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Dietary palmitoleic acid reprograms gut microbiota and improves biological therapy against colitis

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Magnitude and diversity of gut microbiota and metabolic systems are critical in shaping human health and diseases, but it remains largely unclear how complex metabolites may selectively regulate gut microbiota and determine health and diseases. Here, we show that failures or compromised effects of anti-TNF-α therapy in inflammatory bowel diseases (IBD) patients were correlated with intestinal dysbacteriosis with more pro-inflammatory bacteria, extensive unresolved inflammation, failed mucosal repairment, and aberrant lipid metabolism, particularly lower levels of palmitoleic acid (POA). Dietary POA repaired gut mucosal barriers, reduced inflammatory cell infiltrations and expressions of TNF-α and IL-6, and improved efficacy of anti-TNF-α therapy in both acute and chronic IBD mouse models. Ex vivo treatment with POA in cultured inflamed colon tissues derived from Crohn’s disease (CD) patients reduced pro-inflammatory signaling/cytokines and conferred appreciable tissue repairment. Mechanistically, POA significantly upregulated the transcriptional signatures of cell division and biosynthetic process of Akkermansia muciniphila, selectively increased the growth and abundance of Akkermansia muciniphila in gut microbiota, and further reprogrammed the composition and structures of gut microbiota. Oral transfer of such POA-reprogrammed, but not control, gut microbiota induced better protection against colitis in anti-TNF-α mAb-treated recipient mice, and co-administration of POA with Akkermansia muciniphila showed significant synergistic protections against colitis in mice. Collectively, this work not only reveals the critical importance of POA as a polyfunctional molecular force to shape the magnitude and diversity of gut microbiota and therefore promote the intestinal homeostasis, but also implicates a new potential therapeutic strategy against intestinal or abenteric inflammatory diseases.
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Dietary palmitoleic acid reprograms gut microbiota and improves biological
therapy against colitis
Yiwei Chen
a,b
, Qiongdan Mai
a
, Zixu Chen
a
, Tao Lin
a
, Yongjie Cai
a
, Jing Han
c
, Ying Wang
c
, Mudan Zhang
c
,
Shimin Tan
a
, Zhiying Wu
a
, Lingming Chen
a
, Zhiyi Zhang
a
, Yi Yang
a
, Taimei Cui
a
, Beiyin Ouyang
a
, Yue Sun
d
,
Lijia Yang
e
, Lin Xu
a
, Sien Zhang
d
, Jian Li
f
, Hongbo Shen
g
, Linna Liu
h
, Lingchan Zeng
i
, Shenghong Zhang
c
,
and Gucheng Zeng
a
a
Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China;
b
State Key Laboratory of Oncology
in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China;
c
Division of
Gastroenterology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China;
d
Hospital of Stomatology, Guanghua School of
Stomatology, Sun Yat-sen University, Guangzhou, China;
e
College of Stomatology, Jinan University, Guangzhou, China;
f
The Fifth Affiliated
Hospital, Sun Yat-sen University, Zhuhai, China;
g
Clinic and Research Center of Tuberculosis, Shanghai Key Laboratory of Tuberculosis, Tongji
University Shanghai Pulmonary Hospital, Shanghai, China;
h
Guangzhou Eighth People’s Hospital, Guangzhou Medical University, Guangzhou,
China;
i
Clinical Research Center, Department of Medical Records Management, Guanghua School of Stomatology, Hospital of Stomatology,
Sun Yat-sen University, Guangzhou, China
ABSTRACT
Magnitude and diversity of gut microbiota and metabolic systems are critical in shaping human
health and diseases, but it remains largely unclear how complex metabolites may selectively
regulate gut microbiota and determine health and diseases. Here, we show that failures or
compromised eects of anti-TNF-α therapy in inammatory bowel diseases (IBD) patients were
correlated with intestinal dysbacteriosis with more pro-inammatory bacteria, extensive unre-
solved inammation, failed mucosal repairment, and aberrant lipid metabolism, particularly
lower levels of palmitoleic acid (POA). Dietary POA repaired gut mucosal barriers, reduced inam-
matory cell inltrations and expressions of TNF-α and IL-6, and improved ecacy of anti-TNF-α
therapy in both acute and chronic IBD mouse models. Ex vivo treatment with POA in cultured
inamed colon tissues derived from Crohn’s disease (CD) patients reduced pro-inammatory
signaling/cytokines and conferred appreciable tissue repairment. Mechanistically, POA signicantly
upregulated the transcriptional signatures of cell division and biosynthetic process of Akkermansia
muciniphila, selectively increased the growth and abundance of Akkermansia muciniphila in gut
microbiota, and further reprogrammed the composition and structures of gut microbiota. Oral
transfer of such POA-reprogrammed, but not control, gut microbiota induced better protection
against colitis in anti-TNF-α mAb-treated recipient mice, and co-administration of POA with
Akkermansia muciniphila showed signicant synergistic protections against colitis in mice.
Collectively, this work not only reveals the critical importance of POA as a polyfunctional molecular
force to shape the magnitude and diversity of gut microbiota and therefore promote the intestinal
homeostasis, but also implicates a new potential therapeutic strategy against intestinal or aben-
teric inammatory diseases.
ARTICLE HISTORY
Received 28 December 2022
Revised 18 April 2023
Accepted 24 April 2023
KEYWORDS
Gut microbiota; Biological
therapy; Inflammatory bowel
diseases; Akkermansia
muciniphila; TNF-α
Introduction
Physiological functions of gut microbiota and
metabolic systems (including host and microbial
metabolism) play a critical role in modulation of
human health and diseases
1,2
. Unraveling the com-
plexity and diversity of multiplex molecular and
chemical interaction networks in gut microbiota
and metabolic systems have extremely important
implications for understanding pathogenesis
mechanisms and development of better therapies
for diseases
2–4
. However, it is full of great chal-
lenges to achieve in-depth and precise regulation
of microbial and metabolic networks. While pre-
vious studies have shown that metabolite profiles
CONTACT Lingchan Zeng zenglch5@mail.sysu.edu.cn Clinical Research Center, Department of Medical Records Management, Guanghua School of
Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong 510055, China; Shenghong Zhang zhshh3@mail.sysu.edu.cn
Division of Gastroenterology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China; Gucheng Zeng zenggch@mail.sysu.edu.cn
Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China
Y.W.C. and Q.D.M. contributed equally to this work.
Supplemental data for this article can be accessed online at https://doi.org/10.1080/19490976.2023.2211501.
GUT MICROBES
2023, VOL. 15, NO. 1, 2211501
https://doi.org/10.1080/19490976.2023.2211501
© 2023 The Author(s). Published with license by Taylor & Francis Group, LLC.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which
permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been
published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.
may selectively shape the structure, function, or
strain-fitness of gut bacteria
5,6
, it remains largely
unknown how more diverse metabolism pathways
producing highly diverse molecular structures may
selectively regulate growth and physiology of cer-
tain bacterial species or strains and ultimately affect
the entire gut microbiota structure and metabolic
activity.
Inflammatory bowel diseases (IBD), including
ulcerative colitis (UC) and Crohn’s disease (CD),
are chronic gastrointestinal inflammation with dis-
orders of highly-dynamic gut microbiota and
metabolites
4,7
. It is thought that the development,
progression, and poor prognostic outcomes of IBD
result from multiple pathogenesis factors involving
genetic predisposition
8
, intestinal microbiome
disturbance
9
, abnormal immune balance
10
, and
mucosal barrier destruction leading to aberrant
host-microbial interactions
11
. Particularly, IBD
are associated with reductions of gut microbial
diversity, blooms of pro-inflammatory bacteria
and fungi, and perturbations of intestinal
metabolites
4,7,12–14
. However, the exact effects and
mechanisms of molecular and chemical networks
between gut microbiota and metabolic systems on
the development, progression, and prognosis of
IBD remain largely unknown.
Mucosal integrity provides an intact barrier for
colonization and growth of gut microbiota, limit-
ing translocations of pathogenic bacteria or bacter-
ial components and products, and sustaining
immune tolerance
15
, which may serve as
a fundamental anatomic and physiological basis
for maintaining the delicate balance or homeostasis
of chemical networks between gut microbiota and
metabolic systems. Thus, promoting intestinal
mucosal healing is thought to be one of the most
promising therapeutic strategies in IBD
16
.
However, it is unclear how to restore the home-
ostasis of such networks between gut microbiota
and metabolic systems, which may promote the
healing of intestinal mucosal integrity and there-
fore further develop a new therapeutic strategy to
control IBD or other intestinal diseases.
In this study, we found that failure of anti-TNF
treatment in IBD patients attributed to unre-
solved intestinal inflammation, mucosal non-
healing, intestinal dysbacteriosis and lipid abnorm-
alities. Metabolomics analyses revealed that lipid
metabolism abnormalities, particularly decreased
levels of an anti-inflammatory metabolite, palmito-
leic acid (POA), were related to nonresponse to
anti-TNF-α therapy, and oral supplement with pal-
mitoleic acid (POA) could significantly repair
mucosal barriers and reduce inflammation in
both murine models of colitis and highly inflamed
colon tissues derived from Crohn’s disease (CD)
patients. Mechanistically, palmitoleic acid (POA)
rewired disrupted intestinal barrier and repro-
grammed gut microbiota with selectively increas-
ing the abundance of anti-inflammatory gut
bacteria such as Akkermansia muciniphila.
Transcriptomic analysis demonstrated increased
expression of cell division, ATP synthesis coupled
electron transport and biosynthetic process in
Akkermansia muciniphila in response to palmito-
leic acid (POA). Moreover, palmitoleic acid (POA)
dramatically enhanced the efficacy of anti-TNF-α
therapy against colitis in mice.
Therefore, this study not only uncovers pre-
viously unrecognized pathogenesis mechanisms of
IBD but also provides a potential new therapeutic
strategy of IBD and other intestinal or abenteric
inflammatory diseases via promoting the homeos-
tasis of molecular and chemical networks between
gut microbiota and metabolic systems.
Results
Non-responsiveness to anti-TNF-α therapy,
extensive inammation and mucosal injury are
associated with enrichment for pro-inammatory
gut bacteria in IBD patients
Anti-TNF-α therapy is still the most conventionally
and widely used biological therapies of IBD, but
there are still huge unmet clinical needs for
improvement of the efficacy and safety of anti-
TNF-α therapy
14,17,18
. Disruptions of molecular
communications and homeostasis in gut micro-
biota structure and metabolic activity is implicated
in the onset and progression of IBD
7,19
, but it is still
unclear whether and how such disruptions are cor-
related with clinically unmet efficacy of anti-TNF-α
therapy. Thus, we hypothesized that disrupted
homeostasis in gut microbiota and metabolic sys-
tems would have a profound impact on the efficacy
of anti-TNF-α therapy.
2Y. CHEN ET AL.
To address this, we initially assessed the progres-
sion and clinical characteristics of IBD patients
receiving British Society of Gastroenterology con-
sensus guidelines-based anti-TNF-α (Infliximab,
IFX) therapy
20
. Efficacy or responses to therapy of
anti-TNF-α were assessed by the severity of
symptoms
21,22
, classifying patients as either
responders [(R) complete or partial response or
stable disease≥12 weeks; n = 19] or non-
responders [(NR) clinical remission<12 weeks or
progressive disease; n = 12] (see Methods for
details). Endoscopic analysis serves as a golden
standard in examining and assessing the disease
severity in the intestinal mucosa as well as evaluat-
ing the efficacy of treatments
23
. Endoscopic ana-
lyses suggested that, compared with the healthy
controls, anti-TNF-α therapy responders exhibited
clinical remission and mucosal healing while non-
responders still presented moderate or severe dis-
ease with ulcers, stenosis, and fistulas (Figure 1a,
b). Compared with responders, non-responders
showed higher levels of C-reactive protein (CRP)
and erythrocyte sedimentation rate (ESR) in the
serum (Supplementary Figure S1, A to C), more
severe pathology with more extensive mucosal
damages and ulcerations in mucosal biopsies
(Figure 1c, d). Moreover, mucosal biopsies of non-
responders exhibited more infiltrations of inflam-
matory cells (e.g., CD8
+
and CD11b
+
cells) and
glandular destructions characterized by lower
expressions of intestinal mucosal integrity markers,
ZO-1 and Occludin (Figure 1e, f and
Supplementary Figure S1, D and E). Thus, these
data suggest that the compromised effects of anti-
TNF-α therapy were associated with unresolved
intestinal inflammation and failed functional
repairment of colon.
Although the alterations of gut microbiota are
associated with the effects of anti-TNF-α therapy
against IBD
14,18,24,25
, the precise molecular forces
for modulating gut microbiota to improve anti-
TNF-α therapy for IBD remain largely unknown.
To elucidate such molecular forces, we first
addressed the roles of compositional differences
and functional diversity of gut microbiome in the
efficacy of anti-TNF-α therapy. Fecal samples of
anti-TNF-α therapy responders and non-
responders with IBD were collected and sequenced
by 16S rRNA. α-diversity analyses such as Shannon
and Simpson index showed unobservable signifi-
cant difference in microbial community richness
between responders and non-responders
(Supplementary Figure S2A). However, LEfSe ana-
lysis suggested that non-responders showed higher
abundance of Fusobacterium, Paracoccus, Dielma,
Lachnoanaerobaculum, Gemella and Weissella,
while responders exhibited higher abundance of
Candidatus Saccharimonas, Solobacterium at
genus level (Figure 1g, h). Remarkably, compared
with those in responders, there was a higher abun-
dance of pro-inflammatory bacteria that have been
implicated with exacerbation of IBD, such as
Escherichia-Shigella, Fusobacterium and
Gemella
26–28
, in non-responders (Supplementary
Figure S2B). Thus, there is a significant correlation
between shift of gut microbial compositions
toward higher abundance of pro-inflammatory
bacteria and the compromised efficacy of anti-
TNF-α therapy.
We further investigated whether the disease bur-
den and clinical characteristics of IBD patients
were associated with alterations of the gut micro-
biota compositions. We found that most bacterium
enriched in non-responders were positively corre-
lated with characteristic of the inflammatory cell
infiltration and mucosal non-healing in anti-TNF
therapy non-responders with IBD (Figure 1i).
Enriched pathogenic Fusobacterium was positively
associated with inflammatory markers CRP and
ESR, while high abundance of Paracoccus and
Weissella were positively correlated with inflam-
matory infiltrations and intestinal injuries
(Figure 1i). Collectively, these data suggested that
compromised responsiveness to anti-TNF-α ther-
apy was highly associated with increased abun-
dance of pro-inflammatory bacteria and that such
gut dysbacteriosis was positively correlated with
less favorable clinical outcomes of IBD patients.
Metabolomics analyses reveal that lipid metabolism
abnormalities characterized with declined
abundance of palmitoleic acid (POA) are
signicantly related to failure of anti-TNF-α therapy
Since compromised responsiveness to anti-TNF-α
therapy was associated with gut dysbacteriosis, we
then further analyzed whether such alterations of
gut microbial ecosystem may also correlate with
GUT MICROBES 3
Figure 1. Gut dysbacteriosis is correlated with the compromised effects of anti-TNF-α therapy in IBD patients. Note: (a) Representative
endoscopic images of healthy controls (HC), anti-TNF-α therapy (Infliximab, IFX) responders (R) and non-responders (NR) with IBD.
White arrow shows intact intestinal mucosa with clear blood vessels. Green arrow shows the repairment of damaged intestinal mucosa
while yellow arrows show extensive mucosal erosion with bleeding. Blue arrow indicates endoscopic ulcers. (b) Endoscopic subscore
for evaluating mucosal appearance of healthy controls (HC), anti-TNF-α therapy responders (R) and non-responders (NR) with IBD. (c)
Representative images of the Hematoxylin and eosin (H&E)-stained colon biopsy sections of healthy controls (HC), anti-TNF-α therapy
responders (R) and non-responders (NR) with IBD. Top: 10×, scale bar = 100 μm; Bottom: 40×, scale bar = 100 μm. The black-boxed
areas at the top are enlarged in bottom right corner. Green arrow indicates dense and tightly connected intestinal epithelial cells, and
4Y. CHEN ET AL.
alternations of profiles of gut metabolites, which
may further molecularly contribute to disrupted
homeostasis in microbial ecosystems. To address
this, LC-MS was carried out to analyze the meta-
bolism profiles of fecal and serum samples of anti-
TNF-α responders and non-responders with IBD.
We noticed that approximately 34% of fecal meta-
bolites with differential abundance were lipid and
lipid-like molecules, implicating that anti-TNF-α
non-responders showed aberrant compositions
and abundance of lipid metabolites in intestine
(Figure 2, a and b). KEGG pathway classification
also demonstrated that metabolism of lipid such as
cis-12-octadecenoic acid methyl ester and 7-dehy-
drodesmosterol correlated to the responsiveness of
anti-TNF-α therapy (Figure 2, b and c).
To systematically evaluate the lipid metabolic
profiles, we comparatively investigated serum
lipid profiles between anti-TNF-α responders and
non-responders with IBD. We discovered abnor-
mal lipid metabolism in non-responders with
down-regulation of triglycerides (TG), diacylgly-
cerol (DG), lysophospholipid (LPC) and other
compounds containing long-chain fatty acyl-CoA
chain (Figure 2, d and e), which could further be
hydrolyzed into long-chain fatty acids (LCFAs). In
line with these findings, enhanced lipid metabolism
was noted in serum of responders (Figure 2f).
Triglycerides are the most common bioactive
lipid, and triglycerides (16:0/R
2
/R
3
) enriched in
responders could be hydrolyzed into C16:0 (palmi-
tic acid, PA), which could be further desaturated to
C16:1 (palmitoleic acid, POA) (Figure 2, e and g).
Indeed, the relative concentrations of palmitoleic
acid (POA) were significantly lower in plasma from
non-responders of anti-TNF-α therapy than those
from responders (Figure 2h). In agreement with
our findings, long-chain fatty acids (LCFAs) were
found to be the most numerous depleted classes of
metabolites in stools of IBD patients
29
and inflam-
matory intestinal tissues contained much lower
concentrations of palmitoleic acid (POA) than
adjacent non-inflammatory tissues did in colitis
patients
30
.
To validate that these reduced concentrations of
palmitoleic acid (POA) in patients with compro-
mised responsiveness of anti-TNF-α therapy is at
least partially ascribed to gut dysbacteriosis, mice
were treated with a cocktail of broad-spectrum
antibiotics (Abx) before and during dextran sulfate
sodium salt (DSS) treatment. Mice receiving anti-
TNF-α mAb injection underwent rapid loss of
diversity of gut microbiota upon the oral treatment
of broad-spectrum antibiotics (ampicillin, neomy-
cin, vancomycin, and metronidazole), exacerbated
colitis with rapid body weight loss, increased dis-
ease activity index (DAI) score and bloody diar-
rhea, compared with mice treated with IgG
antibody control (Supplementary Figure S4, A to
C). Although anti-TNF-α mAb treatment con-
ferred some benefits, characterized by reduced
colonic inflammation, preserved colon length, and
muted histopathological changes (Supplementary
Figure S4, C to F), the antibiotics treatment
remarkably impaired the benefits of anti-TNF-α
mAb, characterized by more disruptions of epithe-
lial cells, loss of goblet cells, more infiltrations of
inflammatory cells and higher histological scores,
as well as decreased palmitoleic acid (POA) con-
centrations in serum of mice with colitis
yellow arrow shows extensive damage and loss of mucosal epithelial layer. Red arrow marks lesions and infiltration of inflammatory
cells. (d) Histological score for loss of epithelium, crypt damage, depletion of goblet cells and infiltration of inflammatory cells in
healthy controls (HC), anti-TNF-α therapy responders (R) and non-responders (NR) with IBD. (e) Immunohistochemical images of colon
sections stained for epithelial marker including ZO-1 and Occludin in anti-TNF-α therapy responders (R) and non-responders (NR) with
IBD. A high-magnification of the region is shown (bottom right). Scale bar = 50 μm. (f) Immunohistochemical images of colon sections
stained for CD8
+
and CD11b
+
cells in colonic lamina propria in anti-TNF-α therapy responders (R) and non-responders (NR) with IBD.
A high-magnification of the region is shown (bottom right). Scale bar = 50 μm (g) Differences in microbial taxa at genus level between
anti-TNF-α therapy responders (R) and non-responders (NR) with IBD were calculated by LDA effect size (LEfSe). (P < 0.01 and LDA
score>2.0). Higher abundant genera in NR are shaded with red and higher abundant genera in R are shaded with blue. (h) Proportions
of different microbial genus in feces between anti-TNF-α therapy responders (R) and non-responders (NR) with IBD. Significance was
tested using Wilcoxon rank-sum test with FDR multiple test correction. (i) Pearson correlation analysis shows the association between
differential microbial abundance and clinical features. Data represent means ± SEM (HC = 10, R = 19, NR = 12); *P < 0.05; ** P < 0.01;
*** P < 0.001; **** P < 0.0001; NS, no statistical significance. P values were calculated by Kruskal-Wallis test [(b) and (d)], Wilcoxon
rank-sum test (h) and Pearson correlation analysis (i).
GUT MICROBES 5
Figure 2. Differential metabolic profiles between anti-TNF-α therapy responders (R) and non-responders (NR) highlight that aberrant
lipid metabolism with decreased palmitoleic acid (POA) are associated with compromised efficacy of anti-TNF-α therapy. Note: (a)
Volcano plot showing the differential fecal metabolites between anti-TNF-α therapy responders (R) and non-responders (NR) with IBD.
The red points are 24 metabolites enriched in anti-TNF-α non-responders (NR) while the blue points are 27 metabolites enriched in
anti-TNF-α responders (R) (R = 19, NR = 12). (b) Heatmap of differential fecal metabolite profile in anti-TNF-α therapy responders (R)
and non-responders (NR) with IBD. The metabolites with red box were enriched in responders and classified into lipid metabolism
according to KEGG pathway. (c) KEGG pathway classification of differential fecal metabolites between anti-TNF-α therapy responders
(R) and non-responders (NR) with IBD. (d) Volcano plot showing the differential serum metabolites between anti-TNF-α therapy
responders (R) and non-responders (NR) with IBD. The red points are 12 metabolites enriched in anti-TNF-α non-responders (NR) while
the blue points are 19 metabolites enriched in anti-TNF-α responders (R) (R = 6, NR = 6). (e) Heatmap of differential serum metabolite
profiles in anti-TNF-α therapy responders (R) and non-responders (NR) with IBD. The metabolites with red box were enriched in
responders and classified into lipid metabolism according to KEGG pathway. (f) KEGG pathway classification of differential serum
metabolites between anti-TNF-α therapy responders (R) and non-responders (NR) with IBD. (g) Black arrow indicates the metabolic
pathway of triglycerides (16:0/R2/R3) to palmitoleic acid (C16:1). (h) Palmitoleic acid is much higher in plasma samples in anti-TNF-α
6Y. CHEN ET AL.
(Supplementary Figure S4, E to G). Thus, these
data suggested that gut dysbacteriosis led to
reduced abundance levels of palmitoleic acid
(POA) and inefficacy of anti-TNF-α therapy.
Oral administration of palmitoleic acid (POA)
but not palmitic acid (PA) promotes mucosal
healing and attenuates inflammation in DSS-
induced acute and chronic colitis in mice with no
observable toxicity
Next, we explored whether palmitoleic acid
(POA) really played a causal but not correlative
role in regulating the disease outcomes of IBD. To
test the potential anti-inflammatory effects of pal-
mitoleic acid (POA), PBMCs derived from IBD
patients were stimulated with palmitoleic acid
(POA), and palmitic acid (PA) was served as
a control. We observed significant ex vivo effects
reducing the productions of TNF-α and IL-6, two
important cytokines had been implicated to be
involved in promoting IBD pathogenesis
10,11
, by
palmitoleic acid (POA) but not palmitic acid (PA)
in PBMCs from IBD patients (Supplementary
Figure S5). We then employed routine DSS-
induced acute and chronic colitis models, respec-
tively, to evaluate the exact role of palmitoleic acid
(POA) on colitis development and progression.
DSS-treated mice with palmitoleic acid (POA)
treatment showed less colitis development, as
shown by improved body weight regain and
reduced DAI score (Figure 3a, b), slighter bloody
diarrhea and rectal bleeding (Figure 3c), and longer
colons (Figure 3d, f), less infiltrations of inflamma-
tory cells (Figure 3g-i), lower expression of CD11b
in the mucosa and minimal reductions of goblet
cells and crypts, and higher expression of epithelial
markers ZO-1 and Occludin (Figure 3j, and
Supplementary Figure S7A). We also compared
the anti-colitis effects of palmitoleic acid (POA)
and palmitic acid (PA) using DSS-induced model
(Supplementary Figure S6). Palmitoleic acid (POA)
showed much better anti-colitis effects than palmi-
tic acid (PA) (Supplementary Figure S6).
Furthermore, murine colon contained only very
low background levels of palmitoleic acid (POA),
and the concentrations of palmitoleic acid (POA)
in murine colon after palmitoleic acid (POA) treat-
ments increased significantly, with an average
increase of 50% than mice without treatments,
suggesting that a considerable proportion of diet-
ary palmitoleic acid (POA) could successfully reach
the colon and exert anti-colitis effects
(Supplementary Figure S7D).
It is worth to note that mice treated with palmi-
toleic acid (POA) and water control showed
a similar food and water consumption during
DSS administration, suggesting that the attenua-
tion of colitis was not attributed to food and DSS
consumption (Supplementary Figure 8). Also,
although butyrate, a short-chain fatty acid (SCFA)
metabolite, showed some protection effects against
colitis
31
, palmitoleic acid (POA) treatment exhib-
ited a much better curative effect with an increased
colon length and decreased inflammatory infil-
trates than butyrate did (Supplementary Figure
S9). Moreover, DSS-treated mice receiving palmi-
toleic acid (POA) showed significantly lower
expressions of pro-inflammatory IL-6 and TNF-α
in serum and colons (Figure 3j, and Supplementary
Figure S7, B and C). Also, palmitoleic acid (POA)
selectively suppressed TNF-α and IL-6 production
but not IFN-γ/IL-2/IL-4/IL-10/IL-17A in LPS-
stimulated RAW 264.7 cells (Supplementary
Figure 10, A to C). Further immunoblot analysis
also showed that palmitoleic acid (POA) resulted in
the reduced expressions of COX2, pSTAT3, and/or
pNF-κB in either RAW 264.7 cells or peripheral
blood mononuclear cells (PBMCs) derived from
IBD patients (Supplementary Figure S10, D and
E). Thus, palmitoleic acid (POA) might exert anti-
inflammatory activity against colon inflammation
via targeting the IL-6/STAT3 and TNF-α/NF-κB
pathways.
Additionally, we investigated whether palmito-
leic acid (POA) provided protections in chronic
colitis. Mice were treated with DSS to induce
chronic colitis for three consecutive 7-day cycles.
Oral treatments of palmitoleic acid (POA) showed
decreased DAI and longer colons (Supplementary
therapy responders (R) than non-responders (NR) with IBD (R = 26, NR = 24). Data represent mean ± SEM; *P < 0.05. P values were
calculated by two-tailed unpaired Student’s t test (H). [The details for screening statistically differential metabolites in (a) and (d) are
shown in Materials and Methods]
GUT MICROBES 7
Figure 3. Oral supplement with palmitoleic acid (POA) ameliorates colon inflammation in DSS-induced acute colitis in mice. Note: (a)
Changes in the body weight (of initial weight) of mice treated with water, DSS, or DSS plus palmitoleic acid (POA). (b) Disease activity
index (DAI) score for colitis models including body weight loss, stool consistency, and fecal bleeding. (c) Images showing the
development of rectal bleeding and fecal blood of mice treated with water, DSS, or DSS plus palmitoleic acid (POA). Green arrow
indicates normal anus while orange arrow indicates bloody stool. White arrow indicated mild diarrhea with no obvious bloody stools.
(d) Representative endoscopic images of mice treated with water, DSS, or DSS plus palmitoleic acid (POA) at the end of DSS treatment.
Blue arrow showed extensive mucosa damage with bleeding. (e) Gross anatomy of colons of mice treated with water, DSS, or DSS plus
palmitoleic acid (POA). Scale bar = 1.0 cm. (f) Colon lengths of mice treated with water, DSS, or DSS plus palmitoleic acid (POA). (g)
Hematoxylin and eosin (H&E) staining of “Swiss roll” colon sections from mice treated with water, DSS, or DSS plus palmitoleic acid
(POA). Top: original magnification, scale bar = 500 μm. Middle: distal colon sections, scale bar = 100 μm. Bottom: proximal colon
8Y. CHEN ET AL.
Figure 11, A to D), exhibited less epithelial loss,
ulceration, and inflammatory cell infiltration
(Supplementary Figure 11E). Thus, palmitoleic
acid (POA) induced similar protections against
chronic colitis as like in acute colitis. Together,
these data collectively suggest that palmitoleic
acid (POA) could effectively inhibit inflamma-
tion, repair intestinal mucosal barrier, and attenu-
ate both acute and chronic colitis in mouse
models.
Furthermore, the safety profile of palmitoleic
acid (POA) was evaluated in mice by oral
gavage of different doses (0.1 g/kg, 0.5 g/kg, 2.5
g/kg). It showed that mice orally gavaged with
several doses of palmitoleic acid (POA) (0.1 g/
kg, 0.5 g/kg, 2.5 g/kg) and water control showed
no significant differences in weight changes,
colon length and spleen weight (Supplementary
Figure 12, A to C). Compared with the control
group, palmitoleic acid (POA) significantly
increased superoxide dismutase (SOD)
(Supplementary Figure 12D) but not triglycer-
ides (TG), low-density lipoprotein (LDL), high-
density lipoprotein (HDL) and cholesterol
(CHO) expression (Supplementary Figure 12E).
Activities of alkaline phosphatase (ALKP), ala-
nine aminotransferase (ALT), and aspartate
aminotransferase (AST) maintain in the normal
ranges, indicating no observable liver damages
of palmitoleic acid (POA) (Supplementary
Figure 12F). Parameters of renal function
including urea nitrogen (UREA) and creatinine
(CREA) were also within the normal levels as
well (Supplementary Figure 12 G). Also, no
apparent liver histological changes were identi-
fied upon palmitoleic acid (POA) treatments
(Supplementary Figure 12 H). Thus, palmitoleic
acid (POA) had well tolerable safety profiles for
next-step in-depth mechanism and potential
translational research.
Palmitoleic acid (POA) exerts anti-colitis eects in
2,4,6-trinitrobenzenesulfonic acid (TNBS) -induced
colitis model in mice
To explore the breadth of potential therapeutic
applications of palmitoleic acid (POA) against
colitis, we utilized TNBS-induced colitis, which
is recognized as one of the closest models
mimicking with Crohn’s disease (CD)
32
. BALB/
c mice were subjected to intrarectal administra-
tion of TNBS in 50% ethanol to establish TNBS-
induced colitis model. TNBS-treated mice
rapidly developed bloody diarrhea, extensive
wasting syndrome and a weight loss with
a mortality rate of 60% (Supplementary
Figure 13A). However, compared with water
control treatment, TNBS-treated mice with oral
palmitoleic acid (POA) treatment showed higher
survival rate of 83% and recovered body weight,
decreased colonic weight/length ratio (a macro-
scopic indicator of inflammation), and lower
Wallace (indications of extensive inflammatory
lesions) (Supplementary Figure 13, A to C).
Also, compared with TNBS-treated mice
receiving water, TNBS-treated mice receiving
palmitoleic acid (POA) showed a significant
trend of increased colon length (Supplementary
Figure 13D) and displayed less ulceration,
milder colonic tissue damage, much fewer infil-
trations of inflammatory cells in intestinal bar-
rier and less goblet cell loss (Supplementary
Figure 13, E to G), particularly higher expres-
sion of epithelial markers including ZO-1 and
Occludin (Supplementary Figure 13 H), and
lower frequency of CD8
+
, ly6G
+
and CD11b
+
cells in colonic lamina propria (Supplementary
Figure 13, I and J). Collectively, these results
suggest that oral administration with palmitoleic
acid (POA) protected against intestinal inflam-
mation and attenuated TNBS-induced colitis.
sections, scale bar = 100 μm. (h) Histological scores for inflammation, crypt damage, depletion of goblet cells and infiltration of
inflammatory cells. (i) Alcian Blue Periodic acid Schiff (AB-PAS) staining was performed to visualize the presence of goblet cells in the
gut sections (scale bar = 200 μm). (j) The expression of Il-1β, Il6, Tnfα, Occludin and ZO-1 was measured by real-time qPCR. Data
represent mean ± SEM (n = 5-6 mice per group); *P < 0.05; **P < 0.01; ***P < 0.001; NS, no statistical significance. P values were
calculated by unpaired Student’s t test [(a), (b), (f), (h) and (j)]. At least two biological repeats were performed.
GUT MICROBES 9
Palmitoleic acid (POA) ex vivo exerts appreciable
tissue repairment and anti-IL-6/TNF-α eects on
highly inamed colon derived from Crohn’s disease
(CD) patients
Then, we analyzed whether palmitoleic acid (POA)
could confer some therapeutic effects against
human IBD. To address this, we collected highly
inflamed colonic samples from Crohn’s disease
(CD) patients, and these tissues were ex vivo co-
cultured with vehicle control (Ctrl) and palmitoleic
acid (POA), respectively. Histology analysis
revealed reduced pathological impairments in pal-
mitoleic acid (POA)-treated inflamed colon tissues
with less epithelial injury, immune cell infiltration
and edema (Figure 4, b and c). Supplementation
with palmitoleic acid (POA) selectively decreased
colonic production of TNF-α and IL-6, but not IL-
2/IL-4/IL-10/IFN-γ/IL-17A (Figure 4, d to g).
Moreover, Alcian Blue Periodic acid Schiff (AB-
PAS) staining showed that numbers of goblet cells
in palmitoleic acid (POA)-treated group was sig-
nificantly larger than that in the control group
(Figure 4i). Palmitoleic acid (POA) treatment
could support better intestinal integrity and sup-
press neutrophil infiltration as palmitoleic acid
(POA)-treated colon tissue showed increased ZO-
1 expressions and diminished infiltrations of
CD11b
+
cells in colonic tissues (Figure 4, j and k).
Taken together, palmitoleic acid (POA) ex vivo
exerted anti-inflammatory effects and mucosal
repairment in highly inflamed tissues from
Crohn’s disease (CD) patients.
Palmitoleic acid (POA) shapes colonic gut
microbiota composition with selectively increased
abundance of anti-inammatory Akkermansia
muciniphila
Since intestinal metabolites play a significant role
in shaping the gut microbiota
5,33
, we then hypothe-
size that such differentially abundant metabolites
between anti-TNF-α responders and non-
responders with IBD could serve as a molecular
force to regulate the growth and physiology of gut
bacteria. We therefore further investigated how
palmitoleic acid (POA) altered the functional and
compositional diversity of gut microbiota. To
address this, we performed 16S rRNA sequencing
to analyze the colon contents from DSS-treated
mice receiving treatments of palmitoleic acid
(POA) and water, respectively (Figure 5, a to d).
Analysis of β-diversity showed significantly distinct
species diversity between DSS-treated mice receiv-
ing treatments of palmitoleic acid (POA) and water
(Figure 5a), although the α-Diversity using
Shannon and Simpson index showed no significant
species richness between DSS-treated mice receiv-
ing treatments of palmitoleic acid (POA) and water
(Supplementary Figure 14, A and B). Compared
with mice treated with DSS, DSS-treated mice
receiving palmitoleic acid (POA) showed higher
phylum abundance of Firmictues and
Verrucomicrobia, and higher genus abundance of
beneficial genera including Akkermansia,
Lactobacillus and Bifidobacterium (Figure 5b, and
Supplementary Figure 14 C). These data suggest
that oral administrations of palmitoleic acid
(POA) could effectively change the composition
and diversity of gut microbiome.
Importantly, LefSe analysis indicated that nine
bacterial species including Akkermansia mucini-
phila, Lactobacillus johnsonii and Bifidobacterium
pseudolongum were enriched in DSS-treated mice
receiving oral treatments of palmitoleic acid
(POA), while other twenty taxa were enriched in
DSS-treated mice receiving water control
(Figure 5c). Remarkably, Akkermansia muciniphila
was the bacterial species with the most significantly
enriched and highest abundance in DSS-treated
mice receiving oral treatments of palmitoleic acid
(POA) (Figure 5c and Supplementary Figure 14,
D and F).
To verify whether palmitoleic acid (POA)
directly enhances the growth of these bacterial
species observed in colons of DSS-treated mice
receiving oral treatments of palmitoleic acid
(POA), we then performed in vitro co-culture
analyses of Akkermansia muciniphila and
Fusobacterium nucleatum with palmitoleic acid
(POA). Palmitoleic acid (POA) with two different
concentrations (0.7 mM and 7 mM, respectively)
were included in culture medium of these bacter-
ial species to observe the effects of palmitoleic
acid (POA) on the growth of these bacterial spe-
cies. Notably, in consistent with those in vivo
analyses in mice (Figure 5c and Supplementary
Figure 14, D and F), palmitoleic acid (POA) with
10 Y. CHEN ET AL.
Figure 4. Palmitoleic acid (POA) ex vivo exerts tissue repairment and anti-inflammatory effects on highly inflamed colon tissues
derived from Crohn’s disease (CD) patients. Note: (a) Workflow showing the processing of freshly collected surgical highly inflamed
colon resections from Crohn’s disease (CD) patients for pathology, Cytometric Bead Array (CBA) assay and western blotting. (b-c) HE
staining was performed to reveal the pathology of highly inflamed colon tissues co-cultured with vehicle control (Ctrl) or palmitoleic
acid (POA) ex vivo. Top: scale bar = 200 μm; Bottom: scale bar = 50 μm. The black-boxed areas at the top are enlarged below (n = 12).
(d) Representative CBA analysis of culture supernatants of highly inflamed colon tissues co-cultured with vehicle control (Ctrl) or
palmitoleic acid (POA) using human Th1/Th2/Th17 CBA kit. (e-g) The expressions of IL-6, TNF-α, IL-2, IL-4, IL-10, IFN-γ and IL-17A in
culture supernatants of highly inflamed colon tissues co-cultured with vehicle control (Ctrl) or palmitoleic acid (POA). Boxed areas
mark the fluorescent clusters of IL-6 (blue) and TNF-α (red), respectively, and dashed lines mark the shift of fluorescent clusters of IL-6
and TNF-α, respectively. No significant effect on expressions of IL-2, IL-4, IL-10, IFN-γ and IL-17A in inflamed colon tissues between
treatments of palmitoleic acid (POA) and Ctrl. (h) STAT3 and pSTAT3 expressions in highly inflamed colon tissues co-cultured with
GUT MICROBES 11
7 mM concentration significantly facilitated the
growth of Akkermansia muciniphila with acceler-
ated exponential growth and prolonged station-
ary phase growth but inhibited the growth of
Fusobacterium nucleaum in vitro (Figure 5d,
and Supplementary Figure 15), suggesting the
species-specific effects of palmitoleic acid (POA)
on promoting the growth of anti-inflammatory
Akkermansia muciniphila.
Transcriptional changes emphasize responses of
cell division, energy production and biosynthetic
process to palmitoleic acid (POA) in Akkermansia
muciniphila
To elucidate the molecular mechanisms governing
the bacterial responses to palmitoleic acid (POA),
we analyzed the transcriptomes of Akkermansia
muciniphila after co-cultured with palmitoleic
acid (POA). Transcriptomics analyses of
Akkermansia muciniphila revealed that the addi-
tion of palmitoleic acid (POA) led to up-regulation
of 286 genes and down-regulation of 305 genes
(Figure 5e). Significantly altered transcriptional
signatures including cell division, lipid transport
and metabolism, coenzyme transport and metabo-
lism, cell motility, energy production and conver-
sion, post translational modification, and other
uncertain general function (Supplementary
Table 3). In the presence of palmitoleic acid
(POA), genes related to replication (xseA), cell
division (AMUC_RS03580), and nucleotide trans-
port (AMUC_RS00595) were upregulated while
genes related to coenzyme transport and metabo-
lism (AMUC_RS05455), cell motility
(AMUC_RS12080), energy production and con-
version (AMUC_RS11405) and post translational
modification (AMUC_RS07100) were downregu-
lated (Figure 5, f and g, and Supplementary
Table 3). Notably, concomitant with augmented
nucleotide transport metabolism was upregulation
of the genes related to energy production
(AMUC_RS12070) following treatment with pal-
mitoleic acid (POA) (Supplementary Table 3).
These results suggested that palmitoleic acid
(POA) could boost the growth, energy production
and biosynthetic pathway of Akkermansia
muciniphila.
Further GO enrichment analysis showed that
up-regulated differential expressed genes (DEGs)
in Akkermansia muciniphila in response to palmi-
toleic acid (POA) were mainly clustered in cell
division, ATP synthesis coupled electron transport
and biosynthetic process (Figure 5, g and h).
Additionally, down-regulated transcriptional activ-
ities were mainly clustered in calcium ion binding,
RNA helicase activity, anion transport, negative
regulation of cellular process and other biological
process (Supplementary Figure 16). Together, these
data highlighted that palmitoleic acid (POA) could
transcriptionally upregulate genes involving in
pathways of cell division and biosynthetic process
to promote the proliferation of Akkermansia
muciniphila.
Co-administration of palmitoleic acid (POA) and
Akkermansia muciniphila synergistically maintains
intestinal barrier and controls chronic colitis in mice
Given that palmitoleic acid (POA) shapes colonic
gut microbiota composition with increased abun-
dance of Akkermansia muciniphila, and we and
other colleagues have recently shown that
Akkermansia muciniphila could confer significant
anti-inflammatory protection effects
34,35
, we thus
postulated that there should be synergistic effects
between palmitoleic acid (POA) and Akkermansia
muciniphila in control of colitis. Such postulation
is well justified because: (i) palmitoleic acid (POA)
shows a remarkable ability to repair gut mucosal
tissue, which is necessary for bacterial colonization
and growth niches such as Akkermansia mucini-
phila; (ii) palmitoleic acid (POA) may reprogram
the gut microbial structures via favoring the growth
vehicle control (Ctrl) or palmitoleic acid (POA). (i) Alcian Blue Periodic acid Schiff (AB-PAS) staining was performed to visualize the
presence of goblet cells in the gut sections (scale bar = 100 μm). (j-k) IHC staining and relative intensity of ZO-1 and CD11b in highly
inflamed colon tissues co-cultured with vehicle control (Ctrl) or palmitoleic acid (POA) ex vivo (scale bar = 100 μm). Data represents
mean± SEM; *P < 0.05; NS, no statistical significance. P values were calculated by two-tailed unpaired (c) and paired Student’s t test
[(e), (f), (g) and (k)].
12 Y. CHEN ET AL.
Figure 5. Palmitoleic acid (POA) reprograms gut microbiota compositions, enhances the growth of Akkermansia muciniphila via
upregulating transcription signatures of cell division and biosynthetic process, increases the intestinal abundance of Akkermansia
muciniphila. Note: (a) Principal coordinate analysis (PCoA) plots on OUT level were based on weighted and unweighted unifrac. Red
points indicated the microbiota enriched in DSS-treated mice and blue points indicated the microbiota enriched in DSS-treated mice
receiving palmitoleic acid (POA) [analysis of similarities (ANOSIM): R = 0.5720, P = 0.004 and R = 0.8520, P = 0.004 with 999 permuta-
tions]. (b) The relative abundance of bacteria on genus level of DSS-treated mice receiving water or palmitoleic acid (POA). (c) Linear
discriminant analysis (LDA) coupled with effect size measurements (LEfSe) identified the species with different abundance of DSS-
treated mice receiving water or palmitoleic acid (POA). Higher abundant species in DSS-treated mice are shaded with red and higher
abundant species in DSS-treated mice receiving palmitoleic acid (POA) are shaded with blue (P < 0.05 and LDA score>3.0). qPCR
validation of the relative abundance of different microbial taxa was shown in Supplementary Figure 14. (d) The growth curve of
Akkermansia muciniphila cultured with 7 mM palmitoleic acid (POA), 0.7 mM palmitoleic acid (POA), vehicle control (Ctrl), 7 mM butyric
acid or 0.7 mM butyric acid, in an anaerobic cabinet in BHI broth supplemented with 0.25% mucin. The growth curve of other bacteria
was shown in Supplementary Figure 15. (e) Volcano plot of differential expressed genes (DEGs) in Akkermansia muciniphila in response
to 7 mM palmitoleic acid (POA). Red spots represent upregulated genes, and green spots represent downregulated genes (FC ≥ 2 and
GUT MICROBES 13
of beneficial gut bacteria such as Akkermansia
muciniphila.
Indeed, compared with DSS-induced chronic
colitis mice treated with only palmitoleic acid
(POA) or Akkermansia muciniphila, mice receiving
co-administrations of palmitoleic acid (POA) and
Akkermansia muciniphila exhibited higher intest-
inal abundance of Akkermansia muciniphila, much
less ulceration, reduced inflammatory cell infiltra-
tion and more advanced healing of the eroded
mucosal barrier (Supplementary Figure 17, and
Supplementary Figure 18). Thus, co-
administrations of palmitoleic acid (POA) and
Akkermansia muciniphila not only favored the
intestinal growth and abundance of Akkermansia
muciniphila but also synergistically conferred ther-
apeutic effects against colitis with Akkermansia
muciniphila. Thus, these data collectively suggest
that palmitoleic acid (POA) reprogrammed the gut
microbiota with increased abundance of
Akkermansia muciniphila, and co-administration
of palmitoleic acid (POA) and Akkermansia muci-
niphila synergisticallyfacilitated the control of
colitis.
Gut microbiota reprogrammed by palmitoleic acid
(POA) contributes to improved eects of anti-TNF-α
therapy against colitis
We then investigated whether palmitoleic acid
(POA)-reprogrammed gut microbiome character-
ized with higher abundance of Akkermansia muci-
niphila and other beneficial bacteria could really
confer protective effect against colitis. To address
this, littermate mice were orally treated with pal-
mitoleic acid (POA) or water control, respectively,
for 7 days in co-transfer experiments (Figure 6a),
and then the feces from donor mice fed with pal-
mitoleic acid (POA) or water group were trans-
ferred to antibiotic-pretreated recipient mice.
After one-week fecal transplantation, recipient
and donor mice were treated with 2.5% DSS and
Infliximab (IFX, an anti-TNF-α mAb widely used
for biological therapy) injection (Figure 6a).
Recipient mice orally treated with feces derived
from palmitoleic acid (POA)-fed donor mice
gained less weight loss and longer colon length
compared with recipient mice that received feces
from chow-fed donor mice in response to therapy
of anti-TNF-α (Infliximab, IFX) during DSS-
induced colitis (Figure 6, b to d). In addition,
mice transplanted with palmitoleic acid (POA)-
treated gained less weight loss fecal samples devel-
oped lighter colitis features and exhibited alleviated
pathological features with less inflammatory cell
infiltration and more intact colonic architecture
with few observable ulcerations (Figure 6, e and
f). Together, these data suggested that improved
therapeutic benefits of anti-TNF-α against colitis
might be ascribed to reprogrammed gut microbial
community in mice after oral treatments of palmi-
toleic acid (POA).
Palmitoleic acid (POA) promotes mucosal barrier
healing and improves the ecacy of anti-TNF-α
therapy against colitis in mice
Since palmitoleic acid (POA) showed the therapeu-
tic effects of maintaining intestinal epithelial integ-
rity and alleviating inflammation in IBD mouse
models, we next explored whether palmitoleic
acid (POA) increased the efficacy of anti-TNF-α
therapy against colitis. Mice were administrated
with 2.5% DSS and treated with palmitoleic acid
(POA) or water, respectively, followed by intraper-
itoneally (i.p.) injected with anti-TNF-α mAb after
DSS administration. Mice receiving co-
administrations of palmitoleic acid (POA) plus
anti-TNF-α mAb exhibited less weight loss and
lower DAI compared with mice receiving only
p-adjust≤0.05). Each group contains data for three independent samples. (f)Fold changes of the expression of top 10 upregulated or
downregulated genes response to 7 mM palmitoleic acid (POA) compared with control in Akkermansia muciniphila. (g) GO enrichment
analysis of upregulated DEGs in Akkermansia muciniphila in response to 7 mM palmitoleic acid (POA). The plot showed top 10
functional classifications of GO categories including cellular component, biological process, and molecular function. GO enrichment
analysis of downregulated DEGs in Akkermansia muciniphila in response to 7 mM palmitoleic acid (POA) was shown in Supplementary
Figure 16. (h) The expression levels of upregulated DEGs related to functional classifications of GO categories in Akkermansia
muciniphila in response to 7 mM palmitoleic acid (POA). Data represent mean± SEM (n = 5 mice per group); **P < 0.01;
***P < 0.001; NS, no statistical significance. P values were calculated by two-tailed unpaired Student’s t test [(d) and (h)].
14 Y. CHEN ET AL.
Figure 6. Palmitoleic acid (POA)-educated microbiota improves the efficacy of anti-TNF-α therapy against colitis. Note: (a) Experimental
diagram for determining whether the increased anti-TNF-α responsiveness to colitis achieved by palmitoleic acid (POA) treatment is
transferable. Conventionally raised mice were fed with water or palmitoleic acid (POA) for 7 days. Fecal homogenates from water or
palmitoleic acid (POA)-treated mice were orally transmitted into antibiotics-pretreated recipient mice. After one-week transplantation,
all mice were treated with 2.5% DSS and anti-TNF-α mAb (Infliximab, IFX) for 7 days. (b) Changes in the body weight (of initial weight)
of DSS-treated mice, donor mice treated with water or palmitoleic acid (POA), and recipient mice orally transmitted with feces of
donor mice after DSS administration. (c) Gross anatomy of colons of DSS-treated mice, donor mice treated with water or palmitoleic
GUT MICROBES 15
anti-TNF-α mAb or palmitoleic acid (POA)
(Figure 7, a and b). Remarkably, most mice receiv-
ing co-administration of palmitoleic acid (POA)
plus anti-TNF-α mAb showed more significant
attenuation of diarrhea and improved colon length
(Figure 7, c to e). Histopathologically, mice receiv-
ing combination treatments of palmitoleic acid
(POA) with anti-TNF-α mAb showed much better
preservation of mucosal integrity with more obser-
vable goblet cells and crypts, less inflammation and
ulceration (Figure 7, f to i). Taken together, these
data suggested that palmitoleic acid (POA) con-
tributed to better responsiveness of anti-TNF-α
biological therapy against colitis.
Discussion
Due to the challenges of dramatic fluctuations of
diversity and magnitude of microbiota and meta-
bolite network in host
9
, precise orchestration and
underlying mechanisms of chemically and molecu-
larly complex regulation networks in host physiol-
ogy and immune homeostasis remain largely
unraveled
36
. This study has revealed the critical
role of molecular regulation in gut microbiome by
modulating homeostasis among gut microbiota,
metabolic systems, intestinal mucosal barrier, and
immune network. Particularly, this study suggested
the previous unrecognized polyfunctionality and
mechanisms of palmitoleic acid (POA) in both
effectively promoting mucosal healing and selec-
tively modulating abundance, composition, struc-
ture, and function of gut microbiota to alleviate
diseases and enhance biological therapy in the con-
text of this complex host-microbiota interplay net-
works, providing potential novel prevention and
therapeutic strategies against intestinal and aben-
teric diseases.
Molecularly complex regulation of abundance,
composition, structure and function of gut micro-
biota requires exact understanding of the role of
a designated molecule, but most of our previous
understandings are limited in short-chain fatty
acids (SCFAs)
37
, glycans
6
, sugar
38
and other
molecules
39,40
. Increasing evidence has revealed
the important roles of dietary long-chain unsatu-
rated fatty acids in regulating inflammatory
responses
13
. Palmitic acid (PA) is the most com-
mon saturated fatty acid and metabolized to oleic
acid, stearic acid, sphingolipids as well as palmito-
leic acid (POA)
41
. Palmitic acid (PA) might trigger
TLR responses and endoplasmic reticulum (ER)
stress in macrophages, damage gut barriers with
decreased abundance of Akkermansia muciniphila
and increase inflammatory cytokine
productions
42–44
. Excessive intake of long-chain
polyunsaturated unsaturated fatty acids (PUFAs)
such as n-3 and n-6 fatty acids might be associated
with inflammatory diseases including IBD because
of the increased TLR2-mediated ER stress and che-
mokine productions
45
. Different from palmitic
acid (PA) and other LCFAs, palmitoleic acid
(POA) is an omega-7 monounsaturated fatty acid
with a double bond and one of the most abundant
monounsaturated fatty acids in plants and marine
sources
46,47
, which might have anti-inflammatory
capabilities
48
. Our data demonstrated that palmi-
toleic acid (POA) but not palmitic acid (PA) dis-
played anti-inflammatory and anti-colitis
properties via regulating structure and function of
gut microbiota. However, it remains largely
unknown whether and how other LCFAs regulate
gut microbiota and further mediate IBD
pathogenesis.
This work demonstrated in vivo and in vitro
effects of palmitoleic acid (POA) on selectively
increasing the abundance of beneficial gut bacteria
such as Akkermansia muciniphila and significantly
extend the diversity of molecule arsenal and meta-
bolic pathways capable of reprogramming such
highly dynamic and complex bacterial commu-
nities. The concentrations of palmitoleic acid
acid (POA), and recipient mice orally transmitted with feces of donor mice. Scale bar = 1.0 cm. (d) Colon lengths of DSS-treated mice,
donor mice treated with water or palmitoleic acid (POA), and recipient mice orally transmitted with feces of donor mice. (e)
Representative images of pathologic colon sections. Top: original magnification, scale bar = 500 μm. Middle: distal colon sections,
scale bar = 100 μm. Bottom: proximal colon sections, scale bar = 100 μm. (f) Colon histological scores for inflammation, crypt damage,
depletion of goblet cells and infiltration of inflammatory cells. Data represent mean ± SEM (n = 5 mice per group); *P < 0.05; **P <
0.01; ***P < 0.001; ****P < 0.0001; NS, no statistical significance. P values were calculated by two-tailed unpaired Student’s t test [(b),
(d) and (f)]. At least two biological repeats were performed.
16 Y. CHEN ET AL.
Figure 7. Oral administration of palmitoleic acid (POA) enhances therapeutic effects of anti-TNF-α mAb against colitis in mice.
Note: (a) Changes in the body weight (of initial weight) of DSS-induced mouse colitis models treated with water, palmitoleic acid
(POA), anti-TNF-α mAb (Infliximab, IFX), and palmitoleic acid (POA) plus anti-TNF-α mAb (Infliximab, IFX) after DSS administration. (b)
Disease activity index (DAI) score for colitis models including body stool consistency and fecal bleeding. (c) Images showing the
development of severe diarrhea and rectal bleeding of DSS-induced mouse colitis models treated with water, palmitoleic acid (POA),
anti-TNF-α mAb (Infliximab, IFX), and palmitoleic acid (POA) plus anti-TNF-α mAb (Infliximab, IFX) at the end of DSS treatment. Orange
arrow indicates severe diarrhea and bloody stools while white arrow indicates mild diarrhea with no obvious bloody stools. Green
arrow shows no obvious diarrhea with no obvious bloody stools. (d) Gross anatomy of colons of DSS-induced mouse colitis models
GUT MICROBES 17
(POA) in the host should be highly-dynamic and
variable because of the alteration of gut microbiota,
dietary intake, and other physiological or patholo-
gical conditions that may influence metabolic pro-
cesses. It was estimated that approximately 10 ~
17% of palmitic acid (PA), which can later be
metabolized into palmitoleic acid (POA), would
be absorbed in mesenteric lymphatic vessels and
that over 80% of palmitic acid (PA) might reach
colon in adult rats
49
. Given that palmitoleic acid
(POA) is also a microbial metabolite preferentially
produced by some gut bacteria such as
Akkermansia muciniphila
34,50
, this study reveals
previously unknown positive feedback loop from
higher abundance of beneficial gut bacteria, higher
levels of selected bacterial metabolites to preferred
gut microbiota structure for better health and dis-
ease therapies. Also, in other words, the develop-
ment and progression of inflammatory diseases
(e.g., IBD in this study) and limited effects of ther-
apy (e.g., anti-TNF-α mAb) of these inflammatory
diseases may attribute to destructions or failed
reconstructions of this positive feedback loop.
Anti-TNF-α antibody is the earliest, most widely
used, and most well-evaluated biological therapy
for IBD, but the irresponsiveness or therapeutic
resistance is thought to represent the main cause
for the limited success of IBD therapy
51
. Although
treatments for IBD act by inhibiting inflammatory
cytokines or infiltration of inflammatory cell sub-
sets have been made significant progresses
51
,
mucosal healing and regeneration are a most pro-
mising strategy and a most critical clinical indica-
tion during IBD therapy
16
. This study shows that,
palmitoleic acid (POA), a long-chain fatty acid
(LCFA) with higher abundance in responders
receiving anti-TNF-α-based biological therapy, is
a protective metabolite with multi-potency critical
for reprogramming gut microbiota structure and
inducing tissue repair and homeostasis after intest-
inal damage in IBD. Indeed, this study presents
a head-to-head comparison in a highly relevant
mouse IBD model that a long-chain fatty acid
(LCFA) displays a superior capability than butyrate
in intestinal mucosal healing and regeneration.
Such long-chain fatty acid (LCFA)-conferred tissue
repair and homeostasis recovery in intestinal
damage in IBD may be ascribed to: (i) the direct
regulation of expression of molecule components
of the mucosal tight junction (e.g., Occludin, ZO-
1); (ii) suppression of systemic and mucosal
inflammation, which further facilitates the reduc-
tion or inhibition of intestinal damages in IBD; (iii)
reprogramming of gut microbiota toward a more
favorable structures and functions against IBD.
Notably, palmitoleic acid (POA) significantly upre-
gulated the growth fitness of Akkermansia mucini-
phila and inhibited some detrimental pro-
inflammatory bacteria such as Fusobacterium, and
this antibacterial effect of palmitoleic acid (POA)
might have contributed to the gut microbiota-
reprogramming capability of palmitoleic acid
(POA) against colitis. Thus, palmitoleic acid
(POA) and palmitoleic acid (POA)-
reprogrammed gut microbiota may show broader
spectrum roles in enhancing efficacy of other bio-
logical therapies against IBD and other inflamma-
tory disorders. However, more in-depth
mechanisms underlying the palmitoleic acid
(POA)-regulated mucosal healing and regeneration
need to be uncovered.
Accumulating evidence has revealed that IBD
patients and health controls may display signifi-
cantly differential signatures of blood and intestinal
treated with water, palmitoleic acid (POA), anti-TNF-α mAb (Infliximab, IFX), and palmitoleic acid (POA) plus anti-TNF-α mAb
(Infliximab, IFX). Scale bar = 1.0 cm. (e) Colon lengths of DSS-induced mouse colitis models treated with water, palmitoleic acid (POA),
anti-TNF-α mAb (Infliximab, IFX), and palmitoleic acid (POA) plus anti-TNF-α mAb (Infliximab, IFX). (f) Representative images of
pathologic colon sections of DSS-induced mouse colitis models treated with water, palmitoleic acid (POA), anti-TNF-α mAb (Infliximab,
IFX), and palmitoleic acid (POA) plus anti-TNF-α mAb (Infliximab, IFX). Top: original magnification, scale bar = 500 μm. Bottom:
proximal colon sections, scale bar = 100 μm. The black-boxed areas at the top are enlarged below. (g) Colon histological scores for
inflammation, crypt damage, depletion of goblet cells and infiltration of inflammatory cells. (h) Alcian Blue Periodic acid Schiff (AB-
PAS) staining of the colon tissue to visualize goblet cell within the mucosa. Top: low power field, scale bar = 200 μm. Bottom: high
power field, scale bar = 50 μm. The black-boxed areas at the top are enlarged below. (i) Quantification of AB-PAS-positive cell in high
power field of the colon tissue. Data represent mean ± SEM (n = 5-6 mice per group); *P < 0.05; **P < 0.01; ***P < 0.001; ****P <
0.0001; NS, no statistical significance. P values were calculated by two-tailed unpaired Student’s t test [(a), (b), (e), (g)and (i)]. At least
two biological repeats were performed.
18 Y. CHEN ET AL.
metabolites
12,29,52
, and remissions to biological
therapy of IBD patients were associated with
changes of blood and intestinal
metabolome
14,17,18,24,53,54
. For example, higher
fecal butyrate and substrates were significantly cor-
related with clinical benefits of anti-TNF therapy
14
,
and serum secondary bile acids may be a valuable
metabolic signature for prediction of clinical remis-
sion upon receiving anti-TNF or anti-IL12/23 bio-
logical therapeutics
24
. These metabolic signatures
may serve as not only critical biomarkers for deci-
phering the pathogenesis mechanisms of IBD and
predicting disease prognosis and therapeutics out-
comes of IBD but also useful intervention targets
for IBD therapeutics. However, few, if there any,
evidence has been presented that any of these meta-
bolic markers or signatures might successfully
improve biological therapy of IBD. Thus, this
study presents the first evidence demonstrating
that a designated metabolite signature (i.e., palmi-
toleic acid (POA)) may facilitate the biological
therapy of IBD.
Overall, this study reveals the critical role of
palmitoleic acid (POA) in modulating molecular
and chemical network of gut microbiota and gut
metabolites, which determines the magnitude and
diversity of a beneficial gut microbial ecosystem.
Such palmitoleic acid (POA)-modulated magni-
tude and diversity of gut microbial ecosystem
may help us to uncover the pathogenesis mechan-
isms and develop new therapeutics of IBD and
other intestinal and abenteric diseases.
Materials and methods
Ethics statement
IBD patients and healthy controls were recruited at
the First Affiliated Hospital of Sun Yat-sen
University [Ethics Number: (2019)348], confirmed
by radiological, endoscopic, and histological find-
ings. The scoring criteria of mucosal appearance at
endoscopy refers to Chinese Grading System of
Crohn’s Disease (CGSCD) or Mayo Score for
ulcerative colitis. Prior to enrollment, all partici-
pants provided written informed consent.
Response was defined as clinical remission in diar-
rhea, abdominal cramping, endoscopy, and muco-
sal biopsy for at least 12 weeks after initiation. Non-
response was defined as outcomes not meeting
above definitions or adverse effects (for example,
infusion reactions due to immunogenicity) at 12
weeks after initial therapy. Clinical specimens
including stool samples, blood, and colon tissues
were obtained from IBD patients as well as healthy
controls. Feces and serum were frozen at −80°C.
The characteristics of the patients are listed in
Supplementary Table 1. Study procedures were
conducted according to the Declaration of
Helsinki.
Fecal genomic DNA extraction, 16S rRNA gene
sequencing and taxonomic annotation
Fecal DNA was extracted using the EZNA ® soil
DNA Kit (Omega Bio-tek, Norcross, GA, U.S.)
according to manufacturer’s instructions. The
hypervariable region V3-V4 of the bacterial 16S
rRNA gene were amplified with primer pairs 338F
(5’-ACTCCTACGGGAGGCAGCAG-3’) and 806
R(5’-GGACTACHVGGGTWTCTAAT-3’) by an
ABI GeneAmp® 9700 PCR thermocycler (ABI,
CA, USA). PCR products were purified with an
AxyPrep DNA Gel Extraction Kit (Axygen
Biosciences, Union City, CA, USA) and quantified
using QuantiFluor™-ST (Promega, USA). Purified
and pooled amplicon libraries were paired-end
sequenced (2 × 300) on the Illumina MiSeq plat-
form (Illumina, San Diego, USA) according to the
standard protocols by Majorbio Bio-Pharm
Technology Co., Ltd. (Shanghai, China). The raw
reads were demultiplexed, quality-filtered, merged
and clustered into OTUs with a 97% similarity
cutoff using UPARSE (version 7.1, http://drive5.
com/uparse/), and chimeric sequences were identi-
fied and removed using UCHIME. The taxonomy
of each OTU representative sequence was analyzed
by RDP Classifier version 2.2
55
against the 16S
rRNA database (e.g. Silva v137) using confidence
threshold of 0.7.
Fecal UHPLC-MS/MS untargeted metabolism
50 mg solid samples were weighed for the metabo-
lomics study, and the metabolites were extracted
using a 400 µL methanol: water (4:1, v/v) solution.
The mixture was allowed to settle at −20°C and
treated by high throughput tissue crusher
GUT MICROBES 19
Wonbio-96c (Shanghai wanbo biotechnology co.,
LTD) at 50 Hz for 6 min, then followed by vortex
for 30 s and ultrasound at 40 kHz for 30 min at 4°C.
The samples were placed at −20°C for 30 min to
precipitate proteins. After centrifugation at
13,000 × g at 4°C for 15 min, the supernatant was
carefully transferred to sample vials for UHPLC-
MS/MS analysis.
Chromatographic separation of the metabolites
was performed on a Thermo UHPLC system
equipped with an ACQUITY BEH C18 column
(100 mm × 2.1 mm i.d., 1.7 µm; Waters, Milford,
USA). The mass spectrometric data was collected
using a Thermo UHPLC-Q Exactive Mass
Spectrometer equipped with an electrospray ioni-
zation (ESI) source operating in either positive or
negative ion mode. After UPLC-TOF/MS analyses,
the raw data were imported into the Progenesis QI
2.3 (Nonlinear Dynamics, Waters, USA) for peak
detection and alignment. Statistically differential
metabolites between responders’ and non-
responders’ stools were selected with VIP value>1
and p value<0.05. A total of 51 differential peaks
were selected including 24 peaks in ESI+ and 27
peaks in ESI-. Differential metabolites between two
groups were summarized, and mapped into their
biochemical pathways through metabolic enrich-
ment and pathway analysis based on database
search (HMDB, http://www.hmdb.ca/; KEGG,
https://www.kegg.jp/kegg/pathway.html). These
metabolites can be classified according to the path-
ways they involved or the functions they per-
formed. Enrichment analysis was usually to
analyze a group of metabolites in a function node
whether appears or not.
Serum UHPLC-MS/MS lipid metabolism
200 µL liquid sample was extracted using 80 µL
methanol and 400 µL MTBe. After centrifugation
(13000 × g) for 15 min at 4°C and concentration,
80 µL supernatant was transferred to sample vials
for LC-MS/MS analysis. Chromatographic separa-
tion of the lipids was performed on a Thermo
UHPLC Vanquish Horizon system equipped with
an ACQUITY BEH C18 column (100 mm × 2.1
mm i.d., 1.7 µm; Waters, Milford, USA). The
mass spectrometric data was collected using
a Thermo Q-Exactive Mass Spectrometer equipped
with an electrospray ionization (ESI) source oper-
ating in either positive or negative ion mode.
After UPLC-MS analyses, the raw data were
imported into the Lipid Search (Thermo, CA) for
peak detection, alignment, and identification. The
preprocessing results generated a data matrix that
consisted of the lipid class, retention time (RT),
mass-to-charge ratio (m/z) values, and peak inten-
sity. Features which the relative standard deviation
(RSD) of QC > 30% were discarded. Statistically
differential metabolites between responders’ and
non-responders’ serum were selected with VIP
value>1 and p value<0.05. A total of 31 differential
lipids including 24 peaks in ESI+ and 7 peaks in
ESI- were identified and mapped into biochemical
pathways through metabolic enrichment and path-
way analysis based on database search (KEGG,
https://www.kegg.jp/kegg/pathway.html).
Induction of colitis
The animal study was approved by the Animal
Ethics Committee of Sun Yat-sen University
(SYSU-IACUC-2021–000828). To establish an
acute colitis model, oral administration of 2.5%
dextran sodium sulfate (DSS) (Molecular weight
36,000–50000, Yeasen) in drinking water was con-
ducted for 7 days in C57BL/6 female mice. For the
chronic colitis model, mice were treated with 2.0%
DSS to induce chronic colitis for three consecutive
7-day cycles. Each DSS cycle is separated from
normal drinking water. For anti-TNF-α antibody
treatment, mice were injected with 10 mg/kg
Infliximab (170277-31-3) or isotype control IgG
(BE0091, Bio X Cell) for three times a week. For
dietary treatment of metabolites, 0.5 g/kg palmito-
leic acid (Aladdin) or butyrate (Aladdin) was orally
gavaged to mice every two days. For oral bacteria
transfer, mice were orally gavaged with 200 µl sus-
pension containing 2 × 10
8
bacteria for 3 times per
week. Animals were daily monitored for the
appearance of diarrhea, body weight loss, and sur-
vival. The signs of bloody diarrhea were measured
using BASO fecal OB-II (BASO diagnostic Inc.,
Zhuhai, China). Disease activity index (DAI) con-
sisted of the following parameters: body weight loss
(0, < 5% weight loss; 1, 5–10% weight loss; 2, 10–
15% weight loss; 3, 15–20% weight loss; and 4, >
20% weight loss), stool consistency (0, formed
20 Y. CHEN ET AL.
pellets; 2, pasty/semi-formed stool; and 4, liquid
stool) and fecal bleeding (0, no rectal bleeding; 2,
hemoccult-positive; and 4, visible gross bleeding)
as described previously
56
.
For TNBS-induced colitis, anesthetized BALB/c
mice (17–22 g) were sensitized with 2.5% TNBS
(Sigma-Aldrich) together with acetone and olive
oil
57
. After 1 week, the mice were given 100 μl of
2.5% TNBS in 50% ethanol via a flexible feeding
tube that maintained their heads in a vertical posi-
tion for 10 min after an overnight fast. Colon
inflammation was evaluated by the ratio of weight
to length (anus to cecum). The colons were opened
and examined to evaluate macroscopic lesions
according to the Wallace criteria
58
. Colon sections
were stained with H&E and scored according to the
Ameho criteria
59
.
Depletion of commensal bacteria and measurement
of antibiotics eect
Commensal bacteria depletion was achieved by
administering an antibiotic cocktail containing
metronidazole (1 g/L), ampicillin (1 g/L), neomy-
cin (1 g/L) and vancomycin (0.5 g/L) prepared in
autoclaved water (pH 7.2) and filter sterilized, as
we previously performed
60
. To confirm the effect of
antibiotics in mice, fecal samples were collected
and homogenized in PBS. After serial dilutions,
bacterial suspension was plated on brain-heart
infusion agar (BHI) and cultivated under aerobic
and anaerobic conditions.
Safety assessment
C57BL/6 mice were randomly divided into groups
and maintained in a pathogen-free facility under
a day-night cycle. Mice were orally treated with
POA at different doses of 0.1 g/kg, 0.5 g/kg or 2.5
g/kg, and mice in the control group were treated
with an equal volume of vehicle control by gavage.
After 14-day treatment, anesthetized mice were
sacrificed their colons, spleens and livers were col-
lected. Colon length and spleen weight were calcu-
lated while SOD levels and lipid profile in serum
including TG, CHO, HDL, LDL were analyzed.
Serums were also collected to test hepatic enzymes
including AST, ALP, and ALT, and kidney func-
tion UREA and CREA.
Fecal microbiome transplantation (FMT)
Mice were orally administrated with palmitoleic
acid (POA) or water for 1 week. For the fecal
transplants, fresh fecal pellets were collected from
donor groups in PBS with a final concentration of
50 mg feces/ml. Fecal homogenates were centri-
fuged at 1000 rpm to pellet large particles and the
supernatant used for FMT treatment. 200 µL of
fecal supernatant was orally administered into
microbiota-depleted recipient mice. To deplete
microbiota, mice were treated with a cocktail of
antibiotics for 1 week as described above.
Microbial depletion was confirmed by bacterial
colony assays. After two-week fecal transplanta-
tion, donor and recipient mice were treated with
2.5% DSS to induce colitis and injected with anti-
TNF-α mAb (Infliximab, IFX).
Culture of inamed colon explants
Patient colon tissues were weighted, washed three
times with cold PBS with vortex and cultured in 1
ml DMEM (per 100 mg tissue) containing 10% FBS
and 5 × penicillin/streptomycin for 12 hours.
Palmitoleic acid (POA) or vehicle control was
added to the culture medium of colon explants.
The supernatants were centrifuged to remove float-
ing tissue debris, then were analyzed for cytokine
concentrations by Cytometric Bead Array (CBA)
assay and normalized to explant weight.
Histopathological assessment and
immunohistochemistry imaging
Mouse colon tissues and patients’ colon tissues
were harvested. The colon tissues using the Swiss-
roll technique were fixed in 4% paraformaldehyde,
embedded in paraffin. Tissue sections of the whole
colon were stained with hematoxylin & eosin
(H&E) or Alcian Blue Periodic acid Schiff (AB-
PAS). The histopathology was scored by two inde-
pendent pathologists. Three independent para-
meters were measured: the extent of inflammation
(0, none; 1, slight; 2, moderate; 3, severe; 4, mas-
sive), the extent of crypt damage (0, none; 1, the
basal one-third portion damaged; 2, the basal two-
thirds portion damaged; 3, the entire crypt
damaged but the surface epithelium intact; 4, the
GUT MICROBES 21
entire crypt and epithelium lost) and percentage of
involvement (0, none; 1, 0–25% involved; 2, 25–
50% involved; 3, 50–75% involved; 4, 75–100%
involved).
For immunohistochemistry (IHC), 4% parafor-
maldehyde-fixed and paraffin-embedded tissue
sections were de-paraffinized using xylene and
hydrated in an alcohol gradient. The sections
were treated with 10 μM citrate buffer for antigen
retrieval and washed with 1 × PBS (pH 7.2). The
endogenous peroxidase activity was quenched with
methanol and 3% H
2
O
2
, and then the sections were
blocked with 4% goat serum for 30 min. The tissues
were stained with antiZO-1 antibody (AF4144,
Affinity), anti-Occludin antibody (DF7504,
Affinity), anti-CD8 antibody (AF5126, Affinity),
anti-Ly6G antibody (#87048, CST) or anti-CD11b
antibody (ab133347, Abcam), overnight at 4°C fol-
lowed by a biotinylated secondary IgG and then
incubated with streptavidin-peroxidase for 1 hour.
DAB was used for detection. The sections were
counterstained with Mayer’s hematoxylin. All his-
tological assessments were performed by two inde-
pendent blinded observers. The images were
collected using a Zeiss microscope (AxioScan.Z1).
Western blotting
Cells and tissues were lysed with Protein Extraction
Kit containing protease and phosphatase inhibitors
(CW0791). The protein samples with 1 × SDS load-
ing buffer were boiled at 100°C for 10 min. and run
on 10% SDS-page gels (PG112). The gels were
transferred onto a PVDF membrane (Millipore)
by wet transfer. The membranes were incubated
in a blocking solution with 4% nonfat milk in
TBS-T. The membranes were incubated with pri-
mary antibodies in 4% nonfat milk in TBS-T at 4°C
overnight and with the secondary antibodies in
TBST for 1 hour at room temperature. The blots
were visualized by ECL kit.
Cytometric Bead Array (CBA) assay
The levels of cytokines in mice serum or culture
supernatants, including IL-2, IL-4, IL-6, IFN-γ,
TNF, IL-17A, and IL-10, were examined using the
CBA assay (BD Biosciences, San Diego, CA) as we
previously performed
34
. The data were collected on
the Beckman Coulter Gallios (Beckman) using
flowjo software (BD Biosciences). The concentra-
tions of each cytokine were revealed by the fluor-
escence intensity. Cytokine concentrations were
calculated relative to the standard dilution curve.
RNA extraction and real-time qPCR
Total RNA was prepared from tissues using AG
RNAex Pro Reagent (AG21102). The concentra-
tion of RNA was determined by Nanodrop 2000
spectrometer. First-strand cDNA was synthesized
using the Evo M-MLV RT Premix for qPCR
(AG11706) following the manufacturer’s instruc-
tions; qPCR was performed using the SYBR Green
Premix Pro Taq HS qPCR Kit (AG11701) to deter-
mine the expression of target cDNA and bacterial
DNA. GAPDH and 16S rDNA was used as an
endogenous control to normalize gene expression,
respectively. Relative mRNA expression levels were
presented as means ± SEM. Statistical differences
were analyzed by the student’s t-test. For primer
sequences, see Supplementary Table 2.
Liquid culture of Akkermansia muciniphila and
other bacteria
Cultures of Akkermansia muciniphila (ATCC
BAA-835) were grown in brain heart infusion
(BHI) medium with 0.25% mucin for at least 72
hours at 37°C in anaerobic conditions (atmosphere
5% H
2
, 20% CO
2
, 75% N
2
). Cultures of
Fusobacterium nucleatum (ATCC 23,726) were
grown in BHI medium for at least 48 hours at
37°C in anaerobic conditions. The identity of each
strain was confirmed by sequencing the 16S ribo-
somal RNA using primers 27F (5’-
AGAGTTTGATCMTGGCTCAG) and 1492 R (5’-
GGTTACCTTGTTACGACTT). Strains were pre-
served in cryotubes at −80°C in growth medium
containing 20% glycerol. The liquid was shaken to
ensure homogeneity and bacterial growth was
monitored in Spectrophotometer (absorbance at
600 nm).
RNA-seq transcriptomics
Akkermansia muciniphila (ATCC BAA-835)
were grown in brain heart infusion (BHI)
22 Y. CHEN ET AL.
medium with 0.25% mucin with or without pal-
mitoleic acid (POA) at 37°C in anaerobic con-
ditions (atmosphere 5% H
2
, 20% CO
2
, 75% N
2
).
The cells were pelleted by centrifugation at
10,000 × g for 10 min at 4°C. Total RNA was
extracted from the tissue using TRIzol®
Reagent according the manufacturer’s instruc-
tions (Invitrogen) and genomic DNA was
removed using DNase I (Takara). Three inde-
pendently RNA samples from each group were
used for RNA-Seq. RNA-seq transcriptome
library was prepared following TruSeqTM RNA
sample preparation Kit from Illumina (San
Diego, CA) using 2 μg of total RNA. Illumina
sequencing was performed using the Illumina
HiSeq×TEN. Data analyses were performed
using DESeq2. Genes exhibiting 2-fold changes
in expression were statistically significant as
determined by Student’s t-test (p < 0.05). DEGs
GO enrichment analysis is used to identify sta-
tistically significantly enriched GO term using
Fisher’s exact test. The purpose of performing
FDR correction is to reduce the Type-1 error by
Bonferroni, Holm, BY, BH (multiple hypothesis
test method). After multiple testing correction,
GO terms with adjusted p-value≤0.05 are signif-
icantly enriched in DEGs.
Statistical analysis
Statistical analysis was performed using
GraphPad Prism (version 8.0). The statistical sig-
nificance of differences between two groups was
analyzed with the unpaired Student’s t-test or
Mann-Whitney test. For multiple group compar-
isons and repeated measures, analysis of variance
(ANOVA) or Kruskal-Wallis test. All P values
were two-sided. P values less than 0.05 were
considered statistically significant. *P < 0.05; **
P < 0.01; *** P < 0.001; NS, no statistical signifi-
cance. In figure legends, n represents the number
of samples.
Acknowledgments
This work was partially supported by grants from the Natural
Science Foundation of China (82072250 to G.C.Z.).
Author Contributions
Y.W.C., Q.D.M., Z.X.C., T.L. Y.J.C. L.J.Y., Y.S, L.X., and L.N.
L. performed the experiments. Y.W.C., Q.D.M., Z.X.C., T.L.,
Y.J.C., S.M.T., Z.Y.W., T.M.C., B.Y.O., L.M.C., Z.Y.Z., Y.Y., L.
J.Y., Y.S, S.E.Z, L.X., L.N.L., J.L., H.B.S., S.H.Z., L.C.Z., and G.
C.Z. analyzed the data. Y.W.C., J.H., Y.W. and M.D.
Z. contributed materials/analysis tools. Y.W.C., Q.D.M., L.J.
Y., Y.S, L.X., L.N.L., G.C.Z., S.H.Z., and L.C.Z. drafted, dis-
cussed, and revised the manuscript. S.H.Z., L.C.Z., and G.C.
Z. conceived this study.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
The work was supported by the Natural Science Foundation
of China [82072250].
ORCID
Yiwei Chen http://orcid.org/0000-0003-0342-1495
Gucheng Zeng http://orcid.org/0000-0002-2665-2854
Data availability statement
The authors confirm that the data supporting the findings of
this study are available within the article and its supplemen-
tary materials. The datasets generated for this study can be
found in the http://www.ncbi.nlm.nih.gov/bioproject/916399
(BioProject ID: PRJNA916399) and http://www.ncbi.nlm.nih.
gov/bioproject/917464 (BioProject ID: PRJNA917464)
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26 Y. CHEN ET AL.
... Palmitoleic acid (POA) is an omega-7 monounsaturated fatty acid with a double bond and it is one of the most abundant monounsaturated fatty acids in plant and marine sources (Ozogul, Duysak, Ozogul, Ö zkütük, & Türeli, 2008;Yang & Kallio, 2001). Research reports have found that palmitoleic acid has anti-inflammatory, glucose homeostasis regulating, insulin resistance ameliorating, lipid improving, and antibacterial effects (Bolsoni-Lopes et al., 2013;Chen et al., 2023;Frigolet & Gutiérrez-Aguilar, 2017;Tsai et al., 2021). Palmitoleic acid is usually low in oilseed crops, however, it is high in sea buckthorn. ...
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Sea buckthorn is rich in active ingredients, widespread, and has both medicinal and nutritional value. The present comparative study of wild and cultivated species remains insufficient, which is not conducive to their quality control. Therefore, this study aimed to compare the differences of 21 sea buckthorn samples in total phenolic content (TPC), total flavonoid content (TFC), phenolic components content, secondary metabolites, and antioxidant capacity and the fatty acid, to investigate the quality differences of different varieties. The TPC, TFC and antioxidant activity of wild varieties were higher than those of the cultivated. Multivariate statistical analysis revealed large differences in phenolic content, with higher levels of gallic acid and isorhamnetin-3-O-neohesperidin in the wild, whereas the cultivated were characterized by narcissin and kaempferol. These findings provided the scientific basis for the improvement of quality evaluation standards for different varieties and offered new insights for the further development of sea buckthorn resources.
... Importantly, discontinuing anti-TNF therapy often leads to a paradoxical worsening of TB, particularly in patients with disseminated TB (116). However, oral administration of palmitoleic acid has been shown to remodel mucosal barriers and reduce inflammation by promoting the proliferation of anti-inflammatory bacteria such as A. muciniphila (117). The inverse relationship between palmitoleic acid and TNF suggests a molecular mechanism that differs from that of direct TNF blockade, implying that symbiotic bacterial products may offer alternative and gentler therapeutics to deliver anti-TB benefits to patients. ...
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... Queensland fruit oil (17%), Shanxi sea buckthorn fruit oil (13%), hairy dogwood seed oil (3.64%), and any other oils [6]. At the same time, several reports related to the advantages of POA have attracted attention [7,8]. Their results showed that POA could effectively antagonize some chronic sub-health problems, such as metabolic syndrome, glucose and lipid metabolism disorders, and inflammation [9][10][11]. ...
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... Three metabolites were identi ed in our study with the most signi cant causal effects on HD, Cortolone glucuronide (1) levels(p = 0.007), 3-methyl-2-oxobutyrate levels(p = 0.010), and Glycocholate levels(p = 0.017), may potentially trigger HEM through mechanisms such as in ammation. Cortolone glucuronide (1) Palmitoleic acid can inhibit the release of in ammatory factors such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) 29 . It is speculated that, during HD development, Phosphatidylcholine (O-16:1_16:0) might participate in the sequestration of free palmitoleic acid, thereby reducing its antiin ammatory effects. ...
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Both host genetics and the gut microbiome have important effects on human health, yet how host genetics regulates gut bacteria and further determines disease susceptibility remains unclear. Here, we find that the gut microbiome pattern of participants with active tuberculosis is characterized by a reduction of core species found across healthy individuals, particularly Akkermansia muciniphila. Oral treatment of A. muciniphila or A. muciniphila-mediated palmitoleic acid strongly inhibits tuberculosis infection through epigenetic inhibition of tumour necrosis factor in mice infected with Mycobacterium tuberculosis. We use three independent cohorts comprising 6,512 individuals and identify that the single-nucleotide polymorphism rs2257167 ‘G’ allele of type I interferon receptor 1 (encoded by IFNAR1 in humans) contributes to stronger type I interferon signalling, impaired colonization and abundance of A. muciniphila, reduced palmitoleic acid production, higher levels of tumour necrosis factor, and more severe tuberculosis disease in humans and transgenic mice. Thus, host genetics are critical in modulating the structure and functions of gut microbiome and gut microbial metabolites, which further determine disease susceptibility.