Acod1-mediated inhibition of aerobic glycolysis suppresses osteoclast differentiation and attenuates bone erosion in arthritis.

OBJECTIVES: Metabolic changes are crucially involved in osteoclast development and may contribute to bone degradation in rheumatoid arthritis (RA). The enzyme aconitate decarboxylase 1 (Acod1) is known to link the cellular function of monocyte-derived macrophages to their metabolic status. As osteoclasts derive from the monocyte lineage, we hypothesised a role for Acod1 and its metabolite itaconate in osteoclast differentiation and arthritis-associated bone loss. METHODS: Itaconate levels were measured in human peripheral blood mononuclear cells (PBMCs) of patients with RA and healthy controls by mass spectrometry. Human and murine osteoclasts were treated with the itaconate derivative 4-octyl-itaconate (4-OI) in vitro. We examined the impact of Acod1-deficiency and 4-OI treatment on bone erosion in mice using K/BxN serum-induced arthritis and human TNF transgenic (hTNFtg) mice. SCENITH and extracellular flux analyses were used to evaluate the metabolic activity of osteoclasts and osteoclast progenitors. Acod1-dependent and itaconate-dependent changes in the osteoclast transcriptome were identified by RNA sequencing. CRISPR/Cas9 gene editing was used to investigate the role of hypoxia-inducible factor (Hif)-1α in Acod1-mediated regulation of osteoclast development. RESULTS: Itaconate levels in PBMCs from patients with RA were inversely correlated with disease activity. Acod1-deficient mice exhibited increased osteoclast numbers and bone erosion in experimental arthritis while 4-OI treatment alleviated inflammatory bone loss in vivo and inhibited human and murine osteoclast differentiation in vitro. Mechanistically, Acod1 suppressed osteoclast differentiation by inhibiting succinate dehydrogenase-dependent production of reactive oxygen species and Hif1α-mediated induction of aerobic glycolysis. CONCLUSION: Acod1 and itaconate are crucial regulators of osteoclast differentiation and bone loss in inflammatory arthritis.

L-arginine metabolism inhibits arthritis and inflammatory bone loss.

OBJECTIVES: To investigate the effect of the L-arginine metabolism on arthritis and inflammation-mediated bone loss. METHODS: L-arginine was applied to three arthritis models (collagen-induced arthritis, serum-induced arthritis and human TNF transgenic mice). Inflammation was assessed clinically and histologically, while bone changes were quantified by µCT and histomorphometry. In vitro, effects of L-arginine on osteoclast differentiation were analysed by RNA-seq and mass spectrometry (MS). Seahorse, Single Cell ENergetIc metabolism by profilIng Translation inHibition and transmission electron microscopy were used for detecting metabolic changes in osteoclasts. Moreover, arginine-associated metabolites were measured in the serum of rheumatoid arthritis (RA) and pre-RA patients. RESULTS: L-arginine inhibited arthritis and bone loss in all three models and directly blocked TNFα-induced murine and human osteoclastogenesis. RNA-seq and MS analyses indicated that L-arginine switched glycolysis to oxidative phosphorylation in inflammatory osteoclasts leading to increased ATP production, purine metabolism and elevated inosine and hypoxanthine levels. Adenosine deaminase inhibitors blocking inosine and hypoxanthine production abolished the inhibition of L-arginine on osteoclastogenesis in vitro and in vivo. Altered arginine levels were also found in RA and pre-RA patients. CONCLUSION: Our study demonstrated that L-arginine ameliorates arthritis and bone erosion through metabolic reprogramming and perturbation of purine metabolism in osteoclasts.

A new platform for autoimmune diseases. Inducing tolerance with liposomes encapsulating autoantigens.

Autoimmune diseases (AIDs) are caused by the loss of self-tolerance and destruction of tissues by the host's immune system. Several antigen-specific immunotherapies, focused on arresting the autoimmune attack, have been tested in clinical trials with discouraging results. Therefore, there is a need for innovative strategies to restore self-tolerance safely and definitively in AIDs. We previously demonstrated the therapeutic efficacy of phosphatidylserine (PS)-liposomes encapsulating autoantigens in experimental type 1 diabetes and multiple sclerosis. Here, we show that PS-liposomes can be adapted to other autoimmune diseases by simply replacing the encapsulated autoantigen. After administration, they are distributed to target organs, captured by phagocytes and interact with several immune cells, thus exerting a tolerogenic and immunoregulatory effect. Specific PS-liposomes demonstrate great preventive and therapeutic efficacy in rheumatoid arthritis and myasthenia gravis. Thus, this work highlights the therapeutic potential of a platform for several autoimmunity settings, which is specific, safe, and with long-term effects.

Epithelial HIF2α expression induces intestinal barrier dysfunction and exacerbation of arthritis.

OBJECTIVE: To investigate how the mucosal barrier in the intestine influences the development of arthritis, considering that metabolic changes in the intestinal epithelium influence its barrier function. METHODS: Intestinal hypoxia inducible factor (HIF)-2α expression was assessed before, at onset and during experimental arthritis and human rheumatoid arthritis (RA). Intestinal epithelial cell-specific HIF2α conditional knock-out mice were generated (HIF2α∆IEC) and subjected to collagen-induced arthritis. Clinical and histological courses of arthritis were recorded; T-cell and B-cell subsets were analysed in the gut and secondary lymphatic organs; and intestinal epithelial cells were subjected to molecular mRNA sequencing in HIF2α∆IEC and littermate control mice. The gut intestinal HIF2α target genes were delineated by chromatin immunoprecipitation and luciferase experiments. Furthermore, pharmacological HIF2α inhibitor PT2977 was used for inhibition of arthritis. RESULTS: Intestinal HIF2α expression peaked at onset of experimental arthritis and RA. Conditionally, deletion of HIF2α in gut epithelial cells inhibited arthritis and was associated with improved intestinal barrier function and less intestinal and lymphatic Th1 and Th17 activation. Mechanistically, HIF2α induced the transcription of the pore-forming claudin (CLDN)-15, which inhibits intestinal barrier integrity. Furthermore, treatment with HIF2α inhibitor decreased claudin-15 expression in epithelial cells and inhibited arthritis. CONCLUSION: These findings show that the HIF2α-CLDN15 axis is critical for the breakdown of intestinal barrier function at onset of arthritis, highlighting the functional link between intestinal homeostasis and arthritis.

Regulatory eosinophils induce the resolution of experimental arthritis and appear in remission state of human rheumatoid arthritis.

OBJECTIVES: Eosinophils possess pro-inflammatory functions in asthma. However, our recent studies have suggested that innate lymphoid cells type 2 (ILC2s) and eosinophils have proresolving properties in rheumatoid arthritis (RA). Nothing is known yet about the mechanisms determining the double-edged role of eosinophils. Therefore, we investigated whether asthma, a paradigm eosinophilic disease, can elicit resolution of chronic arthritis. METHODS: Ovalbumin-triggered eosinophilic asthma was combined with K/BxN serum-induced arthritis, where lung and synovial eosinophil subsets were compared by single-cell RNA sequencing (scRNA-seq). To investigate the involvement of the ILC2-interleukin-5 (IL-5) axis, hydrodynamic injection (HDI) of IL-25 and IL-33 plasmids, IL-5 reporter mice and anti-IL-5 antibody treatment were used. In patients with RA, the presence of distinct eosinophil subsets was examined in peripheral blood and synovial tissue. Disease activity of patients with RA with concomitant asthma was monitored before and after mepolizumab (anti-IL-5 antibody) therapy. RESULTS: The induction of eosinophilic asthma caused resolution of murine arthritis and joint tissue protection. ScRNA-seq revealed a specific subset of regulatory eosinophils (rEos) in the joints, distinct from inflammatory eosinophils in the lungs. Mechanistically, synovial rEos expanded on systemic upregulation of IL-5 released by lung ILC2s. Eosinophil depletion abolished the beneficial effect of asthma on arthritis. rEos were consistently present in the synovium of patients with RA in remission, but not in active stage. Remarkably, in patients with RA with concomitant asthma, mepolizumab treatment induced relapse of arthritis. CONCLUSION: These findings point to a hitherto undiscovered proresolving signature in an eosinophil subset that stimulates arthritis resolution.

Increased expression of the immunosuppressive interleukin-35 in patients with non-small cell lung cancer.

BACKGROUND: The immunosuppressive role of the cytokine IL-35 in patients with non-small cell lung cancer (NSCLC) is poorly understood. In this study, we analysed the localisation and regulation of IL-35 in the lung of patients with non-small cell lung cancer (NSCLC) to further elucidate the immune-escape of cancer cells in perioperative course of disease. METHODS: Interleukin 35 (IL-35) was measured by ELISA in postoperative serum from 7 patients with NSCLC as well as 8 samples from healthy controls. Immunohistochemistry, FACS analysis, real-time PCR, as well as western blot from samples of the control (CTR), peri-tumoural (PT) and the tumoural (TU) region of the lung derived from patients with NSCLC and 10 controls were performed. RESULTS: Here we found elevated levels of IL-35 in the TU region as well as postoperative serum from patients with lung adenocarcinoma. Consistently, we found an increased expression of IL-35+Foxp-3+ cells, which associated with ARG1 mRNA expression and decreased TNFA in the TU region of the lung of patients with NSCLC as compared to their CTR region. Furthermore, in the CTR region of the lung of patients with NSCLC, CD68+ macrophages were induced and correlated with IL-35+ cells. Finally, IL-35 positively correlated with TTF-1+PD-L1+ cells in the TU region of NSCLC patients. CONCLUSIONS: Induced IL-35+Foxp3+ cell numbers in the TU region of the lung of patients with NSCLC associated with ARG1 mRNA expression and with TTF-1+PD-L1+ cells. In the tumour-free CTR area, IL-35 correlated with CD68+ macrophages. Thus inhibitors to IL-35 would probably succeed in combination with antibodies against immune checkpoints like PD-L1 and PD-1 currently used against NSCLC because they would inhibit immunosuppressive macrophages and T regulatory cells while promoting T cell-mediated anti-tumoural immune responses in the microenvironment as well as the TU region of NSCLC patients.

Interleukin-10-regulated tumour tolerance in non-small cell lung cancer.

BACKGROUND: Lung cancer is the most life-threatening cancer type worldwide. Treatment options include surgery, radio- and chemotherapy, as well as the use of immunomodulatory antibodies. Interleukin (IL)-10 is an immunosuppressive cytokine involved in tumour immune escape. METHODS: Immunohistochemistry (IHC) on human lung surgery tissue as well as human tumour cell line cultures, FACS analysis, real-time PCR and experimental lung cancer. RESULTS: Here we discovered a positive correlation between IL-10 and IL-10 receptor (IL-10R) expression in the lung with tumour diameter in patients with lung cancer (non-small cell lung cancer), the most life-threatening cancer type worldwide. IL-10 and IL-10R were found induced in cells surrounding the lung tumour cells, and IL-10R was mainly expressed on the surface of Foxp-3+ T-regulatory lymphocytes infiltrating the tumour of these patients where its expression inversely correlated with programmed cell death 1. These findings were confirmed in translational studies. In a human lung adenocarcinoma cell line, IL-10R was found induced under metabolic restrictions present during tumour growth, whereby IL-10 inhibited PDL1 and tumour cell apoptosis. CONCLUSIONS: These new findings suggest that IL-10 counteracts IFN-γ effects on PD1/PDL1 pathway, resulting in possible resistance of the tumour to anti-PD1/PDL1 immunotherapy.

Eosinophils preserve bone homeostasis by inhibiting excessive osteoclast formation and activity via eosinophil peroxidase.

Eosinophils are involved in tissue homeostasis. Herein, we unveiled eosinophils as important regulators of bone homeostasis. Eosinophils are localized in proximity to bone-resorbing osteoclasts in the bone marrow. The absence of eosinophils in ΔdblGATA mice results in lower bone mass under steady-state conditions and amplified bone loss upon sex hormone deprivation and inflammatory arthritis. Conversely, increased numbers of eosinophils in IL-5 transgenic mice enhance bone mass under steady-state conditions and protect from hormone- and inflammation- mediated bone loss. Eosinophils strongly inhibit the differentiation and demineralization activity of osteoclasts and lead to profound changes in the transcriptional profile of osteoclasts. This osteoclast-suppressive effect of eosinophils is based on the release of eosinophil peroxidase causing impaired reactive oxygen species and mitogen-activated protein kinase induction in osteoclast precursors. In humans, the number and the activity of eosinophils correlates with bone mass in healthy participants and rheumatoid arthritis patients. Taken together, experimental and human data indicate a regulatory function of eosinophils on bone.

Rheumatoid arthritis and osteoimmunology: The adverse impact of a deregulated immune system on bone metabolism.

The term osteoimmunology describes an interdisciplinary research field that links the investigation of osteology (bone cells) with immunology. The crosstalk between innate and adaptive immune cells and cells involved in bone remodeling, mainly bone-resorbing osteoclasts and bone-forming osteoblasts, becomes particularly obvious in the inflammatory autoimmune disease rheumatoid arthritis (RA). Besides striking inflammation of the joints, RA causes bone loss, leading to joint damage and disabilities as well as generalized osteoporosis. Mechanistically, RA-associated immune cells (macrophages, T cells, B cells etc.) produce high levels of pro-inflammatory cytokines, receptor activator of nuclear factor κB ligand (RANKL) and autoantibodies that promote bone degradation and at the same time counteract new bone formation. Today, antirheumatic therapy effectively ceases joint inflammation and arrests bone erosion. However, the repair of established bone lesions still presents a challenging task and requires improved treatment options. In this review, we outline the knowledge gained over the past years about the immunopathogenesis of RA and the impact of a dysregulated immune system on bone metabolism.

IL-9 Producing Tumor-Infiltrating Lymphocytes and Treg Subsets Drive Immune Escape of Tumor Cells in Non-Small Cell Lung Cancer.

Although lung cancer is the leading cause of cancer deaths worldwide, the mechanisms how lung cancer cells evade the immune system remain incompletely understood. Here, we discovered IL-9-dependent signaling mechanisms that drive immune evasion in non-small cell lung cancer (NSCLC). We found increased IL-9 and IL-21 production by T cells in the tumoral region of the lung of patients with NSCLC, suggesting the presence of Th9 cells in the lung tumor microenvironment. Moreover, we noted IL-9 producing Tregs in NSCLC. IL-9 target cells in NSCLC consisted of IL-9R+ tumor cells and tumor-infiltrating lymphocytes. In two murine experimental models of NSCLC, and in vitro, IL-9 prevented cell death and controlled growth of lung adenocarcinoma cells. Targeted deletion of IL-9 resulted in successful lung tumor rejection in vivo associated with an induction of IL-21 and reduction of Treg cells. Finally, anti-IL-9 antibody immunotherapy resulted in suppression of tumor development even in established experimental NSCLC and was associated with reduced IL-10 production in the lung. In conclusion, our findings indicate that IL-9 drives immune escape of lung tumor cells via effects on tumor cell survival and tumor infiltrating T cells. Thus, strategies blocking IL-9 emerge as a new approach for clinical therapy of lung cancer.

Systemic PPARγ Antagonism Reduces Metastatic Tumor Progression in Adipocyte-Rich Bone in Excess Weight Male Rodents.

Primary tumors are widely associated with an excess in body fat. The role of adipose tissue on tumor cell homing to bone is yet poorly defined. In this study, we aimed to assess whether bone colonization by tumor cells is favored by an adipocyte-rich bone marrow. We delineated the accompanying alterations of the bone microenvironment and established a treatment approach that interferes with high fat diet (HFD)-induced bone metastasis formation. We were able to show that adipocytes affect skeletal tumor growth in a metastatic model of breast cancer in male rats and melanoma in male mice as well as in human breast cancer bone biopsies. Indeed, HFD-induced bone marrow adiposity was accompanied by accelerated tumor progression and increased osteolytic lesions. In human bone metastases, bone marrow adiposity correlated with tumor cell proliferation. By antagonization of the adipocyte differentiation and storage pathway linked to the peroxisome proliferator-activated receptor gamma (PPARγ) with bisphenol-A-diglycidylether (BADGE), we were able to decelerate tumor progression and subsequent osteolytic damage in the bones of two distinct metastatic animal models exposed to HFD. Overall these data show that adipose tissue is a critical factor in bone metastases and cancer-induced bone loss. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Intratumor Heterogeneity Correlates With Reduced Immune Activity and Worse Survival in Melanoma Patients.

BACKGROUND: Human malignant melanoma is a highly aggressive, heterogeneous and drug-resistant cancer. Due to a high number of clones, harboring various mutations that affect key pathways, there is an exceptional level of phenotypic variation and intratumor heterogeneity (ITH) in melanoma. This poses a significant challenge to personalized cancer medicine. Hitherto, it remains unclear to what extent the heterogeneity of melanoma affects the immune microenvironment. Herein, we explore the interaction between the tumor heterogeneity and the host immune response in a melanoma cohort utilizing The Cancer Genome Atlas (TCGA). METHODS: Clonal Heterogeneity Analysis Tool (CHAT) was used to estimate intratumor heterogeneity, and immune cell composition was estimated using CIBERSORT. The Overall Survival (OS) among groups was analyzed using Kaplan-Meier curves with the log-rank test and multivariate cox regression. RNA-seq data were evaluated to identify differentially expressed immunomodulatory genes. The reverse phase protein array (RPPA) data platform was used to validate immune responses at protein level. RESULTS: Tumors with high heterogeneity were associated with decreased overall survival (p = 0.027). High CHAT tumors were correlated with less infiltration by anti-tumor CD8 T cells (p = 0.0049), T follicular cells (p = 0.00091), and M1 macrophages (p = 0.0028), whereas tumor-promoting M2 macrophages were increased (p = 0.02). High CHAT tumors correlated with a reduced expression of immunomodulatory genes, particularly Programmed Cell Death 1 (PD1) and its ligand PD-L1. In addition, high CHAT tumors exhibited lower immune Cytotoxic T lymphocytes (CTLs)-mediated toxicity pathway score (p = 2.9E-07) and cytotoxic pathway score (p = 2.9E-08). High CHAT tumors were also associated with a lower protein level of immune-regulatory kinases, such as lymphocyte-specific protein tyrosine kinase (LCK) (p = 3.4e-5) and spleen tyrosine kinase (SYK) (p = 0.0011). CONCLUSIONS: Highly heterogeneous melanoma tumors are associated with reduced immune cell infiltration and immune response activation as well as decreased survival. Our results reveal that intratumor heterogeneity is an indicative factor for patient survival due to its impact on anti-tumor immune response.

Osteocyte necrosis triggers osteoclast-mediated bone loss through macrophage-inducible C-type lectin.

Although the control of bone-resorbing osteoclasts through osteocyte-derived RANKL is well defined, little is known about the regulation of osteoclasts by osteocyte death. Indeed, several skeletal diseases, such as bone fracture, osteonecrosis, and inflammation are characterized by excessive osteocyte death. Herein we show that osteoclasts sense damage-associated molecular patterns (DAMPs) released by necrotic osteocytes via macrophage-inducible C-type lectin (Mincle), which induced their differentiation and triggered bone loss. Osteoclasts showed robust Mincle expression upon exposure to necrotic osteocytes in vitro and in vivo. RNA sequencing and metabolic analyses demonstrated that Mincle activation triggers osteoclastogenesis via ITAM-based calcium signaling pathways, skewing osteoclast metabolism toward oxidative phosphorylation. Deletion of Mincle in vivo effectively blocked the activation of osteoclasts after induction of osteocyte death, improved fracture repair, and attenuated inflammation-mediated bone loss. Furthermore, in patients with osteonecrosis, Mincle was highly expressed at skeletal sites of osteocyte death and correlated with strong osteoclastic activity. Taken together, these data point to what we believe is a novel DAMP-mediated process that allows osteoclast activation and bone loss in the context of osteocyte death.

The role of Foxp3 and Tbet co-expressing Treg cells in lung carcinoma.

Despite the opposite roles of Tbet and Foxp3 in the immune system as well as in tumour biology, recent studies have demonstrated the presence of of CD4+ T cells, expressing both, Tbet and Foxp3. Although Tbet+Foxp3+ T cells are currently a subject of intense research, less is known about their biological function especially in cancer. Here we found a considerable accumulation of Tbet+Foxp3+CD4+ T cells, mediated by the immunosuppressive cytokine TGFß in the lungs of tumour bearing mice. This is in line with previous studies, demonstrating the important role of TGFß for the immunopathogenesis of cancer. By gathering results both in murine model and in human disease, we demonstrate that, the conversion of IFNγ producing anti-tumoral T-bet+Th1 CD4+ T cells into immunosuppressive Tbet and Foxp3-PD1 co-expressing regulatory cells could represent an additional important mechanism of TGFß-mediated blockade of anti-tumour immunity.

Enhanced Acid Sphingomyelinase Activity Drives Immune Evasion and Tumor Growth in Non-Small Cell Lung Carcinoma.

The lipid hydrolase enzyme acid sphingomyelinase (ASM) is required for the conversion of the lipid cell membrane component sphingomyelin into ceramide. In cancer cells, ASM-mediated ceramide production is important for apoptosis, cell proliferation, and immune modulation, highlighting ASM as a potential multimodal therapeutic target. In this study, we demonstrate elevated ASM activity in the lung tumor environment and blood serum of patients with non-small cell lung cancer (NSCLC). RNAi-mediated attenuation of SMPD1 in human NSCLC cells rendered them resistant to serum starvation-induced apoptosis. In a murine model of lung adenocarcinoma, ASM deficiency reduced tumor development in a manner associated with significant enhancement of Th1-mediated and cytotoxic T-cell-mediated antitumor immunity. Our findings indicate that targeting ASM in NSCLC can act by tumor cell-intrinsic and -extrinsic mechanisms to suppress tumor cell growth, most notably by enabling an effective antitumor immune response by the host. Cancer Res; 77(21); 5963-76. ©2017 AACR.