Functional identification of soluble uric acid as an endogenous inhibitor of CD38

Reviewer #1 (Public Review):

This manuscript describes soluble Uric Acid (sUA) as an endogenous inhibitor of CD38, affecting CD38 activity and NAD+ levels both in vitro and in vivo. Importantly, the inhibition constants calculated support the claim that sUA inhibits CD38 under physiological conditions. These findings are of extreme importance to understanding the regulation of an enzyme that has been shown to be the main NAD+/NMN-degrading enzyme in mammals, which impacts several metabolic processes and has major implications for understanding aging diseases. The manuscript is well written, the figures are self-explanatory, and in the experiments presented, the data is very solid. The authors discuss the main limitations of the study, especially in regard to the in vivo results. As a whole, I believe that this is a very interesting manuscript that will be appreciated by the scientific community and that opens a lot of new questions in the field of metabolism and aging. I found some issues that I believe constitute a weakness in the manuscript, and although they do not require new experiments, they may be considered by the authors for discussion in the final version of the manuscript.

The authors acknowledge the existence of several previous papers involving pharmacological inhibition of CD38 and their impact on several models of metabolism and aging. However, they only cite reviews. Given the focus of the manuscript, I believe that the seminal original papers should be cited.

Related to the previous comment, the authors show that they have identified the functional group on sUA that inhibits CD38, 1,3-dihydroimidazol-2-one. How does this group relate with previous structures that were shown to inhibit CD38 and do not have this chemical structure? Is sUA inhibiting CD38 in a different site? A crystallographic structure of CD38-78c is available in PDB that could be used to study or model these interactions.

Although the mouse model used to manipulate sUA levels is not ideal, the authors discuss its limitations, and importantly, they have CD38 KO mice as control. However, all the experiments were performed in very young mice, where CD38 expression is low in most tissues (10.1016/j.cmet.2016.05.006). This point should be mentioned in the discussion and maybe put in the context of variations of sUA levels during aging.

Reviewer #2 (Public Review):

Summary:

This is an interesting work where Wen et al. aimed to shed light on the mechanisms driving the protective role of soluble uric acid (sUA) toward avoiding excessive inflammation. They present biochemical data to support that sUA inhibits the enzymatic activity of CD38 (Figures 1 and 2). In a mouse model of acute response to sUA and using mice deficient in CD38, they find evidence that sUA increases the plasma levels of nicotinamide nucleotides (NAD+ and NMN) (Figure 3) and that sUA reduces the plasma levels of inflammasome-driven cytokines IL-1b and IL-18 in response to endotoxin, both dependent on CD38 (Figure 4). Their work is an important advance in the understanding of the physiological role of sUA, with mechanistic insight that can have important clinical implications.

Strengths:

The authors present evidence from different approaches to support that sUA inhibits CD38, impacts NAD+ levels, and regulates inflammatory responses through CD38.

Weaknesses:

The authors investigate macrophages as the cells impacted by sUA to promote immunoregulation, proposing that inflammasome inhibition occurs through NAD+ accumulation and sirtuin activity due to sUA inhibition of CD38. Unfortunately, the study still lacks data to support this model, as they could not replicate their in vivo findings using murine bone marrow-derived macrophages, a standard model to assess inflammasome activation. Without an alternative approach, the study lacks data to establish in vitro that sUA inhibition of CD38 reduces inflammasome activation in macrophages - consequently, they cannot determine yet if both NAD+ accumulation and sirtuin activity in macrophages is a mechanism leading to sUA role in vivo.

Reviewer #3 (Public Review):

Summary:

In the present manuscript, the authors propose that soluble Uric acid (sUA) is an enzymatic inhibitor of the NADase CD38 and that it controls levels of NAD modulating inflammatory response. Although interesting the studies are at this stage preliminary and validation is needed.

Strengths:

The study characterizes the potential relevance of sUA in NAD metabolism.

Weaknesses:

(1) A full characterization of the effect of sUA in other NAD-consuming and synthesizing enzymes is needed to validate the statement that the mechanism of regulation of NAD by sUA is mediated by CD38, The CD38 KO may not serve as the ideal control since it may saturate NAD levels already. Analysis of multiple tissues is needed.

(2) The physiological role of sUA as an endogenous inhibitor of CD38 needs stronger validation (sUA deficient model?).

(3) Flux studies would also be necessary to make the conclusion stronger.