Spatial and temporal regulation of cytokine expression in Type 2 immune responses

<p>Type 2 immune responses are generated to provide protection against parasitic helminth infections, however these responses also cause the pathologies associated with allergic inflammation. Studies of the cell types and signalling pathways that mediate Type 2 immune responses have been previously undertaken with the goals of efficient development of vaccines against helminths, and identification of pathways that can be inhibited to decrease the damage caused by allergic inflammation. The cytokines interleukin-4 (IL-4) and interleukin-13 (IL-13) mediate many of the downstream effector functions of the Type 2 immune response. To study the mechanisms that control expression of these two cytokines I have used a novel dual cytokine IL-4 and IL-13 transgenic reporter mouse. Utilising this tool along with other IL-4 reporter mice I have discovered that the amount of T cell receptor (TCR) signalling modulates the allelic expression of IL-4 by CD4⁺ T cells. The transgenic IL-4 reporter mouse has for the first time allowed independent measurement of the effects of IL-4 deficiency on the expression of IL-4 in vivo. Using this system I have found that IL- 4 is not required for the in vivo generation or expansion of IL-4 producing CD4⁺ T cells. Th2 differentiated CD4⁺ T cells also expresses IL-13, however the dual reporter mice have demonstrated that IL-13 is expressed consistently later than IL-4 in vitro, and IL-13 requires constant, or multiple exposures to TCR stimulus for expression to be induced. IL-13 expression is absent from lymph node CD4⁺ T cells during exposure to allergens or helminth infection. Sequestration of CD4⁺ T cells in the lymph node does not impact the number of IL-13 expressing CD4⁺ T cells in the lung during a helminth infection, indicating that adaptive immune cell derived IL-13 may be entirely produced by lung resident cells not requiring transit through the lymph node. I have characterised a population of innate lymphoid cells (ILCs) within the skin and found that the proportion of these cells that constitutively express IL-13 decreases with age. These cells did not drastically change in numbers or IL-13 responses in a range of inflammatory conditions including a model of atopic dermatitis. Basophils were found to respond to the atopic dermatitis model by migrating specifically to the treated skin site and draining lymph node, and producing IL-4 in a thymic stromal lymphopoietin dependant manner. Treatment with exogenous cytokines induced IL-13 expression from group 2 ILCs (ILC2s) in the lung and these cells promoted protective immune responses against Nippostrongylus brasiliensis infection. The immune response generated during a primary infection by Nippostrongylus brasiliensis provides protection from re-infection. Long-term protection is dependent on CD4⁺ T cells but when sufficiently stimulated by cytokine, ILC2s can rescue the protection lost by the depletion of CD4⁺ T cells. This thesis has shown that CD4⁺ T cells and populations of innate immune cells differentially regulate the expression of the closely related Type 2 cytokines IL-4 and IL- 13. These discoveries will help direct future research aiming to boost the effectiveness of anti-helminth vaccines, or decrease the pathology caused by allergic diseases by targeting specific cytokine expression.</p>

Intestinal epithelial tuft cell induction is negated by a murine helminth and its secreted products

Helminth parasites are adept manipulators of the immune system, using multiple strategies to evade the host type 2 response. In the intestinal niche, the epithelium is crucial for initiating type 2 immunity via tuft cells, which together with goblet cells expand dramatically in response to the type 2 cytokines IL-4 and IL-13. However, it is not known whether helminths modulate these epithelial cell populations. In vitro, using small intestinal organoids, we found that excretory/secretory products (HpES) from Heligmosomoides polygyrus blocked the effects of IL-4/13, inhibiting tuft and goblet cell gene expression and expansion, and inducing spheroid growth characteristic of fetal epithelium and homeostatic repair. Similar outcomes were seen in organoids exposed to parasite larvae. In vivo, H. polygyrus infection inhibited tuft cell responses to heterologous Nippostrongylus brasiliensis infection or succinate, and HpES also reduced succinate-stimulated tuft cell expansion. Our results demonstrate that helminth parasites reshape their intestinal environment in a novel strategy for undermining the host protective response.

Spatial and temporal regulation of cytokine expression in Type 2 immune responses

<p>Type 2 immune responses are generated to provide protection against parasitic helminth infections, however these responses also cause the pathologies associated with allergic inflammation. Studies of the cell types and signalling pathways that mediate Type 2 immune responses have been previously undertaken with the goals of efficient development of vaccines against helminths, and identification of pathways that can be inhibited to decrease the damage caused by allergic inflammation. The cytokines interleukin-4 (IL-4) and interleukin-13 (IL-13) mediate many of the downstream effector functions of the Type 2 immune response. To study the mechanisms that control expression of these two cytokines I have used a novel dual cytokine IL-4 and IL-13 transgenic reporter mouse. Utilising this tool along with other IL-4 reporter mice I have discovered that the amount of T cell receptor (TCR) signalling modulates the allelic expression of IL-4 by CD4⁺ T cells. The transgenic IL-4 reporter mouse has for the first time allowed independent measurement of the effects of IL-4 deficiency on the expression of IL-4 in vivo. Using this system I have found that IL- 4 is not required for the in vivo generation or expansion of IL-4 producing CD4⁺ T cells. Th2 differentiated CD4⁺ T cells also expresses IL-13, however the dual reporter mice have demonstrated that IL-13 is expressed consistently later than IL-4 in vitro, and IL-13 requires constant, or multiple exposures to TCR stimulus for expression to be induced. IL-13 expression is absent from lymph node CD4⁺ T cells during exposure to allergens or helminth infection. Sequestration of CD4⁺ T cells in the lymph node does not impact the number of IL-13 expressing CD4⁺ T cells in the lung during a helminth infection, indicating that adaptive immune cell derived IL-13 may be entirely produced by lung resident cells not requiring transit through the lymph node. I have characterised a population of innate lymphoid cells (ILCs) within the skin and found that the proportion of these cells that constitutively express IL-13 decreases with age. These cells did not drastically change in numbers or IL-13 responses in a range of inflammatory conditions including a model of atopic dermatitis. Basophils were found to respond to the atopic dermatitis model by migrating specifically to the treated skin site and draining lymph node, and producing IL-4 in a thymic stromal lymphopoietin dependant manner. Treatment with exogenous cytokines induced IL-13 expression from group 2 ILCs (ILC2s) in the lung and these cells promoted protective immune responses against Nippostrongylus brasiliensis infection. The immune response generated during a primary infection by Nippostrongylus brasiliensis provides protection from re-infection. Long-term protection is dependent on CD4⁺ T cells but when sufficiently stimulated by cytokine, ILC2s can rescue the protection lost by the depletion of CD4⁺ T cells. This thesis has shown that CD4⁺ T cells and populations of innate immune cells differentially regulate the expression of the closely related Type 2 cytokines IL-4 and IL- 13. These discoveries will help direct future research aiming to boost the effectiveness of anti-helminth vaccines, or decrease the pathology caused by allergic diseases by targeting specific cytokine expression.</p>

Mining Anti-Inflammation Molecules From Nippostrongylus brasiliensis-Derived Products Through the Metabolomics Approach

Hookworm is one type of soil-transmitted helminth, which could exert an anti-inflammatory effect in human or animal host, which provides a beneficial possibility for the discovery of inflammatory-related disease interventions. The identification of hookworm-derived anti-inflammatory molecules is urgently needed for future translational research. The emergence of metabolomics has become a powerful approach to comprehensively characterize metabolic alterations in recent times. Herein, excretory and secretory products (ESPs) were collected from cultured adult worm, while small intestinal contents were obtained from Nippostrongylus brasiliensis (N. brasiliensis, Nb)-infected mice. Through ultra-high-performance liquid chromatography coupled with mass spectrometry (UHPLC-MS) platform, metabolomics analysis was used to explore the identification of anti-inflammatory molecules. Out of 45 differential metabolites that were discovered from ESPs, 10 of them showed potential anti-inflammatory properties, which could be subclassed into amino acids, furanocoumarins, linear diarylheptanoids, gamma butyrolactones, and alpha-keto acids. In terms of intestinal contents that were derived from N. brasiliensis-infected mice, 14 out of 301 differential metabolites were discovered to demonstrate anti-inflammatory effects, with possible subclassification into amino acids, benzylisoquinolines, quaternary ammonium salts, pyrimidines, pregnane steroids, purines, biphenyls, and glycerophosphocholines. Furthermore, nine of the differential metabolites appeared both in ESPs and infected intestinal contents, wherein four were proven to show anti-inflammation properties, namely, L-glutamine, glutamine (Gln), pyruvate, and alanine-Gln (Ala-Gln). In summary, we have provided a method for the identification and analysis of parasite-derived molecules with potential anti-inflammatory properties in the present study. This array of anti-inflammatory metabolites could provide clues for future evaluation and translational study of these anti-inflammatory molecules.

Transcriptional Analysis of TH2 Primed Dendritic Cells and T cells

<p><b>Allergy is a condition affecting between 10 and 30% of the world’s population, with incidence rising every year. It is primarily mediated by THelper (TH) 0 cells reacting to an ordinarily harmless environmental antigen to induce an adaptive TH2 response. TH0 cells are presented the antigen by dendritic cells (DC), the immune systems most proficient antigen presenting cell, which act as the bridge between the innate and adaptive immune system. Dendritic cells specific to this study termed Triple Negative (TN) and CD11b+ are able to prime T cells to become TH2 cells, but current research has been unable to fully determine the proteins that mediate this TH2 priming. TN and CD11b+ DC exhibit transcriptional and functional distinction within the TH2 response, but the individual functions they take on during TH2 responses have not fully been determined. Some evidence suggests that the cell surface protein OX40L and the secreted protein TSLP are capable of inducing TH2 priming, but this is not conserved across all TH2 models. In an effort to determine other specific proteins that induce TH2 priming, RNA-sequencing has been utilized on TN and CD11b+ dendritic cells in TH2 inducing conditions. This thesis aims to analyse RNA-sequencing data generated from purified TH2 antigen positive TN and CD11b+ dendritic cells that have taken up a TH2-inducing stimulus – fluorescently labelled (AF488) non-viable Nippostrongylus brasiliensis. Due to the majority of DC-TH0 interactions occurring at the cell surface interface, the bioinformatic analysis was focused on genes belonging to the surface and secreted compartments.</b></p> <p>Here I show that AF488-Nippostrongylus brasiliensis positive TN and CD11b+ DC are transcriptionally distinct from each other. Functional roles of differentially expressed genes (DEG) were also markedly distinct. Superfamily analysis revealed TN genes associated with signal transduction and proteases, whereas CD11b+ DEG were linked to cell adhesion and immune responses. This suggests that the different DC subsets have different roles in an immune response, and potentially different roles in the induction of TH2 immune responses. Network analysis of DEG from DC subsets and proteins expressed by TH0 and TH2 cell surfaces identified over 300 predicted interactions. Notably, 33 identified were known interactions – validating the bioinformatic methods used. Finally, I have been developing a method to assess novel interactions via flow cytometry methods that allows detection of binding and identification of the cell population that is bound. This has shown promise with the detection of generated proteins bound to TN and CD11b+ DC during TH2 stimulating conditions, paving the way for future novel interaction analyses.</p>

Transcriptional Analysis of TH2 Primed Dendritic Cells and T cells

<p><b>Allergy is a condition affecting between 10 and 30% of the world’s population, with incidence rising every year. It is primarily mediated by THelper (TH) 0 cells reacting to an ordinarily harmless environmental antigen to induce an adaptive TH2 response. TH0 cells are presented the antigen by dendritic cells (DC), the immune systems most proficient antigen presenting cell, which act as the bridge between the innate and adaptive immune system. Dendritic cells specific to this study termed Triple Negative (TN) and CD11b+ are able to prime T cells to become TH2 cells, but current research has been unable to fully determine the proteins that mediate this TH2 priming. TN and CD11b+ DC exhibit transcriptional and functional distinction within the TH2 response, but the individual functions they take on during TH2 responses have not fully been determined. Some evidence suggests that the cell surface protein OX40L and the secreted protein TSLP are capable of inducing TH2 priming, but this is not conserved across all TH2 models. In an effort to determine other specific proteins that induce TH2 priming, RNA-sequencing has been utilized on TN and CD11b+ dendritic cells in TH2 inducing conditions. This thesis aims to analyse RNA-sequencing data generated from purified TH2 antigen positive TN and CD11b+ dendritic cells that have taken up a TH2-inducing stimulus – fluorescently labelled (AF488) non-viable Nippostrongylus brasiliensis. Due to the majority of DC-TH0 interactions occurring at the cell surface interface, the bioinformatic analysis was focused on genes belonging to the surface and secreted compartments.</b></p> <p>Here I show that AF488-Nippostrongylus brasiliensis positive TN and CD11b+ DC are transcriptionally distinct from each other. Functional roles of differentially expressed genes (DEG) were also markedly distinct. Superfamily analysis revealed TN genes associated with signal transduction and proteases, whereas CD11b+ DEG were linked to cell adhesion and immune responses. This suggests that the different DC subsets have different roles in an immune response, and potentially different roles in the induction of TH2 immune responses. Network analysis of DEG from DC subsets and proteins expressed by TH0 and TH2 cell surfaces identified over 300 predicted interactions. Notably, 33 identified were known interactions – validating the bioinformatic methods used. Finally, I have been developing a method to assess novel interactions via flow cytometry methods that allows detection of binding and identification of the cell population that is bound. This has shown promise with the detection of generated proteins bound to TN and CD11b+ DC during TH2 stimulating conditions, paving the way for future novel interaction analyses.</p>

Loss of protein tyrosine phosphatase non-receptor type 2 reduces IL-4-driven alternative macrophage activation

Macrophages are a heterogeneous population of innate immune cells that are often divided into two major subsets: classically activated, typically pro-inflammatory (M1) macrophages that mediate host defense, and alternatively activated, tolerance-inducing (M2) macrophages that exert homeostatic and tissue-regenerative functions. Disturbed macrophage function/differentiation results either in inadequate, excessive immune activation or in a failure to induce efficient protective immune responses against pathogens. Loss-of-function variants in protein tyrosine phosphatase non-receptor type 2 (PTPN2) are associated with chronic inflammatory disorders, but the effect of macrophage-intrinsic PTPN2 loss is still poorly understood. Here we report that PTPN2-deficient macrophages fail to acquire an alternatively activated/M2 phenotype. This was the consequence of reduced IL-6 receptor expression and a failure to induce IL-4 receptor in response to IL-6, resulting in an inability to respond to the key M2-inducing cytokine IL-4. Ultimately, failure to adequately respond to IL-6 and IL-4 resulted in increased levels of M1 macrophage marker expression in vitro and exacerbated lung inflammation upon infection with Nippostrongylus brasiliensis in vivo. These results demonstrate that PTPN2 loss interferes with the ability of macrophages to adequately respond to inflammatory stimuli and might explain the increased susceptibility of PTPN2 loss-of-function carriers to developing inflammatory diseases.

IL-4 and helminth infection downregulate MINCLE-dependent macrophage response to mycobacteria and Th17 adjuvanticity

The myeloid C-type lectin receptor (CLR) MINCLE senses the mycobacterial cell wall component trehalose-6,6-dimycolate (TDM). Recently, we found that IL-4 down-regulates MINCLE expression in macrophages. IL-4 is a hallmark cytokine in helminth infections, which appear to increase the risk for mycobacterial infection and active tuberculosis. Here, we investigated functional consequences of IL-4 and helminth infection on MINCLE-driven macrophage activation and Th1/Th17 adjuvanticity. IL-4 inhibited MINCLE and cytokine induction after macrophage infection with Mycobacterium bovis Bacille Calmette-Guerin (BCG). Infection of mice with BCG upregulated MINCLE on myeloid cells, which was inhibited by IL-4 plasmid injection and by infection with the nematode Nippostrongylus brasiliensis in monocytes. To determine the impact of helminth infection on MINCLE dependent immune responses, we vaccinated mice with a recombinant protein together with the MINCLE ligand Trehalose-6,6-dibehenate (TDB) as adjuvant. Concurrent infection with N. brasiliensis or with Schistosoma mansoni promoted T cell-derived IL-4 production and suppressed Th1/Th17 differentiation in the spleen. In contrast, helminth infection did not reduce Th1/Th17 induction by TDB in draining peripheral lymph nodes, where IL-4 levels were unaltered. Upon use of the TLR4-dependent adjuvant G3D6A, N. brasiliensis infection impaired selectively the induction of splenic antigen-specific Th1 but not of Th17 cells. Thus, helminth infection attenuated the Th17 response to MINCLE-dependent immunization in an organ-specific manner. Taken together, our results demonstrate down-regulation of MINCLE expression on monocytes and macrophages by IL-4 as a possible mechanism of thwarted Th17 vaccination responses by underlying helminth infection.

CISH constrains the tuft–ILC2 circuit to set epithelial and immune tone

Innate lymphoid cells (ILCs) are tissue-resident effectors poised to activate rapidly in response to local signals such as cytokines. To preserve homeostasis, ILCs must employ multiple pathways, including tonic suppressive mechanisms, to regulate their primed state and prevent inappropriate activation and immunopathology. Such mechanisms remain incompletely characterized. Here we show that cytokine-inducible SH2-containing protein (CISH), a suppressor of cytokine signaling (SOCS) family member, is highly and constitutively expressed in type 2 innate lymphoid cells (ILC2s). Mice that lack CISH either globally or conditionally in ILC2s show increased ILC2 expansion and activation, in association with reduced expression of genes inhibiting cell-cycle progression. Augmented proliferation and activation of CISH-deficient ILC2s increases basal and inflammation-induced numbers of intestinal tuft cells and accelerates clearance of the model helminth, Nippostrongylus brasiliensis, but compromises innate control of Salmonella typhimurium. Thus, CISH constrains ILC2 activity both tonically and after perturbation, and contributes to the regulation of immunity in mucosal tissue.

IL-13 deficiency exacerbates lung damage and impairs epithelial-derived type 2 molecules during nematode infection

IL-13 is implicated in effective repair after acute lung injury and the pathogenesis of chronic diseases such as allergic asthma. Both these processes involve matrix remodelling, but understanding the specific contribution of IL-13 has been challenging because IL-13 shares receptors and signalling pathways with IL-4. Here, we used Nippostrongylus brasiliensis infection as a model of acute lung damage comparing responses between WT and IL-13-deficient mice, in which IL-4 signalling is intact. We found that IL-13 played a critical role in limiting tissue injury and haemorrhaging in the lung, and through proteomic and transcriptomic profiling, identified IL-13-dependent changes in matrix and associated regulators. We further showed a requirement for IL-13 in the induction of epithelial-derived type 2 effector molecules such as RELM-α and surfactant protein D. Pathway analyses predicted that IL-13 induced cellular stress responses and regulated lung epithelial cell differentiation by suppression of Foxa2 pathways. Thus, in the context of acute lung damage, IL-13 has tissue-protective functions and regulates epithelial cell responses during type 2 immunity.