Characteristics of study participants and sequence of gut microbiome
We enrolled 78 participants including 11 psoriatic patients after 5-month secukinumab treatment, 32 psoriatic patients without treatment, and 35 healthy people as control. There were no significant differences in demographic information between AT, BT, and H group. A summary of related characteristics of above three groups are presented in Table 1, and detailed related information of participants is shown in Table S1.
Table 1 Charasitics of each group
|
|
AT
|
BT
|
H
|
Male/female
|
10/1
|
29/3
|
31/4
|
Age (years)*
|
37.82
|
38.38
|
37.31
|
Types of patients
|
|
|
|
Psoriasis vulgaris
|
11
|
32
|
|
Psoriasis arthritis
|
0
|
0
|
|
Pustular psoriasis
|
0
|
0
|
|
Erythrodermic psoriasis
|
0
|
0
|
|
*Median
AT psoriatic patients after receiving 5-month secukinumab treatment, BT psoriatic patients without therapy, H healthy people.
Then, 78 fecal samples were collected, containing 5,841,838 raw sequences with a mean length of 456 base by 16S rDNA sequencing and analysis. After quality trimming and chimera checking, we obtained 5,585,894 high-quality sequences in total for further analysis. 424 OTUs in all samples were identified, and 382, 412, and 420 OTUs were confirmed in AT, BT, H respectively. The data summary is shown in Table 2. All sequences in three groups were verified high coverage values over 99%. The species accumulation curves of genus level had reached a plateau in all three groups (Fig. 1A). Above results illustrate that most fecal microbiota species were detected, which provided good sequencing depth for fecal microbiota exploration in follow-up analysis.
Table 2 Fecal microbiota community indices
|
Group
|
OTUs
|
Good’s
|
Richness index (M ± SD)
|
Diversity index (M ± SD)
|
ACE
|
Simpson
|
AT
|
382
|
0.9994
|
280.9624 ± 6.0319
|
0.9200 ± 0.0105
|
BT
|
412
|
0.9994
|
235.4373 ± 10.7863
|
0.8019 ± 0.0287
|
H
|
420
|
0.9994
|
246.2601 ± 12.2193
|
0.8452 ± 0.017
|
AT psoriatic patients after receiving 5-month secukinumab treatment, BT psoriatic patients without therapy, H healthy people.
|
Secukinumab treatment leads to significant change in gut microbial diversity
We set out to investigate the richness of fecal microbiota and structural difference among these three groups, the α-diversity (ACE index and Simpson index) of gut microbiota was calculated (Table 2). By ACE indices, we discovered that secukinumab therapy induced significantly increased microbiota richness compared to that of the other two groups (Fig. 1B). Also, secukinumab therapy had an elevated biodiversity than the other two groups by Simpson indices (Fig. 1C). However, there were no significant differences of α-diversity of gut microbiota between psoriatic patients without therapy and healthy people as indicated by both ACE and Simpson indices.
Further, in order to assess the similarity of gut microbial communities among these three groups, beta diversity was analyzed. NMDS based on binary_jaccard was performed, which suggested that the fecal microbiota of secukinumab treatment group was different from the other two groups (stress=0.1480<0.2) (Fig. 1D). The separation trend of secukinumab treatment and psoriasis without treatment is more obvious, and beta diversity of secukinumab treatment is lower, which is validated significantly (p =0.002, PERMANOVA) (Figs. 1D, E).
Secukinumab treatment altered gut microbiota composition
The taxonomy of fecal microbiota was evaluated using the RDP classifier. Two dominant phyla, Firmicutes and Bacteroidetes, based on different relative abundances among three groups were identified. Firmicutes (70.12%) increased and Bacteroidetes (19.38%) decreased in secukinumab treatment group, when compared to psoriasis without therapy group (Firmicutes 51.24%, Bacteroidetes 40.15%) and healthy people (Firmicutes 56.94%, Bacteroidetes 33.95%) (Figs. 2A, B). In family level, higher proportions of Lachnospiraceae (29.32%) and Ruminococcaceae (22.75%) and lower proportions of Bacteroidaceae (14.91%), Prevotellaceae (1.78%) and Veillonellaceae (4.97%) in secukinumab treatment group were verified (Figs. 2C, D). In genus level, the abundance of Faecalibacterium (15.31%) was higher and the abundance of Bacteroides (14.91%) and Prevotella_9 (0.03%) was lower in secukinumab treatment group than the other two groups (Figs. 2E, F).
To further evaluate the differences in the gut microbiota composition between each two groups, Metastats analysis was performed. In phylum level, there was a significant increase in Firmicutes and a decrease in Bacteroidota in secukinumab treatment group than the other two groups. Although the level of Firmicutes seemed lower and Bacteroidota seemed higher in psoriasis without therapy group than healthy control group, there was no statistical significance (Table 3A, placed at the end of the text). Significant decreases in the orders Rhizobiales and Bacteroidales were observed in secukinumab treatment group compared the other two groups, and a significant decrease in unclassified_Firmicutes was confirmed only when compared to healthy control group (Table 3B). In secukinumab treatment group, two families Comamonadaceae and Erwiniaceae were significantly more abundant and one family Xanthobacteraceae was massively less abundant than the other two groups, and three families unclassified_Firmicutes, uncultured_Clostridiales_bacterium and Porphyromonadaceae were less abundant when compared to healthy control group (Table 3C). In genus level, when compared to healthy control group, secukinumab treatment group presented obviously decreased genera Bradyrhizobium, unclassified_Firmicutes and uncultured_Clostridiales_bacterium, but increased genera Hydrogenophaga, Pantoea and unclassified_Comamonadaceae. What’s more, when compared to psoriasis without therapy group, secukinumab treatment group showed statistically decreased genera Bradyrhizobium, Hydrogenophaga and Lactococcus, but increased genera Pantoea and unclassified_Comamonadaceae. Stikingly, lower levels of genera uncultured_Clostridiales_bacterium, Alloprevotella and Butyrivibrio, and higher level of genus Prevotella_7 were identified in psoriasis without therapy compared to healthy control, while there was no profound significance between these two groups in phylum, order and family levels.
Consequently, we investigated the specific significantly different bacterial taxa among these three groups by using LEfSe analysis at genus level (LDA score > 2, p < 0.05). Several genera, including Faecalibacterium, Agathobacter, Subdoligrnulum, Bifidobacterium and Dialister, were significantly enriched in the secukinumab treatment group, while genera Succinivibrio and Paraprevotella were obviously enriched in psoriasis without treatment group (Fig. 3A). Besides, we detected the gut microbiota composition’s alteration between the secukinumab treatment group and the healthy control group. We identified that genera such as Faecalibacterium, Subdoligrnulum and Ruminococcus were significantly increased in secukinumab treatment group, whereas Limosilactobacillus, Desulfovibrio and Paraprevotella were significantly enriched in healthy control group (Fig. 3B). Finally, in Fig. S1, we discovered that genera Prevotella_7, Weissella and Lactococcus were increased in psoriasis without therapy, while Megasphaera, Dialister, Romboutsia and other genera were enriched in healthy control.
Secukinumab treatment altered phenotypic functions of gut microbiota in psoriatic patients
The potential prediction for phenotypic functions in complex communities among these three groups was analyzed by BugBase. 5 predicted phenotypic functions (contains mobile elements, forms biofilms, gram-negative, gram-positive and potentially pathogenic) exhibited significant differences in abundance (Fig. 4). Secukinumab treatment group showed obvious increased abundance in contains mobile elements phenotype than the other two groups, which manifested as increased Lachnospiraceae and Ruminococcaceae but decreased Veillonellaceae abundance (Fig. 4A). Besides, the gut microbiota of secukinumab treatment group demonstrated more significant abundance in form biofilms phenotype, having elevated abundance of Bifidobacteriaceae, Coriobacteriaceae and Verrucomicrobiaceae, and reduced abundance of Enterobacteriaceae and Alcaligenaceae than psoriasis without therapy group (Fig. 4B). What’s more, we observed that the abundance of gut microbiota in secukinumab treatment group was significantly lower in gram negative phenotype (Veillonellaceae, Prevotellaceae, Enterobacteriaceae and Bacteroidaceae) (Fig. 4C), while statistically higher abundance of gut microbiota in gram positive phenotype (Lachnospiraceae and Ruminococcaceae) was confirmed than the other two groups (Fig. 4D). Finally, potentially pathogenic phenotype was evaluated. Interestingly, the gut microbiota abundance of secukinumab treatment group and healthy people group were both obviously reduced in this phenotype than the psoriasis without therapy group. Secukinumab treatment group suggested massively decreased Veillonellaceae, Prevotellaceae and Bacteroidaceae, while healthy control group demonstrated obviously reduced Prevotellaceae and Bacteroidaceae (Fig. 4E).
Secukinumab therapy influenced biological function of gut microbiota in psoriatic patients.
To better understand how biological functions in secukinumab treatment may be affected, we used PICRUSt2 to evaluate the composition of functional gene in fecal microbiota of our samples. As a result, a total of 5 significantly different functional COGs between secukinumab treatment group and healthy control group were found, as well as 7 COGs between secukinumab treatment group and psoriasis without therapy group were predicted (Fig. S2).
Moreover, further analyzation in the context of the KEGG database was performed to better clarify metabolic function changes of the community samples. In secukinumab treatment group, the functional genes for metabolism of terpenoids and polyketides, membrane transport, substance dependence and infectious diseases: viral were high enriched, whereas metabolism of cofactors and vitamins, glycan biosynthesis and metabolism, transport and catabolism and circulatory system were significantly reduced in comparison with healthy people group (Fig. 5A). Detailed information was shown in Table S2. Additionally, compared to psoriasis without therapy group, eight KEGG pathways (including infectious diseases: viral, infectious diseases: parasitic, substance dependence and others) were significantly enriched and 5 KEGG pathways (cardiovascular diseases, circulatory system, glycan biosynthesis and metabolism, amino acid metabolism and metabolism of cofactors and vitamins) were statistically reduced in the secukinumab treatment group (Fig. 5B and Table S2).