Editorial

A disease-promoting role of the intestinal mycobiome in non-

alcoholic fatty liver disease
Wajahat Z. Mehal1,2, Robert F. Schwabe3,4,*
1
Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA; 2VA Connecticut
Healthcare System, West Haven, CT, USA; 3Department of Medicine, Columbia University, New York, NY 10032, USA; 4Institute of Human
Nutrition, New York, NY 10032, USA

See Article, pages 788–799

In less than 4 decades since its entry into the hepatologist’s
lexicon1 and clinical horizon as a potentially progressive and
severe disease,2 non-alcoholic fatty liver disease (NAFLD) has
become the leading cause of chronic liver disease, affecting
about 25% of the adult population worldwide.3 Besides strong
links to diet, obesity and diabetes, a number of liver-intrinsic
pathomechanisms as well as genetic risk factors, such as single
nucleotide polymorphisms in PNPLA3, TM6SF2 and MBOAT7,
contribute to the development and progression of NAFLD and its
more severe form, non-alcoholic steatohepatitis (NASH).4 In
addition to the modulation of hepatic steatosis and subsequent
hepatocyte death, inflammation and fibrogenesis in the liver by
these factors, it has also become apparent that interactions of
the liver with other organs, including the adipose tissue5 and the
gut,6 affect the development and progression of NAFLD. The gut
microbiome has received increasing attention, as it regulates a
wide range of factors that could impact on NAFLD, including
energy extraction, the integrity of the gut barrier, the tone of the
immune system as well as the fine-tuning of metabolic path-
ways with a role in liver physiology.6,7 Strong associations in
patients in conjunction with functional studies in mice have
firmly established the microbiome as a contributor to NAFLD and
a tool/target for the development of non-invasive biomarkers,
novel therapies and personalized medicine approaches.8 How-
ever, it is well-established that our gut is inhabited by a wide
range of bacteria, archaea, fungi and viruses that constantly
interact, compete or collaborate, just as all other species on our
planet do. Moreover, bacteria, fungi and a subtype of viruses
termed bacteriophages have the ability to kill bacteria, thus
ensuring their survival and/or reproduction in the human gut,
and shaping its microbial diversity. Therefore, it is surprising
that current research on the gut microbiota in NAFLD has almost
exclusively focused on bacteria, with only a single study on the
intestinal virome,9 and the role of the mycobiome only studied
Keywords: NASH; fungi; microbiome; microbiota.
Received 13 December 2021; accepted 24 December 2021; available online 21
January 2022
q
DOI of original article: https://doi.org/10.1016/j.jhep.2021.11.029.
* Corresponding author. Address: Department of Medicine, Columbia University, ICRC
Room 926, 1130 St. Nicholas Ave, New York, NY, USA. Tel.: 212-851-5462; fax: 212-
851-4590.
E-mail address: rfs2102@cumc.columbia.edu (R.F. Schwabe).
https://doi.org/10.1016/j.jhep.2021.12.035
in alcohol-related fatty liver disease.10 The multifactorial path-
ogenesis of NAFLD may contribute to the difficulty of finding
effective therapies, as targeting a single mediator or patho-
mechanistic aspect may not be sufficient or may only be appli-
cable to select patients.
The study by Demir, Lang, Hartmann et al.11 now introduces
the mycobiome as a novel and relevant modulator for the
development of NASH. To assess potential correlations between
the mycobiome and NAFLD, the authors analyzed fecal fungi in
a cohort of 78 patients with NAFLD at various stages, including
patients with and without NASH and different degrees of
fibrosis, as well as a control cohort of 16 non-obese patients
without known disease. The authors did not find differences in
the average reads between groups in targeted amplicon
sequencing or the fungal microbiota composition, with similar
beta diversity between controls and patients with NAFLD.
However, the authors observed differences in diversity be-
tween patients with NASH or F2-F4 fibrosis and patients with
NAFL or F0-F1 fibrosis within the subgroup of non-obese pa-
tients, which is consistent with previous studies finding sig-
nificant differences in the bacterial microbiota composition
only in non-obese patients.12 Specific taxa were associated with
the presence of NASH and F2-F4 fibrosis in non-obese patients,
high alanine and aspartate aminotransferase levels, as well as
with histological features such as stage of fibrosis, the grade of
liver inflammation and the NAFLD activity score, which
remained significant after adjustment for potential con-
founders such as age, gender, body mass index, type 2 diabetes,
proton pump inhibitor use and alcohol consumption. In
contrast, there were no significant associations with the grade
of steatosis. After having established strong correlations be-
tween specific taxa of the fecal mycobiome and relevant clin-
ical parameters, the authors tested causation in microbiota-
humanized mice that were kept on a Western diet and
treated with the poorly absorbable antifungal amphotericin B.
Mice receiving amphotericin B displayed reduced liver injury,
as determined by serum alanine aminotransferase, decreased
hepatic cholesterol and triglyceride concentrations, decreased
inflammatory gene expression as well as decreased liver
fibrosis. Importantly, the authors demonstrated successful
mycobiome humanization (by determining the presence of 2
fungi) and the efficacy of amphotericin B (by reduction of

Journal of Hepatology 2022 vol. 76 j 765–767

Editorial
specific fungi). Furthermore, the authors also showed that
amphotericin B did not affect the composition of the bacterial
microbiota. Together, these studies in patients and mice
convincingly show that the intestinal mycobiome has a key role
in NAFLD.
While Demir, Lang, Hartmann et al. convincingly demon-
strated important clinical correlations in patients and causative
studies in mice, their study did not provide clear mechanistic
explanations. The role of the bacterial microbiota in NASH has
been attributed to specific mediators and mechanisms, including
microbe-associated molecular patterns (MAMPs) that are
recognized by specific host receptors and mediate a wide range
of inflammatory responses;7,13 the ability of the host to maintain
an intact gut barrier and thereby minimize the translocation of
bacteria and bacterial MAMPs to the liver, where they could
trigger inflammation and other responses that predispose to
NAFLD;7 metabolites that affect energy extraction;14 metabolic
pathways with high relevance to liver disease, such as the
metabolism of choline, whose bioavailability is reduced through
its conversion into methylamines;15 or bile acids, whose
composition and ability to activate receptors such as farnesoid X
receptor is altered by intestinal bacteria.16,17 It will be important
to understand in similar detail the mechanisms by which in-
testinal fungi affect NAFLD and NASH development. While direct
effects of fungi on NASH, e.g. via alterations of the gut barrier,
inflammation or metabolism appear possible, there could also be
a number of indirect mechanisms through which they affect
NAFLD and NASH. Although the authors excluded major alter-
ations in the bacterial composition by amphotericin B in the
current study using qPCR and in a previous study on alcohol-
related liver disease by principal component analysis of 16s
sequencing data,10 further in-depth analysis is required to
exclude a role of specific bacterial taxa. Furthermore, the authors
pointed out, as a limitation of their study, that amphotericin B
also interacts with cholesterol18 and may affect Toll-like receptor
(TLR) signaling,19 and that this may have contributed to its
beneficial effects on NAFLD and NASH, given that cholesterol and
TLR signaling contribute to NASH and NASH fibrosis.20 Further
investigations of these pathways are needed, particularly
because the authors reported reduced hepatic cholesterol, tri-
glycerides and liver injury in amphotericin B-treated mice,
whereas they did not observe associations of fungi with hepatic
steatosis and ballooning in patients.11 Potential approaches to
address these limitations would be testing if amphotericin B
affects NASH development and liver fibrosis in germ-free mice or
in microbiome-humanized mice colonized with microbiota
without detectable fungi, or studying the effects of other anti-
fungals on NASH.
While it is difficult to translate therapeutic effect from mouse
models of NASH to patients, the observed reduction of fibrosis by
amphotericin B was quite profound.11 Hence, it would be
important to learn more about the role of fungi in NASH, in
particular the identification of the responsible taxa, which may
enable the selection of patients who would benefit from anti-
fungal treatments or achieve a therapeutic modulation of the
intestinal mycobiome via fecal microbiota transplantation. Given
that the human microbiome varies largely by age, sex,
geographic region and nutrition, it would be important to
confirm the findings in patients in a second cohort. Furthermore,
766 Journal of Hepatology 2022 vol. 76 j 765–767
identifying specific pathways and/or receptors through which
the intestinal mycobiome acts in NASH may open up further
options for pharmacologic modulation. So, while Demir, Lang,
Hartmann et al. have put the mycobiome on the NASH horizon,
their work also opens up many relevant questions that need to
be followed up in future studies.
Financial support
The authors received no financial support to produce
this manuscript.
Conflict of interest
The authors declare no conflicts of interest that pertain to
this work.
Please refer to the accompanying ICMJE disclosure forms for
further details.
Authors’ contributions
WZM and RFS contributed to drafting the manuscript.
Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jhep.2021.12.035.
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Author names in bold designate shared co-first authorship
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