CD206+/MHCII− macrophage accumulation at nerve injury site correlates with attenuation of allodynia in TASTPM mouse model of Alzheimer's disease

Chronic pain is undertreated in people with Alzheimer's disease (AD) and better understanding of the underlying mechanisms of chronic pain in this neurodegenerative disease is essential. Neuropathic pain and AD share a significant involvement of the peripheral immune system. Therefore, we examined the development of nerve injury-induced allodynia in TASTPM (APPsweXPS1.M146V) mice and assessed monocytes/macrophages at injury site. TASTPM developed partial allodynia compared to WT at days 7, 14 and 21 days after injury, and showed complete allodynia only after treatment with naloxone methiodide, a peripheralized opioid receptor antagonist. Since macrophages are one of the sources of endogenous opioids in the periphery, we examined macrophage infiltration at injury site and observed that CD206+/MHCII− cells were more numerous in TASTPM than WT. Accordingly, circulating TASTPM Ly6Chigh (classical) monocytes, which are pro-inflammatory and infiltrate at the site of injury, were less abundant than in WT. In in vitro experiments, TASTPM bone marrow-derived macrophages showed efficient phagocytosis of myelin extracts containing amyloid precursor protein, acquired CD206+/MHCII− phenotype, upregulated mRNA expression of proenkephalin (PENK) and accumulated enkephalins in culture media. These data suggest that in TASTPM nerve-injured mice, infiltrating macrophages which derive from circulating monocytes and may contain amyloid fragments, acquire M2-like phenotype after myelin engulfment, and release enkephalins which are likely to inhibit nociceptive neuron activity via activation of opioid receptors

Dorsal root ganglia CX3CR1 expressing monocytes/macrophages contribute to arthritis pain

Pain is a persistent symptom of Rheumatoid Arthritis, and the K/BxN serum transfer model recapitulates both association and dissociation between pain and joint inflammation in RA. Furthermore, this model features monocyte/macrophage infiltration in joints and lumbar dorsal root ganglia (DRG), where these immune cells are close to nociceptive neurons. We focussed on CX3CR1-monocyte/macrophage trafficking and show that at peak paw swelling associated with nociception, CX3CR1 deletion altered neither swelling nor macrophage infiltration/phenotype in paws. However, acute nociception and DRG non-classical monocyte numbers were reduced in CX3CR1GFP/GFP (KO) compared to CX3CR1+/GFP (WT). Nociception that persisted despite swelling had resolved was attenuated in KO and correlated with DRG macrophages displaying M2-like phenotype. Still in the DRG, neurons up-regulated neuropeptide CGRP and olcegepant treatment reduced acute swelling, nociception, and leukocyte infiltration in paws and DRG. We delineate in-vitro a signalling pathway showing that CGRP liberates the CX3CR1 ligand fractalkine (FKN) from endothelium, and in bone marrow-derived macrophages, FKN promotes activation of intracellular kinases, polarisation towards M1-like phenotype and release of pro-nociceptive IL-6. These data implicate non-classical CX3CR1-expressing monocyte and macrophage recruitment into the DRG in initiation and maintenance of arthritis pain

Genetic Depletion or Hyperresponsiveness of Natural Killer Cells Do Not Affect Atherosclerosis Development.

RATIONALE: Chronic inflammation is central in the development of atherosclerosis. Both innate and adaptive immunities are involved. Although several studies have evaluated the functions of natural killer (NK) cells in experimental animal models of atherosclerosis, it is not yet clear whether NK cells behave as protective or proatherogenic effectors. One of the main caveats of previous studies was the lack of specificity in targeting loss or gain of function of NK cells. OBJECTIVES: We used 2 selective genetic approaches to investigate the role of NK cells in atherosclerosis: (1) Ncr1iCre/+R26lsl-DTA/+ mice in which NK cells were depleted and (2) Noé mice in which NK cells are hyperresponsive. METHODS AND RESULTS: No difference in atherosclerotic lesion size was found in Ldlr-/- (low-density lipoprotein receptor null) mice transplanted with bone marrow (BM) cells from Ncr1iCreR26Rlsl-DTA , Noé, or wild-type mice. Also, no difference was observed in plaque composition in terms of collagen content, macrophage infiltration, or the immune profile, although Noé chimera had more IFN (interferon)-γ-producing NK cells, compared with wild-type mice. Then, we investigated the NK-cell selectivity of anti-asialoganglioside M1 antiserum, which was previously used to conclude the proatherogenicity of NK cells. Anti-asialoganglioside M1 treatment decreased atherosclerosis in both Ldlr-/- mice transplanted with Ncr1iCreR26Rlsl-DTA or wild-type bone marrow, indicating that its antiatherogenic effects are unrelated to NK-cell depletion, but to CD8+ T and NKT cells. Finally, to determine whether NK cells could contribute to the disease in conditions of pathological NK-cell overactivation, we treated irradiated Ldlr-/- mice reconstituted with either wild-type or Ncr1iCreR26Rlsl-DTA bone marrow with the viral mimic polyinosinic:polycytidylic acid and found a significant reduction of plaque size in NK-cell-deficient chimeric mice. CONCLUSIONS: Our findings, using state-of-the-art mouse models, demonstrate that NK cells have no direct effect on the natural development of hypercholesterolemia-induced atherosclerosis, but may play a role when an additional systemic NK-cell overactivation occurs

The Dendritic Cell Receptor DNGR-1 Promotes the Development of Atherosclerosis in Mice.

RATIONALE: Necrotic core formation during the development of atherosclerosis is associated with a chronic inflammatory response and promotes accelerated plaque development and instability. However, the molecular links between necrosis and the development of atherosclerosis are not completely understood. Clec9a (C-type lectin receptor) or DNGR-1 (dendritic cell NK lectin group receptor-1) is preferentially expressed by the CD8α+ subset of dendritic cells (CD8α+ DCs) and is involved in sensing necrotic cells. We hypothesized that sensing of necrotic cells by DNGR-1 plays a determinant role in the inflammatory response of atherosclerosis. OBJECTIVE: We sought to address the impact of total, bone marrow-restricted, or CD8α+ DC-restricted deletion of DNGR-1 on atherosclerosis development. METHODS AND RESULTS: We show that total absence of DNGR-1 in Apoe (apolipoprotein e)-deficient mice (Apoe-/-) and bone marrow-restricted deletion of DNGR-1 in Ldlr (low-density lipoprotein receptor)-deficient mice (Ldlr-/-) significantly reduce inflammatory cell content within arterial plaques and limit atherosclerosis development in a context of moderate hypercholesterolemia. This is associated with a significant increase of the expression of interleukin-10 (IL-10). The atheroprotective effect of DNGR-1 deletion is completely abrogated in the absence of bone marrow-derived IL-10. Furthermore, a specific deletion of DNGR-1 in CD8α+ DCs significantly increases IL-10 expression, reduces macrophage and T-cell contents within the lesions, and limits the development of atherosclerosis. CONCLUSIONS: Our results unravel a new role of DNGR-1 in regulating vascular inflammation and atherosclerosis and potentially identify a new target for disease modulation

Selective EGF-Receptor Inhibition in CD4+ T Cells Induces Anergy and Limits Atherosclerosis.

BACKGROUND: Several epidermal growth factor receptor (EGFR) inhibitors have been successfully developed for the treatment of cancer, limiting tumor growth and metastasis. EGFR is also expressed by leukocytes, but little is known about its role in the modulation of the immune response. OBJECTIVES: The aim of this study was to determine whether EGFR expressed on CD4+ T cells is functional and to address the consequences of EGFR inhibition in atherosclerosis, a T cell-mediated vascular chronic inflammatory disease. METHODS: The authors used EGFR tyrosine kinase inhibitors (AG-1478, erlotinib) and chimeric Ldlr-/-Cd4-Cre/Egfrlox/lox mouse with a specific deletion of EGFR in CD4+ T cells. RESULTS: Mouse CD4+ T cells expressed EGFR, and the EGFR tyrosine kinase inhibitor AG-1478 blocked in vitro T cell proliferation and Th1/Th2 cytokine production. In vivo, treatment of Ldlr-/- mice with the EGFR inhibitor erlotinib induced T cell anergy, reduced T cell infiltration within atherosclerotic lesions, and protected against atherosclerosis development and progression. Selective deletion of EGFR in CD4+ T cells resulted in decreased T cell proliferation and activation both in vitro and in vivo, as well as reduced interferon-γ, interleukin-4, and interleukin-2 production. Atherosclerotic lesion size was reduced by 2-fold in irradiated Ldlr-/- mice reconstituted with bone marrow from Cd4-Cre/Egfrlox/lox mice, compared to Cd4-Cre/Egfr+/+ chimeric mice, after 4, 6, and 12 weeks of high-fat diet, associated with marked reduction in T cell infiltration in atherosclerotic plaques. Human blood T cells expressed EGFR and EGFR inhibition reduced T cell proliferation both in vitro and in vivo. CONCLUSIONS: EGFR blockade induced T cell anergy in vitro and in vivo and reduced atherosclerosis development. Targeting EGFR may be a novel strategy to combat atherosclerosis

Genetic and Pharmacological Inhibition of TREM-1 Limits the Development of Experimental Atherosclerosis.

BACKGROUND: Innate immune responses activated through myeloid cells contribute to the initiation, progression, and complications of atherosclerosis in experimental models. However, the critical upstream pathways that link innate immune activation to foam cell formation are still poorly identified. OBJECTIVES: This study sought to investigate the hypothesis that activation of the triggering receptor expressed on myeloid cells (TREM-1) plays a determinant role in macrophage atherogenic responses. METHODS: After genetically invalidating Trem-1 in chimeric Ldlr-/-Trem-1-/- mice and double knockout ApoE-/-Trem-1-/- mice, we pharmacologically inhibited Trem-1 using LR12 peptide. RESULTS: Ldlr-/- mice reconstituted with bone marrow deficient for Trem-1 (Trem-1-/-) showed a strong reduction of atherosclerotic plaque size in both the aortic sinus and the thoracoabdominal aorta, and were less inflammatory compared to plaques of Trem-1+/+ chimeric mice. Genetic invalidation of Trem-1 led to alteration of monocyte recruitment into atherosclerotic lesions and inhibited toll-like receptor 4 (TLR 4)-initiated proinflammatory macrophage responses. We identified a critical role for Trem-1 in the upregulation of cluster of differentiation 36 (CD36), thereby promoting the formation of inflammatory foam cells. Genetic invalidation of Trem-1 in ApoE-/-/Trem-1-/- mice or pharmacological blockade of Trem-1 in ApoE-/- mice using LR-12 peptide also significantly reduced the development of atherosclerosis throughout the vascular tree, and lessened plaque inflammation. TREM-1 was expressed in human atherosclerotic lesions, mainly in lipid-rich areas with significantly higher levels of expression in atheromatous than in fibrous plaques. CONCLUSIONS: We identified TREM-1 as a major upstream proatherogenic receptor. We propose that TREM-1 activation orchestrates monocyte/macrophage proinflammatory responses and foam cell formation through coordinated and combined activation of CD36 and TLR4. Blockade of TREM-1 signaling may constitute an attractive novel and double-hit approach for the treatment of atherosclerosis

Modulation of epidermal growth factor-receptor during atherosclerosis development

Le récepteur à l’EGF (Epidermal Growth Factor) est exprimé, entre autres, par les cellules inflammatoires et vasculaires. Il est impliqué dans la survie et la migration cellulaire. L’EGF-R et ses ligand sont exprimés dans les plaques d’athérosclérose. L’objectif de cette étude est d’évaluer les effets de l’inhibition de l’EGF-R sur les fonctions des lymphocytes T CD4+ et sur les macrophages au cours du développement de l’athérosclérose expérimentale. L’EGFR est exprimé par les lymphocytes T CD4+, ainsi que par les macrophages au sein des plaques d’athérosclérose murines. L’inhibition pharmacologique de l’EGFR (Erlotinib) chez des souris Ldlr-/- sous régime riche en matières grasses réduit le développement et la progression des lésions athéromateuses. Afin d’étudier le rôle spécifique de l’EGFR dans les lymphocytes T CD4+, nous avons généré des souris Cd4Cre Egfrlox/lox. Des souris Ldlr-/- ont été irradiées et retransplantées avec une moelle Cd4Cre Egf-r+/+ ou Cd4Cre Egf-rlox/lox puis mise sous régime riche en matières grasses. L’invalidation spécifique de l’EGFR dans les lymphocytes T CD4+ induit une diminution de la prolifération lymphocytaire T CD4+ in vitro et in vivo, une diminution de la production d’IFN-γ, d’IL-4 et d’IL-2. La transplantation de la moelle Cd4Cre Egf-rlox/lox induit une réduction de la taille des lésions d’athérosclérose sans différence concernant la cholestérolémie, et une diminution de l’infiltration lymphocytaire T dans les plaques. Nous avons ensuite généré des souris LysMCre Egf-rlox/lox pour étudier le rôle spécifique de l’EGF-R exprimé par les cellules myéloides. Des souris Ldlr-/- ont été irradiées et retransplantées avec une moelle LysMCre-Egf-rlox/lox ou LysMCre+Egfrlox/lox. La transplantation de la moelle LysMCre+Egf-rlox/lox induit une réduction de la taille des lésions après 4, 7 et 12 semaines de régime riche en matière grasses. Les plaques des souris chimères Ldlr-/-/LysMCre+Egf rlox/lox sont caractérisées par une diminution significative de l’infiltration macrophagique ainsi qu’une diminution de la taille du noyau nécrotique. L’invalidation génétique de l’EGFR dans la lignée myéloide réduit significativement la production du TNF-α et d’IL-6. Par ailleurs, l’inhibition pharmacologique et l’invalidation génétique d’EGFR réduit la formation des cellules spumeuses par une « down-régulation » du CD36. L’inhibition pharmacologique l’EGF-R diminue l’activité pro-inflammatoire pro-athérogène des lymphocytes T CD4+ et des macrophages, et in fine réduit le développement et la progression de l’athérosclérose expérimentale. Nos résultats suggèrent que l’inhibition de l’EGFR pourrait être une nouvelle stratégie thérapeutique pour le traitement de l’athérosclérose.Background: Several Epidermal Growth Factor receptor (EGF-R) inhibitors have been successfully developed for the treatment of cancer, inhibiting tumor cell survival, proliferation and migration. EGF-R is expressed by leucocytes, but little is known about its role in the modulation of the immune response. The first part of the projet is to determine whether EGFR expressed on myeloid cells is functional, and to address the consequences of EGFR inhibition specifically in myeloid cells on atherosclerosis. The second part is to explore the expression of EGF-R on CD4+ T cells, and to study the effects of the specific EGF-R invalidation on CD4+ T cells during atherosclerosis development. Methods and results: Ldlr-/- mice were orally treated with a specific EGFR inhibitor (Erlotinib, 15mg/kg) for 6 weeks, under a high fat diet. EGFR pharmacological inhibition reduced T cell infiltration, decreases macrophage accumulation within atherosclerotic lesions, and thus, protected against atherosclerosis development in the aortic sinus. In parallel, we generated chimeric Ldlr-/- mice. Ldlr-/- mice were lethally irradiated and reconstituted with LysMCre+ EgfrLox/lox or LysM Cre- EgfrLox/lox bone marrow cells. In addition, irradiated Ldlr-/- mice were also reconstituted with bone marrow from Cd4Cre Egfrlox/lox , or Cd4Cre Egfr+/+ and put under a high fat diet. Animal weight and cholesterolemia were not different between groups. We observed a decrease of atherosclerosis plaque size in the aortic sinus in chimeric Ldlr-/-/LysMCre+ EgfrLox/lox and Ldlr-/-Cd4Cre Egfrlox/lox mice in comparison with chimeric Ldlr-/-/LysMCre- EgfrLox/lox, and Ldlr-/-Cd4Cre Egfr+/+ respectively. Myeloid invalidation of EGFR and pharmacological inhibition using AG-1478, a specific tyrosine kinase inhibitor, affected cytoskeleton reorganization limiting macrophage adhesion, spreading and migration. EGF-R blockage significantly reduced lipid uptake and foam cell formation through the down-regulation of CD36 expression. Selective deletion of Egfr in CD4+ T cells resulted in decreased T cell proliferation and activation both in vitro and in vivo, as well as reduced IFN-γ, IL-17A, IL-4 and IL-10 production. Finally, human blood T cells expressed EGFR and EGFR inhibition reduced T cell proliferation both in vivo and in vitro. Conclusion. EGFR is expressed by human and mouse CD4+ T cells. EGFR pharmacological inhibition or genetic invalidation induced T cell anergy in vitro and in vivo, blocked macrophage activity, and limited atherosclerosis initiation and progression. Our results suggest that targeting EGFR may be a novel strategy to combat atherosclerosis

L’inhibition du récepteur de l’EGF sur les cellules immunitaires réduit l’athérosclérose expérimentale

International audienceNo abstract availabl

Data from: Efferocytosis perpetuates substance accumulation inside macrophage populations

In both cells and animals, cannibalism can transfer harmful substances from the consumed to the consumer. Macrophages are immune cells that consume their own dead via a process called cannibalistic efferocytosis. Macrophages that contain harmful substances are found at sites of chronic inflammation, yet the role of cannibalism in this context remains unexplored. Here we take mathematical and experimental approaches to study the relationship between cannibalistic efferocytosis and substance accumulation in macrophages. Through mathematical modelling, we deduce that substances which transfer between individuals through cannibalism will concentrate inside the population via a coalescence process. This prediction was confirmed for macrophage populations inside a closed system. We used image analysis of whole slide photomicrographs to measure both latex microbead and neutral lipid accumulation inside murine bone marrow-derived macrophages (104-105 cells) following their stimulation into an inflammatory state ex vivo. While the total number of phagocytosed beads remained constant, cell death reduced cell numbers and efferocytosis concentrated the beads among the surviving macrophages. % from small to large numbers per cell. Since lipids are also conserved during efferocytosis, these cells accumulated lipid derived from the membranes of dead and consumed macrophages (becoming macrophage foam cells). Consequently, enhanced macrophage cell death increased the rate and extent foam cell formation. Our results demonstrate that cannibalistic efferocytosis perpetuates exogenous (e.g. beads) and endogenous (e.g. lipids) substance accumulation inside macrophage populations. As such, cannibalism has similar detrimental consequences in both cells and animals