Dear Editor
We thank Kim et al. for their keen interest in recognizing the strengths of our integrative multi-omics approach and its potential to uncover novel biomarker and therapeutic axes in acute liver failure (ALF) [
1,
2].
We also acknowledge the important limitations raised. As noted, our study was conducted at a single tertiary referral center with a relatively small discovery cohort (n=40) and moderate validation cohort (n=230). In this total of 300 participants were recruited between December 2018 and December 2021 at the Institute of Liver and Biliary Sciences, New Delhi, including 270 ALF patients and 30 healthy controls. The first 40 ALF patients and 5 healthy controls were selected for the training cohort, while the remaining 230 ALF patients and 25 healthy controls were assigned to the test cohort. ALF patient samples were prospectively collected, and efforts were made to include all consecutive patients to minimize selection bias. This design allowed us to establish a robust test cohort, ensuring that the predictive models were evaluated on a larger and more representative sample, thereby reducing the risk of over-fitting and providing a better approximation of real-world performance. Furthermore, for the machine learning validation, all the collected patients’ samples were randomly distributed in to a training cohort of 70% and test cohorts of 30% respectively. This approach increases the robustness and the reliability of our analyses, though we agree that validating our results in larger, multicentre cohorts will increase the applicability of our findings. While longitudinal sampling would undoubtedly offer additional insights into temporal dynamics, the present design provides a valid and methodologically sound foundation for the discovery phase, consistent with prior multi-omics studies in ALF and other acute critical illnesses.
It is well reported that altered microbial composition, particularly invasive fungal infections (IFIs) like Candida, Cryptococcus, and Aspergillus, exacerbate inflammation, oxidative stress, and multi-organ dysfunction in Advanced liver disease, significantly raising mortality risk [
3,
4]. Targeting IFIs is therefore critical to improving outcomes and reducing early mortality in liver disease patients. With respect to mechanistic insights, we agree that our network analyses and receptor expression findings are hypothesis-generating and needs validation in a preclinical model of ALF. In the same line we could identify literature that provides evidence in the mouse model, for the role of Clec7a mediated increase in fungal infection and host lipid deposition [
5] and regulate intestinal immunity by controlling Treg cell differentiation through modification of microbiota [
6], We also found that Clec7a / Dectin-1 is essential for CD4⁺ T-cell responses to fungi in the murine gut, highlighting its key role in mucosal adaptive immunity and fungal control [
7]. Validating these studies and the causal contributions of impaired antifungal immunity and Kennedy pathway hyperactivation to ALF pathogenesis in a preclinical mouse model of ALF (APAP induced). Liver proteomics and lipidomics analysis highlight significant dose dependent (APAP; 250 mg/kg and 500 mg/kg) reduction of calcitonin gene-related peptide (Clec7a), a pattern recognition receptor that recognizes β-glucan on fungal surfaces and plays a key role in initiating antifungal immune responses (
Fig. 1) with concordant increase in the lipids particularly liver phosphatidylcholine, choline, and phosphatidic acid associated to the Kennedy pathway. This observation clearly validates our findings and outline important role of fungal surveillance proteins and concordant lipidomics change in ALF pathogenesis (
Fig. 1) which provides attractive therapeutics axis in ALF modulation of these axes can translate into meaningful clinical benefit and provide the mechanistic insights.
We also appreciate the authors’ reference to the complementary work of Zeng et al. [
8] in alcohol-associated liver disease, which reinforces the broader pathogenic role of fungal communities across diverse liver disease etiologies. Such cross-etiology evidence underscores the need for systematic investigation of fungal components in liver disorders beyond ALF. In our study, we further included 200 severe alcoholic hepatitis (SAH) as a diseased control group to explore whether the identified lipidomic alterations were unique to ALF or shared across other acute injuries. Interestingly, ALF patients demonstrated distinct expression patterns of the top five lipid species PC(32:0), PC(26:4/10:0), PC(20:1/14:1), PC(15:0/17:0), TG(4:0/10:0/23:6) compared to SAH patients, suggesting that while both conditions share features of metabolic and inflammatory dysregulation, the lipidomic signatures identified in ALF are unique and disease-specific. This comparative analysis strengthens the biological plausibility of our findings and highlights the potential for lipidomic–mycobiome integration to delineate key contributors of hyper inflammation seen in acute liver failure.
We thank Kim et al. [
2] for recognizing the strengths of our integrative multi-omics approach in identifying potential biomarker panels and guiding targeted interventions to improve ALF outcomes. We emphasize that such integrative analyses spanning proteomics, lipidomics, metabolomics, and metaproteomics should be further encouraged to advance patient stratification and precision therapies in ALF and related diseases.
FOOTNOTES
-
Authors’ contribution
JSM and SKS contributed to the conceptualization of the work. NS and SP participated in data analysis and manuscript preparation. SS, SKS, and JSM critically revised and proofread the manuscript. All authors approved the final version of the letter to the editor.
-
Acknowledgements
This research was supported by ICMR (5/4/8-3/CD/JS/2021-NCD-II and project ID: 2020-4958).
-
Conflicts of Interest
The authors declare no conflicts of interest.
Figure 1.(A) Liver injury after N-acetyl-p-aminophenol (APAP) overdose. Male C57BL/6 mice (10 to 11 weeks old) received two intraperitoneal injections of 250 mg APAP/kg body and 500 mg/kg weight after a fasting period of 12 hours and sacrificed after 24 hours. (B) Liver Proteomics Shows downregulation of CLR Protein “Clec7a” on increasing the dose of APAP from 250 mg/kg to 500 mg/kg (P<0.05). (C) Kennedy cycle (hepatocytes).
Abbreviations
invasive fungal infection
severe alcoholic hepatitis
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