Androgen receptor signaling protects male mice from the development of immune response to peanut.

OBJECTIVES: Peanut (PN) allergy is a major public health concern. Recent research has brought clarity about how individuals become sensitized to PN allergen with routes known through the skin, as well as the airway. Still unclear, however, is the role of sex hormones on the development of allergic immune responses to PN. This study examines the role of androgen receptor (AR) signaling in regulating PN-specific immune responses. METHODS: We utilized a 4-week inhalation mouse model of PN allergy that is known to drive the production of PN-specific antibodies and elicit systemic anaphylaxis following PN challenge. Wildtype (WT) male, female, and androgen receptor-deficient testicular feminization mutant (ARTfm) male mice were examined using this model to document sex differences in PN allergy. To determine if sex differences also existed in the cellular immune response, this study utilized a 3-day inhalation mouse model of PN to examine the response of group 2 innate lymphoid cells (ILC2s). WT male and female mice were examined using this model to document sex differences in ILC2 response within the lungs. RESULTS: AR use is critical in regulating PN-specific antibody levels. We found that ARTfm males have a higher antibody response and significantly worse anaphylactic response following PN challenge relative to WT males. WT males also exhibit a less severe anaphylactic response compared to ARTfm male and female mice. Lastly, we discovered that lung ILC2s from female mice respond more robustly to PN compared to ILC2s within WT male mice. CONCLUSIONS: Taken together, this study suggests that male sex hormones, namely androgens, negatively regulate allergic immune responses to PN.

Understanding sex differences in the allergic immune response to food.

Food allergies are of great public health concern due to their rising prevalence. Our understanding of how the immune system reacts to food remains incomplete. Allergic responses vary between individuals with food allergies. This variability could be caused by genetic, environmental, hormonal, or metabolic factors that impact immune responses mounted against allergens found in foods. Peanut (PN) allergy is one of the most severe and persistent of food allergies, warranting examination into how sensitization occurs to drive IgE-mediated allergic reactions. In recent years, much has been learned about the mechanisms behind the initiation of IgE-mediated food allergies, but additional questions remain. One unresolved issue is whether sex hormones impact the development of food allergies. Sex differences are known to exist in other allergic diseases, so this poses the question about whether the same phenomenon is occurring in food allergies. Studies show that females exhibit a higher prevalence of atopic conditions, such as allergic asthma and eczema, relative to males. Discovering such sex differences in allergic diseases provide a basis for investigating the mechanisms of how hormones influence the development of IgE-mediated reactions to foods. Analysis of existing food allergy demographics found that they occur more frequently in male children and adult females, which is comparable to allergic asthma. This paper reviews existing allergic mechanisms, sensitization routes, as well as how sex hormones may play a role in how the immune system reacts to common food allergens such as PN.

Induction of Peanut Allergy Through Inhalation of Peanut in Mice.

Peanut (PN) allergy is a common life-threatening disease; however, our knowledge on the immunological mechanisms remains limited. Here, we describe the first mouse model of inhalation-driven peanut allergy. We administered PN flour intranasally to naïve wild-type mice twice a week for 4 weeks, followed by intraperitoneal challenge with PN extract. Exposure of mice to PN flour sensitized them without addition of adjuvants, and mice developed PN-specific IgE, IgG1, and IgG2a. After challenge, mice displayed lower body temperature and other clinical signs of anaphylaxis. This inhalation model is an ideal system to allow for future examination of immunological mechanisms critical for the development of PN allergy.

Allergic sensitization to peanuts is enhanced in mice fed a high-fat diet.

The incidence of peanut (PN) allergy is on the rise. As peanut allergy rates have continued to climb over the past few decades, obesity rates have increased to record highs, suggesting a link between obesity and the development of peanut allergy. While progress has been made, much remains to be learned about the mechanisms driving the development of allergic immune responses to peanut. Remaining unclear is whether consuming a Western diet, a diet characterized by overeating foods rich in saturated fat, salt, and refined sugars, supports the development of PN allergy. To address this, we fed mice a high fat diet to induce obesity. Once diet-induced obesity was established, mice were exposed to PN flour via the airways using our 4-week inhalation model. Mice were subsequently challenged with PN extract to induce anaphylaxis. Mice fed a high-fat diet developed significantly higher titers of PN-specific IgE, as well as stronger anaphylactic responses, when compared to their low-fat diet fed counterparts. These results suggest that obesity linked to eating a high-fat diet promotes the development of allergic immune responses to PN in mice. Such knowledge is critical to advance our growing understanding of the immunology of PN allergy.

Airway exposure initiates peanut allergy by involving the IL-1 pathway and T follicular helper cells in mice.

BACKGROUND: Little is currently known regarding the immunologic mechanism(s) that initiate peanut allergy. Notably, peanut proteins have been detected in house dust, and their levels correlate with peanut allergy prevalence. OBJECTIVE: This study aimed to develop a new mouse model for peanut allergy and to investigate the immunologic mechanisms involved in peanut allergen sensitization. METHODS: To mimic environmental exposure, naive mice were exposed to peanut flour by inhalation for up to 4 weeks. We then analyzed serum levels of IgE antibody and challenged mice with peanut proteins. Immunological mechanisms involved in sensitization were analyzed using cytokine reporter mice, an adoptive cell transfer model, and gene knockout mice. RESULTS: When exposed to peanut flour by inhalation, both BALB/c and C57BL/6 mice developed peanut allergy, as demonstrated by the presence of peanut-specific IgE antibodies and manifestation of acute anaphylaxis on challenge. A large number of follicular helper T (Tfh) cells were also detected in draining lymph nodes of allergic mice. These cells produced IL-4 and IL-21, and they more robustly promoted peanut-specific IgE production than Th2 cells did. Genetic depletion of Tfh cells decreased IgE antibody levels and protected mice from anaphylaxis, without affecting Th2 cells. Furthermore, peanut flour exposure increased lung levels of IL-1α and IL-1ß, and mice deficient in the receptor for these cytokines showed a significant decrease in Tfh cells compared with in wild-type mice. CONCLUSIONS: Tfh cells play a key role in peanut allergy, and the IL-1 pathway is involved in the Tfh response to peanut allergen exposure.

Cell extrinsic alterations in splenic B cell maturation in Flt3-ligand knockout mice.

B lymphopoiesis in bone marrow (BM) is critical for maintaining a diverse peripheral B cell pool to fight infection and establish lifelong immunity. The generation of immature B cells is reduced in Flt3-ligand (FL-/-) mice leading to deficiencies in splenic B cells. Here, we sought to understand the cellular basis of the spleen B cell deficiency in FL-/- mice. Significant reductions in transitional (TS) and follicular (FO) B cells were found in FL-/- mice, and increased frequencies, but not absolute numbers, of marginal zone (MZ) B cells. BAFF-R expression on splenic B cells and serum levels of B cell activating factor (BAFF) was comparable to wildtype (WT) mice. Mixed BM chimeras revealed that the reductions in TS and FO B cells were cell extrinsic. FL administration into FL-/- mice restored the deficiency in TS B cells and normalized the MZ compartment. Ki67 analysis revealed a significant decrease in the proliferative capacity of TS B cells in FL-/- mice. A Bcl2 transgene did not rescue TS cells in FL-/- mice, uncoupling FL-deficiency to Bcl2-dependent survival pathways. Upregulation of CD1d expression and adoptive transfer experiments suggested MZ skewing in FL-/- mice. These findings support an integral role for Flt3 signaling in peripheral B cell maturation.

Flt3 signaling regulates the proliferation, survival, and maintenance of multipotent hematopoietic progenitors that generate B cell precursors.

Flt3 signaling plays a crucial role in regulating the survival and differentiation of lymphoid progenitors into B cell precursors (BCPs) in bone marrow. To define further the role of Flt3 signaling in lymphoid progenitor survival, mice deficient in Flt3 ligand that also expressed a Bcl2 transgene (Eµ-bcl2tg flt3l(-/-)) were generated. Intracellular flow cytometry established transgene expression in primitive hematopoietic progenitors, including lineage-negative Sca-1(+) c-kit(+) (LSK(+)) CD27(-) cells enriched for functional hematopoietic stem cells. Compared with flt3l(-/-) mice, Eµ-bcl2tg flt3l(-/-) mice had significantly increased multipotential progenitors (MPPs), IL-7R(+) common lymphoid progenitors, and B cell precursors. To determine whether forced expression of Bcl2 was sufficient to restore lymphoid priming in the absence of Flt3 signaling Eµ-bcl2tg flt3l(-/-)rag1-gfp(+) mice were generated. Analysis of Eµ-bcl2tg flt3l(-/-)rag1-gfp(+) mice revealed that the Bcl2 transgene had no effect on lymphoid priming before CD19 expression. Thus, forced expression of a survival gene can bypass the requirement for threshold levels of Flt3 signaling requisite for lymphoid priming. Temporal Flt3 ligand (FL) replacement therapy in flt3l(-/-) mice revealed specific requirements for Flt3 signaling in the expansion and maintenance of Flt3(+hi) MPP and Flt3(+) all lymphoid progenitors, but not Flt3(+) B lymphoid progenitors (BLPs), the immediate precursors of BCPs. BCPs were restored after temporal in vivo FL treatment, albeit with delayed kinetics. Together, these results show that Flt3 regulates the proliferation, survival, and maintenance of developmental stage-specific hematopoietic progenitors that give rise to BCPs.

Hoxa9 and Flt3 signaling synergistically regulate an early checkpoint in lymphopoiesis.

Hoxa9 and Flt3 signaling are individually important for the generation of lymphoid lineage precursors from multipotent hematopoietic progenitors (MPP) in bone marrow. Mice deficient for Hoxa9, Flt3, or Flt3 ligand (FL) have reduced numbers of lymphoid-primed multipotential progenitors (LMPP), common lymphoid progenitors (CLP), and B/T cell precursors. Hoxa9 regulates lymphoid development, in part, through transcriptional regulation of Flt3. However, it was unclear whether Hoxa9 has functions in lymphopoiesis independent of, or alternatively, synergistically with Flt3 signaling. In this study, we show that Hoxa9(-/-)Flt3l(-/-) mice have more severe deficiencies in all B lineage cells, CLP, LMPP, and total Flt3(+) MPP in bone marrow than the single knockouts. Although LMPP and Flt3(+) CLP contain precursors for NK and dendritic cell lineage cells, no deficiencies in these lineages beyond that in Flt3l(-/-) mice was found. Thymocyte cellularity was significantly reduced in the compound knockout, although peripheral T cell numbers mirrored Flt3l(-/-) mice. Analysis of the hematopoietic progenitor compartment revealed elevated numbers of CD150(+hi)CD34(-)CD41(+) myeloid-biased stem cells in Hoxa9(-/-)Flt3l(-/-) mice. In contrast, CD150(-) MPP enriched for lymphoid potential were synergistically reduced, suggesting Hoxa9 and Flt3 signaling function coordinately to regulate lymphopoiesis at a very early stage. Real-time PCR analysis of CD150(-)Flt3(+) cells from wild-type control, Hoxa9(-/-), and Flt3l(-/-) single knockouts revealed decreased lymphoid transcripts, corroborating the importance of these regulators in lymphoid development. Taken together, these studies reveal a very early checkpoint in lymphopoiesis dependent on the combinatorial activities of Hoxa9 function and Flt3 signaling.

Differential requirement for Hoxa9 in the development and differentiation of B, NK, and DC-lineage cells from Flt3+ multipotential progenitors.

Hoxa9 is a homeodomain transcription factor important for the generation of Flt3+hiIL-7R- lymphoid biased-multipotential progenitors, Flt3+IL-7R+ common lymphoid progenitors (CLPs), and B cell precursors (BCP) in bone marrow (BM). In addition to B-cell, Flt3+IL-7R+ CLPs possess NK and DC developmental potentials, although DCs arise from Flt3+IL-7R- myeloid progenitors as well. In this study, we investigated the requirement for Hoxa9, from Flt3+ or Flt3- progenitor subsets, in the development of NK and DC lineage cells in BM. Flt3+IL-7R+Ly6D- CLPs and their Flt3+IL-7R+Ly6D+ B lineage-restricted progeny (BLP) were significantly reduced in hoxa9-/- mice. Interestingly, the reduction in Flt3+IL-7R+ CLPs in hoxa9-/- mice had no impact on the generation of NK precursor (NKP) subsets, the differentiation of NKP into mature NK cells, or NK homeostasis. Similarly, percentages and numbers of common dendritic progenitors (CDP), as well as their plasmacytoid or conventional dendritic cell progeny in hoxa9-/- mice were comparable to wildtype. These findings reveal distinct requirements for Hoxa9 or Hoxa9/Flt3 molecular circuits in regulation of B versus NK and DC development in BM.

Threshold levels of Flt3-ligand are required for the generation and survival of lymphoid progenitors and B-cell precursors.

The generation of B-cell precursors (BCP) from lymphohematopoietic progenitors (LHP) in bone marrow is dependent on signals provided by the receptor tyrosine kinase Flt3 and its ligand, Flt3-ligand (FL). Mice deficient in FL exhibit striking reductions in LHP and BCP. Currently, the mechanism by which Flt3 regulates lymphoid lineage/B-cell development is unknown. Here, we show that haploinsufficiency of FL (FL(+/) (-) ) reduced the numbers of LHP, common lymphoid progenitors, and pro-B cells, suggesting that FL levels set a threshold for B lymphopoiesis. Limiting dilution analysis confirmed reduced BCP frequency in FL(+/) (-) mice. Real-time PCR of LHP from FL(+/) (-) animals showed increased transcripts of the B lineage inhibitor id1. However, targeted deletion of id1 did not restore the lymphoid/B lineage deficiencies in FL(-/-) mice, supporting Id1-independent mechanisms. BrdU incorporation studies established that FL is not essential for the proliferation of Flt3(+) multipotential progenitors. Analysis of FL(-/-) progenitors expressing low levels of Flt3 revealed decreased levels of the pro-survival factor Mcl1. Consequently, the Flt3(+) LHP progeny of Flt3(low) LSK(+) cells exhibited increased Annexin V staining. Together, these data suggest that Flt3 signaling initiates a cascade of events in Flt3(low) precursors that promote the survival of LHP from which BCP are derived.