This study demonstrates that repeated anti-FcεR1 therapy protects against Type 1 diabetes in the NOD mouse model. Continuous administration of anti-FcεR1 antibody reduced the incidence of diabetes in NOD mice by ~50% compared to controls and was associated with increased total numbers of pancreatic β-islet cells. The protective effects appeared dependent on chronic treatment with anti-FcεR1 antibody, as shorter treatment regimens for 4 weeks did not result in appreciable disease protection. Future studies will optimize anti-FcεR1 antibody concentrations and administration intervals to further improve protection.
In vitro and in vivo assays showed that anti-FcεR1 antibody activates basophils and mast cells and greatly increases circulating levels of histamine and IL-4. This result was not unexpected, as basophils and mast cells both release histamine and IL-4 in response to FcεR1-mediated signaling. We suspect that the bulk of the IL-4 release was due to basophil activation, since basophils are the only cells known to store large quantities of pre-formed IL-4 [30
], are major contributors of IL-4 in helminth infections and allergic diseases [17
], and have been demonstrated to release more IL-4 on a per cell basis than other cell types [32
]. To our knowledge this is the first demonstration that chronic activation of basophils and mast cells is associated with protection against autoimmunity.
Although continuous treatment with anti-FcεR1 antibody did not result in a clear Th2 shift in cytokine production, as measured from spleen and pancreatic lymph node cells, increases in insulin-specific IgG1 revealed a type 2 immune shift in antibody production. Increased serum levels of IL-4 following anti-FcεR1 antibody treatment suggest that this type 2 immune shift in antibody responses may have been driven by IL-4 released from innate cells activated by antibody rather than T-cells. This type 2 immune shift in insulin-specific antibody isotypes is similar to that observed when NOD mice are infected with the filarial nematode Litomosoides sigmodontis
], an intervention that also results in protection against diabetes.
While histamine signaling through H1 and H2 receptors does not appear necessary for protection, as treatment with H1 and H2 receptor blockers did not attenuate the protective effects of anti-FcεR1 injections, results obtained with IL-4 deficient NOD mice demonstrate that IL-4 is partly responsible for therapeutic benefit. The finding that IL-4 protects against autoimmune disease is consistent with prior studies. In NOD mice systemic administration of IL-4 [34
], expression of IL-4 by pancreatic β-islet cells [36
], and transfer of IL-4 expressing DCs [37
] have been shown to prevent the onset of autoimmune diabetes. An important role for IL-4 in the control of Th1-driven autoimmune diseases is further suggested by studies that used helminths to prevent autoimmunity. Helminth or helminth antigen induced protection against autoimmune diabetes is associated with the induction of Th2 immune responses [8
] and studies in experimental autoimmune encephalitis and trinitrobenzene sulfonic acid (TNBS) induced colitis showed that Schistosoma
egg administration failed to protect against autoimmunity in mice deficient in STAT6 or depleted of IL-4 [39
Besides counterbalancing Th1 immune responses, FcεR1-induced IL-4 may protect against Th1 driven autoimmune responses by driving the differentiation of classically (Th1 associated) macrophages into an alternative activated phenotype (AAMØ). AAMØ are anti-inflammatory and are known to be more prevalent during helminth infections [40
]. Future studies will investigate whether FcεR1-treatment induces AAMØ and whether this cell population contributes to the protective effect.
The novelty of this study is that we were able to induce IL-4 release by systemically activating basophils and mast cells with an antibody that directly cross-links FcεR1s. An approach that is based on antibody injections to trigger IL-4 release might be easier to transfer to the bedside compared to therapies that consist of injection with cytokines.
Additionally, given that the protective effects of anti-FcεR1 therapy were only partially reduced in IL-4-deficient NOD mice, it is likely that IL-4 independent mechanisms may also play a role in anti-FcεR1 therapy. These may be directly related to activation of basophils and/or mast cells, or they may be due to induction of negative feedback pathways induced by chronic activation of these cells. Determining the protective mechanisms of repeated anti-FcεR1 injections that are IL-4 independent will be a focus of studies in the future.
One IL-4-independent mechanism may be the induction of IL-13 release by anti-FcεR1 injections as IL-13 has been shown to prevent diabetes onset in NOD mice [41
]. Both basophils as well as mast cells can produce IL-13 after cross linking of FcεR1. Because IL-13 signals through IL-4Rα it is possible that anti-FcεR1-induced IL-13 assumed some of the functions of IL-4 in IL-4 deficient NOD mice and contributed to anti-FcεR1 mediated protection. Evaluating whether IL-13 plays a role in anti-FcεR1 mediated protection against autoimmunity will be the subject of future studies.
In contrast, the observed non-signficant increase of Th17 cells during anti-FcεR1 therapy is very unlikely to be a mechanism by which anti-FcεR1 injections protect against Type 1 diabetes since Th17 responses are thought to have a role in the induction of Type I diabetes, possibly by conversion of Th17 to Th1 cells that can cause diabetes onset [42
Another IL-4-independent mechanism that may be important is the induction of immunoregulatory networks. By repeatedly activating basophils and mast cells, we replicated the immunological phenotype observed in chronic helminth infections and in allergen immunotherapy. In helminth infections, basophils and mast cells are continuously being activated by parasite antigens through parasite-specific IgE on the surface of these cells [46
]. In immunotherapy, patients with allergen-specific IgE are repeatedly given injections of allergen to which they have specific IgE, essentially inducing a chronic state of low level basophil and mast cell activation. While the mechanisms by which chronic helminth infections and allergen immunotherapy modulate the immune system are not completely understood, a number of studies show that both augment key regulators of peripheral tolerance such as IL-10 and T-regulatory cells. Indeed, helminth infections shown to protect against autoimmune diseases in animal models have been repeatedly associated with increases of IL-10 and T-regulatory cells [8
In our study, while not statistically significant, levels of IL-10 production from splenocytes, frequencies of IL-10 producing B cells, and frequencies of regulatory CD4+FoxP3+ T-cells, CD8+FoxP3+ T-cells, and CD1d+CD5+ B-cells were all higher in mice receiving anti-FcεR1 antibody injections. As one of the hallmarks of autoimmune diseases is the loss of peripheral tolerance [49
], it will be important for future studies to more fully evaluate whether anti-FcεR1 therapy functions by augmenting peripheral tolerance. In a similar vein, it would be interesting to evaluate whether allergen immunotherapy in allergic patients has had beneficial effects on patients with concurrent autoimmune diseases.
Future studies will also attempt to further define exactly which cells are involved in anti-FcεR1-mediated protection. In particular, it will be important to determine whether basophils and/or mast cells are necessary for the protective effects of anti-FcεR1 therapy. We have shown that anti-FcεR1 therapy activates these cells, but have not yet demonstrated that these cells are essential. As there are currently no basophil-deficient nor mast-cell deficient mice on a NOD background, it will be necessary to develop techniques of depleting basophils and mast cells without concurrently activating them. Additionally, it has recently been shown that antigen-presenting DCs can express FcεR1 in the setting of Th2 inflammation and be depleted by anti-FCεR1 treatment (15). While it is unlikely these cells play a role in the Th1-mediated pathology of Type 1 diabetes, future studies will also evaluate whether FcεR1+ DCs play a role in FcεR1-mediated protection against Type I diabetes.
Clinically, it is important to ask whether induction of chronic basophil and mast cell activation could actually be used to protect against autoimmune diseases. While at first blush such an approach sounds preposterous given the obvious risk of anaphylaxis, we believe chronic basophil and mast cell activation could be a safe and feasible therapy. We propose that the key to safety would be to start treatment with small amounts of anti-FcεR1 antibody, or another basophil/mast cell activating agent, followed by a gradual increase in treatment dosage. Repeated administration of gradual increasing amounts of anti-FcεR1 did not cause anaphylaxis in the mice we studied, and such an approach would be similar to allergen immunotherapy, in which the dose of allergen given is gradually increased over time. Allergen immunotherapy induces repeated basophil and mast cell activation and is routinely conducted in the outpatient arena for diseases as benign as allergic rhinitis.
Importantly, unlike conventional therapies for autoimmune diseases which predominantly work by incapacitating specific arms of the immune system, a therapeutic approach based on the induction of chronic basophil and mast cell activation has the potential to induce a therapeutic effect without irreversibly inhibiting any pathways of the immune system.
In conclusion, this study demonstrates that repeated administration of anti-FcεR1 antibodies results in activation of basophils and mast cells and protection against Type 1 diabetes in NOD mice by a mechanism that is partially dependent on IL-4. While IL-4 has been previously shown to protect against Th1-driven autoimmune diseases, the utilization of repeated antibody injections to induce IL-4 release represents a novel approach for the treatment of autoimmune diseases. Given the feasibility of developing antibodies for use in humans, such an approach has the potential to be translated into a novel clinical therapy for Th1-driven autoimmune diseases. In addition to evaluating the safety and feasibility of this approach as a therapy for humans with autoimmune diseases, future studies will also focus on obtaining a better understanding of how helminth infections protect against autoimmune diseases to enable development of other novel therapeutic strategies.