The transcription factor STAT6 has been shown to be an important regulator of Th2-type inflammation making it a potential target for therapy in allergic inflammatory disorders such as asthma (38
). Among the many functions of STAT6 is the regulation of chemokine production in the lung during the establishment of allergic airway inflammation (23
). The goal of the current study was to define the critical cellular mediators of STAT6-dependent Th2 cell-active chemokine secretion in the lung during allergic inflammation. Although there are multiple potential cellular sources for these chemokines in the lung (39
), we hypothesized that myeloid-derived cells in the lung, such as pulmonary dendritic cells and/or macrophages, were the primary mediators of chemokine production and Th2 cell recruitment in this model of asthma. Our data suggest that these cells are bone marrow-derived CD11b+
myeloid cells. These cells have already been demonstrated to be critical for initiation of the adaptive immune response in asthma (35
); however our data reveal a novel role for these cells in controlling the effector phase of allergic inflammation by mediating Th2 cell recruitment.
We have previously demonstrated that following adoptive transfer, recruitment of Th2 cells into the airways following antigen exposure occurs via both STAT6-dependent and -independent mechanisms (16
). This is based on the fact that STAT6-/-
mice have reduced Th2 cell recruitment into the airways compared to wild-type mice, but these mice maintain some minimal Th2 cell recruitment that is fully eliminated when chemoattractant receptor signaling is disrupted with pertussis treatment of the Th2 cells (16
). However, STAT6 is clearly necessary for the full dramatic accumulation of Th2 cells in the airways that develops in response to antigen challenge (16
). Since Th2 cells in the lung and airway do not proliferate extensively in response to antigen (14
), the increase in the number of Th2 cells, as well as eosinophils, is dependent on cellular recruitment. These data suggest that in response to IL-4 and IL-13 (produced by the Th2 cells recruited into the lung by STAT6-independent mechanisms) resident lung cells are stimulated via STAT6 to produce chemokines, such as CCL17, CCL22, CCL11, and CCL24. These chemokines then amplify allergic inflammation by increasing the recruitment of Th2 cells and initiating the recruitment of eosinophils into the airways. Consistent with this, studies on humans with asthma have demonstrated increased levels of the Th2-active chemokines CCL17 and CCL22 in the airways (58
), and inhibition of the activity of these chemokines has been shown to reduce allergic inflammation in murine models of asthma (18
). Similarly, the deletion of the eosinophil-active chemokines CCL11 or CCL24 reduced lung eosinophil accumulation in murine models of asthma (44
). These data suggest that STAT6 is a master regulator of cellular recruitment in asthma, and thus may be an effective target to reduce Th2 cell and eosinophil recruitment into the lung. Given the potential therapeutic utility of targeting of STAT6, a more cell-specific approach would be ideal. Thus, we used a strategy that targeted specific cell types in the lung looking for a cell or cells that are necessary and sufficient to lead to Th2 lymphocyte recruitment.
In our experiments, we used the adoptive transfer model of allergic airway inflammation (16
). This model was chosen because the T cell polarization step is performed in vitro
, thus we could eliminate effects on T cell priming/activation and focus on effects on recruitment. In addition, mast cells are not activated in this model, simplifying the analysis of our findings. Previously, we had shown in this model that there was significantly reduced wild-type OVA-specific Th2 cell recruitment into the airways of STAT6-/-
recipient mice compared to wild-type recipient mice following OVA challenge. These results differ from data recently published by King et al.
, which did not demonstrate a Th2 cell recruitment defect in STAT6-/-
mice following naïve T cell transfer and N. brasiliensis
infection with OVA sensitization (60
). However, their model involved T cell priming/activation in vivo
and intact IgE-mast cell responses, so is not completely analogous to our model of allergic inflammation. Furthermore, even though the authors report that there was not a Th2 cell recruitment defect, the percentage of OVA-specific T cells recruited into the BAL in their model was 50% less in STAT6-/-
mice compared to wild-type recipients.
In our first set of experiments, we used mice engineered to express STAT6 under control of the CC10 promoter (31
) to test the role of airway lining cells in the recruitment of Th2 cells and eosinophils. Expression of STAT6 in these cells restored mucus production in the model but did not lead to increased Th2 cell recruitment or eosinophil recruitment compared to STAT6-/-
mice. The EpiSTAT6 mice as well as STAT6-/-
mice had reduced levels of CCL17, CCL11, and CCL24 RNA in the lung. Interestingly, RNA levels of CCL22 were restored but there was no detectable protein in the BAL. It may be that CCL22 is not secreted or remains bound to cellular surfaces as seen with other chemokines (61
). The protein levels of CCL17 and CCL24 were reduced in the BAL of EpiSTAT6 and STAT6-/-
mice compared to wild-type mice, but we were unable to detect significant amounts of CCL11 in the BAL in wild-type, STAT6-/-
mice or the EpiSTAT6 mice. Our experiments demonstrate that expression of STAT6 in CC10 expressing airway lining cells is not sufficient to restore Th2 cell and eosinophil trafficking into the lung in this model. We cannot rule out a role for non-CC10 expressing airway epithelia, however, as these cells would remain STAT6-/-
in the EpiSTAT6 mouse.
We wondered if the eosinophil trafficking defect seen in the STAT6-/- mice and EpiSTAT6 mice was secondary to the Th2 cell trafficking defect or if there was also a coexisting defect in eosinophil trafficking that resulted directly from STAT6-deficiency. In our experiments we bypassed the need for Th2 cell recruitment by transferring the cells directly into the airways via an intratracheal injection. In these experiments, transfer of wild-type OVA-specific Th2 cells directly into the lung of STAT6-/- mice did not restore eosinophil recruitment or the production in the lung of the eosinophil-active chemokines. These data demonstrate that the eosinophil recruitment in this model is also dependent on STAT6-induced chemokine production in the lung.
Voehringer et al
have demonstrated that STAT6 expression in a bone marrow derived myeloid cell is necessary for Th2 cell and eosinophil trafficking into the lung in a model of pulmonary Th2 inflammation induced by N. brasiliensis
). However, these experiments did not identify whether the critical cellular mediator was a lung dendritic cell or macrophage, nor did the studies directly address Th2 cell recruitment in an asthma model. We sought to determine if a similar mechanism was involved in Th2 cell and eosinophil recruitment in a model of allergic airway inflammation. For these experiments, we reconstituted STAT6-/-
mice with wild-type, STAT6-/-
bone marrow. Wild-type or RAG1-/-
bone marrow was able to fully restore the recruitment of Th2 cells and eosinophils in the model. In these experiments, the expression of the chemokines CCL17, CCL22, and CCL24 was restored with wild-type or RAG1-/-
bone marrow transplant into STAT6-/-
mice. Interestingly, CCL11 was not expressed in large amounts at either the RNA or the protein level. Given that STAT6-/-
mice reconstituted with RAG1-/-
bone marrow would only have STAT6 restored in myeloid cells, these data demonstrate that expression of STAT6 in bone marrow derived myeloid cells is sufficient to restore Th2 cell and eosinophil trafficking in a model of allergic airway inflammation.
The bone marrow reconstitution experiments do not fully define the cellular mediator of STAT6-dependent recruitment in this model. For this reason, we wanted to use a system that allowed us to selectively deplete myeloid cell populations in the lung. The identification of dendritic cells and macrophages using cell-type specific markers is different in the lung than in other organs (32
). Specifically, the dendritic cell marker CD11c has been shown to be expressed on both pulmonary macrophages and dendritic cells, and the myeloid marker CD11b, is seen at high levels on pulmonary dendritic cells and at low levels on pulmonary macrophages (32
). Thus, we decided to utilize a transgenic mouse that allowed us to deplete the CD11b+
subpopulation of myeloid cells in the lung using DT, which should have its primary effect on dendritic cells with minimal effects on macrophages. Careful phenotyping of cell types in the lungs of mice after DT administration indicated a very specific reduction in the number of CD11b+
myeloid cells in the lung, and minimal effects on other CD11b+
populations. In these experiments, the reduction in CD11b+
cells significantly attenuated the recruitment of Th2 cells and eosinophils into the lung following adoptive transfer of wild-type Th2 cells and OVA challenge. Furthermore, add back of wild-type bone-marrow derived dendritic cells was able to restore the recruitment of Th2 cells in these mice, demonstrating that these cells were sufficient for Th2 cell recruitment and that the recruitment defect in the DT treated CD11b-DTR mice was due to the deletion of these myeloid cells. In addition, there was a significant reduction in the expression of CCL17, CCL22, CCL11, and CCL24 RNA in the lungs of the CD11b-DTR mice treated with DT compared to those treated with PBS. These data are consistent with the RNA expression profile we generated by isolating these cells from the lung using a cell sorter as well as with data published by others (37
). It is interesting that there is such a dramatic decrease in Th2 cell recruitment and chemokine expression with a 3-fold reduction in this cell population; however, we speculate that we may be depleting the most active CD11b+
cells with DT treatment. Alternatively, we may be reducing the numbers of a subpopulation of CD11b+
myeloid cells below a critical threshold that significantly impairs chemokine production and Th2 cell recruitment. Our results differs somewhat from data published by Beaty et al.
which demonstrated that both CD11c+
dendritic cells and CD11c+
dendritic cells in the lung can produce high levels of the chemokines CCL17 and CCL22 in a murine model of asthma (33
). However, they used a different model of asthma for their experiments. In addition, our findings are in agreement with findings by van Rijt et al.
), which demonstrated that DT-induced depletion of CD11c+
cells in the lungs prevented the development of allergic airway inflammation. This study utilized the OVA immunization model to demonstrate the importance of dendritic cells in the development of allergic airway inflammation. While their study largely focused on the early role of these cells in antigen presentation to T cells, they also examined the effects of CD11c+
depletion on allergic inflammation in the adoptive transfer model. However, in those experiments deletion of CD11c+
cells depleted pulmonary macrophages (CD11c+
) as well as myeloid dendritic cells (CD11c+
), and thus they were not able to define the critical cellular mediator of Th2 cell recruitment in the adoptive transfer model of asthma. In our experiments, we were able to delete only the CD11c+
myeloid cell population and therefore we have extended their observations by more specifically defining the critical cellular mediator of Th2 cell recruitment.
An alternative explanation for our findings is that STAT6 deficiency in CD11b+
myeloid cells affects antigen presentation to Th2 cells, thus the decreased accumulation of Th2 cells in the lung is secondary to a failure to reactivate primed Th2 cells. Recent data has demonstrated that IL-4 and IL-13 can influence the maturation of dendritic cells in the lung (presumably via STAT6 signaling) and can affect their ability to regulate cytokine expression by memory CD4+
T cells (65
). Although this mechanism may be partially responsible for our findings, we believe our findings demonstrate that CD11b+
myeloid cells in the lung have an additional role in directing Th2 cell recruitment into the airways via STAT6-dependent chemokine production. To evaluate the possibility that the phenotype we observed was also secondary to effects on antigen presentation, we examined at the accumulation of the transferred Th2 cells in the draining thoracic lymph nodes. Previous studies had demonstrated that transferred Th2 cells will proliferate in the lymph node only following presentation of inhaled antigen by dendritic cells (14
). The fact that the numbers of transferred Th2 cells in the draining thoracic lymph node were similar in mice following DT-induced depletion of CD11b+
myeloid cells compared to PBS control mice, suggests that antigen presentation was at least partially intact. In addition, we did not see a difference in blood eosinophilia in the two groups of mice suggesting that IL-5 secretion from Th2 cells following CD11b+
cell depletion was not affected. As further evidence, we used IL-13 to stimulate chemokine production (thus bypassing antigen presentation) in CD11b-DTR mice and demonstrate that Th2-active chemokine production was significantly impaired after treatment with DT compared to mice treated with PBS. Thus, we believe these data definitively demonstrate that CD11b+
myeloid cells have a critical role mediating Th2 cell recruitment independent of their role in antigen presentation.
Our data confirm that the recruitment of eosinophils in this model is also dependent on STAT6 expression in a resident lung cell (23
). Consistent with prior data from others (53
), we demonstrate that CD11c+
cells are a source of CCL24 but so are CD11c+
cells (alveolar macrophages), and neither cell makes large amounts of CCL11. In addition to effects on cellular recruitment, STAT6 also is necessary for mucus hypersecretion in this model (23
). In our experiments, we observed that following adoptive transfer and OVA challenge, EpiSTAT6 mice develop mucus hypersecretion similar to wild-type mice, but reconstitution of STAT6 expression in myeloid cells did not restore this endpoint of allergic inflammation. These data are similar to those of Kuperman et al.,
which demonstrated that mucus producing cells are derived from Clara cells in the airways (66
). Our data also suggest that the multiple endpoints of allergic inflammation can be anatomically compartmentalized with different cell-types controlling different STAT6-dependent processes.
In conclusion, our data demonstrate that STAT6 expression in a CD11b+ myeloid cell in the lung is necessary for effective Th2 cell recruitment into the airways in a murine model of allergic airway inflammation. We propose that following STAT6 independent recruitment of Th2 cells into the airway, CD11b+ cells in the lung are stimulated by IL-4 and IL-13 to produce CCL17 and CCL22 via STAT6. These chemokines then help orchestrate the amplification of Th2 cell recruitment. Our studies illuminate a novel link between the innate and adaptive immune systems and suggest that a therapeutic strategy targeting STAT6 in CD11b+ cells may provide a novel means of reducing allergic airway inflammation.