SHIP1 deficiency causes inflammation-dependent retardation in skeletal growth.

Inflammation and skeletal homeostasis are closely intertwined. Inflammatory diseases are associated with local and systemic bone loss, and post-menopausal osteoporosis is linked to low-level chronic inflammation. Phosphoinositide-3-kinase signalling is a pivotal pathway modulating immune responses and controlling skeletal health. Mice deficient in Src homology 2-containing inositol phosphatase 1 (SHIP1), a negative regulator of the phosphoinositide-3-kinase pathway, develop systemic inflammation associated with low body weight, reduced bone mass, and changes in bone microarchitecture. To elucidate the specific role of the immune system in skeletal development, a genetic approach was used to characterise the contribution of SHIP1-controlled systemic inflammation to SHIP1-dependent osteoclastogenesis. Lymphocyte deletion entirely rescued the skeletal phenotype in Rag2 -/- /Il2rg -/- /SHIP1 -/- mice. Rag2 -/- /Il2rg -/- /SHIP1 -/- osteoclasts, however, displayed an intermediate transcriptomic signature between control and Rag2 +/+ /Il2rg +/+ /SHIP1 -/- osteoclasts while exhibiting aberrant in vitro development and functions similar to Rag2 +/+ /Il2rg +/+ /SHIP1 -/- osteoclasts. These data establish a cell-intrinsic role for SHIP1 in osteoclasts, with inflammation as the key driver of the skeletal phenotype in SHIP1-deficient mice. Our findings demonstrate the central role of the immune system in steering physiological skeletal development.

SHIP1 and its role for innate immune regulation-Novel targets for immunotherapy.

Phosphoinositide-3-kinase/AKT (PI3K/AKT) signaling plays key roles in the regulation of cellular activity in both health and disease. In immune cells, this PI3K/AKT pathway is critically regulated by the phosphoinositide phosphatase SHIP1, which has been reported to modulate the function of most immune subsets. In this review, we summarize our current knowledge of SHIP1 with a focus on innate immune cells, where we reflect on the most pertinent aspects described in the current literature. We also present several small-molecule agonists and antagonists of SHIP1 developed over the last two decades, which have led to improved outcomes in several preclinical models of disease. We outline these promising findings and put them in relation to human diseases with unmet medical needs, where we discuss the most attractive targets for immune therapies based on SHIP1 modulation.

Cell-cell interactome of the hematopoietic niche and its changes in acute myeloid leukemia.

The bone marrow (BM) is a complex microenvironment, coordinating the production of billions of blood cells every day. Despite its essential role and its relevance to hematopoietic diseases, this environment remains poorly characterized. Here we present a high-resolution characterization of the niche in health and acute myeloid leukemia (AML) by establishing a single-cell gene expression database of 339,381 BM cells. We found significant changes in cell type proportions and gene expression in AML, indicating that the entire niche is disrupted. We then predicted interactions between hematopoietic stem and progenitor cells (HSPCs) and other BM cell types, revealing a remarkable expansion of predicted interactions in AML that promote HSPC-cell adhesion, immunosuppression, and cytokine signaling. In particular, predicted interactions involving transforming growth factor ß1 (TGFB1) become widespread, and we show that this can drive AML cell quiescence in vitro. Our results highlight potential mechanisms of enhanced AML-HSPC competitiveness and a skewed microenvironment, fostering AML growth.

Immune cell extravasation in an organ-on-chip to model lung inflammation.

Acute respiratory distress syndrome (ARDS) is a severe lung condition with high mortality and various causes, including lung infection. No specific treatment is currently available and more research aimed at better understanding the pathophysiology of ARDS is needed. Most lung-on-chip models that aim at mimicking the air-blood barrier are designed with a horizontal barrier through which immune cells can migrate vertically, making it challenging to visualize and investigate their migration. In addition, these models often lack a barrier of natural protein-derived extracellular matrix (ECM) suitable for live cell imaging to investigate ECM-dependent migration of immune cells as seen in ARDS. This study reports a novel inflammation-on-chip model with live cell imaging of immune cell extravasation and migration during lung inflammation. The three-channel perfusable inflammation-on-chip system mimics the lung endothelial barrier, the ECM environment and the (inflamed) lung epithelial barrier. A chemotactic gradient was established across the ECM hydrogel, leading to the migration of immune cells through the endothelial barrier. We found that immune cell extravasation depends on the presence of an endothelial barrier, on the ECM density and stiffness, and on the flow profile. In particular, bidirectional flow, broadly used in association with rocking platforms, was found to significantly delay extravasation of immune cells in contrast to unidirectional flow. Extravasation was increased in the presence of lung epithelial tissue. This model is currently used to study inflammation-induced immune cell migration but can be used to study infection-induced immune cell migration under different conditions, such as ECM composition, density and stiffness, type of infectious agents used, and the presence of organ-specific cell types.

Temperature-triggered in situ forming lipid mesophase gel for local treatment of ulcerative colitis.

Ulcerative colitis is a chronic inflammatory bowel disease that strongly affects patient quality of life. Side effects of current therapies necessitate new treatment strategies that maximise the drug concentration at the site of inflammation, while minimizing systemic exposure. Capitalizing on the biocompatible and biodegradable structure of lipid mesophases, we present a temperature-triggered in situ forming lipid gel for topical treatment of colitis. We show that the gel is versatile and can host and release drugs of different polarities, including tofacitinib and tacrolimus, in a sustained manner. Further, we demonstrate its adherence to the colonic wall for at least 6 h, thus preventing leakage and improving drug bioavailability. Importantly, we find that loading known colitis treatment drugs into the temperature-triggered gel improves animal health in two mouse models of acute colitis. Overall, our temperature-triggered gel may prove beneficial in ameliorating colitis and decreasing adverse effects associated with systemic application of immunosuppressive treatments.

Natural killer cell-mimic nanoparticles can actively target and kill acute myeloid leukemia cells.

Natural killer (NK) cells play a crucial role in recognizing and killing emerging tumor cells. However, tumor cells develop mechanisms to inactivate NK cells or hide from them. Here, we engineered a modular nanoplatform that acts as NK cells (NK cell-mimics), carrying the tumor-recognition and death ligand-mediated tumor-killing properties of an NK cell, yet without being subject to tumor-mediated inactivation. NK cell mimic nanoparticles (NK.NPs) incorporate two key features of activated NK cells: cytotoxic activity via the death ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and an adjustable tumor cell recognition feature based on functionalization with the NK cell Fc-binding receptor (CD16, FCGR3A) peptide, enabling the NK.NPs to bind antibodies targeting tumor antigens. NK.NPs showed potent in vitro cytotoxicity against a broad panel of cancer cell lines. Upon functionalizing the NK.NPs with an anti-CD38 antibody (Daratumumab), NK.NPs effectively targeted and eliminated CD38-positive patient-derived acute myeloid leukemia (AML) blasts ex vivo and were able to target and kill CD38-positive AML cells in vivo, in a disseminated AML xenograft system and reduced AML burden in the bone marrow compared to non-targeted, TRAIL-functionalized liposomes. Taken together, NK.NPs are able to mimicking key antitumorigenic functions of NK cells and warrant their development into nano-immunotherapeutic tools.

An Innate Checkpoint Determines Immune Dysregulation and Immunopathology during Pulmonary Murine Coronavirus Infection.

Hallmarks of life-threatening, coronavirus-induced disease include dysregulated antiviral immunity and immunopathological tissue injury. Nevertheless, the sampling of symptomatic patients overlooks the initial inflammatory sequela culminating in severe coronavirus-induced disease, leaving a fundamental gap in our understanding of the early mechanisms regulating anticoronavirus immunity and preservation of tissue integrity. In this study, we delineate the innate regulators controlling pulmonary infection using a natural mouse coronavirus. Within hours of infection, the cellular landscape of the lung was transcriptionally remodeled altering host metabolism, protein synthesis, and macrophage maturation. Genetic perturbation revealed that these transcriptional programs were type I IFN dependent and critically controlled both host cell survival and viral spread. Unrestricted viral replication overshooting protective IFN responses culminated in increased IL-1ß and alarmin production and triggered compensatory neutrophilia, interstitial inflammation, and vascular injury. Thus, type I IFNs critically regulate early viral burden, which serves as an innate checkpoint determining the trajectory of coronavirus dissemination and immunopathology.

Active eosinophils regulate host defence and immune responses in colitis.

In the past decade, single-cell transcriptomics has helped to uncover new cell types and states and led to the construction of a cellular compendium of health and disease. Despite this progress, some difficult-to-sequence cells remain absent from tissue atlases. Eosinophils-elusive granulocytes that are implicated in a plethora of human pathologies1-5-are among these uncharted cell types. The heterogeneity of eosinophils and the gene programs that underpin their pleiotropic functions remain poorly understood. Here we provide a comprehensive single-cell transcriptomic profiling of mouse eosinophils. We identify an active and a basal population of intestinal eosinophils, which differ in their transcriptome, surface proteome and spatial localization. By means of a genome-wide CRISPR inhibition screen and functional assays, we reveal a mechanism by which interleukin-33 (IL-33) and interferon-γ (IFNγ) induce the accumulation of active eosinophils in the inflamed colon. Active eosinophils are endowed with bactericidal and T cell regulatory activity, and express the co-stimulatory molecules CD80 and PD-L1. Notably, active eosinophils are enriched in the lamina propria of a small cohort of patients with inflammatory bowel disease, and are closely associated with CD4+ T cells. Our findings provide insights into the biology of eosinophils and highlight the crucial contribution of this cell type to intestinal homeostasis, immune regulation and host defence. Furthermore, we lay a framework for the characterization of eosinophils in human gastrointestinal diseases.

Acute cytomegalovirus infection modulates the intestinal microbiota and targets intestinal epithelial cells.

Primary and recurrent cytomegalovirus (CMV) infections frequently cause CMV colitis in immunocompromised as well as inflammatory bowel disease (IBD) patients. Additionally, colitis occasionally occurs upon primary CMV infection in patients who are apparently immunocompetent. In both cases, the underlying pathophysiologic mechanisms are largely elusive - in part due to the lack of adequate access to specimens. We employed the mouse cytomegalovirus (MCMV) model to assess the association between CMV and colitis. During acute primary MCMV infection of immunocompetent mice, the gut microbial composition was affected as manifested by an altered ratio of the Firmicutes to Bacteroidetes phyla. Interestingly, these microbial changes coincided with high-titer MCMV replication in the colon, crypt hyperplasia, increased colonic pro-inflammatory cytokine levels, and a transient increase in the expression of the antimicrobial protein Regenerating islet-derived protein 3 gamma (Reg3γ). Further analyses revealed that murine and human intestinal epithelial cell lines, as well as primary intestinal crypt cells and organoids represent direct targets of CMV infection causing increased cell death. Accordingly, in vivo MCMV infection disrupted the intestinal epithelial barrier and increased apoptosis of intestinal epithelial cells. In summary, our data show that CMV transiently induces colitis in immunocompetent hosts by altering the intestinal homeostasis.

A combined spatial score of granzyme B and CD68 surpasses CD8 as an independent prognostic factor in TNM stage II colorectal cancer.

BACKGROUND: Previous assessments of peritumoral inflammatory infiltrate in colorectal cancer (CRC) have focused on the role of CD8+ T lymphocytes. We sought to compare the prognostic value of CD8 with downstream indicators of active immune cell function, specifically granzyme B (GZMB) and CD68 in the tumour microenvironment. METHODS: Immunohistochemical (IHC) staining was performed for CD8, GZMB, CD68 and CD163 on next-generation tissue microarrays (ngTMAs) in a primary cohort (n = 107) and a TNM stage II validation cohort (n = 151). Using digital image analysis, frequency of distinct immune cell types was calculated for tumour proximity (TP) zones with varying radii (10 µm-100 µm) around tumour cells. RESULTS: Associations notably of advanced TNM stage were observed for low density of CD8 (p = 0.002), GZMB (p < 0.001), CD68 (p = 0.034) and CD163 (p = 0.011) in the primary cohort. In the validation cohort only low GZMB (p = 0.036) was associated with pT4 stage. Survival analysis showed strongest prognostic effects in the TP25µm zone at the tumour centre for CD8, GZMB and CD68 (all p < 0.001) in the primary cohort and for CD8 (p = 0.072), GZMB (p = 0.035) and CD68 (p = 0.004) in the validation cohort with inferior prognostic effects observed at the tumour invasive margin. In a multivariate survival analysis, joint analysis of GZMB and CD68 was similarly prognostic to CD8 in the primary cohort (p = 0.007 vs. p = 0.002) and superior to CD8 in the validation cohort (p = 0.005 vs. p = 0.142). CONCLUSION: Combined high expression of GZMB and CD68 within 25 µm to tumour cells is an independent prognostic factor in CRC and of superior prognostic value to the well-established CD8 in TNM stage II cancers. Thus, assessment of antitumoral effect should consider the quality of immune activation in peritumoral inflammatory cells and their actual proximity to tumour cells.

IL-33 biology in cancer: An update and future perspectives.

Interleukin-33 (IL-33) is a member of the IL-1 family of cytokines that is constitutively expressed in the nucleus of epithelial, endothelial and fibroblast-like cells. Upon cell stress, damage or necrosis, IL-33 is released into the cytoplasm to exert its prime role as an alarmin by binding to its specific receptor moiety, ST2. IL-33 exhibits pleiotropic function in inflammatory diseases and particularly in cancer. IL-33 may play a dual role as both a pro-tumorigenic and anti-tumorigenic cytokine, dependent on tumor and cellular context, expression levels, bioactivity and the nature of the inflammatory environment. In this review, we discuss the differential contribution of IL-33 to malignant or inflammatory conditions, its multifaceted effects on the tumor microenvironment, while providing possible explanations for the discrepant findings described in the literature. Additionally, we examine the emerging and divergent functions of IL-33 in the nucleus, and aspects of IL-33 biology that are currently under-addressed.

Viral vector-mediated reprogramming of the fibroblastic tumor stroma sustains curative melanoma treatment.

The tumor microenvironment (TME) is a complex amalgam of tumor cells, immune cells, endothelial cells and fibroblastic stromal cells (FSC). Cancer-associated fibroblasts are generally seen as tumor-promoting entity. However, it is conceivable that particular FSC populations within the TME contribute to immune-mediated tumor control. Here, we show that intratumoral treatment of mice with a recombinant lymphocytic choriomeningitis virus-based vaccine vector expressing a melanocyte differentiation antigen resulted in T cell-dependent long-term control of melanomas. Using single-cell RNA-seq analysis, we demonstrate that viral vector-mediated transduction reprogrammed and activated a Cxcl13-expressing FSC subset that show a pronounced immunostimulatory signature and increased expression of the inflammatory cytokine IL-33. Ablation of Il33 gene expression in Cxcl13-Cre-positive FSCs reduces the functionality of intratumoral T cells and unleashes tumor growth. Thus, reprogramming of FSCs by a self-antigen-expressing viral vector in the TME is critical for curative melanoma treatment by locally sustaining the activity of tumor-specific T cells.

Adenovirus vector vaccination reprograms pulmonary fibroblastic niches to support protective inflating memory CD8+ T cells.

Pathogens and vaccines that produce persisting antigens can generate expanded pools of effector memory CD8+ T cells, described as memory inflation. While properties of inflating memory CD8+ T cells have been characterized, the specific cell types and tissue factors responsible for their maintenance remain elusive. Here, we show that clinically applied adenovirus vectors preferentially target fibroblastic stromal cells in cultured human tissues. Moreover, we used cell-type-specific antigen targeting to define critical cells and molecules that sustain long-term antigen presentation and T cell activity after adenovirus vector immunization in mice. While antigen targeting to myeloid cells was insufficient to activate antigen-specific CD8+ T cells, genetic activation of antigen expression in Ccl19-cre-expressing fibroblastic stromal cells induced inflating CD8+ T cells. Local ablation of vector-targeted cells revealed that lung fibroblasts support the protective function and metabolic fitness of inflating memory CD8+ T cells in an interleukin (IL)-33-dependent manner. Collectively, these data define a critical fibroblastic niche that underpins robust protective immunity operating in a clinically important vaccine platform.

PPARÉ£ drives IL-33-dependent ILC2 pro-tumoral functions.

Group 2 innate lymphoid cells (ILC2s) play a critical role in protection against helminths and in diverse inflammatory diseases by responding to soluble factors such as the alarmin IL-33, that is often overexpressed in cancer. Nonetheless, regulatory factors that dictate ILC2 functions remain poorly studied. Here, we show that peroxisome proliferator-activated receptor gamma (PPARγ) is selectively expressed in ILC2s in humans and in mice, acting as a central functional regulator. Pharmacologic inhibition or genetic deletion of PPARγ in ILC2s significantly impair IL-33-induced Type-2 cytokine production and mitochondrial fitness. Further, PPARγ blockade in ILC2s disrupts their pro-tumoral effect induced by IL-33-secreting cancer cells. Lastly, genetic ablation of PPARγ in ILC2s significantly suppresses tumor growth in vivo. Our findings highlight a crucial role for PPARγ in supporting the IL-33 dependent pro-tumorigenic role of ILC2s and suggest that PPARγ can be considered as a druggable pathway in ILC2s to inhibit their effector functions. Hence, PPARγ targeting might be exploited in cancer immunotherapy and in other ILC2-driven mediated disorders, such as asthma and allergy.

Interleukin-33 signaling exacerbates experimental infectious colitis by enhancing gut permeability and inhibiting protective Th17 immunity.

A wide range of microbial pathogens is capable of entering the gastrointestinal tract, causing infectious diarrhea and colitis. A finely tuned balance between different cytokines is necessary to eradicate the microbial threat and to avoid infection complications. The current study identified IL-33 as a critical regulator of the immune response to the enteric pathogen Citrobacter rodentium. We observed that deficiency of the IL-33 signaling pathway attenuates bacterial-induced colitis. Conversely, boosting this pathway strongly aggravates the inflammatory response and makes the mice prone to systemic infection. Mechanistically, IL-33 mediates its detrimental effect by enhancing gut permeability and by limiting the induction of protective T helper 17 cells at the site of infection, thus impairing host defense mechanisms against the enteric pathogen. Importantly, IL-33-treated infected mice supplemented with IL-17A are able to resist the otherwise strong systemic spreading of the pathogen. These findings reveal a novel IL-33/IL-17A crosstalk that controls the pathogenesis of Citrobacter rodentium-driven infectious colitis. Manipulating the dynamics of cytokines may offer new therapeutic strategies to treat specific intestinal infections.

GPR15 Facilitates Recruitment of Regulatory T Cells to Promote Colorectal Cancer.

Colorectal cancer is one of the most frequent malignancies worldwide. Despite considerable progress in early detection and treatment, there is still an unmet need for novel antitumor therapies, particularly in advanced colorectal cancer. Regulatory T cells (Treg) are increased in the peripheral blood and tumor tissue of patients with colorectal cancer. Recently, transient ablation of tumor-associated Tregs was shown to foster CD8+ T-cell-mediated antitumoral immunity in murine colorectal cancer models. However, before considering therapies on targeting Tregs in patients with cancer, detailed knowledge of the phenotype and features of tumor-associated Tregs is indispensable. Here, we demonstrate in a murine model of inflammation-induced colorectal cancer that tumor-associated Tregs are mainly of thymic origin and equipped with a specific set of molecules strongly associated with enhanced migratory properties. Particularly, a dense infiltration of Tregs in mouse and human colorectal cancer lesions correlated with increased expression of the orphan chemoattractant receptor GPR15 on these cells. Comprehensive gene expression analysis revealed that tumor-associated GPR15+ Tregs have a Th17-like phenotype, thereby producing IL17 and TNFα. Gpr15 deficiency repressed Treg infiltration in colorectal cancer, which paved the way for enhanced antitumoral CD8+ T-cell immunity and reduced tumorigenesis. In conclusion, GPR15 represents a promising novel target for modifying T-cell-mediated antitumoral immunity in colorectal cancer. SIGNIFICANCE: The G protein-coupled receptor 15, an unconventional chemokine receptor, directs Tregs into the colon, thereby modifying the tumor microenvironment and promoting intestinal tumorigenesis.See related commentary by Chakraborty and Zappasodi, p. 2817.

The multifaceted role of TRAIL signaling in cancer and immunity.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that can lead to the induction of apoptosis in tumor or infected cells. However, activation of TRAIL signaling may also trigger nonapoptotic pathways in cancer and in nontransformed cells, that is, immune cells. Here, we review the current knowledge on noncanonical TRAIL signaling. The biological outcomes of TRAIL signaling in immune and malignant cells are presented and explained, with a focus on the role of TRAIL for natural killer (NK) cell function. Furthermore, we highlight the technical difficulties in dissecting the precise molecular mechanisms involved in the switch between apoptotic and nonapoptotic TRAIL signaling. Finally, we discuss the consequences thereof for a therapeutic manipulation of TRAIL in cancer and possible approaches to bypass these difficulties.

Heterogeneous bone-marrow stromal progenitors drive myelofibrosis via a druggable alarmin axis.

Functional contributions of individual cellular components of the bone-marrow microenvironment to myelofibrosis (MF) in patients with myeloproliferative neoplasms (MPNs) are incompletely understood. We aimed to generate a comprehensive map of the stroma in MPNs/MFs on a single-cell level in murine models and patient samples. Our analysis revealed two distinct mesenchymal stromal cell (MSC) subsets as pro-fibrotic cells. MSCs were functionally reprogrammed in a stage-dependent manner with loss of their progenitor status and initiation of differentiation in the pre-fibrotic and acquisition of a pro-fibrotic and inflammatory phenotype in the fibrotic stage. The expression of the alarmin complex S100A8/S100A9 in MSC marked disease progression toward the fibrotic phase in murine models and in patient stroma and plasma. Tasquinimod, a small-molecule inhibiting S100A8/S100A9 signaling, significantly ameliorated the MPN phenotype and fibrosis in JAK2V617F-mutated murine models, highlighting that S100A8/S100A9 is an attractive therapeutic target in MPNs.

Cleft lip and cleft palate in Esrp1 knockout mice is associated with alterations in epithelial-mesenchymal crosstalk.

Cleft lip is one of the most common human birth defects. However, there remain a limited number of mouse models of cleft lip that can be leveraged to characterize the genes and mechanisms that cause this disorder. Crosstalk between epithelial and mesenchymal cells underlies formation of the face and palate, but the basic molecular events mediating this crosstalk remain poorly understood. We previously demonstrated that mice lacking the epithelial-specific splicing factor Esrp1 have fully penetrant bilateral cleft lip and palate. In this study, we further investigated the mechanisms leading to cleft lip as well as cleft palate in both existing and new Esrp1 mutant mouse models. These studies included a detailed transcriptomic analysis of changes in ectoderm and mesenchyme in Esrp1-/- embryos during face formation. We identified altered expression of genes previously implicated in cleft lip and/or palate, including components of multiple signaling pathways. These findings provide the foundation for detailed investigations using Esrp1 mutant disease models to examine gene regulatory networks and pathways that are essential for normal face and palate development - the disruption of which leads to orofacial clefting in human patients.