Clinical allergy is caused by dysregulated type 2 immunity, classically characterized by high levels of immunoglobulin E (IgE) and various cytokines, such as IL-4, IL-5, IL-9, and IL-1332. IgE is central to acute allergic reactions and chronic inflammatory allergic diseases33. By contrast, autoinflammatory diseases are a group of clinical syndromes portrayed by constitutive overactivation of innate immune pathways. This results in increased production of or responses to monocyte- and neutrophil-derived cytokines such as interleukin-1β (IL-1β) and Tumor Necrosis Factor-α (TNF-α). Conventionally, type 2 immune cells and autoinflammatory effectors were considered to neutralize each other. However, growing evidence suggests that, in some contexts, autoinflammatory pathways and cytokines may enhance type 2 immune responses. Conversely, type 2 immune cells and cytokines may regulate autoinflammatory responses in complex and context-dependent manners34.
Our longitudinal data shows that 19% of patients had a comorbid autoimmune condition, 24% show persistent high levels of IgE and the suite of cytokines associated with an allergic response, and 14% are characterized by elevated IL-1β associated with several inflammatory cytokines in at least 2 visits and 43% of patients with normal basal levels of cytokines.
Recently, a study described increased levels of IgE, IL-4, IL-5, IL-10, and IL-13 in serum samples from MD patients compared to controls19. Moreover, Flook et al. identified a subgroup of patients with an altered immune response involving IgE and IL4 leading to persistent inflammatory status20. Both studies and the current data support the idea that 24% of MD patients will have type 2 inflammation.
IL-4 is a critical regulatory cytokine during the immune response, particularly important in allergy and asthma. When resting T cells are antigen-activated and expand in response to Interleukin-2 (IL2), they can differentiate as Type 1 (Th1) or Type 2 (Th2) T helper cells. IL-4 influences the Th1 or Th2 fate. Th2 cells secrete IL-4, which both stimulates Th2 in an autocrine fashion and acts as a potent B cell growth factor to promote humoral immunity35. Of note, IL-13 is an immunoregulatory cytokine secreted by activated Th2 cells. It is a crucial mediator in the pathogenesis of allergic inflammation. IL-13 shares many functional properties with IL-4, and IL-13 receptors are expressed in human B cells, basophils, eosinophils, mast cells, endothelial cells, fibroblasts, monocytes, macrophages, respiratory epithelial cells, smooth muscle cells, but unlike IL-4, not in T cells. Thus, IL-13 does not appear necessary in the initial differentiation of CD4 T cells into Th2 cells; instead, it is essential in the effector phase of allergic inflammation36.
To study the potential upstream events driving the release of allergy-related cytokines, we investigated the expression patterns of several receptors and known Th2 response genes. IL4R, IL2RG, JACK2, and JACK3 were significantly higher in the allergy group compared to the Low group, but GATA3, MAPK3, STAT3, and STAT6 were not found to be significant. So, to further validate our TH2 response hypothesis, we studied the ability to polarize macrophages using plasma from MD patients.
Macrophages adopt various functional phenotypes in response to the microenvironment, typically becoming polarized to M1 (classically activated, also known as proinflammatory) or M2 (alternatively activated) subtypes37,38. While classically activated M1 macrophages are induced by TH1 cytokines like interferon γ (IFNγ) or interleukin 1β (IL-1β), alternatively activated M2 macrophages are triggered by TH2 cytokines such as IL-4 and IL-13, inducing an anti-inflammatory phenotype by signaling through IL4R alpha in a STAT6-dependent manner39,40. Thus, in our study, we have studied the potential of patients’ plasma to polarize M0 macrophages derived from THP-1 cells into M1 or M2 macrophages, finding that MD patients with high levels of the allergy cocktail cytokines were able to polarize to M2 macrophages when compared to patients with low levels of cytokines. However, the group of patients with high levels of IL-1β did not have that ability.
As mentioned above, inflammation plays a vital role in wound healing. Monocytes/macrophages and leukocytes coordinate debris removal and initiate wound healing by attracting endothelial cells, epithelial cells, and fibroblasts. However, if chronic inflammation occurs, this process can become dysregulated, leading to pathological wound repair, accumulation of permanent fibrotic scar tissue at the injury site, and failure to restore normal function to the tissue41,42. Nowadays, the role of cytokines as fibrotic mediators has become well established. Initially, several signaling cascades are modified by changes in the microenvironment involving Th1 and Th2 cytokines being released from damaged epithelial or endothelial cells but also because of fibrocyte recruitment and differentiation, activation, and proliferation of pericytes via epithelial to mesenchymal transition (EMT) or endothelial to mesenchymal transition (EnMT).
Cochlear dysfunction, therefore, can be caused by dysfunction of spiral ligament fibrocytes (SLFs), a primary cell type in the lateral wall, essential for maintaining endocochlear potential and blood flow43. SLF dysfunction due to chronic inflammatory responses driving fibrosis can consequently result in impaired hearing.
NLRP3 and IL-1β expression is reduced in M2 cells44, and inhibition of NLRP3 inflammasome drives M2 polarization45. Interestingly, NLRP3 activation can also cause M2 polarization via the upregulation of IL-4 in an inflammasome-independent process46. NLRP3, but not the inflammasome adaptor ASC or caspase-1, may promote the polarization of M2 macrophages by up-regulating the expression of IL-4, thereby contributing to its regulation of asthma. Although NLRP3-controlled M1/M2 polarization is essential, an accurate understanding is yet to be elucidated.
We also studied the potential involvement of sodium in activating the inflammasome in patients with high IL-1β by comparing their gene expression with the ones in macrophage-derived THP-1 cells treated with known concentrations of NaCl.
The mechanism of NaCl triggering endolymphatic hydrops has been associated with a dysregulation of sodium by either the sodium–potassium adenosine triphosphatase (Na/K-ATPase) or the epithelial sodium channel (ENaC), both of which are expressed in the cochlea47,48. The Na/K-ATPase is expressed in the stria vascularis, vestibular dark cells, and the endolymphatic sac, responsible for endolymph secretion49. Glucocorticoid receptors stimulate sodium absorption by upregulation of ENaC1150, which may explain why some MD patients respond to corticosteroids.
Our results show how the central genes associated with the inflammasome are upregulated in both NaCl-treated cells and MD patients PBMC (IL-1β, NLRP3, and CASP1), but intriguingly, GSDMD was found overexpressed in patients and M1 polarized macrophages, but not in NaCl-treated cells throwing new questions in the proinflammatory hypothesis.
Taking everything into account, we can say that we have a group of MD patients with an allergy-type response with high levels of IL-4, IL-13, and IgE, capable of polarizing macrophages into M2-type, a second group with a classical proinflammatory response activated via inflammasome, led by IL-1β with enormous amounts of NFKB and a third group with no disturbance in their cytokine levels. In closing, we are confident that IgE and cytokines levels should be routinely measured and tracked in patients to improve diagnostic and therapeutic management of MD.