In this study, we compared local immune tolerance in BALB/c and C57BL/6 mice, as well as systemic immune differences, through the use of long-term nasal OVA drop stimulation. BALB/c mice were found to develop local tolerance: nasal symptoms were aggravated at first and then relieved with longer nasal drip; the systemic TH2 response was enhanced at first and weakened with time, and the Treg response was continuously enhanced. In contrast, C57BL/6 mice did not develop a local tolerance response even with the prolongation of nasal drip time, and the local symptoms and inflammatory response continued to worsen and remain at a high level; the systemic immune response only maintained a certain degree of TH2 response and the Treg response was not enhanced.
BALB/c and C57BL/6 mice are two inbred strains commonly used in immunological studies. Based on the cytokine production tendency, BALB/c mice are thought to favor TH2-type responses (e.g., IL-4, IL-5, IL-10, and IL-13), whereas C57BL/6 mice favor TH1-type responses (TNF-α and IFN-γ)(16). BALB/c mice are susceptible to viral infections and exhibit lower cytotoxic responses. When stimulated by allergens, the systemic humoral response is stronger, consistent with the cytokine profile. In contrast, C57BL/6 mice are more reactive to viral infection, exhibiting a higher cytotoxic response and a relatively weaker systemic allergic response(17, 18). We observed a local nasal drip AR model in BALB/c and C57BL/6 mice and found that there were significant differences between their immune responses; i.e. BALB/c mice showed relatively mild local allergic inflammatory responses and strong systemic allergic responses. In contrast, C57BL/6 mice show a stronger local allergic inflammatory response and milder systemic allergic response(2).
We observed that the concentrations of OVA-specific IgE, IgG1, and IgG2a in BALB/c mice increased as nasal drip time increased. However, we found that OVA-specific IgE increased with time, sIgG1 increased first and then leveled off at 12th week, and IgG2a did not change throughout the whole course in C57BL/6 mice. Meanwhile, in BALB/c mice, the splenic culture supernatant results showed that IL-4 concentration increased at 8th week then decreased at 15th week, and IL-10 concentration continued to increase with longer nasal drip. In contrast, C57BL/6 mice showed no change in IL-4, IFN-γ, and IL-10 levels during the entire course of nasal OVA drip. These results suggest that when BALB/c mice were stimulated with a certain dose of allergen for a long time, their allergic reaction (TH2 function) was aggravated, but their TH1 and Treg functions also tended to increase with time and eventually suppressed TH2 function. In contrast, although the increase in serum sIgE (TH2 function) in C57BL/6 mice was lower than that in BALB/c mice, Treg function was not significantly enhanced with time, so the overall response was allergic. Some reports have focused on the immunity and tolerance between BALB/c and C57BL/6 mice. For example, Alan et al. found that BALB/c mice produced more specific antibodies than C57BL/6 mice and showed more significant tolerance to enterohemorrhagic Escherichia coli(3). Gera et al. demonstrated that BALB/c mice were more tolerant to colitis than C57BL/6 mice, possibly because of enhanced BALB/c retinoic acid signaling and the consequent increased ability to fight mucosa-associated lesions(19–24). Chen et al. found that BALB/c thymus and peripheral lymphoid organs had more Treg cells and showed a more potent suppressive effect compared to C57BL/6 mice(25). In a study by Ki-Il et al., it was also found that IL-4, IFN-γ, and IL-10 were not significantly increased in the spleen cell culture supernatant of C57BL/6 mice sensitized by dust mites, and only IL-6 was mildly increased. In contrast, all of the above cytokines were significantly increased in BALB/c mice, showing active TH1 responses, TH2 responses, and Treg responses(26).
In this study, we found that eosinophils and goblet cells increased in the nasal mucosa of BALB/c mice at 8th week, while the number of goblet cells decreased and the number of eosinophils stopped increasing at 15th week. The expression of local IL-10 and TGF-β also increased over time, showing the characteristics of local tolerance. In C57BL/6 mice, local eosinophils and goblet cells increased continuously from 8th week to 15th week, and eotaxin expression increased significantly. However, the local IL-10 and TGF-β levels did not change throughout the course. As IL-10 and TGF-β are important cytokines for immune tolerance, the lack of IL-10 and TGF-β may influence the formation of local tolerance(27).
There were 3903 activated genes locally at 15th week in the BALB/c OVA group, indicating that the local immune responses were active in BALB/c mice. It has also been suggested that the formation of local tolerance in BALB/c mice is an active and positive immunoregulatory process. There were 225 activated genes in the C57BL/6 OVA group at 15th week, much lower than that in the BALB/c OVA group. Although the TH2 response was weaker in C57BL/6 mice than in BALB/c mice at 8th week, the lack of regulatory and inhibitory mechanisms led to an increase in symptomatic and inflammatory responses. We compared the genetic alterations in BALB/c and C57BL/6 at 15th week and found that BALB/c mice and C57BL/6 mice differed significantly in the classical Wnt signaling pathway, which has a negative regulatory effect on Treg cells(28).There was also an elevated negative regulation of JUN kinase activity in BALB/c mice. The JUN kinase pathway has a positive regulatory effect on allergic rhinitis. Therefore, the negative regulation of Wnt signaling pathway and JUN kinase pathway may be related to immune tolerance in BALB/c mice.By constructing the PPI network for the BALB/c OVA group with C57BL/6 OVA group DEGS at 15th week, we derived the top 10 HUB nodes: Rps27a, Uba52, Ubc, Kng2, Gnal, C3, Gng7, Gnb3, Reep5, Reep6.
Recent reports have found that Rps27a is involved in protein synthesis, post-translational modification of proteins, and transcription of genes, and is highly expressed in tumor tissues such as breast fibroids, colorectal cancer and kidney(29, 30). Ubiquitin ribosomal fusion protein (UBA52) is sheared into ubiquitin molecules and ribosomal protein L40 by the action of cell-intrinsic enzymes, where ubiquitin molecules are mainly involved in ubiquitination processes in vivo, such as the degradation of proteins, regulation of the TGF-β pathway and immune response(31, 32). UBC genes play key roles in maintaining ubiquitin (Ub) homeostasis(33). CAMP signaling induces kininogen 2 (Kng2) production in brown adipose tissue, which inhibits brown adipose tissue thermogenesis and may be related to local metabolic function(34). Gnal encodes the stimulatory G protein Gαolf, which is essential for activating the cAMP pathway in striatal projection neurons and is associated with dystonia and hyperactivity disorders(35, 36). G protein subunit γ7 (Gng7), a member of the G protein family, was moderately to highly correlated with the degree of infiltration of CD4 + T cells in the peripheral blood of patients with colorectal cancer. Additionally, GNG7 expression positively correlated with the degree of infiltration of B cells, macrophages, neutrophils and dendritic cells(37). Gnb3 encodes the G protein β3 subunit and is associated with obesity, diabetes, depression and tumors(38–40). REEP5 and REEP6 play important roles in IL-8-stimulated activation of CXCR1(41). CXCR1 can be used as a marker of AR(42). Liliane et al. found that C3 induces mice to form a peptide-mediated skin tolerance model by regulating DC function, and that C3-deficient mice fail to produce Treg cells required for the induction of tolerance(43). From the above reports, it appears that C3 and UBA52 may be involved in the regulation of local immune tolerance. However, the role of other genes with a large degree of activation or response in the regulation of local immune tolerance and inflammation needs to be explored in future studies.