Contribution of the Gut Microbiota in P28GST-Mediated Anti-Inflammatory Effects: Experimental and Clinical Insights

Abstract

An original immuno-regulatory strategy against inflammatory bowel diseases based on the use of 28 kDa glutathione S-transferase (P28GST), a unique schistosome protein, was recently proposed. Improvement of intestinal inflammation occurs through restoration of the immunological balance between pro-inflammatory T-helper 1 (Th1) responses and both T-helper 2 (Th2) and regulatory responses. However, detailed mechanisms explaining how P28GST prevents colitis and promotes gut homeostasis remain unknown. Considering the complex interplay between the adaptive and innate immune system and the intestinal microbiota, we raised the question of the possible role of the microbial ecosystem in the anti-inflammatory effects mediated by the helminth-derived P28GST protein. We first analyzed, by 16S rRNA sequencing, the bacterial profiles of mice fecal microbiota at several time points of the P28GST-immunomodulation period prior to trinitrobenzene sulfonic acid (TNBS)-colitis. The influence of gut microbiota in the P28GST-mediated anti-inflammatory effects was then assessed by fecal microbiota transplantation experiments from P28GST-immunized mice to either conventional or microbiota depleted naïve recipient mice. Finally, the experimental data were supplemented by the temporal fecal microbiota compositions of P28GST-treated Crohn's disease patients from a pilot clinical study (NCT02281916). The P28GST administration slightly modulated the diversity and composition of mouse fecal microbiota while it significantly reduced experimental colitis in mice. Fecal microbiota transplantation experiments failed to restore the P28GST-induced anti-inflammatory effects. In Crohn's disease patients, P28GST also induced slight changes in their overall fecal bacterial composition. Collectively, these results provide key elements in both the anti-inflammatory mechanisms and the safe therapeutic use of immunomodulation with such promising helminth-derived molecules.

Keywords: fecal microbiota; fecal transplantation; helminth protein; humans; immunization; inflammatory bowel diseases; mice.

Conflict of interest statement

None of the authors of this manuscript have a financial interest related to this work.

Figures

Figure 1

Prevention of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice by 28 kDa glutathione S-transferase +Adjuvant (P28GST+Adjuvant) but not by adjuvant or P28GST alone. BALB/c mice received three subcutaneous injections of P28GST (5 µg.kg−1) without adjuvant, with adjuvant alone (aluminum hydroxide 0.2% solution), or P28GST combined with adjuvant, at 2 week intervals. Positive and negative colitis controls received saline. TNBS (100 mg.kg−1) was induced in all groups except negative control at day 33 and mice were sacrificed at day 36 (3 days after). Fecal samples were collected before the immune priming (day 1), 1 week after the first boost (day 21), and after the second boost (day 33). (A) Experimental design showing the immunization procedure, the fecal sampling plan and the final colitis schedule in all groups of BALB/c mice. (B) Percentage of loss of initial weight (prior colitis, at day 33 = D0) expressed as the comparison of individual weights recorded at killing (day 36 = D3). (C) Macroscopic clinical scores of colons at day 36 according to the scoring system of Wallace. (D) Histological Ameho’s scoring of colons at day 36. (E) Weight of spleens removed at day 36 expressed in milligrams (mg). Atrophy of spleens observed in positive colitis control, Adjuvant- and P28GST-treated animals was restored in the (P28GST+Adjuvant)-treated group. (F) Enzyme-linked immunosorbent assay was performed to quantify individual murine serum amyloid A protein (SAA) in all groups of mice. SAA levels expressed as mg per ml of blood serum were significantly lower in the (P28GST+Adjuvant)-treated group than others. (G) Representative images from May–Grünwald and Giemsa staining of paraffin-wax-embedded 6 µm sections of the colon of mice in all groups. Bars = 500 µm. Results are expressed as means ± SEM; n = 10 mice per group; * p < 0.05; ** p < 0.01; *** p < 0.001; (*) 0.05 < p < 0.1; NS, not significant is indicated when useful.

Figure 2

Mice receiving P28GST+Adjuvant exhibited a reduced inflammatory transcriptional signature when compared to the positive control, as well as in groups with adjuvant or P28GST alone. (A) Interleukin-6 (IL6), (B) interleukin IL1-β, (C) tumor necrosis factor alpha (TNF-α), (D) peroxisome proliferator activated receptor-γ (Ppar-γ), (E) Zonula occludens-1 (Zo1), (F) Cyclo-oxygenase-2 (Cox-2), (G) Nitric oxide synthase (Nos-2), and (H) Heme oxidase-1 (Hmox1) mRNA expression was assessed by quantitative PCR in the colons from the five groups of mice. Results are expressed as means ± SEM; n = 10 mice per group; * p < 0.05; NS, not significantly different from the positive control group.

Figure 3

Immunizations of mice by Adjuvant, P28GST, and (P28GST+Adjuvant) induce slight changes in the composition of the fecal microbiota in comparison with saline as control. Data were generated by analysis of 16S rRNA gene sequences. (A) Mean relative abundance at the phylum level for each group of mice, n = 8 per group. (B) Individual relative abundance at the phylum level shows consistency of the effects. (C) Mean relative abundance at the family level for each group of mice and (D) focus on the corresponding low-represented mean relative abundance at the family level (below 6%), n = 8 per group. Results are expressed as % of the total operational taxonomic unit (OTU) numbers. * p < 0.05; ** p < 0.01; *** p < 0.001; (*) 0.05 < p < 0.1 versus the control group.

Figure 4

Immunization of BALB/c mice by P28GST+Adjuvant in another set of experiments reproduced subtle changes in fecal microbiota and consistently prevented inflammation in a more severe 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. (A) Rarefaction curves of observed operational taxonomic units (OTUs) as a function of reads show a similar number of sequences in the fecal samples of control (non-immunized) and immunized group of mice. Boxes and whiskers represent ± min/max of observed OTUs (B) Shannon index (C) and Chao 1 (D), respectively, showed no greater differences in alpha diversity and richness. Mean relative abundance at the phylum (E) and family level (F) with a focus on the low-represented OTUs at the family level (below 2%) for each group of mice. Results are expressed in % of the total OTU number, n = 5 per group for microbiota analysis (A–F). (G) Percentage of loss of initial weight (prior colitis, at day 36 = D0) expressed as the comparison of individual weights recorded at killing (day 39 = D3) as means ± SEM. (H) Mortality rate recorded at day 39, expressed in % of initial mouse number and (I) macroscopic clinical (Wallace) scores of colons at day 39 in both the non-immunized and immunized group of mice; means ± SEM. n = 10 per group for colitis evaluation parameters (G–I); * p < 0.05; ** p < 0.01; (*) 0.05 < p < 0.1.

Figure 5

Fecal transplantation fails to induce substantial changes in microbiota and does not transfer resistance to colitis. Mean relative abundance at the phylum (A) and family level (B) for either non-immunized (NI) and immunized (I) feces of donor mice, n = 5 per group; the respective representative pooled fecal transplants (FT) (after resuspension, n = 1) and the corresponding feces of recipient mice, at day 36, n = 5 per group. Results are expressed as % of the total OTU number. (C) Percentage of loss of initial weight (prior colitis, at day 36 = D0) expressed as the comparison of individual weights recorded at killing (day 39 = D3) as means ± SEM, and (D) macroscopic clinical (Wallace) scores of colons at day 39 in both non-immunized and immunized recipient group of mice; means ± SEM. n = 10 per group for colitis evaluation parameters (C and D); NS, not significantly different from the positive control group.

Figure 6

Antibiotic treatment profoundly changed the fecal microbiota to facilitate further recolonization but did not improve the resistance to colitis following fecal transplantation from P28GST-treated mice. (A) Rarefaction curves of observed OTUs as a function of reads showed a huge decrease of sequence numbers in the fecal samples of 5 day antibiotic-treated mice (Abx-treated) compared to the group of control mice. Boxes and whiskers represent ± min/max of observed OTUs. (B) Shannon index (C) and Chao 1 (D) confirms the drop in richness and diversity, n = 4 per group. Mean relative abundances at the phylum (E) and family level (F) from 16S sequence data demonstrated the drastic reduction of diversity while DNA quantification in fecal pellets allowed us to estimate the lowering of total abundance of bacteria with antibiotics at the family level (G). (H) Mean relative abundance at the phylum and family level for antibiotic-treated (Abx) recipient mice following (pooled) fecal transplantation from either non-immunized or immunized mice at day 36, n = 5 per group. Results are expressed as % of the total OTU number. (I) Percentage of loss of initial weight (prior colitis, at day 36 = D0) expressed as the comparison of individual weights recorded at necropsy (day 39 = D3) as means ± SEM and (D) macroscopic clinical (Wallace) scores of colons at day 39 in both non-immunized and immunized recipient group of mice; means ± SEM. n = 10 per group for colitis evaluation parameters (I,J); *** p < 0.001; NS, not significantly different from the positive control group

Figure 7

Fecal microbiota of Crohn’s disease patients was not modified following three subcutaneous injections with P28GST. (A) Rarefaction curves of observed OTUs as a function of reads demonstrated the great heterogeneity of microbiota among the patients at T0 and at T1 (B), n = 5. (C) Principal coordinate analysis (PCoA) of the fecal microbiota collected from 5 CD patients before and after P28GST injections using weighted UniFrac. Colors indicate samples from each individual. Comparison of beta diversity between before and after P28GST injections showed no significant difference with PERMANOVA test (p = 0.81, number of permutations 10,000). Individual relative abundances analyzed from 16S-based metagenomic sequences at the phylum (D) and at the family level (E) before (T0) and after achievement of the treatment protocol (T1) describe the well conserved distribution for each specimen.