A prospective pilot study of a gluten‐free diet for primary sclerosing

Received: 13 March 2022 | First decision: 2 April 2022 | Accepted: 8 October 2022
DOI: 10.1111/apt.17256
A prospective pilot study of a gluten-free diet for primary

sclerosing cholangitis and associated colitis
Timur Liwinski1,2 | Sina Hübener1 | Lara Henze1 | Peter Hübener1 |

Melina Heinemann1 | Marcus Tetzlaff1 | Marie I. Hiller1 | Bettina Jagemann1 |
Rambabu Surabattula3 | Diana Leeming4 | Morten Karsdal4 | Erika Monguzzi3 |
Guido Schachschal5 | Thomas Rösch5 | Corinna Bang6 | Andre Franke6 |
Ansgar W. Lohse1,7 | Detlef Schuppan3,8 | Christoph Schramm1,7,9
1
Department of Medicine, University
Medical Center Hamburg-Eppendorf, Summary
Hamburg, Germany Background: Primary sclerosing cholangitis (PSC) is a progressive bile duct disease
2
Center for Affective, Stress and Sleep
associated with inflammatory bowel disease (PSC-IBD).
Disorders (ZASS), University Psychiatric
Clinics (UPK) Basel, University of Basel, Aim: To investigate whether patients with PSC-IBD benefit from a gluten-free and
Basel, Switzerland
amylase trypsin inhibitor (ATI)-free diet (GFD).
3
Institute of Translational Immunology
and Research Center for Immunotherapy,
Methods: We performed a prospective clinical pilot study administering an eight-
University Medical Center, Johannes week GFD. The primary outcomes were colonic inflammation assessed by proctosig-
Gutenberg University, Mainz, Germany
4
moidoscopy, and liver stiffness (surrogate for fibrosis, inflammation and cholestasis)
Research and Development, Nordic
Bioscience, Biomarkers and Research A/S, measured by transient elastography before and after GFD. Amongst the second-
Herlev, Denmark ary (exploratory) outcomes were colonic mucosal and serum cytokine/chemokine
5
Department of Interdisciplinary Endoscopy,
changes, the intestinal microbiome and transcriptome dynamics, and shifts in serum
University Medical Center Hamburg-
Eppendorf, Hamburg, Germany markers of hepatic fibrogenesis.
6
Institute for Clinical Molecular Biology, Results: Fifteen patients with PSC-IBD completed the study. The study did not meet
Christian Albrechts University of Kiel, Kiel,
Germany its primary outcome: the endoscopic score and liver stiffness remained unchanged.
7
Hamburg Center for Translational However, the expression of pro-inflammatory mucosal cytokines and chemokines
Immunology, Hamburg, Germany
such as IL6, IL8, CCL2, and TNFα was significantly down-regulated. Two critical mark-
8
Division of Gastroenterology, Beth Israel
Deaconess Medical Center, Harvard Medical
ers of liver fibrosis and matrix remodelling, thrombospondin-2 and -4, decreased sig-
School, Boston, Massachusetts, USA nificantly. The microbiota composition changed slightly, including a decrease in the
9
Martin Zeitz Center for Rare Diseases, pathogen Romboutsia ilealis. The intestinal transcriptome indicated a gut barrier im-
University Medical Center Hamburg-
Eppendorf, Hamburg, Germany provement. Pruritus, fatigue, overall well-being, faecal calprotectin levels, and serum
alkaline phosphatase did not change significantly.
Correspondence
Christoph Schramm, Martinistrasse 52, Conclusions: This study did not demonstrate a clinical improvement with short-term
20246 Hamburg, Germany. GFD in patients with PSC-IBD. However, a gluten/ATI-free diet may improve biomark-
Email: c.schramm@uke.de
ers of intestinal inflammation and barrier function in these patients with associated
Timur Liwinski and Sina Hübener contributed equally.Detlef Schuppan and Christoph Schramm contributed equally.
The Handling Editor for this article was Professor Gideon Hirschfield, and it was accepted for publication after full peer-review.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in
any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2022 The Authors. Alimentary Pharmacology & Therapeutics published by John Wiley & Sons Ltd.
224 |
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LIWINSKI et al. 225
Funding information
Deutsche Forschungsgemeinschaft, Grant/ changes in the enteric microbiota. Further investigation of the therapeutic potential
Award Number: CRU306 of the GFD in PSC-IBD is warranted.
1 | I NTRO D U C TI O N steatohepatitis, allergy and Alzheimer's disease,16 but also in first

human pilot studies. 25 Nutritional ATI can also directly compro-
Primary sclerosing cholangitis (PSC) is a chronic and progressive mise the intestinal barrier and alter the intestinal microbiome to-
bile duct disease featuring hepatobiliary inflammation, bile duct fi- wards dysbiosis.17,26
1
brosis, and cholestasis, often resulting in end-stage liver disease. An association between PSC-IBD and celiac disease was first
PSC is strongly associated with a distinct type of inflammatory described by Hay presenting three patients with typical biliary
bowel disease (PSC-IBD; resembling ulcerative colitis), ranging in co- lesions on endoscopic retrograde cholangiography, colitis, severe
2
prevalence from about 20% in Japan to over 70% in Europe. The steatorrhea and total villous atrophy. 27 An increased risk for PSC
inflammatory and fibrotic nature of the disease represents a prema- in celiac disease patients has since been confirmed by population
lignant condition with increased mortality from malignancy, mainly studies28,29 and large-s cale genetic studies have found a striking
cholangiocarcinoma and colorectal cancer.3,4 overlap in genetic risk loci within the human leukocyte antigen
The strong association of PSC with IBD suggests a pathoge- (HLA) complex. 30 However, the effects of a wheat (gluten)-f ree
5
netic link to the gut microenvironment. The human intestine is diet (GFD) on outcomes of PSC-IBD are unknown. We hypothe-
host to a myriad of microorganisms of predominantly bacterial sised that dietary exclusion of pro-inflammatory wheat proteins,
origin. These microbial commensals play a crucial role in host especially ATI, could directly alter the dysbiotic gut microbiota
health and disease due to their interaction with the human im- and alleviate intestinal inflammation in PSC-IBD patients. Because
mune system. 6 Recently, a series of studies have provided strong of the close link between intestinal and liver pathology in PSC, 31
evidence for altered gut microbiota in patients with PSC, yielding we further assumed that restoration of enteric homeostasis might
disease-associated microbes and microbial metabolites that are improve hepatic inflammation and biliary fibrosis markers. We,
7
reproducible across studies. The human diet acts as a substrate therefore, conducted a prospective clinical pilot trial assessing the
for defined microbial species and provides direct signals to the effect of a largely gluten-f ree and thus ATI-f ree diet on patient
digestive and immune systems, thus profoundly impacting the well-b eing, multiple markers of disease activity, gut barrier func-
gut microbial composition and the host's metabolism and immune tion, and the gut microbiota in patients with PSC-IBD with low
system. 8 Therefore, a close and causal relationship between a clinical activity and without celiac disease.
dysregulated mucosal immune response against luminal nutrients
and the intestinal microbiome on the one hand and intestinal and
bile duct inflammation, on the other hand, appears conceivable in 2 | M E TH O DS
9,10
patients with PSC-I BD. In acute and chronic inflammatory in-
testinal diseases, dietary interventions represent an increasingly 2.1 | Trial design
studied therapeutic modality.11,12
Wheat has become a major staple food in most parts of the The present study is an investigator-initiated prospective clinical
world. Modern hexaploid wheat harbours more than 100,000 cod- pilot trial with one interventional arm. We compared established
ing genes, including gluten and a large spectrum of non-gluten pro- clinical markers of disease severity in PSC-I BD plus a compre-
teins, some with the potential to cause adverse reactions, such as hensive set of biological parameters with potential relevance.
allergies. Three types of inflammatory wheat sensitivities can be Our main aim was to assess the efficacy and tolerability of a GFD
distinguished: first, celiac disease, a T helper 1 mediated immune according to a standard operating procedure with >95% dietary
response to specific gluten epitopes in genetically predisposed sub- gluten reduction by entirely excluding wheat, rye and barley from
jects13,14; second, an innate immune response mainly to non-gluten the diet. This approach was chosen, since the innate immunity-
proteins, prominently amylase trypsin inhibitors (ATI)15–17; and third, stimulating activity of ATI proteins is dose-d ependent and a
immediate and delayed-t ype 2 immune responses to a variety of largely (>95%) “gluten”-r educed diet, which is relatively easy to
wheat allergens.18–20 ATI-sensitivity and wheat allergies can explain maintain compared to a strict GFD, would cause no relevant im-
the spectrum of non-celiac wheat sensitivity (NCWS; often incor- mune activation in our (non-celiac) PSC patients. 32 However,
rectly termed non-celiac gluten sensitivity). 21–23 per convention, we are using the term “gluten-f ree diet” (GFD)
Notably, the ATI proteins of wheat that activate intestinal throughout the present work.
myeloid cells via toll-like receptor 4 (TLR4)15,24 have been shown We hypothesised that this GFD over 8 weeks would impact clini-
to promote not only intestinal but also extra-intestinal inflam- cal results and/or biological variables implicated in the pathophysiol-
mation in experimental mouse models of colitis, nonalcoholic ogy of PSC. The study protocol is outlined in Table 1.
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226 LIWINSKI et al.
TA B L E 1 Study protocol
Screening Normal diet Intervention GFD Follow-up
V0 V1 V2 V3 V4 V5 V6 V7
Day (D)/Week (W) of the D1 W8 ± 2d W10 ± 2d W12 ± 2d W14 ± 2d W16 ± 2d W28 ± 7d

study
Confirmation of PSC x
diagnosis
Demographics x
Inclusion and exclusion x
criteria
Written informed consent x
Anamnesis x
Transient elastography x
(FibroScan)
Counselling by a clinical x
dietician
Safety
Physical examination x x x
Vital signs (blood x x x
pressure, heart rate,
body temperature)
Evaluation for adverse x x
events
Laboratory analyses
Blood parameters x x x
Intestinal microbiome x x
(shotgun)
Intestinal transcriptome x x
Faecal calprotectin x x
Gluten peptides (urine) x x x
Clinical impact
Proctosigmoidoscopy with x x
biopsies
Questionnaire diet x x
Questionnaire health x x
status
2.2 | Participants Malassimilation or diarrhoea/steatorrhea was absent in all patients

(Table 2).
Seventeen patients with PSC-IBD were initially enrolled in the study. The ethics committee responsible approved the present study
Fifteen PSC-IBD patients completed the study and were, thus, in- (PV5528). All participants gave a written informed consent statement,
cluded in the analysis. The diagnosis was established based on crite- and the trial was registered at Clinicaltrials.gov: NCT04006886.
ria according to the guidelines of the European Association for the
Study of the Liver.33 Exclusion criteria for this study were acute dis-
ease deterioration, an ongoing GFD, or any other special diets before 2.3 | Dietary intervention
study inclusion, antibiotic treatment within 6 weeks before inclusion
and immunosuppression with >10 mg/d prednisolone or >1.5 mg/kg A >95% gluten-reduced normocaloric balanced diet was advised
body weight azathioprine per day. Concurrent celiac disease was to all participants enrolled in the study—an experienced clinical di-
an exclusion criterion. We ruled out celiac disease using the tissue etician trained study participants on implementing a GFD without
transglutaminase IgA (tTg-IgA) test in the absence of IgA deficiency changing their general nutrition behaviour. Information material
according to national and international guidelines.34 tTg-IgA anti- on sources of gluten-free foods was handed out. The participants
bodies were negative in all instances (with normal total IgA levels). recorded their dietary behaviour throughout the trial using a
|
LIWINSKI et al. 227
TA B L E 2 Clinical patient characteristics buffer to avoid stool contamination for all downstream molecular
analyses. Follow-up visit V7 was carried out 12 weeks after ces-
Age, years (mean [SD]) 42.13 (13.17)
sation of the dietary intervention, with patients resuming diet
Sex, female (%) 8 (53.3)
ad libitum. To verify adherence to the GFD, a point-of-c are test
BMI, kg/m2 (mean [SD]) 24.76 (3.08)
for gluten immunogenic peptides was performed in morning spot
PSC disease duration, months 123.00 [70.00, 264.00] urine samples using the ‘GlutenDetect’ test system (Glutenostics)
(median [IQR])
at visits V2, V4 and V6.
CED, disease duration (median [IQR]) 123.00 [70.00, 264.00]
GOT, U/L (median [IQR]) 30.00 [19.50, 44.25]
GPT, U/L (median [IQR]) 40.50 [20.25, 63.75] 2.5 | Primary outcomes
GGT, U/L (median [IQR]) 67.50 [28.75, 112.50]
AP, U/L (median [IQR]) 121.00 [109.00, 193.00] The primary outcomes were colonic inflammation assessed by
Albumin, g/L (median [IQR]) 38.20 [35.20, 39.80] proctosigmoidoscopy as well as liver stiffness (LSM; proxy of fibro-
Bilirubin, mg/dl (median [IQR]) 0.70 [0.53, 1.08] sis, inflammation, cholestasis) assessed by transient elastography.
PSC Mayo Risk Score (median [range]) 0 [0, 4] At each endoscopic examination, the following variables were re-
Tissue Transglutaminase-IgA 15 (100) corded: erythema, friability, granularity, erosion, ulceration, mucosal
antibodies <20 U/ml (n (%)) detachment and mucosal abrasion. The Mayo endoscopic subscore
Total IgA > 0.80 g/L (n (%)) 15 (100) was reported using a standardised form.36 LSM was performed using
UDCA (n (%)) 14 (93.3) FibroScan (Echosens) as described.37
Azathioprine (n (%)) 5 (33.3)

Vitamin D supplementation (n (%)) 11 (73.3)
2.6 | Secondary outcomes
Mesalazine (n (%)) 6 (40.0)
Transient elastography, kPa (median 5.70 [4.55, 6.95]
Secondary outcomes were the adjunct clinical parameters faecal
[IQR])
calprotectin (gut inflammation marker), serum alkaline phosphatase
Faecal calprotectin, μg/g stool (median 24.60 [10.32, 72.95]
[IQR]) (cholestasis marker), pruritus frequency and severity, fatigue and
overall well-being.
Additional secondary outcomes were translational parame-
standardised questionnaire. The Robert Koch Institute version of ters, including colonic mucosal and serum cytokine and chemo-
the food frequency questionnaire (FFQ) records the portion size kine measurements, intestinal microbiota dynamics, intestinal
and frequency of 53 items consumed within the previous 4 weeks; mucosal transcriptome dynamics and serum markers of hepatic
it was validated for German populations within the German Health fibrogenesis.
Examination Survey for Adults 2008–2011.35 We extracted the di- Faecal calprotectin was analysed, serving as a marker of gross
etary variables total energy intake (kcal/d), consumption of fat (g/d), intestinal inflammation. The local hospital's accredited clinical lab-
carbohydrates (g/d), protein (g/d), sodium (mg/d), potassium (mg/d), oratory determined alkaline phosphatase and other hepatic serum
phosphate (mg/d) and dietary fibre (g/d). parameters. Serum markers of fibrosis and inflammation were mea-
sured as detailed below.
The patients completed a well-validated generic quality of
2.4 | Assessment visits life instrument; the SF-12. 38 A validated add-on questionnaire
was used covering frequent complaints associated with PSC or
The patient follow-up encompassed up to eight visits, including PSC-IBD, including frequency and intensity of pruritus, stool
several examinations and sample collections (Table 1). The assess- frequency, hematochezia, abdominal pain, bloating, fatigue and
ment visits (V) included a screening visit V0 at which the patients sleeping disorder.
were enrolled and informed about the procedures. Before starting Technical details on cytokine and chemokine measurements,
the GFD, patients underwent two further visits (V1, at day 1; V2 serum markers of inflammation and liver fibrosis, 16S and shotgun
after 8 weeks) to undergo several examinations, including the first metagenomic microbiota analysis, and intestinal whole transcrip-
proctosigmoidoscopy with faecal and mucosal sample acquisition tome analysis are provided in the Appendix S1.
at V2. Immediately after V2, which included counselling by a clinical
dietician, the GFD was started for 8 weeks, with four assessment
visits (V3–V6). At visit V6, all analyses performed at V2, including 2.7 | Statistical analysis
proctosigmoidoscopy with biopsy, were repeated. A double biopsy
was performed at each time point (V2 and V6) in both the rectum We performed all statistical analyses using R (R Foundation for
and sigmoid colon. Mucosal samples were carefully washed in a Statistical Computing; v3.6.3). Multiple testing was adjusted for the
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228 LIWINSKI et al.
false discovery rate (FDR), where necessary. Generally, a p-value or 3.4 | Effects of the GFD on circulating markers of
q-value <0.05 was accepted as statistically significant. Details are liver fibrosis and immune cell activation
provided in the Appendix S1.
We analysed several serum markers of liver fibrosis and fibrogenesis
using a panel including established and novel direct fibrosis biomark-
3 | R E S U LT S ers. We observed a significant decrease in the matricellular fibrosis
marker thrombospondin-2 (TSP-2) and thrombospondin-4 (TSP-4) be-
3.1 | Patient characteristics tween V2 and V6 (p < 0.0001 and p = 0.0054; Figure 3A,B). CD163, a
marker of macrophage activation did not change (p > 0.1; Figure 3C).
Detailed patient characteristics are given in Table 2. Eight patients Two crucial collagens-derived serum fibrogenesis markers that reflect
were female (53.3%). The mean age was 42.1 ± 13.2 years. The me- interstitial type III collagen and basement membrane type IV collagen
dian time from the initial diagnosis of PSC was 123 months (IQR, formation (Pro-C3 and Pro-C4, respectively) showed no difference be-
70, 264). All patients had PSC-IBD. At the time of study inclusion, tween V2 and V6 (all p > 0.1; Figure 3D,F).
most patients were classified as Mayo PSC Risk Score category 0
(n = 11, 78.6%). The median initial LSM value was 5.7 kPa (IQR,
4.6, 7.0). 3.5 | Microbiota dynamics under GFD
We then explored microbiota dynamics under the GFD by 16S se-

3.2 | Primary and other clinical outcomes quencing from faecal and sigmoid intestinal mucosal samples. For
the stool dataset, samples from the assessment visits V0, V2, V6
The gluten peptide immunoassay showed a decreased urine excre- and V7 were available. No significant differences between fae-
tion of gluten peptides during the observation period proving adher- cal samples of the four time points were observed as regards in
ence to the GFD (V2 8.26 [0, 14.31], V4 3.56 [0, 8.06], V6 0 [0, 7.11]; between-sample diversity (p = 0.985; Figure S2) and within-sample
repeated measures ANOVA, p = 0.0249). The recording of dietary biodiversity (p = 0.539; Figure S2). Analysing single genera, no dif-
patterns using the FFQ showed no changes during the 8-weeks of ferentially abundant genera could be detected after multiple testing
the intervention period (Hotelling's T-squared test, p = 0.998), and corrections (Figure S2). Similarly, in the matched mucosal samples
the patients' weight remained stable (p = 0.621). We thus assume taken at V2 (normal diet) and V6 (GFD) sample diversity and within-
that any changes in any readouts observed are not explained by al- sample diversity did not differ (p = 0.985 and p = 0.151; Figure S2,
tering the diet's sustenance/nutritional value. respectively). On the level of individual genera, a genus from
The primary clinical endpoints of our study are shown in the Clostridiaceae family labelled 02d06 decreased under a GFD
Figure 1, indicating no statistically significant response regard- (Figure S2; FC = −6.30; FDR = 0.004).
ing the endoscopic histological score (p = 0.398; Figure 1A), To deepen our understanding of microbiota dynamics under
LSM (p = 0.934; Figure 1B), faecal calprotectin levels (p = 0.917; the GFD, we applied metagenomic shotgun sequencing of luminal
Figure 1C) and serum levels of alkaline phosphatase (p = 0.733; samples collected at the time points V2 and V6. Exploring bac-
Figure 1D). Likewise, we did not observe a response to the GFD terial alterations, we could not prove significant differences for
with regard to pruritus frequency (p = 0.981; Figure 1E), pruritus global between-sample (p = 0.951; Figure 4A) or within-sample
intensity (p = 0.811; Figure 1F), frequency of experiencing fatigue diversity (p = 0.561; Figure 4B). Nevertheless, we detected 10
(p = 0.117; Figure 1G), and self-reported overall well-b eing (SF-12; differentially abundant bacterial species (Figure 4C). Amongst
p = 0.344; Figure 1H). these, the GFD was correlated with a decrease in Romboutsia ilea-
lis (FC = – 9.02, FDR < 0.001), Lactobacillus F plantarum (FC = –7.94,
FDR < 0.001), Enterobacter himalayensis (FC = –7.48, FDR < 0.001),
3.3 | Effects of GFD on mucosal markers of and Ruminococcus C sp000437175 (FC = –8.25, FDR < 0.001). In
inflammation reverse, GFD increased the abundance of Haemophilus D parain-
fluenzae N (FC = 6.35, FDR = 0.009), Haemophilus D parainfluen-
The GFD affected multiple mucosal cytokine and chemokine tran- zae (FC = 6.65, FDR = 0.009) and Citrobacter_A amalonaticus_D
scripts in biopsies from the sigmoid colon (qPCR; Figure 2A–F ). (FC = 6.92, FDR = 0.0150).
Specifically, we observed a decrease in the mucosal expression of We additionally explored the dynamics of the intestinal virome.
il8 (p < 0.0001), il6 (p < 0.0001), ccl2 (p = 0.0423), il10 (p < 0.0001;) Here, we observed no statistically significant differences regarding
and tnfa (p < 0.0001), whilst there was no significant change in between-sample diversity (p = 0.469; Figure 4D), within-sample di-
the mucosal expression of il17a (p = 0.0979), il17f (p = 0.215), and versity (p = 0.149; Figure 4E), or single viruses (Figure 4F).
ifng (p = 0.0539). These expression patterns were not reflected by We next studied the functional metabolic capacity of our metag-
systemic changes in serum cytokine/chemokine levels (all p > 0.1; enomic samples. We could not detect an impact of the GFD on
Figure S1). the entire pathway abundance landscape (p = 0.666; Figure 4G),
|
LIWINSKI et al. 229
F I G U R E 1 Primary outcomes.
Clinical parameters and patient-
reported outcomes reflecting PSC-IBD's
liver disease stage and activity. (A)
Histological colitis score; (B) liver stiffness
measurement; (C) Faecal calprotectin
(gross intestinal inflammation); (D) alkaline
phosphatase; (E) pruritus frequency; (F)
pruritus intensity; (G) fatigue frequency;
and (H) overall well-being. All P-values
were calculated using paired Wilcoxon
test. V2, time of the second visit, i.e.,
8 weeks after the screening visit and
immediately before starting the gluten-
free diet; V6, sixth visit, immediately after
completing the 8-week gluten-free diet
course (16 weeks after the screening visit).
and there was no significant change in bacterial genetic richness the GFD several pathways related to the regulation of intercellular
(p = 0.4253; Figure 4H). After correcting the FDR, no significant dif- junctions were significantly increased, including “adherens junction”
ference in a single functional pathway could be detected on the GFD (p = 0.0471), “cell-substrate junction” (p = 0.0262), “cell-substrate
(all FDR > 0.05). adherens junction” (p = 0.0233) and “focal junction” (p = 0.0217;
Figure 5B), suggesting an increase in gut barrier integrity on the GFD.
3.6 | Intestinal transcriptome dynamics under the

gluten-free diet 4 | D I S CU S S I O N
The intestinal cellular response to the GFD on a bulk transcriptome- We investigated the impact of an eight-week GFD on biomarkers of
wide level was assessed. We compared the time points V2 and V6 hepatic and intestinal disease activity, including mucosal transcrip-
in samples from the sigmoid mucosa. In total, the gene expression tome and intestinal faecal and mucosal microbiome composition in
of 157 genes was altered on the GFD (Figure 5A). Gene set enrich- patients with PSC and associated IBD. To our knowledge, this is the
ment analysis unveiled eight altered pathways. Interestingly, on first study assessing the effects of GFD in patients with PSC-IBD. We
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230 LIWINSKI et al.
F I G U R E 2 Mucosal cytokine and chemokine transcript levels under the GFD using qPCR. Both rectal and sigmoid biopsy results are
displayed. Both anatomical sites were analysed together using linear mixed-effects models. V2, time of the second visit, i.e., 8 weeks after
the screening visit and immediately before starting the gluten-free diet; V6, sixth visit, immediately after completing the 8-week gluten-free
diet course (16 weeks after the screening visit).
identified moderate changes in the patient's microbiome configura- chemokines, including IL6, IL8, CCL2 and TNF-alpha. Moreover, the
tion. Moreover, we observed significant potentially favourable effects colonic mucosal transcriptome showed an increase in the expres-
on intestinal mucosal inflammation, barrier function biomarkers, and sion of tight junction proteins, which are an essential component of
serum markers of fibrogenesis. However, the lack of a clinical response the gut barrier.39 We excluded that concurrent celiac disease might
might show that GFD alone is not a sufficient treatment for PSC-IBD. explain the improvement observed. There are shared genetic risk
Eight weeks of gluten and ATI restriction decreased the co- factors between PSC and celiac disease.30 This link is connected
lonic mucosal expression of several proinflammatory cytokines and to the 8.1 ancestral haplotype's (A1, B8, DR3) association with
|
LIWINSKI et al. 231
F I G U R E 3 Changes of circulating
fibrosis markers and macrophage
activation marker CD163. (A)
Thrombospondin-2 (TSP-2); (B)
thrombospondin-4 (TSP-4); (C) CD163; (D)
procollagen type 3 N-terminal propeptide
(pro-C3); and (E) procollagen type 4 N-
terminal propeptide (pro-C4). All P-values
were calculated using paired Wilcoxon
test. V2, time of the second visit, i.e.
8 weeks after the screening visit and
immediately before starting the gluten-
free diet; V6, sixth visit, immediately after
completing the 8-week gluten-free diet
course (16 weeks after the screening visit).
multiple immunopathological diseases,40 including PSC and celiac liver, we could not show that gluten-and ATI-free diet's improvement
41,42
disease. This common genetic background might point to shared of inflammatory and permeability biomarkers translates to clinical
immunological disease mechanisms. However, further research is improvement. This finding may relate to our patient cohort's overall
required to understand how genetic variation might predispose in- low disease activity, small sample size, and relatively short interven-
dividuals with PSC-IBD to immune responses to gluten and other tion period. These findings might also highlight that GFD alone is not
wheat ingredients. a suitable treatment for PSC-IBD and that wheat components are
The immune-attenuating and barrier-strengthening effects ob- not amongst the major drivers of PSC. Nevertheless, given the ob-
served in response to the omission of wheat-containing foods are in served downregulation of inflammation-related biomarkers relevant
line with previous literature: De Palma et al. reported that in healthy for PSC and colitis after the switch to the GFD, we hypothesise an
individuals, TNF-alpha, interferon-gamma, IL-8 and IL-10 production overall beneficial effect, in line with ample experimental evidence
by peripheral blood mononuclear cells following stimulation with fae- that highlights the importance of such enteric immunity and perme-
cal samples was reduced after a GFD.43 Increased intestinal permea- ability dynamics in IBD.45 Notably, we also observed a reduction in
bility after gliadin exposure occurs in patients with celiac disease and two sensitive serum markers of portal hepatic fibrogenesis, TSP-2
44
healthy individuals. Previously, we demonstrated that non-gluten and TSP-4,46,47 under the GFD, whilst the general liver fibrosis mark-
proteins of wheat, in particular ATI, more generally promote intes- ers Pro-C3 and Pro-C4,48,49 and the macrophage marker CD163 did
tinal inflammation by activating the TLR4-MD2-CD14 complex on not change significantly. This highlights a potential favourable im-
myeloid immune cells in the upper and lower gastrointestinal tract. pact of gluten/ATI restriction on fibrogenesis through attenuation
Thus, ATI ingestion upregulates intestinal and circulating proinflam- of inflammatory signalling via the gut-liver axis. However, how these
matory cytokines and chemokines such as IL1β, IL6, IL8 and CCL2 improved fibrosis surrogate markers impact biliary liver fibrosis and
in mice,15,16 and IL8 and IFNγ in intestinal biopsies of celiac patients the long-term disease course needs further exploration. Validation
stimulated with gliadin.15 The ATI species CM3 and 0.19 are the most studies are required to define clinically meaningful changes and cut-
prevalent activators of TLR4 in modern wheat; they retain bioactiv- off values, as well as the long-term prognostic value of these novel
ity regardless of food processing, and baking and are resistant to biomarkers in patients with biliary fibrosis.
enteric proteolysis.17 In an HLA-DQ8 transgenic mouse model, a hu- Our results indicate that omitting gluten-containing food
manised model organism for celiac disease, sensitisation with gluten sources has a less profound effect on the microbiome configura-
and exposure to purified ATI enhanced intestinal barrier defects and tion than starkly altering the macronutrient supply and switching
gluten-induced intestinal inflammation. 26 Notably, purified nutri- the primary energy source. 50 In a previous GFD intervention in
tional ATI worsened inflammation in models of inflammatory bowel 21 healthy volunteers over 4 weeks, the gut microbiome compo-
disease, promoted extra-intestinal inflammation in mouse models of sition remained comparably stable during this short period. The
nonalcoholic steatohepatitis and Alzheimer's disease, and increased authors claimed a more substantial effect on the functional con-
the severity of intestinal and pulmonary allergies.16,17 figuration than taxonomic composition, contrasting our results. 51
In our current study on PSC-IBD, a disease with autoimmune and However, this study used merely 16S sequencing and functional
immune-mediated features that affect both the intestine and the imputation, which is now considered an inappropriate proxy for
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232 LIWINSKI et al.
F I G U R E 4 Analysis of luminal bacterial

metagenome dynamics under GFD using
shotgun metagenomic sequencing. (A)
Biplot illustrating bacterial between-
sample (beta) diversity using bray–Curtis
dissimilarity; (B) bacterial within-sample
(alpha) diversity analysis using Shannon
index; (C) volcano plot showing bacterial
single species differential abundance;
(D) biplot illustrating viral between-
sample (beta) diversity using bray–Curtis
dissimilarity; (E) viral within-sample (alpha)
diversity analysis using Shannon index;
(F) volcano plot showing single viral
species differential abundance; (G) biplot
illustrating the analysis of the bacterial
community metabolic capacity using
metagenomic shotgun sequencing; (H)
bacterial genetic richness analysis using
metagenomic shotgun sequencing. V2,
time of second visit, i.e. 8 weeks after the
screening visit and immediately before
starting the gluten-free diet; V6, sixth
visit, immediately after completing the
8-week gluten-free diet course (16 weeks
after the screening visit).
the actual functional potential profiled by shotgun sequencing. In Previous studies on the effect of the GFD on gut microbiota
a more recent randomised controlled trial with 60 healthy Danish composition in patients with celiac disease and NCWS showed var-
volunteers, an eight-week intervention with a low-gluten diet (2 g ious changes in the abundance of single bacterial taxa, including an
gluten/day) resulted in moderate changes in the intestinal mi- increase of E. coli, Slackia, Enterobacteriaceae and Clostridiaceae,
crobiome profiled by shotgun metagenomic sequencing. Several and a decrease in Lactobacilli, various Bifidobacterium spp., and
functional modules were altered under a low-gluten diet, espe- Veillonellaceae.53 In healthy individuals, an increase in the phyla
cially modules related to carbohydrate metabolism. 52 These re- Proteobacteria and Firmicutes and a decrease in lactobacilli and
sults show that our cohort was potentially underpowered to find a bifidobacteria have been reported on the GFD.53 In accordance,
significant difference in single functional pathways. However, it is the present study observed a reduction of Lactobacillus species
challenging to compare both studies since the participants in the (Lactobacillus F plantarum) under the GFD, and the most prominent
former trial were healthy and because a low-gluten diet may have bacterial signal was a decline of Romboutsia ilealis. In a study on dex-
significantly different sequelae than trying to omit gluten and ATI tran sulfate sodium-induced colitis in mice, the relative abundance of
altogether. Nevertheless, more studies are required to determine Romboutsia was increased compared to the control group, suggest-
the impact of a GFD on microbiome function in healthy individuals ing that Romboutsia may play a role in promoting intestinal inflam-
and PSC patients. mation. The DSS-induced chronic colitis was improved following the
|
LIWINSKI et al. 233
F I G U R E 5 Intestinal whole transcriptome analysis using bulk-RNA-seq. (A) Volcano plot showing differential gene transcript abundance
using mucosal transcriptome profiling (RNA-seq); (B) gene-set enrichment analysis using intestinal RNA-seq data. V2, time of second visit,
i.e. 8 weeks after the screening visit and immediately before starting the gluten-free diet; V6, sixth visit, immediately after completing the
8-week gluten-free diet course (16 weeks after the screening visit).
application of selenium-enriched L. acidophilus, which significantly disease.57 Instead, many patients with clinically evident NCWS do
54
reduced the relative abundance of Lactobacillus and Romboutsia. not appear to react to gluten. 20 Still, amongst non-gluten proteins,
Thus, our data suggest that the GFD may partially alleviate dysbiosis ATI is the best-defined and active component.18,22,23,58
in PSC-IBD. Our results lend further support to the hypothesis that PSC-
The reported effects of a GFD on the gut microbiota are very IBD is an environmental exposure-driven autoimmune disease.59
heterogeneous and partially contradictory between existing stud- However, despite an overlap in genetic architecture with celiac dis-
ies (including ours). The vastly heterogeneous methodological ap- ease,60 the lack of a significant clinical response to GFD renders it
proaches and variation in participants' metadata likely explain these unlikely that PSC-IBD is solely driven by antigens contained in wheat.
incongruencies recently summarised for patients with and without Instead, stimulation of the innate immune response, combined with
celiac disease.14 Moreover, the comparability of our results with pre- other environmental factors, including alimentary, microbial and
vious literature studying the effects of a GFD on the microbiome non-dietary triggers, might instigate PSC-IBD.61 Nevertheless, our
is further limited by the fact that PSC patients have an a priori al- results merit further studies on the outcomes of long-term GFD
tered gut microbiota and intestinal mucosal immunity different from (ATI-free diet) and other exposure-elimination approaches in PSC-
healthy individuals.31 Moreover, results on the effect of a GFD on IBD. The limitation of this study is that it was an uncontrolled, small
health and microbiome configuration cannot be extrapolated from pilot trial. Moreover, the optimal duration of the GFD in PSC-IBD
one population to another due to highly individualised gut micro- and the best time to assess outcome measures are unclear. Thus,
bial compositions, dietary habits, metabolic activity patterns, and a GFD currently cannot be recommended as a treatment for PSC-IBD
plethora of factors associated with the microbial variation. In addi- without further studies. The study's strength is the depth of analysis
tion, there is wide variation in the composition of different GFDs.55 concerning mucosal inflammation, intestinal microbiota composi-
Thus, more collaborative efforts are required to study the impact tion and novel predictive serum markers of liver fibrosis. In addition,
of GFDs in diverse microbial populations to reach biologically and adherence to the diet was controlled using a recently developed
medically sound conclusions that genuinely contribute to improv- urine gluten ELISA. We demonstrated that the gluten-free and thus
ing health in patients. However, the definition of ATI as significant ATI-free diet alleviated colonic mucosal inflammation in PSC-IBD.
contributors to intestinal and extra-intestinal inflammation that are Therefore, our study provides the first evidence for a rationale to
only present and active in gluten-containing foods also permits a investigate this dietary intervention in patients with PSC-IBD fur-
study design that is more focused than considering the contribution ther. Advances in this research field could aid in the development of
of other, likely less relevant components of a complex nutrient like specific nutritional-microbiome-informed interventions to optimise
wheat. Along this line, first studies in mice have begun to disentangle host-microbiota mutualism and improve future PSC treatment.
the microbiome effects of purified wheat ATI.26.
In recent years, adherence to a GFD gained popularity amongst AU T H O R C O N T R I B U T I O N S
individuals without a diagnosis of celiac disease despite the lack of Timur Liwinski: Data curation (lead); formal analysis (lead); investiga-
evidence for a benefit without an adequate medical diagnosis.56 tion (lead); methodology (lead); visualization (lead); writing –original
Whilst gluten is immunogenic, cytotoxic, proapoptotic and proin- draft (lead); writing – review and editing (lead). Sina Hübener:
flammatory in patients with celiac disease, there is contradictory and Conceptualization (lead); investigation (equal); methodology
no conclusive evidence that pure gluten proteins cause or promote (equal); project administration (lead); writing – review and editing
intestinal or extra-intestinal disease in individuals without celiac (equal). Lara Henze: Investigation (equal); methodology (equal).
|
234 LIWINSKI et al.
Peter Huebener: Methodology (equal); validation (equal); writing – bowel disease associated with primary sclerosing cholangitis. Gut.
2005;54(1):91–6. https://doi.org/10.1136/gut.2004.046615
review and editing (equal). Melina Heinemann: Validation (equal);
3. Ali AH, Tabibian JH, Nasser-Ghodsi N, Lennon RJ, DeLeon T, Borad
writing – original draft (equal); writing – review and editing (equal). MJ, et al. Surveillance for hepatobiliary cancers in patients with
Marcus Tetzlaff: Conceptualization (equal); project administration primary sclerosing cholangitis. Hepatology. 2018;67(6):2338–51.
(equal); resources (equal); writing –review and editing (equal). Marie https://doi.org/10.1002/hep.29730
I. Hiller: Funding acquisition (equal); project administration (equal). 4. Soetikno RM, Lin OS, Heidenreich PA, Young HS, Blackstone
MO. Increased risk of colorectal neoplasia in patients with pri-
Bettina Jagemann: Methodology (equal); project administration
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pathogenesis of primary sclerosing cholangitis: evidence and ther-
(equal); validation (equal). Erika Monguzzi: Methodology (equal);
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writing –review and editing (equal). Guido Schachschal: Investigation org/10.1002/hep.31311
(equal); methodology (equal). Thomas Rösch: Investigation (equal); 6. Lloyd-Price J, Abu-Ali G, Huttenhower C. The healthy human mi-
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tigation (equal); methodology (equal); software (equal). Andre
7. Kummen M, Hov JR. The gut microbial influence on cholestatic liver
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Detlef Schuppan: Methodology (equal); resources (equal); supervi-
https://doi.org/10.1016/j.immuni.2020.07.015
sion (equal); validation (equal); writing – review and editing (equal). 9. Gasaly N, de Vos P, Hermoso MA. Impact of bacterial metabolites
Christoph Schramm: Conceptualization (lead); funding acquisi- on gut barrier function and host immunity: a focus on bacte-
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We thank the patients volunteering to participate in the study and
with Crohn's disease. Nutrients. 2021;13(5):1611. https://doi.
all caregivers at the YAEL center for autoimmune liver diseases. We org/10.3390/nu13051611
would also like to thank the staff of the microbiome laboratory, se- 12. Moayyedi P, Simrén M, Bercik P. Evidence-based and mechanistic
insights into exclusion diets for IBS. Nat Rev Gastroenterol Hepatol.
quencing facility and IT department of the IKMB for excellent tech-
2020;17(7):406–13. https://doi.org/10.1038/s41575-020-0270-3
nical support. 13. Lindfors K, Ciacci C, Kurppa K, Lundin KEA, Makharia GK, Mearin
ML, et al. Coeliac disease. Nat Rev Dis Primers. 2019;5(1):3. https://
F U N D I N G I N FO R M AT I O N doi.org/10.1038/s41572-018-0 054-z
14. Verdu EF, Schuppan D. Co-factors, microbes, and immunogenetics
This work was supported by the Helmut and Hannelore Greve
in celiac disease to guide novel approaches for diagnosis and treat-
Foundation, the YAEL Foundation for autoimmune liver diseases, and the ment. Gastroenterology. 2021;161(5):1395–1411.e4. https://doi.
German Research Foundation (DFG; CRU306). MH was supported as a org/10.1053/j.gastro.2021.08.016
postdoctoral fellow by the German Research Foundation (438122637). 15. Junker Y, Zeissig S, Kim S-J, Barisani D, Wieser H, Leffler DA, et al.
Wheat amylase trypsin inhibitors drive intestinal inflammation via
activation of toll-like receptor 4. J Exp Med. 2012;209(13):2395–
C O N FL I C T O F I N T E R E S T 408. https://doi.org/10.1084/jem.20102660
The authors declare no conflict of interest. 16. Zevallos VF, Raker V, Tenzer S, Jimenez-C alvente C, Ashfaq-
Khan M, Rüssel N, et al. Nutritional wheat amylase-trypsin inhib-
itors promote intestinal inflammation via activation of myeloid
ORCID
cells. Gastroenterology. 2017;152(5):1100–1113.e12. https://doi.
Timur Liwinski https://orcid.org/0000-0002-1041-9142 org/10.1053/j.gastro.2016.12.006
Peter Hübener https://orcid.org/0000-0001-7558-7625 17. Pickert G, Wirtz S, Matzner J, Ashfaq-Khan M, Heck R, Rosigkeit S,
Thomas Rösch https://orcid.org/0000-0003-2270-2495 et al. Wheat consumption aggravates colitis in mice via amylase trypsin
inhibitor-mediated dysbiosis. Gastroenterology. 2020;159(1):257–
Detlef Schuppan https://orcid.org/0000-0002-4972-1293
272.e17. https://doi.org/10.1053/j.gastro.2020.03.064
18. Fritscher-Ravens A, Pflaum T, Mösinger M, Ruchay Z, Röcken
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Received: 13 March 2022 | First decision: 2 April 2022 | Accepted: 8 October 2022