Discussion
Basophils are rapidly mobilized after T. muris infection to the inflamed tissue to execute effector functions that support Type 2 inflammation (7, 26, 34). Our previous findings showed that Notch signaling in basophils is required for optimal T. murisinfection-induced basophil gene expression changes and effector function, allowing basophils to support Th2 cell responses in the tissue that promote worm expulsion in C57BL/6 mice (7). However, the regulation of basophil responses following infection and the factors that govern basophil Notch receptor expression remain unclear. Previous studies have leveraged differences in susceptibility to T. muris infection in inbred mouse strains to reveal cellular and molecular players that regulate the Type 2 immune response (9-11, 13-17). Here, we utilized AKR/J mice, which retain adult T. muris worms and mount a Type-1 skewed immune response to infection (9-13, 15), to investigate how basophil responses and basophil Notch expression are regulated in genetically susceptible mice.
Our data show that the size of the basophil population changed dynamically in the cecum and spleen upon T. muris infection in AKR/J mice, similar to the patterns observed in resistant C57BL/6 mice (Fig. 2) (7, 26, 34). However, our studies did not examine levels of factors that promote basophilia, including IL-3, IL-33, TSLP, and basophil-homing chemokines such as CXCL12 and CCL7, nor basophil expression levels of the cognate receptors for these factors (20-29), inT. muris -infected AKR/J vs. C57BL/6 mice. One study has shown that resistant Balb/C but not AKR/J mice infected with T. murishave infection-induced increases in Tslp gene expression in the cecum at 1 and 7 days p.i. (35), suggesting that there are strain-specific differences in the levels of factors that activate basophils. Future experiments that measure the levels and activities of cytokines and chemokines that activate and promote the accumulation of AKR/J vs. C57BL/6 or Balb/C basophils during T. muris infection will be required to determine whether there are strain-dependent differences in the levels and activities of basophil activating factors. Studies that delete IL-3 and TSLP and their receptors in AKR/J mice during T. muris infection will also be required to determine whether these, or other factors, are critical for mobilizing the basophilia that we observe in AKR/J mice upon infection.
In our study, basophil expansion was not associated with Type 2 inflammation and worm clearance in T. muris -infected AKR/J mice (Fig. 1). These data suggest that expansion of the basophil population per se is not sufficient to drive effective Type 2 inflammation and parasite expulsion nor indicative of an optimal Type 2 response. Indeed, there was negligible expression of Type 2 cytokines in the colon of infected AKR/J mice, but considerable expression of Ifng (Fig. 1C-E). Thus, basophils, while expanded in AKR/J mice, may not be receiving proper activation signals and may not be optimally functional. Further studies are required to investigate whether AKR/J basophils demonstrate similar infection-induced basophil degranulation, tissue positioning, cytokine and chemokine production, and interactions with other cell types, similar to what is seen in C57BL/6 mice.
We also found that basophils in AKR/J mice do not upregulate Notch2 on day 14 p.i. with T. muris as basophils in C57BL/6 mice do (Fig. 3) (7). One caveat of our study is that we did not measure expression of all four Notch receptors (30); we focused on Notch2, as this receptor was most strongly upregulated on C57BL/6 basophils following T. muris infection (7) and Notch receptor expression is highly tissue- and context-dependent (30). It is possible that AKR/J basophils upregulate other Notch receptors that control basophil effector function in infection. Regardless, our data suggest that AKR/J basophils may not be fully competent to receive critical Notch signals that program these cells to support Type 2 responses in C57BL/6 mice (7). It is notable that in the naïve state, C57BL/6 cecum basophils have lower Notch2 expression than AKR/J basophils (Fig. 4E-G), suggesting that C57BL/6 and AKR/J mice have different baseline levels of basophil Notch signaling activity. Further studies should focus on whether AKR/J basophils show evidence of proper Notch programming during T. muris infection to determine if the Notch pathway is active in mobilizing basophil responses in a genetically susceptible inbred mouse strain.
Interestingly, our IFN-γ neutralization experiment (Fig. 4) suggests that IFN-γ is not the primary player in suppressing Notch2 expression by AKR/J cecum basophils following T. muris infection on day 14 p.i. However, AKR/J basophils may upregulate Notch2 during infection and IFN-γ neutralization at other timepoints. If Type 2-associated factors are important in upregulating Notch2 on basophils, then it is not surprising that we did not observe increased Notch2 expression on basophils in infected AKR/J mice, in light of our findings that IFN-γ neutralization did not provoke increased colonic gene expression of Il4 andIl13 on day 14 p.i. (SFig. 3). Thus, a kinetic analysis of AKR/J basophil Notch receptor expression should be performed. Future studies could also assess whether treatment with IL-4 complexes, which also provide protection to AKR/J mice (31), are sufficient to upregulate Notch2 on AKR/J basophils.
Indeed, previous studies have shown that IFN-γ neutralization does not control all facets of the host response to T. muris across susceptible mouse models. IFN-γ neutralization results in increased worm clearance and a decrease in IFN-γ-associated IgG2a in AKR/J mice (31), worm clearance and IL-13 upregulation in susceptible B cell-depleted C57BL/6 mice (36), and Type 2 cytokine responses on day 21 p.i. in susceptible TSLPR-deficient mice (37). However, it does not alter worm burden in C57BL/6 mice infected with a low dose of T. muris that retain worms (38) nor susceptible Muc5ac -deficient mice that have elevated IFN-γ levels (39) Thus, it is likely that Notch2 expression by basophils is governed by factors other than IFN-γ in AKR/J mice. C57BL/6 and AKR/J mice differ substantially at a wide array of loci that may regulate basophil responses during helminth infection (9-11, 13-15, 17). Further investigation and genetic analyses will be needed to determine why AKR/J basophils do not upregulate Notch2 receptor expression compared to C57BL/6 basophils during T. muris infection. Such studies could potentially illuminate new factors that promote basophil upregulation of Notch receptors, in addition to IL-3 and IL-33 (7).
In summary, our data reveal that the basophil population expands in the spleen and cecum during T. muris infection but does not upregulate Notch2 expression in susceptible AKR/J mice, even when IFN-γ is neutralized. Continued comparative studies in AKR/J and C57BL/6 mice could be leveraged to determine how and when Notch signaling shapes anti-helminth basophil responses. Our study emphasizes the significance of utilizing inbred mouse models to dissect the correlates of an effective Type 2 immune response and reveals new insight into how basophil responses are regulated during intestinal helminth infection.