Nir Yogev

Caspase-8 Serves Both Apoptotic and Nonapoptotic Roles

Knockout of caspase-8, a cysteine protease that participates in the signaling for cell death by receptors of the TNF/nerve growth factor family, is lethal to mice in utero. To explore tissue-specific roles of this enzyme, we established its conditional knockout using the Cre/loxP recombination system. Consistent with its role in cell death induction, deletion of caspase-8 in hepatocytes protected them from Fas-induced caspase activation and death. However, application of the conditional knockout approach to investigate the cause of death of caspase-8 knockout embryos revealed that this enzyme also serves cellular functions that are nonapoptotic. Its deletion in endothelial cells resulted in degeneration of the yolk sac vasculature and embryonal death due to circulatory failure. Caspase-8 deletion in bone-marrow cells resulted in arrest of hemopoietic progenitor functioning, and in cells of the myelomonocytic lineage, its deletion led to arrest of differentiation into macrophages and to cell death. Thus, besides participating in cell death induction by receptors of the TNF/nerve growth factor family, caspase-8, apparently independently of these receptors, also mediates nonapoptotic and perhaps even antiapoptotic activities.

Lysozyme M–Positive Monocytes Mediate Angiotensin II–Induced Arterial Hypertension and Vascular Dysfunction

Background— Angiotensin II (ATII), a potent vasoconstrictor, causes hypertension, promotes infiltration of myelomonocytic cells into the vessel wall, and stimulates both vascular and inflammatory cell NADPH oxidases. The predominant source of reactive oxygen species, eg, vascular (endothelial, smooth muscle, adventitial) versus phagocytic NADPH oxidase, and the role of myelomonocytic cells in mediating arterial hypertension have not been defined yet. Methods and Results— Angiotensin II (1 mg · kg−1 · d−1 for 7 days) increased the number of both CD11b+Gr-1lowF4/80+ macrophages and CD11b+Gr-1highF4/80− neutrophils in mouse aorta (verified by flow cytometry). Selective ablation of lysozyme M-positive (LysM+) myelomonocytic cells by low-dose diphtheria toxin in mice with inducible expression of the diphtheria toxin receptor (LysMiDTR mice) reduced the number of monocytes in the circulation and limited ATII-induced infiltration of these cells into the vascular wall, whereas the number of neutrophils was not reduced. Depletion of LysM+ cells attenuated ATII-induced blood pressure increase (measured by radiotelemetry) and vascular endothelial and smooth muscle dysfunction (assessed by aortic ring relaxation studies) and reduced vascular superoxide formation (measured by chemiluminescence, cytochrome c assay, and oxidative fluorescence microtopography) and the expression of NADPH oxidase subunits gp91phox and p67phox (assessed by Western blot and mRNA reverse-transcription polymerase chain reaction). Adoptive transfer of wild-type CD11b+Gr-1+ monocytes into depleted LysMiDTR mice reestablished ATII-induced vascular dysfunction, oxidative stress, and arterial hypertension, whereas transfer of CD11b+Gr-1+ neutrophils or monocytes from gp91phox or ATII receptor type 1 knockout mice did not. Conclusions— Infiltrating monocytes with a proinflammatory phenotype and macrophages rather than neutrophils appear to be essential for ATII-induced vascular dysfunction and arterial hypertension.

A transgenic mouse model of inducible macrophage depletion: effects of diphtheria toxin-driven lysozyme M-specific cell lineage ablation on wound inflammatory, angiogenic, and contractive processes.

Whether the wound macrophage is a key regulatory inflammatory cell type in skin repair has been a matter of debate. A transgenic mouse model mediating inducible macrophage depletion during skin repair has not been used to date to address this question. Here, we specifically rendered the monocyte/macrophage leukocyte lineage sensitive to diphtheria toxin by expressing the lysozyme M promoter-driven, Cre-mediated excision of a transcriptional STOP cassette from the simian DT receptor gene in mice (lysM-Cre/DTR). Application of diphtheria toxin to lysM-Cre/DTR mice led to a rapid reduction in both skin tissue and wound macrophage numbers at sites of injury. Macrophage-depleted mice revealed a severely impaired wound morphology and delayed healing. In the absence of macrophages, wounds were re-populated by large numbers of neutrophils. Accordingly, macrophage-reduced wound tissues exhibited the increased and prolonged persistence of macrophage inflammatory protein-2, macrophage chemoattractant protein-1, interleukin-1beta, and cyclooxygenase-2, paralleled by unaltered levels of bioactive transforming growth factor-beta1. Altered expression patterns of vascular endothelial growth factor on macrophage reduction were associated with a disturbed neo-vascularization at the wound site. Impaired wounds revealed a loss of myofibroblast differentiation and wound contraction. Our data in the use of lysM-Cre/DTR mice emphasize the pivotal function of wound macrophages in the integration of inflammation and cellular movements at the wound site to enable efficient skin repair.

Nonredundant Roles for B Cell-Derived IL-10 in Immune Counter-Regulation

IL-10 plays a central role in restraining the vigor of inflammatory responses, but the critical cellular sources of this counter-regulatory cytokine remain speculative in many disease models. Using a novel IL-10 transcriptional reporter mouse, we found an unexpected predominance of B cells (including plasma cells) among IL-10-expressing cells in peripheral lymphoid tissues at baseline and during diverse models of in vivo immunological challenge. Use of a novel B cell-specific IL-10 knockout mouse revealed that B cell-derived IL-10 nonredundantly decreases virus-specific CD8+ T cell responses and plasma cell expansion during murine cytomegalovirus infection and modestly restrains immune activation after challenge with foreign Abs to IgD. In contrast, no role for B cell-derived IL-10 was evident during endotoxemia; however, although B cells dominated lymphoid tissue IL-10 production in this model, myeloid cells were dominant in blood and liver. These data suggest that B cells are an underappreciated source of counter-regulatory IL-10 production in lymphoid tissues, provide a clear rationale for testing the biological role of B cell-derived IL-10 in infectious and inflammatory disease, and underscore the utility of cell type-specific knockouts for mechanistic limning of immune counter-regulation.

Transepithelial Pathogen Uptake into the Small Intestinal Lamina Propria

The lamina propria that underlies and stabilizes the gut lining epithelium is densely populated with strategically located mononuclear phagocytes. Collectively, these lamina propria macrophages and dendritic cells (DC) are believed to be crucial for tissue homeostasis as well as the innate and adaptive host defense. Lamina propria DC were recently shown to gain direct access to the intestinal lumen by virtue of epithelium-penetrating dendrites. However, the role of these structures in pathogen uptake remains under debate. In this study, we report that entry of a noninvasive model pathogen (Aspergillus fumigatus conidia) into the murine small intestinal lamina propria persists in the absence of either transepithelial dendrites or lamina propria DC and macrophages. Our results suggest the existence of multiple pathogen entry pathways and point at the importance of villus M cells in the uptake of gut lumen Ags. Interestingly, transepithelial dendrites seem altogether absent from the small intestine of BALB/c mice suggesting that the function of lamina propria DC extensions resides in their potential selectivity for luminal Ags, rather than in general uptake or gut homeostasis.

Modulation of dendritic cell properties by laquinimod as a mechanism for modulating multiple sclerosis.

Laquinimod is an orally administered compound that is under investigation in relapsing-remitting multiple sclerosis. To understand the mechanism by which laquinimod exerts its clinical effects, we have performed human and murine studies assessing its immunomodulatory properties. In experimental autoimmune encephalomyelitis, the therapeutic administration of laquinimod beginning during the recovery of SJL mice, prevented further relapses as expected and strongly reduced infiltration of CD4+ and CD8+ T cells in the central nervous system. We hypothesized that this beneficial effect was mediated by dendritic cells, since we and others found a modulation of different dendritic cell subsets under treatment. According to the findings on antigen-presenting cells in the murine system, we found a reduced capacity of human monocyte-derived dendritic cells treated with therapeutic concentrations of laquinimod, upon maturation with lipopolysaccharide, to induce CD4+ T cell proliferation and secretion of pro-inflammatory cytokines. Furthermore, laquinimod treatment of mature dendritic cells resulted in a decreased chemokine production by both murine and human dendritic cells, associated with a decreased monocyte chemo-attraction. In laquinimod-treated patients with multiple sclerosis we consistently found reduced chemokine and cytokine secretion by conventional CD1c+ dendritic cells upon lipopolysaccharide stimulation. Similarly to the animal model of relapsing-remitting multiple sclerosis, dendritic cell subsets were altered in patients upon laquinimod treatment, as the number of conventional CD1c+ and plasmacytoid CD303+ dendritic cells were decreased within peripheral blood mononuclear cells. Moreover, laquinimod treatment in patients with multiple sclerosis and mice modified the maturation of dendritic cells demonstrated by an upregulation of CD86 expression in vivo. Our data suggest that inhibition of the NF-κB pathway is responsible for the changes observed in dendritic cell maturation and functions. These findings indicate that laquinimod exhibits its disease-modulating activity in multiple sclerosis by downregulating immunogenicity of dendritic cell responses. We suggest that monitoring dendritic cell properties in multiple sclerosis should be implemented in future therapeutic trials.

Interleukin 17 Drives Vascular Inflammation, Endothelial Dysfunction, and Arterial Hypertension in Psoriasis-Like Skin Disease

Objective— Interleukin (IL)-17A is regarded as an important cytokine to drive psoriasis, an inflammatory skin disease marked by increased cardiovascular mortality. We aimed to test the hypothesis that overproduction of IL-17A in the skin leading to dermal inflammation may systemically cause vascular dysfunction in psoriasis-like skin disease. Approach and Results— Conditional overexpression of IL-17A in keratinocytes caused severe psoriasis-like skin inflammation in mice (K14-IL-17Aind/+ mice), associated with increased reactive oxygen species formation and circulating CD11b+ inflammatory leukocytes in blood, with endothelial dysfunction, increased systolic blood pressure, left ventricular hypertrophy, and reduced survival compared with controls. In K14-IL-17Aind/+ mice, immunohistochemistry and flow cytometry revealed increased vascular production of the nitric oxide/superoxide reaction product peroxynitrite and infiltration of the vasculature with myeloperoxidase+CD11b+GR1+F4/80− cells accompanied by increased expression of the inducible nitric oxide synthase and the nicotinamide dinucleotide phosphate (NADPH) oxidase, nox2. Neutrophil depletion by anti-GR-1 antibody injections reduced oxidative stress in blood and vessels. Neutralization of tumor necrosis factor-&agr; and IL-6 (both downstream of IL-17A) reduced skin lesions, attenuated oxidative stress in heart and blood, and partially improved endothelial dysfunction in K14-IL-17Aind/+ mice. Conclusions— Dermal overexpression of IL-17A induces systemic endothelial dysfunction, vascular oxidative stress, arterial hypertension, and increases mortality mainly driven by myeloperoxidase+CD11b+GR1+F4/80− inflammatory cells. Depletion of the GR-1+ immune cells or neutralization of IL-17A downstream cytokines by biologicals attenuates the vascular phenotype in K14-IL-17Aind/+ mice.

Effective treatment of allergic airway inflammation with Helicobacter pylori immunomodulators requires BATF3-dependent dendritic cells and IL-10

Significance Allergic asthma represents an increasingly common public health problem. Here, we provide preclinical evidence for the efficacy of active tolerization using Helicobacter pylori components as a viable strategy for asthma prevention. We use a mouse model of allergic asthma to show that regular treatment with H. pylori extract effectively alleviates all hallmarks of the disease. Successful treatment depends on the regulatory cytokine IL-10 and on basic leucine zipper ATF-like 3 (BATF3)-dependent dendritic cell lineages. H. pylori extracts lacking the γ-glutamyl-transpeptidase GGT or the vacuolating cytotoxin VacA fail to protect against asthma; conversely, both factors can be administered in purified form to achieve protection. In conclusion, the immunomodulatory properties of the common infectious agent H. pylori can be exploited for therapeutic purposes in an allergy model. The prevalence of allergic asthma and other atopic diseases has reached epidemic proportions in large parts of the developed world. The gradual loss of the human indigenous microbiota has been held responsible for this trend. The bacterial pathogen Helicobacter pylori is a constituent of the normal gastric microbiota whose presence has been inversely linked to allergy and asthma in humans and experimental models. Here we show that oral or i.p. tolerization with H. pylori extract prevents the airway hyperresponsiveness, bronchoalveolar eosinophilia, pulmonary inflammation, and Th2 cytokine production that are hallmarks of allergen-induced asthma in mice. Asthma protection is not conferred by extracts from other enteropathogens and requires a heat-sensitive H. pylori component and the DC-intrinsic production of IL-10. The basic leucine zipper ATF-like 3 (BATF3)-dependent CD103+CD11b− dendritic cell lineage is enriched in the lungs of protected mice and strictly required for protection. Two H. pylori persistence determinants, the γ-glutamyl-transpeptidase GGT and the vacuolating cytotoxin VacA, are required and sufficient for asthma protection and can be administered in purified form to prevent asthma. In conclusion, we provide preclinical evidence for the concept that the immunomodulatory properties of H. pylori can be exploited for tolerization strategies aiming to prevent allergen-induced asthma.

Tolerance without Clonal Expansion: Self-Antigen-Expressing B Cells Program Self-Reactive T Cells for Future Deletion

B cells have been shown in various animal models to induce immunological tolerance leading to reduced immune responses and protection from autoimmunity. We show that interaction of B cells with naive T cells results in T cell triggering accompanied by the expression of negative costimulatory molecules such as PD-1, CTLA-4, B and T lymphocyte attenuator, and CD5. Following interaction with B cells, T cells were not induced to proliferate, in a process that was dependent on their expression of PD-1 and CTLA-4, but not CD5. In contrast, the T cells became sensitive to Ag-induced cell death. Our results demonstrate that B cells participate in the homeostasis of the immune system by ablation of conventional self-reactive T cells.

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.