Gut microbiota facilitate chronic spontaneous urticaria

Chronic spontaneous urticaria (CSU) comes with gut dysbiosis, but its relevance remains elusive. Here we use metagenomics sequencing and short-chain fatty acids metabolomics and assess the effects of human CSU fecal microbial transplantation, Klebsiella pneumoniae, Roseburia hominis, and metabolites in vivo. CSU gut microbiota displays low diversity and short-chain fatty acids production, but high gut Klebsiella pneumoniae levels, negatively correlates with blood short-chain fatty acids levels and links to high disease activity. Blood lipopolysaccharide levels are elevated, link to rapid disease relapse, and high gut levels of conditional pathogenic bacteria. CSU microbiome transfer and Klebsiella pneumoniae transplantation facilitate IgE-mediated mast cell(MC)-driven skin inflammatory responses and increase intestinal permeability and blood lipopolysaccharide accumulation in recipient mice. Transplantation of Roseburia hominis and caproate administration protect recipient mice from MC-driven skin inflammation. Here, we show gut microbiome alterations, in CSU, may reduce short-chain fatty acids and increase lipopolysaccharide levels, respectively, and facilitate MC-driven skin inflammation.


Supplementary Methods Study participants and conduct
We obtained detailed data on the medical history, comorbidities, and clinical manifestation of all the patients.We also performed laboratory tests such as routine blood analyses, autoantibodies (e.g., anti-thyroid autoantibodies, SLE-related antibodies), sedimentation, CRP, and thyroid hormone levels.
Tlr4 -/-mouse genotyping Mouse tail tissues (~0.5 g) were clipped with sterile scissors, and genomic DNA was extracted using the Mouse Tail Direct PCR Kit (Selleck, China).Extracted DNA was subjected to PCR using primers(Forward:AGGGAGATGTGTGTGTGAAGAAGCCT; Reverse:TTCCAGCTATGGCCCAGATGAACT).The 416 bp band represents the pure heterozygote and no band at this position represents wild type.
Western Blot BMMC cell samples were lysed with RIPA buffer containing 1 mM protease inhibitor (Beyotime Technology, China) at 4°C for 30 min, and centrifuged at 14,000 rpm for 10 min.The supernatant was collected and the protein concentration was determined using the BCA Protein Concentration Assay Kit (Beyotime Technology, China).Protein extracts were mixed with the uploading buffer and heated at 95°C for 10 min to denature the proteins.Denatured proteins were separated by 10% SDS-PAGE gel electrophoresis and transferred to a 0.45um PVDF membrane(Millipore, USA).The membrane was closed with 5% BSA for 1 h at room temperature and incubated with primary antibody for 16 h at 4°C, followed by incubation with horseradish peroxidasecoupled secondary antibody for 1 h at room temperature.The bands were visualized using a chemiluminescence kit (NCM Biotech, China) and a chemiluminescence imaging system, ChemiDoc MP (BIORAD, USA).Molecular Weight of glycosylated TLR4: 95/120 kDa, Molecular Weight of Actin: 43 kDa.Antibodies and dilution ratios used were as follows: TLR4 antibody, (Santa Cruz, Cat#: sc-293072; Clone:25, dilution ratio: 1:500); actin antibody, (Santa Cruz, Cat#: sc-8432; Clone:C-2, dilution ratio: 1:1000).Supplementary Fig. 1 Details of participants' screening and research process.A total of 77 participants were enrolled, including 38 HC and 39 CSU patients.Of them, 26 HC and 26 CSU patients received metagenomic sequencing in fecal samples; 38 HC and 29 CSU patients received targeted metabolomics in plasma; 26 HC and 16 CSU patients receiving both metagenomic sequencing in fecal samples and targeted metabolomics in plasma.Of the HC and CSU patients that received metagenomic sequencing in fecal samples, LPS in plasma were examined in 16 HC and 16 CSU patients.16 HC and 11 CSU patients received metagenomic sequencing in fecal samples, targeted metabolomics in plasma, and LPS examination in plasma.26 CSU patients who had undergone fecal metagenomic sequencing were followed up, and 22 patients completed follow-up for remission and relapse.HC: healthy controls; CSU: chronic spontaneous urticaria.Supplementary Fig. 2 Analysis between CSU patients and HC based on metagenomic sequencing and the association analysis of CSU recurrence with clinical features.Differential families a and genus b identified by MaAsLin2 with BH correction between HC and CSU group (P values were provided in Source Data File).#P<0.2, +P<0.1, *P<0.05,**P<0.01.c Two-tailed spearman's correlation between the sum of SCFAproducing bacteria and the sum of opportunistic pathogens in all samples (HC=26, CSU=26, P=0.00014).The line represents centre of overall linear fit, with grey areas standing for the standard error of 95% confidence interval.d The Spearman's correlation networks based on the core species in HC (upper panel) and CSU (lower panel), respectively (r>0.5, P<0.05), which was constructed by R package NetCoMi and "eigenvector" was used to identify hubs to each group.Core species were those that were detected in at least 50% of samples in this cohort.Each node represents a core species, and nodes of the same color are derived from the same phylum.An edge between nodes denotes the different association in CSU and HC, and the color and size of edges represent the different association types and association intensity, respectively."+" indicates a positive correlation, and "-" indicates a negative correlation.e The differential Spearman's correlations network between HC (n=26) and CSU (n=26).fCladogram of significant differences between the two groups from the family level to the species level.HC(n=26) vs CSU(n=26).The blue dot indicates that the taxon presents a higher relative abundance in HC(n=26), and the red dot indicates that the taxon presents a higher relative abundance in CSU.g Receiver operating characteristic curves of Klebsiella pneumoniae for correctly distinguishing HC(n=26) and CSU(n=26, AUC=0.742).The shape and whiskers of violin plot respectively represent density distribution and overall range of the data in (a, b).AUC: area under curve.h Relapse-free analysis based on the 5 SCFA-producing bacteria (Left: n=22, P=0.024) or 4 opportunistic pathogens (Right: n=22, P=0.Supplementary Fig. 7 Representative gating strategies for mouse skin tissue flow cytometry analysis.The first gate circled single cells, the second gate circled live cells, the third gate circled CD45 + cells, and the fourth gate circled CD117 + FcɛRI + cells.Source data are provided as a Source Data file.
Supplementary Table 2 047) was determined by logrank test.**P<0.01.Source data are provided as a Source Data file.Supplementary Fig.3 Alteration of gut microbiota in mice before and after antibiotic treatment by 16S rDNA sequencing.a Number of OTUs shared and unique between Abx-before and Abx-after.b Comparison of β-diversity between Abx-before and Abx-after (n=5, P=0.007).Significance was determined by PERMANOVA.c Left panel: comparison of Shannon index between Abx-before and Abx-after (n=5, P=0.0079);Right panel: comparison of Simpson index between Abx-before and Abxafter (n=5, P=0.0079).Box plots indicate median (middle line), 25th, 75th percentile (box) and 5th and 95th percentile (whiskers) as well as outliers (single points).Significance was determined by two-tailed Wilcoxon test.**P<0.01.Abx-before: mice before antibiotic treatment.Abx-after: mice after antibiotic treatment.Source data are provided as a Source Data file.Supplementary Fig. 4 Comparison of gut microbiota between fecal microbial transplantation (FMT)-HC mice and FMT-CSU mice after FMT by metagenomic sequencing.a Comparison of β-diversity between Control, FMT-HC and FMT-CSU.(n=5).b Left panel: comparison of Shannon indexes between Control, FMT-HC and FMT-CSU (n=5, Control vs FMT-HC, P=0.0079,Control vs FMT-CSU, P=0.0079); Right panel: comparison of Simpson indexes between Control, FMT-HC and FMT-CSU(n=5, Control vs FMT-HC, P=0.0079,Control vs FMT-CSU, P=0.0079).Significance was determined by Wilcoxon test.Box plots indicate median (middle line), 25th, 75th percentile (box) and 5th and 95th percentile (whiskers) as well as outliers (single points).cDifferent species between FMT-HC and FMT-CSU(n=5, P values were provided in Source Data File).#P<0.2, +P<0.1, *P<0.05,**P<0.01.The shape and whiskers of violin plot respectively represent density distribution and overall range of the data.Significance was determined by PERMANOVA(a) or two-tailed Wilcoxon test(b, c).Control: gavage with solvents; FMT-HC: FMT from healthy control; FMT-CSU: FMT from CSU patients.ns: no significant.Source data are provided as a Source Data file.
The bacteria alteration between HC and CSU