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

  1. Yuki Hamada
  2. Takehide Sakamoto
  3. Daisuke Yarimizu
  4. Hikari Uehara
  5. Xinyan Shao
  6. Tom Macpherson
  7. Emi Hasegawa
  8. Masao Doi

  • Curated by eLife

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    eLife Assessment

    This valuable study links NAD⁺-dependent HSD3B6 steroidogenic activity, androgen receptor signaling, and CD38-associated inflammation to meibomian gland atrophy, suggesting a potentially targetable feedback cycle relevant to dry eye disease. The revised work provides solid evidence for this relationship, with additional measurements showing no significant differences in circulating testosterone or aldosterone and persistence of elevated Cd38 expression and reduced glandular NAD⁺ levels after castration, strengthening support for a local steroidogenic mechanism. However, the global knockout does not definitively establish a gland-specific intracrine mechanism, and the absence of tear-film and ocular-surface functional measurements limits conclusions about the consequences for dry eye disease.

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Abstract

Vicious cycles —reciprocal cause-and-effect loops that progressively intensify dysfunction —are implicated in many diseases. Here, we examined whether “intracrine” steroidogenesis, a tissue-local mode of hormone production, participates in a pathological vicious cycle in eyelid meibo-mian gland dysfunction (MGD), the most common cause of evaporative dry eye disease with incidence increasing with age. We show that loss of meibomian intracrine androgen production triggers inflammatory remodeling of the eyelid tarsal plate. Conversely, inflammation suppresses nicotinamide adenine dinucleotide (NAD)-dependent 3β-hydroxysteroid dehydrogenase activity in the meibomian gland, thereby further weakening intracrine steroidogenesis. Mechanistically, we identified inflammation-associated accumulation of the NAD-degrading enzyme CD38 as a key down-regulator for the NAD-dependent intracrine activity. Accordingly, pharmacological inhibition of CD38 (using 78c) in aged mice, restores meibomian cellular NAD levels, rescues intracrine activity, suppresses inflammatory signatures, and promotes recovery of gland size. Thus, our data suggest that boosting NAD availability not only ameliorates meibomian gland dysfunction but also induces a reciprocal “virtuous” cycle, in which suppression of inflammation and restoration of intracrine activity mutually reinforce each other. These findings constitute a previously unknown example in which the reversal of a pathological vicious cycle into a bene-ficial virtuous cycle underlies the therapeutic effects of NAD boosting and may help combat age-associated meibomian gland disorder.

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  1. eLife

    eLife Assessment

    This valuable study links NAD⁺-dependent HSD3B6 steroidogenic activity, androgen receptor signaling, and CD38-associated inflammation to meibomian gland atrophy, suggesting a potentially targetable feedback cycle relevant to dry eye disease. The revised work provides solid evidence for this relationship, with additional measurements showing no significant differences in circulating testosterone or aldosterone and persistence of elevated Cd38 expression and reduced glandular NAD⁺ levels after castration, strengthening support for a local steroidogenic mechanism. However, the global knockout does not definitively establish a gland-specific intracrine mechanism, and the absence of tear-film and ocular-surface functional measurements limits conclusions about the consequences for dry eye disease.

  2. eLife

    Reviewer #1 (Public review):

    Summary:

    The authors provide a compelling story linking NAD dependent HSD3b6 gene expression in the eyelid to a viscous cycle involving decreased steroidogenesis and AR signaling, pro-inflammatory cytokine release, inflammation, CD38 activation and further NAD+ decline that induces meibomian gland atrophy leading to dry eye disease. The work follows on studies published by the authors in Nature Aging and uses a previously reported HSD3b6 knockout mouse to try and show meibomian gland atrophy, inflammation and CD38+ cell recruitment. Overall the data presented does not appear to support the authors conclusions, and the described interactions seem to be overstated, in that if HSD3b6 was critical to meibomian gland function, one might expect complete loss of the meibomian gland in knockout mice, which is not the case.

    Comments on revised version:

    It should be noted that while the authors indicate the CD38 is significantly up-regulated in the HSD3b6 KO mouse, the increase was not sufficient to show a significant adjusted P-value after revision of the manuscript. 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, that 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 or with 78c+NMN treatment as identified by adjusted P-value.

  3. eLife

    Reviewer #2 (Public review):

    Summary:

    In the manuscript entitled, "NAD boosting mediated by CD38 inhibition drives reversal of a pathological vicious cycle of intracrine activity and inflammation in eyelid meibomian gland dysfunction", 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:

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

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

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

    Comments on the revised version:

    The title of the manuscript is a bit deceiving, particularly because it mentions "meibomian gland dysfunction". The authors would need to demonstrate abnormalities in lipid synthesis (quantity or quality) and ocular surface changes associated with loss of this intracrine signaling. The data presented are strong for demonstrating the role of NAD synthesis and utilization in meibomian gland homeostasis, but do not explain the role in ophthalmic diseases, such as MGD. This could possibly be applicable to forms of blepharitis or conjunctivitis as well given their findings of CD38+ cells present in the eyelids of the KO mice. Therefore, the authors should remove the claims of meibomian gland dysfunction in the title and comments in the abstract. Instead, they could use meibomian gland homeostasis for both sections.

  4. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    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 of our work and for the constructive comments regarding the interpretation of the transcriptomic data and the molecular basis of meibomian gland dysfunction.

    First, regarding the statistical significance of Cd38 expression in the microarray analysis, we performed additional validation using gene-specific quantitative PCR in independent eyelid tarsal plate samples. These new data are now presented in Fig. 2C, and the Results section has been revised as follows:

    “… To validate the upregulation of Cd38, we performed real-time PCR analysis using gene-specific primers in independent eyelid tarsal plate samples, which confirmed a significant increase in Cd38 expression in Hsd3b6−/− mice compared to Hsd3b6+/+ controls. This difference was also observed in castrated mice; thus, the effect of Hsd3b6 deletion on Cd38 expression is independent of testicular testosterone (Fig. 2C).”

    Second, we apologize for the error in the original supplementary DEG file, which caused confusion during the review process. We have now replaced it with a complete RNA-seq dataset that includes all expression values for aged mice treated with 78c+NMN (please see Supplementary Data 2). As shown in Fig. 4E, F, treatment with 78c+NMN significantly increased the expression of Elovl4 and Elovl7, elongases responsible for the synthesis of very long-chain fatty acids in the meibomian gland. We also re-examined representative meibum lipid-related genes (Awat2, Far2, Soat1, Plin2, and Scd1). Although none reached statistical significance individually, collective analysis of these representative lipid-related genes by two-way ANOVA with Bonferroni's post hoc test demonstrated a significant overall increase following 78c+NMN treatment in aged mice, compared with controls, supporting coordinated upregulation of the meibomian gland lipid synthesis program.

    These analyses indicate that, although the individual lipid-related genes showed only modest changes, they exhibit a coordinated increase following 78c+NMN treatment in aged mice. By contrast, the same gene set showed no significant overall change in young (3-month-old) mice, consistent with the original RNA-seq results presented in Fig. 4E. To better illustrate these findings, we have summarized the expression of these representative genes in a new Supplementary Fig. S5 and revised the Results accordingly, as follows:

    “…Furthermore, representative meibum lipid-related genes, including Awat2, Far2, Soat1, Plin2, and Scd1, exhibited a coordinated increase following 78c-NMN treatment (Fig. S5). While the changes in individual genes were modest and did not reach statistical significance (Fig. 4E), analysis of the gene set as a whole demonstrated a significant overall increase compared with vehicle-treated controls.”

    “In contrast, the same treatment in young mice did not significantly alter gene expression profiles (Fig. 4E, young), and no coordinated increase was observed for Awat2, Far2, Soat1, Plin2, or Scd1 (Fig. S5). This result is consistent with previous studies [44,47,48] showing that 78c (or NMN) exerts minimal effects in young mice compared with aged mice across multiple physiological parameters, including glucose homeostasis, exercise tolerance, and cardiac function.”

    We also would like to note that the complete RNA-seq datasets provided in Supplementary Data 2 allow readers to examine the expression profiles of additional genes of interest.

    Finally, regarding the observation that meibomian glands remain partially preserved despite the presence of gland atrophy in Hsd3b6−/− mice, together with the limited significant changes in classical lipid-related genes, we agree that this is an important point for interpreting the mechanism(s) of meibomian gland dysfunction. Our previous transcriptomic analysis of FACS-purified meibomian acinar cells in Hsd3b6−/− mice (PMID: 37117756) demonstrated reduced expression of genes involved in cell proliferation/differentiation, despite relatively preserved expression of canonical Pparg-dependent lipid genes. Our findings therefore extend the current framework by suggesting that meibomian gland atrophy can occur, at least in part, independently of detectable changes in Pparg expression, supporting the idea that intracrine steroidogenesis contributes to maintaining the proliferative activity of basal acinar cells alongside the Pparg-dependent pathway. Accordingly, we have revised the Discussion to clarify the relationship between our findings and the established Pparg-centred model of meibomian gland maintenance, as follows:

    “Although 78c+NMN treatment effectively restored meibomian gland size, the molecular mechanisms underlying this recovery remain incompletely understood. Treatment with 78c+NMN significantly increased the expression of Elovl4, Elovl7, and Hsd3b6. However, representative meibum lipid-related genes, including Awat2, Far2, Soat1, Plin2, and Scd1, exhibited only a modest coordinated increase following treatment. Interestingly, the expression of Pparg, a key transcriptional regulator of meibocyte differentiation and lipid synthesis [88-90], remained unchanged. These findings suggest that restoration of meibomian gland size by 78c+NMN does not require detectable changes in Pparg mRNA expression, although Pparg activity may instead be regulated at the level of protein abundance, subcellular localization, and/or post-translational modifications such as phosphorylation or SUMOylation [88,91].

    Our previous transcriptomic analysis of FACS-purified meibomian gland basal acinar cells (meibomian precursor cells expressing androgen receptors) from Hsd3b6−/− mice demonstrated impaired expression of genes involved in cell proliferation and differentiation despite relatively preserved expression of lipid-related genes [6]. In contrast, a recent single-cell study showed that Pparg expression progressively increases during meibocyte differentiation, although it is already detectable in basal acinar cells [92]. These observations suggest that Hsd3b6-dependent intracrine activity primarily supports the maintenance and proliferation of basal acinar cells, whereas the canonical Pparg pathway predominantly regulates subsequent meibocyte differentiation and lipid synthesis. Together, these findings suggest that Hsd3b6 dependent intracrine activity contributes to meibomian gland homeostasis through mechanisms that complement the Pparg-associated pathway. Thus, our findings extend the current framework by indicating that meibomian gland homeostasis is maintained not only through canonical PPARγ-mediated regulation but also through intracrine steroidogenesis.

    Newly added references:

    (88) Jester, J.V., Potma, E., and Brown, D.J. (2016). PPARγ Regulates Mouse Meibocyte Differentiation and Lipid Synthesis. Ocul Surf 14, 484-494. 10.1016/j.jtos.2016.08.001.

    (89) Kim, S.W., Xie, Y., Nguyen, P.Q., Bui, V.T., Huynh, K., Kang, J.S., Brown, D.J., and Jester, J.V. (2018). PPARγ regulates meibocyte differentiation and lipid synthesis of cultured human meibomian gland epithelial cells (hMGEC). Ocul Surf 16, 463-469. 10.1016/j.jtos.2018.07.004.

    (90) Hwang, H.S., Parfitt, G.J., Brown, D.J., and Jester, J.V. (2017). Meibocyte differentiation and renewal: Insights into novel mechanisms of meibomian gland dysfunction (MGD). Exp Eye Res 163, 37-45. 10.1016/j.exer.2017.02.008.

    (91) Ahmadian, M., Suh, J.M., Hah, N., Liddle, C., Atkins, A.R., Downes, M., and Evans, R.M. (2013). PPARγ signaling and metabolism: the good, the bad and the future. Nat Med 19, 557-566. 10.1038/nm.3159.

    (92) Zhu, X., Xu, M., Portal, C., Lin, Y., Ferdinand, A., Peng, T., Morrisey, E.E., Dlugosz, A.A., Castellano, J.M., Lee, V., et al. (2025). Identification of Meibomian gland stem cell populations and mechanisms of aging. Nat Commun 16, 1663. 10.1038/s41467-02556907-6.

    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 proinflammatory 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 3BHSD 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 careful evaluation of our work and for recognizing the novelty and potential therapeutic implications of the relationship among intracrine signaling, inflammation, and NAD metabolism in meibomian gland dysfunction. We also appreciate the reviewer’s constructive comments on the limitations of the present study and areas for improvement in the manuscript.

    Regarding the lack of information on meibomian gland health, tear film, and ocular surface, we have added a limitations section to explicitly acknowledge that these functional outcomes were not directly assessed in the present study. As the reviewer kindly noted, we only focused on a key pathological hallmark of MGD, namely age-associated tissue atrophy, and based on this we explored the NAD/3β-HSD/inflammation-mediated mechanism. Thus, a limitation of the present study is the lack of assessment of the downstream consequences of MGD. This point is now explicitly discussed in the manuscript; please note that additional details are provided in our responses to Reviewer #2’s Recommendations for the authors.

    Regarding the human data, we added two additional human eyelid samples, increasing the sample size of the older group from 2 to 4. Nevertheless, we agree that the sample size remains limited and have now explicitly acknowledged this limitation in Discussion as follows:

    “A caveat of this study is that the number of human eyelid samples analyzed was limited. Larger human cohorts will be necessary to establish the translational relevance of the age-associated CD38 accumulation observed in human tissues.”

    Taken together, we believe that these revisions have clarified the scope and limitations of our present study.

    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 of our work and for highlighting the importance of distinguishing local intracrine mechanisms from systemic hormonal effects. In response, we performed several additional experiments and substantially revised the manuscript. Specifically, we measured plasma testosterone and aldosterone levels and found no significant differences between Hsd3b6−/− and Hsd3b6+/+ mice. In addition, we repeated key experiments in castrated mice and observed that the effects of Hsd3b6 deletion on tarsal plate Cd38 expression and meibomian NAD levels were preserved despite the absence of testicular testosterone. These new findings provide additional support for a local intracrine mechanism in the eyelid. We also revised the Introduction and Discussion to better explain the proposed intracrine–inflammation vicious cycle and clarified the rationale and limitations of the LPS-based inflammation model, including its inability to fully recapitulate the chronic low-grade inflammation characteristic of MGD. Further details are provided in our responses to Reviewer #3’s Recommendations for the authors. We hope that these additions and revisions have strengthened the manuscript and addressed the reviewer’s concerns.

    Recommendations for the authors:

    Reviewer #1 (Recommendations for the authors):

    Overall, the authors need to provide bulk RNA sequencing data for all treatment groups and provide some explanation of the measurements in individual mice for gland growth.

    We thank the reviewer for this helpful comment. We have now replaced the original supplementary file with a complete RNA-seq dataset that includes all treatment groups (Supplementary Data 2). In addition, as described in our response to the Public Review comment above, we extensively revised our manuscript to provide explanation of gene expression profiles (particularly, representative meibum lipid-related genes) in the dataset.

    Accordingly, we have expanded the Results and reconstructed the Discussion by incorporating two new discussion paragraphs. The relevant revisions are provided below for your perusal:

    “… Furthermore, representative meibum lipid-related genes, including Awat2, Far2, Soat1, Plin2, and Scd1, exhibited a coordinated increase following 78c-NMN treatment (Fig. S5). While the changes in individual genes were modest and did not reach statistical significance (Fig. 4E), analysis of the gene set as a whole demonstrated a significant overall increase compared with vehicle-treated controls.”

    “In contrast, the same treatment in young mice did not significantly alter gene expression profiles (Fig. 4E, young), and no coordinated increase was observed for Awat2, Far2, Soat1, Plin2, or Scd1 (Fig. S5). This result is consistent with previous studies [44,47,48] showing that 78c (or NMN) exerts minimal effects in young mice compared with aged mice across multiple physiological parameters, including glucose homeostasis, exercise tolerance, and cardiac function.”

    “Although 78c+NMN treatment effectively restored meibomian gland size, the molecular mechanisms underlying this recovery remain incompletely understood. Treatment with 78c+NMN significantly increased the expression of Elovl4, Elovl7, and Hsd3b6. However, representative meibum lipid-related genes, including Awat2, Far2, Soat1, Plin2, and Scd1, exhibited only a modest coordinated increase following treatment. Interestingly, the expression of Pparg, a key transcriptional regulator of meibocyte differentiation and lipid synthesis [88-90], remained unchanged. These findings suggest that restoration of meibomian gland size by 78c+NMN does not require detectable changes in Pparg mRNA expression, although Pparg activity may instead be regulated at the level of protein abundance, subcellular localization, and/or post-translational modifications such as phosphorylation or SUMOylation [88,91].

    Our previous transcriptomic analysis of FACS-purified meibomian gland basal acinar cells (meibomian precursor cells expressing androgen receptors) from Hsd3b6−/− mice demonstrated impaired expression of genes involved in cell proliferation and differentiation despite relatively preserved expression of lipid-related genes [6]. In contrast, a recent single-cell study showed that Pparg expression progressively increases during meibocyte differentiation, although it is already detectable in basal acinar cells [92]. These observations suggest that Hsd3b6-dependent intracrine activity primarily supports the maintenance and proliferation of basal acinar cells, whereas the canonical Pparg pathway predominantly regulates subsequent meibocyte differentiation and lipid synthesis. Together, these findings suggest that Hsd3b6-dependent intracrine activity contributes to meibomian gland homeostasis through mechanisms that complement the Pparg-associated pathway. Thus, our findings extend the current framework by indicating that meibomian gland homeostasis is maintained not only through canonical PPARγ-mediated regulation but also through intracrine steroidogenesis.”

    Reviewer #2 (Recommendations for the authors):

    The main suggestion for the study is the correlation with meibomian gland function. While the study correlates changes with 3B-HSD activity, pro-inflammatory cytokines, and NAD cofactor levels, there is little support describing the pathological changes to the meibomian glands and the impact on the tear film and ocular surface. The changes in the lipid profiles, tear film stability, aqueous tear production, ocular surface staining properties, corneal epithelial transcriptional abnormalities, and leukocyte infiltration into the corneal stroma could all be assessed. While the authors have done a thorough job of demonstrating the correlation of the three major factors listed above, the correlation with meibomian gland function is not completely assessed in the study.

    We thank the reviewer for this insightful comment. We agree that functional assessments such as tear film stability, ocular surface pathology, lipid composition, and other downstream manifestations of MGD would complement the mechanistic findings reported here and provide additional insight into their physiological consequences.

    However, the primary objective of the present study was to define the molecular mechanism underlying MGD, specifically the pathological interaction between meibomian gland intracrine activity and inflammation, and the role of the CD38–NAD axis in this process. Accordingly, our experiments were designed to focus on mechanistic endpoints directly related to this hypothesis, including 3β-HSD activity, NAD availability, inflammatory gene expression, and CD38 accumulation. In addition, we assessed a key pathological hallmark of MGD, namely age-associated meibomian gland atrophy and its recovery following 78c/NMN treatment.

    While direct evaluation of tear film and ocular surface parameters was beyond the scope of the present mechanistic study, we agree that such analyses represent important future directions. To acknowledge this, we have added the following statement to the Discussion:

    “… Firstly, while this study focused on defining the molecular and structural mechanisms underlying meibomian gland dysfunction, functional outcomes such as tear film stability, ocular surface integrity, meibum lipid composition, and other clinically relevant manifestations of disease were not directly assessed. Given the well-established link between meibomian gland dysfunction and tear film instability [57-59], future studies incorporating these functional assessments will be important to determine how the molecular and structural changes identified here contribute to ocular surface pathology and disease progression.”

    The description of the vicious cycle seemed a bit out of place in the introduction. This would have been most appropriate in the conclusion/discussion in the manuscript. The general nature of the description lacked focus on the eyelids and meibomian glands, and the narrative took time to get to the interactions that would be assessed in the study.

    We thank the reviewer for this insightful suggestion. We agree that the original Introduction placed excessive emphasis on the general concept of vicious cycles and that the discussion of disease-wide examples diverted attention from the specific focus of the present study on the meibomian gland.

    In response, we substantially shortened and refocused the Introduction. Specifically, we removed the broad descriptions of vicious cycles in neurodegenerative, metabolic, and cardiovascular diseases. The revised Introduction now introduces the concept of a vicious cycle only briefly and moves more directly to the biological context of meibomian gland dysfunction and local intracrine steroidogenesis.

    At the same time, we agree that the broader implications of this concept are better suited for the Discussion. We therefore added a brief concluding paragraph to discuss how our findings may inform the understanding of age-associated vicious-cycle processes in other diseases and may also provide a framework for considering intracrine-related pathology beyond the meibomian gland. The corresponding text has been added to the Discussion, as follows.

    “Finally, although the present study focused on the eyelid meibomian gland, the concept of an intracrine–inflammation vicious cycle may have broader relevance. Self-reinforcing pathological processes are implicated in several age-associated diseases, and altered intracrine activity has been described in multiple extra gonadal and cancerous tissues [5,7,8,93,94]. Our findings may therefore provide a useful framework for understanding how local hormone metabolism contributes to the initiation and maintenance of tissue-specific pathological cycles during ageing.”

    Reviewer #3 (Recommendations for the authors):

    (1) Introduction:

    (a) The first paragraph of the introduction defines the term "vicious cycle," but its relevance to the current study is low. It is not until line 54 that the specific tissue of interest is introduced. It is suggested that the beginning of the introduction be shortened to emphasize information that is directly relevant to the study.

    We thank the reviewer for this helpful suggestion. We agree that the original opening of the Introduction was too general and that the specific focus of the study on the meibomian gland should be introduced earlier.

    In response, we substantially shortened the first part of the Introduction. Specifically, we removed the broad discussion of vicious cycles across multiple disease contexts and revised the opening so that the concept of a vicious cycle is introduced only briefly. The revised Introduction now moves directly to the relevance of local intracrine steroidogenesis in the eyelid meibomian gland, thereby better emphasizing the biological context and focus of the present study.

    At the same time, we recognized that some of the broader implications of the study remain important. Therefore, we incorporated these points into the Discussion, where we briefly discuss the potential relevance of intracrine–inflammation vicious cycles to other age-associated diseases and intracrine-related pathologies beyond the meibomian gland, as follows:

    “Finally, although the present study focused on the eyelid meibomian gland, the concept of an intracrine–inflammation vicious cycle may have broader relevance. Self-reinforcing pathological processes are implicated in several age-associated diseases, and altered intracrine activity has been described in multiple extragonadal and cancerous tissues [5,7,8,93,94]. Our findings may therefore provide a useful framework for understanding how local hormone metabolism contributes to the initiation and maintenance of tissue-specific pathological cycles during ageing.”

    (b) In the introduction, the authors should also include common types of intracrine hormones, such as DHT.

    We thank the reviewer again for this valuable suggestion. In response, we revised the Introduction to include representative examples of intracrine steroid hormones, including 5α-dihydrotestosterone (DHT) and estrogens, together with a brief description of their local production and action within peripheral tissues. We believe that this addition helps clarify the concept of intracrinology for readers less familiar with the field.

    “Common examples of intracrine steroid hormones include 5α-dihydrotestosterone (DHT) produced in the skin, prostate, and eyelid meibomian glands, as well as estrogens synthesized within the breast and endometrium [3-8]. These locally produced hormones act within the tissue in which they are synthesized.”

    (c) The hypothesis of a "vicious cycle" in MGD is not fully explained. Specifically, how intracrine steroids drive the meibomian gland into a vicious cycle is not described. The current information only discusses a possible association between steroid hormones and meibomian gland health.

    According to this important suggestion, we performed additional experiments and rewrote our introduction. Please note that plasma testosterone levels were not impaired in Hsd3b6−/− mice and their eyelid proinflammatory phenotypes (compared to wt mouse eyelid) remained preserved in castrated mice. These data are now provided, by performing additional experiments (please see our replies to your comments, below). Taking these observations into account, we have tempered our introduction.

    (2) Results:

    (a) One major concern is that sex hormones have long been recognized to be associated with the meibomian gland and have been extensively studied (see the TFOS guidelines). There have also been clinical trials using androgens to treat MGD. With the global knockout of Had3b6, systemic sex hormone levels could also be altered, which may result in MGD (estrogens are synthesized from androgens and would therefore also be affected).

    This thoughtful concern is well received, as we were not attentive to describe systemic testosterone in Hsd3b6+/+ and Hsd3b6−/− mice in our original manuscript (for which we should apologize). The corresponding plasma testosterone data are now incorporated as Fig. S1A and presented below for your perusal.

    Importantly, plasma testosterone levels in Hsd3b6−/− mice were comparable with those in wt Hsd3b6+/+ mice, consistent with the known compensatory role of the paralogous enzyme Hsd3b1 in the mouse testis [21], which was confirmed to be expressed in Hsd3b6−/− mice (see new Fig. S1B). This type of compensation by paralogous steroidogenic enzymes has also been reported in other knockout model, such as Hsd17b3−/− mice, in which circulating testosterone levels were preserved due to a compensatory role of Hsd17b12 in the testis (new refs [83,84]). Based on these results, we rewrote our Results and Discussion, as follows.

    “plasma testosterone levels were indistinguishable between Hsd3b6−/− and Hsd3b6+/+ mice (Fig. S1A), consistent with the known compensatory role of the paralogous enzyme Hsd3b1 in the mouse testis [21], which was confirmed to be expressed in Hsd3b6−/− mice (Fig. S1B). These results indicate that local steroidogenic activity in the eyelid exerts anti-inflammatory effects independent of circulating testosterone levels.”

    “For the interpretation of our meibomian intracrine activity model, it is important to note that in Hsd3b6−/− mice, plasma testosterone levels were maintained at the levels comparable with those in Hsd3b6+/+ mice (Fig. S1A). This appears due to a redundant expression of Hsd3b1 in the testis [6], although in the meibomian gland, Hsd3b6 is the sole enzyme (Fig. S1B) and accordingly its deletion causes the complete abolition of meibomian 3β-HSD activity [6]. The resilience of plasma testosterone levels has also been reported in a knockout mouse model deficient in Hsd17b3, a member of 17-ketosteroid reductase enzyme family required for the formation of testosterone [83], likely due to a potential compensatory role of the paralogous enzyme Hsd17b12 in the testis [84]. The Hsd3b6−/− vs. Hsd3b6+/+ phenotypes in the tarsal plate Cd38 expression and meibomian NAD levels were preserved in castrated mice (Fig. 2C, F), which provide additional evidence to support that non-systemic local steroidogenic activity in the eyelid, rather than circulating testicular testosterone, underlies the phenotypes observed in Hsd3b6−/− mice.”

    Newly added references:

    (83) Lawrence, B.M., O'Donnell, L., Gannon, A.L., Smith, S., Curley, M.K., Darbey, A., Mackay, R., O'Shaughnessy, P.J., Smith, L.B., and Rebourcet, D. (2024). Compensatory mechanisms that maintain androgen production in mice lacking key androgen biosynthetic enzymes. FASEB J 38, e70177. 10.1096/fj.202402093R.

    (84) Rebourcet, D., Mackay, R., Darbey, A., Curley, M.K., Jørgensen, A., Frederiksen, H., Mitchell, R.T., O'Shaughnessy, P.J., Nef, S., and Smith, L.B. (2020). Ablation of the canonical testosterone production pathway via knockout of the steroidogenic enzyme HSD17B3, reveals a novel mechanism of testicular testosterone production. FASEB J 34, 1037310386. 10.1096/fj.202000361R.

    - For context, please note that plasma testosterone levels showed substantial inter-individual variability in both genotypes. Such variability is a well-established physiological feature of male mice, reflecting episodic testosterone secretion. Indeed, classical studies showed that variation in testosterone levels within the same mouse sampled on different occasions is comparable to that observed between individual mice, and a recent longitudinal LC–MS/MS study confirmed 10- to 100-fold fluctuations within individual adult male mice (Bartke and Dalterio, 1975, PMID: 1216260; Kettering et al., 2026, PMID: 41880754). Thus, the variability observed in our dataset is consistent with the known physiological dynamics of circulating testosterone in male mice.

    (b) To support the hypothesis of intracrine regulation in MGD, the following would be essential:

    - a local or targeted knockout model, and

    - a global knockout with appropriate controls, such as Had3b6+/+ mice combined with gonadectomy.

    Otherwise, the current study reflects the role of general steroid hormones in the meibomian gland rather than an intracrine-specific mechanism.

    We repeated our experiments using castrated Hsd3b6−/− mice. As shown in new Fig. 2C, we observed a significant increase in Cd38 expression in the tarsal plate of Hsd3b6−/− mice (n = 6, castrated) as compared to Hsd3b6+/+ control mice (n = 4, castrated). In addition, in Fig. 2F, we observed a significant decrease in NAD levels in the meibomian gland of Hsd3b6−/− mice (n = 3, castrated) as compared to Hsd3b6+/+ control mice (n = 5, castrated). These results provide additional evidence to support that local steroidogenic activity in the eyelid, rather than circulating testicular testosterone, underlies the phenotypes observed in Hsd3b6−/− mice. Based on these results and consideration, we have modified our manuscript as follows.

    “… This difference was also observed in castrated mice; thus, the effect of Hsd3b6 deletion on Cd38 expression is independent of testicular testosterone (Fig. 2C).”

    “… Note that regardless of castration, Hsd3b6 deletion reduced meibomian gland NAD levels.”

    “…The Hsd3b6−/− vs. Hsd3b6+/+ phenotypes in the tarsal plate Cd38 expression and meibomian NAD levels were preserved in castrated mice (Fig. 2C, F), which provide additional evidence to support that non-systemic local steroidogenic activity in the eyelid, rather than circulating testicular testosterone, underlies the phenotypes observed in Hsd3b6−/− mice.”

    (c) With the knockout of Had3b6, other steroid hormones may also be altered systemically, such as increased synthesis of the mineralocorticoid aldosterone. These hormonal changes could cause systemic effects such as hypertension, with unknown consequences for the meibomian gland. Please provide data on systemic steroid hormone levels to better interpret the current findings.

    We thank the reviewer for raising this important point. To address the possibility that systemic mineralocorticoid alterations contribute to the observed meibomian gland phenotypes, we measured plasma aldosterone levels in Hsd3b6−/− and Hsd3b6+/+ mice. These new data are now included as Fig. S6.

    Importantly, plasma aldosterone levels were not significantly different between Hsd3b6−/− and Hsd3b6+/+ mice (Fig. S6). This finding is consistent with the known expression pattern of steroidogenic enzymes in the adrenal zona glomerulosa, where both Hsd3b1 and Hsd3b6 are redundantly expressed and contribute to aldosterone biosynthesis (refs. 85-87). Thus, similar to the compensatory role of Hsd3b1 in maintaining systemic testosterone production in the testis, the redundant expression of Hsd3b1 in aldosterone-producing cells likely preserves systemic aldosterone levels in Hsd3b6−/− mice.

    Based on these new data, we have added the plasma aldosterone measurements as Fig. S6 and revised the Discussion accordingly. These results further support our conclusion that the phenotypes observed in Hsd3b6−/− mice are unlikely to be secondary to systemic endocrine alterations and instead reflect the loss of local steroidogenic activity within the eyelid.

    “…Plasma aldosterone levels were likewise preserved in Hsd3b6−/− mice (Fig. S6), consistent with the redundant expression of Hsd3b6 and Hsd3b1 in the adrenal gland zona glomerulosa cells [85-87].”

    (d) What is the age of the mice that underwent microarray analysis? The Methods section only specifies "young" and "old" without providing the actual ages. Although the ages can be inferred from the figures, they should also be clearly stated in the Methods section.

    We thank the reviewer for this comment. We have now clarified the age of the mice in the Methods section. All mice used for the microarray analysis were age-matched and were 9 weeks old at the time of tissue collection (please see page 35, lines 818-819).

    (e) Line 148: CD11b+ cells in the eye are usually referred to as dendritic cells derived from monocytes that function as antigen-presenting cells (APCs), rather than as phagocytes.

    (f) Line 149: How was it determined that the CD38+ cells are macrophages? CD38 can also be expressed by T cells, plasma cells, NK cells, and other immune cell types.

    Heeding these important instructions and based on their co-labeling with the myeloid marker CD11b, we have rewritten our Results on the cell identity of CD38 cells, as follows.

    “… We also found that the myeloid cell marker CD11b stained the same cells (Fig. S2), indicating an increased number of myeloid immune cells that express CD38 in the tarsal plate of Hsd3b6−/− mice (Fig. 2E, vs. Hsd3b6+/+).”

    In addition, to clarify that multiple immune cell types can contribute to CD38 expression in the tarsal plate and ocular tissues, we have extensively revised our manuscript by adding a new paragraph to the Discussion as follows.

    “Secondly, the cellular identity of the CD38-expressing cells observed in the tarsal plate warrants careful interpretation. In the present study, we observed co-localization of CD38 with the myeloid marker CD11b, indicating that these cells are of myeloid origin. However, CD11b+ cells in ocular tissues such as in conjunctiva are often described as monocyte-derived dendritic cells functioning as antigen-presenting cells, rather than as classical phagocytic macrophages [95,96]. Therefore, our data do not allow us to further resolve the specific myeloid subpopulation (e.g., macrophages, dendritic cells, or other subsets) responsible for CD38 expression. In addition, CD38 is known to be expressed across multiple immune cell types, including T cells, plasma cells, and NK cells [97-99], suggesting that non-myeloid immune cells may also contribute to the overall CD38 signal in the tarsal plate. While previous studies in other tissues, such as in adipose tissue and liver [39,40], have attributed age-associated CD38 upregulation primarily to macrophages, our findings highlight the need for more precise cell-type–resolved analyses in ocular tissues. Future studies using lineage-specific markers or single-cell approaches will be required to define the exact cellular sources of CD38 and their respective contributions to NAD metabolism and inflammation in the eyelid.” Added references:

    (94) Khandelwal, P., Blanco-Mezquita, T., Emami, P., Lee, H.S., Reyes, N.J., Mathew, R., Huang, R., and Saban, D.R. (2013). Ocular mucosal CD11b+ and CD103+ mouse dendritic cells under normal conditions and in allergic immune responses. PLoS One 8, e64193. 10.1371/journal.pone.0064193.

    (95) Ahadome, S.D., Mathew, R., Reyes, N.J., Mettu, P.S., Cousins, S.W., Calder, V.L., and Saban, D.R. (2016). Classical dendritic cells mediate fibrosis directly via the retinoic acid pathway in severe eye allergy. JCI Insight 1. 10.1172/jci.insight.87012.

    (96) Malavasi, F., Deaglio, S., Funaro, A., Ferrero, E., Horenstein, A.L., Ortolan, E., Vaisitti, T., and Aydin, S. (2008). Evolution and function of the ADP ribosyl cyclase/CD38 gene family in physiology and pathology. Physiol Rev 88, 841-886. 10.1152/physrev.00035.2007.

    (97) Katsuyama, E., Humbel, M., Suarez-Fueyo, A., Satyam, A., Yoshida, N., Kyttaris, V.C., Tsokos, M.G., and Tsokos, G.C. (2024). CD38 in SLE CD4 T cells promotes Ca2+ flux and suppresses interleukin-2 production by enhancing the expression of GM2 on the surface membrane. Nat Commun 15, 8304. 10.1038/s41467-024-52617-7.

    (98) Peralta Ramos, J.M., Castellani, G., Kviatcovsky, D., Croese, T., Tsitsou-Kampeli, A., Burgaletto, C., Abellanas, M.A., Cahalon, L., Phoebeluc Colaiuta, S., Salame, T.M., et al. (2025). Targeting CD38 immunometabolic checkpoint improves metabolic fitness and cognition in a mouse model of Alzheimer's disease. Nat Commun 16, 3736. 10.1038/s41467-025-58494-y.

    (g) LPS-induced inflammation is acute and severe and may not accurately model MGD, which is typically characterized by chronic, low-grade inflammation. It is suggested to use a more relevant model, such as benzalkonium chloride exposure or a desiccating stress chamber.

    We thank the reviewer for this important suggestion. We agree that benzalkonium chloride exposure and desiccating stress are widely used and well-established models for ocular surface inflammation and dry eye [30-35], and we have also tested these approaches in our experimental system. However, while these treatments reproducibly induced inflammatory responses in the cornea, conjunctiva, and meibomian orifice, we did not observe detectable inflammatory changes within the tarsal plate (i.e., the internal eyelid tissue containing the meibomian glands). To our knowledge, induction of inflammation in the tarsal plate by these approaches has not been reported in the literature. As such, in our current study, we adopted subcutaneous LPS injection into the eyelid. However, as noted by the reviewer, this approach represents an acute and relatively strong inflammatory stimulus and does not fully recapitulate the chronic low-grade inflammation characteristic of MGD. Please note that, by describing both the rationale and limitations of this approach, we have improved our manuscript as follows:

    “Eye-drop instillation of inflammatory stimuli such as those by lipopolysaccharide (LPS) or benzalkonium chloride has been shown to be effective to cause inflammation in the cornea and conjunctiva [30-35]. However, we found this approach ineffective to induce inflammation in the tarsal plate (inside the eyelid tissue). Therefore, we performed subcutaneous injection of LPS to the eyelids (see Methods).”

    Methods [Page 30, lines 694-701] “LPS (Sigma) was reconstituted in saline and injected subcutaneously (5 µg/eyelid) 8 h before tissue collection for qRT-PCR analysis. LPS was administered every other day for a total of three or six doses before the measurement of meibomian NAD⁺ concentration or meibomian 3β-HSD enzymatic activity, respectively. Subcutaneous microinjection was performed using an insulin syringe (Becton Dickinson, 30G). This LPS-based approach represents an acute inflammatory stimulus and does not fully recapitulate the chronic low-grade inflammation characteristic of MGD — while it provides a practical means to induce inflammation in the tarsal plate.” Added references:

    (30) Lin, Z., Liu, X., Zhou, T., Wang, Y., Bai, L., He, H., and Liu, Z. (2011). A mouse dry eye model induced by topical administration of benzalkonium chloride. Mol Vis 17, 257-264.

    (31) Lin, Z., He, H., Zhou, T., Liu, X., Wang, Y., Wu, H., and Liu, Z. (2013). A mouse model of limbal stem cell deficiency induced by topical medication with the preservative benzalkonium chloride. Invest Ophthalmol Vis Sci 54, 6314-6325. 10.1167/iovs.12-10725.

    (32) Wu, M., Downie, L.E., Hill, L.J., and Chinnery, H.R. (2023). Topical Decorin Reduces Corneal Inflammation and Imparts Neuroprotection in a Mouse Model of Benzalkonium Chloride-induced Corneal Neuropathy. Invest Ophthalmol Vis Sci 64, 20. 10.1167/iovs.64.2.20.

    (33) Wu, C.Y., Wu, M., Huang, X., Gu, B.J., Maldonado-Codina, C., Morgan, P.B., Downie, L.E., and Chinnery, H.R. (2022). Topographical Distribution and Phenotype of Resident Meibomian Gland Orifice Immune Cells (MOICs) in Mice and the Effects of Topical Benzalkonium Chloride (BAK). Int J Mol Sci 23. 10.3390/ijms23179589.

    (34) Simmons, K.T., Xiao, Y., Pflugfelder, S.C., and de Paiva, C.S. (2016). Inflammatory Response to Lipopolysaccharide on the Ocular Surface in a Murine Dry Eye Model. Invest Ophthalmol Vis Sci 57, 2443-2451. 10.1167/iovs.15-18396.

    (35) Corrales, R.M., Villarreal, A., Farley, W., Stern, M.E., Li, D.Q., and Pflugfelder, S.C. (2007). Strain-related cytokine profiles on the murine ocular surface in response to desiccating stress. Cornea 26, 579-584. 10.1097/ICO.0b013e318033a729.

  5. Version published to 10.7554/elife.110696.2 on eLife
  6. Version published to 10.7554/elife.110696 on eLife
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    Authors used C57BL/6J from #InternationalMouseSt in their study. Including #RRIDs will make this less ambiguous. SciScore made a table with this resource, see “Automated Services” module (download as csv, xml or #jats) #methodsmatter #OpenScience

  8. Version published to 10.64898/2026.02.08.704637v3 on bioRxiv
  9. Version published to 10.64898/2026.02.08.704637v2 on bioRxiv
  10. eLife

    eLife Assessment

    The authors provide valuable data linking NAD+ dependent HSD3b6 gene expression in the eyelid to a vicious cycle involving decreased steroidogenesis and AR signaling, pro-inflammatory cytokine release, inflammation, CD38 activation, and further NAD+ decline, which induces meibomian gland atrophy leading to dry eye disease. Overall, the presented work provides evidence for the pathologic relationship between a pro-inflammatory environment, intracrine activity, and the NAD+ cofactor. However, the current study does not clearly establish the proposed intracrine mechanism and may largely reflect systemic hormonal effects resulting from the global Had3b6 knockout, leading to an incomplete narrative.

  11. eLife

    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.

  12. eLife

    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.

  13. eLife

    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.

  14. eLife

    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.

  15. Version published to 10.7554/elife.110696.1 on eLife
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    NAD boosting mediated by CD38 inhibition drives reversal of a pathological vicious cycle of intracrine activity and inflammation in eyelid meibomian gland dysfunction https://www.biorxiv.org/content/10.64898/2026.02.08.704637v1

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    NAD boosting mediated by CD38 inhibition drives reversal of a pathological vicious cycle of intracrine activity and inflammation in eyelid meibomian gland dysfunction https://www.biorxiv.org/content/10.64898/2026.02.08.704637v1

  18. Version published to 10.64898/2026.02.08.704637v1 on bioRxiv