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.