Regulation of B cell homeostasis and activation by the tumor suppressor gene CYLD

B cell homeostasis is regulated by multiple signaling processes, including nuclear factor-κB (NF-κB), BAFF-, and B cell receptor signaling. Conditional disruption of genes involved in these pathways has shed light on the mechanisms governing signaling from the cell surface to the nucleus. We describe a novel mouse strain that expresses solely and excessively a naturally occurring splice variant of CYLD (CYLDex7/8 mice), which is a deubiquitinating enzyme that is integral to NF-κB signaling. This shorter CYLD protein lacks the TRAF2 and NEMO binding sites present in full-length CYLD. A dramatic expansion of mature B lymphocyte populations in all peripheral lymphoid organs occurs in this strain. The B lymphocytes themselves exhibit prolonged survival and manifest a variety of signaling disarrangements that do not occur in mice with a complete deletion of CYLD. Although both the full-length and the mutant CYLD are able to interact with Bcl-3, a predominant nuclear accumulation of Bcl-3 occurs in the CYLD mutant B cells. More dramatic, however, is the accumulation of the NF-κB proteins p100 and RelB in CYLDex7/8 B cells, which, presumably in combination with nuclear Bcl-3, results in increased levels of Bcl-2 expression. These findings suggest that CYLD can both positively and negatively regulate signal transduction and homeostasis of B cells in vivo, depending on the expression of CYLD splice variants

Smad7 in T cells drives T helper 1 responses in multiple sclerosis and experimental autoimmune encephalomyelitis

Autoreactive CD4+ T lymphocytes play a vital role in the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Since the discovery of T helper 17 cells, there is an ongoing debate whether T helper 1, T helper 17 or both subtypes of T lymphocytes are important for the initiation of autoimmune neuroinflammation. We examined peripheral blood CD4+ cells from patients with active and stable relapsing–remitting multiple sclerosis, and used mice with conditional deletion or over-expression of the transforming growth factor-β inhibitor Smad7, to delineate the role of Smad7 in T cell differentiation and autoimmune neuroinflammation. We found that Smad7 is up-regulated in peripheral CD4+ cells from patients with multiple sclerosis during relapse but not remission, and that expression of Smad7 strongly correlates with T-bet, a transcription factor defining T helper 1 responses. Concordantly, mice with transgenic over-expression of Smad7 in T cells developed an enhanced disease course during experimental autoimmune encephalomyelitis, accompanied by elevated infiltration of inflammatory cells and T helper 1 responses in the central nervous system. On the contrary, mice with a T cell-specific deletion of Smad7 had reduced disease and central nervous system inflammation. Lack of Smad7 in T cells blunted T cell proliferation and T helper 1 responses in the periphery but left T helper 17 responses unaltered. Furthermore, frequencies of regulatory T cells were increased in the central nervous system of mice with a T cell-specific deletion and reduced in mice with a T cell-specific over-expression of Smad7. Downstream effects of transforming growth factor-β on in vitro differentiation of naïve T cells to T helper 1, T helper 17 and regulatory T cell phenotypes were enhanced in T cells lacking Smad7. Finally, Smad7 was induced during T helper 1 differentiation and inhibited during T helper 17 differentiation. Taken together, the level of Smad7 in T cells determines T helper 1 polarization and regulates inflammatory cellular responses. Since a Smad7 deletion in T cells leads to immunosuppression, Smad7 may be a potential new therapeutic target in multiple sclerosis

Thymus-Derived Regulatory T Cells Are Positively Selected on Natural Self-Antigen through Cognate Interactions of High Functional Avidity

Regulatory T (Treg) cells expressing Foxp3 transcripton factor are essential for immune homeostasis. They arise in the thymus as a separate lineage from conventional CD4(+)Foxp3(-) T (Tconv) cells. Here, we show that the thymic development of Treg cells depends on the expression of their endogenous cognate self-antigen. The formation of these cells was impaired in mice lacking this self-antigen, while Tconv cell development was not negatively affected. Thymus-derived Treg cells were selected by self-antigens in a specific manner, while autoreactive Tconv cells were produced through degenerate recognition of distinct antigens. These distinct modes of development were associated with the expression of T cell receptor of higher functional avidity for self-antigen by Treg cells than Tconv cells, a difference subsequently essential for the control of autoimmunity. Our study documents how self-antigens define the repertoire of thymus-derived Treg cells to subsequently endow this cell type with the capacity to undermine autoimmune attack

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

IL-17A mediated endothelial breach promotes metastasis formation

The role of the IL-23/IL-17A axis in tumor-immune interactions is a matter of controversy. While some suggest that IL-17A producing T cells (TH17) can suppress tumor growth, others report that IL-17A and IL-23 accelerate tumor growth. Here, we systematically assessed the impact of IL-17A-secreting lymphocytes in several murine models of tumor lung metastasis. Genetic fate-mapping revealed that IL-17A was secreted within lung metastases predominantly by γδ T cells, while TH17 cells were virtually absent. Using different tumor models, we found Il17a-/- mice to consistently develop fewer pulmonary tumor colonies. IL-17A specifically increased blood vessel permeability and the expression of E-selectin and VCAM-1 by lung endothelial cells in vivo. In transgenic mice, specific targeting of IL-17A to the endothelium increased the number of tumor foci. Moreover, the direct impact of IL-17A on lung endothelial cells resulted in impaired endothelial barrier integrity showing that IL-17A promotes the formation of lung metastases through tumor-endothelial transmigration

Functionality of <i>Il6ra</i>^{<i>Δmyel</i>} mice.

<p>(A) Flow cytometric analysis of IL-6Rα expression of different organs. CD4⁺ cells are pre-gated on living CD90.2⁺ cells. Neutrophils (Gr-1^hi F4/80^-), monocytes (Gr-1^int F4/80⁺) and macrophages (Gr-1^- F4/80⁺) are pre-gated on living CD90.2^-/B220^- and CD11b⁺ cells. Gray histograms represent IgG2b isotype control for the IL-6R staining. Numbers in upper right corner represent the mean Mean Fluorescent Intensity (MFI) values of Il6ra^Δmyel or <i>wt</i> cells. Shown are representative histograms (n = 7 or 8 (3 independent experiments). (B) MFI of IL-6Rα expression pre-gated on CD4⁺ cells, neutrophils (Gr-1^hi F4/80^-), monocytes (Gr-1^int F4/80⁺) or macrophages (Gr-1^- F4/80⁺). Data are shown as bar graphs with mean and SEM. *p ≤ 0,05 Significance was calculated using Mann Whitney test. (C) Quantitative RT-PCR from CD11b⁺ MACS purified bone marrow cells for the IL-6Rα gene in <i>wt</i> and <i>Il6ra</i>^{<i>Δmyel</i>} mice. Expression levels are shown relative to the housekeeping gene HPRT (n = 5). Data are shown as bar graphs with mean and SEM. * p ≤ 0,05 Significance was calculated using Mann Whitney test. (D) Serum concentrations of sIL-6Rα examined by ELISA in <i>wt</i> (n = 6) and <i>Il6ra</i>^{<i>Δmyel</i>} (n = 10) mice at the age of 5 weeks to 5 months. Data are shown as bar graphs with mean and SEM. *** p ≤ 0,001 Significance was calculated using Mann Whitney test.</p

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 carcinogenesi

No differences of <i>Il6ra^Δmyel</i> mice in myelomonocytic cells and T cells compared to <i>wt</i> in IMQ-induced psoriasis.

<p>(A) Flow cytometric analysis of CD11b⁺ cells of the indicated organs. Cells are pre-gated on living CD90.2^-/B220^- cells. (B) Total cell numbers of CD11b⁺ cells of the indicated organs. Bar graphs are shown with mean and SEM. Significance was calculated using Kruskal-Wallis test. (C) Flow cytometric analysis of Ly6C⁺ and Ly6G⁺ cells of the indicated organs. Cells are pre-gated on living CD90.2^-/B220^- and CD11b⁺ cells. (D) Total cell numbers of Ly6G⁺ cells, pro-inflammatory monocytes (Ly6G^-Ly6C^hi) and resident monocytes (Ly6G^-Ly6C^int). Bar graphs of the indicated organs are shown with mean and SEM. Significances of the spleen were calculated using Kruskal-Wallis test (IMQ groups n = 6, sham groups n = 3). Significances of the ears were calculated with Student’s t-Test (IMQ groups n = 6, sham groups n = 1). (E) Flow cytometric analysis of IL-17A producing γδ T cells of the indicated organs. Cells are pre-gated on living CD45.2⁺/CD3⁺ cells. (F) Total cell numbers of γδ TCR⁺ and IL-17A⁺ γδ TCR⁺. Data are shown as bar graphs with mean and SEM. Significance was calculated using Kruskal-Wallis test.</p

<i>Il6ra</i>^{<i>Δmyel</i>} mice show no differences in myelomonocytic cell compartments and T cells compared to <i>wt</i>.

<p>(A) Flow cytometric analysis of CD11b⁺ cells of the indicated organs. Cells are pre-gated on living CD90.2^-/CD19^- cells (n = 5). (B) Percentages and total cell numbers of CD11b⁺ cells of the indicated organs. Bar graphs are shown with mean and SEM. Significance was calculated using Mann Whitney test (n = 5). (C) Flow cytometric analysis of Gr-1⁺ and F4/80⁺ cells of the indicated organs. Cells are pre-gated on living CD90.2^-/CD19^- and CD11b⁺ cells (n = 5). (D) Total cell numbers of neutrophils (Gr-1^hi F4/80^-), monocytes (Gr-1^int F4/80⁺) and macrophages (Gr-1^- F4/80⁺). Data are shown as bar graphs with mean and SEM. Significance was calculated using Mann Whitney test (n = 5). (E) Flow cytometric analysis of IL-17A producing γδ T cells of indicated organs. Cells are pre-gated on living CD45.2⁺/CD3⁺ cells (n = 5). (F) Total cell numbers of γδ TCR⁺ and IL-17A⁺ γδ TCR⁺. Data are shown as bar graphs with mean and SEM. Significance was calculated using Mann Whitney test (n = 5).</p