The Mechanisms Of Chemokines CCL6/15/23 And CCR1 In Regulating Allergic Airway Inflammation

Bronchial asthma(abbreviated as asthma)is one of the most common chronic diseases in the world.There are about 400 million asthma patients in the world,accounting for 1-18% of the total population in different countries.The incidence of asthma has been rising.More than half of asthma in patients are allergen-induced type 2 immune responses dominated by type 2 helper T cells(TH2)cells,which are characterized as recurrent wheeze,shortness of breath,chest tightness,and coughing,together with variable expiratory airflow limitation.One of the predictive risk factors and biomarkers for acute asthma attacks is blood or sputum eosinophilia.Eosinophils are differentiated from hematopoietic stem cells(HSC)and mature in the bone marrow.They are recruited to the site of airway inflammation during allergic responses and release a series of cytokines,chemokines,and degranulated proteins to be involved in the pathogenesis of airway inflammation.Directly targeting the differentiation of eosinophils is an effective treatment strategy to control the clinical symptoms and reduce the severity of asthma.Chemokines are a class of small molecule secreted proteins.After binding to G protein-coupled receptors(GPCRs),they are widely involved in various immune responses such as immune cell interaction and migration.The important chemokines known in asthma are including eosinophil chemokine CCL11(also known as Eotaxin-1)and its receptor CCR3,etc.The clinical trials about these chemokines have not achieved the expected effect.Continue to explore the key regulatory mechanism of chemokines in allergic asthma inflammation will provide more evidence and basis for the targeted therapy of asthma.Mouse CCL6(also known as C10),human CCL15(also known as MIP-5,MIP-1δ),and CCL23(also known as MPIF-1)are chemokines belonging to the same subfamily.These are reported to play an important role in a variety of inflammatory diseases.,including pulmonary fibrosis,allergic bronchopulmonary aspergillosis,sepsis,and demyelinating disease.Mouse CCL6 may be the same as human CCL15 and CCL23 that can activate CCR1,but it has not been confirmed by experiments.A recent study has found that mouse eosinophils disrupt the HSC homeostasis by secreting CCL6 in allergic inflammation.But the function and mechanism of m CCL6 in allergic asthma are still unclear.The role of h CCL15 and h CCL23 in asthma patients remains to be further explored.Objectives To determine the expression of h CCL15,h CCL23,and m CCL6 and their correlation with asthma.To investigate the role of the m CCL6 in allergic asthma pathogenesis.To clarify the interaction between m CCL6 and the receptor CCR1,and the effect of blocking CCR1 on asthma inflammation for providing a new therapeutic target for asthma prevention and treatment.Methods 1.To detect the expression of h CCL15,h CCL23,and m CCL6 in asthma patients and asthma model mice.To confirm the secretion sources by using flow cytometry,immunofluorescence.2.To establish the Ccl6 gene knockout(Ccl6-/-)mice,and establish OVA-sensitized and challenge-induced asthma models.To analyze the changes of eosinophilia,airway inflammation,TH2 immune response,and HSC hematopoietic function in OVA-induced Ccl6-/-mice by using immunohistochemistry,immunofluorescence,flow cytometry.3.To verify whether CCR1 is the receptor of m CCL6 by using the Glo Sensor experiment.Making a series of N-terminal truncation of CCR1 to detect the change in the efficiency of activation by m CCL6.Use Western blot to analyze the level of phosphorylated protein in the downstream MAPK signaling pathway after CCR1 was activated.To detect the expression of CCR1 on the membrane of bone marrow stem and progenitor cells in the mouse OVA model,or after m CCL6 treatment in vitro by using flow cytometry.4.To evaluate the effect of CCR1 on eosinophil differentiation by using CCR1 inhibitor BX471 and Ccr1 knockout(Ccr1-/-)mice in bone marrow eosinophil differentiation(BMDE)experiments in vitro.After using bone marrow cells from WT and Ccr1-/-mice for total or competitive bone marrow transplantation,an OVA-induced asthma model was then established to analyze the changes in the bone marrow differentiation lineage of eosinophils.BX471 was administered in an asthma model for assessing the therapeutic effect of CCR1 inhibitor on airway inflammation.Results 1.The h CCL15 and h CCL23 are highly expressed in the plasma of asthma patients and are mainly secreted by eosinophils.In the mouse asthma model,m CCL6 in the bronchial alveolar lavage fluid(BALF)supernatant,lung tissue,and peripheral blood is elevated,and it also mainly comes from eosinophils.In the OVA-induced asthma model,the level of m CCL6 in eosinophils is increased.IL-5 can promote the expression of m CCL6 in eosinophils through the MAPK pathway.2.There is no physiological defect in Ccl6-/-mice except m CCL6 deletion.m CCL6 deletion significantly reduces OVA-induced eosinophilia,airway inflammation,mucus hypersecretion,and TH2 immune response in asthmatic mice.The OVA-induced bone marrow eosinophil differentiation is inhibited in Ccl6-/-mice.On the other hand,the absence of m CCL6 can also decrease the recruitment of eosinophils to airway inflammation in the pulmonary alveoli.3.m CCL6 can directly activate CCR1 which depends on the N-terminal structure of CCR1.After CCR1 is activated by m CCL6,the downstream signaling G protein pathway and β-arrestin pathway are also activated,which is manifested by increased phosphorylation of ERK1/2 and p38 proteins and internalization of CCR1 membrane protein.CCR1 is also activated on bone marrow stem progenitor cells in the OVA model in vivo,and on stem progenitor cells and eosinophils that interfered with m CCL6 in vitro.4.The CCR1 inhibitor BX471 significantly reduces the differentiation of eosinophils in vitro,and inhibits the OVA-induced pulmonary eosinophilia in vivo,and relieves airway inflammation.After bone marrow transplantation in vivo,in the OVA-induced inflammatory environment,the ability of stem progenitor cells lacking CCR1 to differentiate into eosinophils is significantly reduced compared with normal stem progenitor cells.Conclusions Eosinophil-derived m CCL6 and its orthologs h CCL23 and h CCL15 are increased in allergic mice and asthma patients,which interacts with receptor CCR1 to promote eosinophil differentiation in the OVA-induced allergic inflammation,constituting a feedforward loop of asthma exacerbation.It provides a new mechanism for eosinophils to participate in the regulation of allergic inflammation,revealing that blocking the m CCL6-CCR1 signal axis can be a potential therapeutic strategy for the treatment of asthma.