The varying regional structure and functionality in the intestine serve to influence the local mucosal-associated microbiota along the gastrointestinal tract. In turn, the microbiome plays an important role in host nutrition, intestinal homeostasis and the development of disease. A number of studies have highlighted the particular importance of the resident ileal microbiota in relation to these processes[1-5]. The ileum supports the largest microbial community in the small intestine, with up to 108 bacteria/g luminal material. The predominant phyla are Firmicutes and Actinobacteria. It has been shown previously that segmented filamentous bacteria (Firmicutes, proposed genera Savagella) influences susceptibility to colitis, and promote the differentiation of mucosal Th17 cells in the intestine. Intestinal mucosal Th17 responses are associated with the development of colitis-associated cancer and are also proposed to influence systemic immune responses and inflammatory diseases[11-13].
A recent paper by Roberti and colleagues (2020) has demonstrated further evidence for the influence of the ileal microbiota on regulating mucosal immunity, in the context of disease prognosis for patients with proximal colorectal cancer (pCC). The initial focus of this publication is a clinical study examining samples collected from two cohorts of patients (157 patients in total), collected from three clinical centres. The patients had all undergone right hemicolectomy with ileal resection and were not receiving antibiotic therapy; their ages ranged from 26-97. The first “discovery” cohort was of 97 patients who were suspected of having pCC, which was subsequently confirmed at surgery. The second “validation” cohort of 60 patients had a confirmed diagnosis of pCC prior to surgery (this cohort had the highest proportion of patients with stage IV disease).
The investigators compared the frequency of apoptosis in ileal and colonic mucosal samples from patients who had received pre-operative chemotherapy, to those that had not (approximately 25% had received pre-operative chemotherapy). A significant difference in apoptotic frequency in ileal crypt epithelial cells between the two groups of patients was found, as demonstrated by immunostaining for cleaved caspase 3. This was shown for patients in both the discovery and validation cohorts. For patients with stage IV cancers that received no pre-operative chemotherapy, the extent of Ileal crypt epithelial apoptosis in response to chemotherapy correlated with improved disease prognosis. An increased number of TFH cells (defined as CD4+BCL-6+ T cells) in tumour-draining lymph nodes was also associated with pre-operative chemotherapy in both patient cohorts, with a corresponding decrease in the number of these cells in the ileal lamina propria (data from the discovery cohort only).
Terminal ileal mucus samples (from health sites upstream of the ileo-caecal valve) were obtained from 70 individuals in the discovery cohort, with a subset of 30 having matching data for ileal crypt apoptosis. The data from this patient subset demonstrated that variation in ileal microbiota was associated with apoptotic sensitivity, with a positive correlation in relation to levels of Erysipelotrichaeceae (a class of gram-positive bacteria within the Firmicute phylum) and a negative correlation with Fusobacteriaceae (gram-negative rods, belonging to the Fusobacteria phylum).
The relative abundance of different bacterial families in the ileal microbiota was also found to have association with what was happening downstream, in the tumour site located in the proximal colon (analysis of 60 paired samples from patients in the discovery cohort). Proprionibacteriaceae, Corynebacteriaceae and Fusobacteriaceae were all associated with reduced T cell numbers in the tumour core, with the abundance of Fusobacteriaceae having the strongest association with reduced T cell numbers. Conversely, Carnobacteriaceae, Sutterellaceae, Eubacteriaceae and Veillonellaceae were all associated with increased T cell numbers, principally in the invasive margin of the tumours. Follow-up of all 70 patients in the discovery cohort from which the ileal microbiome was profiled, demonstrated that the presence of several bacterial species had a strong negative influence on progression-free survival, including Bacteroides nordii, Bacteroides uniformi, Bacteroides oxioreductans and Paraprevotella clara (all from the Bacteroidetes phylum). In contrast, Bacteroides fragilis was associated with a more positive outcome for patients.
These clinical observations were complemented with the use of a mouse model of colon cancer. MC38 mouse colorectal adenocarcinoma cells were implanted subcutaneously (SC) in syngeneic C57BL/6J mice; MC38 cells are derived from dimethylhydrazine-induced colorectal tumours, with this model commonly used to examine tumour immune responses. Treatment with oxaliplatin was effective in reducing tumour growth; this was also associated with increased ileal and colonic epithelial cell apoptosis (measured at 24 hrs post-oxaliplatin treatment) and decreased proliferation (measured at 72 hrs post-treatment). Treatment of MC38-recipient mice with broad spectrum antibiotics (for two weeks prior to tumour cell implantation and through to study termination) significantly reduced the efficacy of oxaliplatin (administered IP at 10 mg/kg, once per week, from 7 days post-cell implantation) and the extent of crypt epithelial cell apoptosis in the ileum and colon. The antibiotic-treated mice were shown to have very low levels of culturable bacteria in their faeces, thus confirming the effectiveness of the treatment in eliminating the microbiota. Based on this evidence, the authors proposed that the intestinal microbiota were able to influence the anti-tumour efficacy of oxaliplatin, even in the case of the tumour being extra-intestinal.
The influence of the microbiome on tumour growth and oxaliplatin efficacy was further investigated by inoculating groups of germ-free mice with microbiota derived from faecal samples collected from the proximal colon of 12 patients (5 male, 7 female; age range 51-80; 6 had received 5‑FU-based chemotherapy in combination with oxaliplatin (4) or irinotecan (1) or both (1), together with anti‑VEGF). Two weeks after faecal microbiome transplant, the mice were implanted with MC38 cells and treated as previously. Tumours grew at a similar rate in mice with the patient-derived microbiomes as they did in mice with conventional flora; however, inhibition of tumour growth by oxaliplatin demonstrated microbiome donor-specific effects, with four of the twelve donor groups showing no efficacy of oxaliplatin treatment. When the faeces of the mice were analysed after the study, there was no obvious difference across the groups in relation to the diversity of the isolated flora, although between the responders and non-responders some differences in the relative abundance of certain species could be observed. The presence of Ruthenibacterium lactatiformans, Blaultia faecis (both Clostridia from the Firmicutes phylum) and Paraprevotella clara (shown by the authors to be associated with poor prognosis in pCC patients) was associated with no response to oxaliplatin; in contrast, organisms like Butyricimonas faecihominis (Bacteroidetes) and Escherichia fergusonii (Proteobacteria) were associated with the expected level of tumour growth inhibition. Stool collected immediately prior to treatment also demonstrated a difference in the presence of Bacteroides fragilis in oxaliplatin-responsive and non-responsive groups, with responders showing a higher relative abundance of this bacterium. To complement these data, phenotypic profiling of T cells isolated from the spleen and tumour draining lymph nodes was performed. This profiling demonstrated numbers of TFH cells were increased and TH17 cells were decreased in responder groups, as compared to non-responders.
In order to investigate the role of ileal epithelial cell apoptosis in modulating the anti-tumour effect of oxaliplatin, both the ileal and colonic epithelium was isolated from mice pre-treated with oxaliplatin or PBS. The isolated epithelial cells were then used to pre-immunise groups of recipient mice, prior to SC implantation of MC38 tumour cells. It was found that immunisation with intestinal epithelial cells inhibited tumour cell growth in recipient mice, regardless of donor mouse treatment or whether the cells were from the ileum or colon. There was some suggestion that pre-treatment of donor mice with oxaliplatin enhanced the anti-tumour effect of the immunisation with ileal cells, and that ileal cells from oxaliplatin-treated donors had more of an effect than colonic cells from the same donors.
Oxaliplatin treatment resulted in more of the donor cells being apoptotic (and less necrotic), as compared to cells from PBS-treated mice. Physiological apoptotic cell death is generally thought to be immunosuppressive. This prevents the immune system being exposed to self-antigens derived from intracellular components, with the dead cell being neatly packaged and tagged for phagocytosis by neighbouring cells, or by professional phagocytes like macrophages, which can secrete TGF‑β1 in response to engagement of their phosphatidylserine (PS) receptor during engulfment of apoptotic bodies. In contrast, necroptosis, a programmed form of cell death, which can be initiated for example in cells infected with a virus that codes for apoptotic inhibitors, is considered to be inflammatory.
The authors showed the importance of the apoptotic cell fraction within the donor epithelial cell population by cell sorting, prior to immunisation, on the basis of labelling with 7‑aminoactinomycin D (7‑AAD) and annexin‑V to discriminate cell membrane integrity and PS exteriorisation, respectively. Immunisation with annexin‑V-positive cells (i.e. apoptotic cells) was associated with a strong anti-tumour response, whereas injection of live cells had less effect and dead cells disrupted by freeze-thaw had no effect at all. Differential isolation of villus and crypt epithelium showed that the crypt fraction functioned well in the vaccination model of tumour protection, whereas the villus fraction did not. This is because only the small intestinal crypt epithelial cells are sensitive to apoptosis induction in response to ionising radiation and chemotherapeutic agents, whereas villus epithelial cells are refractory, a property that is cell cycle-independent.
In addition, intestinal epithelial-specific deletion of the downstream apoptotic effectors, caspases 3 and 7, in intestinal epithelial donor mice abolished the anti-tumour effect of vaccination, including when donor mice were pre-treated with oxaliplatin. In contrast, vaccination of MC38 tumour-recipient mice with intestinal epithelial cells from mice null for expression of RIP Kinase 3, which is required for necroptosis, was still effective in preventing tumour growth. The protective effect of intestinal epithelial cell vaccination was confirmed using another syngeneic colon tumour model, CT26 cells grown in BALB/c mice.
The deletion of caspases 3 and 7 also prevented the increase in TFH cells observed within the mesenteric lymph nodes of mice immunised with ileal epithelial cells and subsequently treated with oxaliplatin. The key role of the lymph node TFH cells in effecting the protective effect of the epithelial cell vaccination was supported by studies in which CXCR5/CCL13-dependent TFH cell migration was blocked, either using CXCR5 null mice or CCL13 neutralising antibodies. Antibody-mediated depletion of B cells, which receive help from the TFH cells, also inhibited the inhibition of tumour growth observed in ileal epithelial cell-vaccinated mice. Interestingly, depletion of CD4+ T cells in MC38-recipient mice only resulted in a partial inhibition of the protection afforded by ileal epithelial cell vaccination; in contrast, depletion of CD8+ T cells completely abolished this protection. CD4+ TFH cells control germinal centre development and the maturation of immature B cells into memory B cells and plasma cells; however, there is also a sub-set of CD8+ TFH cells. CD8+ TFH cells may be important in regulating adaptive immune responses to viral infection and, in the absence of adequate regulatory T cell activity, have been shown to promote autoimmunity.
The tumour protective effect of ileal epithelial cell immunisation was maintained for a period of weeks. For intestinal epithelial cell-immunised mice, which demonstrated complete rejection of the initially-implanted MC38 cells, re-implantation of MC38 cells (at two weeks post-original implantation) failed to elicit tumour growth. In contrast, secondary implantation of other syngeneic tumours (fibrosarcoma and cholangiocarcinoma) resulted in 100% tumour incidence. Again, similar results could be demonstrated in BALB/c mice, where intestinal epithelial cell immunisation failed to protect against tumour growth following secondary implantation of a syngeneic breast cancer cell line. Additionally, it was shown that extending the period between immunisation with intestinal epithelial cells and implantation of MC38 cells, enhanced the anti-tumour effect associated with the immunisation. The function of TFH cells in the maintenance of ileal epithelial cell vaccination was demonstrated using mice with CD4+ T cell-specific deletion of the TFH cell transcription factor, Bcl6. These mice showed a high incidence of tumour regrowth, following a second implantation of MC38 cells, in mice that were originally tumour-free after initially receiving MC38 cells and immunised with ileal epithelial cells from oxaliplatin-treated donor mice.
The importance for the intestinal microbiome in the protective effect of ileal epithelial cell immunisation was shown in studies using both specific pathogen-free and germ-free (GF) donor mice. Immunisation with cells from GF mice was not associated with an inhibition of MC38 tumour growth regardless of whether the donor mice had been treated with oxaliplatin (to enhance the proportion of apoptotic cells). Ileal epithelial cells grown in vitro as organoids and treated with oxaliplatin, were also used to immunise mice prior to MC38 cell implantation. Like primary cells derived from GF animals, the absence of any associated microbiota meant that the oxaliplatin-treated organoids were not immunogenic; however, the addition of the ileal microbiota derived from certain patients with pCC (6 out of 10 tested) was able to confer immunogenicity and an anti-tumour response in the recipient mice. Injection of patient-derived ileal microbiome alone, in the absence of organoids, was not sufficient for an anti-tumour response. As with the differential prognosis of pCC patients, specific bacterial species were associated with the immunogenic response to the oxaliplatin-treated organoid/microbiome injection. Microbiome samples that conferred response (to immunisation) showed an increase in the relative abundance of organisms such as Propionobacterium acnes and Erysipelatoclostridium ramosum, with non-responders showing increased levels of organisms such as Streptococcus gallolyticus and Corynebacterium amycolatum, using culturomics. Metagenomic analysis showed that the response was associated with increased Faecalibacterium prausnitzi and Bacteroides faecis, with a broader range of organisms associated with no response, including Paraprevotella clara and Fusobacterium canifelinum.
The authors sought to determine whether the role of the microbiome (in promoting the immunogenicity of the ileal epithelial cells) was dependent on intestinal epithelial sensing of specific molecular patterns, either microbiome-associated and/or (host) damage-associated molecular patterns. This was achieved by comparing the anti-tumour effect of immunisation with ileal epithelial cells derived from a range of donor mouse strains that were genetically-modified to be deficient in a range of specific molecular pattern signalling pathways. These included mice null for the expression of Ectonucleoside triphosphate diphosphohydrolase‑1, which hydrolyses ATP (and subsequently, ADP) released from damaged cells, thereby modulating signalling through the range of purinergic receptors; mice null for both TLR2 and TLR4, used to sense bacterial cell wall products such as zymozan, LPS and peptidoglycan; mice null for TLR9, which senses bacterial CpG DNA, and mice with individual deficiencies in the expression of the inflammatory cytokines, IL‑18 and IL‑1α/β. In addition, immunisation was performed using ileal epithelial cells from oxaliplatin-treated wild-type donor mice, which were additionally treated with chemical agents to deplete cellular ATP or block its release following cellular damage, or neutralising antibodies to the damage-associated molecular patterns, HMGB1 and calreticulin. The results showed that the most important factors for the effectiveness of the immunisation were TLR2/4 signalling, and IL‑1 secretion and ATP release in the ileal epithelial cell donor mice. Previously, the authors had demonstrated that oxaliplatin-treated CT26 cells were immunogenic and had an anti-tumour effect when used as a vaccine, and that this effect was dependent on calreticulin recognition and HMGB1‑TLR2/4 signalling.
Immunohistochemistry demonstrated that IL‑1β expression was increased in the ileal villus lamina propria of oxaliplatin-treated mice, relative to mice treated with PBS, and that this increase in IL‑1β was reduced following treatment with broad spectrum antibiotics. Blockade of IL‑1β signalling by an anti‑IL‑1R1 antibody prevented the anti-tumour efficacy of ileal epithelial cells from oxaliplatin treated mice and conversely, supplementation by IL‑1β conferred immunogenicity to oxaliplatin-treated organoids.
IL‑1β is known to have a role in dendritic cell activation and their subsequent migration to local lymph nodes. Following treatment of mice with oxaliplatin, a reduction in CD45+CD11+MHCIIhi dendritic cells (DCs) was observed in the ileum, with a corresponding increase in the mesenteric lymph nodes (mLN) and increase in transcript levels of a range of inflammatory cytokines including Il23, Il12b, Il6 and Il1b. Neutralisation of either of the IL‑12 subunits (p70 or p40) was found to inhibit the anti-tumour efficacy of immunisation in the MC38 tumour model. Subsequently, the authors examined how specific bacterial species, associated with response or no response to ileal epithelial immunisation, effected IL‑12p70 and IL‑1β secretion by bone marrow-derived DCs that were cultured under two different conditions for differentiation into type 1 conventional DCs (induced by culture with FLT3L), or monocyte-derived inflammatory DCs (induced by culture with GM‑CSF and IL‑4).
Both immunogenic species of bacteria (Alistepes Onderdonkii and a non-toxigenic strain of Bacillus fragilis) and non-immunogenic species (Fusobacterium nucleatum, Paraprevotella clara and Streptococcus gallolyticus) induced the secretion of IL‑12p70 and IL‑1β by FLT3L-cultured DCs (although IL‑12p70 induction by F. nucleatum was poor compared to the other species). In contrast, Alistipes Onderdonkii was the only bacterial species that demonstrated a robust IL‑12p70 and IL‑1β response from GM‑CSF/IL‑4-cultured DCs. Monocolonisation of GF mice, which were subsequently used for production of ileal epithelial cell organoids, was also used to demonstrate that the immunogenic species of bacteria enhanced the anti-tumour action of an oxaliplatin-treated organoid-based vaccine, as compared to organoids derived from conventional SPF donors or organoids derived from mice that were monocolonised by non-immunogenic bacterial species. The immunogenic bacteria also enhanced the anti-tumour efficacy of a dual chemotherapy regimen of oxaliplatin and anti‑PD‑1 IgG, when given to both MC38 tumour-bearing C57BL/6 mice and CT26 tumour-bearing BALB/c mice. The latter model was used to show that faecal microbiome transplant from a chemotherapy non-responder patient compromised oxaliplatin/anti‑PD‑1 IgG-mediated inhibition of tumour growth, and that this could be restored by supplementation with a monoculture of an immunogenic bacterial species (B. fragilis).
In summary, the authors proposed that activation of caspases 3 and 7 in ileal epithelial cells, through the triggering of apoptosis by chemotherapeutic agents such as oxaliplatin, leads to the production of self-antigens that, in the presence of immunogenic bacterial species, result in increased numbers of CD8+ TFH cells (in mLNs and tdLNs) specific to these self-antigens. Subsequently, these TFH cells help to direct anti-tumour immunity. This work further broadens our appreciation of the interdependent relationship between the intestinal epithelium, the intestinal microbiome and host immunity, and their combined influence on homeostasis and disease.
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