The roles of IL-36γ and CXCR6 in the immune response to respiratory viral infection Open Access
Wein, Alexander (Summer 2018)
Abstract
The immune response to infection requires coordination between many cell types for effective pathogen clearance. Cytokines and chemokines serve as secreted proteins that recruit, activate, and regulate immune cells and have been described as the language of the immune system. Just as the study of language can give insight into the human experience and interactions, the study of cytokines and chemokines allows for a better understanding of the immune system.
Influenza virus causes seasonal epidemics that kill 50,000 people in the US each year and occasional pandemics that have killed as many as 100 million people. Many complicated cases of influenza result from dysregulation of cytokine production leading to an inappropriate and deleterious immune response. Defining the roles of individual cytokines is key to understanding the sequelae of influenza infection.
IL-36g is an important cytokine in the immune response to influenza. It served to promote survival of alveolar macrophages during influenza infection by skewing the macrophages towards an M1 phenotype. The rapid loss of alveolar macrophages in Il36g-/- mice led to increased viral titers, pathology, morbidity, and mortality and transfer of WT alveolar macrophages rescued the Il36g-/- mice. Thus, IL-36g acts on alveolar macrophages to enable their survival during respiratory viral infection, decreasing viral replication and pathology.
Preexisitng immunity to influenza can prevent infection, lead to asymptomatic infection, or decrease the severity of symptoms. Unlike neutralizing antibodies, resident memory T cells (TRM) in the lung provide protection across strains of influenza and were found to decrease disease severity during the 2009 H1N1 pandemic. While the factors that lead to the generation of lung TRM cells remain unknown, the chemokine receptor CXCR6 serves to recruit antigen-specific T cells to the airways following respiratory viral infection. CXCR6 is expressed by T cells in the lung and its ligand, CXCL16, is expressed by the airway epithelium in mice and humans. Mice lacking CXCR6 or CXCL16 have decreased airway TRM cells after influenza infection and Cxcr6-/- mice have greater morbidity following secondary infection. The data show the importance of CXCR6 for trafficking of T cells to the airways.
Table of Contents
Chapter 1. Cytokines, Chemokines, and Influenza................................................... 1
Cytokines and Chemokines.......................................................................................1
IL-1 family................................................................................................................2
IL-36 Discovery and Characterization......................................................................3
IL-36 Protein Regulation, Structure, and Signaling.................................................6
Effects of IL-36 by Cell Type.................................................................................... 10
IL-36 in Disease.......................................................................................................16
Chemokines.............................................................................................................27
CXCR6 Discovery and Characterization...................................................................28
Discovery and characterization of CXCL16..............................................................32
Structure and non-canonical functions of CXCL16.................................................. 34
Function of CXCR6/CXCL16 in various tissues......................................................... 36
CXCR6/CXCL16 in Disease.........................................................................................40
Influenza virus...........................................................................................................47
Influenza and human health......................................................................................48
Immune response to influenza...................................................................................49
Alveolar macrophages during influenza infection.....................................................52
Lung-resident memory T cells.................................................................................. 55
Cytokine storm..........................................................................................................56
Disease modifying agents in influenza......................................................................57
IL-36 in the lung........................................................................................................ 57
IL-36 in influenza......................................................................................................60
CXCR6/CXCL16 in the lung....................................................................................... 60
Chapter 2. IL-36g protects against severe influenza infection by promoting lung alveolar macrophage survival and limiting viral replication 63
Materials and Methods..............................................................................................66
Results.......................................................................................................................70
Figure 1: RT-PCR of whole lung homogenate or sorted cells for expression of Il36 cytokines. 72
Figure 2: Survival and weight loss of WT and Il36-/- mice following influenza infection. 73
Figure 3: Viral titers, cytokine levels, and pathology of WT and Il36-/- mice following influenza infection. 74
Figure 4: Analysis of innate immune subsets in the lung by flow cytometry. 76
Figure 5: Apoptosis and surface marker expression of WT and Il36-/- mice...... 78
Figure 6: Transfer of WT alveolar macrophages rescues mortality in Il36-/- mice. 80
Discussion..............................................................................................................81
Supplemental Materials......................................................................................... 86
Supplemental Figure 1: Expression of Il36 family members in naïve mice as reported in Figure 1 A-C. 86
Supplemental Figure 2: Gating strategy for innate immune cell subsets........ 86
Supplemental Table 1: SNP analysis of Il36g-/- mice.......................................89
Chapter 3. CXCR6 regulates partitioning of lung-resident memory CD8 T cells between the airways and parenchyma and maintains the airway T cell memory pool..........................................................................................91
Results............................................................................................................94
Figure 7: CXCR6 is upregulated on lung-resident memory CD8 T cells.............. 97
Figure 8: CXCR6 expression on lung-resident memory T cells occurs throughout infection and is antigen-dependent. 99
Figure 9: Mice lacking Cxcr6 have decreased airway-resident cells following influenza infection. 102
Figure 10: Mixed bone-marrow chimeras show a defect in airway recruitment in Cxcr6-/- T cells. 104
Figure 11: CXCL16 is expressed in the lung and is required for T cell recruitment to the airways. 106
Figure 12: OT-I T cells lacking Cxcr6 are located further from the airways than WT cells. 109
Figure 13: CXCR6 expression is downregulated upon entry into the airways. 111
Discussion......................................................................................................111
Supplemental Materials..................................................................................127
Supplemental Figure 3: Gating strategy for human lung T cells..................... 127
Supplemental Figure 4: Fluorescence microscopy of naïve mouse lungs........ 128
Supplemental Figure 5: Method of measuring distance to the nearest airway by fluorescence microscopy. 129
Chapter 4. Discussion, insights, and future directions................................. 130
Function of individual IL-36 members............................................................131
CXCR6 and HIV/AIDS......................................................................................134
Relative roles of CXCR3 and CXCR6 in airway homing of T cells.....................136
Potential for vaccine design...........................................................................139
CXCR6 as a mechanism of “one mucosa”........................................................ 140
Conclusion....................................................................................................... 143
References........................................................................................................144
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