Akkermansia muciniphila - friend or foe in colorectal cancer?

Akkermansia muciniphila is a gram-negative anaerobic bacterium, which represents a part of the commensal human microbiota. Decline in the abundance of A. muciniphila among other microbial species in the gut correlates with severe systemic diseases such as diabetes, obesity, intestinal inflammation and colorectal cancer. Due to its mucin-reducing and immunomodulatory properties, the use of probiotics containing Akkermansia sp. appears as a promising approach to the treatment of metabolic and inflammatory diseases. In particular, a number of studies have focused on the role of A. muciniphila in colorectal cancer. Of note, the results of these studies in mice are contradictory: some reported a protective role of A. muciniphila in colorectal cancer, while others demonstrated that administration of A. muciniphila could aggravate the course of the disease resulting in increased tumor burden. More recent studies suggested the immunomodulatory effect of certain unique surface antigens of A. muciniphila on the intestinal immune system. In this Perspective, we attempt to explain how A. muciniphila contributes to protection against colorectal cancer in some models, while being pathogenic in others. We argue that differences in the experimental protocols of administration of A. muciniphila, as well as viability of bacteria, may significantly affect the results. In addition, we hypothesize that antigens presented by pasteurized bacteria or live A. muciniphila may exert distinct effects on the barrier functions of the gut. Finally, A. muciniphila may reduce the mucin barrier and exerts combined effects with other bacterial species in either promoting or inhibiting cancer development.

Macrophages from naked mole-rat possess distinct immunometabolic signatures upon polarization.

The naked mole-rat (NMR) is a unique long-lived rodent which is highly resistant to age-associated disorders and cancer. The immune system of NMR possesses a distinct cellular composition with the prevalence of myeloid cells. Thus, the detailed phenotypical and functional assessment of NMR myeloid cell compartment may uncover novel mechanisms of immunoregulation and healthy aging. In this study gene expression signatures, reactive nitrogen species and cytokine production, as well as metabolic activity of classically (M1) and alternatively (M2) activated NMR bone marrow-derived macrophages (BMDM) were examined. Polarization of NMR macrophages under pro-inflammatory conditions led to expected M1 phenotype characterized by increased pro-inflammatory gene expression, cytokine production and aerobic glycolysis, but paralleled by reduced production of nitric oxide (NO). Under systemic LPS-induced inflammatory conditions NO production also was not detected in NMR blood monocytes. Altogether, our results indicate that NMR macrophages are capable of transcriptional and metabolic reprogramming under polarizing stimuli, however, NMR M1 possesses species-specific signatures as compared to murine M1, implicating distinct adaptations in NMR immune system.

HDM induces distinct immunometabolic phenotype in macrophages in TLR4-dependent manner.

Asthma is one of the most common chronic diseases. In many cases it is preceded by the development of an immune response to allergens such as animal fur, dust, pollens and etc. In human population this disease is heterogeneous, and no selective drugs are available at the moment for some endotypes of asthma. The role of the adaptive immune system in the pathogenesis of asthma was extensively studied, while the role of innate immune cells, in particular myeloid cells, was not sufficiently addressed. Myeloid cells, such as macrophages and dendritic cells, are characterized by high plasticity, heterogenicity and ability to undergo polarization in response to various pathogenic stimuli, including those engaging innate immune receptors. Recently, special attention was drawn to the link between polarization of macrophages and cell metabolism. We hypothesized that immunometabolic reprogramming of myeloid cells, in particular, of macrophages and dendritic cells during sensitization with an allergen may affect further immune response and asthma development. To test this hypothesis, we generated distinct types of myeloid cells in vitro from murine bone marrow and analyzed their immunometabolic profiles upon activation with house dust mite extract (HDM) and its key active components. We found that the combination of lipopolysaccharide (LPS) and beta-glucan is sufficient to upregulate proinflammatory cytokine production as well as respiratory and glycolytic capacity of myeloid cells, comparably to HDM. This specific immunometabolic phenotype was associated with altered mitochondrial morphology and possibly with increased ROS production in macrophages. Moreover, we found that both TNF production and metabolic remodeling of macrophages in response to HDM are TLR4-dependent processes. Altogether, these results expand our understanding of molecular mechanisms underlying asthma induction and pathogenesis and may potentially lead to new therapeutic strategies for the treatment of this disease.

Therapeutic Potential of Combining IL-6 and TNF Blockade in a Mouse Model of Allergic Asthma.

Combined anti-cytokine therapy is a promising therapeutic approach for uncontrolled steroid-resistant asthma. In this regard, simultaneous blockade of IL-4 and IL-13 signaling by Dupilumab (anti-IL-4Ra monoclonal antibody) was recently approved for severe eosinophilic asthma. However, no therapeutic options for neutrophilic asthma are currently available. Recent advances in our understanding of asthma pathogenesis suggest that both IL-6 and TNF may represent potential targets for treatment of severe neutrophilic asthma. Nevertheless, the efficacy of simultaneous pharmacological inhibition of TNF and IL-6 in asthma was not yet studied. To evaluate the potency of combined cytokine inhibition, we simultaneously administrated IL-6 and TNF inhibitors to BALB/c mice with HDM-induced asthma. Combined IL-6/TNF inhibition, but not individual blockade of these two cytokines, led to complex anti-inflammatory effects including reduced Th2-induced eosinophilia and less prominent Th17/Th1-mediated neutrophilic infiltrate in the airways. Taken together, our results provide evidence for therapeutic potential of combined IL-6/TNF inhibition in severe steroid-resistant asthma.

Macrophages acquire a TNF-dependent inflammatory memory in allergic asthma.

BACKGROUND: Infectious agents can reprogram or "train" macrophages and their progenitors to respond more readily to subsequent insults. However, whether such an inflammatory memory exists in type 2 inflammatory conditions such as allergic asthma was not known. OBJECTIVE: We sought to decipher macrophage-trained immunity in allergic asthma. METHODS: We used a combination of clinical sampling of house dust mite (HDM)-allergic patients, HDM-induced allergic airway inflammation in mice, and an in vitro training setup to analyze persistent changes in macrophage eicosanoid, cytokine, and chemokine production as well as the underlying metabolic and epigenetic mechanisms. Transcriptional and metabolic profiles of patient-derived and in vitro trained macrophages were assessed by RNA sequencing or metabolic flux analysis and liquid chromatography-tandem mass spectrometry analysis, respectively. RESULTS: We found that macrophages differentiated from bone marrow or blood monocyte progenitors of HDM-allergic mice or asthma patients show inflammatory transcriptional reprogramming and excessive mediator (TNF-α, CCL17, leukotriene, PGE2, IL-6) responses upon stimulation. Macrophages from HDM-allergic mice initially exhibited a type 2 imprint, which shifted toward a classical inflammatory training over time. HDM-induced allergic airway inflammation elicited a metabolically activated macrophage phenotype, producing high amounts of 2-hydroxyglutarate (2-HG). HDM-induced macrophage training in vitro was mediated by a formyl peptide receptor 2-TNF-2-HG-PGE2/PGE2 receptor 2 axis, resulting in an M2-like macrophage phenotype with high CCL17 production. TNF blockade by etanercept or genetic ablation of Tnf in myeloid cells prevented the inflammatory imprinting of bone marrow-derived macrophages from HDM-allergic mice. CONCLUSION: Allergen-triggered inflammation drives a TNF-dependent innate memory, which may perpetuate and exacerbate chronic type 2 airway inflammation and thus represents a target for asthma therapy.

Dual Role of TNF and LTα in Carcinogenesis as Implicated by Studies in Mice.

Tumor necrosis factor (TNF) and lymphotoxin alpha (LTα) are two related cytokines from the TNF superfamily, yet they mediate their functions in soluble and membrane-bound forms via overlapping, as well as distinct, molecular pathways. Their genes are encoded within the major histocompatibility complex class III cluster in close proximity to each other. TNF is involved in host defense, maintenance of lymphoid tissues, regulation of cell death and survival, and antiviral and antibacterial responses. LTα, known for some time as TNFß, has pleiotropic functions including control of lymphoid tissue development and homeostasis cross talk between lymphocytes and their environment, as well as lymphoid tissue neogenesis with formation of lymphoid follicles outside the lymph nodes. Along with their homeostatic functions, deregulation of these two cytokines may be associated with initiation and progression of chronic inflammation, autoimmunity, and tumorigenesis. In this review, we summarize the current state of knowledge concerning TNF/LTα functions in tumor promotion and suppression, with the focus on the recently uncovered significance of host-microbiota interplay in cancer development that may explain some earlier controversial results.

Multi-dimensional immunoproteomics coupled with in vitro recapitulation of oncogenic NRASQ61R identifies diagnostically relevant autoantibody biomarkers in thyroid neoplasia.

Tumor-associated antigen (TAA)-specific autoantibodies have been widely implicated in cancer diagnosis. However, cancer cell lines that are typically exploited as candidate TAA sources in immunoproteomic studies may fail to accurately represent the autoantigen-ome of lower-grade neoplasms. Here, we established an integrated strategy for the identification of disease-relevant TAAs in thyroid neoplasia, which combined NRASQ61R oncogene expression in non-tumorous thyroid Nthy-ori 3-1 cells with a multi-dimensional proteomic technique DISER that consisted of profiling NRASQ61R-induced proteins using 2-dimensional difference gel electrophoresis (2D-DIGE) coupled with serological proteome analysis (SERPA) of the TAA repertoire of patients with thyroid encapsulated follicular-patterned/RAS-like phenotype (EFP/RLP) tumors. We identified several candidate cell-based (nicotinamide phosphoribosyltransferase NAMPT, glutamate dehydrogenase GLUD1, and glutathione S-transferase omega-1 GSTO1) and autoantibody (fumarate hydratase FH, calponin-3 CNN3, and pyruvate kinase PKM autoantibodies) biomarkers, including NRASQ61R-induced TAA phosphoglycerate kinase 1 PGK1. Meta-profiling of the reactivity of the identified autoantibodies across an independent SERPA series implicated the PKM autoantibody as a histological phenotype-independent biomarker of thyroid malignancy (11/38 (29%) patients with overtly malignant and uncertain malignant potential (UMP) tumors vs 0/22 (p = 0.0046) and 0/20 (p = 0.011) patients with non-invasive EFP/RLP tumors and healthy controls, respectively). PGK1 and CNN3 autoantibodies were identified as EFP/RLP-specific biomarkers, potentially suitable for further discriminating tumors with different malignant potential (PGK1: 7/22 (32%) patients with non-invasive EFP/RLP tumors vs 0/38 (p = 0.00044) and 0/20 (p = 0.0092) patients with other tumors and healthy controls, respectively; СNN3: 9/29 (31%) patients with malignant and borderline EFP/RLP tumors vs 0/31 (p = 0.00068) and 0/20 (p = 0.0067) patients with other tumors and healthy controls, respectively). The combined use of PKM, CNN3, and PGK1 autoantibodies allowed the reclassification of malignant/UMP tumor risk in 19/41 (46%) of EFP/RLP tumor patients. Taken together, we established an experimental pipeline DISER for the concurrent identification of cell-based and TAA biomarkers. The combination of DISER with in vitro oncogene expression allows further targeted identification of oncogene-induced TAAs. Using this integrated approach, we identified candidate autoantibody biomarkers that might be of value for differential diagnostic purposes in thyroid neoplasia.

Non-redundant Functions of IL-6 Produced by Macrophages and Dendritic Cells in Allergic Airway Inflammation.

Asthma is a common inflammatory disease of the airway caused by a combination of genetic and environmental factors and characterized by airflow obstruction, wheezing, eosinophilia, and neutrophilia of lungs and sputum. Similar to other proinflammatory cytokines, IL-6 is elevated in asthma and plays an active role in this disease. However, the exact molecular mechanism of IL-6 involvement in the pathogenesis of asthma remains largely unknown and the major cellular source of pathogenic IL-6 has not been defined. In the present study, we used conditional gene targeting to demonstrate that macrophages and dendritic cells are the critical sources of pathogenic IL-6 in acute HDM-induced asthma in mice. Complete genetic inactivation of IL-6 ameliorated the disease with significant decrease in eosinophilia in the lungs. Specific ablation of IL-6 in macrophages reduced key indicators of type 2 allergic inflammation, including eosinophil and Th2 cell accumulation in the lungs, production of IgE and expression of asthma-associated inflammatory mediators. In contrast, mice with deficiency of IL-6 in dendritic cells demonstrated attenuated neutrophilic, but regular eosinophilic response in HDM-induced asthma. Taken together, our results indicate that IL-6 plays a pathogenic role in the HDM-induced asthma model and that lung macrophages and dendritic cells are the predominant sources of pathogenic IL-6 but contribute differently to the disease.

Citrobacter rodentium-induced colitis: A robust model to study mucosal immune responses in the gut.

Citrobacter rodentium is a natural mouse pathogen which reproducibly infects mice and causes intestinal disease. The C. rodentium model of infection is very useful for investigating host-pathogen immune interactions in the gut, and can also be used to understand the pathogenesis of several important human intestinal disorders, including Crohn's disease, ulcerative colitis, dysbiosis and colon tumorigenesis. Both innate and adaptive immune responses play a critical role in protection against C. rodentium. Here, we summarize the role of immune components in protection against C. rodentium and describe techniques for the analysis of innate and adaptive mucosal immune responses, including setting up the infection, analysis of colonic hyperplasia and bacterial dissemination, evaluation of antibody responses, and purification and analysis of intestinal epithelial and lymphoid cells.