C-C chemokine receptor 4 deficiency exacerbates early atherosclerosis in mice

Reviewer #1 (Public review):

Summary:

The article provides valuable information on the role of CCR4 in an inflammatory condition, namely, the arteriosclerosis plaque. The data demonstrated that in the absence of CCR4, the Th1 cells infiltrated the plaque and Tregs lost its functions. The data are clear and well-presented. Mostly importantly, the data on CCR4-specific deficiency in Regulatory T cells is more impressive.

Strengths:

The data are clear, well performed, and interesting in focusing on the plaque and compared to peripheral organs. The disease is relevant and the data could be used to understand the risk of patients under immunomodulator use.

Weaknesses:

Still, we don't know the mechanism, besides migration.

Reviewer #2 (Public review):

Summary:

Tanaka et al. investigated the role of CCR4 in early atherosclerosis, focusing on the immune modulation elicited by this chemokine receptor under hypercholesterolemia. The study found that Ccr4 deficiency led to qualitative changes in atherosclerotic plaques, characterized by an increased inflammatory phenotype. The authors further analyzed the CD4 T cell immune response in para-aortic lymph nodes and atherosclerotic aorta, showing an increase mainly in Th1 cells and the Th1/Treg ratio in Ccr4-/-Apoe-/- mice compared to Apoe-/- mice. They then focused on Tregs, demonstrating that Ccr4 deficiency impaired their immunosuppressive function in in-vitro assays and elegantly showed that Ccr4-deficient Tregs had, as expected, impaired migration to the atherosclerotic aorta. Adoptive cell transfer of Ccr4-/- Tregs to Apoe-/- mice mimicked early atherosclerosis development in Ccr4-/-Apoe-/- mice. Therefore, this work shows that CCR4 plays an important role in early atherosclerosis but not in advanced stages.

Strengths:

Several in vivo and in vitro approaches were used to address the role of CCR4 in early atherosclerosis. Particularly, through the adoptive cell transfer of CCR4+ or CCR4- Tregs, the authors aimed to directly demonstrate the role of CCR4 in Tregs' protection against early atherosclerosis.

Weaknesses:

The isolation of Tregs was inadequately controlled; they were isolated based solely on CD4 and CD25 expression. CD25 is also expressed by activated effector T cells, meaning the analyzed cells could be a pool of mainly Tregs but also include effector T cells.

The study primarily focused on Th1 and Tregs without thoroughly investigating other CD4 T cell subsets. Th17 cells are known to play an important role in atherosclerosis; non-pathogenic Th17 cells express CCR4, while pathogenic Th17 cells do not. Considering that Figure 3 shows an increased frequency of IL17-expressing CD4 T cells compared to Apoe-/- mice, and given the imprecise Treg isolation, differences in non-pathogenic Th17 cells could be contributing to the observed effects.

Furthermore, the clinical relevance of these findings is not discussed. As an initial approach, the authors could analyze public datasets to determine if certain Ccr4 single nucleotide polymorphisms correlate with a higher incidence of atherosclerosis.

Reviewer #3 (Public review):

Summary:

In this paper, Tanaka and colleagues address the role played by the C-C chemokine receptor 4 (CCR4) in developing early atherosclerotic plaques using ApoE-deficient mice fed with a standard chow diet as a model. Since CCR4 is expressed in several T CD4+ lymphocyte subsets, the authors examined the consequences of CCR4 deficiency on the differentiation profile and traffic of T CD4+ lymphocytes. By histological analysis of aortic lesions, they demonstrated that the absence of CCR4 promoted the development of early atherosclerosis, characterized by an inflammatory reaction with increased levels of macrophages and T CD4+ inflammatory lymphocytes while decreased collagen content. Using flow cytometry together with mRNA expression analysis for identifying T CD4+ cell subsets, the authors found that the accelerated aortic inflammation induced by CCR4 deficiency correlated with higher proliferation of T CD4+ cells in lymphoid tissues, favouring the expansion of the pro-inflammatory effector Th1 cell subset, typically found in atherosclerotic lesions. Interestingly, the increased T CD4+ cell response occurred despite the expansion of T CD4+ Foxp3+ regulatory cells (Treg), which were in higher numbers in the lymphoid tissues of CCR4-deficient mice, suggesting the absence of CCR4 interfered with the regulatory actions of Treg cells. Using in vitro and or in vivo approaches, the authors found evidence of CCR4 requirement for Treg suppressive activity and migratory capacity to inflamed aortic areas, contributing to why CCR4 deficiency induced an augmented Th1/Treg ratio in the aortic lesions. These findings might not be surprising considering the demonstrated involvement of CCR4 in driving Treg migration to inflamed tissues in immune-related pathological models and Treg-dendritic cell contact for imprinting suppressive signals. However, in previous studies using a murine model of advanced atherosclerosis, neither hematopoietic nor systemic CCR4 deficiency altered the development of the aortic lesions. The authors included a thoughtful discussion about hypothetical mechanisms explaining these contrasting results, highlighting putative differences in the role played by the CCL17/CCL22-CCR4 axis along the stages of atherosclerosis development in this murine model.

Major strengths and weaknesses:

The main effects of CCR4 deficiency on early atherosclerosis development and Treg functional loss are valuable and supported by collected data. In vivo studies for comparing Treg-tissue accumulation or atherosclerotic lesions in Apoe-/- mice that received Treg derived from Apoe-/- or Apoe-/-Ccr4-/- mice, strengthening results. However, an incomplete description of methods (particularly flow cytometry) and data analysis weakens some conclusions of this study. Readers should note some inconsistencies in the T CD4+ response analysis in different tissues. In aortic lesions, but not in lymphoid tissues (peripheral, para-aortic, and spleen), the ratio Th1/Treg was used for evaluating the effect of CCR4 deficiency on the profile of Th cell subsets. In lymphoid tissues, increments in the frequency of both effector Th1 and Treg were observed in CCR4-deficient Apoe-/- mice compared to CCR4-sufficient Apoe-/- mice. Therefore, it is not convincing that CCR4-deficiency shifts Th1 cell/Treg balance toward Th1 cell responses in all lymphoid tissues; this claim needs to be revised by the authors. The Treg dysfunction, caused by CCR4 deficiency, enhanced T CD4+ activation and might have amplified rather than shifted, the typical biased Th1-mediated inflammatory response observed in the lymphoid tissues of hypercholesterolemic mice. A different scenario emerged in aortic lesions, where recruitment of effector Th1 cells, but not of additional effector T CD4+ cell subsets expanded in lymphoid tissues, leading to a higher Th1/Treg balance. Also, effector Th17 cells seem to predominate among effector TCD45+CD3+CD4+ cells in the aorta of Apoe-/- mice, and the Th1/Th17 balance appears to have increased as a consequence of CCR4 deficiency as well. Modulation of Th1/Th17 balance might be responsible for changes in the type and functional properties of recruited inflammatory cells in the aorta.

Study limitations:

This investigation has some limitations. Current tools for single-cell characterization have revealed the phenotypic heterogeneity and dynamics of aortic leukocytes, including T cells, which are among the principal aortic leukocytes found in mouse and human atherosclerotic lesions (doi:10.1161/CIRCRESAHA.117.312513). The flow cytometry analysis applied in this study cannot distinguish the generation of particular phenotypes within T CD4+ subsets, including putative phenotypes of no-suppressive T cells expressing low levels of Foxp3, as seems could occur in other chronic inflammatory disorders (doi: 10.1038/nm.3432; doi: 10.1172/JCI79014). Limitations due to the use of a complete CCR4 knockout mouse and putative differences in CCR4-mediated mechanisms along atherosclerosis stages and in human atherosclerosis were commented on by the authors in the discussion.

Global Impact

This work opens the way for a deeper analysis of the contribution of CCR4 and its ligands to the activation and differentiation of T CD4+ lymphocytes during atherosclerosis development, with these lymphocytes being fundamental players in the generation of pro-atherogenic and anti-atherogenic immune responses. Differences in the mechanisms mediated by the CCL17/CCL22-CCR4 axis among early and advanced atherosclerosis highlight the complex landscape to examine and validate in human samples and the need to achieve a deep knowledge for identifying genuine and safe targets capable of promoting protective anti-atherogenic immune responses.