Astrocytic Fas ligand expression is required to induce T‐cell apoptosis and recovery from experimental autoimmune encephalomyelitis

In T‐cell‐mediated autoimmune diseases of the CNS, apoptosis of Fas⁺ T cells by FasL contributes to resolution of disease. However, the apoptosis‐inducing cell population still remains to be identified. To address the role of astrocytic FasL in the regulation of T‐cell apoptosis in experimental autoimmune encephalomyelitis, we immunized C57BL/6 glial fibrillary acid protein (GFAP)‐Cre FasL^fl/fl mice selectively lacking FasL in astrocytes with MOG_35–55 peptide. GFAP‐Cre FasL^fl/fl mice were unable to resolve EAE and suffered from persisting demyelination and paralysis, while FasL^fl/fl control mice recovered. In contrast to FasL^fl/fl mice, GFAP‐Cre FasL^fl/fl mice failed to induce apoptosis of Fas⁺ activated CD4⁺ T cells and to increase numbers of Foxp3⁺ Treg cells beyond day 15 post immunization, the time point of maximal clinical disease in control mice. The persistence of activated and GM‐CSF‐producing CD4⁺ T cells in GFAP‐Cre FasL^fl/fl mice also resulted in an increased IL‐17, IFN‐γ, TNF, and GM‐CSF mRNA expression in the CNS. In vitro, FasL⁺ but not FasL^? astrocytes induced caspase‐3 expression and apoptosis of activated T cells. In conclusion, FasL expression of astrocytes plays an important role in the control and elimination of autoimmune T cells from the CNS, thereby determining recovery from EAE.     

Macrophage-dependent neutrophil recruitment is impaired under conditions of increased intestinal permeability in JAM-A-deficient mice

Junctional adhesion molecule-A (JAM-A) is a transmembrane glycoprotein expressed on leukocytes, endothelia, and epithelia that regulates biological processes including barrier function and immune responses. While JAM-A has been reported to facilitate tissue infiltration of leukocytes under inflammatory conditions, the contributions of leukocyte-expressed JAM-A in vivo remain unresolved. We investigated the role of leukocyte-expressed JAM-A in acute peritonitis induced by zymosan, lipopolysaccharide (LPS), or TNFα using mice with selective loss of JAM-A in myelomonocytic cells (LysM-Cre;Jam-a^fl/fl). Surprisingly, in LysM-Cre;Jam-a^fl/fl mice, loss of JAM-A did not affect neutrophil (PMN) recruitment into the peritoneum in response to zymosan, LPS, or TNFα although it was significantly reduced in Jam-a^KO mice. In parallel, Jam-a^KO peritoneal macrophages exhibited diminished CXCL1 chemokine production and decreased activation of NF-kB, whereas those from LysM-Cre;Jam-a^fl/fl mice were unaffected. Using Villin-Cre;Jam-a^fl/fl mice, targeted loss of JAM-A on intestinal epithelial cells resulted in increased intestinal permeability along with reduced peritoneal PMN migration as well as lower levels of CXCL1 and active NF-kB similar to that observed in Jam-a^KO animals. Interestingly, in germ-free Villin-Cre;Jam-a^fl/fl mice, PMN recruitment was unaffected suggesting dependence on gut microbiota. Such observations highlight the functional link between a leaky gut and regulation of innate immune responses.     

Protective dendritic cell responses against listeriosis induced by the short form of the deubiquitinating enzyme CYLD are inhibited by full‐length CYLD

The deubiquitinating enzyme CYLD is an important tumor suppressor and inhibitor of immune responses. In contrast to full‐length CYLD, the immunological function of the naturally occurring short splice variant of CYLD (sCYLD) is insufficiently described. Previously, we showed that DCs, which lack full‐length CYLD but express sCYLD, exhibit augmented NF‐κB and DC activation. To explore the function of sCYLD in infection, we investigated whether DC‐specific sCYLD regulates the pathogenesis of listeriosis. Upon Listeria monocytogenes infection of CD11c‐Cre Cyld^{ex7/8 fl/fl} mice, infection of CD8α⁺ DCs, which are crucial for the establishment of listeriosis in the spleen, was not affected. However, NF‐κB activity of CD11c‐Cre Cyld^{ex7/8 fl/fl} DCs was increased, while activation of ERK and p38 was normal. In addition, CD11c‐Cre Cyld^{ex7/8 fl/fl} DCs produced more TNF, IL‐10, and IL‐12 upon infection, which led to enhanced stimulation of IFN‐γ‐producing NK cells. In addition CD11c‐Cre Cyld^{ex7/8 fl/fl} DCs presented Listeria Ag more efficiently to CD8⁺ T cells resulting in a stronger pathogen‐specific CD8⁺ T‐cell proliferation and more IFN‐γ production. Collectively, the improved innate and adaptive immunity and survival during listeriosis identify the DC‐specific FL‐CYLD/sCYLD balance as a potential target to modulate NK‐cell and Ag‐specific CD8⁺ T‐cell responses.     

A20 expression in dendritic cells protects mice from LPS‐induced mortality

DCs contribute to immune homeostasis under physiological conditions and regulate the immune activation during infection. The deubiquitinase A20 inhibits the activation of NF‐κB‐dependent immune reactions, and prevents the hyperactivation of DCs under steady‐state conditions. However, the role of DC‐specific A20 under pathological conditions is unknown. Here, we demonstrate that upon injection of low‐dose LPS, mice with DC‐specific A20 deletion (CD11c‐Cre A20^fl/fl) died within 6 h, whereas A20^fl/fl controls survived. LPS‐induced mortality in CD11c‐Cre A20^fl/fl mice was characterized by increased serum levels of IL‐2, IL‐10, IL‐12, IFN‐γ, and TNF. Upon LPS stimulation, the activation of NF‐κB and ERK‐NFATc3 pathways were enhanced in A20‐deficient DCs, resulting in an increased production of IL‐2, IL‐12, and TNF both in vitro and in vivo. Targeted inhibition of ERK in A20‐deficient DCs abolished the increased production of IL‐2. A20‐deficient DCs failed to induce LPS tolerance, which was independent of T cells and the intestinal flora, since T‐cell depletion and decolonization of CD11c‐Cre A20^fl/fl mice could not prevent death of LPS‐challenged CD11c‐Cre A20^fl/fl mice. In conclusion, these findings show that DC‐specific A20 preserves immune homeostasis in steady‐state conditions and is also required for LPS tolerance.     

Protein kinase 2 (CK2) controls CD4+ T cell effector function in the pathogenesis of colitis

Crohn's disease (CD), one of the major forms of inflammatory bowel disease (IBD), is characterized by chronic inflammation of the gastrointestinal tract and associated with aberrant CD4⁺ T-helper type 1 (Th1) and Th17 responses. Protein kinase 2 (CK2) is a conserved serine–threonine kinase involved in signal transduction pathways, which regulate immune responses. CK2 promotes Th17 cell differentiation and suppresses the generation of Foxp3⁺ regulatory T cells. The function of CK2 in CD4⁺ T cells during the pathogenesis of CD is unknown. We utilized the T cell-induced colitis model, transferring CD45RB^hi-naive CD4⁺ T cells from CK2α^fl/fl controls and CK2α^fl/fldLck-Cre mice into Rag1^−/− mice. CD4⁺ T cells from CK2α^fl/fldLck-Cre mice failed to induce wasting disease and significant intestinal inflammation, which was associated with decreased interleukin-17A-positive (IL-17A⁺), interferon-γ-positive (IFN-γ⁺), and double-positive IL-17A⁺IFN-γ⁺ CD4⁺ T cells in the spleen and colon. We determined that CK2α regulates CD4⁺ T cell proliferation through a cell-intrinsic manner. CK2α is also important in controlling CD4⁺ T cell responses by regulating NFAT2, which is vital for T cell activation and proliferation. Our findings indicate that CK2α contributes to the pathogenesis of colitis by promoting CD4⁺ T cell proliferation and Th1 and Th17 responses, and that targeting CK2 may be a novel therapeutic treatment for patients with CD.     

MZB1 is a GRP94 cochaperone that enables proper immunoglobulin heavy chain biosynthesis upon ER stress

MZB1 (pERp1) is a B-cell-specific and endoplasmic reticulum (ER)-localized protein implicated in antibody secretion and integrin-mediated cell adhesion. Here, we examine the role of MZB1 in vivo by conditional gene inactivation in the mouse germline and at different stages of B lymphopoiesis. Deletion of MZB1 impairs humoral immune responses and antibody secretion in plasma cells that naturally undergo ER stress. In addition, we found that experimental induction of ER stress by tunicamycin injections in mice results in a block of pro-B-cell to pre-B-cell differentiation specifically in Mzb1^−/− mice. A similar developmental block was observed in Mzb1^fl/flmb1^Cre mice, whereby a Cre recombinase-induced genotoxic stress unmasks a role for MZB1 in the surface expression of immunoglobulin µ heavy chains (µHCs). MZB1 associates directly with the substrate-specific chaperone GRP94 (also called HSP90B1 or gp96) in an ATP-sensitive manner and is required for the interaction of GRP94 with µHCs upon ER stress. Thus, MZB1 seems to act as a substrate-specific cochaperone of GRP94 that enables proper biosynthesis of µHCs under conditions of ER stress.     

Granzyme-Mediated Regulation of Host Defense in the Liver in Experimental Leishmania donovani Infection

In the livers of susceptible C57BL/6 (B6) mice infected with Leishmania donovani, CD8⁺ T cell mechanisms are required for granuloma assembly, macrophage activation, intracellular parasite killing, and self-cure. Since gene expression of perforin and granzymes A and B (GzmA and GzmB), cytolytic proteins linked to CD8⁺ cell effector function, was enhanced in infected liver tissue, B6 mice deficient in these granular proteins were used to gauge host defense roles. Neither perforin nor GzmA was required; however, mice deficient in GzmB (GzmB^−/−, GzmB cluster^−/−, and GzmA×B cluster double knockout [DKO] mice) showed both delayed granuloma assembly and initially impaired control of parasite replication. Since these two defects in B6 mice were limited to early-stage infection, innately resistant 129/Sv mice were also tested. In this genetic setting, expression of both innate and subsequent T (Th1) cell-dependent acquired resistance, including the self-cure phenotype, was entirely derailed in GzmA×B cluster DKO mice. These results, in susceptible B6 mice for GzmB and in resistant 129/Sv mice for GzmA and/or the GzmB cluster, point to granzyme-mediated host defense regulation in the liver in experimental visceral leishmaniasis.     

Self-Reactivity and the Expression of Memory Markers Vary Independently in MRL-Mp+/+ and MRL-Mp-lpr/lpr Mice

MRL-Mp-lpr/lpr mice contain phenotypically abnormal populations of T cells, and exhibit an SLE-like autoimmune disease in which autoantibodies are a prominent feature. We analyzed the phenotype and T-cell receptor Vß expression pattern in CD4⁺ T cells of this mutant mouse strain to detect abnormalities that could explain the autoimmunity. The CD4⁺ T cells contain two distinct abnormal populations. One of these expresses B220 and HSA, and in these and other respects closely resembles the accumulating CD4^–CD8^– population. The other expresses a high level of CD44 (Pgp-1), and a high level of the 16A epitope of CD45, and so resembles post-activation T cells. Both of these cell types are exclusive to MRL-Mp-lpr/lpr. We also identified V ß5- and V ß11-positive CD4+ T cells, in both MRL-Mp-lpr/lpr and MRL-Mp-+/+ mice. We conclude that autoimmune T cells can be detected in these mice, but that they are not the cause of the accumulation of abnormal CD4⁺ and CD4^–CD8^–cells.     

Kidney‐specific knockout of Sav1 in the mouse promotes hyperproliferation of renal tubular epithelium through suppression of the Hippo pathway

We have previously reported that Salvador homologue 1 (SAV1), a component of the Hippo pathway, is significantly down‐regulated in high‐grade clear cell renal cell carcinoma (ccRCC) due to 14q copy number loss, and that this down‐regulation contributes to the proliferation and survival of renal tubular epithelial cells through activation of Yes‐associated protein 1 (YAP1), a downstream target of the Hippo pathway. However, the impact of SAV1 loss on the proliferation and survival of kidney cells in vivo remained to be determined. To address this issue, we generated kidney‐specific Sav1‐knockout (Cdh16‐Cre;Sav1^fl/fl) mice. Sav1 deficiency enhanced the proliferation of renal tubular epithelial cells in Cdh16‐Cre;Sav1^fl/fl mice, accompanied by nuclear localization of Yap1, suggesting suppression of the Hippo pathway. Sav1 deficiency in renal tubules also caused structural and cellular abnormalities of the epithelial cells, including significant enlargement of their nuclei. Furthermore, Cdh16‐Cre;Sav1^fl/fl mice developed both glomerular and tubular cysts. Although lining cells of the glomerular cysts showed no atypia, those of the tubular cysts showed variations in cell size and nuclear shape, which became more severe as the mice aged. In aged Cdh16‐Cre;Sav1^fl/fl mice, we observed focal disruption of proximal tubules and perivascular lymphocytic infiltration. In conclusion, Sav1 is required for the maintenance of growth, nuclear size and structure of renal tubules under physiological conditions, and its deficiency leads to the acquisition of enhanced proliferation of renal epithelial cells through suppression of Hippo signalling. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Interleukin-10 from T Cells,but Not Macrophages and Granulocytes,Is Required for Chronic Disease in Leishmania mexicana Infection

Chronic cutaneous disease of mice caused by the protozoan parasite Leishmania mexicana requires interleukin-10 (IL-10) and FcγRIII (an activating IgG receptor). Macrophages readily secrete IL-10 in response to IgG-coated amastigotes, making macrophages a prime candidate as the critical source of IL-10. However, indirect evidence suggested that macrophage IL-10 is not essential for chronic disease. I now show directly that mice lacking IL-10 from macrophages and granulocytes still have chronic disease, like wild-type C57BL/6 mice. However, T cell-derived IL-10 is required for chronic disease. CD4-cre IL-10^flox/flox mice lack IL-10 from T cells (both CD4⁺ and CD8⁺) and heal their L. mexicana lesions, with parasite control. I had previously shown that depletion of CD25⁺ T cells had no effect on chronic disease, and thus, T cells other than CD25⁺ regulatory T (T_reg) cells should be the important source of IL-10. Given that conventional T cells do not express FcγRs, there is likely to be an indirect pathway by which FcγRIII on some other cell engaged by IgG1-amastigote immune complexes induces IL-10 from T cells. Further work is needed to delineate these pathways.     

β2 Integrin deficiency yields unconventional double-negative T cells distinct from mature classical natural killer T cells in mice

Expressed on leucocytes, β₂ integrins (CD11/CD18) are specifically involved in leucocyte function. Using a CD18-deficient (CD18^−/−) mouse model, we here report on their physiological role in lymphocyte differentiation and trafficking. CD18^−/− mice present with a defect in the distribution of lymphocytes with highly reduced numbers of naïve B and T lymphocytes in inguinal and axillary lymph nodes. In contrast, cervical lymph nodes were fourfold enlarged harbouring unconventional T-cell receptor-αβ (TCR-αβ) and TCR-γδ CD3⁺ CD4⁻ CD8⁻ (double-negative; DN) T cells that expanded in situ. Using adoptive transfer experiments, we found that these cells did not home to peripheral lymph nodes of CD18^wt recipients but, like antigen-experienced T or natural killer (NK) T cells, recirculated through non-lymphoid organs. Lacking regulatory functions in vitro, CD18^−/− TCR-αβ DN T cells did not suppress the proliferation of polyclonally activated CD4⁺ or CD8⁺ (single-positive; SP) T cells. Most interestingly, CD18^−/− TCR-αβ DN T cells showed intermediate TCR expression levels, an absent activation through allogeneic major histocompatibility complex and a strong proliferative dependence on interleukin-2, hence, closely resembling NKT cells. However, our data oppose former reports, clearly showing that, because of an absent reactivity with CD1d-αGalCer dimers, these cells are not mature classical NKT cells. Our data indicate that CD18^−/− TCR-αβ DN T cells, like NKT and TCR-γδ T cells, share characteristics of both adaptive and innate immune cells, and may accumulate as a compensatory mechanism to the functional defect of adaptive immunity in CD18^−/− mice.     

Accelerated induction of mycobacterial antigen-specific CD8+ T cells in the Mycobacterium tuberculosis-infected lung by subcutaneous vaccination with Mycobacterium bovis bacille Calmette–Guérin

Both CD4⁺ and CD8⁺ T cells are important in protection against Mycobacterium tuberculosis infection. To evaluate the effect of vaccination with Mycobacterium bovis bacille Calmette–Guérin (BCG) on the CD8⁺ T-cell response to pulmonary M. tuberculosis infection, we analyzed the kinetics of CD8⁺ T cells specific to the mycobacterial Mtb32a_309–318 epitope, which is shared by M. tuberculosis and M. bovis BCG, in the lung of mice infected with M. tuberculosis. The CD8⁺ T cells were detected by staining lymphocytes with pentameric major histocompatibility complex (MHC) class I H-2D^b–Mtb32a_209–318 peptide complex and were analysed by flow cytometry. Mtb32a-specific CD8⁺ T cells became detectable on day 14, and reached a plateau on day 21, in the lung of M. tuberculosis-infected unvaccinated mice. Subcutaneous vaccination with M. bovis BCG in the footpads induced Mtb32a-specific CD8⁺ T cells in the draining lymph nodes (LNs) on day 7 and their numbers further increased on day 14. When M. bovis BCG-vaccinated mice were exposed to pulmonaryinfection with M. tuberculosis 4 weeks after vaccination, the Mtb32a-specific CD8⁺ T cells in the infected lung became detectable on day 7 and reached a plateau on day 14, which was 1 week earlier than in the unvaccinated mice. The pulmonary CD8⁺ T cells from the BCG-vaccinated M. tuberculosis-infected mice produced interferon-γ in response to Mtb32a_209–318 peptide on day 7 of the infection, whereas those of unvaccinated mice did not. The results demonstrate that induction of mycobacterial antigen-specific protective CD8⁺ T cells in the M. tuberculosis-infected lung is accelerated by subcutaneous vaccination with M. bovis BCG.     

Conditional ablation of NKp46+ cells using a novel Ncr1greenCre mouse strain: NK cells are essential for protection against pulmonary B16 metastases

To study gene functions specifically in NKp46⁺ cells we developed novel Cre mice allowing for conditional gene targeting in cells expressing Ncr1 (encoding NKp46). We generated transgenic Ncr1^greenCre mice carrying an EGFPcre fusion under the control of a proximal Ncr1 promoter that faithfully directed EGFPcre expression to NKp46⁺ cells from lymphoid and nonlymphoid tissues. This approach allowed for direct detection of Cre‐expressing NKp46⁺ cells via their GFP signature by flow cytometry and histology. Cre was functional as evidenced by the NKp46⁺ cell‐specific expression of RFP in Ncr1^greenCreRosa‐dtRFP reporter mice. We generated Ncr1^greenCreIl2rg^fl/fl mice that lack NKp46⁺ cells in an otherwise intact hematopoietic environment. Il2rg encodes the common gamma chain (γ_c), which is an essential receptor subunit for cytokines (IL‐2, ‐4, ‐7, ‐9, ‐15, and ‐21) that stimulate lymphocyte development and function. In Ncr1^greenCreIl2rg^fl/fl mice, NK cells are severely reduced and the few remaining NKp46⁺ cells escaping γc deletion failed to express GFP. Using this new NK‐cell‐deficient model, we demonstrate that the homeostasis of NKp46⁺ cells from all tissues (including the recently described intraepithelial ILC1 subset) requires Il2rg. Finally, Ncr1^greenCreIl2rg^fl/fl mice are unable to reject B16 lung metastases demonstrating the essential role of NKp46⁺ cells in antimelanoma immune responses.     

Evidence that Meningeal Mast Cells Can Worsen Stroke Pathology in Mice

Stroke is the leading cause of adult disability and the fourth most common cause of death in the United States. Inflammation is thought to play an important role in stroke pathology, but the factors that promote inflammation in this setting remain to be fully defined. An understudied but important factor is the role of meningeal-located immune cells in modulating brain pathology. Although different immune cells traffic through meningeal vessels en route to the brain, mature mast cells do not circulate but are resident in the meninges. With the use of genetic and cell transfer approaches in mice, we identified evidence that meningeal mast cells can importantly contribute to the key features of stroke pathology, including infiltration of granulocytes and activated macrophages, brain swelling, and infarct size. We also obtained evidence that two mast cell-derived products, interleukin-6 and, to a lesser extent, chemokine (C-C motif) ligand 7, can contribute to stroke pathology. These findings indicate a novel role for mast cells in the meninges, the membranes that envelop the brain, as potential gatekeepers for modulating brain inflammation and pathology after stroke.Stroke, the leading cause of adult disability and the fourth most common cause of death in the Unites States,^1,2 occurs when there is insufficient blood flow to the brain, and the resultant injury initiates a cascade of inflammatory events, including immune cell infiltration into the brain.^3–5 This post-stroke inflammation is a critical determinant of damage and recovery after stroke; understanding the interplay between the immune system and the brain after stroke holds much promise for therapeutic intervention.^4–7 However, successfully exploiting this therapeutic potential requires a detailed understanding of the interplay between the immune system and the brain after stroke.⁴An understudied but important aspect of this interplay is the role of meningeal-located immune cells in modulating brain pathology. The meninges have long been recognized as an anatomical barrier that protects the central nervous system (CNS). However, accumulating evidence suggests that the meninges are important for communication between the CNS and immune system during health and disease.^8–10 All blood vessels pass through the meningeal subarachnoid space before entering the brain, and this vascular connection and the close proximity of the meninges to the underlying parenchymal nervous tissue make them ideally located to act as a gatekeeper to modulate immune cell trafficking to the CNS. To support this gatekeeper function is evidence that the meninges modulate brain infiltration of T cells, neutrophils, and monocytes during meningitis and autoimmune conditions,^11–14 with immune cells observed in some instances accumulating in the meninges before they infiltrate into the parenchyma.^11,13Emerging evidence suggests that the actions of immune cells resident in the meninges are important for this gatekeeper function.^11,12,15 Mast cells (MCs), best known as proinflammatory effector cells, can play critical roles in the development of inflammation in many disease settings.^16–18 MCs reside in high numbers within the meninges, but their function in this site has not been fully investigated in stroke pathology. Unlike most immune cells, mature MCs do not circulate in the blood but are long-term residents of tissues, often in perivascular locations, and can rapidly perform their functions in situ. CNS MCs are found in the brain parenchyma and the meninges of rodents and humans.¹⁸ It has been proposed that brain parenchymal MCs can enhance brain neutrophil numbers after stroke and can exacerbate stroke pathology.^19–24 However, much of the evidence to support such conclusions is indirect. For example, some of the studies that implicate MCs in stroke pathology used pharmacologic approaches to interfere with MC activation,^19,20,22 but such drugs can have effects on other cell types.²⁵ Moreover, the role of the meningeal MCs in modulating post-stroke inflammation and pathology is unknown. Finally, little is understood about which among the many MC-derived mediators may be important in stroke pathology.^17,26To address these questions, we used genetic and cell transfer approaches to study the role of MCs in the pathology of ischemic stroke in mice. Specifically, we tested a c-kit–mutant mouse model (ie, WBB6F₁-Kit^W/W-v mice) which is profoundly MC deficient and can be repaired of this deficiency by engraftment of in vitro-derived MCs from wild-type (WT) mice. This MC knock-in approach enables the MC-dependent effects in the mutant mice to be separated from effects due to other abnormalities associated with their mutation,^11,17,26,27 because only the MC deficiency is repaired by MC engraftment. Furthermore, one can investigate the mechanisms by which MCs influence stroke pathology by engrafting MCs from transgenic mice that lack specific MC-associated products. We also tested our newly described Cpa3-Cre; Mcl-1^fl/fl mice, in which MC (and basophil) numbers are reduced constitutively via Cre-mediated depletion of the anti-apoptotic factor, myeloid cell leukemia sequence 1 (Mcl-1), in the affected lineages.²⁸ Cpa3-Cre; Mcl-1^fl/fl mice lack the other abnormalities associated with the c-kit mutations in WBB6F₁-Kit^W/W-v mice.²⁸With the use of these in vivo models, we identified meningeal MCs as important contributors to key features of stroke pathology, including increased numbers of brain granulocytes and activated macrophages, brain swelling, and infarct size. We also obtained evidence that two potentially proinflammatory MC-derived products, IL-6 and, to a lesser extent, chemokine (C-C motif) ligand 7 (CCL7), can contribute to pathology in this setting.     

Impact of peroxisome proliferator-activated receptor γ on angiotensin II type 1 receptor-mediated insulin sensitivity,vascular inflammation and atherogenesis in hypercholesterolemic mice

Introduction

The angiotensin II type 1 receptor (AT1R) and the peroxisome proliferator-activated receptor γ (PPARγ) have been implicated in the pathogenesis of atherosclerosis. A number of studies have reported that AT1R inhibition or genetic AT1R disruption and PPARγ activation inhibit vascular inflammation and improve glucose and lipid metabolism, underscoring a molecular interaction of AT1R and PPARγ. We here analyzed the hypothesis that vasculoprotective anti-inflammatory and metabolic effects of AT1R inhibition are mediated by PPARγ.

Material and methods

Female ApoE^–/–/AT1R^–/– mice were fedwith a high-fat and cholesterol-rich diet and received continuous treatment with the selective PPARγ antagonist GW9662 or vehicle at a rate of 700 ng/kg/min for 4 weeks using subcutaneously implanted osmotic mini-pumps. Additionally, one group of female ApoE^–/– mice served as a control group. After treatment for 4 weeks mice were sacrificed and read-outs (plaque development, vascular inflammation and insulinsensitivity) were performed.

Results

Using AT1R deficient ApoE^–/– mice (ApoE^–/–/AT1R^–/– mice) we found decreased cholesterol-induced endothelial dysfunction and atherogenesis compared to ApoE^–/– mice. Inhibition of PPARγ by application of the specific PPARγ antagonist GW9662 significantly abolished the anti-atherogenic effects of AT1R deficiency in ApoE^–/–/AT1R^–/– mice (plaque area as % of control: ApoE^–/–: 39 ±5%; ApoE^–/–/AT1R^–/–: 17 ±7%, p = 0.044 vs. ApoE^–/–; ApoE^–/–/AT1R^–/– + GW9662: 31 ±8%, p = 0.047 vs. ApoE^–/–/AT1R^–/–). Focusing on IL6 as a pro-inflammatory humoral marker we detected significantly increased IL-6 levels in GW9662-treated animals (IL-6 in pg/ml: ApoE^–/–: 230 ±16; ApoE^–/–/AT1R^–/–: 117 ±20, p = 0.01 vs. ApoE^–/–; ApoE^–/–/AT1R^–/– + GW9662: 199 ±20, p = 0.01 vs. ApoE^–/–/AT1R^–/–), while the anti-inflammatory marker IL-10 was significantly reduced after PPARγ inhibition in GW9662 animals (IL-10 in pg/ml: ApoE^–/–: 18 ±4; ApoE^–/–/AT1R^–/–: 55 ±12, p = 0.03 vs. ApoE^–/–; ApoE^–/–/AT1R^–/– + GW9662: 19 ±4, p = 0.03 vs. ApoE^–/–/AT1R^–/–). Metabolic parameters of glucose homeostasis (glucose and insulin tolerance test) were significantly deteriorated in ApoE^–/–/AT1R^–/– mice treated with GW9662 as compared to vehicle-treated ApoE^–/–/AT1R^–/– mice. Systolic blood pressure and plasma cholesterol levels were similar in all groups.

Conclusions

Genetic disruption of the AT1R attenuates atherosclerosis and improves endothelial function in an ApoE^–/– mouse model of hypercholesterolemia-induced atherosclerosis via PPARγ, indicating a significant role of PPARγ in reduced vascular inflammation, improvement of insulin sensitivity and atheroprotection of AT1R deficiency.