IL-35 secretion correlates positively with the percentage of CD4+CD25+Foxp3+ Treg cells in the blood of patients with allergic rhinitis (AR).
PBMCs were isolated from whole blood of patients with AR or from healthy controls and magnetically sorted for CD4 expression followed FoxP3 and CD25 quantification by flow cytometry to identify Tregs. CD25+/FoxP3+ cells were significantly less abundant in AR patients when compared to normal controls (2.5% vs 7%); interestingly the reduction in double-positive cells can be accounted for by the loss of FoxP3 expression alone (3.9% vs 12% in AR and healthy samples, respectively, Fig. 1A). This is particularly noteworthy due to the well-defined role of FoxP3 in enabling proper Treg function. Moreover, IL-35 secretion from cultured PBMCs isolated from AR patients was significantly reduced compared to that from that secreted by PBMCs isolated from healthy controls, as measured by ELISA (Fig. 1B, left). We found a strong positive correlation (r = 0.7941, p < 0.001) between the percentage CD4+CD25+FoxP3+ cells and IL-35 secretion (Fig. 1B, right). Based on this correlation, the role of IL-35 was examined further in a murine model.
Antibody targeting IL-35 partly disrupted the function of taurine in a mouse AR model.
Taurine administration has been demonstrated to reduce AR-induced physiological response 19,22, increase the percentage of Tregs 37, and reduce phosphorylation of STAT1 38, however the role of taurine in influencing IL-35 in AR has not been described. To investigate the interplay between taurine and the immunoregulatory cascade it is known to impact, we administered taurine with and without co-administration of an IL-35 neutralizing antibody to an OVA-induced allergic rhinitis mouse model. Mice sensitized with only saline were used as a non-AR control and saline-treated AR mice served as a vehicle-treated control. First, sternutation was quantified for a 30-minute observation period as marker for general AR symptomology. Saline-sensitized negative control mice exhibited minimal sneezing. As expected, saline (vehicle) treated AR mice demonstrated frequent sneezing, a significant increase over control mice (p < 0.001). AR mice treated with taurine DOSE, however experienced a robust repression ( p < 0.001) confirming an effective reduction in symptoms. To establish a potential role of IL-35 in this AR model an IL-35 neutralizing antibody was administered to AR mice also treated with taurine; IL-35 neutralization restored AR symptoms to nearly that of vehicle treated AR mice (p < 0.05) (Fig. 2A) strongly implicating IL-35 as mediator of sneezing in AR mice treated with taurine.
To delve in the mechanistic impact of cytokine release in a taurine-treated AR model, the concentrations of four key immunoregulatory cytokines were measured: IL-4, IL-5, IL-13, and IL-35. IL-4 and IL-13 are known to be increased in model AR 39 and IL-5 induces eosinophil infiltration during allergic response 40. As expected, the serum concentration of all three of the type 2 cytokines (IL-4, IL-5, and IL-13) were increased in the AR model compared to the saline control mouse group indicating an immune response (Fig. 2B, p < 0.001). In alignment with a reduction in AR symptoms, the concentrations of these cytokines decreased following taurine treatment (Fig. 2B, p < 0.05). Notably, the concentration of immunoregulatory IL-35 decreased significantly in the AR model and was restored to a nearly normal concentration with taurine treatment. Importantly, the AR symptomology directly correlates to serum IL-35 levels throughout this experiment and the concentration of IL-35 in the serum itself is amenable to taurine treatment, thus confirming the potential of taurine as an AR therapeutic and indicating the mechanism of action is via IL-35.
To verify the role of IL-35 in attenuating AR following taurine treatment, an anti-IL-35 antibody was employed to sequester the cytokine in mice also given taurine. Following anti-35 antibody treatment IL-4, IL-5, and IL-13 concentrations were partially restored (p < 0.05), indicating the anti-inflammatory effects of taurine were lost and suggesting taurine suppresses AR via an IL-35-dependent mechanism (Fig. 2B). Moreover, the resulting serum concentration of IL-35 in taurine plus antibody treated AR mice was lower compared to the taurine alone condition (Fig. 2B). This reduction is expected to be the result of a feedback loop resulting from the alterations in the cytokine cascade as well as the resulting change Treg populations (discussed below), although we cannot conclusively rule out an interaction between the neutralizing antibody and the ELISA assay itself.
Taurine alleviates AR damage to nasal mucosa
Comparison of H&E stains of nasal mucosa from vehicle treated AR mice show extensive infiltration of mucus-secreting goblet cells indicating both a successful creation of an AR model and providing a tissue-level explanation for the increase in sneezing noted above. In contrast, taurine-treated AR mice were found to have mucosa histology which was highly similar to that of non-AR (healthy) control mice with minimal goblet cell presence. Administration of an anti-IL-35 antibody in combination with taurine treatment in AR mice resulted in goblet cell-rich mucosa with morphology nearly identical to that of the saline-treated AR mice (Fig. 2C). The restoration of the goblet cell infiltration following antibody treatment parallels and explains the increase in sneezing frequency in these mice and confirms the therapeutic effects are dependent on IL-35. In addition to phenotypic and histologic response to taurine treatment we sought to examine the effects of taurine and anti-IL-35 antibody treatment on the Treg population.
In agreement with the patient samples analyzed, the percentage of CD4+CD25+FoxP3+ Tregs within the CD4+ population decreased from approximately 6% in non-AR mice to 2% in AR model mice. Administration of taurine rescued the Treg population in AR mice to nearly 6% of CD4+ cells. Also, in alignment of our previous results, addition of an anti-IL-35 antibody plus taurine treatment inhibited the increase in Treg numbers induced by taurine, resulting in approximately 4% CD4+CD25+FoxP3+ indicating taurine restores Treg populations though an induction of IL-35 (Fig. 2D).
To determine the tissue-level alterations in protein expression which may account for the impact of taurine we measured protein expression in the nasal tissues of AR and control mice. As expected, based on flow cytometry and ELISA data, both IL-35 and FoxP3 abundance were reduced in the AR model compared to control mice while STAT1 phosphorylation was increased. Taurine treatment rescued IL-35 and FoxP3 levels and reduced STAT1 phosphorylation to nearly that of non-AR tissue (Fig. 2E). To delve into the role of IL-35 on protein abundance we examined FoxP3 and pSTAT1 in nasal mucosa tissue from mice treated with both taurine and an anti-IL-35 antibody. FoxP3 levels were reduced compared to taurine-treated AR mice while STAT1 phosphorylation was increased indication a partial reinstatement of the AR model state (Fig. 2E). The loss of IL-35 following antibody treatment is suspected to be the result of a feedback loop in which IL-35 production is dependent upon IL-35 stimulation. As STAT1 phosphorylation correlates inversely with IL-35 levels and positively with AR symptoms we expect that STAT1 plays a role in the regulation of AR by IL-35.
There was a robust decrease in FoxP3 abundance in AR nasal mucosa compared to non-AR mice, indicating a loss of CD4+CD25+FoxP3+ Tregs in this tissue. Importantly, AR mice treated with taurine had a complete restoration for FoxP3 levels, a clear indication of normalization of immune response (p < 0.001, Fig. 2E). Anti-IL-35 antibody diminished FoxP3 restoration following taurine treatment, resulting in protein abundance equidistant from the AR model and normal control mice (p < 0.001, Fig. 2E).
IL-35 targeting antibody disrupted taurine response in AR CD4 + T cells
We sought to delineate the interplay of taurine and IL-35 on CD4+ AR cells. Murine AR CD4+ cells were exposed to taurine with or without an anti-IL-35 antibody; cytokine release was measured by ELISA. Secretion of the pro-inflammatory cytokines IL-4, IL-5, and IL-13 were all reduced with taurine treatment suggesting a reduction in AR pathology. In contrast, anti-inflammatory IL-35 secretion was increased. Each of these effects were reversed when an IL-35 neutralizing antibody was administered with the taurine treatment, a clear indication that the anti-inflammatory impact of taurine is mediated via IL-35 (Fig. 3A). Accordingly, STAT1 phosphorylation was strongly reduced in CD4+ cells following taurine treatment and was fully restored when cells were cultured in the presence of an IL-35 neutralizing antibody (Fig. 3B). The correlation of STAT1 phosphorylation with IL-35 availability suggests that STAT1 is the downstream target of IL-35 in AR.
Recombinant IL-35 mimics taurine in AR CD4 T cells.
To elucidate the role of IL-35 in regulating the autoimmune cytokine cascade, recombinant IL-35 or taurine was administered to CD4+ cells isolated from AR mouse model blood. Measurement of the type 2 cytokines IL-4, IL-5, and IL-13 showed all were reduced significantly with either taurine or recombinant IL-35 treatment (p < 0.001, Fig. 4A). Recombinant IL-35 also induced further IL-35 secretion (Fig. 4A). Moreover, both taurine and recombinant IL-35 reduced STAT1 phosphorylation in accordance with a known IL-35 signaling pathway (Fig. 4B) 41. The fact that taurine induced IL-35 release and that IL-35 presence, either secreted from CD4+ cells or administered exogenously, shows a dose-dependent trend in reducing STAT1 phosphorylation strongly suggests that taurine-induced IL-35 secretion controls STAT1 phosphorylation in AR.
STAT1 inhibition suppressed the IL-35 antibody function in normal CD4 T cells.
To further investigate the role of STAT1 in immunoregulation we employed a STAT1 inhibitor, fludarabine phosphate, in conjugation with anti- IL-35 antibody treatment of normal mouse CD4+ cells. As expected, exposure to anti-IL-35 antibody alone produced an increase in secretion of IL-4, IL-5, and IL-13 (p < 0.001, Fig. 5A). Treatment with the STAT1 inhibitor along an anti-IL-35 antibody diminished the response to the antibody and resulted in a reduction in cytokine release (p < 0.001, Fig. 5A). Accordingly, western blotting showed an increase in STAT1 phosphorylation (activation) in cells subjected to antibody treatment which was reversable with STAT1 inhibitor treatment (Fig. 5B). Interestingly, while STAT1 inhibitor treatment reduced STAT1 phosphorylation to below that of untreated cells, type 2 cytokine release was only partly blunted by the presence of the inhibitor, suggesting a parallel pathway may also be at play.