A gut microbial signature for combination immune checkpoint blockade across cancer types.

Immune checkpoint blockade (ICB) targeting programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte protein 4 (CTLA-4) can induce remarkable, yet unpredictable, responses across a variety of cancers. Studies suggest that there is a relationship between a cancer patient's gut microbiota composition and clinical response to ICB; however, defining microbiome-based biomarkers that generalize across cohorts has been challenging. This may relate to previous efforts quantifying microbiota to species (or higher taxonomic rank) abundances, whereas microbial functions are often strain specific. Here, we performed deep shotgun metagenomic sequencing of baseline fecal samples from a unique, richly annotated phase 2 trial cohort of patients with diverse rare cancers treated with combination ICB (n = 106 discovery cohort). We demonstrate that strain-resolved microbial abundances improve machine learning predictions of ICB response and 12-month progression-free survival relative to models built using species-rank quantifications or comprehensive pretreatment clinical factors. Through a meta-analysis of gut metagenomes from a further six comparable studies (n = 364 validation cohort), we found cross-cancer (and cross-country) validity of strain-response signatures, but only when the training and test cohorts used concordant ICB regimens (anti-PD-1 monotherapy or combination anti-PD-1 plus anti-CTLA-4). This suggests that future development of gut microbiome diagnostics or therapeutics should be tailored according to ICB treatment regimen rather than according to cancer type.

The heightened importance of the microbiome in cancer immunotherapy.

The human microbiome is recognized as a key factor in health and disease. This has been further corroborated by identifying changes in microbiome composition and function as a novel hallmark in cancer. These effects are exerted through microbiome interactions with host cells, impacting a wide variety of developmental and physiological processes. In this review, we discuss some of the latest findings on how the bacterial component of the microbiome can influence outcomes for different cancer immunotherapy modalities, highlighting identified mechanisms of action. We also address the clinical efforts to utilize this knowledge to achieve better responses to immunotherapy. A refined understanding of microbiome variations in patients and microbiome-host interactions with cancer therapies is essential to realize optimal clinical responses.

The gut microbial metabolic capacity of microbiome-humanized vs. wild type rodents reveals a likely dual role of intestinal bacteria in hepato-intestinal schistosomiasis.

Increasing evidence shows that the host gut microbiota might be involved in the immunological cascade that culminates with the formation of tissue granulomas underlying the pathophysiology of hepato-intestinal schistosomiasis. In this study, we investigated the impact of Schistosoma mansoni infection on the gut microbial composition and functional potential of both wild type and microbiome-humanized mice. In spite of substantial differences in microbiome composition at baseline, selected pathways were consistently affected by parasite infection. The gut microbiomes of infected mice of both lines displayed, amongst other features, enhanced capacity for tryptophan and butyrate production, which might be linked to the activation of mechanisms aimed to prevent excessive injuries caused by migrating parasite eggs. Complementing data from previous studies, our findings suggest that the host gut microbiome might play a dual role in the pathophysiology of schistosomiasis, where intestinal bacteria may contribute to egg-associated pathology while, in turn, protect the host from uncontrolled tissue damage.

'Know thyself' - host factors influencing cancer response to immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICIs) have revolutionised oncology and are now standard-of-care for the treatment of a wide variety of solid neoplasms. However, tumour responses remain unpredictable, experienced by only a minority of ICI recipients across malignancy types. Therefore, there is an urgent need for better predictive biomarkers to identify a priori the patients most likely to benefit from these therapies. Despite considerable efforts, only three such biomarkers are FDA-approved for clinical use, and all rely on the availability of tumour tissue for immunohistochemical staining or genomic assays. There is emerging evidence that host factors - for example, genetic, metabolic, and immune factors, as well as the composition of one's gut microbiota - influence the response of a patient's cancer to ICIs. Tantalisingly, some of these factors are modifiable, paving the way for co-therapies that may enhance the therapeutic index of these treatments. Herein, we review key host factors that are of potential biomarker value for response to ICI therapy, with a particular focus on the proposed mechanisms for these influences. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The MITRE trial protocol: a study to evaluate the microbiome as a biomarker of efficacy and toxicity in cancer patients receiving immune checkpoint inhibitor therapy.

BACKGROUND: The gut microbiome is implicated as a marker of response to immune checkpoint inhibitors (ICI) based on preclinical mouse models and preliminary observations in limited patient series. Furthermore, early studies suggest faecal microbial transfer may have therapeutic potential, converting ICI non-responders into responders. So far, identification of specific responsible bacterial taxa has been inconsistent, which limits future application. The MITRE study will explore and validate a microbiome signature in a larger scale prospective study across several different cancer types. METHODS: Melanoma, renal cancer and non-small cell lung cancer patients who are planned to receive standard immune checkpoint inhibitors are being recruited to the MITRE study. Longitudinal stool samples are collected prior to treatment, then at 6 weeks, 3, 6 and 12 months during treatment, or at disease progression/recurrence (whichever is sooner), as well as after a severe (≥grade 3 CTCAE v5.0) immune-related adverse event. Additionally, whole blood, plasma, buffy coat, RNA and peripheral blood mononuclear cells (PBMCs) is collected at similar time points and will be used for exploratory analyses. Archival tumour tissue, tumour biopsies at progression/relapse, as well as any biopsies from body organs collected after a severe toxicity are collected. The primary outcome measure is the ability of the microbiome signature to predict 1 year progression-free survival (PFS) in patients with advanced disease. Secondary outcomes include microbiome correlations with toxicity and other efficacy end-points. Biosamples will be used to explore immunological and genomic correlates. A sub-study will evaluate both COVID-19 antigen and antibody associations with the microbiome. DISCUSSION: There is an urgent need to identify biomarkers that are predictive of treatment response, resistance and toxicity to immunotherapy. The data generated from this study will both help inform patient selection for these drugs and provide information that may allow therapeutic manipulation of the microbiome to improve future patient outcomes. TRIAL REGISTRATION: NCT04107168 , ClinicalTrials.gov, registered 09/27/2019. Protocol V3.2 (16/04/2021).

A purine metabolic checkpoint that prevents autoimmunity and autoinflammation.

Still's disease, the paradigm of autoinflammation-cum-autoimmunity, predisposes for a cytokine storm with excessive T lymphocyte activation upon viral infection. Loss of function of the purine nucleoside enzyme FAMIN is the sole known cause for monogenic Still's disease. Here we discovered that a FAMIN-enabled purine metabolon in dendritic cells (DCs) restrains CD4+ and CD8+ T cell priming. DCs with absent FAMIN activity prime for enhanced antigen-specific cytotoxicity, IFNγ secretion, and T cell expansion, resulting in excessive influenza A virus-specific responses. Enhanced priming is already manifest with hypomorphic FAMIN-I254V, for which ∼6% of mankind is homozygous. FAMIN controls membrane trafficking and restrains antigen presentation in an NADH/NAD+-dependent manner by balancing flux through adenine-guanine nucleotide interconversion cycles. FAMIN additionally converts hypoxanthine into inosine, which DCs release to dampen T cell activation. Compromised FAMIN consequently enhances immunosurveillance of syngeneic tumors. FAMIN is a biochemical checkpoint that protects against excessive antiviral T cell responses, autoimmunity, and autoinflammation.

A novel technique capable of taking 'protected' biopsies for reliable assessment of the distribution of microbiota along the colonic mucosa.

We evaluated a novel 'protected' biopsy method to reliably ascertain the spatial distribution of the mucosa-adherent colonic microbiota. Apart from minor differences at genus level, overall similarities along the colon were high between the various areas, irrespective of protected or unprotected sampling.

Identification of bacteria-derived HLA-bound peptides in melanoma.

A variety of species of bacteria are known to colonize human tumours1-11, proliferate within them and modulate immune function, which ultimately affects the survival of patients with cancer and their responses to treatment12-14. However, it is not known whether antigens derived from intracellular bacteria are presented by the human leukocyte antigen class I and II (HLA-I and HLA-II, respectively) molecules of tumour cells, or whether such antigens elicit a tumour-infiltrating T cell immune response. Here we used 16S rRNA gene sequencing and HLA peptidomics to identify a peptide repertoire derived from intracellular bacteria that was presented on HLA-I and HLA-II molecules in melanoma tumours. Our analysis of 17 melanoma metastases (derived from 9 patients) revealed 248 and 35 unique HLA-I and HLA-II peptides, respectively, that were derived from 41 species of bacteria. We identified recurrent bacterial peptides in tumours from different patients, as well as in different tumours from the same patient. Our study reveals that peptides derived from intracellular bacteria can be presented by tumour cells and elicit immune reactivity, and thus provides insight into a mechanism by which bacteria influence activation of the immune system and responses to therapy.

Lipopolysaccharide O structure of adherent and invasive Escherichia coli regulates intestinal inflammation via complement C3.

Gut dysbiosis associated with intestinal inflammation is characterized by the blooming of particular bacteria such as adherent-invasive E. coli (AIEC). However, the precise mechanisms by which AIEC impact on colitis remain largely unknown. Here we show that antibiotic-induced dysbiosis worsened chemically-induced colitis in IL-22-deficient mice, but not in wild-type mice. The increase in intestinal inflammation was associated with the expansion of E. coli strains with genetic and functional features of AIEC. These E. coli isolates exhibited high ability to out compete related bacteria via colicins and resistance to the host complement system in vitro. Mutation of wzy, the lipopolysaccharide O polymerase gene, rendered AIEC more sensitive to the complement system and more susceptible to engulfment and killing by phagocytes while retaining its ability to outcompete related bacteria in vitro. The wzy AIEC mutant showed impaired fitness to colonize the intestine under colitic conditions, but protected mice from chemically-induced colitis. Importantly, the ability of the wzy mutant to protect from colitis was blocked by depletion of complement C3 which was associated with impaired intestinal eradication of AIEC in colitic mice. These studies link surface lipopolysaccharide O-antigen structure to the regulation of colitic activity in commensal AIEC via interactions with the complement system.

FAMIN Is a Multifunctional Purine Enzyme Enabling the Purine Nucleotide Cycle.

Mutations in FAMIN cause arthritis and inflammatory bowel disease in early childhood, and a common genetic variant increases the risk for Crohn's disease and leprosy. We developed an unbiased liquid chromatography-mass spectrometry screen for enzymatic activity of this orphan protein. We report that FAMIN phosphorolytically cleaves adenosine into adenine and ribose-1-phosphate. Such activity was considered absent from eukaryotic metabolism. FAMIN and its prokaryotic orthologs additionally have adenosine deaminase, purine nucleoside phosphorylase, and S-methyl-5'-thioadenosine phosphorylase activity, hence, combine activities of the namesake enzymes of central purine metabolism. FAMIN enables in macrophages a purine nucleotide cycle (PNC) between adenosine and inosine monophosphate and adenylosuccinate, which consumes aspartate and releases fumarate in a manner involving fatty acid oxidation and ATP-citrate lyase activity. This macrophage PNC synchronizes mitochondrial activity with glycolysis by balancing electron transfer to mitochondria, thereby supporting glycolytic activity and promoting oxidative phosphorylation and mitochondrial H+ and phosphate recycling.

High-resolution mapping reveals that microniches in the gastric glands control Helicobacter pylori colonization of the stomach.

Lifelong infection of the gastric mucosa by Helicobacter pylori can lead to peptic ulcers and gastric cancer. However, how the bacteria maintain chronic colonization in the face of constant mucus and epithelial cell turnover in the stomach is unclear. Here, we present a new model of how H. pylori establish and persist in stomach, which involves the colonization of a specialized microenvironment, or microniche, deep in the gastric glands. Using quantitative three-dimensional (3D) confocal microscopy and passive CLARITY technique (PACT), which renders tissues optically transparent, we analyzed intact stomachs from mice infected with a mixture of isogenic, fluorescent H. pylori strains with unprecedented spatial resolution. We discovered that a small number of bacterial founders initially establish colonies deep in the gastric glands and then expand to colonize adjacent glands, forming clonal population islands that persist over time. Gland-associated populations do not intermix with free-swimming bacteria in the surface mucus, and they compete for space and prevent newcomers from establishing in the stomach. Furthermore, bacterial mutants deficient in gland colonization are outcompeted by wild-type (WT) bacteria. Finally, we found that host factors such as the age at infection and T-cell responses control bacterial density within the glands. Collectively, our results demonstrate that microniches in the gastric glands house a persistent H. pylori reservoir, which we propose replenishes the more transient bacterial populations in the superficial mucosa.

GPR35 promotes glycolysis, proliferation, and oncogenic signaling by engaging with the sodium potassium pump.

The sodium potassium pump (Na/K-ATPase) ensures the electrochemical gradient of a cell through an energy-dependent process that consumes about one-third of regenerated ATP. We report that the G protein-coupled receptor GPR35 interacted with the α chain of Na/K-ATPase and promotes its ion transport and Src signaling activity in a ligand-independent manner. Deletion of Gpr35 increased baseline Ca2+ to maximal levels and reduced Src activation and overall metabolic activity in macrophages and intestinal epithelial cells (IECs). In contrast, a common T108M polymorphism in GPR35 was hypermorphic and had the opposite effects to Gpr35 deletion on Src activation and metabolic activity. The T108M polymorphism is associated with ulcerative colitis and primary sclerosing cholangitis, inflammatory diseases with a high cancer risk. GPR35 promoted homeostatic IEC turnover, whereas Gpr35 deletion or inhibition by a selective pepducin prevented inflammation-associated and spontaneous intestinal tumorigenesis in mice. Thus, GPR35 acts as a central signaling and metabolic pacesetter, which reveals an unexpected role of Na/K-ATPase in macrophage and IEC biology.

Interleukin-22 promotes phagolysosomal fusion to induce protection against Salmonella enterica Typhimurium in human epithelial cells.

Intestinal epithelial cells (IECs) play a key role in regulating immune responses and controlling infection. However, the direct role of IECs in restricting pathogens remains incompletely understood. Here, we provide evidence that IL-22 primed intestinal organoids derived from healthy human induced pluripotent stem cells (hIPSCs) to restrict Salmonella enterica serovar Typhimurium SL1344 infection. A combination of transcriptomics, bacterial invasion assays, and imaging suggests that IL-22-induced antimicrobial activity is driven by increased phagolysosomal fusion in IL-22-pretreated cells. The antimicrobial phenotype was absent in hIPSCs derived from a patient harboring a homozygous mutation in the IL10RB gene that inactivates the IL-22 receptor but was restored by genetically complementing the IL10RB deficiency. This study highlights a mechanism through which the IL-22 pathway facilitates the human intestinal epithelium to control microbial infection.

Pathogen Resistance Mediated by IL-22 Signaling at the Epithelial-Microbiota Interface.

Intestinal colonization resistance to bacterial pathogens is generally associated, among other factors, with mucosal homeostasis that preserves the integrity of the intestinal barrier. Mucosal homeostasis depends on physical and molecular interactions between three components: the resident microbiota, the epithelial layer and the local immune system. The cytokine IL-22 helps to orchestrate this three-way interaction. IL-22 is produced by immune cells present beneath the epithelium and is induced by bacteria present in the intestine. IL-22 stimulates the epithelial cells via the IL-22RA1-IL-10R2 receptor complex inducing changes in the expression of genes involved in the maintenance of epithelial barrier integrity, with a variety of functions in pathogen resistance such as mucus layer modifications and hydration, tight junction fortification and the production of a broad range of bactericidal compounds. These mechanisms of pathogen resistance, in turn, affect the microbiota composition and create an environment that excludes pathogens. Here we highlight the role of IL-22 as key mediator in the give-and-take relationship between the microbiota and the host that impacts pathogen resistance.

Distinct Commensals Induce Interleukin-1ß via NLRP3 Inflammasome in Inflammatory Monocytes to Promote Intestinal Inflammation in Response to Injury.

The microbiota stimulates inflammation, but the signaling pathways and the members of the microbiota involved remain poorly understood. We found that the microbiota induces interleukin-1ß (IL-1ß) release upon intestinal injury and that this is mediated via the NLRP3 inflammasome. Enterobacteriaceae and in particular the pathobiont Proteus mirabilis, induced robust IL-1ß release that was comparable to that induced by the pathogen Salmonella. Upon epithelial injury, production of IL-1ß in the intestine was largely mediated by intestinal Ly6C(high) monocytes, required chemokine receptor CCR2 and was abolished by deletion of IL-1ß in CCR2(+) blood monocytes. Furthermore, colonization with P. mirabilis promoted intestinal inflammation upon intestinal injury via the production of hemolysin, which required NLRP3 and IL-1 receptor signaling in vivo. Thus, upon intestinal injury, selective members of the microbiota stimulate newly recruited monocytes to induce NLRP3-dependent IL-1ß release, which promotes inflammation in the intestine.

Neutrophils recruited by IL-22 in peripheral tissues function as TRAIL-dependent antiviral effectors against MCMV.

During primary infection, murine cytomegalovirus (MCMV) spreads systemically, resulting in virus replication and pathology in multiple organs. This disseminated infection is ultimately controlled, but the underlying immune defense mechanisms are unclear. Investigating the role of the cytokine IL-22 in MCMV infection, we discovered an unanticipated function for neutrophils as potent antiviral effector cells that restrict viral replication and associated pathogenesis in peripheral organs. NK-, NKT-, and T cell-secreted IL-22 orchestrated antiviral neutrophil-mediated responses via induction in stromal nonhematopoietic tissue of the neutrophil-recruiting chemokine CXCL1. The antiviral effector properties of infiltrating neutrophils were directly linked to the expression of TNF-related apoptosis-inducing ligand (TRAIL). Our data identify a role for neutrophils in antiviral defense, and establish a functional link between IL-22 and the control of antiviral neutrophil responses that prevents pathogenic herpesvirus infection in peripheral organs.

Therapeutic modulation of intestinal dysbiosis.

The human gastrointestinal tract is home to an extremely numerous and diverse collection of microbes, collectively termed the "intestinal microbiota". This microbiota is considered to play a number of key roles in the maintenance of host health, including aiding digestion of otherwise indigestible dietary compounds, synthesis of vitamins and other beneficial metabolites, immune system regulation and enhanced resistance against colonisation by pathogenic microorganisms. Conversely, the intestinal microbiota is also a potent source of antigens and potentially harmful compounds. In health, humans can therefore be considered to exist in a state of natural balance with their microbial inhabitants. A shift in the balance of microbiota composition such that it may become deleterious to host health is termed "dysbiosis". Dysbiosis of the gut microbiota has been implicated in numerous disorders, ranging from intestinal maladies such as inflammatory bowel diseases and colorectal cancer to disorders with more systemic effects such as diabetes, metabolic syndrome and atopy. Given the far reaching influence of the intestinal microbiota on human health a clear future goal must be to develop reliable means to alter the composition of the microbiota and restore a healthy balance of microbial species. While it is clear that much fundamental research remains to be done, potentially important therapeutic options include narrow spectrum antibiotics, novel probiotics, dietary interventions and more radical techniques such as faecal transplantation, all of which aim to suppress clinical dysbiosis, restore intestinal microbiota diversity and improve host health.

Comparative whole genome sequence analysis of the carcinogenic bacterial model pathogen Helicobacter felis.

The gram-negative bacterium Helicobacter felis naturally colonizes the gastric mucosa of dogs and cats. Due to its ability to persistently infect laboratory mice, H. felis has been used extensively to experimentally model gastric disorders induced in humans by H. pylori. We determined the 1.67 Mb genome sequence of H. felis using combined Solexa and 454 pyrosequencing, annotated the genome, and compared it with multiple previously published Helicobacter genomes. About 1,063 (63.6%) of the 1,671 genes identified in the H. felis genome have orthologues in H. pylori, its closest relative among the fully sequenced Helicobacter species. Many H. pylori virulence factors are shared by H. felis: these include the gamma-glutamyl transpeptidase GGT, the immunomodulator NapA, and the secreted enzymes collagenase and HtrA. Helicobacter felis lacks a Cag pathogenicity island and the vacuolating cytotoxin VacA but possesses a complete comB system conferring natural competence. Remarkable features of the H. felis genome include its paucity of transcriptional regulators and an extraordinary abundance of chemotaxis sensors and restriction/modification systems. Helicobacter felis possesses an episomally replicating 6.7-kb plasmid and harbors three chromosomal regions with deviating GC content. These putative horizontally acquired regions show homology and synteny with the recently isolated H. pylori plasmid pHPPC4 and homology to Campylobacter bacteriophage genes (transposases, structural, and lytic genes), respectively. In summary, the H. felis genome harbors a variety of putative mobile elements that are unique among Helicobacter species and may contribute to this pathogen's carcinogenic properties.

Use of purified Clostridium difficile spores to facilitate evaluation of health care disinfection regimens.

Clostridium difficile is a major cause of antibiotic-associated diarrheal disease in many parts of the world. In recent years, distinct genetic variants of C. difficile that cause severe disease and persist within health care settings have emerged. Highly resistant and infectious C. difficile spores are proposed to be the main vectors of environmental persistence and host transmission, so methods to accurately monitor spores and their inactivation are urgently needed. Here we describe simple quantitative methods, based on purified C. difficile spores and a murine transmission model, for evaluating health care disinfection regimens. We demonstrate that disinfectants that contain strong oxidizing active ingredients, such as hydrogen peroxide, are very effective in inactivating pure spores and blocking spore-mediated transmission. Complete inactivation of 106 pure C. difficile spores on indicator strips, a six-log reduction, and a standard measure of stringent disinfection regimens require at least 5 min of exposure to hydrogen peroxide vapor (HPV; 400 ppm). In contrast, a 1-min treatment with HPV was required to disinfect an environment that was heavily contaminated with C. difficile spores (17 to 29 spores/cm²) and block host transmission. Thus, pure C. difficile spores facilitate practical methods for evaluating the efficacy of C. difficile spore disinfection regimens and bringing scientific acumen to C. difficile infection control.

Comparative genome and phenotypic analysis of Clostridium difficile 027 strains provides insight into the evolution of a hypervirulent bacterium.

BACKGROUND: The continued rise of Clostridium difficile infections worldwide has been accompanied by the rapid emergence of a highly virulent clone designated PCR-ribotype 027. To understand more about the evolution of this virulent clone, we made a three-way genomic and phenotypic comparison of an 'historic' non-epidemic 027 C. difficile (CD196), a recent epidemic and hypervirulent 027 (R20291) and a previously sequenced PCR-ribotype 012 strain (630). RESULTS: Although the genomes are highly conserved, the 027 genomes have 234 additional genes compared to 630, which may contribute to the distinct phenotypic differences we observe between these strains relating to motility, antibiotic resistance and toxicity. The epidemic 027 strain has five unique genetic regions, absent from both the non-epidemic 027 and strain 630, which include a novel phage island, a two component regulatory system and transcriptional regulators. CONCLUSIONS: A comparison of a series of 027 isolates showed that some of these genes appeared to have been gained by 027 strains over the past two decades. This study provides genetic markers for the identification of 027 strains and offers a unique opportunity to explain the recent emergence of a hypervirulent bacterium.