91 x10^3 cfu/g) and MLN(369 cfu/g±112 cfu/g)(P>0.05). But we found less bacteria in Atg16L1f/f mice in the stools(2.20x10^6 cfu/g±0.50x10^6 cfu/g) and MLN(20 cfu/g±2.00cfu/g) compared to Atg16L1f/f CD11c-Cre mice in the stools(10.7x10^6 cfu/g±1.71x10^6 cfu/g) and MLN(36 cfu/g±1.10 cfu/g) in 3% DSS actue model(P<0.05). In 2.5% DSS chronic model, the same result was got as in 3% DSS actue model. There are much more bacteria in Atg16L1f/f CD11c-Cre mice in the stools(435.51x10^6 cfu/g±43.9 x10^6 cfu/g), ileum(89 x10^3 cfu/g±3.54 x10^3 cfu/g), colon(246 x10^3 cfu/g±71 x10^3 cfu/g) and MLN(76 cfu/g±8.4 cfu/g) compared to Atg16L1f/f mice in the stools(27.33 x10^6 cfu/g±1.23 x10^6 cfu/g), ileum(37 x10^3 cfu/g±1.37 x10^3 cfu/g), colon(36x10^3 cfu/g±3.30 x10^3 cfu/g) and MLN(18 cfu/g±2.80 cfu/g)(P>0.05).Part three: Regulatory effect of Atg16L1 on bone marrow drived dentritic cells(BMDC)Objective:To intvestigate regulation effects of ATG16L1 in BMDC.Methods: 1) The expression levels of LC3 II and P40 were detected by western blot in BMDC. 2)Autohphage fuctions in BMDC were detected by ROS, gentomycin protection assay and bacteria uptake assay. 3) Autophagesosome and phagesosome were counted by Transmission Electron Microscope(TEM). 4) The expressions of Beclin-1, ATG16L1, Lamp1, rab5 and rab7 proteins were detected at the same time with LC3 and salmonella by immunohistochemistry to check the trafficking of salmonella. 5) Antigen presentation assay were preceded in Atg16L1f/f mice and Atg16L1f/f CD11c-Cre mice to test out na?ve T cells proliferation.Results: 1) We show that LC3 II by Western blot that autophagy is functionally knocked out in dentritic cells carrying the CD11c-Cre. 2) Cytochalasin D is a potent inhibitor of actin polymerization and will inhibit active uptake of salmonella. After we add cytochalasin D to the cells before infection, thers is almost no bacteria uptake. And there were no differences in uptake of salmonella between Atg16L1f/f CD11c-Cre dentritic cells and Atg16L1f/f dentritic cells at 15 min, 30 min, and 60 min post infection in both RPMI and EBSS conditions. But ATG16L1 deficient dentritic cells have increased ROS production when stimulated with salmonella. Neutrophil cytosol factor 40 or p40 Phox is a cytosolic regulatory component of the superoxide-producing phagocyte NADPH-oxidase. Higher p40 Phox is correlated with increased ROS production. Consistent results were showed that hihger P40 were found in ATG16L1 deficient dentritic cells.Despite the increase in ROS production in autophagy deficient dentritic cells, we found that there is actually increased intracellular survival of salmonella in autophagy deficient dentritic cells. 3) The results of immunofluorescence staining showed that there seems to be co-localization of salmonella with beclin-1 in both ATG16L1f/f CD11c-Cre dentritic cells and ATG16L1f/f dentritic cells, which is the early autophagy vesicle marker, prior to the ATG16L1 step. There is no co-localization of salmonella with ATG16L1 in KO dentritic cells as expected. But there is intact co-localization of salmonella with LC3(40%).There is higer co-localization of salmonella with Lamp1 in ATG16L1f/f dentritic cells compared to ATG16L1f/f CD11c-Cre dentritic cells. We next assessed whether there is any alterations of co-localization of salmonella with phagocytosis markers Rab5 and Rab7. There is increased co-localization of salmonella with Rab 5 and Rab7 showing a shift in the trafficking of intracellular salmonella to the phagocytosis pathway with dentritic cells that are deficient in autophagy. 4) The results of SEM revealed that at baseline, there are very little autophagy and phagocytosis vesicles. Infection with salmonella increases the number of phagosomes and autophagosomes in the floxed dentritic cells but only phagosomes in the autophagy deficient dentritic cells. Under starvation condition, there is a shifting of vesicles to the autophagy pathway in the floxed dentritic cells but not in the autophagy deficient macropahges. Very rarely, we see an autophagosome in the autophagy deficient dentritic cells. This may reflect autophagy vesicles that are formed prior to deletion of the ATG16L1 gene. With salmonella infection in starvation condition, we observe salmonella in the autophagosome, but in the autophagy deficient dentritic cells, salmonella are only found in phagosomes. Also, there is larger number of mitochondria in ATG16L1f/f CD11c-Cre dentritic cells compared to ATG16L1f/f dentritic cells(P<0.05), which is consistent with the results of ROS production. 5) Under Ova-peptide conditions, ATG16L1 deficiency in DCs and ATG16L1f/f resulted in similar antigen presentation to CD4+ T cells and the proliferations of CD4+T cells are respectively(92.6% ± 4.6% vs 91.4% ± 5.3%, P > 0.05); while under Ova-protein conditions, proliferation of CD4+T cells was reduced in ATG16L1f/f CD11c-Cre mice compared to ATG16L1f/f CD11c-Cre mice(65.8%±1.7% vs 81.7%±2.6%, P<0.05).Conclusions: 1 We generated Atg16l1f/f mice and Atg16l1f/f Cre-DC mice successfully. 2 Loss of Atg16L1 in myeloid cells exacerbated DSS-induced murine models of colitis. But there is no effect on the salmonella-induced murine model of cilitis.3 Atg16L1 deficiency in dentritic cells leads to increased ROS production, reduced intracellular killing of salmonella, shift of salmonella to phagocytosis vesicles and reduced antigen presentation. 4 Atg16L1 can keep the ability of antigen presetation of cells after OVA-protien stimulation. |
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