Title: Excess fatty acids induce pancreatic acinar cell pyroptosis through macrophage M1
polarization
Version: 0 Date: 14 Aug 2021
Reviewer's report:
The manuscript by Xia et al. deals with an important topic in the course of pancreatitis. The
immune response during acute pancreatitis represents an increasingly important mechanism for
the therapy of the disease. Activation of the inflammasome complex plays an important role
in the macrophage-mediated immune response and represents a promising target for treatment.
The manuscript is detailed, and the illustrations are well presented. However, there are some
concerns about the techniques used.
1. If I understand correctly, the pancreatitis model used in this paper is the taurocholate induced
pancreatitis model. Taurocholate solution was injected into the pancreatic duct at a concentration
of 5%. This is quite a high concentration, previous work showed lethality in animals even at
lower doses (Gut. 2007 Nov;56(11):1590-8., Nat Protoc. 2010 Feb;5(2):335-41.). What is the
situation in these experiments? Was there ligation of the pancreatic duct after injection of the
taurocholate? What amount of fluid was injected into the pancreatic duct? Histology shows very
little infiltration of leukocytes into the organ, this is unusual for such a severe disease model.
Taurocholate acts via the bile salt receptors on the one hand and via the pressure building up in
the pancreatic duct on the other, which directly influences Ca2+ signalling.
2. The model used here is ultimately not a model of hyperlipidemia-induced pancreatitis but of
taurocholate-induced pancreatitis, so it is also surprising that a therapy with CTSS inhibitor has
such a huge effect.
3. A second model would be helpful to verify the results obtained here.
4. The authors write that the animals were processed 48 h after surgery and induction of
pancreatitis. However, treatment with the inhibitor for CTSS occurred over 1 week. A more
detailed description of the models and time points used is needed to understand what exactly
was studied.
5. Figure 2 shows the flow cytometry analysis of isolated and PA-treated macrophages, where
a clear shift towards M1 can be observed. Can an induction of the inflammasome signalling
pathway (ASC, IL1beta, IL18, NLRP3 or caspase-1) already be observed here? Inflammasome
activation is significantly influenced by NFkB, can nuclear localisation of NFkBp65 be observed
in addition to IRF5?
6. Figure 6 and 7 shows the effect of CTSS exosomes on the induction of the inflammasome
complex in isolated acinar cells or AR42J. It is described that acinar cells do not express
any components of the inflammasome complex. Furthermore, no control staining has been
performed to show that these are indeed cells that possess a secretory compartment. A clearer
magnification, as well as a control staining of e.g. amylase, would give certainty. AR42J
cells behave only after treatment with dexamethasone (Am J Physiol. 1996 May;270(5 Pt
1):G746-51. ). These experiments should be repeated in freshly isolated acinar cells to rule out
the possibility that this is an artefact of the cell line.
7.  AR42J cells are not acinar cells, this should be clearly stated in the manuscript.
8. As far as I know, apoptosis of acinar cells is rare, mostly acinar cells die necrotic or necroptotic
cell death (Cell Mol Gastroenterol Hepatol. 2016 Jul;2(4):519-535.).
Title: Excess fatty acids induce pancreatic acinar cell pyroptosis through macrophage M1
polarization
Version: 0 Date: 14 Aug 2021
Reviewer's report:
The manuscript by Xia et al. deals with an important topic in the course of pancreatitis. The
immune response during acute pancreatitis represents an increasingly important mechanism for
the therapy of the disease. Activation of the inflammasome complex plays an important role
in the macrophage-mediated immune response and represents a promising target for treatment.
The manuscript is detailed, and the illustrations are well presented. However, there are some
concerns about the techniques used.
1. If I understand correctly, the pancreatitis model used in this paper is the taurocholate induced
pancreatitis model. Taurocholate solution was injected into the pancreatic duct at a concentration
of 5%. This is quite a high concentration, previous work showed lethality in animals even at
lower doses (Gut. 2007 Nov;56(11):1590-8., Nat Protoc. 2010 Feb;5(2):335-41.). What is the
situation in these experiments? Was there ligation of the pancreatic duct after injection of the
taurocholate? What amount of fluid was injected into the pancreatic duct? Histology shows very
little infiltration of leukocytes into the organ, this is unusual for such a severe disease model.
Taurocholate acts via the bile salt receptors on the one hand and via the pressure building up in
the pancreatic duct on the other, which directly influences Ca2+ signalling.
2. The model used here is ultimately not a model of hyperlipidemia-induced pancreatitis but of
taurocholate-induced pancreatitis, so it is also surprising that a therapy with CTSS inhibitor has
such a huge effect.
3. A second model would be helpful to verify the results obtained here.
4. The authors write that the animals were processed 48 h after surgery and induction of
pancreatitis. However, treatment with the inhibitor for CTSS occurred over 1 week. A more
detailed description of the models and time points used is needed to understand what exactly
was studied.
5. Figure 2 shows the flow cytometry analysis of isolated and PA-treated macrophages, where
a clear shift towards M1 can be observed. Can an induction of the inflammasome signalling
pathway (ASC, IL1beta, IL18, NLRP3 or caspase-1) already be observed here? Inflammasome
activation is significantly influenced by NFkB, can nuclear localisation of NFkBp65 be observed
in addition to IRF5?
6. Figure 6 and 7 shows the effect of CTSS exosomes on the induction of the inflammasome
complex in isolated acinar cells or AR42J. It is described that acinar cells do not express
any components of the inflammasome complex. Furthermore, no control staining has been
performed to show that these are indeed cells that possess a secretory compartment. A clearer
magnification, as well as a control staining of e.g. amylase, would give certainty. AR42J
cells behave only after treatment with dexamethasone (Am J Physiol. 1996 May;270(5 Pt
1):G746-51. ). These experiments should be repeated in freshly isolated acinar cells to rule out
the possibility that this is an artefact of the cell line.
7.  AR42J cells are not acinar cells, this should be clearly stated in the manuscript.
8. As far as I know, apoptosis of acinar cells is rare, mostly acinar cells die necrotic or necroptotic
cell death (Cell Mol Gastroenterol Hepatol. 2016 Jul;2(4):519-535.).
Please review this paper.
user
Title: Excess fatty acids induce pancreatic acinar cell pyroptosis through macrophage M1
polarization
Version: 0 Date: 14 Aug 2021
Reviewer's report:
The manuscript by Xia et al. deals with an important topic in the course of pancreatitis. The
immune response during acute pancreatitis represents an increasingly important mechanism for
the therapy of the disease. Activation of the inflammasome complex plays an important role
in the macrophage-mediated immune response and represents a promising target for treatment.
The manuscript is detailed, and the illustrations are well presented. However, there are some
concerns about the techniques used.
1. If I understand correctly, the pancreatitis model used in this paper is the taurocholate induced
pancreatitis model. Taurocholate solution was injected into the pancreatic duct at a concentration
of 5%. This is quite a high concentration, previous work showed lethality in animals even at
lower doses (Gut. 2007 Nov;56(11):1590-8., Nat Protoc. 2010 Feb;5(2):335-41.). What is the
situation in these experiments? Was there ligation of the pancreatic duct after injection of the
taurocholate? What amount of fluid was injected into the pancreatic duct? Histology shows very
little infiltration of leukocytes into the organ, this is unusual for such a severe disease model.
Taurocholate acts via the bile salt receptors on the one hand and via the pressure building up in
the pancreatic duct on the other, which directly influences Ca2+ signalling.
2. The model used here is ultimately not a model of hyperlipidemia-induced pancreatitis but of
taurocholate-induced pancreatitis, so it is also surprising that a therapy with CTSS inhibitor has
such a huge effect.
3. A second model would be helpful to verify the results obtained here.
4. The authors write that the animals were processed 48 h after surgery and induction of
pancreatitis. However, treatment with the inhibitor for CTSS occurred over 1 week. A more
detailed description of the models and time points used is needed to understand what exactly
was studied.
5. Figure 2 shows the flow cytometry analysis of isolated and PA-treated macrophages, where
a clear shift towards M1 can be observed. Can an induction of the inflammasome signalling
pathway (ASC, IL1beta, IL18, NLRP3 or caspase-1) already be observed here? Inflammasome
activation is significantly influenced by NFkB, can nuclear localisation of NFkBp65 be observed
in addition to IRF5?
6. Figure 6 and 7 shows the effect of CTSS exosomes on the induction of the inflammasome
complex in isolated acinar cells or AR42J. It is described that acinar cells do not express
any components of the inflammasome complex. Furthermore, no control staining has been
performed to show that these are indeed cells that possess a secretory compartment. A clearer
magnification, as well as a control staining of e.g. amylase, would give certainty. AR42J
cells behave only after treatment with dexamethasone (Am J Physiol. 1996 May;270(5 Pt
1):G746-51. ). These experiments should be repeated in freshly isolated acinar cells to rule out
the possibility that this is an artefact of the cell line.
7.  AR42J cells are not acinar cells, this should be clearly stated in the manuscript.
8. As far as I know, apoptosis of acinar cells is rare, mostly acinar cells die necrotic or necroptotic
cell death (Cell Mol Gastroenterol Hepatol. 2016 Jul;2(4):519-535.).
assistant