Group 2 innate lymphocytes (ILC2s) are developmentally programmed to generate type 2 cytokines, perhaps creating the opportunity for understanding the origins of dysregulated allergic immune responses accompanied by adaptive Th2 responses. We used a fate-mapping approach to more fully investigate the ontogeny of these cells in the mouse during prenatal, neonatal, and adult life. Using several approaches, we define the neonatal window as a period of vigorous de novo ILC2 generation accompanied by ILC2 activation and proliferation in all tissues of the mouse. ILC2s generated during the neonatal period remain the major constituents of adult ILC2s in the lung and visceral adipose tissue. In contrast, neonatal-labeled ILC2s are progressively replaced by adult-derived ILC2s in skin and small intestine with age, suggesting that different tissue microenvironments influence the survival and/or turnover of ILC2s over the course of adult life. Despite increased ILC2 numbers generated in response to helminthic infection, the proportion of fate-mapped cells does not significantly change, suggesting that proliferation of existing cells rather than generation of new cells is the main mechanism by which ILC2 numbers increase under inflammatory conditions. These data uncover 3 distinct phases of ILC2 ontogeny and reveal unsuspected tissue-specific effects driving the maintenance and turnover of these potentially long-lived tissue resident cells.

In humans, allergic disease bears striking resemblance to the host response to helminth infection. Thus, we studied N. brasiliensis infection in mice to identify factors that initiate host immunity and identified three that bind the environmental polysaccharide chitin. We further demonstrated that purified chitin acutely induces lung inflammation in mice, similar to that observed in human allergic disease, dependent on resident group 2 innate lymphoid cells (ILC2s). Given their number, distribution and function, lung ILC2s likely coordinate responses with resident epithelial cells that monitor barrier interactions with the environment. In this thesis, we demonstrate that alveolar type II (ATII) cells are the main source of interleukin (IL)-33 and thymic stromal lymphopoietin (TSLP) generated in response to chitin or N. brasiliensis challenge. IL-33 and TSLP synergistically activate ILC2s to produce IL-5, IL-13 and IL-9, and autocrine IL-9 promotes an ILC2 transcriptional program associated with rapid and robust cytokine production necessary to optimize epithelial gene responses in the conducting airways. Thus, ILC2s link a specialized, distal, epithelial population involved in alveolar function with more proximal epithelia that regulate airway flow, revealing a key role for ILC2s in tissue homeostasis that might underlie similar organizational hierarchies for innate lymphoid cells in other organs.

In metazoan organisms ranging from simple to the complex, the innate immune system is the first line of defense against damage or infection. Vertebrates have the particular challenge of dealing with parasitism and allergen exposure, to which Type 2 responses are critical at mucosal barrier sites. While there is some overlap between innate and adaptive Type 2 effector cell functions, their unique contributions are unclear. Additionally, the broader role of innate immunity in tissue regulation as it relates to the conserved mechanisms of metabolic homeostasis is unexplored.

We targeted the IL-4/13 locus in mice to generate a Th2-specific model of IL-4/13 deficiency (CD4Cre x IL-4/13 fl/fl). Consistent with prior studies, we verified that CD4 T cell-derived IL-4/13 was not required for the clearance of acute Nippostrongylus brasiliensis helminth infection. However, in response to chronic conditions, such as OVA-induced airway inflammation and Schistosoma mansoni infection, conditional knockout mice had an intermediate phenotype. The conditional CD4Cre x IL-4/13 fl/fl model suggests that while innate IL-4/13 can mediate the clearance of acute helminth infection, they may interact with Th2 cells in chronic inflammation.

As IL-4/13 have been implicated as important for adipose tissue macrophage alternative activation in the lean metabolic state, we identified eosinophils as the major IL-4-producing cells in white adipose tissue. In eosinophil-deficient mice, adipose tissue macrophage alternative activation was impaired but could be restored by reconstituting the eosinophil lineage. Additionally, eosinophil-deficient mice, when maintained on a high fat diet, developed increased adiposity and glucose intolerance. Thus, we find that eosinophils play a role in metabolic regulation by sustaining alternatively activated macrophages that promote the lean phenotype.

Our findings confirm that Type 2 innate effector cells are important for the clearance of acute helminth infection, but may be interacting with Th2 cells during chronic infection and airway inflammation. We have also uncovered a surprising observation that eosinophils, a major feature of Type 2 immune responses, sustain macrophage alternative activation in adipose tissue. This suggests that innate cells have evolved functions not only for immunity, but for also for metabolic homeostasis as part of a highly integrated survival strategy.

Cytokines play a pivotal role in the maintenance of homeostasis and in the coordination of immune responses. Our lab has designed a variety of cytokine reporter mice, which allow for the determination of cellular sources of cytokines directly in vivo. In this dissertation, reporter mice designed to mark cells expressing IL-4, IL-5, IL-13, IFN-gamma, and IL-17A are described.

First, we focused on type 2 immunity, an immune program that is induced in response to allergens and parasitic helminths. We used IL-4 and IL-13 reporter mice to identify and characterize a subset of lineage-negative cells called Ih2 cells that participate in type 2 immune responses in vivo. We demonstrated that Ih2 cells were widely distributed at rest. We further showed that these cells expanded robustly in response to the epithelial cytokines IL-25 or IL-33 and after infection with the helminth Nippostrongylus brasiliensis. Finally, we demonstrated that activation of Ih2 cells with IL-25 is sufficient to mediate worm clearance, even in the absence of adaptive immunity.

Next, we used an IL-17A reporter mouse to identify cells that participate in IL-17-associated immune responses, which are involved in host defense against extracellular bacteria and fungi and also in the development of certain autoimmune disorders. We demonstrated that during Klebsiella pneumoniae infection or experimental autoimmune encephalomyelitis, a substantial proportion of gamma-delta T cells, invariant (i)NKT cells, and other CD4- CD3+ cells and a smaller percentage of CD4+ T cells were marked with the reporter in vivo. Administration of IL-1beta and/or IL-23 elicited rapid expression of the reporter by resident gamma-delta T cells, iNKT cells and other CD4- CD3+ cells in vivo, demonstrating that these innate-like T cells are poised for rapid IL-17A production and likely comprise major sources of this cytokine during infection and autoimmunity.

Taken together, the results of these studies suggest that the cytokines associated with type 2 or IL-17-associated immune responses can be produced by a wide variety of cell types, including innate cells, innate-like T cells, and adaptive immune cells. Coordination of the effector functions of these various cell types is likely critical for the efficacy of these immune programs.

Allergic and parasitic helminth immunity is characterized by infiltration of tissues with IL-4- and IL-13-expressing cells, including Th2 cells, eosinophils and basophils. Additionally, tissue macrophages assume a distinct phenotype, designated alternatively activated macrophages. Relatively little is known regarding the early factors that trigger these host responses, however we do know that these host responses depend on Stat6 signaling downstream of IL-4 and IL-13 receptors.

We identified two mammalian chitinase-like proteins, AMCase and Ym2 as being Stat6-dependent and upregulated during an immune response to the helminth, Nippostrongylus brasiliensis. AMCase is an enzymatically active chitinase that degrades chitin, while Ym2 is a chitinase-like protein that has lost enzymatic activity. In experiments using transgenic mice that overexpressed AMCase or Ym2, as well as antibody blockade experiments we concluded that neither AMCase nor Ym2 were proinflammatory. Thus, we took the approach of exploring the effects of chitin, a carbohydrate ligand of AMCase and Ym2 to ascertain their functions in immunity to chitin-containing pathogens and allergens.

Chitin is the second most abundant polysaccharide in nature, and it confers structural rigidity to fungi, crustaceans, helminths and insects. We found that chitin induced the tissue accumulation of IL-4-expressing innate immune cells, including eosinophils and basophils, when given to mice. Moreover, chitin mediated alternative macrophage activation in vivo and induced leukotriene B4 production, which was required for optimal immune cell recruitment. Importantly, we found that tissue infiltration of effector cells and alternative macrophage activation was abolished when chitin was treated with AMCase, but not Ym2. Therefore, we conclude that chitin is a recognition element for tissue infiltration by innate cells implicated in allergic and helminth immunity and this process is negatively regulated by a vertebrate chitinase.

This work implicates chitin, a common environmental biopolymer and component of aerosolized antigens as an pathogen associated molecular pattern that can trigger early inflammatory responses associated with allergic and parasitic immunity. In our model chitin induces alternative macrophage activation and recruitment of cells that can produce IL-4 and IL-13. IL-4/IL-13 expression in tissues leads to activation of Stat6-dependent genes, including AMCase and Ym2. While the function of Ym2 remains unclear at this time, AMCase appears to act in a negative feedback fashion to degrade chitin and dampen chitin-induced inflammation.