Expression of TLR5 and Muc1 by DC
By Western immunoblotting, we confirmed that wild-type but not Muc1−/− knockout DC expressed abundant Muc1 under resting conditions (fig. ). In addition, the expression of Muc1 was not affected after activation with LPS or flagellin in either Muc1−/− DC or Muc1+/+ DC (fig. ). We noted a faint Muc1 immunoreactive band in DC derived from Muc1−/− mice, which may likely represent cross-reactivity with other Mucin species by the polyclonal antibody used in these studies. By contrast, both Muc1−/− and Muc1+/+ DC expressed intact TLR5 protein at rest and on activation with either LPS or flagellin (fig. ). Thus, we confirmed that Muc1+/+ DC expressed both TLR5 and Muc1, whereas Muc1−/− DC expressed TLR5 normally with disrupted Muc1 expression.
Fig. 1 Western immunoblotting images of Muc1 mucin isoform 7 (clone EP1024Y), TLR5 (clone M300) and β-actin as an internal control of equivalent protein loading as detected by enhanced chemiluminescence. The expected positively staining bands of Muc1 (more ...)
Analysis of Extracellular Antigen Uptake by DC
One of the key functions of DC is to sample their environment by taking up extracellular proteins and particulate antigens by mechanisms that include phagocytosis and endocytosis. We assessed antigen uptake by exposing resting and activated DC to exogenous low molecular weight (40 kDa) FITC-conjugated dextran and measuring DC MFI using flow cytometry in a comprehensive analysis of time-dependent antigen uptake over a 4-hour period.
Unexpectedly, we found that resting Muc1+/+
DC more efficiently took up FITC-dextran as compared with their Muc1−/−
DC counterparts both at baseline and after cell stimulation (fig. ). For example, whereas Muc1+/+
DC continued to take up exogenous FITC-dextran for the duration of the assay (up to 240 min), by comparison this ability remained dampened in Muc1−/−
DC between 180 and 240 min (fig. , data expressed as geometric MFI). Although there were trends for suppressed uptake of FITC-dextran by DC after exposure to LPS and flagellin, these did not reach statistical significance (fig. ). Flow cytometric histograms from one of the experiments completed for BM-DC are also shown (fig. ). It can be appreciated that Muc1−/−
DC show a dampened uptake of exogenous antigen over the entire time period of this assay as compared with their wild-type DC counterparts. These data suggest that Muc1 plays a previously under-appreciated role in the uptake of exogenous antigens by DC and are in keeping with a recent study implicating Muc1 in lectin-mediated endocytosis [30
] (see Discussion).
Fig. 2 Time-dependent uptake of FITC-conjugated dextran (molecular weight 40 kDa) by Muc1+/+ and Muc1−/− DC generated in vitro from bone-marrow precursors. The uptake of FITC-dextran was assessed in resting DC (a) and on activation by LPS (b (more ...)
A series of flow histograms of FITC-dextran uptake of immature BM-DC from Muc1+/+ and Muc1−/− mice from a typical experiment is also shown for the whole time course of the experiment (d).
To confirm intracellular accumulation of FITC-dextran using an alternative approach, we next employed imaging fluorescence cytofluorography, which involves quantitative analysis of subcellular localization using an ImageStream flow cytometer (see Materials and Methods and [27
]). Using this approach and the software associated with this technique (IDEAS version 3.0.245) we were able to distinguish membrane-bound FITC-dextran from intracellularly accumulated and taken up FITC-dextran, and confirmed that this material is endocytosed by both Muc1+/+
BM-DC (fig. ). In the first series of measurements (fig. ) we validated the technique for measuring internalization of FITC-conjugated dextran particles (see Materials and Methods). In the second series of measurements, we confirmed that both Muc1+/+Muc1−/−
BM-DC took up FITC-dextran from their extracellular environment and in the panels shown (fig. for wild-type DC, fig. for knockout DC), FITC-dextran uptake after 60 min following exposure of DC to this material is clearly shown. For comparison, BM-DC that have not taken up FITC-dextran are also shown (red rectangular gate, fig. , ). A summary of the data employing this technique is also shown (fig. ) and describes the percent internalized FITC-dextran by Muc1+/+
as compared with Muc1−/−
BM-DC at t = 0, t = 30 and t = 60 min after exposure of DC to FITC-dextran. For comparison, the percent internalized FITC-dextran of BM-DC held at 4°C and analyzed under identical conditions is also shown (fig. ). As can be seen, BM-DC analyzed for FITC-dextran uptake at 37°C show 8–10 times greater internalized fluorescence than their control counterparts held at 4°C. Thus, using 2 distinct approaches we concluded that FITC-dextran endocytosis, a receptor-mediated event in DC, is unexpectedly attenuated in the absence of Muc1.
Fig. 3 Validation of the time-dependent internalization of FITCdextran by ImageStream cytoflourographic analysis of Muc1+/+ and Muc1−/− DC. In experiments of FITC-dextran uptake by endocytic and phagocytic mechanism described above, aliquots (more ...)
Fig. 3 Representative data from Muc1+/+ DC is shown (c) as well as from Muc1−/− DC (d) sampled 60 min after pulsing DC with FITC-dextran. Also included are representative images, with the overlay showing the FITC signal (green) clearly inside (more ...)
Muc1−/− DC Exhibit Heightened Co-Stimulatory Molecule Expression
In addition to displaying processed antigen peptides in the context of MHC, activated DC up-regulate the expression of co-stimulatory molecules that provide the essential second signal for T cell activation. Using immunofluorescence and flow cytometry, we found that the constitutive expression of CD40, CD80 and CD86 was significantly elevated on Muc1−/− BM-DC as compared with their wild-type counterparts (p < 0.01; fig. ). The expression of CD40, CD80 and CD86 was further enhanced on Muc1+/+ DC in response to LPS or flagellin, consistent with a maturing phenotype (fig. , ). In contrast, only CD40 and CD80 were significantly increased on Muc1−/− DC in response to LPS (fig. , ), and although there were trends towards increased expression of CD86 in response to LPS stimulation, these differences were not statistically significant. Interestingly, Muc1−/− DC also failed to show enhanced expression of any of the 3 co-stimulatory molecules examined after stimulation with flagellin (fig. , ). This was in sharp contrast to flagellin-stimulated wild-type DC, which up-regulated CD40, CD80 and CD86 compared to resting cells (fig. , ). One possible explanation for these data is that DCs depend in part on intact expression of Muc1 for enhanced expression of co-stimulatory molecules in response to flagellin.
Fig. 4 Cell surface-expression of co-stimulatory molecules in Muc1+/+ and Muc1−/− BM-DC as quantified by multi-parameter flow cytometry. Data are expressed (in a and b) as geometric mean fluorescence intensity (MFI) ± 1 standard deviation (more ...)
c Typical sets of flow cytometric histograms of the data described in . Data represent geometric mean fluorescence intensity (MFI) and percent (%) of cells positive of the described data.
In order to ensure that the BM-DC used in these experiments faithfully represented DC in situ, we next isolated highly pure primary lung (fig. ) and splenic (fig. ) DC from Muc1−/− DC and Muc1+/+ mice. In these experiments, we studied expression of the co-stimulatory molecules CD40, CD54 and CD86, and found that in contrast to BM-DC, the expression of CD40 by lung DC was similar between genotypes in the resting state and after LPS stimulation (fig. ). However, Muc1−/− lung DC displayed enhanced expression of CD40 on activation by flagellin while their wild-type counterparts did not respond under these conditions (p < 0.05; fig. ). We also found that CD86 expression by lung DC was similar between genotypes and did not change on either Muc1−/− or Muc1+/+ lung DC after stimulation by either LPS or flagellin (fig. ). By contrast, expression of CD54 was markedly greater on lung Muc1−/− DC as compared to their wild-type counterparts both before and after stimulation with LPS or flagellin. Both Muc1−/− and Muc1+/+ lung DC exhibited marginal but statistically significant enhanced expression of CD54 on activation by LPS and flagellin (p < 0.05; fig. ).
Fig. 5 Cell surface-expression of co-stimulatory molecules CD40, CD54 (ICAM-1) and CD86 by Muc1+/+ and Muc1−/− primary lung DC as quantified by multi-parameter flow cytometry. Data are expressed (in a and b) as geometric mean fluorescence intensity (more ...)
Fig. 6 Cell surface-expression of co-stimulatory molecules CD40, CD54 (ICAM-1) and CD86 by Muc1+/+ and Muc1−/− primary splenic DC as quantified by multi-parameter flow cytometry. Data are expressed (in a and b) as geometric mean fluorescence (more ...)
We also observed that expression of CD40 by Muc1−/− and Muc1+/+ splenic DC was similar and only partially enhanced on activation by LPS (p < 0.05; fig. ). Additionally, while the expression of CD86 was also similar between genotypes, LPS enhanced expression of CD86 in both Muc1−/− and Muc1+/+ splenic DC (p < 0.05), while only Muc1−/− DC responded to flagellin, further supporting our hypothesis of enhanced sensitivity to exogenous activation of Muc1−/− DC by this TLR 5 agonist (p < 0.05; fig. ). Although the expression of CD40 and CD86 was similar on splenic DC from both genotypes, the expression of CD54 was markedly greater on splenic Muc1−/− DC than their wild-type counterparts both in the resting state and following stimulation (fig. ). However, both Muc1−/− and their wild-type counterparts exhibited enhanced expression of CD54 on activation by LPS or flagellin.
Activation of Muc1−/− DC by Flagellin Magnifies Pro-Inflammatory Cytokine Production
When examining cytokine secretion, we found that deletion of Muc1−/− in BM-DC promoted an unusual pattern of constitutive cytokine secretion as well as altered responsiveness upon activation by LPS or flagellin (fig. ). Constitutive secretion of IL-12p40 by either Muc1+/+ or Muc1−/− BM-DC was very low. However, secretion of IL-12p40 by Muc1−/− DC was markedly enhanced on activation of both Muc1−/− and Muc1+/+ DC (p < 0.01; fig. ). Interestingly, on activation by flagellin, Muc1−/− BM-DC secreted far greater levels of IL-12p40 than their wild-type counterparts in several independent experiments (fig. ). Under these circumstances, Muc1−/− DC secreted ~2.5-fold more IL-12p40 than their wild-type counterparts in response to flagellin (p < 0.01; fig. ). We observed a very similar pattern when assessing the secretion of TNF-α by Muc1−/− as compared with Muc1+/+ DC (fig. ). Muc1−/− DC secreted far greater levels of TNF-α on activation by flagellin as compared with their wild-type counterparts (fig. ), while the levels secreted by LPS stimulated Muc1−/− and Muc1+/+ DC were highly comparable (fig. ). In contrast, whereas both LPS and flagellin increased IL-10 secretion in Muc1+/+ DC (fig. ), only LPS modestly enhanced IL-10 production in Muc1−/− DC but at levels that were at least half of those produced by there wild-type counterparts (fig. , p < 0.05). Moreover, while flagellin provoked enhanced secretion of IL-10 by Muc1+/+ DC, it failed to stimulate enhanced release of IL-10 by Muc1−/− BM-DC counterparts (fig. ).
Fig. 7 Secretion of inflammatory cytokine production by Muc1+/+ and Muc1−/− BM-DC (see Materials and Methods). Data are described as pg of cytokine secreted per million DC (pg/106 cells) ± 1 standard deviation about the mean (n = 59 independent (more ...)
We also studied secretion of the chemokine KC (or CXCL1) by DC and determined that under resting conditions the spontaneous secretion of KC was similar for Muc1−/− and Muc1+/+ DC and that the magnitude of enhanced secretion of KC was similar between genotypes on activation by LPS (p < 0.01; fig. ). By contrast, Muc1−/− BM-DC exhibited significantly enhanced production of KC as compared their wild-type counterparts on activation by flagellin (p < 0.05; fig. ).
A striking finding was that Muc1−/− DC secreted constitutively greater levels of the pleiotropic cytokine VEGF than Muc1+/+ DC (p < 0.01). VEGF secretion was further increased in Muc1−/− DC in response to both LPS and flagellin, whereas only LPS significantly increased VEGF secretion by Muc1+/+ DC (p < 0.05). Additionally, Muc1−/− DC also secreted constitutively greater levels of the cytokine IL-6 than Muc1+/+ DC (p < 0.05). IL-6 secretion was further increased in Muc1−/− DC and Muc1+/+ DC in response to both LPS and flagellin, although the levels secreted by Muc1−/− DC were again greater than their wild-type BM-DC counterparts (p < 0.05).
Activation of Lung or Splenic DC and Differential Pro-Inflammatory Cytokine Secretion
Next, we investigated cytokine secretion using primary lung (fig. ) or splenic (data not shown) DC from both genotypes. Since in vitro propagated BM-DC displayed hyper-responsiveness to in vitro stimulation by LPS and/or flagellin, we wanted to establish to what extent these important observations would be reproduced by primary DC freshly isolated from lung and spleen of Muc1−/− and Muc1+/+ mice.
Fig. 8 Secretion of inflammatory cytokine production by Muc1+/+ and Muc1−/− primary lung DC (see Materials and Methods; ad). Data are described as pg of cytokine secreted per million DC (pg/106 cells) ± 1 standard deviation about the (more ...)
We found that many of our observations established with BM-DC were reproduced in DC purified from lung and spleen, and in many examples, Muc1−/− DC isolated from these organs were found to be even more sensitive to ex vivo stimulation with LPS or flagellin. For example, we found that lung Muc1−/− DC constitutively secreted greater levels of IL-12p40 than their wild-type counterparts both at baseline and following LPS and flagellin exposure (p < 0.05; fig. ). Similar observations were made for the secretion of TNF-α (fig. ) by lung DC, although we could not detect significant levels of constitutive secretion by either Muc1−/− and Muc1+/+ lung DC (fig. ).
When considering secretion of IL-10 (fig. ), there was little constitutive IL-10 secretion whereas both LPS and flagellin enhanced secretion of IL-10 by wild-type Muc1+/+ DC more than Muc1−/− DC (p < 0.05; fig. ), similar to the case of bone marrow-derived DC (fig. ). Finally, both Muc1−/− and Muc1+/+ lung DC secreted similarly low levels of the chemokine KC/CXCL1 that could be markedly enhanced on activation by LPS or flagellin (p < 0.01; fig. ). Concordant with our observations of IL-12p40 and TNF secretion by lung DC, LPS- or flagellin-stimulated Muc1−/− DC tended to secrete greater levels of KC than their Muc1+/+ DC counterparts (fig. ).
We observed very similar responses using primary spleen DC isolated from wild-type and Muc1−/− mice (data not shown). For example, similar to primary lung DC, spleen DC from Muc1+/+ mice secreted significantly more IL-12p40, and TNF, but less IL-10 than DC from Muc1−/− mice after activation with LPS and/or flagellin (not shown). Finally, concordant with the observations for lung DC, we found that Muc1−/− splenic DC secreted more KC on activation by LPS or flagellin as compared their Muc1+/+ DC counterparts (not shown, p < 0.01). Although there were some stimulus-specific differences in cytokine secretion when comparing BM-DC with primary lung or spleen DC (fig. , ), taken together these data strongly support the idea that Muc1 normally dampens pro-inflammatory cytokine secretion in DC in response to different TLR ligands (see also Discussion).
Muc1−/− DC Constitutively Drive Greater Naive CD4+ T Cell Proliferation and Altered T Cell Responsiveness
One of the most important functions of DC is to migrate to the draining regional lymph nodes and form stimulatory synapses with naive CD4+ T cells. DC-mediated activation of T cell activation can be estimated in vitroby titrating variable numbers of DC against a constant number of CD4+ T cells and measuring cell cycle progression and cytokine production. In order to examine the effects of Muc1 deficiency on the ability of DC to stimulate T cells, we titrated 5-fold dilutions of resting or flagellin-stimulated DC from Muc1−/− mice or their wild-type counterparts with highly pure naive allogeneic responder T cells. After 4 days of co-culture, specific CD4+ T cell proliferation was quantified by the incorporation of BrdU (see Methods). Consistent with greater expression of costimulatory molecules (fig. ), we found that Muc1−/− BM-DC exhibited a constitutive ability to more potently stimulate naive CD4+ T cell proliferation than wild-type DC (fig. ). Activation of Muc1+/+ DC with flagellin enhanced CD4+ T cell proliferation compared with unstimulated Muc1+/+ DC (fig. ). In contrast, flagellin-exposed Muc1−/− DC did not induce greater T cell proliferation than resting Muc1−/− DC implicating that such DC were already at a heightened state of CD4+ T cell allo-stimulation.
Fig. 9 Quantitation of naive CD4+ T cell proliferation on contact with resting (a) or flagellin-activated (b) Muc1+/+ and Muc1−/− BM-DC. Resting or flagellin-activated DC were reciprocally titrated (1 to 5 dilution series) against a constant (more ...)
We also contrasted the stimulatory ability of BM-DC with their lung (fig. ) and splenic (fig. ) counterparts. Concordantly, we found that in the resting state, both lung and splenic Muc1−/− DC exhibited a heightened ability to stimulate naive CD+ T cell proliferation as compared with Muc1+/+ lung or splenic DC. In addition, activation of either Muc1+/+ lung DC (fig. ) or Muc1+/+ splenic DC (fig. ) with flagellin, enhanced CD4+ T cell proliferation compared with unstimulated Muc1+/+ DC (fig. , respectively). By contrast however, unlike in vitro propagated BM-DC, flagellin stimulated Muc1−/− lung DC (fig. ) and Muc1−/− splenic DC (fig. ) indeed showed an increased ability to stimulate CD4+ T cell proliferation as compared to unstimulated resting DC.
By also measuring the secretion of both DC- and T cell-derived cytokines (e.g. IL-12p40 vs. IFN-γ and IL-13, respectively) by multiplex cytokine array assays (Bioplex; Bio-Rad) in co-cultures of DC with naive CD4+ T cells, we determined whether Muc1 deficiency altered cytokine secretion during DC-T cell cross-talk (fig. ). We note that these assays were performed after extensive washing of TLR ligand-exposed DC, and thus reflect indirect effects of DC maturation on T cell responses. Using resting DC, Muc1+/+ DC/T cell co-cultures secreted low amounts of TNF-α that was enhanced on stimulation of naive CD4+ T cells with LPS or flagellin-exposed DC (p < 0.01; fig. ). While co-cultures using Muc1−/− DC promoted a similar pattern of cytokine secretion, the amount of TNF-α secreted was markedly greater as compared with co-cultures using Muc1+/+ DC (fig. ). In contrast, IL-12p40 secretion was much higher in co-cultures of CD4+ T cells and Muc1+/+ DC as compared with Muc1−/− DC, even after stimulation of those DC with LPS or flagellin (p < 0.05; fig. ).
Fig. 10 Quantitation of cytokine production in co-cultures of naive CD4+ CD62L+ T cells with resting, LPS or flagellin-activated Muc1+/+ and Muc1−/− BM-DC. Culture supernatants were harvested after 4 days from the DC and naive CD4+ CD62L+ T cell (more ...)
The secretion of IFN-γ (a Th1 type cytokine) followed a similar pattern as seen for TNF-α production in DC/T cell co-cultures (fig. ). Under conditions of pre-stimulating Muc1−/− DC with LPS or flagellin, we observed greater levels of IFN-γ secreted from the co-culture as compared with Muc1+/+ DC-stimulated CD4+ T cells (p < 0.05; fig. ). However, in both Muc1−/− DC and wild-type counterparts, both LPS and flagellin enhanced the ability of DC to promote increased IFN-γ secretion in the co-culture (p < 0.01). This was in sharp contrast to the secretion of IL-13, which was only significantly increased in DC/T cell co-cultures using flagellin-stimulated wild-type Muc1+/+ or to a lesser degree Muc1−/− DC (fig. ).
Using multiplex cytokine array assays, we repeated these studies using naive CD62L+ CD4+ T cells co-cultured with freshly isolated primary lung or splenic DC ex vivo (table ). In many respects, stimulation of CD4+ T cells in co-culture with lung or spleen Muc1−/− DC and wild-type counterparts recapitulated many of the cytokine secretion readouts that we observed using BM-DC. In the case of TNF-α and IL-6 secretion, both LPS and flagellin pre-stimulated DC provoked enhanced levels of both of these cytokines to be secreted by co-cultures of CD4+ T cells and lung or spleen DC, with significantly more TNF-α and IL-6 secreted using CD4+ T cells co-cultured with Muc1−/− DC compared to wild-type spleen or lung DC (p < 0.05; table ). However, while the secretion of IL-12p40 was augmented in co-cultures of CD4+ T cells and either LPS or flagellin pre-stimulated lung or spleen DC (table ), Muc1−/− DC promoted much lower amounts of IL-12p40 to be secreted in co-culture than their wild-type counterparts (table ), similar to the case of BM-DC (fig. ). Flagellin-exposed Muc1−/− spleen and lung DC also induced the secretion of more IFN-γ (and less IL-13) in co-culture with naive T cells than wild-type DC (p < 0.05; table ), similar to results obtained using BM-DC (fig. ).
Cytokine secretion by co-cultures of lung or splenic DC-stimulated naive CD4+ T cells