NAD boosting mediated by CD38 inhibition drives reversal of a pathological vicious cycle of intracrine activity and inflammation in eyelid meibomian gland dysfunction

Reviewer #1 (Public review):

Summary:

While the results show some loss in the eyelid meibomian glands, there is significant gland retention in HSD3b6 KO mice, as shown in Figure 2. This is supported by the lack of DEG patterns showing downregulation of Meibum lipid genes (AWAT2, Far2, Soat1, Plin2, SCD, etc.), and no decrease in Pparg expression, known to be critical for meibomian gland lipid gene expression.

Weaknesses:

It should be noted that while the authors indicate that CD38 is significantly up-regulated in the HSD3b6 KO mouse, the increase was not sufficient to show a significant adjusted P-value. Bulk RNA sequencing also shows no significant change in meibum lipid gene expression for aged mice that are treated with 78c, an inhibitor of CD38, which the authors indicate increases NAD levels, leading to increased meibomian gland size compared to vehicle-treated mice. Unfortunately, there was no increase in meibum lipid gene expression with 78c, as identified by adjusted P-value. However, it should be noted that the supplemental file covering DEG expression was labeled as a Microarray analysis. This did not include the 78c+NMN treated mice, which the authors contend show a more impactful effect on the meibomian gland.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors demonstrate strong correlations between a pro-inflammatory state, the activity of an intracrine hormone (3 beta-hydroxysteroid dehydrogenase, 3B-HSD), and the NAD co-factor. Specifically, in a 3B-HSD knockout mouse, there was an upregulation in pro-inflammatory cytokines and increased CD38+ cells (CD38 is an enzyme that depletes NAD, a necessary cofactor for 3B-HSD activity). Conversely, induction of inflammation in the eyelids resulted in reductions in 3B-HSD activity. Supplementation with 5 alpha-dihydrotestosterone (DHT) or the NAD precursor NMN, and inhibition of CD38 activity (78c), corrected the pathologies observed in both the 3B-HSD knockout mouse and the pro-inflammatory model (LPS injection into eyelids).

Strengths:

The experiments were performed with good rigor, assessing the impact of inflammation and 3B-HSD activity using multiple model systems. The endpoints represented a combination of transcriptional changes, protein quantification, enzymatic activity, and immunofluorescent microscopy. The authors use human tissue from both younger and older individuals to justify their hypotheses that increased CD38 + cells and reduced 3B-HSD quantity exist in older individuals. The data provide the foundation for assessing more global changes to the tear film and ocular surface.

Weaknesses:

The main weaknesses of the study include the following:

(1) An absence of information on meibomian gland health, tear film, and ocular surface.

(2) Too few human subjects to validate the hypotheses.

Conclusion:

Overall, this study demonstrates an important relationship that exists between intracrine signaling, inflammation, and cofactor signaling. It represents a novel approach in therapeutic design for patients with meibomian gland dysfunction.

Reviewer #3 (Public review):

Summary:

The authors aimed to investigate whether disruption of intracrine steroid hormone metabolism contributes to meibomian gland dysfunction and proposed a "vicious cycle" of gland dysfunction and inflammation, using a global Had3b6 knockout mouse model. The work addresses an important aspect of MGD, but its impact may be limited unless the intracrine mechanism can be more clearly distinguished from systemic hormonal effects.

Strengths:

This study addressed an important question. The hormonal regulation of the meibomian gland has long been recognized. If clarified, the concept of local steroid metabolism influencing gland homeostasis could have implications for understanding disease mechanisms and identifying therapeutic targets.

Weaknesses:

The use of a global knockout makes it difficult to separate local intracrine effects from systemic hormonal changes, and key controls and hormone measurements are lacking.
LPS-induced inflammation may not reflect the chronic nature of MGD.

Author Response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

While the results show some loss in the eyelid meibomian glands, there is significant gland retention in HSD3b6 KO mice, as shown in Figure 2. This is supported by the lack of DEG patterns showing downregulation of Meibum lipid genes (AWAT2, Far2, Soat1, Plin2, SCD, etc.), and no decrease in Pparg expression, known to be critical for meibomian gland lipid gene expression.

Weaknesses:

It should be noted that while the authors indicate that CD38 is significantly up-regulated in the HSD3b6 KO mouse, the increase was not sufficient to show a significant adjusted P-value. Bulk RNA sequencing also shows no significant change in meibum lipid gene expression for aged mice that are treated with 78c, an inhibitor of CD38, which the authors indicate increases NAD levels, leading to increased meibomian gland size compared to vehicle-treated mice. Unfortunately, there was no increase in meibum lipid gene expression with 78c, as identified by adjusted P-value. However, it should be noted that the supplemental file covering DEG expression was labeled as a Microarray analysis. This did not include the 78c+NMN treated mice, which the authors contend show a more impactful effect on the meibomian gland.

We thank the reviewer for the careful evaluation and insightful comments regarding the interpretation of meibomian gland phenotypes and gene expression profiles.

Regarding the point on the apparent retention of meibomian gland structure and the lack of downregulation of key lipid-related genes (e.g., Awat2, Far2, Soat1, Plin2, Scd, and Pparg), we agree that these observations are important for interpreting the extent of gland dysfunction. In the revised manuscript, we will more clearly present and discuss the RNA-seq data, including the expression profiles of representative meibomian gland lipid genes (and other DEGs), to better contextualize these findings.

With respect to Cd38 expression, we acknowledge that the statistical significance based on adjusted P-values was limited in the current microarray dataset. To address this point, we will perform additional validation using targeted quantitative PCR with specific primers to more accurately assess Cd38 expression changes.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors demonstrate strong correlations between a pro-inflammatory state, the activity of an intracrine hormone (3 beta-hydroxysteroid dehydrogenase, 3B-HSD), and the NAD co-factor. Specifically, in a 3B-HSD knockout mouse, there was an upregulation in pro-inflammatory cytokines and increased CD38+ cells (CD38 is an enzyme that depletes NAD, a necessary cofactor for 3B-HSD activity). Conversely, induction of inflammation in the eyelids resulted in reductions in 3B-HSD activity. Supplementation with 5 alpha-dihydrotestosterone (DHT) or the NAD precursor NMN, and inhibition of CD38 activity (78c), corrected the pathologies observed in both the 3B-HSD knockout mouse and the pro-inflammatory model (LPS injection into eyelids).

Strengths:

The experiments were performed with good rigor, assessing the impact of inflammation and 3B-HSD activity using multiple model systems. The endpoints represented a combination of transcriptional changes, protein quantification, enzymatic activity, and immunofluorescent microscopy. The authors use human tissue from both younger and older individuals to justify their hypotheses that increased CD38 + cells and reduced 3B-HSD quantity exist in older individuals. The data provide the foundation for assessing more global changes to the tear film and ocular surface.

Weaknesses:

The main weaknesses of the study include the following:

(1) An absence of information on meibomian gland health, tear film, and ocular surface.

(2) Too few human subjects to validate the hypotheses.

Conclusion:

Overall, this study demonstrates an important relationship that exists between intracrine signaling, inflammation, and cofactor signaling. It represents a novel approach in therapeutic design for patients with meibomian gland dysfunction.

We thank the reviewer for the positive evaluation of our study and for recognizing the rigor of the experiments, the use of multiple model systems, and the potential of the data to provide a foundation for further investigation.

Regarding the points raised under weaknesses, we agree that evaluation of meibomian gland function, tear film, and ocular surface phenotypes would provide important additional insight. In the present study, we focused primarily on the structural phenotype of the meibomian gland, particularly gland size, as a primary feature of MGD. We acknowledge that pathological assessments of gland function and ocular surface conditions have not been fully addressed. We will clearly state this limitation and expand the Discussion to position these aspects as important directions for future investigation.

With respect to the limited number of human samples, we acknowledge that this is an important consideration for validating the translational relevance of our findings. We will revise the manuscript to more explicitly address this limitation and interpret the human data with appropriate caution.

Reviewer #3 (Public review):

Summary:

The authors aimed to investigate whether disruption of intracrine steroid hormone metabolism contributes to meibomian gland dysfunction and proposed a "vicious cycle" of gland dysfunction and inflammation, using a global Had3b6 knockout mouse model. The work addresses an important aspect of MGD, but its impact may be limited unless the intracrine mechanism can be more clearly distinguished from systemic hormonal effects.

Strengths:

This study addressed an important question. The hormonal regulation of the meibomian gland has long been recognized. If clarified, the concept of local steroid metabolism influencing gland homeostasis could have implications for understanding disease mechanisms and identifying therapeutic targets.

Weaknesses:

The use of a global knockout makes it difficult to separate local intracrine effects from systemic hormonal changes, and key controls and hormone measurements are lacking.

LPS-induced inflammation may not reflect the chronic nature of MGD.

We thank the reviewer for the thoughtful evaluation and for highlighting the importance of distinguishing intracrine mechanisms from systemic hormonal effects.

We agree that, as currently presented, the use of a global Hsd3b6 knockout model makes it difficult to fully separate local intracrine effects from systemic hormonal changes. This point is also consistent with the major concern raised in the editorial assessment regarding the need to more clearly establish the proposed intracrine mechanism. To address this issue, we will strengthen the evidence for intracrine regulation by incorporating additional analyses. Specifically, we will assess systemic testosterone levels in Hsd3b6 knockout mice and include appropriate controls using orchidectomized (ORX) mice. These analyses will help to better distinguish local intracrine mechanisms from systemic hormonal influences.