The Modulation of Cholesterol Metabolism Is Involved in the Antiviral Effect of Nitazoxanide
, Salomè Valentina Ibba 2,†, Claudia Vanetti 1,2, Sergio Strizzi 2, Jean-François Rossignol 3, Mara Biasin 2, Daria Trabattoni 2 and Mario Clerici 1,4,*Round 1
Reviewer 1 Report
The authors therefore present a study on “The modulation of cholesterol metabolism is involved in the antiviral effect of thiazolides”.
Having previously studied the role of NTZ, a thialozide exhibiting antiviral activity on HIV via the activation of the type I interferon pathway, the authors in this article wanted to understand the mechanisms of action involved. NTZ is shown to be active on different families of RNA and DNA viruses and increases the expression of proteins involved in cholesterol metabolism. In this study, the authors were more particularly interested in Cholesterol-25-hydrolase (CH25H) which is able to catalyze cholesterol. NTZ treatment-induced CH25H formation repress cholesterol biosynthetic pathways and promote cholesterol mobilization and efflux from the cell.
The authors use the influenza virus (Flu) as an infectious model inducing an interferon response and use the NTZ in parallel to study the cholesterol intracellular homeostasis.
It is difficult to understand the rationale for viral choice in this study. Part of the description of the virus is non-existent and the infectious model is not described. The authors speak of "Flu-stimulated cells" but do not describe this stimulation. Are the cells directly infected? If yes, how long are cells infected before treatment? Is the level of infection controlled? Did the authors quantified the IFN activation of the cells?
On line 182, the authors explained that the expression of MSR1 is upregulated upon NTZ treatment in unstimulated cells and is slightly reduced under FLU stimulation. This trend is hardly credible: the standard deviation of the Flu stimulated cells is quite high, and the difference is not significant.
The synoptic summary is a very good idea to help the reader to get an overview of the metabolic pathway considered. Perhaps some colors could help to see the different level of regulation and what is up or down regulated.
Minor correstions
On line 197, / to be deleted.
On the different graph, the use of the same scale for each graph of the figure would help to determine the importance of the different gene expression tested.
Author Response
Reviewer 1
The authors therefore present a study on “The modulation of cholesterol metabolism is involved in the antiviral effect of thiazolides”.
Having previously studied the role of NTZ, a thialozide exhibiting antiviral activity on HIV via the activation of the type I interferon pathway, the authors in this article wanted to understand the mechanisms of action involved. NTZ is shown to be active on different families of RNA and DNA viruses and increases the expression of proteins involved in cholesterol metabolism. In this study, the authors were more particularly interested in Cholesterol-25-hydrolase (CH25H) which is able to catalyze cholesterol. NTZ treatment-induced CH25H formation repress cholesterol biosynthetic pathways and promote cholesterol mobilization and efflux from the cell.
The authors use the influenza virus (Flu) as an infectious model inducing an interferon response and use the NTZ in parallel to study the cholesterol intracellular homeostasis.
We are thankful to the Reviewer 1 for her/his overall positive assessment, and for her/his time and efforts in reviewing the manuscript, which we believe is now ameliorated.
It is difficult to understand the rationale for viral choice in this study. Part of the description of the virus is non-existent and the infectious model is not described. The authors speak of "Flu-stimulated cells" but do not describe this stimulation. Are the cells directly infected? If yes, how long are cells infected before treatment? Is the level of infection controlled? Did the authors quantified the IFN activation of the cells?
We are sorry if the procedure was not clearly stated. In the Materials and Methods section the FLU antigen stimulation is accurately described. Indeed, in “2.1 Reagents” (lines 80-88) the preparation employed to ex-vivo stimulate the cells is specified. Moreover, in “2.2 PBMC isolation and culture” (lines 91-102) the protocol for ex-vivo stimulation is described (i.e. concentration, duration, ..). No infectious virus was employed, but just viral antigens for specific viral stimulation, avoiding the detrimental effects of an actual productive infection, as it was not the purpose of the present study.
In order to make it more explicit, we started a new line with the description of the antigens and specified “viral antigens” in line 88.
The two sections now read as follow: “Stimulation with FLU antigens was performed with viruses kindly provided by Dr. Shearer, National Institutes of Health (NIH, WDC, USA). Two live UV-inactivated influenza viruses (FLU) were used: an influenza A virus (A/Bangkok/RX73 and A/Puerto Ri-co/8/34 strains; 1:800) and the 1998–1999 formula of influenza virus vaccine (1:5000; Wy-eth Laboratories Inc., Marietta, PA, USA). The influenza virus vaccine was an inactivated trivalent subunit formulation that contains the hemagglutinin antigens of influenza A H1N1, influenza A H3N1, and influenza B virus strains (each at 30 mg/ml). Such preparation of viral antigens was diluted 1:1000 in complete medium for FLU stimulated conditions.”,
“After viability assessment, cells were washed and re-suspended in RPMI complete medium and seeded simultaneously in the following conditions: unstimulated, unstimulated in the presence of NTZ (10 µg/mL), FLU-stimulated (1:1000), FLU-stimulated in the presence of NTZ. PBMCs were collected for gene expression analyses at 4 hours. In order to establish the suitable NTZ concentration, dose-responses experiments were performed and 10 µg/mL was identified, consistently with how previously observed”.
Moreover, the IFN-related activation was measured by analyzing multiple interferon-stimulated genes. Not only this was the aim of the study, but is commonly taken into the practice as the outcome of the actual IFN production, as the interferon family includes numerous members and their analysis might result as non-informative.
On line 182, the authors explained that the expression of MSR1 is upregulated upon NTZ treatment in unstimulated cells and is slightly reduced under FLU stimulation. This trend is hardly credible: the standard deviation of the Flu stimulated cells is quite high, and the difference is not significant.
We understand the Reviewer concern and it was not meant to over-estimate our results. Therefore, we rephrased that sentence, which now reads: “but it had no significant effect on the expression of this gene upon FLU stimulation”
The synoptic summary is a very good idea to help the reader to get an overview of the metabolic pathway considered. Perhaps some colors could help to see the different level of regulation and what is up or down regulated.
We noticed that a visual summary is recently becoming more and more common, and we agree with Reviewer 1 that it greatly helps to deliver the take-home message at a glance. As suggested, we introduced color-coded arrows, corresponding to the different levels of regulation for an enhanced clarity.
Minor correstions
On line 197, / to be deleted.
We are sorry for the typo, which is now removed.
On the different graph, the use of the same scale for each graph of the figure would help to determine the importance of the different gene expression tested.
We agree with the Reviewer 1 that a single univocal scale might be helpful to grasp the overall picture. However, we prefer to keep the graphs the way they are in order to better appreciate the relative increases/decreases among the different culture conditions, and we believe that the mutual abundance might not meaningful by itself, while stimuli-related increases/decreases are.
Reviewer 2 Report
The manuscript reports some new aspects of nitazoxanide's mechanism of action to justify its antiviral action. In particular the ability of Nitazoxanide to alter cholesterol metabolism pathways targeting some interferon-stimulated genes (f.i. cholesterol-25-hydrolase) has been investigated.
The manuscript is well organized, references are adequate and pertinent. Figures 1-3 are clear.
The presented results referes to NTZ, the only drug used in the experiments. No other thiazolide derivative has been tested. Therefore I don’t think it it is completly correct to generically speak of a class effect (thiazolides class effect), when the study is limited to NTZ. I think that the title should be modified, accordingly.
The Authors have to spell out the acronyms (PBMCs, OSBP etc..) the first time they are reported in the manuscript, and then make use of the acronyms in the following parts. Please revise the manuscript.
I believe this manuscript is of interest for scientists working in this field, thus I suggest its publication after these minor revisions.
Author Response
Reviewer 2
The manuscript reports some new aspects of nitazoxanide's mechanism of action to justify its antiviral action. In particular the ability of Nitazoxanide to alter cholesterol metabolism pathways targeting some interferon-stimulated genes (f.i. cholesterol-25-hydrolase) has been investigated.
The manuscript is well organized, references are adequate and pertinent. Figures 1-3 are clear.
We would like to thank the Reviewer 2 for the forthcoming comments.
The presented results referes to NTZ, the only drug used in the experiments. No other thiazolide derivative has been tested. Therefore I don’t think it it is completly correct to generically speak of a class effect (thiazolides class effect), when the study is limited to NTZ. I think that the title should be modified, accordingly.
We agree with the Reviewer 2 on the inaccuracy of the title, which is now modified as follows: “The modulation of cholesterol metabolism is involved in the antiviral effect of Nitazoxanide“”
The Authors have to spell out the acronyms (PBMCs, OSBP etc..) the first time they are reported in the manuscript, and then make use of the acronyms in the following parts. Please revise the manuscript.
We are thankful for the suggestion, which surely increases the fluency, making it more reader friendly. In particular, we spelled out the following acronyms:
Line 36 - cholesterol-25-hydroxylase (CH25H)
Line 64 - 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA)
Line 93 - peripheral blood mononuclear cells (PBMCs)
Line 109 - oxysterol binding protein (OSBP)
Line 110 - acetyl-CoA acetyltransferase 2 (ACAT2)
Line 111 - macrophage scavenger receptor 1 (MSR1)
Line 116 - glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
I believe this manuscript is of interest for scientists working in this field, thus I suggest its publication after these minor revisions.
