Abstract

Introduction

The recognition of the role of the immune system in cancer development has opened up novel therapeutic opportunities for cancer immunotherapy. Several strategies, including the use of chimeric antigen receptor T cells and immune checkpoint blockade, have represented major milestones in the therapy against cancer. Despite the great success of cancer immunotherapy using immune checkpoint inhibitors (ICIs), their therapeutic benefits are limited—60%–70% of patients do not respond to single agent immunotherapy—by either various resistance mechanisms1 or ICI-associated toxic effects, including frequent gastrointestinal, endocrine, and dermatological toxicities and fatal neurotoxicity and cardiotoxicity.2 Thus, novel therapeutic strategies that provide manageable side effects to existing ICI would enhance and expand their therapeutic efficacy and application, as recently suggested.3

Due to its proven role in cancer development and immune regulation, gut microbiome has gained increasing expectation as a potential armamentarium to improve cancer immunotherapy.4 5 Studies have shown that the effectiveness of immunotherapy against different tumors requires the presence of commensal bacteria.6–8 Clinical studies have corroborated these findings with compelling evidence that microbial richness and diversity are associated with a durable response to immunotherapy5 9 with gut microbiota signatures predicting toxicity associated with combined cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1) blockade.10 However, there is no identifiable pattern among the bacteria found to influence cancer therapies. Both Gram-positive and Gram-negative, both facultative and obligate anaerobes, and both coccus and rod-shaped bacteria are found to influence ICI treatment outcome and adverse effects (AEs).11 Bacteroides, for instance, have shown a positive effect with anti-CTLA-4 therapies8 but negative effects with anti-PD-1 therapies, being enriched in non-responders9 and in patients with ICI-related toxicity.10 Thus, understanding how the repertoire of commensal microbes can be specifically manipulated to improve ICI therapeutic effectiveness has remained elusive. Considering that the gut microbiota potentially affects the efficacy of ICI through several mechanisms, including the production of critical metabolites,12 it is not surprising that the expression of certain metabolites is different in responders compared with non-responders to immunotherapy.13 This implicates that microbial metabolites can be used as predictive biomarkers of response to immunotherapy and points to a mean to optimize the efficacy of immune checkpoint blockade.

Microbiota-dependent tryptophan catabolites are abundantly produced within the gastrointestinal tract and are known to exert profound effects on host physiology, including the maintenance of epithelial barrier function and immune homeostasis.14 These metabolites are known to act as ligands of the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor first identified for its role in the binding and metabolism of xenobiotics and now implicated in a multiplicity of activities, such as the regulation of the immune response and the maintenance of the intestinal barrier homeostasis and microbial symbiosis.15 We have shown that indole-3-carboxaldehyde (3-IAld), produced in condition of unrestricted tryptophan availability in both mice16 17 and humans,17–21 is a ligand of both murine and human AhR16 and evidence has since accumulated to link defective production of 3-IAld in certain clinical22–24 and preclinical25 settings involving impaired barrier function and mucosal homeostasis.25 26

Based on these premises, in this study, we have evaluated whether and how 3-IAld could prevent intestinal toxicity, a common side effect of CTLA-4 inhibitors,2 while preserving the ICI therapeutic efficacy. To this purpose, we have administered an enteric formulation of 3-IAld, recently shown to increase its efficacy while minimizing unwanted toxicity through localized delivery in the small intestine,27 to mice with anti-CTLA-4-induced colitis.28 We found that 3-IAld protected mice from intestinal damage via a dual action on both the host and the microbe sides. Indeed, paralleling the activation of the host AhR/interleukin (IL)-22-dependent pathway, 3-IAld also affected the composition and function of the microbiota such that fecal microbiome transplantation (FMT) from 3-IAld-treated mice protected against ICI-induced colitis likely via butyrate-producing bacteria. Importantly, while preventing intestinal damage, 3-IAld did not impair the antitumor activity of anti-CTLA-4 in a murine model of melanoma. Therefore, enteric formulated 3-IAld in combination therapy with ICI may preserve the mucosal integrity in the gastrointestinal tract without negatively affecting ICI therapeutic effectiveness.

Methods

Results

3-IAld protects against ICI-induced colitis

To evaluate whether enteric formulated 3-IAld would rescue from intestinal damage and toxicity in ICI-induced colitis, we resorted to a murine model of ICI-induced colitis in which the vigorous T-cell response promoted by anti-CTLA-4 treatment exacerbated the DSS-induced intestinal damage.33 34 Administration of 3-IAld starting the week before and continuously until the end of the experiment (figure 1A) improved survival (figure 1B), prevented weight loss (figure 1C), ameliorated disease activity index (figure 1D), and improved gross pathology (figure 1E) in colitic mice. Of note, the protective effects of 3-IAld were comparable with those observed on the administration of the high-affinity AhR ligand, FICZ (figure 1B–E). Similar results were obtained in models of immune-mediated colitis obtained by infusion of human PBMCs or murine CD4⁺ T cells in NSG (figure 1F–J) or RAG1-deficient mice (online supplemental figure S2), respectively, and subsequent treatment with anti-CTLA-4. The treatment with 3-IAld increased survival (figure 1F), reduced weight loss (figure 1G), improved inflammatory histopathology and barrier function, as revealed by the expression of the tight junction zonula occludens 1 (ZO-1) (figure 1H, I), and decreased inflammatory cytokine production (figure 1J) in NSG and RAG1-deficient mice (online supplemental figure S2). Similarly, the protective effects of 3-IAld were associated with an improved colon histopathology (figure 2A, B) and intestinal barrier integrity in mice with DSS-colitis, as revealed by ZO-1 expression and proliferation of intestinal stem cells, revealed by Ki-67 staining (figure 2C) and Lrg5 expression (figure 2D). Of interest, 3-IAld also promoted the expression of Nfil3, a transcription factor that directs the development of innate lymphoid cells (ILC)35 known to maintain epithelia barrier integrity36 as well as of Muc1, a cell surface mucin that functions as a barrier to infection and as regulator of inflammation37 (figure 2D). In agreement with these data, the passage of dextran-FITC across the intestinal barrier was reduced (figure 2E), and the levels of soluble CD14, a marker of gut permeability,38 were decreased (figure 2F). These changes were paralleled by a switch towards an anti-inflammatory profile with reduced levels of TNF-α, IL-1β and IL-17A, and increased amounts of IL-10 (figure 2G). Consistent with the anti-inflammatory profile, the levels of calprotectin were also reduced (figure 2H). Considering that 3-IAld is defective in mice with colitis,25 these results indicate that 3-IAld supplementation may protect against ICI-induced colitis by maintaining epithelial barrier integrity and dampening the inflammatory response, an activity consistent with its AhR-agonistic activity and comparable to FICZ.

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Figure 1

3-IAld protects mice from ICI-induced colitis. C57BL/6 mice were treated with DSS in drinking water for 1week followed by a recovery period of another week, and administered 100µg of anti-CTLA-4 mAb or isotype control two times (at days 0, 4, and 8 following DSS administration). 3-IAld (18mg/kg) was administered intragastrically every other day starting 4days before DSS treatment. FICZ was used as control as depicted in the experimental schedule (A). Mice were evaluated for (B) % survival, (C) % weight change, (D) disease activity index, and (E) rectal bleeding. NSG mice infused intraperitoneally with hPBMCs were treated with αhCTLA-4 and 3-IAld. Mice were sacrificed at 21 days and evaluated for (F) % survival, (G) % weight change, (H) histology score, (I) colon histology (PAS staining) and ZO-1 protein expression and (J) inflammatory cytokine expression. Photographs were taken with a high-resolution microscope (Olympus BX51), ×20 magnification (scale bars, 200µm). White arrow indicates rectal bleeding. Yellow arrows indicate inflammatory cells recruitment. For histology, data are representative of two or three independent experiments. Each in vivo experiment includes four to six mice per group (16–24 mice in each experiment). Data are represented as mean±SEM. (B–D) Anti-CTLA-4-treated mice versus none (isotype control), anti-CTLA-4+3-IAld- versus anti-CTLA-4-treated mice, (F–J) hPBMCs+αhCTLA-4 versus hPBMCs (+humanIgG) or hPBMCs+αhCTLA-4+3IAld. Two-way analysis of variance, Bonferroni post hoc test. H₂O, untreated mice. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. 3-IAld, indole-3-carboxaldehyde; FICZ, 6-formylindolo(3,2-b)carbazole; hPBMC, human peripheral blood mononuclear cell; ICI, immune checkpoint inhibitor; PAS, periodic acid–Schiff.

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Figure 2

3-IAld prevents epithelial damage in DSS plus anti-CTLA-4-induced colitis. C57BL/6 mice were subjected to DSS plus anti-CTLA-4-induced colitis and administered 3-IAld as described in the legend of figure 1. Mice were evaluated for (A) colon histology (periodic acid–Schiff staining), (B) histology score, (C) ZO-1 and Ki-67 protein expression, (D) intestinal stem cells genes expression, (E) Dextran-FITC and (F) sCD14 levels in the serum, (G) cytokine and (H) calprotectin levels in colon homogenates. For immunofluorescence, nuclei were counterstained with Hoechst 33342. Photographs were taken with a high-resolution microscope (Olympus BX51), ×40 magnification (scale bars, 100µm). Yellow arrows indicate inflammatory cell recruitment. For histology and immunofluorescence, data are representative of three independent experiments. Each in vivo experiment includes three to six mice per group (15–30 mice in each experiment). Data are represented as mean±SEM, (B) Kruskal-Wallis test. (D–H) Anti-CTLA-4- versus none (isotype control) or anti-CTLA-4+3-IAld-treated mice. One-way analysis of variance, Bonferroni post hoc test. H₂O, untreated mice. *P<0.05, **P<0.01, ***P<0.001. 3-IAld, indole-3-carboxaldehyde; FICZ, 6-formylindolo(3,2-b)carbazole; IL, interleukin; TNF-α, tumour necrosis factor alpha.

Beneficial effects of 3-IAld are not dependent on IDO1 and IL-10

We have previously demonstrated that IDO1, an enzyme involved in the kynurenine pathway of tryptophan metabolism,39 mediated protection from intestinal pathology in a murine model of ICI-induced colitis with increased production of IL-10.28 We evaluated whether the IDO1/IL-10 pathway was involved in the therapeutic activity of 3-IAld. As shown in figure 3, 3-IAld increased neither IDO1 gene or protein expression (figure 3A, B) nor its enzymatic activity (figure 3C), as measured by the kynurenine/tryptophan ratio. Accordingly, the downstream genes Haao and Kynu were not increased on administration of 3-IAld (figure 3D), likely indicating that the activity of 3-IAld occurs independently of IDO1 engagement. Experiments in IDO1-deficient mice with ICI–colitis confirmed that the protective effects of 3-IAld did not involve IDO1 (figure 3E, F). Likewise, experiments in IL-10-deficient mice revealed that the activity of 3-IAld did not involve IL-10 either. Due to the existing epithelial damage, a chronic enterocolitis develops in these mice by 2–3 months of age.40 Therefore, we administered 3-IAld, as indicated in figure 1A, to 10-week-old mice treated with a lower dosage of DSS with and without concomitant anti-CTLA-antibody. We first noticed that IL-10-deficient mice at variance with wild-type mice developed signs of colitis on anti-CTLA-4 treatment alone (figure 3G), one first finding demonstrating that anti-CTLA4 treatment alone can drive intestinal inflammation de novo. As expected, the severity of colitis increased on concomitant DSS and anti-CTLA-4 treatment (figure 3I, J). Strikingly, 3-IAld not only promoted mucin homeostasis as seen on PAS/Alcian blue staining and the resolution of the rectal prolapse (online supplemental figure S3) in IL-10-deficient mice with DSS-colitis but also prevented weight loss (figure 3H) and improved histopathology (figure 3G, I, J) in these mice on anti-CTLA-4 treatment alone or combined with DSS. These results would indicate that 3-IAld protects from epithelial damage and immune-mediated damage in the relative absence of IL-10.

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Figure 3

Protective activity of 3-IAld is not dependent on IDO1 and IL-10. C57BL/6 (A–D, G), Indo1 ^–/– (E–F) and Il10 ^–/– (G–J) mice were subjected to anti-CTLA-4 with (A–F, H–J) or without (G) DSS and administered 3-IAld as described in the legend of figure 1. Mice were evaluated for (A) IDO1 gene and (B) protein expression, (C) Trp, Kyn levels and Kyn:Trp ratio, (D) Haao and Kynu expression, (E, H) % weight change, (F–J) colon histology (periodic acid–Schiff staining), (I) histology score. For immunofluorescence, nuclei were counterstained with Hoechst 33342. Photographs were taken with a high-resolution microscope (Olympus BX51), ×20 magnification (scale bar, 200µm). For histology and immunofluorescence, data are representative of three independent experiments. Each in vivo experiment includes 3 mice per group (9–18 mice total). Data are represented as mean±SEM. Anti-CTLA-4+3-IAld- versus anti-CTLA-4-treated mice. One-way analysis of variance, Bonferroni post hoc test, (I) Kruskal-Wallis test. H₂O, untreated mice. *P<0.05, **P<0.01. 3-IAld, indole-3-carboxaldehyde; IDO1, indoleamine 2,3-dioxygenase 1; IL, interleukin; Kyn, kynurenine; ns, not significant; Trp, tryptophan.

Beneficial effects of 3-IAld involve the AhR/IL-22 axis

It has been suggested that signaling by indoles in response to inflammation induced by acute stressors occurs through IL-22,41 a cytokine that depends on AhR42 and partners with IL-10 in maintaining mucosal homeostasis.43 We found that administration of 3-IAld to DSS +anti-CTLA-4 antibody-treated mice induced the expression of the AhR-dependent genes Cyp1a1 and Ahrr (figure 4A), a finding suggesting AhR engagement. Experiments in AhR-deficient mice confirmed the lack of 3-IAld’s activity in these mice (figure 4B, C) as already described.16 Moreover, 3-IAld also induced IL-22 gene and protein expression (figure 4A, D), as well as the downstream target Reg3γ (figure 4E), an antimicrobial peptide produced by intestinal epithelial cells. In agreement with previous findings,16 experiments in IL-22-deficient mice directly proved that IL-22 is instrumental for 3-IAld’s activity in colitis. We found that the weight loss (figure 4F), disease activity index (figure 4G), and colon histopathology (figure 4H, I) were not rescued by 3-IAld in these mice. Similarly, 3-IAld neither prevented epithelial barrier damage, as shown by ZO-1 staining (figure 4I), nor reverted the inflammatory profile (figure 4J) in the relative absence of IL-22. These results indicate that the protective activity of 3-IAld against colitis depends on AhR and IL-22, while IDO1 and IL-10 are dispensable.

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Figure 4

Beneficial effect of 3-IAld involves the AhR/IL-22 axis. C57BL/6 (A, D, E), AhR ^–/– (B, C), Il22 ^–/– (F–J) mice were subjected to DSS plus anti-CTLA-4-induced colitis and administered 3-IAld as described in the legend of figure 1. Mice were evaluated for (A) AhR-related genes, (B, F) % weight change, (C, I) colon histology (periodic acid–Schiff staining), (D) IL-22 levels, (E) Reg3γ gene expression, (G) disease activity index, (H) histology score, (I) ZO-1 expression, and (J) cytokine production in colon homogenates. For immunofluorescence, nuclei were counterstained with Hoechst 33342. Photographs were taken with a high-resolution microscope (Olympus BX51), ×20 magnification (scale bars, 200µm). For histology and immunofluorescence, data are representative of three independent experiments. Each in vivo experiment includes 3 mice per group (9–12 mice in each experiment). Data are represented as mean±SEM. Anti-CTLA-4+3-IAld- versus anti-CTLA-4-treated mice. One-way analysis of variance, Bonferroni post hoc test, (F) Kruskal-Wallis test. H₂O, untreated mice. None, DSS+isotype control. **P<0.01. 3-IAld, indole-3-carboxaldehyde; IL, interleukin; ns, not significant; TNF-α, tumour necrosis factor alpha.

3-IAld modifies intestinal microbiota composition and function

In addition to preserving an intact epithelium, the AhR/IL-22 axis maintains a balanced microbiota and a functioning defense system.44 45 We therefore evaluated whether treatment with 3-IAld would affect the fecal microbial composition. Bacteroidota represented the most abundant phyla in murine feces followed by Firmicutes, while Proteobacteria and Actinobacteria were present at very low abundances (figure 5A). At genus level, Muribaculaceae (former S24–7) were the most abundant, followed by members of Clostridia (Lachnospiraceae and Oscillospiraceae) and Bacteroidia (Prevotellaceae, Rikenellaceae, and Bacteroidaceae) (online supplemental figure S4). The analysis of alpha diversity did not reveal significant differences in richness and evenness between untreated and 3-IAld-treated groups, as measured by observed operational taxonomic units (OTUs), Chao1 and Shannon indexes (figure 5B). On the contrary, significant differences were observed in compositional structure, as measured by Jaccard and Bray-Curtis indexes (figure 5C). Indeed, PCoA analysis derived from Jaccard and Bray-Curtis distances revealed the presence of distinct clusters (online supplemental figure S5). In order to identify putative 3-IAld microbial signatures, we performed high-dimensional class comparisons using LEfSe. As shown in figure 5D, E, we observed that treatment with 3-IAld was associated with a predominance of members of Bacteroidota (Prevotellaceae, Bacteroidaceae, and Muribaculaceae), Desulfobacterota and, among Firmicutes, of Lachnospiraceae (Roseburia genus). Increased levels of alphaproteobacteria and different members of Bacteroidota (Rikenellaceae and Bacteroidales F082) were instead observed in untreated mice. We then resorted to Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2 (PICRUSt2) and the KEGG database to determine the abundance of functional modules inferred from 16S data, and LEfSe was applied in order to evaluate significant association in either untreated or 3-IAld-treated samples. As shown in figure 5F, pectin degradation followed by transport systems, such as phosphate, lipopolysaccharide, tungstate, glutamine and the phosphoenolpyruvate–carbohydrate phosphotransferase (PTS) system, and sulfur reduction, were the modules most increased by 3-IAld, while biosynthetic pathways, that is, ubiquinone, fatty acids, polyamine, cysteine and threonine biosynthesis, were reduced. The relative abundance of pectin-degrading and sugar-degrading anaerobic bacteria on 3-IAld treatment led us to assess the fecal and serum levels of SCFAs that, together with different gases (including hydrogen sulfide, H₂S), are known end products of bacterial fermentation of pectin.46 Consistent with the selective increase of the genus Roseburia, known to produce high levels of butyrate,47 the fecal levels of n-butyric acid were significantly increased along with those of valeric acid and, to a lesser extent, propionic acid (figure 5G). However, SCFAs were not increased, if not decreased for the acetic acid, at the systemic level (figure 5G). Thus, the combined metagenomics and metaproteomic analysis revealed that 3-IAld is able to shift the microbial composition toward sugar-fermenting bacteria and localized SCFA production.

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Figure 5

3-IAld modifies intestinal microbiota composition and function. (A) Barplot showing bacterial composition (abundance percentage) of each sample at phylum level. Taxa are differentiated by colors. Samples are ranked based on the abundance of the most abundant Phylum (Bacteroidota) and grouped by None and 3-IAld-treated. (B) Boxplots of observed features, Chao1 and Shannon alpha diversity indexes. Significance was evaluated by applying a Kruskal-Wallis test (ns). (C) Boxplots of Jaccard and Bray-Curtis beta diversity indexes evaluating distances within or between none and 3-IAld-treated samples. Significance was evaluated by applying a Kruskal-Wallis test (the p value is indicated). (D, E) LEfSe at genus level. LEfSe emphasizes a set of features that significantly discriminate between none and 3-IAld treatments. A p value of <0.05and an LDA score of ≥3.5 were regarded as significant in Kruskall-Wallis and pairwise Wilcoxon tests, respectively. The cladogram (D) simultaneously highlights both phyla and specific genera. Taxa (circles) are colored green when significantly associated to none, red when significantly associated with 3-IAld-treated samples, and yellow when not significantly associated to either groups. The size of each circle is proportional to the abundance of the corresponding taxon in all samples. The barplot of the LDA scores (E) shows genera significantly associated with either none and 3-IAld-treated samples accordingly to legend colors. LEfSe has been applied with default alpha values. (F) LEfSe on KEGG modules. Barplot of the LDA score computed on metabolic functions inferred by PICRUSt2 analysis (KEGG modules) and significantly associated with either none or 3-IAld-treated samples accordingly to legend colors. The threshold value of the logarithmic LDA score was set to 2.0. (G) Levels of SCFA measured in the feces and serum by mass spectrometry. (A–F) Six mice per group (12 mice total); (G) 3–6 mice per group (9 mice total). Data are represented as mean±SD. None versus 3-IAld, t test. *P<0.05, **P<0.01. 3-IAld, indole-3-carboxaldehyde; LDA, linear discriminant analysis; LEfSe, linear discriminant analysis together with effect size evaluation; ns; not significant.

3-IAld-modified microbiota provides protection to ICI-induced colitis

We resorted to fecal microbiota transplantation to evaluate the activity of 3-IAld-modified microbiota in colitis. Feces were collected from untreated or 3-IAld-treated mice for 6days and transplanted (FMT) into recipient mice before and at the onset of colitis.32 At variance with transfer of feces from untreated mice, the transplantation of feces from 3-IAld-treated mice prevented weight loss (figure 6A), and ameliorated colon histopathology (figure 6B–D) in DSS-treated mice and similarly in DSS +anti-CTLA-4-treated mice (figure 6E). The reduced colonic inflammation was accompanied by the induction of IL-10-producing regulatory T cells (Treg) (figure 6F), as revealed by the reversal of DNA hypermethylation of Foxp3 promoter, likely occurring via butyrate (figure 6F), as already shown.44 Indeed, the finding that the protective activity of feces from 3-IAld-treated mice involved host IL-22 more than IL-10 (figure 6G–I), indicates that the beneficial activity of 3-IAld may occur through different pathways that include the modification of the composition and function of the microbiota, the increase intestinal barrier via the AhR/IL-22 axis and the control of inflammation via Treg cells.

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Figure 6

3-IAld-modified microbiota provides protection to ICI-induced colitis. C57BL/6 (A–F), Il22 ^–/– and Il10 ^–/– (G–I) mice were subjected to DSS-colitis with (E) or without (A–D, F–I) anti-CTLA-4 and transplanted with fresh fecal pellets from control or 3-IAld-treated mice 1day before and 2days after colitis induction. One group of mice was treated with 1% butyrate in drinking water 1week before starting DSS administration. Mice were sacrificed 7 (A–D, F–I) or 14 (E) days after colitis induction and evaluated for (A, E) % weight change, (B) gross pathology, (C, H) histology score, (D, G) colon histopathology (periodic acid–Schiff staining), (F) methylation/demethylation status of Foxp3 promoter in mesenteric lymph nodes, and (I) sCD14 serum levels. Photographs were taken with a high-resolution microscope (Olympus BX51), ×10 and ×20 magnification (scale bars, 500 and 200µm). For histology, data are representative of three independent experiments. Each in vivo experiment includes 3 mice per group (6–12 mice in each experiment). Data are represented as mean±SD. Treated versus control FMT mice. One-way analysis of variance, Bonferroni post hoc test, (C) Kruskal-Wallis test. H₂O, untreated mice. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. 3-IAld, indole-3-carboxaldehyde; FMT, fecal microbiota transplantation. ns, not significant.

3-IAld does not interfere with the antitumor activity of anti-CTLA-4 antibody

The potential application of 3-IAld requires that the protection against side effects does not compromise the therapeutic efficacy of ICI. For this reason, we assessed the effect of 3-IAld in the anti-CTLA-4-responsive B16 melanoma model. 3-IAld neither modified tumor growth nor interfered with the therapeutic efficacy of anti-CTLA-4 antibody (figure 7A, B) and did not affect recruitment of CD4⁺ and CD8⁺ tumor-infiltrating lymphocytes (figure 7C, D), consistent with the increased expression of leukocyte-recruiting chemokine Cxcl9 and effector Perforin (figure 7E). Likewise, 3-IAld did not interfere with the therapeutic efficacy of anti-PD-1 antibody in a model of Lewis lung carcinoma. Indeed, 3-IAld did not prevent the ability of anti-PD-1 antibody to increase survival (figure 7F), decrease tumor growth (figure 7G), improve gross pathology (figure 7H), and reduce the recruitment of Foxp3⁺CD25⁺Treg cells in the lung (figure 7I, J). Thus, the beneficial activity of 3-IAld may extend to combination immunotherapy.

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Figure 7

3-IAld does not interfere with the antitumor activity of anti-CTLA-4 antibody. (A–E) C57BL/6 mice were subcutaneously injected with B16 tumor cells and administered 100µg anti-CTLA-4 mAb or isotype control intraperitoneally four times at 3-day intervals up to 16 days. 3-IAld was administered intragastrically every other day. Mice were evaluated for (A) tumor growth, (B) histology (periodic acid–Schiff staining), (C) immunohistochemistry for CD8⁺ and CD4⁺ tumor-infiltrating cells and (D) number of positive cells per HPF, and (E) Cxcl9 and Perforin expression in melanoma. (F–J) C57BL/6 mice were orthotopically injected with LLC cells and administered 200µg anti-PD-1 mAb or isotype control intraperitoneally five times at 3-day intervals up to 18 days. 3-IAld was administered intragastrically every other day. Mice were evaluated for (F) % survival, (G) lung weight, (H) lung gross pathology, (I, J) CD4⁺CD25⁺Foxp3⁺ cells. Photographs were taken with a high-resolution microscope (Olympus BX51), ×40 magnification (scale bars, 100µm). For histology, data are representative of two independent experiments. Each in vivo experiment includes three mice per group (nine mice in each experiment). Data are represented as mean±SEM. Treated versus none (isotype control) or αCTLA-4/αPD-1+3-IAld-treated versus αCTLA-4/αPD-1-treated mice. One-way analysis of variance, Bonferroni post hoc test. *P<0.05, **P<0.01, ***P<0.001. 3-IAld, indole-3-carboxaldehyde; HPF, high-power field; TIL, tumor-infiltrating lymphocyte.

Discussion

This study is a proof-of-concept demonstration of the therapeutic potential of bacterial metabolites as biologics capable of alleviating ICI-induced intestinal toxicity without interfering with ICI’s antitumor activity. We found that 3-IAld, directly delivered in the intestine, activated the AhR/IL-22 pathway for epithelial barrier function and immune homeostasis during colitis. Despite the ability of 3-IAld to promote IL-10R1 expression in the gut25 and the ability of the IDO1/IL-10 pathway to afford protection from ICI-induced intestinal damage,28 we did not find the involvement of this pathway in our setting. Interestingly, however, the chronic administration of 3-IAld (online supplemental figure S6A) only slightly prevented weight loss (online supplemental figure S6B) and improved colon histopathology in DSS colitis of aged IL-10-deficient mice (online supplemental figure S6C, D), implicating a possible role for IL-10 in mediating the clinical response of 3-IAld during chronic intestinal epithelial damage.

Gastrointestinal inflammation is a common immune-related AE of anti-CTLA-4 treatment due to alterations in gut barrier integrity.48 However, decreased gut barrier function induced by immunotherapy increased the therapeutic efficacy of ICI by favoring systemic translocation of microbes and microbial metabolites that act through innate immune pathways contribute to the therapeutic success of immunotherapy.11 It has been found that patients who experience some grade of intestinal inflammation had a significantly higher chance of responding to ICI treatment.49 Studies in mice and humans with melanoma have shown that members of the Bacteroidales order were instrumental for the T helper 1 (Th1) antitumor immunity elicited by anti-CTLA-4.8 Paradoxically, however, transplantation of Th1-promoting Bacteroides fragilis was associated with a reduced deterioration of the enteric mucosa,33 thus segregating its role in immunotherapeutic effectiveness from that in toxicity. Our results are consistent with this observation as 3-IAld supplementation during immunotherapy, while strengthening the mucosal barrier function, did not impede the recruitment of CD4⁺ and CD8⁺ lymphocytes at the tumor site. Thus, the therapeutic success of anti-CTLA-4 antibody may occur independently of bacteria translocation and accumulation at the tumor site and likely occurs through alternative mechanisms, including the systemic translocation of metabolites, as recently proposed.11 In this regard, we found that 3-IAld itself, which is systemically detected after enteric administration,50 did not show a direct antitumor activity in vivo or in vitro on cultured B16 cells (data not shown), a finding consistent with the disparate role of AhR ligands in cancer.51 Thus, metabolites other than 3-IAld may contribute to the antitumor activity of CTLA-4 inhibitors in our setting. This is likely to occur as we have found that the levels of n-butyric and valeric acid were significantly increased after 3-IAld’s treatment. That indoles are able to induce butyrate, known to promote IL-22 production52 while repressing IDO1 expression in the gut53 has already been reported.41 However, while protecting from intestinal epithelial damage, systemic butyrate limited the antitumor effect of CTLA-4 blockade in hosts with cancer.54 Neither the antitumor activity of anti-CTLA-4 antibody was impaired nor the serum levels of butyrate increased in 3-IAld-treated mice, as opposed to butyrate-treated mice (data not shown), a finding pointing to the potential clinical utility of 3-IAld in finely regulating the local versus systemic butyrate levels. Of interest, we found that valeric acid, a potent histone deacetylase inhibitor endowed with antitumor activity55 was increased after 3-IAld treatment. Although an attractive working hypothesis, whether and how the valeric acid contributes to the overall effects of 3-IAld is presently unknown.

It is clear that 3-IAld modulates the composition and function of the gut microbiome, an activity consistent with the ability of indole and indolyl compounds to act as trans-kingdom signaling molecules.56 We have indeed shown that 3-IAld exhibited potent antimicrobial activity in vitro57 and modulated the composition of airway and gut microbiota in murine model of cystic fibrosis.58 Alternatively, 3-IAld may affect the microbiota composition via the regulatory activity of IL-22 on the microbiota.45 This mechanism has been recently reported for indole-3-carbinol that, by inducing IL-22 from ILC type 3, promoted the expansion of butyrate-producing bacteria, such as Roseburia spp.41 Consistent with the ability of 3-IAld to promote ILC3 (16), we obtained similar results here by showing the predominant expansion of sugar-degrading members of Bacteroidota and Firmicutes by 3-IAld. While Bacteroidota are involved in multiple activities, in addition to monosaccharide metabolism, Firmicutes rely on the PTS system for transport and metabolism of disaccharides, monosaccharides and other sugar derivatives,59 eventually converging on the butyrate biosynthesis pathway and butyrate production.60 Growing experimental evidences support the importance of butyrate, a four-carbon SCFA, for intestinal health,61 even though diverging results were obtained on butyrate induction or supplementation in patients with inflammatory bowel disease.62 Transplantation of feces from 3-IAld-treated mice or butyrate supplementation was able to protect from colitis in IL-22-sufficient mice, thus directly proving that 3-IAld at the forefront of host–microbe interactions coordinates the activity of the microbiota and the host during ICI therapy. Likely as a result of the cross-feeding networks, we also found that sulfate reducing bacteria belonging to the Desulfobacterota phylum, abundantly present in the human gut,63 were expanded after 3-IAld treatment. It is known that dietary components provide fiber and macromolecules that are degraded by bacterial enzymes to monomers, and these are fermented by intestinal bacteria with the production to molecular hydrogen which promotes the metabolic dominance by sulfate-reducing bacteria.64 As one of the three mammalian gasotransmitters, H₂S plays a major role in maintaining physiological homeostasis. Numerous homeostatic functions have been proposed for H₂S, including cytoprotection and anti-inflammatory activity in the gut, neuromodulation, and cardiovascular function.65 Therefore, 3-IAld appears to modulate the function of the microbiota to engage multiple protective functions, mainly centered on carbohydrate metabolism, which raises the possibility of conceptualizing 3-IAld as a prebiotic substance.

Much has to be learned to fully harness gut microbiome for clinical applicability, including the definition of a shared gut microbiome across various solid tumors treated with ICI. Our study provides a piece of evidence that moving past bacterial phylogeny and focusing on bacterial metabolome may lead to a new class of discrete molecules that working at the interface between microbes and the host immune system may optimize ICI therapy via a precision enteric medicine that targets the epithelial barrier for mucosal homeostasis and prevention of distant immunopathology.66 Future human studies are needed to confirm whether 3-IAld, commonly detected in humans, could be used as a modifier of the mucosal milieu to provide an allied microbiota to optimize ICI regimens and/or as a biomarker for predicting ICI efficacy. In this regard, it is encouraging that 3-IAld attenuated the increase in epithelial permeability caused by inflammatory stimuli in an in vitro preclinical screen using the human Caco-2 epithelial cell line.67

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