The Generation of Mice that Are Homozygous for a Disrupted Ndfip1 Locus
ES cells harboring a disruption of the Ndfip1
gene were obtained from BayGenomics (cell line code RRD002). The targeted ES cells contain a gene-trapping vector that was inserted within intron 2 of the gene encoding Ndfip1 (Stryke et al., 2003
). The gene trap vector is composed of an artificial intron (En2), a splice acceptor site, and a βGeo cassette (). This disruption of the Ndfip1
gene results in a truncation of the mRNA transcript just beyond exon 2 (). To confirm the presence of the gene trap vector, ES cells were tested by PCR. PCR with primers “a” and “b” () produces the 1.0 kb bp band, indicating the presence of the wild-type locus. In contrast, PCR with primers “a” and “c” yielded a band of 0.3 bp, indicating disruption of the Ndfip1
locus. ES cells carrying this mutation were injected into mouse blastocysts to generate chimeras as described previously (McDonald et al., 1999
). Two male chimeras transmitted to the germline. The resulting agouti progeny were tested for the presence of the disrupted Ndfip1
allele by PCR (data not shown).
The Generation of Mice Homozygous for a Disrupted Ndfip1 Gene
Mice heterozygous for the disrupted locus were inter-crossed to produce homozygous Ndfip1−/− animals. The PCR protocol described above was used to genotype the resulting progeny (). Once identified, homozygous mice were tested by RT-PCR to see whether they expressed any full-length Ndfip1 mRNA (). These data show that two kinds of transcripts were produced in Ndfip1−/− tissues. One of them (EX2-βGeo) was a truncated transcript that consisted of exons 1 and 2 and βGeo. The second one (Ndfip1-AST), based on mRNA sequencing, was an alternatively spliced transcript consisting of the full-length Ndfip1 with 206 bp from the ampicillin resistance gene inserted in the reverse orientation between exons 2 and 3 (data not shown). The βGeo was not included in this transcript. This Amp fragment introduced a translation stop site in each of the three possible reading frames. Taken together, these data suggest that insertion of the gene trap vector into the Ndfip1 locus results in a disruption of the Ndfip1 gene.
Mice Lacking Ndfip1 Develop Spontaneous Inflammation of the Skin and Die Prematurely
mice appeared normal at birth. Furthermore, the number of Ndfip1−/−
mice produced from inter-crosses of Ndfip1+/−
animals conformed, for the most part, to normal Mendelian expectations (see Table S1
in the Supplemental Data available online). At 6 weeks, Ndfip1−/−
began to develop skin lesions on their ears (data not shown), and by 8 weeks of age, all Ndfip1−/−
mice had these lesions. Gross inspection of the mice revealed a profound hepatomegally and splenomegally. Organ size was increased from a liver to body weight ratio of 48 ± 4 mg/g for Ndfip1+/+
animals to 101 ± 11 mg/g for Ndfip1−/−
mice (p < 0.008) and from a spleen to body weight ratio of 3.4 ± 0.5 mg/g for Ndfip1+/+
mice to 16.9 ± 2.7 mg/g for Ndfip1−/−
animals (p < 0.003). Additionally, over time, the tails of Ndfip1−/−
became segmented in appearance and tended to be shorter then the tails of their Ndfip1+/+
littermates (data not shown).
In an effort to determine the underlying cause of the increased spleen and liver size and inflammation of the ear, tissue sections were examined. Hematoxylin and eosin (H&E) staining of paraffin-embedded sections of organs from Ndfip1−/− mice revealed multiple defects. Ear sections revealed a high degree of inflammation with a predominantly eosinophilic and lymphocytic infiltrate (). The liver contained intrahepatic bile ducts (). Apparent in both liver and spleen were extensive foci of extramedullary hematopoiesis (). Lungs of Ndfip1−/− mice also displayed signs of inflammation with goblet cell hyperplasia and an inflammatory infiltrate in the perivascular regions (). Kidneys in the Ndfip1−/− mice appeared normal (data not shown).
Loss of Ndfip1 Leads to Inflammatory Disease, Splenomegally, Hepatomegally, and Extramedullary Hematopoiesis of the Spleen and Liver
Because the phenotype showed both an alteration in hematopoiesis and was inflammatory in nature, we characterized hematopoietic-derived cells of primary and secondary lymphoid organs by flow cytometry. We found that Ndfip1−/−
mice had fewer B cells (B220+
) and more myeloid lineage cells (GR1+
) in their bone marrow as compared to age-matched Ndfip1+/+
animals, whereas pre-erythroid cells (ter119+
) were equal in number (Figure S1A
). The spleens of mice lacking Ndfip1 also showed elevated numbers of myeloid lineage cells and, in keeping with histological evidence of splenic hematopoiesis, pre-erythroid cells (Figure S1B
). T cells in Ndfip1−/−
animals, particularly those that expressed CD4, were increased in number and were activated as shown by their increased expression of CD44.
Although some Ndfip1−/− mice died soon after weaning, many of the mice survived longer (). The persistent inflammation of the ear resulted in destruction of much of the ear tissue, and once inflammation was established, mice began to appear cachectic. Beginning at 10 weeks, there was a dramatic decrease in the survival of Ndfip1−/− mice, and none of these mice survived beyond 14 weeks of age.
The Ndfip−/− Inflammatory Phenotype Is Due to a Defect in Cells of the Hematopoietic Lineage
Flow cytometric analysis revealed multiple changes in cells from the hematopoietic lineage; however, these changes either could have been due to a primary defect caused by the loss of Ndfip1 or could have been caused by inflammation. To find out whether Ndfip1 deficiency causes a defect in bone marrow-derived cells that initiates inflammation, we transferred Ndfip1−/− or Ndfip1+/+ bone marrow cells into lethally irradiated C57BL/6 recipients and monitored the mice for signs of inflammation.
Mice receiving Ndfip1−/− cells, but not those that were reconstituted with Ndfip1+/+ cells, developed skin lesions beginning approximately 5 weeks postreconstitution and, like Ndfip1−/− mice, died within 8 weeks of the onset of inflammation. Recipients of Ndfip1−/− bone marrow cells also developed splenomegaly and hepatomegaly (data not shown). Again, the inflammatory infiltrate in the skin was predominantly lymphocytic and eosinophilic (), and extramedullary hematopoiesis was observed in the enlarged spleen and liver (). However, some of the characteristics of the Ndfip1−/− mice were less severe or not recapitulated in the bone marrow chimeras. Splenomegally and hepatomegally was less pronounced in the chimeras, and the reconstituted mice did not develop a segmented tail (data not shown) or intrahepatic bile ducts (). Thus, nonhematopoietic cells are required for these phenotypes in the Ndfip1−/− mice.
Reconstitution of Wild-Type Mice with Ndfip1−/− Marrow Leads to Inflammatory Disease
However, these data show that bone marrow-derived cells are responsible for the inflammatory disease and premature deaths observed in Ndfip1−/− mice.
T Cells Lacking Ndfip1 Are Increased in Number and Are Activated
To find out which bone marrow-derived cells were responsible for promoting inflammation in the Ndfip1−/−
mice, we reconstituted lethally irradiated mice with a mixture of equal numbers of GFP+
(Schaefer et al., 2001b
and GFP− Ndfip1−/−
bone marrow. This experimental design allowed study of Ndfip1+/+
cells exposed to the same inflammatory conditions. Additionally, this experiment allowed us to distinguish between primary events (which would occur only in the Ndfip1−/−
cells) and secondary events (which would affect both Ndfip1−/−
Mice reconstituted with Ndfip1−/− bone marrow developed disease approximately 6 weeks after bone marrow transfer (see above), and therefore we chose to analyze the mixed chimeras between 5 and 6 weeks after reconstitution. This time frame allowed us to see changes in immune system cells that preceded any outward signs of inflammation. Because GFP expression in the Ndfip1+/+ cells limited the fluorimeter channels available to characterize the cells, we sorted live GFP+ and live GFP− cells from each tissue and stained cells with the various antibodies, including some that would register in the same channel as GFP. We then treated each sample with saponin, thereby releasing all of the GFP from the cells, which allowed us to detect antibody staining that would otherwise be obscured.
With this technique, data were collected from six mice, and in each case, Ndfip1+/+ cells were compared to Ndfip1−/− cells isolated from the same mouse. The percentages of the Ndfip1+/+ and Ndfip1−/− cells in the thymuses and bone marrow of the mixed chimeras were similar (). Likewise, in the spleens of the chimeras, the numbers of macrophages (CD11b+), a subset of dendritic cells (CD11b+ and CD11c+), and B cells of both origins were comparable and there was no evidence that B cells of either type were abnormally activated, as tested by expression of CD69 (). However, a greater percentage of the Ndfip1−/− cells in spleens (and lymph nodes, data not shown) were T cells (15.5% ± 3% of Ndfip1−/− cells versus 9.2% ± 1.8% of Ndfip1+/+ cells). In addition, many more of the Ndfip1−/− cells were activated, as defined by increased expression of CD44 and Ly6c (data not shown). This difference was true for both CD4+ and CD8+ T cells (). Thus, T cells lacking Ndfip1 were activated prior to any outward signs of inflammation.
Mixed Bone Marrow Chimeras Reveal a Profound Defect in Ndfip1−/− T Cells
To ensure that the T cell defects were not due to differences in the genetic background between the Ndfip1−/−
(C57BL/6 and 129.ola) and GFP Tg. mice (C57BL/6), we compared these mixed chimeras to those made with a mixture of cells from GFP Tg. mice and Ndfip1+/−
mice. The Ndfip1+/−
mice have a mixed genetic background that is similar to that of the Ndfip1−/−
mice. Comparison of these two sets of mixed chimeras revealed that the T cell defect was a direct consequence of loss of Ndfip1 (Figure S2
Based on these data, we could not rule out the possibility that cells other then T cells are affected by the loss of Ndfip1. However, our data clearly indicate that T cells lacking Ndfip1 are activated before any outward signs of disease and that this activation is intrinsic to the mutant T cell.
Ndfip1−/− T Cells Proliferate More and Readily Produce IL-4 In Vitro
Inflammatory disorders of the skin, particularly those with eosinophilic involvement, are often potentiated by Th2 CD4+
T cells (Del Prete, 1992
; Ricci et al., 1994
; Romagnani et al., 1991
). Accordingly, we tested whether Ndfip1−/−
T cells were capable of responding effectively to TCR-mediated signals that lead to proliferation and/or the production of the Th2 cytokine, IL-4, or the Th1 cytokine, IFN-γ. We again used T cells isolated from mixed chimera mice to ensure that the T cells were exposed to the same environment prior to analysis. T cells from the mixed chimeras were sorted for GFP expression, labeled with CFSE, and cultured for 3 days in the presence or absence of the TCR-stimulating reagents, anti-CD3 and anti-CD28. We then stained cells with antibodies against CD4 and CD8 and treated the cells with saponin to remove GFP.
Unstimulated cells did not divide regardless of Ndfip1 expression, demonstrating that Ndfip1−/− cells were still dependent on TCR stimulation to divide. On the other hand, when cells were stimulated, Ndfip1−/− CD4+ T cells proliferated more readily than wild-type cells (). These data imply that Ndfip1 might affect how T cells respond to activation signals.
T Cells Lacking Ndfip1 Are More Likely to Proliferate and Produce Th2 Cytokines
We then wanted to see whether Ndfip1−/− T cells were capable of producing cytokines after culture in Th1 or Th2-polarizing conditions. T cells were isolated from the spleens of 5- to 6-week-old Ndfip1+/+ and Ndfip1−/− mice, and activated T cells (CD44+) were depleted from each sample. Cells were then cultured for 6 days under either Th1- or Th2-polarizing conditions or activated in the absence of cytokine polarization.
When cells were activated in the absence of polarizing conditions (control), neither type of cell produced much IL-4 or IFN-γ (). Furthermore, when cells were cultured under Th1-polarizing conditions, Ndfip1−/− T cells were no more likely to produce IFN-γ than control cells. In contrast, when cells were cultured in Th2-polarizing conditions, Ndfip1−/− T cells were much more likely to make IL-4. These data support the hypothesis that loss of Ndfip1 biases T cells toward a Th2 phenotype and might help to explain why mice lacking Ndfip1 are prone to develop an inflammatory condition with high numbers of infiltrating eosinophils.
Ndfip1−/− T Cells Are Much More Likely to Drive a Th2 Response In Vivo
The presence of eosinophils at the inflammatory sites suggests that Ndfip1−/− mice develop a Th2-mediated disease. Knowing that loss of Ndfip1 led to a defect in T cells suggested to us that these T cells might drive disease because of an uncontrolled bias toward production of Th2 cytokines. Thus, we wished to test whether Ndfip1−/− T cells were Th2 biased in vivo and whether this bias resulted in increased Th2-dependent immunoglobulin switching.
For this experiment, we made bone marrow chimera mice to study a large number of animals that were healthy at the time of immunization. We immunized the mice with ovalbumin (OVA) mixed with an adjuvant that induces either a Th2-polarized response (Alum) or a Th1-polarized response (complete Freund’s adjuvant, CFA). Mice reconstituted with Ndfip1−/−
bone marrow typically began to show signs of inflammation 6 weeks after the transfer of bone marrow, and their condition worsened over the next 4-6 weeks. We found that when these same mice were immunized with Alum 5 weeks after reconstitution, they became very sick within 8 days such that the experiment had to be terminated prematurely. Chimeras made from Ndfip1+/+
bone marrow remained healthy even after they were immunized with OVA + Alum and they showed no signs of inflammation in either their skin or lung (Figures S2A and S2B
). In contrast, chimeras made from Ndfip1−/−
bone marrow that received OVA + Alum had visible lesions on their skin (data not shown) and inflammation in the skin and lungs (Figures S2C and S2D
T cells from all groups were analyzed 8 days after antigen stimulation for cytokine production. Very few T cells from Ndfip1+/+ bone marrow chimeras made IL-4 in response to in vitro challenge with antigen (). This was true regardless of the adjuvant used and is probably due to the short duration of the experiment. In contrast, the Ndfip1+/+ cells were able to produce IFN-γ in response to ex vivo antigen exposure but only if they had come from animals immunized with OVA + CFA, in line with the known Th1-polarizing capacity of this adjuvant. In contrast to these results, T cells lacking Ndfip1 were consistently less likely to produce IFN-γ as compared to wild-type cells and instead produced IL-4. This was true regardless of the adjuvant used to prime the animal and occurred even in the absence of antigen challenge in vitro.
Figure 6 Immunization of Ndfip1−/− Mice with OVA+CFA or OVA+ALUM Leads to IL-4 Production and Increased Antigen-Specific IgE Ndfip1+/+ and Ndfip1−/− bone marrow chimeras were immunized with OVA+CFA or OVA+ALUM 5 weeks after reconstitution. (more ...)
To find out whether the Th2 bias of Ndfip1−/− T cells were reflected in the antibody isotypes generated in mice containing these cells, we measured the amounts of various immunoglobulin isotypes in unimmunized chimeras and the isotypes of ovalbumin antibodies in the immunized animals (). In comparison with Ndfip1+/+ chimeras, Ndfip1−/− chimeras contained high amounts of the Th2-dependent immunoglobulin class IgE and low amounts of the Th1-dependent isotypes IgG2a/b. Similar results were observed for ovalbumin antibodies after immunization with OVA + CFA or OVA + Alum, with amounts of IgE higher, and levels of IgG2a/b and IgG3 lower in the Ndfip1−/− chimeras than the Ndfip1+/+ mice.
These results support the conclusion that lack of Ndfip1 predisposes T cells toward a Th2 phenotype, regardless of the conditions under which they are activated.
Ndfip1 Binds Itch after T Cell Stimulation and Promotes Itch Function
Given that Ndfip1−/−
and Itch mutant mice have a similar phenotype and because a WW domain portion of Itch has been shown to bind Ndfip1 in vitro (Harvey et al., 2002
), we postulated that Ndfip1 might regulate Itch. To test this idea, we first needed to know whether Ndfip1 protein is expressed in T cells and whether its expression affects Itch expression.
T cells were isolated from 6- to 8-week-old Ndfip+/+ and Ndfip1−/− mice, the cells were cultured in media or stimulated for 24 hr, and their whole-cell lysates were analyzed by immunoblot for expression of Itch and Ndfip1. Ndfip1+/+ and Ndfip1−/− T cells contained equivalent amounts of Itch, indicating that expression of Ndfip1 does not regulate Itch expression in T cells.
Unstimulated T cells expressed negligible amounts of Ndfip1 protein. After 2 hr of stimulation, Ndfip1 protein increased in amount (), suggesting that Ndfip1 function may be particularly relevant in activated T cells.
Ndfip1 Interacts with Itch and Promotes Degradation of JunB
To find out whether Ndfip1 could physically associate with Itch, we immunoprecipitated Itch from lysates of T cells that were unstimulated or stimulated for 24 hr. We found that isolates of Itch contained Ndfip1 in stimulated T cells (). This was specific for the Itch IP and did not occur in isotype controls (Figure S4
); thus, Ndfip1 does bind Itch in activated T cells.
To determine whether these interactions could occur after lysis, we chose to look at whether the proteins colocalized in activated T cells. Itch and Ndfip1 localization was examined in unstimulated T cells or in cells that had been stimulated for 2 or 24 hr. In unstimulated cells, Ndfip1 was not expressed, and Itch was found in intracellular vesicles (). 2 hr after stimulation, Ndfip1 could be detected and was localized near the plasma membrane. Because we did not see staining with this antibody in nonpermeabilized cells (data not shown), we believe this region to represent cytoplasm near the plasma membrane. At this time point, some of the Itch colocalized near the plasma membrane with Ndfip1. Colocalization of Itch with Ndfip1 was more evident by 24 hr when nearly all the Itch and Ndfip1 polarized into a region near the inner surface of the cell. Interestingly, in cells lacking Ndfip1, Itch remained localized within the cytoplasmic vesicles for the duration of this experiment. This would suggest that Ndfip1 is required to recruit Itch to a discrete region within the cell.
That Itch and Ndfip1 are physically associated after T cell stimulation supports the hypothesis that Ndfip1 might promote Itch function. One well-described function of Itch is ubiquitination of JunB, a phenomenon that leads to degradation of the protein. JunB expression is increased 1–2 hr after T cell stimulation and then wanes (Foletta et al., 1998
). This timing is consistent with expression of Ndfip1 and its colocalization with Itch. Therefore, we postulated that Ndfip1 might promote Itch-dependent degradation of JunB. This would predict that JunB could have a longer half-life in cells lacking Ndfip1.
To test this idea, JunB expression was measured in unstimulated T cells, in T cells that had been stimulated for 2 or 6 hr, and in T cells that had been stimulated for 6 hr, but incubated in cyclohexamide for the last 4 of these 6 hr, to block protein synthesis. As predicted by previous reports, JunB amounts increased after 2 hr of stimulation, and this was also true in cells lacking Ndfip1 (, compare lanes 1 and 2). Amounts of JunB subsequently declined in Ndfip1+/+ cells (), but this decline did not occur in cells lacking Ndfip1. The maintenance of JunB in Ndfip1−/− cells was mainly due to lack of JunB degradation, rather than increased synthesis of the protein because amounts of JunB remained high in these cells even if the cells were cultured in cyclohexamide. Thus, Ndfip1 controls amounts of JunB in activated T cells by inducing its degradation, probably via association of Ndfip1 with Itch.
To determine whether the reduced JunB degradation was a direct result of the loss of Ndfip1 rather than a by-product of the activation status of the cells, we retrovirally re-expressed Ndfip1 in an Ndfip1−/− T cell line. As was the case in primary T cells that lack Ndfip1, cells from an Ndfip1−/− T cell line that were transduced with an empty vector showed prolonged JunB expression after stimulation (, top left). In contrast, cells transduced with an Ndfip1-containing vector degraded JunB to the same extent as did Ndfip1+/+ cells.
We also wanted to know whether increasing Ndfip1 in wild-type cells would alter their JunB degradation. To do this, we overexpressed Ndfip1 in an Ndfip1+/+ T cell line, again via the retroviral system. Like primary T cells, cells from the Ndfip1+/+ cell line transduced with an empty vector show degradation of JunB 6 hr after stimulation (, bottom left). When Ndfip1 expression was increased in these cells, by expressing a Flag-tagged Ndfip1, JunB expression was reduced. Cells that expressed the Flag-tagged Ndfip1 contained less JunB protein 2 hr after stimulation when compared to empty vector controls. 6 hr after stimulation, JunB expression had returned to prestimulation amounts in cells overexpressing Ndfip1, while their wild-type counterparts continued to express elevated amounts of JunB.
These data predict that JunB expression might be unusually high in T cells from mice lacking Ndfip1. To test this, we isolated T cells from 8- to 10-week-old Ndfip1+/+ and Ndfip1−/− mice and tested their cell lysates for JunB by immunoblot. JunB expression was increased in T cells lacking Ndfip1 (). These amounts were quantified in several different experiments, normalized to β-actin, and compared to Ndfip1+/+ T cells (normalizing wild-type to 1). We found that Ndfip1−/− T cells contained approximately 5-fold more JunB than wild-type cells; it is possible, however, that some of the increased JunB in these cells results from their increased activation status. Taken together, these data support our hypothesis that the loss of Ndfip1 results in reduced degradation of JunB, likely the result of reduced Itch function.