B cell expansion hinders the stroma-epithelium regenerative cross talk during mucosal healing.

Therapeutic promotion of intestinal regeneration holds great promise, but defining the cellular mechanisms that influence tissue regeneration remains an unmet challenge. To gain insight into the process of mucosal healing, we longitudinally examined the immune cell composition during intestinal damage and regeneration. B cells were the dominant cell type in the healing colon, and single-cell RNA sequencing (scRNA-seq) revealed expansion of an IFN-induced B cell subset during experimental mucosal healing that predominantly located in damaged areas and associated with colitis severity. B cell depletion accelerated recovery upon injury, decreased epithelial ulceration, and enhanced gene expression programs associated with tissue remodeling. scRNA-seq from the epithelial and stromal compartments combined with spatial transcriptomics and multiplex immunostaining showed that B cells decreased interactions between stromal and epithelial cells during mucosal healing. Activated B cells disrupted the epithelial-stromal cross talk required for organoid survival. Thus, B cell expansion during injury impairs epithelial-stromal cell interactions required for mucosal healing, with implications for the treatment of IBD.

Dual roles of B lymphocytes in mouse models of diet-induced nonalcoholic fatty liver disease.

BACKGROUND AND AIMS: Growing evidence suggests an important role of B cells in the development of NAFLD. However, a detailed functional analysis of B cell subsets in NAFLD pathogenesis is lacking. APPROACH AND RESULTS: In wild-type mice, 21 weeks of high fat diet (HFD) feeding resulted in NAFLD with massive macrovesicular steatosis, modest hepatic and adipose tissue inflammation, insulin resistance, and incipient fibrosis. Remarkably, Bnull (JHT) mice were partially protected whereas B cell harboring but antibody-deficient IgMi mice were completely protected from the development of hepatic steatosis, inflammation, and fibrosis. The common feature of JHT and IgMi mice is that they do not secrete antibodies, whereas HFD feeding in wild-type mice led to increased levels of serum IgG2c. Whereas JHT mice have no B cells at all, regulatory B cells were found in the liver of both wild-type and IgMi mice. HFD reduced the number of regulatory B cells and IL-10 production in the liver of wild-type mice, whereas these increased in IgMi mice. Livers of patients with advanced liver fibrosis showed abundant deposition of IgG and stromal B cells and low numbers of IL-10 expressing cells, compatible with our experimental data. CONCLUSIONS: B lymphocytes have both detrimental and protective effects in HFD-induced NAFLD. The lack of secreted pathogenic antibodies protects partially from NAFLD, whereas the presence of certain B cell subsets provides additional protection. IL-10-producing regulatory B cells may represent such a protective B cell subset.

Active Akt signaling triggers CLL toward Richter transformation via overactivation of Notch1.

Richter's transformation (RT) is an aggressive lymphoma that occurs upon progression from chronic lymphocytic leukemia (CLL). Transformation has been associated with genetic aberrations in the CLL phase involving TP53, CDKN2A, MYC, and NOTCH1; however, a significant proportion of RT cases lack CLL phase-associated events. Here, we report that high levels of AKT phosphorylation occur both in high-risk CLL patients harboring TP53 and NOTCH1 mutations as well as in patients with RT. Genetic overactivation of Akt in the murine Eµ-TCL1 CLL mouse model resulted in CLL transformation to RT with significantly reduced survival and an aggressive lymphoma phenotype. In the absence of recurrent mutations, we identified a profile of genomic aberrations intermediate between CLL and diffuse large B-cell lymphoma. Multiomics assessment by phosphoproteomic/proteomic and single-cell transcriptomic profiles of this Akt-induced murine RT revealed an S100 protein-defined subcluster of highly aggressive lymphoma cells that developed from CLL cells, through activation of Notch via Notch ligand expressed by T cells. Constitutively active Notch1 similarly induced RT of murine CLL. We identify Akt activation as an initiator of CLL transformation toward aggressive lymphoma by inducing Notch signaling between RT cells and microenvironmental T cells.

Endogenous CD83 Expression in CD4+ Conventional T Cells Controls Inflammatory Immune Responses.

The glycoprotein CD83 is known to be expressed by different immune cells including activated CD4+Foxp3+ regulatory T cells (Tregs) and CD4+Foxp3- conventional T cells. However, the physiological function of endogenous CD83 in CD4+ T cell subsets is still unclear. In this study, we have generated a new CD83flox mouse line on BALB/c background, allowing for specific ablation of CD83 in T cells upon breeding with CD4-cre mice. Tregs from CD83flox/flox/CD4-cretg/wt mice had similar suppressive activity as Tregs from CD83flox/flox/CD4-crewt/wt wild-type littermates, suggesting that endogenous CD83 expression is dispensable for the inhibitory capacity of Tregs. However, CD83-deficient CD4+ conventional T cells showed elevated proliferation and IFN-γ secretion as well as an enhanced capacity to differentiate into Th1 cells and Th17 cells upon stimulation in vitro. T cell-specific ablation of CD83 expression resulted in aggravated contact hypersensitivity reaction accompanied by enhanced CD4+ T cell activation. Moreover, adoptive transfer of CD4+CD45RBhigh T cells from CD83flox/flox/CD4-cretg /wt mice into Rag2-deficient mice elicited more severe colitis associated with increased serum concentrations of IL-12 and elevated CD40 expression on CD11c+ dendritic cells (DCs). Strikingly, DCs from BALB/c mice cocultured with CD83-deficient CD4+ conventional T cells showed enhanced CD40 expression and IL-12 secretion compared with DCs cocultured with CD4+ conventional T cells from CD83flox/flox/CD4-crewt/wt wild-type mice. In summary, these results indicate that endogenous CD83 expression in CD4+ conventional T cells plays a crucial role in controlling CD4+ T cell responses, at least in part, by regulating the activity of CD11c+ DCs.

Chronic intestinal inflammation drives colorectal tumor formation triggered by dietary heme iron in vivo.

The consumption of red meat is associated with an increased risk for colorectal cancer (CRC). Multiple lines of evidence suggest that heme iron as abundant constituent of red meat is responsible for its carcinogenic potential. However, the underlying mechanisms are not fully understood and particularly the role of intestinal inflammation has not been investigated. To address this important issue, we analyzed the impact of heme iron (0.25 µmol/g diet) on the intestinal microbiota, gut inflammation and colorectal tumor formation in mice. An iron-balanced diet with ferric citrate (0.25 µmol/g diet) was used as reference. 16S rRNA sequencing revealed that dietary heme reduced α-diversity and caused a persistent intestinal dysbiosis, with a continuous increase in gram-negative Proteobacteria. This was linked to chronic gut inflammation and hyperproliferation of the intestinal epithelium as attested by mini-endoscopy, histopathology and immunohistochemistry. Dietary heme triggered the infiltration of myeloid cells into colorectal mucosa with an increased level of COX-2 positive cells. Furthermore, flow cytometry-based phenotyping demonstrated an increased number of T cells and B cells in the lamina propria following heme intake, while γδ-T cells were reduced in the intraepithelial compartment. Dietary heme iron catalyzed formation of fecal N-nitroso compounds and was genotoxic in intestinal epithelial cells, yet suppressed intestinal apoptosis as evidenced by confocal microscopy and western blot analysis. Finally, a chemically induced CRC mouse model showed persistent intestinal dysbiosis, chronic gut inflammation and increased colorectal tumorigenesis following heme iron intake. Altogether, this study unveiled intestinal inflammation as important driver in heme iron-associated colorectal carcinogenesis.

Alternative Splice Forms of CYLD Mediate Ubiquitination of SMAD7 to Prevent TGFB Signaling and Promote Colitis.

BACKGROUND & AIMS: The CYLD lysine 63 deubiquitinase gene (CYLD) encodes tumor suppressor protein that is mutated in familial cylindromatosus, and variants have been associated with Crohn disease (CD). Splice forms of CYLD that lack exons 7 and 8 regulate transcription factors and functions of immune cells. We examined the expression of splice forms of CYLD in colon tissues from patients with CD and their effects in mice. METHODS: We performed immunohistochemical analyses of colon tissues from patients with untreated CD and patients without inflammatory bowel diseases (controls). We obtained mice that expressed splice forms of CYLD (sCYLD mice) without or with SMAD7 (sCYLD/SMAD7 mice) from transgenes and CYLD-knockout mice (with or without transgenic expression of SMAD7) and performed endoscopic analyses. Colitis was induced in Rag1-/- mice by transfer of CD4+ CD62L+ T cells from C57/Bl6 or transgenic mice. T cells were isolated from mice and analyzed by flow cytometry and quantitative real-time polymerase chain reaction and intestinal tissues were analyzed by histology and immunohistochemistry. CYLD forms were expressed in mouse embryonic fibroblasts, primary T cells, and HEK293T cells, which were analyzed by immunoblot, mobility shift, and immunoprecipitation assays. RESULTS: The colonic lamina propria from patients with CD was infiltrated by T cells and had higher levels of sCYLD (but not full-length CYLD) and SMAD7 than tissues from controls. Incubation of mouse embryonic fibroblasts and T cells with transforming growth factor ß increased their production of sCYLD and decreased full-length CYLD. Transgenic expression of sCYLD and SMAD7 in T cells prevented the differentiation of regulatory T cells and T-helper type 17 cells and increased the differentiation of T-helper type 1 cells. The same effects were observed in colon tissues from sCYLD/SMAD7 mice but not in those from CYLD-knockout SMAD7 mice. The sCYLD mice had significant increases in the numbers of T-helper type 1 cells and CD44high CD62Llow memory-effector CD4+ T cells in the spleen and mesenteric lymph nodes compared with wild-type mice; sCYLD/SMAD7 mice had even larger increases. The sCYLD/SMAD7 mice spontaneously developed severe colitis, with infiltration of the colon by dendritic cells, neutrophils, macrophages, and CD4+ T cells and increased levels of Ifng, Il6, Il12a, Il23a, and Tnf mRNAs. Co-transfer of regulatory T cells from wild-type, but not from sCYLD/SMAD7, mice prevented the induction of colitis in Rag1-/- mice by CD4+ T cells. We found increased levels of poly-ubiquitinated SMAD7 in sCYLD CD4+ T cells. CYLD formed a nuclear complex with SMAD3, whereas sCYLD recruited SMAD7 to the nucleus, which inhibited the expression of genes regulated by SMAD3 and SMAD4. We found that sCYLD mediated lysine 63-linked ubiquitination of SMAD7. The sCYLD-SMAD7 complex inhibited transforming growth factor ß signaling in CD4+ T cells. CONCLUSIONS: Levels of the spliced form of CYLD are increased in colon tissues from patients with CD. sCYLD mediates ubiquitination and nuclear translocation of SMAD7 and thereby decreases transforming growth factor ß signaling in T cells. This prevents immune regulatory mechanisms and leads to colitis in mice.

Nuclear Translocation of RELB Is Increased in Diseased Human Liver and Promotes Ductular Reaction and Biliary Fibrosis in Mice.

BACKGROUND & AIMS: Cholangiocyte proliferation and ductular reaction contribute to the onset and progression of liver diseases. Little is known about the role of the transcription factor nuclear factor-κB (NF-κB) in this process. We investigated the activities of the RELB proto-oncogene NF-κB subunit in human cholangiocytes and in mouse models of liver disease characterized by a ductular reaction. METHODS: We obtained liver tissue samples from patients with primary sclerosing cholangitis, primary biliary cholangitis, hepatitis B or C virus infection, autoimmune hepatitis, alcoholic liver disease, or without these diseases (controls) from a tissue bank in Germany. Tissues were analyzed by immunohistochemistry for levels of RELB and lymphotoxin ß (LTB). We studied mice with liver parenchymal cell (LPC)-specific disruption of the cylindromatosis (CYLD) lysine 63 deubiquitinase gene (Cyld), with or without disruption of Relb (CyldΔLPC mice and Cyld/RelbΔLPC mice) and compared them with C57BL/6 mice (controls). Mice were fed 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or standard chow diets to induce biliary injury or were given injections of CCl4 to induce non-cholestatic liver fibrosis. Liver tissues were analyzed by histology, immunohistochemistry, immunoblots, in situ hybridization, and quantitative real-time polymerase chain reaction. Cholangiocytes were isolated from normal human liver, incubated with LTB receptor agonist, and transfected with small interfering RNAs to knock down RELB. RESULTS: In liver tissues from patients with primary sclerosing cholangitis, primary biliary cholangitis, chronic infection with hepatitis B or C virus, autoimmune hepatitis, or alcoholic liver disease, we detected increased nuclear translocation of RELB and increased levels of LTB in cholangiocytes that formed reactive bile ducts compared with control liver tissues. Human cholangiocytes, but not those with RELB knockdown, proliferated with exposure to LTB. The phenotype of CyldΔLPC mice, which included ductular reaction, oval cell activation, and biliary fibrosis, was completely lost from Cyld/RelbΔLPC mice. Compared with livers from control mice, livers from CyldΔLPC mice (but not Cyld/RelbΔLPC mice) had increased levels of mRNAs encoding cytokines (LTB; CD40; and tumor necrosis factor superfamily [TNFSF] members TNFSF11 [RANKL], TNFSF13B [BAFF], and TNFSF14 [LIGHT]) produced by reactive cholangiocytes. However, these strains of mice developed similar levels of liver fibrosis in response to CCl4 exposure. CyldΔLPC mice and Cyld/RelbΔLPC mice had improved liver function on the DDC diet compared with control mice fed the DDC diet. CONCLUSION: Reactive bile ducts in patients with chronic liver diseases have increased levels of LTB and nuclear translocation of RELB. RELB is required for the ductular reaction and development of biliary fibrosis in CyldΔLPC mice. Deletion of RELB and CYLD from LPCs protects mice from DDC-induced cholestatic liver fibrosis.

mTORC1 activation in B cells confers impairment of marginal zone microarchitecture by exaggerating cathepsin activity.

Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell metabolism and lymphocyte proliferation. It is inhibited by the tuberous sclerosis complex (TSC), a heterodimer of TSC1 and TSC2. Deletion of either gene results in robust activation of mTORC1. Mature B cells reside in the spleen at two major anatomical locations, the marginal zone (MZ) and follicles. The MZ constitutes the first line of humoral response against blood-borne pathogens and undergoes atrophy in chronic inflammation. In previous work, we showed that mice deleted for TSC1 in their B cells (TSC1BKO ) have almost no MZ B cells, whereas follicular B cells are minimally affected. To explore potential underlying mechanisms for MZ B-cell loss, we have analysed the spleen MZ architecture of TSC1BKO mice and found it to be severely impaired. Examination of lymphotoxins (LTα and LTß) and lymphotoxin receptor (LTßR) expression indicated that LTßR levels in spleen stroma were reduced by TSC1 deletion in the B cells. Furthermore, LTα transcripts in B cells were reduced. Because LTßR is sensitive to proteolysis, we analysed cathepsin activity in TSC1BKO . A higher cathepsin activity, particularly of cathepsin B, was observed, which was reduced by mTORC1 inhibition with rapamycin in vivo. Remarkably, in vivo administration of a pan-cathepsin inhibitor restored LTßR expression, LTα mRNA levels and the MZ architecture. Our data identify a novel connection, although not elucidated at the molecular level, between mTORC1 and cathepsin activity in a manner relevant to MZ dynamics.

Hepatocyte-specific deletion of IL1-RI attenuates liver injury by blocking IL-1 driven autoinflammation.

BACKGROUND & AIMS: Interleukin (IL)-1-type cytokines including IL-1α, IL-1ß and interleukin-1 receptor antagonist (IL-1Ra) are among the most potent molecules of the innate immune system and exert biological activities through the ubiquitously expressed interleukin-1 receptor type 1 (IL-1R1). The role of IL-1R1 in hepatocytes during acute liver failure (ALF) remains undetermined. METHODS: The role of IL-1R1 during ALF was investigated using a novel transgenic mouse model exhibiting deletion of all signaling-capable IL-1R isoforms in hepatocytes (Il1r1Hep-/-). RESULTS: ALF induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS) was significantly attenuated in Il1r1Hep-/- mice leading to reduced mortality. Conditional deletion of Il1r1 decreased activation of injurious c-Jun N-terminal kinases (JNK)/c-Jun signaling, activated nuclear factor-kappa B (NF-κB) p65, inhibited extracellular signal-regulated kinase (ERK) and prevented caspase 3-mediated apoptosis. Moreover, Il1r1Hep-/- mice exhibited reduced local and systemic inflammatory cytokine and chemokine levels, especially TNF-α, IL-1α/ß, IL-6, CC-chemokine ligand 2 (CCL2), C-X-C motif ligand 1 (CXCL-1) and CXCL-2, and a reduced neutrophil recruitment into the hepatic tissue in response to injury. NLRP3 inflammasome expression and caspase 1 activation were suppressed in the absence of the hepatocellular IL-1R1. Inhibition of IL-1R1 using IL-1ra (anakinra) attenuated the severity of liver injury, while IL-1α administration exaggerated it. These effects were lost ex vivo and at later time points, supporting a role of IL-1R1 in inflammatory signal amplification during acute liver injury. CONCLUSION: IL-1R1 in hepatocytes plays a pivotal role in an IL-1-driven auto-amplification of cell death and inflammation in the onset of ALF. LAY SUMMARY: Acute liver injury which can cause lethal liver failure is medicated by a class of proteins called cytokines. Among these, interleukin-1 (IL-1) and the corresponding receptor IL-1R1 play a prominent role in the immune system, but their role in the liver is undetermined. In the current study, a novel mouse model with defective IL-1R1 in liver cells was studied. Mice lacking this receptor in liver cells were protected from cell death to a certain extent. This protection occurred only in the presence of other, neighboring cells, arguing for the involvement of proteins derived from these cells. This effect is called paracrine signaling and the current study has for the first time shown that the IL-1R1 receptor on hepatocytes is involved in acute liver failure in this context. The approved drug anakinra - which blocks IL-1R1 - had the same effect, supporting the proposed mechanism of action. The findings of this study suggest new treatment options for patients with acute liver failure by blocking defined signals of the immune system.

Balanced Bcl-3 expression in murine CD4+ T cells is required for generation of encephalitogenic Th17 cells.

The function of NF-κB family members is controlled by multiple mechanisms including the transcriptional regulator Bcl-3, an atypical member of the IκB family. By using a murine model of conditional Bcl-3 overexpression specifically in T cells, we observed impairment in the development of Th2, Th1, and Th17 cells. High expression of Bcl-3 promoted CD4+ T-cell survival, but at the same time suppressed proliferation in response to TCR stimulation, resulting in reduced CD4+ T-cell expansion. As a consequence, T-cell-specific overexpression of Bcl-3 led to reduced inflammation in the small intestine of mice applied with anti-CD3 in a model of gut inflammation. Moreover, impaired Th17-cell development resulted in the resistance of Bcl-3 overexpressing mice to EAE, a mouse model of multiple sclerosis. Thus, we concluded that fine-tuning expression of Bcl-3 is needed for proper CD4+ T-cell development and is required to sustain Th17-cell mediated pathology.

NF-κB-inducing kinase is essential for B-cell maintenance in mice.

NF-κB-inducing kinase (NIK) is a key mediator of the noncanonical NF-κB signaling pathway, which is critical for normal B-cell development and function. It is well established that the complete deletion of NIK in mice results in defective B cells and impaired secondary lymphoid organogenesis. To address the role of NIK deficiency specifically in B cells, we generated a new mouse strain for the conditional deletion of this kinase. Deletion of NIK during B-cell development results in a drastic reduction of mature B cells from the transitional 2 stage on, while B-1 B cells are less affected. Moreover, deletion of NIK in the germinal centers decreases the numbers of germinal center B cells and impairs the ability of NIK-deficient B cells to develop into class-switched cells in vivo. This new mouse strain will be helpful for studying the role of NIK in different cell types of the body.

Hepatic B cell leukemia-3 promotes hepatic steatosis and inflammation through insulin-sensitive metabolic transcription factors.

BACKGROUND & AIMS: The pathomechanisms underlying non-alcoholic fatty liver disease (NAFLD) and the involved molecular regulators are incompletely explored. The nuclear factor-kappa B (NF-κB)-cofactor gene B cell leukemia-3 (Bcl-3) plays a critical role in altering the transcriptional capacity of NF-κB - a key inducer of inflammation - but also of genes involved in cellular energy metabolism. METHODS: To define the role of Bcl-3 in non-alcoholic steatohepatitis (NASH), we developed a novel transgenic mouse model with hepatocyte-specific overexpression of Bcl-3 (Bcl-3Hep) and employed a high-fat, high-carbohydrate dietary feeding model. To characterize the transgenic model, deep RNA sequencing was performed. The relevance of the findings was confirmed in human liver samples. RESULTS: Hepatocyte-specific overexpression of Bcl-3 led to pronounced metabolic derangement, characterized by enhanced hepatic steatosis from increased de novo lipogenesis and uptake, as well as decreased hydrolysis and export of fatty acids. Steatosis in Bcl-3Hep mice was accompanied by an augmented inflammatory milieu and liver cell injury. Moreover, Bcl-3 expression decreased insulin sensitivity and resulted in compensatory regulation of insulin-signaling pathways. Based on in vivo and in vitro studies we identified the transcription factors PPARα, PPARγ and PGC-1α as critical regulators of hepatic metabolism and inflammation downstream of Bcl-3. Metformin treatment improved the metabolic and inflammatory phenotype in Bcl-3Hep mice through modulation of PPARα and PGC-1α. Remarkably, these findings were recapitulated in human NASH, which exhibited increased expression and nuclear localization of Bcl-3. CONCLUSIONS: In summary, Bcl-3 emerges as a novel regulator of hepatic steatosis, insulin sensitivity and inflammation in NASH. LAY SUMMARY: Non-alcoholic fatty liver disease (NAFLD) is considered the most prevalent liver disease worldwide. Patients can develop end-stage liver disease resulting in liver cirrhosis or hepatocellular carcinoma, but also develop complications unrelated to liver disease, e.g., cardiovascular disease. Still there is no full understanding of the mechanisms that cause NAFLD. In this study, genetically engineered mice were employed to examine the role of a specific protein in the liver that is involved in inflammation and the metabolism, namely Bcl-3. By this approach, a better understanding of the mechanisms contributing to disease progression was established. This can help to develop novel therapeutic and diagnostic options for patients with NAFLD.

Overexpression of Bcl-3 inhibits the development of marginal zone B cells.

The transcription factor Bcl-3 functions as a proto-oncogene via regulation of cell proliferation and apoptosis. Bcl-3 is an atypical member of the IκB family and plays a central role in the immune response through interactions with the NF-κB subunits p50 and p52. To investigate the impact of Bcl-3 on B-cell maturation and regulation, we generated mice that overexpress Bcl-3 specifically in B cells. Interestingly, these mice lack marginal zone B cells and exhibit a significant reduction in the number of B-1 B cells. Further, B cells from these mice are impaired in their proliferative capacity. Our data demonstrate that the overexpression of the transcription factor Bcl-3 inhibits germinal center formation, marginal zone B-cell development, and affects the B-1 B-cell compartment.

The deubiquitinating enzyme CYLD regulates the differentiation and maturation of thymic medullary epithelial cells.

The cross talk between thymocytes and the thymic epithelium is critical for T-cell development and the establishment of central tolerance. Medullary thymic epithelial cells (mTECs) are located in the thymic medulla and mediate the elimination of self-reactive thymocytes, thereby preventing the onset of autoimmunity. Previous studies identified the deubiquitinating enzyme CYLD as a critical regulator of T-cell development by activating proximal T-cell receptor signaling during the transition of double-positive to single-positive thymocytes. Here we evaluated the impact of the naturally occurring short-splice variant of the cyld gene (sCYLD) on the development and maturation of mTECs. We found that thymi of CYLD(ex7/8) mice, solely expressing sCYLD, displayed a reduced number of mature mTECs caused by a developmental block during the transition of immature to mature mTECs. Further, we could demonstrate an impaired negative selection of thymocytes in these mice. Our data demonstrate that inefficient negative selection in the thymus of CYLD(ex7/8) mice result from a defect in mTEC maturation.

The tumor suppressor CYLD controls the function of murine regulatory T cells.

CYLD was originally identified as a tumor suppressor gene mutated in familial cylindromatosis, an autosomal dominant predisposition to multiple benign neoplasms of the skin known as cylindromas. The CYLD protein is a deubiquitinating enzyme that acts as a negative regulator of NF-κB and JNK signaling through its interaction with NEMO and TNFR-associated factor 2. We have previously described a novel mouse strain that expresses solely and excessively a naturally occurring splice variant of CYLD (CYLD(ex7/8)). In this study, we demonstrate that CYLD plays a critical role in Treg development and function. T cells of CYLD(ex7/8) mice had a hyperactive phenotype manifested by increased production of inflammatory cytokines and constitutive activation of the NF-κB pathway. Furthermore, the amount of Foxp3(+) regulatory T cells in these mice was markedly enhanced in thymus and peripheral organs. Importantly, these regulatory T cells displayed decreased expression levels of CD25 and CTLA-4 associated with impaired suppressive capacity. Hence, our data emphasize an essential role of CYLD in maintaining T cell homeostasis as well as normal T regulatory cell function, thereby controlling abnormal T cell responses.

Isolation and high-dimensional flow cytometric analysis of tumor-infiltrating leukocytes in a mouse model of colorectal cancer.

Colorectal cancer (CRC) is a complex and heterogeneous disease characterized by dysregulated interactions between tumor cells and the immune system. The tumor microenvironment plays a pivotal role in cancer initiation as well as progression, with myeloid immune cells such as dendritic cell and macrophage subsets playing diverse roles in cancer immunity. On one hand, they exert anti-tumor effects, but they can also contribute to tumor growth. The AOM/DSS colitis-associated cancer mouse model has emerged as a valuable tool to investigate inflammation-driven CRC. To understand the role of different leukocyte populations in tumor development, the preparation of single cell suspensions from tumors has become standard procedure for many types of cancer in recent years. However, in the case of AOM/DSS-induced colorectal tumors, this is still challenging and rarely described. For one, to be able to properly distinguish tumor-associated immune cells, separate processing of cancerous and surrounding colon tissue is essential. In addition, cell yield, due to the low tumor mass, viability, as well as preservation of cell surface epitopes are important for successful flow cytometric profiling of tumor-infiltrating leukocytes. Here we present a fast, simple, and economical step-by-step protocol for isolating colorectal tumor-associated leukocytes from AOM/DSS-treated mice. Furthermore, we demonstrate the feasibility of this protocol for high-dimensional flow cytometric identification of the different tumor-infiltrating leukocyte populations, with a specific focus on myeloid cell subsets.

Natural antibodies are required for clearance of necrotic cells and recovery from acute liver injury.

Background & Aims: Hepatocellular necrosis is common in both acute and chronic liver injury and may evolve to fibrosis and liver failure. Injury leads to accumulation of necrotic cell debris in the liver, which drives persistent inflammation and poor recovery. This study investigated the role of natural antibodies (NAbs) in the clearance of necrotic cells in the injured liver, their impact on tissue regeneration and their potential as a therapy for acute liver injury. Methods: We used murine models of drug-induced liver injury and focal thermal injury in immunocompetent and antibody-deficient mice (Rag2-/- and IgMi). Intravital microscopy was used to investigate the role of NAbs in the phagocytosis of necrotic cells in the liver in vivo. Immunostainings were used to quantify the extent of liver necrosis (fibrin), antibody deposition (IgM and IgG) and cellular proliferation (Ki67). Results: Both IgM and IgG NAbs bound necrotic liver areas and opsonized multiple debris molecules released during hepatocellular necrosis such as DNA, histones, actin, phosphoinositides and mitochondrial cardiolipin, but not phosphatidylserine. Rag2-/- and IgMi mice presented impaired recovery from liver injury, which was correlated to the sustained presence of necrotic debris in the tissue, prolonged inflammation and reduced hepatocellular proliferation. These defects were rescued by treating mice with NAbs after the induction of injury. Mechanistically, in vitro and in vivo, phagocytosis of necrotic debris was dependent on NAbs via Fcγ receptors and CD11b. Moreover, NAb-mediated phagocytosis of necrotic cell debris occurs in two waves, firstly driven by neutrophils and then by recruited monocytes. Importantly, supplementation of immunocompetent mice with NAbs also improved liver regeneration significantly, demonstrating the therapeutic potential of natural IgM and IgG. Conclusion: NAbs drive the phagocytosis of necrotic cells in liver injury and promote liver regeneration and recovery. Impact and implications: Treatment with natural antibodies after acute liver injury improved recovery by increasing the clearance of necrotic debris and by improving cellular proliferation in the liver. This preclinical study provides a basis for the development of an immunotherapy for patients with early-stage, reversible, liver injury that aims to prevent disease chronification into fibrosis and liver failure.

Myeloid cell-derived interleukin-6 induces vascular dysfunction and vascular and systemic inflammation.

Aims: The cytokine interleukin-6 (IL-6) plays a central role in the inflammation cascade as well as cardiovascular disease progression. Since myeloid cells are a primary source of IL-6 formation, we aimed to generate a mouse model to study the role of myeloid cell-derived IL-6 in vascular disease. Methods and results: Interleukin-6-overexpressing (IL-6OE) mice were generated and crossed with LysM-Cre mice, to generate mice (LysM-IL-6OE mice) overexpressing the cytokine in myeloid cells. Eight- to 12-week-old LysM-IL-6OE mice spontaneously developed inflammatory colitis and significantly impaired endothelium-dependent aortic relaxation, increased aortic reactive oxygen species (ROS) formation, and vascular dysfunction in resistance vessels. The latter phenotype was associated with decreased survival. Vascular dysfunction was accompanied by a significant accumulation of neutrophils, monocytes, and macrophages in the aorta, increased myeloid cell reactivity (elevated ROS production), and vascular fibrosis associated with phenotypic changes in vascular smooth muscle cells. In addition to elevated Mcp1 and Cxcl1 mRNA levels, aortae from LysM-IL-6OE mice expressed higher levels of inducible NO synthase and endothelin-1, thus partially accounting for vascular dysfunction, whereas systemic blood pressure alterations were not observed. Bone marrow (BM) transplantation experiments revealed that vascular dysfunction and ROS formation were driven by BM cell-derived IL-6 in a dose-dependent manner. Conclusion: Mice with conditional overexpression of IL-6 in myeloid cells show systemic and vascular inflammation as well as endothelial dysfunction. A decrease in circulating IL-6 levels by replacing IL-6-producing myeloid cells in the BM improved vascular dysfunction in this model, underpinning the relevant role of IL-6 in vascular disease.

AKT activity orchestrates marginal zone B cell development in mice and humans.

The signals controlling marginal zone (MZ) and follicular (FO) B cell development remain incompletely understood. Here, we show that AKT orchestrates MZ B cell formation in mice and humans. Genetic models that increase AKT signaling in B cells or abolish its impact on FoxO transcription factors highlight the AKT-FoxO axis as an on-off switch for MZ B cell formation in mice. In humans, splenic immunoglobulin (Ig) D+CD27+ B cells, proposed as an MZ B cell equivalent, display higher AKT signaling than naive IgD+CD27- and memory IgD-CD27+ B cells and develop in an AKT-dependent manner from their precursors in vitro, underlining the conservation of this developmental pathway. Consistently, CD148 is identified as a receptor indicative of the level of AKT signaling in B cells, expressed at a higher level in MZ B cells than FO B cells in mice as well as humans.

Liver specific deletion of CYLDexon7/8 induces severe biliary damage, fibrosis and increases hepatocarcinogenesis in mice.

BACKGROUND & AIMS: CYLD is a tumor suppressor gene that is mutated in familial cylindromatosis, an autosomal dominant predisposition to tumors of skin appendages. Reduced CYLD expression has been observed in other tumor entities, including hepatocellular carcinoma. In the present study, we analyzed the role of CYLD in liver homeostasis and hepatocarcinogenesis in vivo. METHODS: Mice with liver-specific deletion of CYLDexon7/8 (CYLD(FF)xAlbCre) were generated. Liver tissues were histologically analyzed and oval cell activation was investigated. Hepatocarcinogenesis was induced by diethylnitrosamine/phenobarbital (DEN/PB). Microarray expression profiling of livers was performed in untreated as well as DEN/PB-treated mice. NF-κB signaling was assessed by ELISA, quantitative real-time PCR, and Western blotting. RESULTS: CYLD(FF)xAlbCre hepatocytes and cholangiocytes did not express full-length CYLD (FL-CYLD) protein but showed increased expression of the naturally occurring short-CYLD splice variant (s-CYLD). CYLD(FF)xAlbCre mice exhibited a prominent biliary phenotype with ductular reaction and biliary-type fibrosis. In addition, CYLD(FF)xAlbCre mice showed a significantly increased sensitivity towards DEN/PB-induced hepatocarcinogenesis. Moreover, we could observe the development of cholangiocellular carcinoma, in line with enhanced oval cell activity. NF-κB-signaling was increased in livers of CYLD(FF)xAlbCre mice and likely contributed to the inflammatory and fibrotic response. CONCLUSIONS: The deletion of exon7/8 of the CYLD gene activates oval cells, leads to a biliary phenotype, and increases the susceptibility towards carcinogenesis in the liver. Thus, our study presents a novel model of biliary damage and liver fibrosis, followed by cancer development.