Involvement of an IgE/Mast cell/B cell amplification loop in abdominal aortic aneurysm progression.

AIMS: IgE type immunoglobulins and their specific effector cells, mast cells (MCs), are associated with abdominal aortic aneurysm (AAA) progression. In parallel, immunoglobulin-producing B cells, organised in tertiary lymphoid organs (TLOs) within the aortic wall, have also been linked to aneurysmal progression. We aimed at investigating the potential role and mechanism linking local MCs, TLO B cells, and IgE production in aneurysmal progression. METHODS AND RESULTS: Through histological assays conducted on human surgical samples from AAA patients, we uncovered that activated MCs were enriched at sites of unhealed haematomas, due to subclinical aortic wall fissuring, in close proximity to adventitial IgE+ TLO B cells. Remarkably, in vitro the IgEs deriving from these samples enhanced MC production of IL-4, a cytokine which favors IgE class-switching and production by B cells. Finally, the role of MCs in aneurysmal progression was further analysed in vivo in ApoE-/- mice subjected to angiotensin II infusion aneurysm model, through MC-specific depletion after the establishment of dissecting aneurysms. MC-specific depletion improved intramural haematoma healing and reduced aneurysmal progression. CONCLUSIONS: Our data suggest that MC located close to aortic wall fissures are activated by adventitial TLO B cell-produced IgEs and participate to their own activation by providing support for further IgE synthesis through IL-4 production. By preventing prompt repair of aortic subclinical fissures, such a runaway MC activation loop could precipitate aneurysmal progression, suggesting that MC-targeting treatments may represent an interesting adjunctive therapy for reducing AAA progression.

Monocytes are the main source of STING-mediated IFN-α production.

BACKGROUND: Type I interferon (IFN-I) production by plasmacytoid dendritic cells (pDCs) occurs during viral infection, in response to Toll-like receptor 7 (TLR7) stimulation and is more vigorous in females than in males. Whether this sex bias persists in ageing people is currently unknown. In this study, we investigated the effect of sex and aging on IFN-α production induced by PRR agonist ligands. METHODS: In a large cohort of individuals from 19 to 97 years old, we measured the production of IFN-α and inflammatory cytokines in whole-blood upon stimulation with either R-848, ODN M362 CpG-C, or cGAMP, which activate the TLR7/8, TLR9 or STING pathways, respectively. We further characterized the cellular sources of IFN-α. FINDINGS: We observed a female predominance in IFN-α production by pDCs in response to TLR7 or TLR9 ligands. The higher TLR7-driven IFN-α production in females was robustly maintained across ages, including the elderly. The sex-bias in TLR9-driven interferon production was lost after age 60, which correlated with the decline in circulating pDCs. By contrast, STING-driven IFN-α production was similar in both sexes, preserved with aging, and correlated with circulating monocyte numbers. Indeed, monocytes were the primary cellular source of IFN-α in response to cGAMP. INTERPRETATION: We show that the sex bias in the TLR7-induced IFN-I production is strongly maintained through ages, and identify monocytes as the main source of IFN-I production via STING pathway. FUNDING: This work was supported by grants from Région Occitanie/Pyrénées-Méditerranée (#12052910, Inspire Program #1901175), University Paul Sabatier, and the European Regional Development Fund (MP0022856).

Accuracy of citrulline, I-FABP and D-lactate in the diagnosis of acute mesenteric ischemia.

Early diagnosis of acute mesenteric ischemia (AMI) remains a clinical challenge, and no biomarker has been consistently validated. We aimed to assess the accuracy of three promising circulating biomarkers for diagnosing AMI-citrulline, intestinal fatty acid-binding protein (I-FABP), and D-lactate. A cross-sectional diagnostic study enrolled AMI patients admitted to the intestinal stroke center and controls with acute abdominal pain of another origin. We included 129 patients-50 AMI and 79 controls. Plasma citrulline concentrations were significantly lower in AMI patients compared to the controls [15.3 µmol/L (12.0-26.0) vs. 23.3 µmol/L (18.3-29.8), p = 0.001]. However, the area under the receiver operating curves (AUROC) for the diagnosis of AMI by Citrulline was low: 0.68 (95% confidence interval = 0.58-0.78). No statistical difference was found in plasma I-FABP and plasma D-lactate concentrations between the AMI and control groups, with an AUROC of 0.44, and 0.40, respectively. In this large cross-sectional study, citrulline, I-FABP, and D-lactate failed to differentiate patients with AMI from patients with acute abdominal pain of another origin. Further research should focus on the discovery of new biomarkers.

I-FABP is decreased in COVID-19 patients, independently of the prognosis.

BACKGROUND: Severe acute respiratory syndrome caused by the novel coronavirus (SARS-CoV-2) is frequently associated with gastrointestinal manifestations. Herein we evaluated the interest in measuring the intestinal fatty acid-binding protein (I-FABP), a biomarker of intestinal injury, in COVID-19 patients. METHODS: Serum I-FABP was analyzed in 28 consecutive patients hospitalized for a PCR-confirmed COVID-19, in 24 hospitalized patients with non-COVID-19 pulmonary diseases, and 79 patients admitted to the emergency room for abdominal pain. RESULTS: I-FABP serum concentrations were significantly lower in patients with COVID-19, as compared to patients with non-COVID-19 pulmonary diseases [70.3 pg/mL (47-167.9) vs. 161.1 pg/mL (88.98-305.2), respectively, p = 0.008]. I-FABP concentrations in these two populations were significantly lower than in patients with abdominal pain without COVID-19 [344.8 pg/mL (268.9-579.6)]. I-FABP was neither associated with severity nor the duration of symptoms. I-FABP was correlated with polymorphonuclear cell counts. CONCLUSIONS: In this pilot study, we observed a low I-FABP concentration in COVID-19 patients either with or without gastrointestinal symptoms, of which the pathophysiological mechanisms and clinical impact remain to be established. Further explorations on a larger cohort of patients will be needed to unravel the molecular mechanism of such observation, including the effects of malabsorption and/or abnormal lipid metabolism.

A CD31-Derived Peptide Prevents the Development of Antibody-Mediated Lesions in a Rat Model of Aortic Allograft.

BACKGROUND: Antibody-mediated rejection (AMR) is a major cause of graft loss. The development of donor-specific antibodies (DSAs) directed against the allogeneic HLA molecules expressed by the graft also leads to accelerated arteriosclerosis. CD31 is a protein expressed on endothelial and immune cells, ensuring homeostasis at this interface. When strong immune stimulation occurs, the regulatory function of CD31 is lost owing to cleavage of its extracellular portion. P8RI, a synthetic peptide that binds to the ectodomain of CD31, is able to restore the CD31 immunomodulatory function. In this study, we hypothesized that CD31 could represent an attractive molecular target in AMR and investigated whether P8RI could prevent the development of vascular antibody-mediated lesions. MATERIALS AND METHODS: A rat model of orthotopic aortic allograft was used, and P8RI was administered for 28 days. Circulating DSAs were quantified to assess the alloimmune humoral response, and histologic and immunohistochemical analyses of aortic allografts were performed to estimate antibody-mediated lesions in the allograft. RESULTS: Aorta-allografted rats receiving P8RI developed fewer DSAs than control animals (mean fluorescence intensity 344 vs 741). The density of nuclei in the media (3.4 x 10-5 vs 2.2 x 10-5 nuclei/px2) and media surface area (2.33 x 106 vs 2.02 x 106 px2) were higher in animals treated with P8RI than in control animals. CONCLUSIONS: These data support a therapeutic potential for molecules able to restore the CD31 signaling to fight AMR. P8RI, an agonist synthetic peptide targeting CD31, might prevent DSA production and have a beneficial effect in limiting arterial antibody-mediated lesions. CD31 agonists may become therapeutic tools to prevent and treat solid organ transplant arteriosclerosis.

Early Detection of Localized Immunity in Experimental Autoimmune Myocarditis Using [99mTc]Fucoidan SPECT.

PURPOSE: The aim of the study was to evaluate the ability of technetium-99m-fucoidan ([99mTc]fucoidan), a molecular imaging agent specific for selectins, in the assessment of early localized immunity in a rat model of experimental autoimmune myocarditis (EAM). PROCEDURES: EAM was induced in Lewis rats and troponin T; brain natriuretic peptide (BNP) and anti-myosin antibodies were measured in plasma. Separately, [99mTc]fucoidan single-photon emission computed tomography (SPECT)/x-ray computed tomography (CT) was performed in the very early phase of myocarditis at 10, 15, and 21 days after immunization. Then, hearts were collected and used for autoradiography, well counting, histology, and flow cytometry analysis. RESULTS: The EAM acute phase is characterized by extensive myocardial necrosis, release of troponin and BNP, and pericardial effusion. [99mTc]Fucoidan uptake was significantly increased in EAM compared with controls starting from D15. There was a close relationship between uptake of the tracer and myocardial content in CD45+, CD8+, CD11b+, and CD31+ cells. CONCLUSIONS: [99mTc]Fucoidan SPECT/CT accurately diagnosed the autoimmune attack in the early steps of EAM and could be used to monitor disease evolution and therapy efficiency.

Adipocytes orchestrate the formation of tertiary lymphoid organs in the creeping fat of Crohn's disease affected mesentery.

The formation of tertiary lymphoid organs (TLOs) is orchestrated by the stromal cells of tissues chronically submitted to inflammatory stimuli, in order to uphold specific adaptive immune responses. We have recently shown that the smooth muscle cells of the arterial wall orchestrate the formation of the TLOs associated with atherosclerosis in response to the local release of TNF-α. Observational studies have recently documented the presence of structures resembling TLOs the creeping fat that develops in the mesentery of patients with Crohn's disease (CD), an inflammatory condition combining a complex and as yet not elucidated infectious and autoimmune responses. We have performed a comprehensive analysis of the TLO structures in order to decipher the mechanism leading to their formation in the mesentery of CD patients, and assessed the effect of infectious and/or inflammatory inducers on the potential TLO-organizer functions of adipocytes. Quantitative analysis showed that both T and B memory cells, as well as plasma cells, are enriched in the CD-affected mesentery, as compared with tissue from control subjects. Immunohistochemistry revealed that these cells are concentrated within the creeping fat of CD patients, in the vicinity of transmural lesions; that T and B cells are compartmentalized in clearly distinct areas; that they are supplied by post-capillary high endothelial venules and drained by lymphatic vessels indicating that these nodules are fully mature TLOs. Organ culture showed that mesenteric tissue samples from CD patients contained greater amounts of adipocyte-derived chemokines and the use of the conditioned medium from these cultures in functional assays was able to actively recruit T and B lymphocytes. Finally, the production of chemokines involved in TLO formation by 3T3-L1 adipocytes was directly elicited by a combination of TNF-α and LPS in vitro. We therefore propose a mechanism in which mesenteric adipocyte, through their production of key chemokines in response to inflammatory/bacterial stimuli, may orchestrate the formation of functional TLOs developing in CD-affected mesentery.

Macrophage CD31 Signaling in Dissecting Aortic Aneurysm.

BACKGROUND: The authors recently found that a CD31 agonist peptide reaches macrophages in injured aortas and exerts beneficial effects on apolipoprotein E-knockout (Apo E-/-) mice subjected to angiotensin (Ang) II infusion, a model of experimental acute aortic dissection and intramural hematoma (ADIM). OBJECTIVES: The purpose of this study was to evaluate the therapeutic potential of a drug-suitable agonist peptide in experimental ADIM. METHODS: P8RI, a retro-inverso sequence of the best candidate identified by functional in vitro screening of a peptide library, passed an absorption, distribution, metabolism, excretion and toxicology analysis. Apo E-/- mice (male, 28-week-old) implanted with Ang II-releasing pumps received P8RI (2.5 mg/kg/day) or vehicle from day 14 (n = 10/group). Leukocytes were analyzed by flow cytometry. Healing features of human and mouse dissected aortic segments were assessed by histology and immunofluorescence. The effect of CD31 on macrophages was evaluated using cells from CD31-/- mice and P8RI, in vitro. RESULTS: Human and experimental ADIM were characterized by the infiltration of proinflammatory macrophages. The absence of CD31 enhanced the proinflammatory polarization of macrophages, whereas the CD31 agonist P8RI favored reparative macrophages both in vitro and in vivo. The administration of P8RI after the occurrence of ADIM prevented aneurysmal transformation by promoting the resolution of intramural hematoma and the production of collagen in dissected aortas in vivo, associated with enrichment of M2 macrophages at the site of injury. CONCLUSIONS: CD31 signaling promotes the switching of proinflammatory macrophages to the reparative phenotype and favors the healing of experimental dissected aortas. Treatment with a drug-suitable CD31 agonist may facilitate the clinical management of ADIM.

Distinct effect of age, sex, and CMV seropositivity on dendritic cells and monocytes in human blood.

We analyzed the impact of age, sex, and CMV on blood monocyte and dendritic cell (DC) subpopulations in 256 healthy individuals aged from 19 to 96 years. Flow cytometry was performed on whole blood within the 4 h following blood drawing. Myeloid (mDC) and plasmacytoid DC (pDC), classical, intermediate, and nonclassical monocytes were enumerated by means of TruCount tubes (BD Biosciences). We provided reference values for mDC, pDC and the three monocyte subpopulations. The numbers of classical, intermediate, and nonclassical monocytes slightly increased with age while the numbers of mDC and pDC did not vary significantly. The level of expression of CD64 and CD163 on monocytes significantly increased with age while HLA-DR expression did not vary significantly. More precisely, CD163 expression level on intermediate monocyte slightly increased with age in women only (Spearman P = 0.019) while CD64 expression increased on monocytes in CMV-positive individuals only. We observed that sex had almost no impact on the numbers of monocytes and DC and on their expression level of CD64 and HLA-DR. We observed a significant decrease in the numbers of pDC with age in CMV-positive individuals, but not in CMV negative individuals. This suggests that the lifelong subclinical infection by CMV could influence the number of circulating DC of lymphoid origin. In contrast, CMV serostatus had no significant impact on absolute numbers of mDC and monocytes.

Diagnosis biomarkers in acute intestinal ischemic injury: so close, yet so far.

Acute intestinal ischemic injury (i3) is a life-threatening condition with disastrous prognosis, which is currently difficult to diagnose at the early stages of the disease; a rapid diagnosis is mandatory to avoid irreversible ischemia, extensive bowel resection, sepsis and death. The overlapping protein expression of liver and gut related to the complex physiopathology of the disease, the heterogeneity of the disease and its relative rarity could explain the lack of a useful early biochemical marker of i3. Apart from non-specific biological markers of thrombosis, hypoxia inflammation, and infection, several more specific biomarkers in relation with the gut barrier dysfunction, the villi injury and the enterocyte mass have been used in the diagnosis of acute i3. It includes particularly D-lactate, intestinal fatty acid-binding protein (FABP) and citrulline. Herein, we will discuss leading publications concerning these historical markers that point out the main limitations reagrding their use in routine clinical practice. We will also introduce the first and limited results arising from omic studies, underlying the remaining effort that needs to be done in the field of acute i3 biological diagnosis, which remains a challenge.

Erythrocyte Efferocytosis by the Arterial Wall Promotes Oxidation in Early-Stage Atheroma in Humans.

BACKGROUND: Since red blood cells (RBCs) are the predominant cellular blood component interacting with the arterial wall, we explored the role of RBCs efferocytosis by vascular smooth muscle cells (vSMCs) in the initiation of human atheroma. METHODS AND RESULTS: The comparison of human healthy aortas with aortic fatty streaks or fibroatheromas revealed that RBC angiophagy is implicated from the earliest stages of atherogenesis, as documented by the concomitant detection of redox-active iron, hemoglobin, glycophorin A, and ceroids. RBCs infiltration in the arterial wall was associated with local lipid and protein oxidation, as well as vascular response (expression of heme oxygenase-1 and of genes related to iron metabolism as well as those encoding for phagocytosis). These effects were recapitulated in vitro when vSMCs were co-cultured with phosphatidyl-exposing senescent (s) RBCs but not with fresh RBCs. VSMCs engulfing sRBC increased their intracellular iron content, accumulated hemoglobin, lipids, and activated their phagolysosomes. Strikingly, injections of sRBCs into rats promoted iron accumulation in the aortic wall. In rabbits, hypercholesterolemia increased circulating senescent RBCs and induced the subendothelial accumulation of iron-rich phagocytic foam cells. RBCs bring cholesterol and iron/heme into the vascular wall and interact with vSMCs that phagocytize them. CONCLUSION: This study presents a previously unforeseen mechanism of plaque formation that implicates intimal RBC infiltration as one of the initial triggers for foam cell formation and intimal oxidation. Pathogenic effects exerted by several metabolic and hemodynamic factors may rely on their effect on RBC biology, thereby impacting how RBCs interact with the vascular wall.

Atrial fibrillation is associated with the fibrotic remodelling of adipose tissue in the subepicardium of human and sheep atria.

AIMS: Accumulation of atrial adipose tissue is associated with atrial fibrillation (AF). However, the underlying mechanisms remain poorly understood. We examined the relationship between the characteristics of fatty infiltrates of the atrial myocardium and the history of AF. METHODS AND RESULTS: Atrial samples, collected in 92 patients during cardiac surgery and in a sheep model of persistent AF, were subjected to a detailed histological analysis. In sections of human right atrial samples, subepicardial fatty infiltrations were commonly observed in the majority of patients. A clear difference in the appearance and fibrotic content of these fatty infiltrations was observed. Fibro-fatty infiltrates predominated in patients with permanent AF (no AF: 37 ± 24% vs. paroxysmal AF: 50 ± 21% vs. permanent AF: 64 ± 23%, P < 0.001). An inverse correlation between fibrotic remodelling and the amount of subepicardial adipose tissue suggested the progressive fibrosis of fatty infiltrates with permanent AF. This hypothesis was tested in a sheep model of AF. In AF sheep, an increased accumulation of peri-atrial fat depot was observed on cardiac magnetic resonance imaging and dense fibro-fatty infiltrations predominated in the left atria of AF sheep. Cellular inflammation, mainly consisting of functional cytotoxic T lymphocytes, was observed together with adipocyte cell death in human atria. CONCLUSION: Atrial fibrillation is associated with the fibrosis of subepicardial fatty infiltrates, a process in which cytotoxic lymphocytes might be involved. This remodelling of the atrial subepicardium could contribute to structural remodelling forming a substrate for AF.

CD4+CXCR3+ T cells and plasmacytoid dendritic cells drive accelerated atherosclerosis associated with systemic lupus erythematosus.

Cardiovascular disease due to accelerated atherosclerosis is the leading cause of death in patients with systemic lupus erythematosus (SLE). Noteworthy, accelerated atherosclerosis in SLE patients appears to be independant of classical Framingham risk factors. This suggests that aggravated atherosclerosis in SLE patients may be a result of increased inflammation and altered immune responses. However, the mechanisms that mediate the acceleration of atherosclerosis in SLE remain elusive. Based on experimental data which includes both humans (SLE patients and control subjects) and rodents (ApoE-/- mice), we herein propose a multi-step model in which the immune dysfunction associated with SLE (i.e. high level of IFN-α production by TLR 9-stimulated pDCs) is associated with, first, an increased frequency of circulating pro inflammatory CD4+CXCR3+ T cells; second, an increased production of CXCR3 ligands by endothelial cells; third, an increased recruitment of pro-inflammatory CD4+CXCR3+ T cells into the arterial wall, and fourth, the development of atherosclerosis. In showing how SLE may promote accelerated atherosclerosis, our model also points to hypotheses for potential interventions, such as pDCs-targeted therapy, that might be studied in the future.

Control of the T follicular helper-germinal center B-cell axis by CD8⁺ regulatory T cells limits atherosclerosis and tertiary lymphoid organ development.

BACKGROUND: The atheromodulating activity of B cells during the development of atherosclerosis is well documented, but the mechanisms by which these cells are regulated have not been investigated. METHODS AND RESULTS: Here, we analyzed the contribution of Qa-1-restricted CD8(+) regulatory T cells to the control of the T follicular helper-germinal center B-cell axis during atherogenesis. Genetic disruption of CD8(+) regulatory T cell function in atherosclerosis-prone apolipoprotein E knockout mice resulted in overactivation of this axis in secondary lymphoid organs, led to the increased development of tertiary lymphoid organs in the aorta, and enhanced disease development. In contrast, restoring control of the T follicular helper-germinal center B-cell axis by blocking the ICOS-ICOSL pathway reduced the development of atherosclerosis and the formation of tertiary lymphoid organs. Moreover, analyses of human atherosclerotic aneurysmal arteries by flow cytometry, gene expression analysis, and immunofluorescence confirmed the presence of T follicular helper cells within tertiary lymphoid organs. CONCLUSIONS: This study is the first to demonstrate that the T follicular helper-germinal center B-cell axis is proatherogenic and that CD8(+) regulatory T cells control the germinal center reaction in both secondary and tertiary lymphoid organs. Therefore, disrupting this axis represents an innovative therapeutic approach.

Upholding the T cell immune-regulatory function of CD31 inhibits the formation of T/B immunological synapses in vitro and attenuates the development of experimental autoimmune arthritis in vivo.

CD31, a trans-homophilic inhibitory receptor expressed on both T- and B-lymphocytes, drives the mutual detachment of interacting leukocytes. Intriguingly, T cell CD31 molecules relocate to the immunological synapse (IS), where the T and B cells establish a stable interaction. Here, we show that intact CD31 molecules, which are able to drive an inhibitory signal, are concentrated at the periphery of the IS but are excluded from the center of the IS. At this site, were the cells establish the closest contact, the CD31 molecules are cleaved, and most of the extracellular portion of the protein, including the trans-homophilic binding sites, is shed from the cell surface. T cells lacking CD31 trans-homophilic binding sites easily establish stable interactions with B cells; at the opposite, CD31 signaling agonists inhibit T/B IS formation as well as the ensuing helper T cell activation and function. Confocal microscopy and flow cytometry analysis of experimental T/B IS shows that the T cell inhibitory effects of CD31 agonists depend on SHP-2 signaling, which reduces the phosphorylation of ZAP70. The analysis of synovial tissue biopsies from patients affected by rheumatoid arthritis showed that T cell CD31 molecules are excluded from the center of the T/B cell synapses in vivo. Interestingly, the administration of CD31 agonists in vivo significantly attenuated the development of the clinical signs of collagen-induced arthritis in DBA1/J mice. Altogether, our data indicate that the T cell co-inhibitory receptor CD31 prevents the formation of functional T/B immunological synapses and that therapeutic strategies aimed at sustaining CD31 signaling will attenuate the development of autoimmune responses in vivo.

Inflammatory micro-environmental cues of human atherothrombotic arteries confer to vascular smooth muscle cells the capacity to trigger lymphoid neogenesis.

BACKGROUND: Experimental atherosclerosis is characterized by the formation of tertiary lymphoid structures (TLOs) within the adventitial layer, which involves the chemokine-expressing aortic smooth muscle cells (SMCs). TLOs have also been described around human atherothrombotic arteries but the mechanisms of their formation remain poorly investigated. Herein, we tested whether human vascular SMCs play the role of chemokine-expressing cells that would trigger the formation of TLOs in atherothrombotic arteries. RESULTS: We first characterized, by flow cytometry and immunofluorescence analysis, the prevalence and cell composition of TLOs in human abdominal aneurysms of the aorta (AAAs), an evolutive form of atherothrombosis. Chemotaxis experiments revealed that the conditioned medium from AAA tissues recruited significantly more B and T lymphocytes than the conditioned medium from control (N-AAA) tissues. This was associated with an increase in the concentration of CXCL13, CXCL16, CCL19, CCL20, and CCL21 chemokines in the conditioned medium from AAA tissues. Immunofluorescence analysis of AAA cryosections revealed that α-SMA-positive SMCs were the main contributors to the chemokine production. These results were confirmed by RT-qPCR assays where we found that primary vascular SMCs from AAA tissues expressed significantly more chemokines than SMCs from N-AAA. Finally, in vitro experiments demonstrated that the inflammatory cytokines found to be increased in the conditioned medium from AAA were able to trigger the production of chemokines by primary SMCs. CONCLUSION: Together, these results suggest that human vascular SMCs in atherothrombotic arteries, in response to inflammatory signals, are converted into chemokine-expressing cells that trigger the recruitment of immune cells and the formation of aortic TLOs.

CD31 is a key coinhibitory receptor in the development of immunogenic dendritic cells.

CD31 is a transhomophilic tyrosine-based inhibitory motif receptor and is expressed by both dendritic cells (DCs) and T lymphocytes. Previous studies have established that the engagement of CD31 drives immune-inhibitory signaling in T lymphocytes, but the effect exerted by CD31 signaling in DCs remains elusive. Here, we show that CD31 is a key coinhibitory receptor on stimulated DCs, favoring the development of tolerogenic functions and finally resulting in T-cell tolerance. The disruption of CD31 signaling favored the immunogenic maturation and migration of resident DCs to the draining lymph nodes. In contrast, sustaining the CD31/SHP-1 signaling during DC maturation resulted in reduced NF-κB nuclear translocation, expression of costimulatory molecules, and production of immunogenic cytokines (e.g., IL-12, IL-6), whereas the expression of TGF-ß and IL-10 were increased. More importantly, CD31-conditioned DCs purified from the draining lymph nodes of ovalbumin-immunized mice favored the generation of antigen-specific regulatory T cells (CD25(+) forkhead box P3(+)) at the expense of effector (IFN-γ(+)) cells upon coculture with naive ovalbumin-specific CD4(+) T lymphocytes ex vivo. Finally, the adoptive transfer of CD31-conditioned myelin oligodendrocyte glycoprotein-loaded DCs carried immune tolerance against the subsequent development of MOG-induced experimental autoimmune encephalomyelitis in vivo. The key coinhibitory role exerted by CD31 on DCs highlighted by the present study may have important implications both in settings where the immunogenic function of DCs is desirable, such as infection and cancer, and in settings where tolerance-driving DCs are preferred, such as autoimmune diseases and transplantation.

M1 macrophages act as LTßR-independent lymphoid tissue inducer cells during atherosclerosis-related lymphoid neogenesis.

AIMS: The goal of this study was to characterize the role of inflammatory macrophages in the induction of the vascular smooth muscle cell (VSMC)-mediated formation of aortic tertiary lymphoid organs (TLOs). METHODS AND RESULTS: Mouse bone marrow-derived M1 macrophages acted as lymphoid tissue inducer cells. Indeed, they expressed high levels of tumour necrosis factor (TNF)-α and membrane-bound lymphotoxin (LT)-α, two inducing cytokines that triggered expression of the chemokines CCL19, CCL20, and CXCL16, as did M1 supernatant. The blockade of LTßR signalling with LTßR-Ig had no effect, whereas that of TNFR1/2 signalling reduced chemokine expression by VSMCs in both wild-type (WT) and LTßR KO mice, demonstrating that LTßR signalling is dispensable for the M1-inducing effect. This effect was corroborated by the development of TLOs observed in LTßR KO->apolipoprotein E knockout (ApoE KO) aortic segments after orthotopic transplantation. Furthermore, treatment of ApoE KO mice with anti-TNF-α antibody decreased the number and incidence of aortic TLOs. Finally, lymphoid nodules composed of T and B cells formed in in vivo-implanted scaffolds seeded with VSMCs previously stimulated ex vivo by M1-conditioned medium. CONCLUSIONS: These results are the first to identify M1 macrophages as inducer cells that trigger the expression of chemokines by VSMCs independently of LTßR signalling. We propose that the dialogue between macrophages and VSMCs-established across the vascular wall-contributes to the formation of aortic TLOs.

A CD31-derived peptide prevents angiotensin II-induced atherosclerosis progression and aneurysm formation.

AIMS: The loss of the inhibitory receptor CD31 on peripheral T lymphocytes is associated with the incidence of atherosclerotic complications such as abdominal aortic aneurysms (AAA) in patients and plaque thrombosis in mice. However, we have recently discovered that a small fragment of extracellular CD31 remains expressed on the surface of the apparently 'CD31-negative' T-cells and that it is possible to restore the CD31-mediated T-cell inhibition in vivo by using a synthetic CD31-derived peptide. Here, we wanted to evaluate the therapeutic potential of the peptide in an experimental model of accelerated atherosclerosis and AAA formation. METHODS AND RESULTS: The effect of the murine CD31-derived peptide (aa 551-574, 1.5 mg/kg/day, sc) was evaluated on the extent of atherosclerotic plaques and the incidence of AAA in 28-week-old apolipoprotein E knockout mice (male, n ≥ 8/group) submitted to chronic angiotensin II infusion. The therapeutic mechanisms of the peptide were assessed by evaluating its effect on immune cell functions in vivo and in vitro. The prevalence of angiotensin II-induced AAA correlated with the loss of extracellular CD31 on T-cells. CD31 peptide treatment reduced both aneurysm formation and plaque size (P < 0.05 vs. control). Protection was associated with reduced perivascular leucocyte infiltration and T-cell activation in vivo. Functional in vitro studies showed that the peptide is able to suppress both T-cell and macrophage activation. CONCLUSION: CD31 peptides could represent a new class of drugs intended to prevent the inflammatory cell processes, such as those underlying progression of atherosclerosis and development of AAA.