DISCUSSION
The eLTs can produce antibodies locally and therefore perpetuate inflammation in NPs.8 However, the mechanisms underlying the formation and expansion of eLTs in NPs remain unexplored. In this study, for the first time, we identified a critical role for IL-17A-induced stromal cell remodeling in the initiation, and crosstalk between B cells and stromal cells via CXCL13 and LTα1β2 in the enlargement and maintenance of eLTs in NPs.
Expression of homeostatic chemokines is central to the initiating events that lead to lymphoneogenesis.13-15, 39, 40 In NPs without eLTs, we found that stromal cells were the major cellular source of CXCL13, indicating that stromal cells are critical for the initiation of B cell recruitment and compartmentalization in NPs. Evidence that IL-17A may play an important role in eLT formation by inducing the production of lymphoid chemokine CXCL12 and CXCL13 emerge from recent animal studies of bronchial infection and autoimmune encephalitis.18, 32 In this study, we found that IL-17A expression correlated with CXCL13 expression in NPs. We further discovered that nasal stromal cells had the expression of IL-17RA and IL-17A induced CXCL13 production in nasal stromal cells. Although IL-17A induced the production of CXCL12, another important B cell chemokine, during bacteria-induced lung lymphoid neogenesis,18 we failed to find an induction of CXCL12 in nasal stromal cells by IL-17A, which is consistent with our previous finding of no association between CXCL12 expression and eLT formation in NPs.8Therefore, distinct mechanisms may underline the effect of IL-17A in promoting eLT development in different organs and pathological conditions.
Stromal cells have a complex role at local microenvironments, which induce immune cell migration, activation and survival, and support lymphoid enlargement. FRCs provide homeostatic chemokines, and secrete extracellular matrix proteins to form the structural framework for immune cell interaction in SLOs.41 BECs and LECs regulate lymphocyte entry into SLOs. DNs have recently been shown to contain a novel subset of fibroblastic contractile pericytes.42 Although the phenotype and function of stromal cells are well documented in SLOs, little is known of their role in eLT formation. In this study, we revealed an expansion of FRC population of stromal cells in both NPs with and without eLTs, which was likely induced by IL-17A and LTα1β2. The expansion of FRCs in NPs without eLTs indicates a role of FRCs, but not other types of stromal cells, in eLT formation in its infant stage given to the findings that stromal cells were the main producer of CXCL13 in NPs without eLTs and FRCs were the major source for CXCL13 in stromal cells in NPs. LECs is also a fundamental compartment in controlling SLOs organogenesis.43 Nevertheless, LECs were only expanded in NPs with eLTs, and the expansion of LECs was induced by LTα1β2 but not IL-17A. Since LTα1β2 was only upregulated in NPs with eLTs, LECs are more likely involved in the enlargement rather than the initiation of eLTs in NPs by facilitating the entry of lymphocytes into NPs.
In NPs with eLTs, we found that B cells were main producer of CXCL13. After the B cell recruitment under the control of CXCL13 derived from stromal cells, B cells themselves may provide a “second wave” of supply of CXCL13. LTα3 and LTα1β2are reported to be involved in eLT formation.44, 45Compared with control tissues, the mRNA expression of LTβ was only upregulated in NPs with eLTs, suggesting an involvement of membrane form of LT in the later stage of eLT formation in NPs. Previous studies show that CXCL13 induces murine splenic B cells to upregulate membrane-bound LT via Grb2, and CXCL13 expression induction is dependent on LTβR pathway in SLOs.37, 38, 46 In this study, we demonstrated a positive feedback loop between CXCL13 and LTα1β2 on B cells in eLTs in NPs, which obviously exaggerates the B cell recruitment and compartmentalization. In addition to B cells, 36% reduction of CXCL13 expression was founded in NPs with eLTs after depletion of stromal cells. Nasal stromal cells also had LTβR expression. We found that LTα1β2 reshaped stromal cells to FRC and LEC type and promoted their CXCL13 production. Thus, the crosstalk between stromal cells and B cells further perpetuate the eLT development in NPs.
Using a murine model with high nasal type 17 inflammation, we confirmed that IL-17A was able to induce eLT formation in nasal mucosa and this process was dependent on CXCL13 and LTα1β2 in vivo .In the animal study, we found that only high levels of IL-17A induced by 100 μg curdlan led to eLT formation. In contrast, the comparatively lower IL-17A levels induced by curdlan at 20 μg only induced lymphocyte clusters. This is in line with the finding in humans that the IL-17A levels were more prominently elevated in NPs with eLTs than those without eLTs.
There are several limitations in this study. We established a mouse model by using curdlan, which elicits a high local IL-17A inflammation but may not mirror the pathogenesis of NPs in humans. It is considered that the type 17 response is less important for Caucasian patients with NPs than for Chinese patients. Nevertheless, eLTs have also been reported in Caucasian patients,47 indicating that additional mechanisms may underlie eLT formation in Caucasian patients.
These comments notwithstanding, for the first time, we have established a paradigm of how eLTs are formed in NPs in Chinese patients. We suspect that targeting IL-17A, CXCL13 and LTα1β2may provide opportunities for the design of therapies to manipulate eLT formation and alleviate inflammation in patients with NPs.