Platelets promote acute liver injury via extracellular vesicles-mediated Aldolase A

Reviewer #1 (Public review):

Summary:

In this manuscript, Yang et al expand on their previous work showing that platelet recruitment to the liver via liver macrophages is important for APAP-induced liver injury. Here, they show that platelets induce a glycolytic switch in liver non-parenchymal cells, including Kupffer cells, and that this is mediated by the protein Aldolase A produced by platelet-derived extracellular vesicles (PEV). They show that targeting Aldolase A may be a valid therapeutic strategy for severe APAP injury.

Strengths:

(1) They nicely showed that platelet effects in APAP are mediated by Aldoa via platelet-derived extracellular vesicles.

(2) Their data show that one of the effects of platelets in APAP liver injury is inducing metabolic switch to the glycolytic pathway, including in KCs.

(3) Their data points to the therapeutic potential of targeting ALDOA in severe APAP liver injury.

Weaknesses:

(1) They have not shown that the platelet-induced glycolytic switch is only in KCs.

(2) They also have not shown that KC's role in APAP injury is primarily mediated by their interaction with platelets and the subsequent glycolytic switch.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors have investigated the role of platelet-derived ALDOA in liver injury induced acetaminophen (APAP) induced acute liver injury. There are some major flaws in data interpretation as described below. While a decrease in liver injury due to platelet depletion and lower injury in platelet-specific ALDOA KO mice seems real, the claims related to EVs and Platelet-KC crosstalk are not well supported.

Strengths:

Core findings are interesting and supported by the data

Weaknesses:

(1) At least two additional timepoints, one at 6 hr and another at 24 hr should be performed in the APAP model to better understand the dynamics of liver injury, especially after platelet depletion.

(2) Interpretation of the experiments in Figure 2 with clodronate is flawed. 2-DG pretreatment and CLDN administration alone both seem to decrease liver injury substantially, so it is not surprising to see very little injury in the 2-DG+CLDN group.

(3) Since both 2-DG and CLDN were administered pre-APAP, it is possible that they may interfere with APAP metabolism. This should be checked by looking at GSH depletion at 30 min post APAP treatment. The same question goes for S2 figure data.

(4) There are no data on specific steps of APAP toxicity, such as GSH depletion, JNK activation, mitochondrial injury, etc., which are all well characterized in any of the studies. Rather, only injury endpoints are measured. It is critical to measure the mechanistic steps. This applies to all studies, but most importantly to the ALDOA-PF-KO mice in Figure 6.

(5) Interpretation of data in Figure 5F is flawed. Since depletion of platelets also decreases liver injury along with the platelets, it can not be deduced that the decrease in ALDOA is only in platelets. Many other things are changing.

Reviewer #3 (Public review):

Summary:

The authors address the possibility that platelet (PLT) derived EVs are important mediators of acute liver injury. Furthermore, KCs are important mediators of inflammation and are noted to need to undergo metabolic reprogramming to achieve their effects during injury. They use an APAP-induced liver injury model (AILI). They show that PLTs are recruited and that they interact with KCs in this model system. RNA-seq of KCs showed upregulation of glycolysis and gluconeogenesis. PLT depletion led to reduced liver injury. RNA-seq of KCs showed downregulation of glycolysis. In vitro co-culture of KCs and pets recapitulated the glycolysis findings. In vivo, 2DG inhibited liver injury, but not in the setting of KC depletion. They went on to show that PLT-derived EVs mediate this effect on KCs using a mix of in vitro and in vivo assays, although control EVs were lacking. After doing mass spec on EVs, they find that ALDOA is the critical payload of the PEVs that mediates the pro-glycolytic effect in vivo. They both delete ALDOA from PLTs, and they use an ALDOA inhibitor to show that injury in AILI requires ALDOA.

Strengths:

This is generally an interesting series of observations with an elegant mechanism. Many of the experiments are done in vivo with highly rigorous KO models. However, in many of the EV experiments, there are concerns about a lack of appropriate controls that might limit the rigor of those aspects of the study. 

Weaknesses:

(1) There is strong variability in the gene expression between mice in Figure 1B. I worry that the signals may not be statistically significant. The authors should assess the statistical significance.

(2) In Figure 2B, the necrosis areas that are circled in the image do not seem to resemble the quantitation on the right. For example, I don't see 60% necrosis in the APAP PBS group. Also, I don't see 5-10% necrosis in the CLDN APAP group. More images that are clearer are needed, and circled necrosis areas should be shown.

(3) In Figure 2D, a higher N should be shown. The number of mice (3) is different from the other experiments, so the exclusion of those mice should be explained.

(4) In general, control EVs from a non-PLT source should be used for all EV-related experiments. EVs derived from AML12 hepatocytes would seem to be a reasonable control for some of the experiments. Otherwise, it is hard to know if this is a general EV effect or one that is specific to PLT-derived EVs. In Figure 3B, EVs from non-PLTs should be used as a control. Since it is possible that all EVs express some level of TSG101 or CD63. In addition, control EVs should be used to test effects on KC metabolism, since the claim is that the effects are specific to PLT-derived EVs. Similarly, Figure 4 needs some kind of EV control that is not from PLTs.

(5) Figure 5B should include an EV control in the blot. Most of the blots need controls from AML12 EVs or from another in vivo source.

(6) It is a little difficult to imagine how enough ALDOA protein could be transmitted from PEVs to influence KC glycolysis on the gene expression level. It is possible that ALDOA is required for PLT-induced activation of KCs, or that EVs from PLTs can induce a metabolic shift in KCs. However, it has not been definitively shown that ALDOA from PEVs is directly causing the KC activation. Ultimately, it would be good to obtain PEVs from ALDOA WT and KO mice, then provide these PEVs to AILI mice without PLTs to see if they have differential effects on the AILI model. This would really demonstrate that the ALDOA in the PEVs is mediating the glycolytic, injurious effect.