There was no evidence (reduction in water consumption, change in stool type, weight loss, or scoring on the Mouse Grimace Scale [22]) that daily I.G. gavage of PBS or treatment caused any toxicity or adverse events during the first seven days prior to initiating DSS administration. Water consumption (with or without DSS) for each group was monitored during throughout the experiment, via weighing of the water bottle, with no significant difference in consumption between the groups.

Severity of ColitisWeight Loss

When compared to the naive control, the only two groups that lost significant weight were the “DSS-Only” group with a mean weight change of − 9.5% (p =  < 0.0001 [95% CI 6.848 to 16.58]) and EHv5HeLM- group with a mean weight change of − 6.9% (p = 0.0007 [95% CI 3.106 to 15.12]). EHv5WT and HeLM both showed a non-significant weight loss of − 2.92% (p = 0.1074 [95% CI − 0.6731 to 10.96]) and − 0.68% (p = 0.5586 [95% CI − 2.494 to 8.307]), respectively, Fig. 2A.

Fig. 2

Clinical assessment of colitis severity. A Weight loss was pronounced in the DSS-Only and EHv5HeLM- groups, and the EHv5WT and HeLM groups demonstrated no significant loss compared to the naïve control. B Colonic shortening was significantly less in the EHv5WT and HeLM groups compared to DSS-Only. No difference was seen in caecum size. Representative sections on the right with red line dividing colonic length and caecum measurements. C Administration of EHv5WT and HeLM resulted in significantly lower DAI scores when compared to the DSS-only. ns Non-significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001

Colonic Length

Colonic length has an inverse relationship to colonic inflammation and is established as a marker for the severity of inflammation in murine models of colitis [14]. Compared to the naïve control group (10.7 cm), all groups had significant shortening of the colon. However, compared to the DSS-only group (7.4 cm), EHv5WT and HeLM did significantly better with an average length of 9.3 (p =  < 0.0001 [95% CI − 2.588 to − 1.112]) and 9.2 cm (p =  < 0.0001 [95% CI − 2.439 to − 1.069]), respectively. There was significant shortening in the EHv5HeLM- group, 7.0 cm, which was not significant to the DSS-only group (p = 0.5137), Fig. 2B. No significant difference in the cecal length was found.

Disease Activity Index (DAI)

Symptoms of colitis, defined as a DAI score ≥ 2, were first displayed 3 days after administration of DSS, for both DSS-only and EHv5HeLM- groups. The severity of colitis in these two groups increased thereafter and became significantly divergent to the naïve control after five days of DSS (p = 0.0002 and 0.0004, respectively). In contrast, EHv5WT and HeLM remained protected from colitis, only reaching significant change from the naïve control after 7 days of DSS (p = 0.0170 and 0.0197), Fig. 2C. Importantly, when compared to the DSS-only group (the positive control), with a mean DAI score of 9.5 on day 15, EHv5WT and HeLM provided significantly successful treatment against colitis such that their scores were 3.8 (p =  < 0.0001 [95% CI 3.105 to 8.161]) and 3.9 (p =  < 0.0001 [95% CI 3.345 to 8.029]), respectively. This protective effect was not seen in EHv5HeLM- which had a mean score of 8.7 (p = 0.8659 [95% CI =  − 2.185 to 3.919]). EHv5WT, 3.9, and HeLM, 3.85, showed no significant increase in DAI when compared to the DSS-Only control, (p = 0.1731 and 0.0643, respectively), Supplementary Fig. 4. While the male mice in all groups demonstrated a more severe pattern of colitis, this was still significantly ameliorated in the EHv5WT and HeLM groups. When repeated measures from the analysis are removed, these two groups remain statistically non-divergent from the naïve control—i.e., their DAI score was unchanged despite receiving DSS.

Histological Scoring

The MCHI was developed to standardize histological assessment of colitis in the murine model and has been validated with a high inter-rater reliability [16]. The score range is 0 (no inflammation) to 22 (severe disease). The score for the naïve group on the MCHI histological scoring system was 0. The EHv5WT group and the HeLM groups scored 2.8 and 3, respectively, this was not significantly different from the naïve group (p = 0.5212 and 0.4429), Fig. 3A. The DSS-Only group had the highest score of 15.5 (p =  < 0.0001), EHv5HeLM- scored 15.2 (p =  < 0.0001). While the MCHI scoring system does not use categories (e.g., mild, moderate, or severe), it is our opinion that the EHv5WT and HeLM scores would represent a mild histological grading, equivalent to a Nancy Index score of 1. Accordingly, while there is no statistical difference in these scores and the naïve group, it should be noted that there is still a degree of “mild” inflammation that is seen on histology that could have clinical significance.

Fig. 3

Histological and biochemical severity of colitis. A Analysis of the Mouse Colitis Histology Index (MCHI) score shows that administration of EHv5WT or HeLM significantly reduced the histological severity of colitis seen, an effect not seen in the mutant EHv5HeLM-. Representative slides are on the right. B Fecal calprotectin levels were higher in the DSS-only and EHv5HeLM- groups compared to EHv5WT and HeLM, which when elevated demonstrated no significant difference to the naïve control. ns Non-significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001

Fecal Calprotectin

The heterodimer complex of S100A8/S100A9 proteins (Calprotectin) are pro-inflammatory metal sequesters found in abundance in the neutrophil cytoplasm. Upon neutrophil apoptosis, these proteins are released into the colonic lumen. Measurement of calprotectin has an established history in clinical practice as an objective measurement of colitis severity.

While both Ehv5WT and the HeLM groups demonstrated elevated calprotectin levels, compared to the naïve control (5.6 ng/mL), there was non-significant difference with mean concentrations of 16.3 (p = 0.4006, 95% CI − 28.27 to 6.850) and 15.1 ng/mL (p = 0.5178, 95% CI − 27.06 to 8.059), respectively, Fig. 3B. The DSS-only group measured 39.6 ng/mL, while EHv5HeLM- was 41.0 ng/mL (p =  < 0.0001 for both).

Gene Expression Analysis“Pro-inflammatory” Genes

All groups receiving 4% DSS had an upregulation in TNF-α; however, EHv5WT and HeLM had a significantly diminished response compared to the DSS-Only group, Fig. 4A. The expression fold change were as follows: DSS-Only = 2.72, EHv5WT = 1.64 (p = 0.0014), HeLM = 2.10 (p = 0.0445), and EHv5HeLM- = 2.31 (p = 0.3286). IL-6 gene expression was also increased across all groups. However, there was a significant difference, with a moderated expression change, in the EHv5WT = 0.53 (p = 0.0007) and HeLM = 1.13 (p = 0.0172) compared to the EHv5HeLM- = 3.06 (p = 0.1165) and the DSS-Only group = 2.24, Fig. 4B. IL-1β was increased in the DSS-Only and EHv5HeLM- groups (1.78 and 1.05, respectively). The HeLM group had a dampened level of expression change, 0.82 with the CI crossing 0 (p = 0.2408). The EHv5WT group had a marginal decrease in gene expression, − 0.08 (p = 0.0127), Fig. 4C. We found no significant difference in IL-17 or IL-23 expression across the groups (data not shown).

Fig. 4

Gene expression analysis. The following rectosigmoid RT-qPCR results are displayed as expression fold change to the log2, comparative to the naïve control. A Demonstrates that EHv5WT and HeLM reduced the expression of TNF-α. B IL-6 was also found to be downregulated for these two groups. C Expression of IL-1β was only significantly different in the EHv5WT group. D Shows that expression of IL-10 was upregulated in EHv5WT and HeLM while being downregulated in the DSS-Only and EHv5HeLM- groups. E All groups demonstrated downregulation of Claudin-I apart from the HeLM group which showed marginal increased expression. F and G Demonstrates an increased expression of TLR-2 and decreased expression of TLR-4, DSS-Only, and EHv5HeLM- had the opposite gene expression to this. ns Non-significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001

Anti-inflammatory and “Protective” Genes

Gene expression for IL-10 was markedly divergent between the treatment groups. The gene expression change for DSS-Only was − 0.849, similar to EHv5HeLM- =  − 0.98 (p = 0.9896). The other two groups however had significantly increased expression with EHv5WT = 2.14 (p =  < 0.0001) and HeLM = 2.71 (p =  < 0.0001), Fig. 4D. We found a non-significant general trend of a reduced expression of the Claudin-1 gene across all groups other than the HeLM group that showed a marginal increase in expression, albeit with a broad range. The expression fold changes were as follows: DSS-Only =  − 1.20, EHv5WT =  − 0.75 (p = 0.7572), HeLM = 0.33 (p = 0.0127), and EHv5HeLM- =  − 0.42 (p = 0.03723, Fig. 4E. There was no significant difference found for MUC-2 (data not shown).

Toll-Like Receptors (TLRs)

The DSS-Only and EHv5HeLM- groups were similar to each other, with the expression fold change for TLR-4 being increased 0.27 and 0.22 (p = 0.9933), respectively, while that of TLR-2 was decreased − 0.42 and − 1.11 (p = 0.5236), respectively. The inverse was true for EHv5WT and HeLM-treated groups with TLR-4 showing a reduced expression − 0.67 (p = 0.0011) and − 0.40 (p = 0.0090) and TLR-2 being increased 0.98 (p = 0.0360) and 1.69 (p = 0.0005), respectively, Fig. 4F, G, suggesting that the HeLM molecule may be agonistic to TLR-2.

Cell Culture Analysis

Purified HeLM was found to be toxic to the HEK-Blue cell culture lines at high concentrations (150 μg/mL). HeLM, and its cyclic lipopeptides, have strong biosurfactant properties that disrupt membranes and have long been known to be cytotoxic at higher concentrations to a variety of cell lines in vitro [23]. Accordingly, HeLM required a 1/200 dilution (0.75 μg/mL) for incubation to be successful. Results were deemed significant if the fold induction was ≥ 2. There was no agonism of the HEK-Blue Null1 cell culture (negative control), furthermore HeLM did not inhibit the agonism of TNFα (positive control). HeLM demonstrated significant agonism to the HEK-Blue hTLR2 cell line. This was seen down to a dilution of 0.5 × 10−7 (0.00075 ng/mL), Table 1. No significant agonistic effects were seen on the HEK-Blue hTLR4 cell line, Table 1. At higher concentrations (0.75–0.075 μg/mL), HeLM was found to have a significant antagonistic effect on HEK-Blue hTLR4. This appears to be due to HeLM having an inhibitory effect directly on the control agonist, LPS (derived from Escherichia coli), rather than on the hTLR4 complex, Table 1.

Table 1 HEK-blue cell culture analysisMetagenomics

The relative abundance of the bacterial species were proto-typical of mammalian feces, with the phyla Firmicutes Bacteroidota, Proteobacteria, and Actinobacteriota being predominant, Fig. 5A, B. Results show groups before treatment/colitis (panel A) and after (panel B). When looking at the individual group’s species richness (α diversity), both the Simpson Diversity and Shannon indices showed no significant difference when looking at the intra-group time points. While not reaching significance, a decrease in the α diversity, by both metrics, was seen in the DSS group which was not apparent in the EHv5WT and HeLM groups. For example, the Shannon Index shows a decrease of 0.74 for the DSS-Only group, compared to an increase of 1.175 and 1.45 for the EHv5WT and HeLM groups, respectively, Fig. 5C. We next looked at the variability in community composition (β diversity) by calculating weighted and unweighted UniFrac distances. These were constructed on Principle Coordinate Analysis (PCoA), Fig. 5D, and Non-Metric Multidimensional Scaling (NMDS) graphs, Fig. 5E. There was no apparent clustering of the groups by their time point (before or after administration of DSS) during the experiment. Statistical analysis on both of these measures, with post hoc analysis, showed no significant intra-group difference between the time points of before colitis induction to after. The same was true of the NMDS plot for the Bray–Curtis value of dissimilarity, Supplementary Fig. 5.

Fig. 5

Metagenomic analysis. a Refers to samples taken at the start of the experiment (day − 1); b samples taken on the final day (day 15). A Relative abundance of Phyla according to group. B Phylogenic evolutionary tree by the top 100 genera. C Boxplot of the Shannon Index of Alpha-diversity—all results were non-significant from time point a to b. D 3D visualization of the Principal Coordinate Analysis (PCOA) of Weighted UniFrac distance. E Non-metric Multidimensional Scaling (NMDS) plots, with stress results of 0.12 and 0.1 for weighted and unweighted UniFrac, respectively

To detect any differences that may have been missed by UniFrac measurements, t tests were performed at the Phyla level. This showed the DSS-Only group had a significantly reduced Firmicutes population [− 0.11 (− 0.0059 to − 0.2317) p = 0.0422], along with a significant increase in Actinobacteriota. Of the other groups, the only Phyla to reach significant difference was in the HeLM group where a 0.024 difference was noted in Actinobacteria (p = 0.03215; 0.0048–0.04258), but given the small effect size the clinical significance of this is uncertain. Interestingly, when taken down to the genus and species level, the DSS-Only group demonstrated a reduced abundance in bacteria of the genus Blautia and Lachnospiraceae species both of which are characterized as producers of butyrate [24, 25]—a short chain fatty acid that is essential for the normal function of colonocytes.