BACKGROUND
invariant natural killer t  cells participate in both innate and adaptive immune functions .
inkt cells develop from cd4+ cd8+ double positive  thymocytes that express an invariant t-cell receptor .
in mice tcrα chain vα14-jα <dig> pairs with vβ <dig>  vβ <dig> or vβ <dig> to generate a tcr repertoire that recognizes glycolipid antigens presented by the mhc class i-like molecule cd1d .
recognition of lipid antigens presented by cd1d expressed on dp thymocytes leads to commitment to the inkt lineage.
immature inkt <dig> cells then receive developmental cues that control differentiation into type <dig> inkt <dig>  type <dig> inkt <dig> and type <dig> inkt <dig> cells that produce cytokines that are similar to those produced by mature effector cd <dig> t cells after encounter with antigen .
however, in contrast to effector cd <dig> t cells, inkt cells gain the ability to express cytokines during development and respond rapidly when stimulated  <cit> .
whereas the specific transcription factors that regulate cytokine production by each subset of inkt cells are well defined, the transcriptional programs that control inkt cell generation and differentiation into effector cells remain to be delineated.
the connection between inkt cells and the development of asthma or allergic lung inflammation in mice was revealed and non-adaptive immune cellular candidates were sought to understand how non-allergic environmental triggers could lead to asthma.
typical allergen sensitization and challenge models could not explain why air pollution, viruses and obesity enhanced the disease.
these studies uncovered roles for inkt and innate lymphoid cell populations in the lung in modulating allergic lung inflammation.
since then, numerous human studies have attempted to correlate frequency, function and location of inkt cells to asthma severity.
akbari et al.
showed that lung cd4+ inkt cells from asthmatics secreted both il- <dig> and il- <dig> but little ifn-γ on αgalcer stimulation  <cit> .
however, conclusions about the contribution of peripheral blood inkt cells from asthmatics to the severity of asthma have been equivocal .
nonetheless, inkt cells play an essential role in viral and pediatric exacerbations of asthma and in severe asthma  <cit> .
defining their contribution to allergic lung inflammation is of critical clinical importance to finding novel approaches to regulate the number and function of inkt cells.
furthermore, understanding the molecular mechanisms that regulate how inkt cells develop and differentiate is key to controlling the number and frequency of these cells.
β-catenin is ubiquitously expressed and in t cells is augmented in response to tcr signals and regulates t cell development in the thymus .
in addition to conventional t cells, the thymus is also the site for innate immune cell development  <cit> .
we have demonstrated that β-catenin expression regulates the generation of innate-like cd <dig>  thymocytes  <cit> .
however, a role for β-catenin in the generation and differentiation of inkt cells in the thymus remains to be defined.
in this study we report that mice with enforced expression of β-catenin in inkt cell precursors  experience augmented il-25-dependent lung inflammation.
inkt cells in the lung produced increased amounts of il- <dig> and il- <dig> and presented intensified inflammation at alveolar sites and surrounding blood vessels.
we demonstrate that augmented lung inflammation correlated with enhanced generation of inkt <dig> and inkt <dig> cells in β-cat-tg thymus at the expense of inkt <dig> cells.
using mice in which β-catenin was deleted in inkt cell precursors  we show that expression of β-catenin is selectively required for differentiation of inkt <dig> and inkt <dig> cells as inkt <dig> cells developed normally.
in conclusion, this study demonstrates that β-catenin expression was required to promote differentiation and functional maturation of inkt <dig> and inkt <dig> cells that contributed to augmented il-25-dependent lung inflammation.
methods
mice
β-cat-tg mice, expressing an activated form of β-catenin in thymocytes and t cells from the proximal lck promoter, have been previously described  <cit> .
β-cat-cko mice were generated by breeding mice bearing a loxp-flanked gene encoding β-catenin   <cit>  with mice expressing the cre recombinase under the control of the cd <dig> promoter .
all the mice used are on a c57bl/ <dig> genetic background.
cd <dig> + c57bl/ <dig> sjl mice were purchased from taconic.
cd <dig>  + 2+ mice were generated by breeding c57bl/ <dig> sjl mice with c57bl/ <dig> mice.
age-matched  littermate controls or c57bl/ <dig> mice were used in all experiments.
all mice were bred and maintained in animal facility at the national institute on aging .
the studies were carried out in accordance with the recommendations in the guide for the care and use of laboratory animals .
the protocol was approved by the animal care and use committee of the nia intramural research program, nih.
this program is fully accredited by the association for assessment and accreditation of laboratory animal care international , registered by the united states department of agriculture  and maintains an assurance with the public health service .
flow cytometry
single-cell suspensions were prepared from thymus and spleens as per standard protocols.
hepatic lymphocytes were isolated from livers that were homogenized, filtered through nylon mesh and washed in pbs with 1 % fbs.
cells were then resuspended in 44 % percoll , underlaid with 66 % percoll, and centrifuged for 20 min at 2000 rpm.
cells at the interface were collected and washed.
cells were stained, acquired on a facscantoii  and analyzed with flowjo .
dead cells were excluded using the fixable viability dye efluor® <dig> .
the following antibodies and their isotype controls conjugated to fitc, pe, percp-cy <dig> , pe-cy <dig>  apc, apc-cy <dig> or pacific blue  were used for staining: anti-cd <dig> , anti-cd8α , anti-tcrβ , anti-cd1d , anti-siglec-f , anti-ly6g , anti-cd11c , anti-cd11b , anti-cd <dig> , anti-ifn-γ , anti-il- <dig> , anti-il- <dig>  and anti-il-17a .
anti-il-17rb-apc  and its isotype control were purchased from r&d systems.
pe- or apc- conjugated mouse cd1d tetramers loaded with glycolipid pbs- <dig>  were obtained from the tetramer facility of the us national institutes of health.
in brief, cells were incubated with fc block and stained with antibodies, and then fixed with 2 % paraformaldehyde.
for ifn-γ, il- <dig>  il- <dig> and il-17a intracellular staining, cells were permeabilized, stained and fixed using the bd cytofix/cytoperm kit .
for plzf and t-bet intracellular staining, cells were permeabilized and stained with anti-plzf   plus fitc anti-mouse  and apc-conjugated anti-t-bet  purchased from ebioscience, using the foxp <dig> staining buffer kit .
bone marrow chimeras
for the bm chimera experiments, each recipient mouse received 2 ×  <dig> whole bone marrow cells from a single donor in 400 μl of pbs through i.v.
injection.
for the bm mixed chimera experiments, bm cells from two different types of donor mice were mixed at 1: <dig> ratio.
each recipient mouse received 9 ×  <dig> cells in 250 μl of pbs through i.v.
injection.
in all experiments, cd <dig> congenic markers were used to distinguish cells derived from the different sources.
all bm chimeras were reconstituted for at least 7 weeks before analysis.
in vitro pma- and ionomycin-induced activation assay
for in vitro stimulation, thymocytes were cultured in t cell medium  and stimulated for 5 h with phorbol 12-myristate 13-acetate   and ionomycin .
for intracellular cytokine staining, brefeldin a was added for the final  <dig>  h.
in vivo α-galactosylceramide stimulation
three micrograms of α-galactosylceramide in 200 μl pbs were intraperitoneally injected into mice.
mice were sacrificed to obtain spleen 3 h after injection and splenocytes were stained for cd1d-tet, tcrβ, cd <dig>  cd8α, il-17rb and il- <dig>
ahr induction
the ova/il-25-induced ahr model was performed as previously described  <cit>  with modification.
mice were immunized with 50 μg/2 mg i.p.
ova/alum twice , then treated with pbs or 2 μg il- <dig> via i.p.
at day  <dig> and finally challenged twice with 50 μg intranasal ova .
at day  <dig>  analysis was performed.
the mouse trachea was cannulated and lungs were lavaged with  <dig>  ml cold pbs to obtain the bal fluid from which, after centrifugation, cells were stained for flow cytometry with anti-tcrβ, anti-cd <dig>  anti-ly6g, anti-cd11b, anti-cd11c and anti-siglec-f. the left lung lobe was inflated with 1 % low-melt agarose and fixed in formalin until embedded in paraffin to do sections that were stained with hematoxylin and eosin and periodic acid schiff.
photographs were taken with a zeiss axiophot microscope  with a prog res c <dig> digital camera attached to the scope.
camera software was adobe photoshop  <dig> .
lymphocytes from the other lung lobes were isolated by digestion with rpmi medium containing collagenase ii and dnase i followed by a lympholyte separation prior to staining for flow cytometry analysis.
statistics
statistical significance was determined by the student’s t-test.
RESULTS
β-cat-tg mice show an exacerbated response to il-25-dependent ahr induction
inkt cells that migrate to the lung tissue are known to cause il-25-dependent airway hyperresponsiveness .
the receptor for il- <dig> is il-17rb, which is present in certain inkt cell subsets  <cit> .
il- <dig> is both necessary and sufficient for th <dig> cytokine production, eosinophilic airway inflammation and ahr .
to determine if enforced expression of β-catenin affects inkt cell function we analyzed transgenic mice that express a proximal lck-driven stabilized mutant form of β-catenin in thymocytes and t cells .
we sensitized mice twice with ova, along with treatment with il- <dig> and finally challenged the mice intranasally with ova.
at the end of the protocol, we evaluated several hallmark characteristics of allergic lung inflammation.
histological analysis of lung sections from sensitized and challenged control and β-cat-tg mice showed typical features of allergic lung inflammation, which were particularly exacerbated in the β-cat-tg animals that were treated with il- <dig> .
hematoxylin and eosin and periodic acid schiff  staining of the lung tissue revealed greater infiltration of immune cells and a significant increase in the number of mucus-producing cells in the lungs of il- <dig> treated β-cat-tg mice compared to control mice .
we quantified the immune cells infiltrating the airways by counting cells in the bronchoalveolar lavage fluid .
we noticed an increase in all of these infiltrating cell types in the balf from the challenged β-cat-tg mice .
we wished to analyze the effect of the loss-of-function of β-catenin in ahr.
to this end, we generated β-catflox/flox cd4-cre+ mice .
when the same experiment was performed in β-cat-cko mice, we did not see an increase in infiltration of cells compared to treated controls .
these data suggest that β-cat-tg mice experience greater inflammation when challenged with ova and il- <dig> compared to control.fig.
<dig> β-cat-tg mice show an exacerbated response to il-25-dependent ahr induction.
β-cat-tg, β-cat-cko and control mice were sensitized with ova, treated with il- <dig> or pbs and finally challenged with ova to induce airway hyperreactivity .
a histological analysis of lung tissues with hematoxylin and eosin  and periodic acid schiff  staining.
representative control and β-cat-tg sections are shown.
h&e bars, 100 μm; pas bars, 100 μm.
graph shows percentage of pas-positive cells per total number of cells in the epithelium of the airway, counting 5– <dig> airways per lung section .
b total cellularity  and cell numbers of eosinophils , alveolar macrophages , neutrophils  and lypmhocytes  present in bronchoalveolar lavage  fluid  from control and β-cat-tg  and control and β-cat-cko .
c il-4+, il-13+ and il-17a+ cell percentages of lung inkt cells after il-25-dependent ahr induction from control and β-cat-tg  and control and β-cat-cko  .
ahr experiment was performed twice, with 3– <dig> mice per group in each independent experiment.
* p < .05; ** p < .01
we further analyzed lung cell suspensions to detect the cytokine production by inkt cells in the tissue.
ifn-γ and il- <dig> production was comparable in control, β-cat-tg and β-cat-cko mice challenged with ova and il- <dig> .
strikingly, il-13-expressing inkt cells that have been specially implicated in il-25-dependent ahr induction  <cit>  were significantly enhanced in inkt cells from lung cell suspensions from β-cat-tg mice .
by contrast il-13-producing inkt cells in β-cat-cko mice were fewer and unresponsive .
finally, il-17a was also largely produced by β-cat-tg inkt cells .
thus, β-cat-tg il-17rb+ inkt cells contribute to a more robust allergic inflammatory response in the lungs of allergen-challenged β-cat-tg mice compared to control mice.
the allergic response was particularly aggravated in cat-tg mice in the presence of il- <dig> .
these data demonstrate that in mice with enforced expression of β-catenin functional inkt cells lead to a more robust response to il-25-dependent ahr challenge, with all measured parameters of allergic inflammation enhanced, including higher inkt production of th <dig> and th <dig> cytokines.
β-catenin expression regulates the differentiation of effector inkt <dig> and inkt <dig> cells
to determine the specific role of β-catenin in inkt cell development, we studied thymic inkt cells in mice with t-cell conditional deletion or enforced expression of β-catenin.
β-cat-cko mice showed similar proportions and total cell numbers of thymocytes  and mature t cells compared to control mice .
however, β-cat-cko mice had a significant 50 % reduction in the number of inkt cells .
β-cat-tg mice, on the other hand, presented a remarkable increase in the number of thymic inkt cells compared to control mice .
in light of these data, we conclude that β-catenin expression is limiting for the generation of thymic inkt cells.fig.
<dig> β-catenin expression regulates the differentiation of effector inkt <dig> and inkt <dig> cells.
a-b frequency and total inkt cell numbers from control and β-cat-cko mice  and from control and β-cat-tg mice  .
numbers over dot plots refer to total thymocyte cell numbers.
c-d representative dot plots showing inkt <dig>  inkt <dig> and inkt <dig> populations among inkt cells  and total numbers  from control and β-cat-cko mice  and from control and β-cat-tg mice .
gating strategies are shown in right .
* p < .05; ** p < .01; *** p < .001
analysis of expression of transcription factors plzf and t-bet in inkt cell populations from control, β-cat-cko and β-cat-tg mice showed that the frequency and number of inkt <dig> and inkt <dig> cells was significantly affected by β-catenin expression.
specifically inkt <dig> and inkt <dig> cells, but not inkt <dig> cells, accounted for the major decrease in β-cat-cko and the greatest increase in β-cat-tg mice .
we conclude that β-catenin expression is required for the differentiation and expansion of inkt <dig> and inkt <dig> cells.
β-catenin regulates cytokine production by inkt cells
inkt cells acquire functional capabilities in the thymus as part of the developmental program.
to determine if β-cat-tg inkt cells were functionally skewed towards a th2/th <dig> phenotype, we cultured β-cat-tg thymocytes in vitro with pma  and ionomycin  for 5 h and then assayed ifn-γ, il- <dig>  il- <dig> and il- <dig> production by thymic inkt cells.
the results showed that the frequency of ifn-γ-producing inkt cells from β-cat-tg mice was significantly reduced compared to inkt cells from control mice .
strikingly, a much higher percentage of inkt cells from β-cat-tg mice expressed il- <dig>  compared to control inkt cells, even in unstimulated conditions .
likewise, we found enhanced production of il- <dig> and also il-13 + il-17a+ double-producers by β-cat-tg inkt cells compared to control inkt cells .
we conclude that enforced expression of β-catenin promotes a higher frequency of th2- and th17-like inkt cells.
importantly, we note that in the absence of stimulation approximately 26 % β-cat-tg thymic inkt cells produced il- <dig> compared to  <dig>  % control thymic inkt cells .
these data demonstrate that β-catenin promotes the acquisition of effector functionality in developing inkt cells in the thymus.fig.
<dig> β-catenin regulates cytokine production by inkt cells.
frequency of ifn-γ- and il-4-producing  and il-13- and il-17a-producing  inkt cells from control and β-cat-tg mice in in vitro unstimulated or pma + ionomycin stimulated inkt cells.
left, representative experiment and right, graph showing mean ± sem, n =  <dig>  * p < .05; *** p < .001
β-catenin regulation of inkt <dig> and inkt <dig> cells is maintained in the periphery
we then examined the distribution of inkt cells in the periphery in the β-catenin mutant mice.
we observed that spleen and liver from β-cat-cko mice showed a specific reduction of the inkt <dig> and inkt <dig> cells in the spleen, and β-cat-tg spleens had reduced inkt <dig> and increased inkt <dig> and inkt <dig> cells .
analysis of liver from control, β-cat-cko and β-cat-tg mice showed comparable numbers of inkt <dig> cells .
by contrast, the frequency of cytokine-producing cells was significantly diminished in β-cat-cko mice .
we conclude that altered inkt cell development in β-cat-cko and β-cat-tg mice is reflected in the β-catenin-dependent skewing towards inkt <dig> and inkt <dig> cells in the distribution of these cells in the periphery.fig.
<dig> β-catenin regulation of inkt <dig> and inkt <dig> cells is maintained in the periphery.
a total cell numbers of inkt <dig>  inkt <dig> and inkt <dig> cells from control, β-cat-cko and β-cat-tg mice in spleen and liver .
b gating strategy for cd4+ il-17rb+ inkt cells is shown in left.
right, frequency of il-4-expressing cd4+ il-17rb+ inkt cells from spleens of control and β-cat-tg mice 3 h after injection with pbs or 3 μg α-galactosylceramide .
* p < .05; ** p < .01; *** p < .001
to further test the efficacy of induced response of th2-like inkt <dig> cells expanded in β-cat-tg mice we tested cytokine production in vivo.
to address this we administered 3 μg α-galactosylceramide  intraperitoneally to β-cat-tg and control mice and 3 h post-injection analyzed cytokine production by splenic inkt cells.
αgc is known to activate inkt cells in vivo  <cit> .
importantly, we found that αgc-stimulated cd4+ il-17rb+ inkt cells  in β-cat-tg mice produced high levels of il- <dig>  .
therefore, we conclude that enforced expression of β-catenin promotes th2-biased cytokine production by inkt <dig> cells in response to stimulation in vivo.
enhanced generation of β-catenin-dependent inkt <dig> and inkt <dig> cells is cell autonomous
to determine if the effect of enforced β-catenin expression on inkt cell generation was cell intrinsic, we generated chimeric mice using either control or β-cat-tg cells as bone marrow donor cells.
analysis of chimeric mice showed that the frequency and numbers of inkt <dig> and inkt <dig> cells was significantly greater when the donor bone marrow was derived from β-cat-tg mice compared to control mice .
furthermore, mixed chimeric mice generated with bone marrow cells derived from wild-type control  and from β-cat-tg  transferred into irradiated wild-type mouse acting as recipient  also showed significantly increased generation of inkt <dig> and inkt <dig> cells .
thus, this data demonstrates that β-catenin controls differentiation of effector inkt <dig> and inkt <dig> cells in a cell-intrinsic manner.fig.
<dig> enhanced generation of β-catenin-dependent inkt <dig> and inkt <dig> cells is cell autonomous.
a flow cytometry of a bone-marrow chimera experiment analyzed after 7– <dig> weeks, in which cd <dig> + recipient c57bl/ <dig> sjl mice were irradiated and  <dig> million cells isolated from bone marrow were i.v.
injected from cd <dig> + donors, either control or β-cat-tg mice.
top, total inkt cells gated on cd <dig> + cells.
middle, inkt <dig>  inkt <dig> and inkt <dig> cells within the inkt populations.
bottom, number of total inkt, inkt <dig>  inkt <dig> and inkt <dig> cells from donor control and donor β-cat-tg cells .
b flow cytometry of cd <dig>  and cd <dig>  on thymic cells of mixed bone-marrow chimeras containing cd <dig> + recipient c57bl/ <dig> sjl cells, cd <dig>  + cd <dig> + donor control cells and cd <dig> + donor β-cat-tg cells.
inkt cells from both donor cells  and inkt <dig>  inkt <dig> and inkt <dig> cells within the inkt populations .
representative data out of three mice analyzed.
bottom, number of total inkt, inkt <dig>  inkt <dig> and inkt <dig> cells from donor control and donor β-cat-tg cells .
mice were analyzed 7 weeks after mixed bone-marrow chimeras were performed.
* p < .05; ** p < .01; *** p < .001
discussion
in this study we demonstrate that β-catenin regulates the differentiation of inkt cells to inkt <dig> and inkt <dig>  but not inkt <dig>  effector subsets.
conditional deletion of β-catenin in inkt cell-precursors impaired differentiation of inkt <dig> and inkt <dig>  but not inkt <dig> cells.
by contrast, enforced expression of β-catenin promoted the generation of inkt <dig> cells and inkt <dig> cells without affecting generation of inkt <dig> cells.
the effect of enhanced β-catenin expression in inkt cell precursors to promote development of inkt <dig> and inkt <dig> cells was cell-autonomous.
importantly, we show that β-cat-tg inkt cells acquired the ability to produce enhanced levels of il- <dig> and il- <dig> during development that augmented lung inflammation in a mouse model for asthma.
cytokine production by inkt <dig>  inkt <dig> and inkt <dig> effector subsets has been shown to be controlled by transcription factors t-bet, gata <dig> and rorγt that also regulate ifn-γ, il- <dig> and il- <dig> production by t helper cells .
however, transcriptional control of differentiation of inkt <dig> cells to inkt <dig>  inkt <dig> and inkt <dig> subsets has remained to be defined.
this report shows that β-catenin deficiency resulted in a profound decrease in inkt <dig> and inkt <dig> subsets of inkt cells whereas inkt <dig> cells developed normally.
by contrast, enforced expression of β-catenin promoted the development of inkt <dig> and inkt <dig> cells.
it was important to note that the majority of inkt cells in the thymus of c57bl/ <dig> mice were inkt <dig> cells and the observation that enforced expression of β-catenin altered the pattern to inkt <dig> and inkt <dig> cells suggests that β-catenin may be a major factor in the distinct pathways that critically direct differentiation of inkt effector subsets.
these observations are congruent with the observation that a cohort of genes  that have been previously implicated in inkt cell development were up-regulated in mice with enforced expression of β-catenin and diminished in expression in cells with deletion of β-catenin.
unlike effector cd <dig> t cells, inkt cells acquire functional capabilities during the differentiation and maturation process.
it was interesting to note that β-cat-tg inkt cells developed the capacity to produce enhanced levels of il- <dig>  which was reminiscent of the report that tcf <dig> and β-catenin promote expression of gata <dig> and il- <dig> in t helper  <dig> cells  <cit> .
thus, we posit that β-catenin-dependent gene expression regulates the differentiation and functional maturation of inkt <dig> and inkt <dig> cells.
finally, our results obtained from the allergic lung inflammation mouse model may be of some clinical importance.
although the precise role for the different human inkt subsets in the lung and peripheral blood has yet to be clearly defined, correlative studies suggest that inkt cells are involved in exacerbations of asthma  <cit> .
furthermore, il- <dig> has been reported to be increased in asthmatic humans  <cit> .
patients with high il-25- and il-17rb-containing airway epithelial cells present a more prominent th <dig> reaction and respond better to inhaled corticosteroids treatment  <cit> .
we hypothesize that inkt <dig> cells may also be increased in il-25-high asthmatic patients facilitating the generation of “th2-high” asthma, as opposed to a severe, neutrophil-mediated, steroid-insensitive disease.
modula ting the numbers of inkt cells may be beneficial in reducing asthma symptoms and exacerbations.
targeting novel pathways or molecules involved in inkt differentiation, such as β-catenin as we have described here, could be a new approach.
in conclusion, this study shows that β-catenin expression regulates the differentiation of inkt cells that augment lung inflammation associated with asthma and provides insights that may be of use in designing regimens to limit at least one type of lung inflammation.
CONCLUSIONS
in conclusion data presented in this paper indicate that β-catenin regulates the differentiation of inkt cells to inkt <dig> and inkt <dig>  but not inkt <dig>  effector subsets.
we propose that inkt <dig> and inkt <dig> cells promote asthma and posit that modulating the development and function of these cells provides novel means to control of lung inflammation.
abbreviations
inktinvariant natural killer t
dpdouble positive thymocytes
tcrt-cell receptor
icd8innate-like cd8
ilinterleukin
ahrairway hyperresponsiveness
ifn-γinterferon gamma
αgcα-galactosylceramide
competing interests
the authors declare that they have no competing interests.
authors’ contributions
jms, rb-b and as designed the experiments; rb-b, as and das performed experiments; fcs, kp, y-hk, c-hc and h-hx contributed chimera experiments; jxf and nmh contributed to the lung inflammation experiments.
all authors read and approved the final manuscript.
authors’ information
not applicable.

 invariant natural killer t cells participate in both innate and adaptive immune functions. inkt cells develop from cd4+ cd8+ double positive thymocytes that express an invariant t-cell receptor. in mice tcrα chain vα14-jα pairs with vβ vβ or vβ to generate a tcr repertoire that recognizes glycolipid antigens presented by the mhc class i-like molecule cd1d. recognition of lipid antigens presented by cd1d expressed on dp thymocytes leads to commitment to the inkt lineage. immature inkt cells then receive developmental cues that control differentiation into type inkt type inkt and type inkt cells that produce cytokines that are similar to those produced by mature effector cd t cells after encounter with antigen. however, in contrast to effector cd t cells, inkt cells gain the ability to express cytokines during development and respond rapidly when stimulated. whereas the specific transcription factors that regulate cytokine production by each subset of inkt cells are well defined, the transcriptional programs that control inkt cell generation and differentiation into effector cells remain to be delineated. the connection between inkt cells and the development of asthma or allergic lung inflammation in mice was revealed and non-adaptive immune cellular candidates were sought to understand how non-allergic environmental triggers could lead to asthma. typical allergen sensitization and challenge models could not explain why air pollution, viruses and obesity enhanced the disease. these studies uncovered roles for inkt and innate lymphoid cell populations in the lung in modulating allergic lung inflammation. since then, numerous human studies have attempted to correlate frequency, function and location of inkt cells to asthma severity. akbari et al. showed that lung cd4+ inkt cells from asthmatics secreted both il- and il- but little ifn-γ on αgalcer stimulation. however, conclusions about the contribution of peripheral blood inkt cells from asthmatics to the severity of asthma have been equivocal. nonetheless, inkt cells play an essential role in viral and pediatric exacerbations of asthma and in severe asthma. defining their contribution to allergic lung inflammation is of critical clinical importance to finding novel approaches to regulate the number and function of inkt cells. furthermore, understanding the molecular mechanisms that regulate how inkt cells develop and differentiate is key to controlling the number and frequency of these cells. β-catenin is ubiquitously expressed and in t cells is augmented in response to tcr signals and regulates t cell development in the thymus. in addition to conventional t cells, the thymus is also the site for innate immune cell development. we have demonstrated that β-catenin expression regulates the generation of innate-like cd thymocytes. however, a role for β-catenin in the generation and differentiation of inkt cells in the thymus remains to be defined. in this study we report that mice with enforced expression of β-catenin in inkt cell precursors experience augmented il-25-dependent lung inflammation. inkt cells in the lung produced increased amounts of il- and il- and presented intensified inflammation at alveolar sites and surrounding blood vessels. we demonstrate that augmented lung inflammation correlated with enhanced generation of inkt and inkt cells in β-cat-tg thymus at the expense of inkt cells. using mice in which β-catenin was deleted in inkt cell precursors we show that expression of β-catenin is selectively required for differentiation of inkt and inkt cells as inkt cells developed normally. in conclusion, this study demonstrates that β-catenin expression was required to promote differentiation and functional maturation of inkt and inkt cells that contributed to augmented il-25-dependent lung inflammation. methods mice β-cat-tg mice, expressing an activated form of β-catenin in thymocytes and t cells from the proximal lck promoter, have been previously described. β-cat-cko mice were generated by breeding mice bearing a loxp-flanked gene encoding β-catenin with mice expressing the cre recombinase under the control of the cd promoter. all the mice used are on a c57bl/ genetic background. cd + c57bl/ sjl mice were purchased from taconic. cd  + 2+ mice were generated by breeding c57bl/ sjl mice with c57bl/ mice. age-matched littermate controls or c57bl/ mice were used in all experiments. all mice were bred and maintained in animal facility at the national institute on aging. the studies were carried out in accordance with the recommendations in the guide for the care and use of laboratory animals. the protocol was approved by the animal care and use committee of the nia intramural research program, nih. this program is fully accredited by the association for assessment and accreditation of laboratory animal care international, registered by the united states department of agriculture and maintains an assurance with the public health service. flow cytometry single-cell suspensions were prepared from thymus and spleens as per standard protocols. hepatic lymphocytes were isolated from livers that were homogenized, filtered through nylon mesh and washed in pbs with 1 % fbs. cells were then resuspended in 44 % percoll, underlaid with 66 % percoll, and centrifuged for 20 min at 2000 rpm. cells at the interface were collected and washed. cells were stained, acquired on a facscantoii and analyzed with flowjo. dead cells were excluded using the fixable viability dye efluor®. the following antibodies and their isotype controls conjugated to fitc, pe, percp-cy, pe-cy apc, apc-cy or pacific blue were used for staining: anti-cd, anti-cd8α, anti-tcrβ, anti-cd1d, anti-siglec-f, anti-ly6g, anti-cd11c, anti-cd11b, anti-cd, anti-ifn-γ, anti-il-, anti-il- and anti-il-17a. anti-il-17rb-apc and its isotype control were purchased from r&d systems. pe- or apc- conjugated mouse cd1d tetramers loaded with glycolipid pbs- were obtained from the tetramer facility of the us national institutes of health. in brief, cells were incubated with fc block and stained with antibodies, and then fixed with 2 % paraformaldehyde. for ifn-γ, il- il- and il-17a intracellular staining, cells were permeabilized, stained and fixed using the bd cytofix/cytoperm kit. for plzf and t-bet intracellular staining, cells were permeabilized and stained with anti-plzf plus fitc anti-mouse and apc-conjugated anti-t-bet purchased from ebioscience, using the foxp staining buffer kit. bone marrow chimeras for the bm chimera experiments, each recipient mouse received 2 ×  whole bone marrow cells from a single donor in 400 μl of pbs through i.v. injection. for the bm mixed chimera experiments, bm cells from two different types of donor mice were mixed at 1: ratio. each recipient mouse received 9 ×  cells in 250 μl of pbs through i.v. injection. in all experiments, cd congenic markers were used to distinguish cells derived from the different sources. all bm chimeras were reconstituted for at least 7 weeks before analysis. in vitro pma- and ionomycin-induced activation assay for in vitro stimulation, thymocytes were cultured in t cell medium and stimulated for 5 h with phorbol 12-myristate 13-acetate and ionomycin. for intracellular cytokine staining, brefeldin a was added for the final  h. in vivo α-galactosylceramide stimulation three micrograms of α-galactosylceramide in 200 μl pbs were intraperitoneally injected into mice. mice were sacrificed to obtain spleen 3 h after injection and splenocytes were stained for cd1d-tet, tcrβ, cd cd8α, il-17rb and il- ahr induction the ova/il-25-induced ahr model was performed as previously described with modification. mice were immunized with 50 μg/2 mg i.p. ova/alum twice, then treated with pbs or 2 μg il- via i.p. at day and finally challenged twice with 50 μg intranasal ova. at day analysis was performed. the mouse trachea was cannulated and lungs were lavaged with  ml cold pbs to obtain the bal fluid from which, after centrifugation, cells were stained for flow cytometry with anti-tcrβ, anti-cd anti-ly6g, anti-cd11b, anti-cd11c and anti-siglec-f. the left lung lobe was inflated with 1 % low-melt agarose and fixed in formalin until embedded in paraffin to do sections that were stained with hematoxylin and eosin and periodic acid schiff. photographs were taken with a zeiss axiophot microscope with a prog res c digital camera attached to the scope. camera software was adobe photoshop. lymphocytes from the other lung lobes were isolated by digestion with rpmi medium containing collagenase ii and dnase i followed by a lympholyte separation prior to staining for flow cytometry analysis. statistics statistical significance was determined by the student’s t-test.  β-cat-tg mice show an exacerbated response to il-25-dependent ahr induction inkt cells that migrate to the lung tissue are known to cause il-25-dependent airway hyperresponsiveness. the receptor for il- is il-17rb, which is present in certain inkt cell subsets. il- is both necessary and sufficient for th cytokine production, eosinophilic airway inflammation and ahr. to determine if enforced expression of β-catenin affects inkt cell function we analyzed transgenic mice that express a proximal lck-driven stabilized mutant form of β-catenin in thymocytes and t cells. we sensitized mice twice with ova, along with treatment with il- and finally challenged the mice intranasally with ova. at the end of the protocol, we evaluated several hallmark characteristics of allergic lung inflammation. histological analysis of lung sections from sensitized and challenged control and β-cat-tg mice showed typical features of allergic lung inflammation, which were particularly exacerbated in the β-cat-tg animals that were treated with il-. hematoxylin and eosin and periodic acid schiff staining of the lung tissue revealed greater infiltration of immune cells and a significant increase in the number of mucus-producing cells in the lungs of il- treated β-cat-tg mice compared to control mice. we quantified the immune cells infiltrating the airways by counting cells in the bronchoalveolar lavage fluid. we noticed an increase in all of these infiltrating cell types in the balf from the challenged β-cat-tg mice. we wished to analyze the effect of the loss-of-function of β-catenin in ahr. to this end, we generated β-catflox/flox cd4-cre+ mice. when the same experiment was performed in β-cat-cko mice, we did not see an increase in infiltration of cells compared to treated controls. these data suggest that β-cat-tg mice experience greater inflammation when challenged with ova and il- compared to control.fig. β-cat-tg mice show an exacerbated response to il-25-dependent ahr induction. β-cat-tg, β-cat-cko and control mice were sensitized with ova, treated with il- or pbs and finally challenged with ova to induce airway hyperreactivity. a histological analysis of lung tissues with hematoxylin and eosin and periodic acid schiff staining. representative control and β-cat-tg sections are shown. h&e bars, 100 μm; pas bars, 100 μm. graph shows percentage of pas-positive cells per total number of cells in the epithelium of the airway, counting 5– airways per lung section. b total cellularity and cell numbers of eosinophils, alveolar macrophages, neutrophils and lypmhocytes present in bronchoalveolar lavage fluid from control and β-cat-tg and control and β-cat-cko. c il-4+, il-13+ and il-17a+ cell percentages of lung inkt cells after il-25-dependent ahr induction from control and β-cat-tg and control and β-cat-cko. ahr experiment was performed twice, with 3– mice per group in each independent experiment. * p <.05; ** p <.01 we further analyzed lung cell suspensions to detect the cytokine production by inkt cells in the tissue. ifn-γ and il- production was comparable in control, β-cat-tg and β-cat-cko mice challenged with ova and il-. strikingly, il-13-expressing inkt cells that have been specially implicated in il-25-dependent ahr induction were significantly enhanced in inkt cells from lung cell suspensions from β-cat-tg mice. by contrast il-13-producing inkt cells in β-cat-cko mice were fewer and unresponsive. finally, il-17a was also largely produced by β-cat-tg inkt cells. thus, β-cat-tg il-17rb+ inkt cells contribute to a more robust allergic inflammatory response in the lungs of allergen-challenged β-cat-tg mice compared to control mice. the allergic response was particularly aggravated in cat-tg mice in the presence of il-. these data demonstrate that in mice with enforced expression of β-catenin functional inkt cells lead to a more robust response to il-25-dependent ahr challenge, with all measured parameters of allergic inflammation enhanced, including higher inkt production of th and th cytokines. β-catenin expression regulates the differentiation of effector inkt and inkt cells to determine the specific role of β-catenin in inkt cell development, we studied thymic inkt cells in mice with t-cell conditional deletion or enforced expression of β-catenin. β-cat-cko mice showed similar proportions and total cell numbers of thymocytes and mature t cells compared to control mice. however, β-cat-cko mice had a significant 50 % reduction in the number of inkt cells. β-cat-tg mice, on the other hand, presented a remarkable increase in the number of thymic inkt cells compared to control mice. in light of these data, we conclude that β-catenin expression is limiting for the generation of thymic inkt cells.fig. β-catenin expression regulates the differentiation of effector inkt and inkt cells. a-b frequency and total inkt cell numbers from control and β-cat-cko mice and from control and β-cat-tg mice. numbers over dot plots refer to total thymocyte cell numbers. c-d representative dot plots showing inkt inkt and inkt populations among inkt cells and total numbers from control and β-cat-cko mice and from control and β-cat-tg mice. gating strategies are shown in right. * p <.05; ** p <.01; *** p <.001 analysis of expression of transcription factors plzf and t-bet in inkt cell populations from control, β-cat-cko and β-cat-tg mice showed that the frequency and number of inkt and inkt cells was significantly affected by β-catenin expression. specifically inkt and inkt cells, but not inkt cells, accounted for the major decrease in β-cat-cko and the greatest increase in β-cat-tg mice. we conclude that β-catenin expression is required for the differentiation and expansion of inkt and inkt cells. β-catenin regulates cytokine production by inkt cells inkt cells acquire functional capabilities in the thymus as part of the developmental program. to determine if β-cat-tg inkt cells were functionally skewed towards a th2/th phenotype, we cultured β-cat-tg thymocytes in vitro with pma and ionomycin for 5 h and then assayed ifn-γ, il- il- and il- production by thymic inkt cells. the results showed that the frequency of ifn-γ-producing inkt cells from β-cat-tg mice was significantly reduced compared to inkt cells from control mice. strikingly, a much higher percentage of inkt cells from β-cat-tg mice expressed il- compared to control inkt cells, even in unstimulated conditions. likewise, we found enhanced production of il- and also il-13 + il-17a+ double-producers by β-cat-tg inkt cells compared to control inkt cells. we conclude that enforced expression of β-catenin promotes a higher frequency of th2- and th17-like inkt cells. importantly, we note that in the absence of stimulation approximately 26 % β-cat-tg thymic inkt cells produced il- compared to  % control thymic inkt cells. these data demonstrate that β-catenin promotes the acquisition of effector functionality in developing inkt cells in the thymus.fig. β-catenin regulates cytokine production by inkt cells. frequency of ifn-γ- and il-4-producing and il-13- and il-17a-producing inkt cells from control and β-cat-tg mice in in vitro unstimulated or pma + ionomycin stimulated inkt cells. left, representative experiment and right, graph showing mean ± sem, n =  * p <.05; *** p <.001 β-catenin regulation of inkt and inkt cells is maintained in the periphery we then examined the distribution of inkt cells in the periphery in the β-catenin mutant mice. we observed that spleen and liver from β-cat-cko mice showed a specific reduction of the inkt and inkt cells in the spleen, and β-cat-tg spleens had reduced inkt and increased inkt and inkt cells. analysis of liver from control, β-cat-cko and β-cat-tg mice showed comparable numbers of inkt cells. by contrast, the frequency of cytokine-producing cells was significantly diminished in β-cat-cko mice. we conclude that altered inkt cell development in β-cat-cko and β-cat-tg mice is reflected in the β-catenin-dependent skewing towards inkt and inkt cells in the distribution of these cells in the periphery.fig. β-catenin regulation of inkt and inkt cells is maintained in the periphery. a total cell numbers of inkt inkt and inkt cells from control, β-cat-cko and β-cat-tg mice in spleen and liver. b gating strategy for cd4+ il-17rb+ inkt cells is shown in left. right, frequency of il-4-expressing cd4+ il-17rb+ inkt cells from spleens of control and β-cat-tg mice 3 h after injection with pbs or 3 μg α-galactosylceramide. * p <.05; ** p <.01; *** p <.001 to further test the efficacy of induced response of th2-like inkt cells expanded in β-cat-tg mice we tested cytokine production in vivo. to address this we administered 3 μg α-galactosylceramide intraperitoneally to β-cat-tg and control mice and 3 h post-injection analyzed cytokine production by splenic inkt cells. αgc is known to activate inkt cells in vivo. importantly, we found that αgc-stimulated cd4+ il-17rb+ inkt cells in β-cat-tg mice produced high levels of il-. therefore, we conclude that enforced expression of β-catenin promotes th2-biased cytokine production by inkt cells in response to stimulation in vivo. enhanced generation of β-catenin-dependent inkt and inkt cells is cell autonomous to determine if the effect of enforced β-catenin expression on inkt cell generation was cell intrinsic, we generated chimeric mice using either control or β-cat-tg cells as bone marrow donor cells. analysis of chimeric mice showed that the frequency and numbers of inkt and inkt cells was significantly greater when the donor bone marrow was derived from β-cat-tg mice compared to control mice. furthermore, mixed chimeric mice generated with bone marrow cells derived from wild-type control and from β-cat-tg transferred into irradiated wild-type mouse acting as recipient also showed significantly increased generation of inkt and inkt cells. thus, this data demonstrates that β-catenin controls differentiation of effector inkt and inkt cells in a cell-intrinsic manner.fig. enhanced generation of β-catenin-dependent inkt and inkt cells is cell autonomous. a flow cytometry of a bone-marrow chimera experiment analyzed after 7– weeks, in which cd + recipient c57bl/ sjl mice were irradiated and million cells isolated from bone marrow were i.v. injected from cd + donors, either control or β-cat-tg mice. top, total inkt cells gated on cd + cells. middle, inkt inkt and inkt cells within the inkt populations. bottom, number of total inkt, inkt inkt and inkt cells from donor control and donor β-cat-tg cells. b flow cytometry of cd and cd on thymic cells of mixed bone-marrow chimeras containing cd + recipient c57bl/ sjl cells, cd  + cd + donor control cells and cd + donor β-cat-tg cells. inkt cells from both donor cells and inkt inkt and inkt cells within the inkt populations. representative data out of three mice analyzed. bottom, number of total inkt, inkt inkt and inkt cells from donor control and donor β-cat-tg cells. mice were analyzed 7 weeks after mixed bone-marrow chimeras were performed. * p <.05; ** p <.01; *** p <.001 discussion in this study we demonstrate that β-catenin regulates the differentiation of inkt cells to inkt and inkt but not inkt effector subsets. conditional deletion of β-catenin in inkt cell-precursors impaired differentiation of inkt and inkt but not inkt cells. by contrast, enforced expression of β-catenin promoted the generation of inkt cells and inkt cells without affecting generation of inkt cells. the effect of enhanced β-catenin expression in inkt cell precursors to promote development of inkt and inkt cells was cell-autonomous. importantly, we show that β-cat-tg inkt cells acquired the ability to produce enhanced levels of il- and il- during development that augmented lung inflammation in a mouse model for asthma. cytokine production by inkt inkt and inkt effector subsets has been shown to be controlled by transcription factors t-bet, gata and rorγt that also regulate ifn-γ, il- and il- production by t helper cells. however, transcriptional control of differentiation of inkt cells to inkt inkt and inkt subsets has remained to be defined. this report shows that β-catenin deficiency resulted in a profound decrease in inkt and inkt subsets of inkt cells whereas inkt cells developed normally. by contrast, enforced expression of β-catenin promoted the development of inkt and inkt cells. it was important to note that the majority of inkt cells in the thymus of c57bl/ mice were inkt cells and the observation that enforced expression of β-catenin altered the pattern to inkt and inkt cells suggests that β-catenin may be a major factor in the distinct pathways that critically direct differentiation of inkt effector subsets. these observations are congruent with the observation that a cohort of genes that have been previously implicated in inkt cell development were up-regulated in mice with enforced expression of β-catenin and diminished in expression in cells with deletion of β-catenin. unlike effector cd t cells, inkt cells acquire functional capabilities during the differentiation and maturation process. it was interesting to note that β-cat-tg inkt cells developed the capacity to produce enhanced levels of il- which was reminiscent of the report that tcf and β-catenin promote expression of gata and il- in t helper cells. thus, we posit that β-catenin-dependent gene expression regulates the differentiation and functional maturation of inkt and inkt cells. finally, our results obtained from the allergic lung inflammation mouse model may be of some clinical importance. although the precise role for the different human inkt subsets in the lung and peripheral blood has yet to be clearly defined, correlative studies suggest that inkt cells are involved in exacerbations of asthma. furthermore, il- has been reported to be increased in asthmatic humans. patients with high il-25- and il-17rb-containing airway epithelial cells present a more prominent th reaction and respond better to inhaled corticosteroids treatment. we hypothesize that inkt cells may also be increased in il-25-high asthmatic patients facilitating the generation of “th2-high” asthma, as opposed to a severe, neutrophil-mediated, steroid-insensitive disease. modula ting the numbers of inkt cells may be beneficial in reducing asthma symptoms and exacerbations. targeting novel pathways or molecules involved in inkt differentiation, such as β-catenin as we have described here, could be a new approach. in conclusion, this study shows that β-catenin expression regulates the differentiation of inkt cells that augment lung inflammation associated with asthma and provides insights that may be of use in designing regimens to limit at least one type of lung inflammation.  in conclusion data presented in this paper indicate that β-catenin regulates the differentiation of inkt cells to inkt and inkt but not inkt effector subsets. we propose that inkt and inkt cells promote asthma and posit that modulating the development and function of these cells provides novel means to control of lung inflammation. abbreviations inktinvariant natural killer t dpdouble positive thymocytes tcrt-cell receptor icd8innate-like cd8 ilinterleukin ahrairway hyperresponsiveness ifn-γinterferon gamma αgcα-galactosylceramide competing interests the authors declare that they have no competing interests. authors’ contributions jms, rb-b and as designed the experiments; rb-b, as and das performed experiments; fcs, kp, y-hk, c-hc and h-hx contributed chimera experiments; jxf and nmh contributed to the lung inflammation experiments. all authors read and approved the final manuscript. authors’ information not applicable. 
this report shows that β-catenin deficiency resulted in a profound decrease in inkt and inkt subsets of inkt cells whereas inkt cells developed normally. this effect of expression of β-catenin in inkt cell precursors was cell autonomous. a role for β-catenin expression in promoting inkt and inkt subsets was confirmed when we noted that enforced transgenic expression of β-catenin in inkt cell precursors enhanced the frequency and number of inkt and inkt cells at the cost of inkt cells. thus, we demonstrate that β-catenin expression in inkt cell precursors promotes differentiation toward inkt and inkt effector subsets and supports enhanced capacity to produce type and cytokines which in turn augment lung inflammation in mice. it was important to note that the majority of inkt cells in the thymus of c57bl/ mice were inkt cells and enforced expression of β-catenin altered the pattern to inkt and inkt cells suggesting that β-catenin may be a major factor in the distinct pathways that critically direct differentiation of inkt effector subsets. inkt cells differentiate and acquire functional characteristics during development in the thymus. however, the correlation between development of inkt cells in the thymus and role in lung inflammation remains unknown. in addition, transcriptional control of differentiation of inkt cells into inkt cell effector subsets in the thymus during development is also unclear. beta-catenininkt cellsinkt2inkt il-17rbissue-copyright-statement© the author 2015 invariant natural killer t cells have been implicated in lung inflammation in humans and also shown to be a key cell type in inducing allergic lung inflammation in mouse models. to study the role for β-catenin in lung inflammation we utilize mice with conditional deletion and enforced expression of β-catenin in a well-established mouse model for il-25-dependen lung inflammation. furthermore, inkt cells acquired greater capability to produce type- cytokines when β-catenin expression was enhanced. specifically, we demonstrate that conditional deletion of β-catenin permitted development of mature inkt cells while impeding maturation of inkt and cells. in this report we show that β-catenin dependent mechanisms direct differentiation of inkt and inkt subsets but not inkt cells.
 invariant natural killer t cells have been implicated in lung inflammation in humans and also shown to be a key cell type in inducing allergic lung inflammation in mouse models. inkt cells differentiate and acquire functional characteristics during development in the thymus. however, the correlation between development of inkt cells in the thymus and role in lung inflammation remains unknown. in addition, transcriptional control of differentiation of inkt cells into inkt cell effector subsets in the thymus during development is also unclear. in this report we show that β-catenin dependent mechanisms direct differentiation of inkt and inkt subsets but not inkt cells. methods to study the role for β-catenin in lung inflammation we utilize mice with conditional deletion and enforced expression of β-catenin in a well-established mouse model for il-25-dependen lung inflammation.  specifically, we demonstrate that conditional deletion of β-catenin permitted development of mature inkt cells while impeding maturation of inkt and cells. a role for β-catenin expression in promoting inkt and inkt subsets was confirmed when we noted that enforced transgenic expression of β-catenin in inkt cell precursors enhanced the frequency and number of inkt and inkt cells at the cost of inkt cells. this effect of expression of β-catenin in inkt cell precursors was cell autonomous. furthermore, inkt cells acquired greater capability to produce type- cytokines when β-catenin expression was enhanced. discussion this report shows that β-catenin deficiency resulted in a profound decrease in inkt and inkt subsets of inkt cells whereas inkt cells developed normally. by contrast, enforced expression of β-catenin promoted the development of inkt and inkt cells. it was important to note that the majority of inkt cells in the thymus of c57bl/ mice were inkt cells and enforced expression of β-catenin altered the pattern to inkt and inkt cells suggesting that β-catenin may be a major factor in the distinct pathways that critically direct differentiation of inkt effector subsets.  thus, we demonstrate that β-catenin expression in inkt cell precursors promotes differentiation toward inkt and inkt effector subsets and supports enhanced capacity to produce type and cytokines which in turn augment lung inflammation in mice. keywords beta-catenininkt cellsinkt2inkt il-17rbissue-copyright-statement© the author 2015 