Respirovirus such as influenza virus infection induces pulmonary anti-viral immune response, orchestration of innate and adaptive immunity restrain viral infection, otherwise causes severe diseases such as pneumonia. Chemokines regulate leukocyte recruitment to the inflammation site. One chemokine CXCL5, plays a scavenging role to regulate pulmonary host defense against bacterial infection, but its role in pulmonary influenza virus infection is underdetermined. Here, using an influenza (H1N1) infected CXCL5-/- mouse model, we found that CXCL5 not only responds to neutrophil infiltration into infected lungs at the innate immunity stage, but also affects B lymphocyte accumulation in the lungs by regulating the expression of the B cell chemokine CXCL13. Inhibition of CXCL5-CXCR2 axis markedly induces CXCL13 expression in CD64+CD44hiCD274hi macrophages/monocytes in infected lungs, and in vitro administration of CXCL5 to CD64+ alveolar macrophages suppresses CXCL13 expression via the CXCL5-CXCR2 axis upon influenza challenge. CXCL5 deficiency leads to increased B lymphocyte accumulation in infected lungs, contributing to an enhanced B cell immune response and facilitating induced bronchus-associated lymphoid tissue formation in the infected lungs during the late infection and recovery stages. These data highlight multiple regulatory roles of CXCL5 in leukocyte chemotaxis during pulmonary influenza infection.
Keywords: CXCL5, neutrophil, B lymphocyte, CXCL13, influenza, pulmonary infection
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Introduction
Respirovirus infection is one of the main causes of respiratory diseases worldwide. Every year, the influenza epidemic causes illness in millions of patients, and one in ten patients dies. Influenza virus enter the host through the upper respiratory tract, and directly infect airway epithelial cells, alveolar epithelial cells, and immune cells. Virus infection triggers cellular immune pathways to express abundant inflammatory cytokines and chemokines, including TNF-α, IL-1α/β, IL-2, IL-4, IL-6, IFN-α/β, CXCL-8 family members, CXCL-9/10, and MIP-1/2, in the respiratory tract and alveolar spaces (1, 2). These cytokines, in turn, recruit innate immune cells such as macrophages, granulocytes, dendritic cells (DCs), and natural killer (NK) cells into the infected lungs to exert anti-viral innate immune responses. Following infection, antigen-presenting cells (APCs) travel to the lung-draining lymph nodes and activate adaptive immune T cells and B cells. Activated lymphocytes then migrate to the infected lungs to exert their antiviral cellular immune and humoral immune effects, and ultimately clear viral infection and provide long-term protection (3). Fine-tuned pulmonary antiviral innate and adaptive immunity is critical for elimination of viral infection and control of lung inflammation; otherwise severe conditions such as pneumonia can develop.
Glutamic acid+leucine+arginine (ELR+) CXC chemokines are neutrophil chemoattractants. Seven members of this class of chemokines (IL-8 family) have been identified to date in humans, including IL-8 (CXCL8), CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, and CXCL7 (4). No human CXCL8 homologs have been identified in rodents. However, CXCL1, CXCL2, CXCL3, CXCL5, and CXCL7 are important neutrophil chemoattractants in mice (5). Receptors for the IL-8 family include CXCR1, CXCR2, and Duffy Antigen Receptor for Chemokines (DARC); CXCR2 and DARC bind almost all the IL-8 family members (5, 6). CXCR1 and CXCR2 are members of the G protein-coupled receptor family that contain 7 transmembrane domains, and their expression can be detected on most leukocytes (at high levels on polymorphonuclear leukocytes) and structural cells, such as epitehlial and endothelial cells. Signals triggered via CXCR2 activate G protein and subsequent kinases to induce distinct biological outcomes that are often correlated with leukocyte migration and inflammation (7, 8). In contrast, DARC, which is highly expressed on the surfaces of erythrocytes and endothelial cells, lacks the intracellular motif to enable G protein coupling and is unable to transmit chemokine-mediated intracellular signals. Thus, DARC is considered a chemokine sink or a chemokine reservoir (9).
CXCL5, implicated in a variety of inflammatory diseases and tumorigenesis (10–12), is produced by lung resident cells, such as lung epithelial cells, and platelets during lung inflammation (13). A previous study by Mei et al. uncovered a critical role of CXCL5 in the control of chemokine scavenging and innate immunity against severe bacterial pneumonia (14). CXCL5 enhances the concentrations of other neutrophil chemokines in blood, in part by competing for binding to scavenger molecules, such as red cell DARC and heparan sulfate proteoglycans. During severe inflammation, high CXCL1 and CXCL2 plasma concentrations relative to those at the site of inflammation impair an effective chemokine gradient and desensitize CXCR2. Thus, CXCL5 negatively regulates neutrophil influx to the lung during severe Escherichia coli (E. coli) pneumonia by influencing the concentrations of other chemokines in the blood (14, 15). In addition to its prominent role in regulating innate immune cell trafficking in pulmonary bacterial infection models, how is CXCL5 function in respirovirus infection, such as influenza virus infection, which requires an intricate network of innate and adaptive immune mechanism. In this study, we generated an influenza (H1N1) infected CXCL5-knockout (CXCL5-/-) mouse model to elucidate the potential role of CXCL5 in viral infection-induced inflammatory pulmonary disease.