Airway inflammation is the most common hallmark of allergic asthma. Chemokine receptors involved in leukocyte recruitment are closely related to the pathology in asthma. CCR9 has been described as a homeostatic and inflammatory chemokine receptor, but its role and that of its ligand CCL25 during lung inflammation remain unknown. To investigate the role of CCR9 as a modulator of airway inflammation, we established an OVA-induced allergic inflammation model in CCR9-deficient mice. Here, we report the expression of CCR9 and CCL25 as early as 6 hours post-OVA challenge in eosinophils and T-lymphocytes. Moreover, in challenged CCR9-deficient mice, cell recruitment was impaired at peribronchial and perivenular levels. OVA-administration in CCR9-deficient mice leads to a less inflammatory cell recruitment, which modifies the expression of IL-10, CCL11, and CCL25 at 24 hours after OVA challenge. In contrast, the secretion of IL-4 and IL-5 was not affected in CCR9-deficient mice compared to WT mice. These results demonstrate for the first time that CCR9 and CCL25 expressions are induced in the early stages of airway inflammation and they have an important role modulating eosinophils and lymphocytes recruitment at the first stages of inflammatory process, suggesting that they might be a potential target to regulate inflammation in asthma.

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Growing evidence demonstrates a strong correlation between obesity and an increased risk of breast cancer, although the mechanisms involved have not been completely elucidated. Some reports have described a crosstalk between adipocytes, cancer cells, and immune cells within the tumor microenvironment, however, it is currently unknown whether obesity can promote tumor growth by inducing systemic alterations of the immune cell homeostasis in peripheral lymphoid organs and adipose tissue. Here, we used the E0771 breast cancer cell line in a mouse model of diet-induced obesity to analyze the immune subpopulations present in the tumors, visceral adipose tissue (VAT), and spleen of lean and obese mice. Our results showed a significant reduction in the frequency of infiltrating CD8+ T cells and a decreased M1/M2 macrophage ratio, indicative of the compromised anti-tumoral immune response reported in obesity. Despite not finding differences in the percentage or numbers of intratumoral Tregs, phenotypic analysis showed that they were enriched in CD39+, PD-1+ and CCR8+ cells, compared to the draining lymph nodes, confirming the highly immunosuppressive profile of infiltrating Tregs reported in established tumors. Analysis of peripheral T lymphocytes showed that tumor development in obese mice was associated to a significant increase in the percentage of peripheral Tregs, which supports the systemic immunosuppressive effect caused by the tumor. Interestingly, evaluation of immune subpopulations in the VAT showed that the characteristic increase in the M1/M2 macrophage ratio reported in obesity, was completely reversed in tumor-bearing mice, resembling the M2-polarized profile found in the microenvironment of the growing tumor. Importantly, VAT Tregs, which are commonly decreased in obese mice, were significantly increased in the presence of breast tumors and displayed significantly higher levels of Foxp3, indicating a regulatory feedback mechanism triggered by tumor growth. Altogether, our results identify a complex reciprocal relationship between adipocytes, immune cells, and the tumor, which may modulate the immune macroenvironment that promotes breast cancer development in obesity.

The study of tumor exosomes has gained relevance in the last decades due to their potential use for therapeutic and diagnostic application. Although there is extensive knowledge of exosome biology, some biological samples like tumor-derived exosomes have been difficult to characterize due to their complexity and heterogeneity. This distinctive feature makes difficult the identification of specific exosome subpopulations with a shared molecular signature that could allow for targeting of exosomes with therapeutic and diagnostic potential use in cancer patients. Nanoscale flow cytometry has lately emerged as an alternative tool that can be adapted to the study of nanoparticles, such as exosomes. However, the physicochemical properties of these particles are an important issue to consider as nanoparticles need the application of specific settings which differ from those used in conventional flow cytometry of cells. Therefore, in the last few years, one of the main aims has been the optimization of technical and experimental protocols to improve exosome analysis. In this chapter, we discuss several aspects of cytometric systems with a special emphasis in technical considerations of samples and equipment.