Multi-omics analysis reveals gut microbiota-metabolite interactions and their association with liver function in autoimmune overlap syndrome

Table 1 Demographic and clinical data of patients in the study

Characteristic	PBC (n = 16)	AIH (n = 9)	OS (n = 7)
Age (years)	58.00 (42.25-61.00)	52.00 (28.00-57.50)	57.00 (42.00-60.00)
Sex (male/female)	2/14	1/8	1/6
RBC (× 1012/L)	3.89 (3.67-4.04)	3.98 (3.32-5.19)	4.40 (4.23-4.55)a
WBC (× 109/L)	4.17 (3.05-5.73)	4.74 (3.58-6.69)	6.09 (4.75-8.37)a
PLT (× 109/L)	138.50 (46.00-219.00)	132.00 (60.00-245.00)	181.00 (111.00-281.00)
ALT (IU/L)	57.50 (31.75-76.00)	109.00 (39.00-275.00)	82.00 (16.00-259.00)
AST (IU/L)	95.00 (63.00-126.00)	97.00 (39.00-275.00)	52.00 (49.00-199.00)
TBIL (μmol/L)	35.05 (24.80-49.60)	31.60 (16.85-54.95)	30.80 (19.50-33.90)
GGT (IU/L)	190.50 (93.75-485.00)	66.00 (57.00-132.5)	186.00 (29.00-494.00)
ALP (IU/L)	324 (176.50-583.00)	138.00 (94.00-294.50)	216.00 (90.00-310.00)
ALB (g/dL)	37.9 (32.68-45.00)	38.40 (26.20-42.70)	37.30 (34.50-46.80)
TG (mmol/L)	0.97 (0.76-1.24)	1.27 (0.73-1.70)	1.02 (0.64-1.36)
CHOL (mmol/L)	4.63 (3.59-8.12)	4.48 (2.94-5.49)	5.14 (4.03-7.22)
TBIL (μmol/L)	35.05 (24.8-49.6)	31.60 (16.85-54.95)	30.80 (19.50-33.90)
INR	1.04 (0.98-1.13)	1.13 (1.05-1.24)	1.00 (0.91-1.14)
IGA (mg/dL)	3.59 (3.17-4.40)	2.49 (2.12-3.91)	3.50 (2.70-7.87)
IGG (mg/dL)	16.85 (14.63-20.70)	16.00 (15.00-29.55)	17.05 (13.80-18.10)
IGM (mg/dL)	4.45 (2.75-5.26)	1.34 (0.83-2.38)	1.85 (1.06-4.48)
ANA (positive/total)	16/16	8/9	6/7
AMA (positive/total)	12/16	2/9	5/7
ACA (positive/total)	4/16	1/9	1/7

^aP < 0.05.

P vs primary biliary cholangitis. PBC: Primary biliary cholangitis; AIH: Autoimmune hepatitis; OS: Overlap syndrome; RBC: Red blood cell; WBC: White blood cell; PLT: Platelets; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; TBIL: Total bilirubin; ALB: Albumin; GGT: Gamma-glutamyl transferase; ALP: Alkaline phosphatase; TG: Triglyceride; CHOL: Cholesterol; INR: International normalized ratio; IGA: Immunoglobulin A; IGG: Immunoglobulin G; IGM: Immunoglobulin M; ANA: Antinuclear antibody; AMA: Antimitochondrial antibody; ACA: Anticardiolipin antibody.

Table 2 Top 10 phylum-level shifts in gut microbiota composition among three patient groups

Gut microbiome	AIH (%)	PBC (%)	OS (%)
Firmicutes	61.287655	53.938722	49.503265
Proteobacteria	11.205756	19.142401	28.905284
Bacteroidetes	20.802816	21.928249	15.823387
Actinobacteria	5.998074	2.806389	1.049066
Verrucomicrobia	0.516567	1.83022	3.527271
Fusobacteria	0.04227	0.275033	1.138819
Candidatus Saccharibacteria	0.037415	0.019463	0.017548
Synergistetes	0.005823	0.011598	0.012459
Cyanobacteria	0.004831	0.002569	0.009026
Tenericutes	4.87 × 10-4	9.63 × 10-4	0.0

PBC: Primary biliary cholangitis; AIH: Autoimmune hepatitis; OS: Overlap syndrome.

Table 3 Significantly altered metabolites in overlap syndrome compared to primary biliary cholangitis

Name	FC	P value	VIP
4-(anilinomethylidene)-3-methyl-4,5-dihydroisoxazol-5-one	1.552696	0.000721	2.639853
2-hydroxy-6-[(8Z,11Z)-pentadeca-8,11,14-trien-1-yl]benzoic acid	0.370792	0.003842	1.818916
Ergosterol peroxide	3.118903	0.004243	2.925869
Cholest-4-en-3-one	0.40443	0.004499	1.984777
Propionylcarnitine	1.925051	0.004683	2.61661
Fludrocortisone acetate	0.248968	0.005297	1.854885
N-Methyldioctylamine	1.585333	0.005887	2.162885
Proscillaridin A	3.2096	0.009332	2.315147
(+/-)-CP 47,497-C7-hydroxy metabolite	0.544798	0.012931	2.197074
PC (16:1e/22:1)	1.747451	0.014132	2.307943
Octadec-9-ynoic acid	0.440369	0.014988	2.076044
PC (16:1e/22:0)	0.48038	0.016027	2.072384
17beta-Trenbolone	0.574612	0.01804	2.224429
SM (d14:0/14:1)	2.716597	0.018685	2.053585
alpha-farnesene	0.647487	0.022544	1.773355
Trigonelline	0.421173	0.023757	1.652406
LPE 18:2	1.567382	0.025836	1.859335
9-HOTrE	0.616145	0.029622	1.97427
Verapamil hydrochloride	0.563206	0.036	1.803812
Cer-NS (d18:1/16:0)	0.549991	0.037082	1.795717
1,3-dimethyluracil	0.471792	0.037551	1.671353
6-(7-methyloctyl)-1H,3H,4H,6H-furo[3,4-c]furan-1-one	1.971003	0.037965	2.152845
(+/-)5(6)-DiHET	0.366881	0.042184	1.46635
Pilocarpine	0.489733	0.043371	1.922503
PC (3:0/16:4)	1.628991	0.044805	1.521527
Testosterone	0.573249	0.046918	1.426691

FC: Fold change; VIP: Variable importance in projection; PC: Phosphatidylcholine; SM: Sphingomyelin; LPE: Lyso-phosphatidylethanolamine; Cer-NS: Ceramide n-acyl-sphingosine.

Table 4 Significantly altered metabolites in overlap syndrome compared to autoimmune hepatitis

Name	FC	P value	VIP
All-Trans-13,14-dihydroretinol	0.155118	0.002634	2.637447
Alpha-farnesene	0.544136	0.001544	2.616726
PC (14:1e/18:0)	2.137244	0.003936	2.522067
(+/-)-CP 47,497-C7-hydroxy metabolite	0.473641	0.01921	2.325252
PC (16:1e/22:1)	1.939505	0.00826	2.30595
Methyl nicotinate	0.063976	0.045424	2.14672
Octadec-9-ynoic acid	0.502862	0.011726	1.993845
Homo-gamma-linolenic acid (C20:3)	0.49057	0.027711	1.964745
PC (16:1e/22:0)	0.585272	0.012906	1.906264
PC (16:0e/16:0)	1.635313	0.029478	1.902553
Linolelaidic acid (C18:2N6T)	0.649017	0.029729	1.902188
Homoarginine	0.481561	0.016852	1.82961
Mupirocin	0.563021	0.046233	1.817926
Phenylglyoxylic acid	0.286324	0.03155	1.786545
Tetranor-12(S)-HETE	0.433141	0.004515	1.741955
N-Methyldioctylamine	1.530444	0.024254	1.708007
Jasmonic acid	0.545306	0.024253	1.438671
(2S)-2-(2-hydroxypropan-2-yl)-2H,3H,7H-furo[3,2-g]chromen-7-one	0.647968	0.021451	1.313058

FC: Fold change; VIP: Variable importance in projection; PC: Phosphatidylcholine.