Epithelial regulation of macrophage function within the pulmonary micro-environment

Abstract

The lungs play a major role in gas exchange, yet the continuous and direct exposure of pulmonary epithelium to infections, toxins, and airborne pollutants requires a robust response to maintain homeostasis and prevent extensive damage that could impact tissue function. Lung epithelium protects against infections and injury by functioning as a physical barrier to pathogens and potentially harmful particles. It can also secrete mucus which traps dust, irritants and pathogens and is then cleared to prevent noxious material from damaging tissue; airway mucus has also been found to have antibacterial and antiviral properties. Unregulated acute pulmonary inflammation, including conditions such as acute respiratory distress syndrome (ARDS) and pneumonitis, are associated with epithelial damage and frequently associated with poor patient outcomes and impacts upon morbidity and mortality. Exacerbated inflammation can also lead to chronic conditions such as airway allergy and chronic obstructive pulmonary disease (COPD) and can impact upon pulmonary fibrosis and irreparable tissue damage. Apoptosis is a form of programmed cell death to remove unneeded or damaged cells and tissue and thus is necessary for normal embryonic development, maintenance of tissue homeostasis, ageing, and many diseases. Damage to the epithelium, neighbouring stromal cells and recruited immune cells frequently results in apoptosis. Apoptosis is considered an anti-inflammatory form of cell death, preventing damage to surrounding cells for example by the accumulation of DNA which leads to inflammation and autoimmunity. During apoptosis, intracellular components are dismantled in a controlled manner which is regulated by the activation of selected proteases known as caspases. Hallmark characteristics of apoptosis include nuclear chromatin condensation and significant reduction in cell size. The dying cells release “find me” signals to recruit phagocytes such as the small nucleotides ATP and UTP. The release of these signals is mediated by various channels, for example, pannexin channels of which pannexin 1 (Panx1) is the most extensively studied. Panx1 channels are activated by caspases during apoptosis and Panx1 has been found to regulate epithelial proliferation and efficient epithelial repair post tissue injury as well as regulating inflammation. Following the release of “find me” signals, structural and molecular changes also occur called “eat me” signals that promote engulfment of the apoptotic cells by macrophages. Efferocytosis is the process of quick and efficient engulfment of apoptotic cells by phagocytes, particularly macrophages and is therefore indispensable for the normal functioning of tissue. It is required for organ development, maintaining tissue homeostasis, resolution of inflammation and regeneration following injury. Defects in efferocytosis are linked to various autoimmune diseases, chronic inflammatory conditions and even cancer. Failure to eliminate damaged cells by efferocytosis is undesirable as the dying cells can release pro-inflammatory and immunogenic cell components which are harmful to the tissue microenvironment. The process of efferocytosis involves four stages i) attraction of the macrophage towards the apoptotic cell due to the released “find-me” signals, ii) recognition and tethering of the apoptotic cells by receptors on the macrophages iii) engulfment of apoptotic cells and finally, iv) degradation of apoptotic cells and induction of an anti-inflammatory response by these macrophages. Efferocytosis is one of the major functions of macrophages and engulfment of these apoptotic cells alters macrophage phenotype to be more anti-inflammatory. These anti-inflammatory macrophages secrete anti-inflammatory cytokines, pro-resolving mediators and reduce recruitment of other immune cells. Little is known about the molecular mechanisms and pathways that regulate the communication between epithelial cells and nearby inflammatory cells at homeostasis. Furthermore, pulmonary epithelium is known to produce various signals that influence the inflammatory response after injury, but less is known about the impact of this communication on immune cell phenotype and function. The central hypothesis for this thesis is that pulmonary epithelial cells can regulate macrophage function and phenotype to maintain homeostasis and promote tissue repair after injury. To investigate this hypothesis, we measured: i. the effect of pulmonary epithelium secreted mediators on macrophage phagocytosis of apoptotic cells at homeostasis, ii. changes to pulmonary macrophage phenotype after epithelial injury and iii. the effect of disrupting Panx1 on the development of fibrosis. Epithelial cell supernatant was used to determine the role epithelial cells play in the regulation of macrophage phenotype and phagocytic clearance of apoptotic cells during homeostasis. Human monocyte-derived macrophages were cultured, and an increase in efferocytosis was measured following incubation with lung and colonic epithelial cell supernatant. Further experiments showed the contributing factors in the secretome are likely involved in apoptotic corpse internalisation through cytoskeletal rearrangement. This highlights one communication network between epithelial cells and macrophages and its influence on efferocytosis at homeostasis. In addition, pulmonary macrophages were isolated and phenotypes assessed using single-cell RNA sequencing and cellular indexing of transcriptomes and epitopes sequencing (CITE-Seq) which synchronously quantifies cell surface protein and transcriptomic data in an in vivo model of acute airway epithelial injury induced by naphthalene. Naphthalene leads to early airway epithelial cell death and disruption of normal airway epithelial architecture. Following injury, macrophages are known to be required for epithelial regeneration, but their specific roles are yet to be elucidated. Macrophage phenotype after epithelial injury was altered to promote wound healing, regeneration and engulfment by the upregulation of genes related to growth factors and phagocytosis receptors. Both bleomycin and silica are known to cause epithelial cell death by inducing double strand breaks in the cell’s DNA and epithelial regeneration is modified to lead to fibrosis. As the Panx1 channel has been shown to regulate epithelial repair post tissue injury, Panx1 regulation of fibrogenesis was investigated as its effects on tissue fibrosis have not yet been studied. Lack of Panx1 expression had no effect on immune cell infiltration into the lungs. However, Panx1 knockout mice had exacerbated lung fibrosis after the initial inflammatory phase and ongoing experiments are underway to understand the molecular mechanism by which Panx1 regulates tissue fibrosis. The data in this thesis demonstrates epithelial cell regulation of macrophage function (efferocytosis) and phenotype (transcriptome) both at homeostasis and following epithelial injury to promote a pro-reparative environment