Critical Involvement of CD44 in T Helper Type 2 Cell-Mediated Eosinophilic Airway Inflammation in a Mouse Model of Acute Asthma

Interactions between CD44 and hyaluronan (HA) are crucial for recruiting leukocytes to inflamed tissues. This review summarizes findings from our studies of the roles of CD44-HA interactions in leukocyte trafficking, with a particular focus on airway T helper type 2 (Th2) cells in mouse models of acute asthma. In a mite allergen-induced model of acute asthma, intraperitoneal injection of anti-CD44 monoclonal antibodies blocked lymphocytes and eosinophils from accumulating in the lung, and suppressed both the antigen-induced increase in Th2 cytokines in the bronchoalveolar lavage fluid (BALF) and airway hyperresponsiveness (AHR). CD44 deficiency was associated with decreased mite allergen-induced Th2 cell-mediated airway inflammation and AHR in sensitized mice. Asthmatic responses to antigen-sensitized splenic CD4+ T cells transferred from CD44-deficient mice were weaker than in wild-type mice. Administration of anti-CD44 monoclonal antibodies preferentially suppressed the airway accumulation of antigen-specific Th2 cells induced by antigen challenge, without affecting Th1 and Th17 cells. Increased HA-binding ability of CD44 and expression of Neu1 sialidase were observed on antigen-specific Th2 cells compared with antigen-specific Th1 and Th17 cells. Finally, in a mouse model of acute asthma, neuraminidase 1-deficient SM/J mice exhibited a lower Th2 cytokine concentration and a lower absolute Th2 cell number in the BALF, as well as an attenuated AHR. Our findings indicate that CD44 critically contributes to the antigen challenge-induced airway accumulation of antigen-specific Th2 cells, without affecting Th1 and Th17 cells, in mice. Furthermore, neuraminidase 1 activity is necessary for the interaction between HA and CD44, and Th2 cell-mediated airway inflammation.

IFNγ suppresses the expression of GFI1 and thereby inhibits Th2 cell proliferation

While IFNγ is a well-known cytokine that actively promotes the type I immune response, it is also known to suppress the type II response by inhibiting the differentiation and proliferation of Th2 cells. However, the mechanism by which IFNγ suppresses Th2 cell proliferation is still not fully understood. We found that IFNγ decreases the expression of growth factor independent-1 transcriptional repressor (GFI1) in Th2 cells, resulting in the inhibition of Th2 cell proliferation. The deletion of the Gfi1 gene in Th2 cells results in the failure of their proliferation, accompanied by an impaired cell cycle progression. In contrast, the enforced expression of GFI1 restores the defective Th2 cell proliferation, even in the presence of IFNγ. These results demonstrate that GFI1 is a key molecule in the IFNγ-mediated inhibition of Th2 cell proliferation.

Dual Activation of Estrogen Receptor Alpha and Glucocorticoid Receptor Upregulate CRTh2-Mediated Type 2 Inflammation; Mechanism Driving Asthma Severity in Women?

Background: Type 2-high asthma is characterized by elevated levels of circulating Th2 cells and eosinophils, cells that express chemoattractant-homologous receptor expressed on Th2 cells (CRTh2). Severe asthma is more common in women than men; however, the underlying mechanism(s) remain elusive. Here we examined whether the relationship between severe asthma and type 2 inflammation differs by sex and if estrogen influences Th2 cell response to glucocorticoid (GC). Methods: Type 2 inflammation and the proportion of blood Th2 cells (CD4 CRTh2 ) were assessed in whole blood from subjects with asthma (n = 66). The effects of GC and estrogen receptor alpha (ERα) agonist on in vitro differentiated Th2 cells were examined. Expression of CRTh2, type 2 cytokines and degree of apoptosis (Annexin V , 7-AAD) were determined by flow cytometry, qRT-PCR, western blot and ELISA. Results: In severe asthma, the proportion of circulating Th2 cells and hospitalizations were higher in women than men. Women with severe asthma also had more Th2 cells and serum IL-13 than women with mild/moderate asthma. Th2 cells, eosinophils and CRTh2 mRNA correlated with clinical characteristics associated with asthma control in women but not men. In vitro, GC and ERα agonist treated Th2 cells exhibited less apoptosis, more CRTh2 as well as IL-5 and IL-13 following CRTh2 activation than Th2 cells treated with GC alone. Conclusion: Women with severe asthma had higher levels of circulating Th2 cells than men, which may be due to estrogen modifying the effects of GC, enhancing Th2 cell survival and type 2 cytokine production. (249)

Development of the Th2 Immune Response Models to Evaluate Allergenicity of Milk Proteins

<p>Food allergy, defined as an adverse immune response to food, is increasing in prevalence. It can be broadly separated into phases of sensitization, in which allergy-triggering Immunoglobulin E (IgE) is generated, and the post-sensitization allergic response, in which the allergic response is triggered by sensitizing allergen. While much is known about the specific mediators that cause allergies, the immune processes that underlie disease progression are less clear. This project has employed mouse models of Th2 immunity to clarify the factors involved in the initiation and maintenance of allergic disease. At the centre of allergic disease is the Interleukin (IL)-4-producing CD4+ T helper type 2 (Th2) cell. One of the key inducers of Th2 cell development in vitro is IL-4, but its involvement in Th2 cell development in vivo is controversial. In our studies, we saw that Th2 cell development could be initiated in vivo by primary, adjuvant-free allergen immunisation in the absence of IL-4. However, Th2 cells were more frequent in IL-4-sufficient conditions. We also determined that genetic lesions that result in loss of one, or both, IL-4 alleles impaired the Th2 cell-mediated allergic process, such that IL-4-heterozygous mice can be considered haplo-insufficient for IL-4 in allergic disease contexts. In addition to the generation of IgE antibody, Th2 cells are implicated in the post-sensitization phase of allergy. Multiple oral challenges of sensitized mice induces elevations in Th2-associated cytokines and elevates intestinal mast cell frequencies. It was the second aim of this project to clarify the role of CD4+ T cells in the post-sensitization intestinal allergic process. We demonstrate a key role for CD4+ T cells in this jejunal mast cell recruitment, and identify that this is required in addition to their established contribution to IgE production. Our investigations also reveal a previously unappreciated role for the CD4+ T cell-derived cytokine IL-3 in oral food allergy. These findings suggest that intestinally localised mast cell-inducer Th2 (Th2m) cells are required for allergic responses generated in the intestine. We also investigated whether specific components of ruminant milks influence the allergic process. While goat and cow milks share significant protein homology, goat milk has lower sensitizing and response-evoking capacity, or allergenicity, than cow milk, in numerous experimental systems. In this project, we compared dominant allergens purified from cow and goat milks for their ability to initiate Th2 cell development. We also examined the ability of one of these allergens to initiate the intestinal allergic process. In these studies, we observed similar Th2 cell development and intestinal mast cell activity in response to both cow and goat milk proteins. These responses indicate that the intrinsic allergenicity of the proteins analysed is not sufficient to explain the differential allergenicity attributed to cow and goat milk. These studies examine the endogenous and exogenous factors that contribute to the development of allergic disease. This project clarifies the role of IL-4 in in vivo Th2 cell development, identifies functional segregation of CD4+ Th2 cells in the intestinal allergic process and further illustrates some of the similarities in the allergenicity of isolated cow and goat milk proteins. Collectively, these studies uncover fundamental aspects of the allergic process which may be useful targets for disease intervention in both prophylactic and therapeutic settings.</p>

Development of the Th2 Immune Response Models to Evaluate Allergenicity of Milk Proteins

<p>Food allergy, defined as an adverse immune response to food, is increasing in prevalence. It can be broadly separated into phases of sensitization, in which allergy-triggering Immunoglobulin E (IgE) is generated, and the post-sensitization allergic response, in which the allergic response is triggered by sensitizing allergen. While much is known about the specific mediators that cause allergies, the immune processes that underlie disease progression are less clear. This project has employed mouse models of Th2 immunity to clarify the factors involved in the initiation and maintenance of allergic disease. At the centre of allergic disease is the Interleukin (IL)-4-producing CD4+ T helper type 2 (Th2) cell. One of the key inducers of Th2 cell development in vitro is IL-4, but its involvement in Th2 cell development in vivo is controversial. In our studies, we saw that Th2 cell development could be initiated in vivo by primary, adjuvant-free allergen immunisation in the absence of IL-4. However, Th2 cells were more frequent in IL-4-sufficient conditions. We also determined that genetic lesions that result in loss of one, or both, IL-4 alleles impaired the Th2 cell-mediated allergic process, such that IL-4-heterozygous mice can be considered haplo-insufficient for IL-4 in allergic disease contexts. In addition to the generation of IgE antibody, Th2 cells are implicated in the post-sensitization phase of allergy. Multiple oral challenges of sensitized mice induces elevations in Th2-associated cytokines and elevates intestinal mast cell frequencies. It was the second aim of this project to clarify the role of CD4+ T cells in the post-sensitization intestinal allergic process. We demonstrate a key role for CD4+ T cells in this jejunal mast cell recruitment, and identify that this is required in addition to their established contribution to IgE production. Our investigations also reveal a previously unappreciated role for the CD4+ T cell-derived cytokine IL-3 in oral food allergy. These findings suggest that intestinally localised mast cell-inducer Th2 (Th2m) cells are required for allergic responses generated in the intestine. We also investigated whether specific components of ruminant milks influence the allergic process. While goat and cow milks share significant protein homology, goat milk has lower sensitizing and response-evoking capacity, or allergenicity, than cow milk, in numerous experimental systems. In this project, we compared dominant allergens purified from cow and goat milks for their ability to initiate Th2 cell development. We also examined the ability of one of these allergens to initiate the intestinal allergic process. In these studies, we observed similar Th2 cell development and intestinal mast cell activity in response to both cow and goat milk proteins. These responses indicate that the intrinsic allergenicity of the proteins analysed is not sufficient to explain the differential allergenicity attributed to cow and goat milk. These studies examine the endogenous and exogenous factors that contribute to the development of allergic disease. This project clarifies the role of IL-4 in in vivo Th2 cell development, identifies functional segregation of CD4+ Th2 cells in the intestinal allergic process and further illustrates some of the similarities in the allergenicity of isolated cow and goat milk proteins. Collectively, these studies uncover fundamental aspects of the allergic process which may be useful targets for disease intervention in both prophylactic and therapeutic settings.</p>

Understanding the Generation and Regulation of IL-4 Producing T Helper 2 Cells

<p>A keenly sought therapeutic approach for the treatment of allergic disease is the identification and neutralization of the cytokine that regulates the differentiation of Th2 cells. Th2 cells are CD4 T cells differentiated to secrete IL-4. These cells are exciting new targets for asthma therapies due to the key role they play in allergic airway diseases. Recently the cytokine IL-25 has been shown to enhance Th2 cell activity and play important roles in mediating allergic inflammatory responses. To investigate this further we crossed IL-25 deficient mice with GFP-IL-4 reporter mice and developed an assay of in vitro and in vivo IL-4 independent Th2 differentiation. These assays were used to determine whether IL-25 was critical for the formation of Th2 cells. We found there was no physiological role for IL-25 in either the differentiation of Th2 cells or their development to effector or memory Th2 subsets. In the strong Th2 setting of a helminth infection the absence of IL-25 resulted in no defects in the effector type 2 responses associated with T helper type 2 immunity including, mucous hyperplasia, class switching to IgE and protection against challenge infections. Importantly this data challenges the newly found and growing status of the cytokine IL-25 and its proposed role in promoting Th2 responses. The second part of this thesis investigated whether the genomic organisation, which reflects commitment to Th2 cytokine expression, could provide a clearer definition of a functional in vivo Th2 cell. Two distinct IL-4 reporter mice were crossed and Th2 in vivo assays were developed that allowed tracking of the individual alleles of IL-4 in a variety of tissue types and Th2 subsets. Interestingly in vivo expression of IL-4 is mostly monoallelic yet there is a small highly activated population of biallelic IL-4 expressing Th2 cells. Physiologically each allele of IL-4 was required for a functional Th2 response with total serum IgE titres up to 4 fold lower in IL-4+/- heterozygous compared to the IL-4+/+ sufficient animals and a significant loss in protective immunity against challenge infections with helminths occurred in the IL-4+/- heterozygous animals. The similarity in deficiencies in Th2 immunity in the IL-4+/- heterozygous and IL-4-/- deficient compared to the IL-4+/+ sufficient animals lead to the proposal that the generation of biallelic Th2 cells may be required for specialised cell-to-cell mediated delivery of tailored activation signals and higher quantities of IL-4 required to mediate fully developed Th2 immune responses.</p>

Understanding the Generation and Regulation of IL-4 Producing T Helper 2 Cells

<p>A keenly sought therapeutic approach for the treatment of allergic disease is the identification and neutralization of the cytokine that regulates the differentiation of Th2 cells. Th2 cells are CD4 T cells differentiated to secrete IL-4. These cells are exciting new targets for asthma therapies due to the key role they play in allergic airway diseases. Recently the cytokine IL-25 has been shown to enhance Th2 cell activity and play important roles in mediating allergic inflammatory responses. To investigate this further we crossed IL-25 deficient mice with GFP-IL-4 reporter mice and developed an assay of in vitro and in vivo IL-4 independent Th2 differentiation. These assays were used to determine whether IL-25 was critical for the formation of Th2 cells. We found there was no physiological role for IL-25 in either the differentiation of Th2 cells or their development to effector or memory Th2 subsets. In the strong Th2 setting of a helminth infection the absence of IL-25 resulted in no defects in the effector type 2 responses associated with T helper type 2 immunity including, mucous hyperplasia, class switching to IgE and protection against challenge infections. Importantly this data challenges the newly found and growing status of the cytokine IL-25 and its proposed role in promoting Th2 responses. The second part of this thesis investigated whether the genomic organisation, which reflects commitment to Th2 cytokine expression, could provide a clearer definition of a functional in vivo Th2 cell. Two distinct IL-4 reporter mice were crossed and Th2 in vivo assays were developed that allowed tracking of the individual alleles of IL-4 in a variety of tissue types and Th2 subsets. Interestingly in vivo expression of IL-4 is mostly monoallelic yet there is a small highly activated population of biallelic IL-4 expressing Th2 cells. Physiologically each allele of IL-4 was required for a functional Th2 response with total serum IgE titres up to 4 fold lower in IL-4+/- heterozygous compared to the IL-4+/+ sufficient animals and a significant loss in protective immunity against challenge infections with helminths occurred in the IL-4+/- heterozygous animals. The similarity in deficiencies in Th2 immunity in the IL-4+/- heterozygous and IL-4-/- deficient compared to the IL-4+/+ sufficient animals lead to the proposal that the generation of biallelic Th2 cells may be required for specialised cell-to-cell mediated delivery of tailored activation signals and higher quantities of IL-4 required to mediate fully developed Th2 immune responses.</p>

Basophils and Mast Cells in COVID-19 Pathogenesis

Basophils and mast cells are among the principal inducers of Th2 responses and have a crucial role in allergic and anti-parasitic protective immunity. Basophils can function as antigen-presenting cells that bind antigens on their surface and boost humoral immune responses, inducing Th2 cell differentiation. Their depletion results in lower humoral memory activation and greater infection susceptibility. Basophils seem to have an active role upon immune response to SARS-CoV-2. In fact, a coordinate adaptive immune response to SARS-CoV-2 is magnified by basophils. It has been observed that basophil amount is lower during acute disease with respect to the recovery phase and that the grade of this depletion is an important determinant of the antibody response to the virus. Moreover, mast cells, present in a great quantity in the nasal epithelial and lung cells, participate in the first immune response to SARS-CoV-2. Their activation results in a hyperinflammatory syndrome through the release of inflammatory molecules, participating to the “cytokine storm” and, in a longer period, inducing pulmonary fibrosis. The literature data suggest that basophil counts may be a useful prognostic tool for COVID-19, since their reduction is associated with a worse prognosis. Mast cells, on the other hand, represent a possible therapeutic target for reducing the airway inflammation characteristic of the hyperacute phase of the disease.