Gliadin Intake Causes Disruption of the Intestinal Barrier and an Increase in Germ Cell Apoptosis in A Caenorhabditis Elegans Model
Round 1
Reviewer 1 Report
I confirm that the paper is very interesting and I haven't to make any corretction
Reviewer 2 Report
The authors have adaequately addressed all previous concerns.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
This is a very interesting paper and it may open new perspective to understand better the ethiopathogenesis of Celiac Disease.
page 3 - 94 wavelengtts may become wave-lenghts
page 3- 118 a better description of "soaking method"
page 8-251 a better description of different types of ROS in the pathogenesis of intestinal disorders
page 16 - 500 the same as above: a better description of different type of ROS
Author Response
Responses to Reviewer’s comments
Reviewer 1:
Comments and Suggestions for Authors
This is a very interesting paper and it may open new perspective to understand better the ethiopathogenesis of Celiac Disease.
(1) page 3 - 94 wavelengths may become wave-lengths
Response: Yes, we corrected as indicated in the page 3 line 100, line 107 and 108 where it is applied.
(2) page 3- 118 a better description of "soaking method"
Response: We added description of soaking method in the page 4 line 131-139 as follows:
RNAi experiments were performed using the soaking method as described previously [28]. dsRNA for cep-1 and mpk-1 genes was synthesized in vitro from respective cDNA templates. The cDNA templates flanked by T7 promoter sequences were generated by PCR using T7 primer, 5’-GTAATACGACTCACTATAGGGC-3’ and CMo422 primer, 5’-GCGTAATACGACTCACTATAGGGAACAAAAGCTGGAGCT-3’. Soaking buffer without dsRNA was used as the negative mock RNAi control. L1-stage worms were soaked in dsRNA solution for 24 h, then transferred to OP50-seeded NGM plates to be grown until the worms reached the L4 stage. Worms were then either treated or not treated with gliadin, incubated for an additional 24 h, and the adult stage worms were examined using the germ cell apoptosis assay.
(3) page 8-251 a better description of different types of ROS in the pathogenesis of intestinal disorders
Response: Because meaning of ROS (reactive oxygen species) itself contains different types of reactive oxygen, we changed the sentence from different types of ROS to ROS in the page 9 line 74.
(4) page 16 - 500 the same as above: a better description of different type of ROS
Response: Same as (3)
Reviewer 2 Report
In the current manuscript the authors elucidated the link between gliadin-induced intestinal stress and reproductionin germ cells of C. elegans. The authors conclude that gliadin increased reactive oxygen species (ROS) production in the intestine, decreased intestinal F-actin levels, and increased germ cell apoptosis. They also found that gliadin-triggered germ cell apoptosis was suppressed by depletion of cep-1, ced-13, egl-1, or mpk-1.
Major:
In Fig.1 “reproter” needs to be changed into reporter. The assays shown need to be better described in the materials and methods section as well as what the respective reporters, contain. It is important within the context of this manuscript. The conclusion from these experiments cannot be that oxidative stress induced these reporters. The conclusion can only be that gliadin induced these reporters and that gliadin induced DCFDA fluorescence.
Experiments in figure 3 need to be redone without LPS treatment. The argumentation in the results section is simply incorrect if Raw cells are pretreated with LPS that alone is a ROS producer.
Measurements in figure 6 need to be accompanied with NAC only treatment group and ROS measurements.
To link data in figure 8 with ROS, NAC treatment experiments are recommended.
Fig. 11, again, the NAC only treatment group is absent.
Author Response
Responses to Reviewer’s comments
Reviewer 2:
Comments and Suggestions for Authors
In the current manuscript the authors elucidated the link between gliadin-induced intestinal stress and reproduction in germ cells of C. elegans. The authors conclude that gliadin increased reactive oxygen species (ROS) production in the intestine, decreased intestinal F-actin levels, and increased germ cell apoptosis. They also found that gliadin-triggered germ cell apoptosis was suppressed by depletion of cep-1, ced-13, egl-1, or mpk-1.
Major:
(1) In Fig.1 “reproter” needs to be changed into reporter.
Response: We appreciate your correction. Yes, we changed as indicated.
(2) The assays shown need to be better described in the materials and methods section as well as what the respective reporters, contain. It is important within the context of this manuscript. The conclusion from these experiments cannot be that oxidative stress induced these reporters. The conclusion can only be that gliadin induced these reporters and that gliadin induced DCFDA fluorescence.
Response: We appreciate the reviewer’s comment. According to the reviewer’s suggestion, we have described the assay method separately under “2.2 Live images observation of fluorescence-tagged transgenic worms” in the Materials and Methods section on page 2 line 74-81 as follows:
“2.2 Live images observation of fluorescence-tagged transgenic worms
To observe the expression of glutathione S-transferase 4 (GST-4) and cytochrome P450 oxidase (CYP-35) by gliadin treatment, the transgenic strains, CL2166 tagged with GFP reporter to gst-4 gene, a general indicator of oxidative stress responses and CY573 tagged with GFP reporter to cyp-35B1, a reporter of oxidase detoxification, were used. The synchronized L4-stage of worms expressing GFP were treated with gliadin for 24 h at 20°C. The worms were then mounted into 0.2 mM tetramisole hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) in M9 buffer on a poly-L-lysine (Sigma-Aldrich, St. Louis, MO, USA) coated glass slide. Live images of worms were observed under a fluorescence microscope (Zeiss Axioscope, Germany).”
We understand the reviewer’s concern about conclusion of the experiments with transgenic worms in figure 1. However, it was previously reported that oxidative stress or xenobiotic stress can induce the expression of the phase II detoxification genes, which encode enzymes that synthesized glutathione, scavenge free radicals, and detoxify reactive products of phase I (P450) system (Hayes and McMahon, 2001; Toone et al., 2001; Nguyen et al., 2003; Motohashi and Yamamoto, 2004). In addition, it was also reported that C. elegans glutathione S-transferase is upregulated in response to oxidative stress and it is evident that expression of reporter in the transgenic strain CL2166 is altered accordingly (Leiers et al., 2003). Based on the previous reports, there seems to be a strong correlation between oxidative stress responses and induction of phase II detoxification genes including cyp-35 and gst-4. Furthermore, as we mentioned in the text (in the result section, page 5), we have already described that the gst-4 reporter can be considered as an indicator of oxidative stress responses in our previous paper (Lim et al, 2018). Therefore, we think that it is reasonable to conclude that oxidative stress responses were induced by gliadin intake through these experiments.
(3) Experiments in figure 3 need to be redone without LPS treatment. The argumentation in the results section is simply incorrect if Raw cells are pretreated with LPS that alone is a ROS producer.
Response: We appreciated the reviewer’s comments and we performed additional experiments without LPS treatment to avoid LPS effect on cells, and newly obtained results are presented in the figure 3.
(4) Measurements in figure 6 need to be accompanied with NAC only treatment group and ROS measurements.
Response: In figure 6, we described effects of gliadin intake on intestinal F-actin intensity. As reviewer suggested, we tested NAC only treatment group which is named as “control” in the graph (Fig 6B). To make a clear definition of the control that is the NAC only treated group, we added a phrase as follows: “…..and either no fed (control), fed gliadin, WGH…..” in the figure 6 legend, page 11 line 332-333. Reviewer also suggested to measure ROS in this experiment. However, we have already shown measurement of ROS in figure 1 (group with gliadin treatment) and figure 2 (groups with synthetic gliadin peptides and with WGH).
(5) To link data in figure 8 with ROS, NAC treatment experiments are recommended.
Response: Yes, it is an important issue to link gliadin-induced germ cell apoptosis (GIGA) with ROS production and an antioxidant, NAC treatment. In fact, this is the main finding in this study. In figure 8, we attempted to investigate the possible molecular pathway regulating GIGA by using different genetic backgrounds (mutant worms). Furthermore, the link between GIGA and NAC treatment was described in figure 10. As shown in figure 10, GIGA was suppressed by NAC treatment. We further confirmed the link between GIGA and ROS production, NAC treatment in figure 11 by examining mev-1, a hypersensitive mutant worm to gliadin treatment. In figure 8, we examined mutant worms containing loss-of-function mutations of genes that are involved in germ cell apoptosis pathway in C. elegans. As shown in figure 8, GIGA was abolished in ced-4, ced-3, and was not increased in cep-1, egl-1, ced-13 and mpk-1 mutant worms, suggesting that depletion of these genes suppressed GIGA, and GIGA were occurred through the general germ cell apoptosis pathway in C. elegans. In contrast, GIGA was increased in lip-1 mutants in figure 8. lip-1 is a negative regulator of mpk-1. In figure 8, lip-1 shows hypersensitivity to gliadin treatment like mev-1. As reviewer recommended, we could measure ROS production and do NAC treatment in lip-1 mutants to see if effect of gliadin is suppressed as we found in mev-1 mutants in figure 11. However, we could not perform this experiment because of the limited time for revision of the manuscript. Instead, we showed the link between GIGA and ROS, NAC treatment using either N2 or mev-1 mutants in figure 10 and figure11, respectively. In addition, we added the sentence that hypersensitivity to gliadin treatment in the lip-1 mutants is possibly suppressed by NAC treatment as shown in mev-1 mutants and its possibility remains to be determined in the result section as follows in the page 12 line 406-408:
“This result demonstrates that lip-1 mutants show hypersensitivity to gliadin treatment. Whether this hypersensitivity in the lip-1 mutants is possibly suppressed by NAC treatment remains to be determined.”
(6) Fig. 11, again, the NAC only treatment group is absent.
Response: We appreciate the reviewer’s comment. According to the reviewer’s suggestion, we performed additional experiments and changed figure 11 including the NAC only treatment group.
Finally, we appreciate the reviewer very much. Reviewer’s comments surely improve our manuscript and we hope that the revised manuscript will be suitable for publication in the Nutrients.
Round 2
Reviewer 2 Report
In the revised version the authors improved the manuscript.
Although I understand the authors response, the conclusion from experiments in Fig.1 can still not be not be that oxidative stress induced these reporters. Oxidative stress would imply an imbalance in the oxidative vs antioxidant systems with an advantage of the former. The authors did not assess the antioxidant status at the time of the experiments in Fig.1 nor did they include antioxidants in these particular experiments. Hence, the conclusion can only be that gliadin induced these reporters and that gliadin induced DCFDA fluorescence.
Moreover, the data in Fig 8 are crucial for the overall conclusion of the study and need to be accompanied by the NAC treatment as already outlined in the previous revision round. The authors should negotiate with the editors about more or additional time for revision.
Minor:
l 268 mammalian cell cultures
