Experiencia específica en el municipio de Arauca

Inflammatory bowel disease (IBD) is a chronic, relapsing inflammation of the intestinal mucosa that results in impaired epithelial barrier integrity and a failure to regulate mucosal wound healing (Hisamatsu et al., 2013; Kaser et al., 2010; Krishnan et al., 2011; Neurath, 2014; Strober et al., 2007). There are multiple genetic and inducible models of intestinal injury that are designed to mimic some of the features of human IBD (Melgar et al., 2008; te Velde et al., 2007; Wirtz et al., 2007), although no model can completely recapitulate the complexity of this multi-factorial human disease. Studies in this thesis will utilize the dextran sodium sulfate (DSS)-induced model of acute intestinal damage and inflammation. Oral administration of DSS in the drinking water is toxic to epithelial cells at the base of the colon crypts, leading to barrier leakage and subsequent exposure to commensal bacteria that results in epithelial damage and increased expression of pro-inflammatory cytokines that activate immune cell populations (Melgar et al., 2008; Perse and Cerar, 2012; te Velde et al., 2007). Symptoms of DSS-induced intestinal injury include lethargy, weight loss and bloody stool that can eventually result in death. Depending upon the dose, molecular weight and duration of DSS administration, the mice can develop acute or chronic inflammation with varying degrees of mucosal lesions and immune infiltrate, making DSS an attractive model to examine multiple stages of disease severity (Melgar et al., 2005; Melgar et al., 2008; te Velde et

al., 2007; Wirtz et al., 2007). Tissue injury specifically during acute DSS exposure, as employed in Chapter 4, is characterized by goblet cell depletion with accompanying loss of mucins, epithelial degeneration and necrosis, loss of crypt architecture and extensive immune cell infiltration consisting primarily of neutrophilia (Melgar et al., 2005; Melgar et al., 2008; te Velde et al., 2007; Wirtz et al., 2007). Importantly, unlike other intestinal injury models such as T cell transfers into immuno-deficient mice, the progression of DSS-induced intestinal inflammation does not require the adaptive immune response (Perse and Cerar, 2012).

Notably, expression of IL-33 and its receptor ST2 has been shown to be dysregulated in patients diagnosed with IBD (Kobori et al., 2010; Lopetuso et al., 2012; Pastorelli et al., 2011; Pastorelli et al., 2010). However, whether IL-33 plays a tissue- protective or pathologic role in the colonic intestinal mucosa during disease is controversial (Duan et al., 2012; Garcia-Miguel et al., 2013; Grobeta et al., 2012; Imaeda et al., 2011; Lopetuso et al., 2012; Pastorelli et al., 2011; Pushparaj et al., 2013; Sedhom et al., 2013) and the mechanisms which act downstream of IL-33 to regulate disease severity are poorly understood. Additionally, the etiology of IBD has been linked to multiple growth factor pathways, including EGFR-associated family members (Hisamatsu et al., 2013; Lu et al., 2014; Neurath, 2014; Sipos et al., 2010; Yan et al., 2011). Collectively, these attributes make DSS a useful model to assess the contribution of ILC2s to intestinal inflammation and tissue repair. In Chapter 4 of this thesis, I will employ oral administration of 3% DSS to induce acute intestinal injury over a one week period and assess the role of the IL-33-ILC2-AREG pathway in regulating intestinal inflammation and repair of the barrier.

1.7 Outline of the thesis

The focus of this thesis will be to explore how populations of innate lymphoid cells orchestrate restoration of tissue homeostasis at epithelial barrier surfaces of the lung and intestine in response to acute tissue damage. Chapter 2 will characterize the population of ILCs constitutively present in the respiratory tract of humans and mice and examine whether lung ILC2s can influence airway epithelial barrier integrity and tissue homeostasis following influenza virus-induced pulmonary damage. The mechanism of ILC-mediated tissue repair will be examined in Chapter 3 through interrogation of the role of the AREG-EGFR growth factor pathway in host recovery from influenza virus infection. Lastly, data presented in Chapter 4 will explore the relevance of this pathway in sites outside the lung and uncover an IL-33-dependent innate immune mechanism of intestinal tissue repair dependent on AREG-EGFR interactions. Collectively, the data presented in this thesis reveal a conserved innate mechanism of barrier repair by which the mammalian immune system responds to damage cues from the local microenvironment to promote restoration of tissue homeostasis.

Figure 1: The type 2 immune response at barrier surfaces

Figure 1: The type 2 immune response at barrier surfaces

At mucosal surfaces, a single layer of epithelial cells serves as a critical barrier between the host immune system and outside envrionmental stimuli. Upon exposure to pathogens or allergens, epithelial cells release cytokine signals such as Interleukin (IL)- 25, IL-33 and thymic stromal lymphopoietin (TSLP) to elicit and activate the innate and adaptive immune cells of the type 2 inflammatory response. While activation of these cells is essential for host protective immunity and tissue repair to restore homeostasis, dysregulation of the type 2 immune response can result in development of pathologic chronic inflammation and tissue fibrosis.

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Figure 2: The ILC family

Figure 2: The ILC family

The innate lymphoid cell (ILC) family is composed of three main groups that all derive from Id2+ ILC precursors. Group 1 ILCs (orange) require T-bet, IL-7 and IL-15 for development and respond to IL-12 to produce IFNγ. Group 2 ILCs (green) are comprised of Nuocytes (Nuo), Natural Helper (NH) cells, Innate Type 2 Helper (Ih2) cells and lung- resident ILCs that express GATA3 and depend upon RORα, TCF-1 and Gfi1 for development. Upon IL-25, IL-33 and/or TSLP stimulation, Group 2 ILCs can produce IL- 5, IL-13, IL-9 and amphiregulin (Areg). Group 3 ILCs (blue) consist of a heterogeneous population of RORγt-dependent ILCs that respond to IL-23, IL-1β and AhR ligands to express IL-22 and/or IL-17A.

Group 2 (ILC2) Group 3 (ILC3) Group 1 (ILC1) NH ILC17 Nuo LTI- like Ih2 ILC22 NCR- 22 Lung ILC IL-17A IL-22 IL-5 IL-13 IL-9 Areg IFN!" Id2+ _ILC Precursor(s) IL-7

IL-15 IL-2 IL-7

IL-25 IL-33 IL-12 IL-2 IL-7 IL-23 IL-1#" T-bet ROR$" Tcf-1 Gfi1 ROR!t Tcf-1 AhR ligands TSLP ILC1 PLZF GATA3 Nfil3

Figure 3: ILC2s regulate immunity, inflammation and tissue homeostasis in the lung

Figure 3: ILC2s regulate immunity, inflammation and tissue homeostasis in the lung

Contact with allergens, viruses or parasitic helminth worms causes inflammation of the airway epithelium, resulting in production of IL-33, IL-25 and/or TSLP (red arrow) that activates Group 2 ILCs to express a variety of effector cytokines that can drive either pathologic airway hyper-reactivity (AHR) responses (blue arrow) or can promote

IL-5 IL-13 IL-9 Areg ILC Eosinophil recruitment IL-33 IL-25 TSLP

Airway Inflammation Airway Repair

Airway Hyper-reactivity Epithelial cell & goblet cell proliferation AAMac + IL-4 Th2 helminth Influenza allergen CD4 !"#

beneficial tissue-protective responses to repair the damaged epithelium (green arrow). IL-5-mediated recruitment of eosinophils and IL-13-mediated epithelial cell/goblet cell proliferation can coordinately drive AHR responses. IL-9 acts in an autocrine manner to promote optimal IL-5/IL-13 expression in ILC2s. ILC2-derived IL-13 also influences differentiation of alternatively-activated macrophages and polarization of Th2 CD4 T cells (via DCs). Additionally, data presented in Chapters 2 and 3 of this thesis demonstrate that ILC2 expression of amphiregulin (Areg) can promote epithelial cell proliferation to repair injured airway epithelia that has been damaged by virus infection (green arrow).