The Nucleoporin Nup96 Is Required for Proper Expression of Interferon-Regulated Proteins and Functions

Immunity 24, 295–304, March 2006 ª2006 Elsevier Inc.

DOI 10.1016/j.immuni.2006.01.014

The Nucleoporin Nup96 Is Required for Proper Expression of Interferon-Regulated Proteins and Functions Ana M.C. Faria,3 Agata Levay,1,2 Yaming Wang,4 Alice O. Kamphorst,3 Magda L.P. Rosa,3 Daniel R. Nussenzveig,1 Wayne Balkan,2 Yuh Min Chook,5 David E. Levy,4 and Beatriz M.A. Fontoura1,2,6,* 1 Department of Cell Biology University of Texas Southwestern Medical Center Dallas, Texas 75390 2 Department of Molecular and Cellular Pharmacology Sylvester Comprehensive Cancer Center University of Miami Miller School of Medicine Miami, Florida 33136 3 Departamento de Bioquı´mica e Imunologia Instituto the Ciencias Biologicas Universidade Federal de Minas Gerais Belo Horizonte Minas Gerais Brazil 4 Department of Pathology New York University School of Medicine New York, New York 10016 5 Department of Pharmacology University of Texas Southwestern Medical Center Dallas, Texas 75390

Summary Nup98 and Nup96 are components of the nuclear transport machinery and are induced by interferons (IFN). Nup98 is a constituent of an mRNA export pathway that is targeted by viruses and regulated by IFN. However, the role of Nup96 in IFN-related mechanisms has not been established. To investigate the function of Nup96 in vivo, we generated Nup96+/2 mice that express low levels of Nup96, as Nup962/2 mice are lethal. The Nup96+/2 mice presented selective alterations of the immune system, which resulted in downregulation and impaired IFN a- and g-mediated induction of MHC I and IFNg induction of MHC II, ICAM-1, and other proteins. Frequency of TCRab+ and CD4+ T cells, which depends on MHC function, is reduced in NUP96+/2 mice. Upon immunization, Nup96+/2 mice showed impaired antigen presentation and T cell proliferation. Nup96+/2 cells and mice were highly susceptible to viral infection, demonstrating a role for Nup96 in innate and adaptive immunity. Introduction Nuclear pore complex proteins (nucleoporins or Nups) are constituents of the nuclear pore complex (NPC), which mediates nuclear entrance and exit of molecules and is regulated by signaling pathways and viruses (Bednenko et al., 2003; Fontoura et al., 2005; Rout et al., 2003; Suntharalingam and Wente, 2003). Although *Correspondence: [email protected] 6 Present address: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390.

several nucleoporins are structural components of the NPC, some Nups, such as Nup98, are more dynamically associated with the NPC and with the nuclear interior (Griffis et al., 2002). We have shown that Nup98 and Nup96 are encoded by the same gene (Fontoura et al., 1999) and are upregulated by interferons (Enninga et al., 2002). Studies in yeast and vertebrate cells showed that both proteins are involved in mRNA export (Dockendorff et al., 1997; Emtage et al., 1997; Powers et al., 1997; Teixeira et al., 1997). In fact, inhibition of mRNA export mediated by the vesicular stomatitis virus (VSV) matrix (M) protein is reverted by treating cells with interferon or is partially reverted by overexpression of Nup98/Nup96 (Enninga et al., 2002). In addition, we found that the mRNA export factor Rae1, which is a binding partner of Nup98 and is regulated by IFN, mediates the interaction between VSV M protein and Nup98 (Faria et al., 2005). These interactions result in inhibition of mRNA export that can be fully reverted by increased expression of Rae1 (Faria et al., 2005). Thus, regulation of specific nuclear transport pathways may constitute a defense mechanism against viral infection that facilitates expression of antiviral genes. Nup96 is localized at both sides of the NPC and is a constituent of the Nup107-160 complex that contains Nup107, Nup160, Nup133, Nup85, Nup96, Sec13, Nup43, Nup37, and Seh1 (Belgareh et al., 2001; Fontoura et al., 1999; Harel et al., 2003; Loiodice et al., 2004; Walther et al., 2003). This complex has a crucial role in nuclear pore assembly (Harel et al., 2003; Walther et al., 2003) and mRNA export (Vasu et al., 2001). Nup96 interacts with Sec13 (Enninga et al., 2003; Lutzmann et al., 2002), which is a NPC protein and a component of the COPII-coated vesicles at the endoplasmic reticulum (Bickford et al., 2004). The Nup96 homolog in S. cerevisiae is C-Nup145p, which has functions related to mRNA export and chromatin metabolism (Dockendorff et al., 1997; Emtage et al., 1997; Galy et al., 2000; Teixeira et al., 1997). Recently, a putative homolog of Nup96 in Arabidopsis thaliana was shown to be required for basal defense and constitutive resistance responses to pathogens mediated by the resistance (R) gene (Zhang and Li, 2005). Based on our previous observation that Nup96 is induced by interferons (Enninga et al., 2002) and that the Nup98/Nup96 gene is involved in antiviral functions, we investigated whether Nup96 has a role in immune response. We have developed the Nup96+/2 mice, which express low levels of Nup96 and analyzed expression and function of interferon-regulated genes such as MHC class I, MHC class II, and others. MHC I and MHC II are key players in immune responses. MHC class I antigens are cell surface glycoproteins constituted of an a chain and a nonpolymorphic chain (b2 microglobulin) and are expressed in all nucleated cells (Natarajan et al., 1999). MHC II antigens are membrane glycoproteins composed by two polymorphic a and b chains that are expressed selectively in antigen-presenting cells such as macrophages, dendritic cells, and B lymphocytes (Hedge et al., 2003). Both MHC class I and class II are regulated by cytokines, including IFNs,

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TNF, IL-4, and TGF-b as well as by mitogens such as LPS, phorbol esters, and concanavalin A (David-Watine et al., 1990; Hedge et al., 2003). Presentation of antigenic peptides in the pockets of MHC class I and II is an essential step for the generation and selection of CD8+ and CD4+ T lymphocytes, respectively, in the thymus as well as for the activation of mature T cells in the peripheral lymphoid tissue. Therefore, we also investigated the generation of the main T cell subsets in the thymus of the Nup96+/2 mice. Furthermore, we have studied the susceptibility of Nup96+/2 mice to viral infection. Results Reduced Levels of Nup96 In Vivo Did Not Show Gross Pathological Alterations in the Whole Animal or Defects in Nuclear Structure at the Cellular Level To study the function of Nup96 in vivo, we attempted to generate Nup962/2 mice. Nup98 and Nup96 are encoded by the same gene. This gene yields several alternatively spliced mRNAs that encode precursor proteins that are autocatalytically proteolysed (Figure 1A) (Enninga et al., 2003; Fontoura et al., 1999; Rosenblum and Blobel, 1999). Therefore, to knock out exclusively the Nup96 gene, we have knocked in three stop codons at the 50 end of exon 21 of the Nup98-Nup96 gene (Figure 1B). In this manner, expression of the unique sequence of Nup96 is abolished, and exon 20, which encodes the 6 kDa polypeptide that is shared by the Nup98-6 kDa precursor and the amino terminus of Nup96, is maintained to allow proper expression of the Nup98-6 kDa precursor and, therefore, of Nup98 (Figure 1A). No Nup962/2 mice were generated, indicating that Nup96 is essential. Instead, Nup96 heterozygous and wild-type mice were born at a normal Mendelian frequency and were genotyped by southern blot analysis (Figure 1C). Pathological evaluation was performed in each organ of 2-month-old Nup96+/2 mice, and no alterations were observed (data not shown). Immunoblot analysis showed that the Nup96+/2 mice express low levels of Nup96 as compared to their wild-type littermates (Figures 1D and 1E). However, the Nup98 levels in Nup96+/2 mice are similar to the wild-type mice, indicating that disruption of the Nup96 gene did not alter the biogenesis of Nup98 (Figures 1D and 1E). In addition, the levels of other nucleoporins such as Sec13 and Nup133, which are in complex with Nup96 at the NPC, were not significantly altered in the Nup96+/2 mice (Figure 1E). Also, the levels of Nup62, which is a constituent of the central cylinder of the NPC, and actin were not affected in the Nup96+/2 mice (Figure 1E). To examine potential defects in nuclear structure due to low levels of Nup96, we analyzed Nup96+/2 cells by electronmicroscopy and found no obvious alterations of the nucleus, including absence of nuclear pore clustering and lack of nuclear envelope abnormalities (Figure 2A). We have also tested bulk poly(A) RNA nuclear export (Figure 2B), nuclear import of proteins through the Kapa/Kapb1 (Figure 2C) and the Kapb2/transportin1-mediated pathways (Figure 2D), and found that they occurred similarly in both Nup96+/+ and Nup96+/2 cells. We then assessed the intracellular localization of nucleoporins by confocal microscopy and showed that Nup62, Nup98, and the remaining pool of Nup96 were

Figure 1. Targeted Disruption of the Mouse Nup96 Gene Yielded Nup96 Mice Expressing Low Levels of Nup96 (A) Schematic representation of the Nup98-Nup96 and Nup98-6 kDa precursor proteins. These precursors undergo autocleavage, which generates Nup98, Nup96, and a 6 kDa polypeptide. (B) The exon 21 region of the wild-type allele is depicted at the top. Shown at the bottom is the mutant allele containing a three-way stop codon at the 50 end of exon 21 of Nup96 and a Neo cassette upstream of exon 21 that was deleted by using the Cre recombinase. The 30 probe used for Southern blot analysis is indicated. (C) Southern blot of genomic DNA from Nup96+/+ and Nup96+/2 mice digested with BglI and NcoI and hybridized to a 30 probe shows the 1.4 kb fragment for the wild-type and the 2.2 and 1.4 kb fragments for the mutant. (D and E) Immunoblot analysis was performed with tissue extracts from kidney and liver or with cell extracts from macrophages, using antibodies against Nup98, Nup96, Nup133, Sec13, Nup62, and actin.

all localized at the expected sites (Figure 2E). Thus, these findings indicate that there were no major alterations in nuclear structure or gross defects in some of the major nuclear transport pathways. However, these findings do not exclude potential transport defects of specific classes of proteins or RNAs, as shown below in Figure 4. Downregulation of Nup96 In Vivo Induced a Decrease in the Expression of IFN-Regulated Gene Products Because Nup96 is induced by interferon-g prior to the upregulation of other interferon-inducible genes, such as the MHC class I and class II (Enninga et al., 2002; Giroux et al., 2003), and that IFN-g is able to facilitate mRNA export that is blocked by a viral protein, at least in part through upregulation of the Nup98-Nup96 gene, we investigated whether Nup96 has any role on the expression of interferon-g-inducible genes. To test this possibility, we obtained macrophages and dendritic cells from spleen and thymus of Nup96+/+ and Nup96+/2 mice and measured MHC class I (H-2Db) and MHC class II (I-Ab) expression by FACs analysis. We found that both MHC class I and class II levels are reduced in macrophages and dendritic cells from the spleen and thymus (Figures 3A–3C, 3H, and 3I). In addition, a decrease in MHC class I and MHC class II levels was also observed in T and B lymphocytes from lymph nodes (data not shown). We then tested whether other interferon-induced genes were altered in macrophages from Nup96+/2 mice.

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Figure 2. Nuclear Structure and Some of the Major Nuclear Transport Pathways Are Not Altered in Cells from Nup96+/2 Mice (A) Livers from Nup96+/+ and Nup96+/2 mice were processed for thin sectioning and observed by electron microscopy. Arrowheads show NPCs. (B) Oligo-dT in situ hybridization was performed in MEFs from Nup96+/+ and Nup96+/2 mice. (C and D) (C) Nuclear import assays were performed with NLS I-BSA or MBP-M3-GFP (D) with MEFs from Nup96+/+ and Nup96+/2 mice. (E) Intracellular localization of Nup98 and Nup96 and of other nucleoporins that are recognized by the mAb414, such as Nup62 and, to a lesser extent, Nup153, was assessed by immunofluorescence followed by deconvolution and apotome microscopy.

In fact, the levels of CD54 (ICAM-1) and CD86 (B7.2), which are plasma membrane proteins known to be induced by interferon-g, are also reduced in macrophages expressing low levels of Nup96 (Figures 3E and 3G), whereas the constitutive levels of CD11b, which is a surface protein that is regulated by IFN, did not change (Figure 3F). We did not observe any difference on the expression levels of several plasma membrane proteins that are not regulated by interferon but are important in immune response, including CD4, CD8, CD25, and CD62L (Figure 3J). Altogether, these results indicate that Nup96 facilitate expression of major interferon-regulated gene products. To investigate whether the induction levels of MHC expression by IFN were also altered in Nup96+/2 mice, we isolated macrophages from Nup96+/+ and Nup96+/2 mice and stimulated them in vitro with IFN. As shown in Figure 3D, upregulation of MHC II and MHC I by interferon-g was considerably reduced in Nup96+/2 mice as compared to Nup96+/+ mice. In addition, IFNa-mediated induction of MHC I is impaired in Nup96+/2 mice, indicating that the response to both IFNs is defective in these

mice (Figure 3K). IFNg induced expression of CD54 and CD11b, and CD86 was also diminished in macrophages of Nup96+/2 mice (Figures 3E–3G). Interestingly, the constitutive levels of CD11b were not altered in Nup96+/2 macrophages, whereas its induction by interferon-g was abnormal (Figure 3F). Thus, these results show that macrophages of Nup96+/2 mice are refractory to IFN mediated upregulation of several gene products, indicating that this effect of IFNs is NUP96 dependent. The reduced levels of these IFN-regulated proteins were observed in Nup96 mice with a C57BL/6J and 129/SvEv mixed background as well as with Nup96+/2 mice with a homogenous BALB/c background (data not shown), indicating that this regulation is not strain specific but is rather due to lower levels of Nup96. To investigate whether impaired expression of interferon-regulated gene products occurs at the RNA level, we quantified specific RNA species derived from total cell extract or nuclear and cytoplasmic fractions of peritoneal macrophages from Nup96+/+ and Nup96+/2 mice. Whereas no difference in the total levels of MHC I, MHC II (Eb), b2 microglobulin, TAP1, or CIITA mRNAs were

Figure 3. Expression of Interferon-Regulated Proteins Is Altered in Nup96+/2 Mice (A–I) Macrophages and dendritic cells isolated from thymus and spleen of Nup96+/+ and Nup96+/2 mice were stained with antibodies against MHC I (H-2Db), MHC II (I-Ab), CD54 (ICAM-1), CD11b, and CD86 and followed by flow cytometry analysis. In IFNg-treated samples, incubation was performed with 100 U/ml IFNg for 48 hr. (J) Lymphocytes isolated from Nup96+/+ and Nup96+/2 mice were stained with antibodies against CD4, CD8, CD25, and CD62L, and their levels of expression were analyzed by flow cytometry. Numbers represent the average of the mean fluorescence intensity for the molecule in macrophages isolated from a group of mice (n = 7–8). Results represent mean values 6 SD. Cells were gated initially by light scatter and finally by a macrophage marker (Mac3). *p < 0.05. (K) Peritoneal macrophages, untreated or treated with IFNs a (50 U/ml) and g as above, were stained with antibodies against MHC I (H-2Db), MHC II (I-Ab), and CD54 (ICAM-1) and followed by flow cytometry analysis. M.F.I., mean fluorescence intensity. PLN, peripheral lymph nodes. C, control.

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Figure 4. Nuclear Export of mRNAs Encoding a Specific Class of Immune-Related Genes Is Altered in Macrophages from Nup96+/2 Mice Total RNA obtained from nuclear (N) and cytoplasmic fractions (C) of peritoneal macrophages was amplified and quantified by real-time RT-PCR using primers specific to the depicted mRNAs.

observed (data not shown), the ratios of nuclear/cytoplasmic levels of these mRNAs were partially but significantly increased in macrophages from Nup96+/2 mice as compared to Nup96+/+ mice (Figure 4). This partial nuclear retention likely contributes to the lower expression levels of these proteins at the plasma membrane. In contrast, RPS11, a-tubulin, and ICAM-1 mRNAs were not retained in the nucleus, although the levels of ICAM-1 protein at the plasma membrane were lower in macrophages from Nup96+/2 mice. It is possible that ICAM-1 protein or cell surface expression is affected at a posttranscriptional/transport step, possibly due to reduced expression of other IFNg-induced proteins. Overall, these results show that nuclear export of specific classes of mRNAs is disrupted due to lower levels of

Nup96. Since these mRNAs encode major players of immune response, such as MHC I and MHC II, Nup96+/2 mice may present alterations related to MHC functions, as we show below. NUP96+/2 Mice Have Altered Numbers of T and B Cell Subpopulations in Lymphoid Organs Since MHC expression is essential not only for immune responses but also for lymphopoiesis and physiological interactions between lymphocytes and antigen-presenting cells in the periphery, we analyzed the frequency of T cells, B cells, macrophages, and dendritic cells in a central lymphoid organ (thymus) as well as in a peripheral organ (spleen) (Figure 5A). There is a significant decrease in TCRab+ T cells and in CD4+ T cells in the

Figure 5. Frequency of TCRab+ T Cells and CD4+ T Cells Is Reduced in the Thymus of NUP96+/2 Mice, and Frequency of B Cells Is Diminished in the Spleen of the Nup96+/2 Mice; T Cell Proliferation Is Decreased in Nup96+/2 Mice Immunized with CFA-Emulsified Ovalbumin (A) Cell subsets were analyzed by flow cytometry using antibodies against CD3 for T lymphocyte, CD19 for B lymphocytes, Mac3 for macrophages, and CD11c for dendritic cells. For T cell development study, thymic CD3+ cells were analyzed for expression of either CD4 or CD8. *p < 0.05. Results are the mean 6 SD (n = 8). (B) Nup96+/+ and Nup96+/2 mice were immunized with OVA emulsified in CFA. Eight days postimmunization, cells from the draining lymph nodes were cultured in the presence of ovalbumin. T cell proliferation was measured by thymidine incorporation. Results are the mean 6 SD (n = 8). (C and D) Intracellular staining of IFN-g and IL-4 produced by CD4+ and CD8+ T cells is shown in primary and secondary responses to OVA. (E) Proliferation of T cells after in vitro stimulation with anti-CD3 mAb is increased in spleen, MLN (mesenteric lymph nodes), and PLN of NUP96+/2 mice. *p < 0.05. Results are the mean 6 SD (n = 7).

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Figure 6. Cytokine Production by Splenic T Cells Is Altered in Nup96+/2 Mice; Immunoglobulin Levels Are Slightly but Significantly Reduced in NUP96+/2 Mice (A and B) T cells isolated from the spleen of Nup96+/+ and Nup96+/2 mice were stimulated in vitro with ConA for 48 hr. IL-4, IL-2, and IFN-g production were measured in the culture supernants by ELISA. *p < 0.05. Results are the mean 6 SD (n = 8). (C) Intracellular staining of IFN-g and IL-4, produced by CD4+ and CD8+ T cells, is shown in response to 24 hr ConA treatment. (D and E) Immunoglobulin levels were determined in sera of Nup96+/+ and Nup96+/2 mice by ELISA. *p < 0.05. Results represent mean values 6 SD (n = 8).

thymus of NUP96+/2 mice, indicating that the defect on MHC II expression may affect the maturation of CD4+ cells in the thymus. No alterations were observed in immature DN cells or in mature TCRgd+ or in CD8+ T cells. In the spleen, a significant decrease in the frequency of B cells was observed. The other analyzed splenic populations, including resting and activated T cells as well as antigen-presenting cells, were unchanged. Reduced Levels of Nup96 Alter T and B Cell Functions Since the interaction of MHCs with TCRs is crucial to activate T cells, we tested whether the reduced levels of MHC expression would affect two major T cell functions, including T cell proliferation and cytokine production by T cells. Nup96+/+ and Nup96+/2 mice were immunized with ovalbumin and a potent Th1 adjuvant, Mycobacterium tuberculosis-containing CFA, and specific T cell proliferation was measured by thymidine incorporation. We have chosen this type of stimulation because it is Th1 mediated and induces an IFNg response, which would probably be affected by the reduced levels of Nup96. Indeed, the reduced levels of Nup96 yielded a specific decrease in T cell proliferation (Figure 5B) without change in the production of specific Th1 cytokines, IFNg, and IL-2 (data not shown). Furthermore, IFN-gpositive CD4+ T cells were present in lower frequency in Nup96+/2 cells as compared to Nup96+/+ cells (Figure 5C). This effect was more significant in the secondary response to the antigen than in the primary response. No significant trend was observed in the prevalence of IL4-producing CD4+ or CD8+ T cells (Figure 5D). Since immunization with CFA-emulsified OVA triggers a MHCdependent response, these results indicate that presentation of soluble antigen is deficient in Nup96+/2 mice. Since MHC-dependent T cell proliferation was diminished in Nup96+/2 mice, we investigated whether this defect is intrinsic and possibly independent on MHC expression. Therefore, we performed polyclonal MHCindependent stimulation of T cells using anti-CD3 monoclonal antibody. As shown in Figure 5E, T cells derived from spleen, mesenteric lymph nodes, and peripheral lymph nodes showed an increase in T cell proliferation when stimulated with anti-CD3 antibody. In addition, nonstimulated control T cells also showed an increased ability to proliferate (see Discussion). Thus, the observed decrease in MHC-dependent T cell proliferation is likely

a consequence of the diminished levels of MHC expression in Nup96+/2 mice, as the intrinsic ability of these cells to proliferate is enhanced. To further evaluate T cell function, we examined cytokine production by T cells from Nup96+/+ and Nup96+/2 mice. We obtained splenic cells from these mice, stimulated in vitro with ConA for 48 hr, a T cell mitogen, and measured cytokine production. As shown in Figure 6A, IFNg production is increased and IL-4 production is reduced in Nup96+/2 mice (Figure 6B), suggesting that these mice are skewed toward a Th1 profile of cytokine production. Interestingly, CD8+ T cells seem to be important in this process, since they are the major source of IFN-g produced after polyclonal stimulation with ConA for 24 hr (Figure 6C). In contrast, IL-2 production was not altered (Figure 6A). Because the production of postswitch immunoglobulins by B cells depends on CD4-T cell help, we tested the levels of serum Ig in Nup96+/+ and Nup96+/2 mice. We observed a modest but a significant reduction in the total Ig levels in Nup96+/2 mice (Figure 6D). This reduction may be related to the lower number of B cells shown in Figure 5A and is likely due to a decrease in IgG levels, since IgM and IgA levels were not altered. When we examined subclasses of IgG, we observed that IgG1 level was reduced (Figure 6E) (see Discussion). Thus, physiological B cell function is slightly impaired in Nup96+/2 mice, and MHC-dependent T cell functions are highly altered in these mice. These abnormalities are reflected in the susceptibility of Nup96+/2 mice to viral disease upon intranasal infection with VSV (Figure 7C). In addition, infection of Nup96+/+ and Nup96+/2 cells with VSV showed that Nup96+/2 cells express viral proteins earlier than Nup96+/+ cells and are more susceptible to cell death (Figures 7A and 7B). These results strongly indicate a role for Nup96 in antiviral response and in both innate and adaptive immunity. Discussion We showed here that Nup96 is selectively involved in the expression of key IFNg-regulated genes. Nup96 is a constituent of the Nup107-Nup160 complex, and experiments that have knocked down members of this complex other than Nup96 showed a major disruption of NPC assembly yielding defective pores (Boehmer et al., 2003; Harel et al., 2003; Walther et al., 2003). These

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Figure 7. Low Levels of Nup96 Increase Susceptibility to Viral Infection (A and B) Nup96+/+ and Nup96+/2 cells were infected with VSV-GFP, and cell viability was determined as described in Experimental Procedures. For each condition, 66 images were analyzed. Results represent mean values 6 SD. (C) Nup96+/+ and Nup96+/2 mice were infected with VSV at 105 plaque-forming units and monitored for morbidity (n = 13). Results are the mean 6 SD.

results were obtained at the cellular level. Here, we chose to use an in vivo system in which Nup96 is expressed at low levels, but, probably due to redundancies present in a whole-animal model, nuclear structure was not disrupted. However, it was possible to select for prominent defects that cannot be compensated in the presence of low levels of Nup96 and that are related to Nup96 function in vivo. We observed a defect in the immune system of Nup96+/2 mice, which showed that Nup96 is not only regulated by interferons as we previously shown (Enninga et al., 2002) but actively par-

ticipates in the immune response mediated by interferons. Studies that depleted Nup107 in cells either showed no alteration of Nup96 assembly at the NPC (Boehmer et al., 2003) or affected Nup96 localization at the NPC (Walther et al., 2003). This difference may be related to the degree of depletion achieved in each system. Regarding the effect of Nup107 depletion on mRNA export, Boehmer et al. found a partial inhibition of mRNA export (Boehmer et al., 2003). Here we showed that, in the context of a whole-animal model, reduced levels of

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Nup96 did not significantly alter the levels or the localization of various nucleoporins. In our studies, upon downregulation of Nup96 expression, the levels of other constituents of the Nup107-160 complex were not affected, probably due to redundancies or compensatory mechanisms present in a whole-animal model. However, these low levels of Nup96 may still have caused NPC abnormality, which was reflected on nucleocytoplasmic transport functions related to mRNA export of key regulators of immunity but not of bulk mRNA. In addition, we cannot exclude defects related to intranuclear functions of Nup96, since a pool of Nup96 is found inside the nucleus (Enninga et al., 2003; Fontoura et al., 1999). Although we did not see a significant change in total mRNA levels of these immune-related genes, we cannot rule out a potential role for Nup96 in regulating mRNA levels of additional genes. The reduced levels of MHC I and MHC II in Nup96+/2 mice pointed to a role for NUP96 in constitutive as well as IFN-induced expression of these proteins. In the thymus, a selective decrease in TCRab+ and CD4+ T cell populations (Figure 5A) suggests that impairment in MHC II expression by thymic macrophages and DCs have a major role in the development of these populations. Interestingly, although the expression of MHC class I is also reduced in thymic macrophages and DCs (Figure 3), frequency of local CD8+ T cells is not altered (Figure 5A). Probably the lower numbers of mature CD8+ T cells as compared to CD4+ T cells in the thymus can explain the fact that defects in T cell development are detectable only in the latter population. Another possibility that may account for the reduction in thymic CD4+ T cells is the poor expression of the costimulatory molecule CD86 (B7.2) in thymic antigen-presenting cells. Since CD86 is downmodulated in peritoneal macrophages of NUP96+/2 mice, there is a possibility that its expression is also downregulated in thymic cells. CD86 is usually expressed in the thymus, notably on dendritic and medulary epithelial cells. Alterations of B7 expression such as the ones present in CD80/CD86-deficient mice or CD86-transgenic mice result in profound defects in the frequency of thymic CD4+ and CD8+ mature T cells (Yu et al., 2000) . On the other hand, the normal frequency of CD4+ cells in the spleen (Figure 5A) suggests that peripheral mechanisms of maintenance of the T cell pool such as homeostatic proliferation operate to compensate the defect in the thymic output of these cells (Almeida et al., 2001). Another piece of evidence for a role of NUP96 in MHC expression in these mice is the fact that expression of MHC I and II by peritoneal macrophages is not properly upregulated even in the presence of high levels of IFN in vitro (Figure 3). This result suggests that NUP96 is required for the inductive effect of IFNs in MHC expression. Interestingly, spleen cells from NUP96+/2 mice produce significantly higher levels of IFNg when stimulated in vitro with ConA than control wild-type mice (Figure 6A). A feedback mechanism may be operating in these mice to compensate for the defect in MHC expression. MHC II-dependent immune response to a soluble antigen is also impaired in NUP96+/2 (Figure 5B). This defective response is not due to a constitutive impaired ability for proliferation, since polyclonally stimulated splenic T cells as well as nonstimulated T cells from

NUP96+/2 mice showed higher indexes of proliferation when compared to control mice. Therefore, the impaired immune response to ovalbumin is likely due to low levels of MHC II in the Nup96+/2 mice, since antigen presentation is required for specific immune responses. However, NUP96+/2 mice have also reduced levels of CD86 and CD54 (ICAM-1) expression. Interaction between CD28 and the costimulatory molecules of the B7 family, B7.1 (CD80) and B7.2 (CD86), is a critical step in signaling for activation and differentiation after antigen-specific stimulation of T cells (Hathcock et al., 1993; McAdam et al., 1998). On the other hand, interaction between T cell-mounted LFA-1 and its ligands, ICAM-1 (CD54), -2, and -3, is important for T cell surveillance and migration, T cell-APC crosstalk, and cytotoxic T cell function. LFA-1 coengagement by APC-expressed ICAM-1 triggers T cell proliferation and IL-2 secretion and induces transmembrane signals distinct from those delivered through the TCR-CD3 complex alone, suggesting a costimulatory role for this interaction in T cell activation (Zuckerman et al., 1998). The increased proliferation of T cells from Nup96+/2 mice stimulated via CD3 may be related to additional functions of Nups related to regulation of cell growth. It has been shown that Nup96 is localized in specific mitotic structures including the spindles and the kinetochores (Enninga et al., 2003; Loiodice et al., 2004). Thus, the function or functions of Nups in mitotic processes remain to be elucidated, and this does not exclude potential effects of Nups on cell growth during interphase. Frequency of B cells is also reduced in the spleen of NUP96+/2 mice. Several factors may contribute to maintain B cells numbers in normal mice including homeostatic proliferation (Woodland and Schmidt, 2005) and the production of IL-4, a B cell growth factor, by T cells (O’Garra et al., 1986). We observed that downregulation of Nup96 is associated with a reduction in the IL-4 production by splenic T cells upon stimulation with ConA (Figure 6B). At the same time, IFNg production is significantly upregulated (Figure 6A). IFNg inhibits proliferation and differentiation of B cells and also downmodulates IL-4 production by T cells (Reynolds et al., 1987). Thus, high levels of endogenous IFNg may act directly on B cell proliferation or may indirectly affect the physiological levels of B cell proliferation by reducing IL-4 production in the spleen. This upregulation of IFN-g production may represent a mechanism to compensate for the downregulation of interferon-induced genes. In fact, IFNg has also been shown to regulate constitutive expression of MHC I (Lee et al., 1999). B cell function is also altered following NUP96 downregulation in vivo. Reduction in serum immunoglobulin levels in Nup96+/2 mice may be due to a defective interaction of B cells with CD4+ T cells, since IgG is the isotype affected. Production of IgG antibodies is highly dependent on germinal center formation, cytokine secretion by activated CD4+ T cells, and CD40-CD40 ligand interaction (Cerutti et al., 1998; Stavnezer, 2000). MHC class II downmodulation in NUP96+/2 mice may interfere with IgG production in two levels: (1) by reducing activation of CD4+ T cells and altering cytokine production by these cells and (2) and by a direct interference in T-B cell interaction that occurs via MHC II and costimulatory molecules. We observed evidence for both mechanisms,

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since CD4+ T cell response is altered due to the impaired expression of MHC class II in antigen-presenting cells. Cytokine production by T cells is also relevant for this process, since the main isotype affected is IgG1, an immunoglobulin that is highly dependent on IL-4 production by Th2 cells. We also observed a reduction on the levels of MHC II expression in B cells of NUP96+/2 mice (data not shown), suggesting that direct interaction of these cells with CD4+ T cells may be also altered. In addition, it is possible that, similar to macrophages, B cells of NUP96+/2 mice present a defective expression of costimulatory molecules, namely CD86. Studies of mice rendered deficient in B7.1 and B7.2 have indicated an essential role for B7 costimulation in humoral responses, including immunoglobulin class switch and germinal center formation (Borriello et al., 1997). Importantly, the higher susceptibility of Nup96+/2 cells and Nup96+/2 mice to VSV infection (Figure 7), together with impaired responses to type I and type II IFNs (Figure 3), shows a role for Nup96 in both innate and adaptive immunity. In addition, a recent work in Arabidopsis thaliana carrying a mutation in a Toll Interleukin1 receptornucleotide binding-Leu-rich repeat-type R gene has pointed to a function for Nup96 in basal defense and constitutive resistance responses to pathogens (Zhang and Li, 2005). Altogether, these findings underline the importance of nucleocytoplasmic trafficking and/or of additional unknown functions of nucleoporins in regulating immunity. Experimental Procedures Generation of Knockin Mice A 1.6 kb DNA fragment corresponding to a region 0.5–2 kb upstream of exon 21 of the Nup98/Nup96 gene was generated as short arm and inserted into the 30 end of a Neo gene cassette, which was flanked by loxP sites. A three-way stop codon, which stops translation in any reading frame, was then introduced at the 50 end of exon 21. The stop codon was created by PCR using primers corresponding to a region located at the 50 end of exon 21 and a region approximately 0.56 kb upstream of exon 21. The PCR product was then cloned into PspMOI site at the 50 end of the neo gene cassette. A long arm of 8 kb genomic region was then generated and inserted downstream of the introduced stop codons at the 50 end of exon 21. The targeting vector was linearized with NotI and then transfected by electroporation into 129/SvEv iTL1 embryonic stem cells at InGenious Targeting Laboratory. After selection in G418, surviving colonies were expanded, and PCR analysis was performed to identify clones that had undergone homologous recombination. The correctly targeted ES cell lines were microinjected into C57BL/6J blastocysts. The chimeric mice were generated and gave germline transmission of the stop codon introduced at the 50 end of the Nup96 gene. Mice were genotyped by Southern blot analysis. Genomic DNA was digested with NcoI and BglI, and hybridization was performed using a probe 600 bp upstream the 30 end of exon 21 of the Nup98/Nup96 gene with the AlkPhos Direct kit (Amersham). Heterozygous mice were generated with deletion of the Neo cassette by crossbreeding with Jackson EIIA TG mice (a loxP deleter cre mouse). The presence of the mutation was confirmed by PCR using a 50 end primer complementary to the site of the mutation and a 30 end primer 800 bp downstream of this site. Mice analyzed in these studies included Nup96+/+ and Nup96+/2 mice with 129/SvEv and C57BL/6J strains background as well as Nup96+/+ and Nup96+/2 mice that were backcrossed to a homogenous BALB/c strain background. Mice were housed under specific pathogen-free conditions in our facilities. Transmission Electron Microscopy Mice were anesthetized by intraperitoneal injection of an anesthetic cocktail containing ketamine/xylazine. Livers were perfused with

PBS followed by ice-cold 2.5% glutaraldehyde in 100 mM cacodylate (pH 7.4). Tissues were fixed overnight in the same solution and postfixed in 1% osmium tetroxide in the same buffer and then treated with uranyl acetate, dehydrated in ethanol and propylene oxide, and embedded in Epon. Grids were analyzed in an electron microscope (100 CX; Jeol Ltd., Tokyo, Japan) operated at 80 kV. Immunoblot Analysis Immunoblots were performed with anti-Nup98 (Radu et al., 1995), anti-Nup96 (Fontoura et al., 1999), anti-Sec13 (Enninga et al., 2003), or mAb14 antibodies (Davis and Blobel, 1986) as previously described (Faria et al., 2005). Oligo-dT In Situ Hybridization, Nuclear Import Assay, and Deconvolution/Apotome Microscopy MEFs grown on coverslips were washed in PBS, fixed with electron microscopy grade formaldehyde, and permeabilized with Triton X-100 as previously described (Bastos et al., 1996). Oligo-dT in situ hybridization was performed as previously described (Bastos et al., 1996). Samples were examined on a Zeiss Axiovert 200M with Deconvolution and Apotome Systems, Video System, Axiovision 4.0. Nuclear import assays were performed as previously described (Moore and Blobel, 1992). TMBP-M3-GFP or NLS-BSA/ tetramethylrhodamine B isothiocyanate-NLS-bovine serum albumin was used as substrate (Chook et al., 2002; Moore and Blobel, 1992). RNA Quantification by Real-Time PCR Perinoteal macrophages were lyzed for 10 min at 4ºC in a buffer containing 25 mM Tris (pH 7.4), 15 mM NaCl, 12.5 mM MgCl2, 5% sucrose, 0.7% Igepal, and 1000 U/ml RNAsin. Nuclear fractions were collected by centrifugation at 1000 3 g for 3 min and supernatants constituted the cytoplasmic fractions. Total RNA from each fraction was purified using the PicoPure isolation kit (Arcturus). Quality and size distribution of RNAs were determined by the Agilent 2100 Bioanalyzer (Agilent Technologies). RNAs were amplified according to the Amino Allyl MessageAmp aRNA kit (Ambion). The resulting cDNA was diluted in water and used in PCR reactions using the iCycler iQ Real-time PCR Detection System (Bio-Rad) according to the manufacturer’s instructions and as previously described (Chang et al., 2004). Antigens Hen egg ovalbumin (grade III) and Concanavalin A were purchased from Sigma. Immunization Mice were immunized subcutaneously (s.c.) at the base of the tail with 100 mg OVA emulsified in complete Freund’s adjuvant (CFA) containing 50 mg Mycobacterium tuberculosis H37RA (DIFCO). Eight days postimmunization, mice were sacrificed, and the draining lymph nodes (inguinal) were harvested for proliferation and cytokine assays. Cell Preparations Spleen and lymph nodes (inguinal, brachial, and mesenteric) were removed, and cell suspensions were prepared using a tissue homogenizer and gently centrifuged. Spleen cells were depleted of erythrocytes by resuspending them in 9 ml of water and immediately adding 1 ml 103 PBS to recover tonicity. Peritoneal macrophages were collected 3 days after injection of 2 ml 3% thioglycolate solution i.p. Analysis of Cell Markers and Intracellular Cytokines by Flow Cytometry Rat FITC-labeled antibodies used for labeling mouse markers were anti-TCRa, anti-TCRd, anti-CD19, anti-CD11c, anti-Mac3, and antiCD11b. Rat PE-labeled antibodies were anti-CD4, anti-CD8a, antiH-2kb, anti-I-Ab, anti-CD54, anti-CD11a, anti-CD14, anti-CD16, anti-CD80, and anti-CD86. Cychrome-labeled anti-CD3 antibody was also used. Intracellular cytokines were detected in cells labeled with FITC-labeled CD4 or CD8 antibodies and PE-labeled anti-IFN-g or anti-IL-4 antibodies. Cells were applied to a FACScan. Cell subsets were also analyzed by their specific cell-surface markers (CD3 for T lymphocyte, CD19 for B lymphocytes, Mac3 for macrophages,

Role of Nup96 in Interferon-Regulated Functions 303

and CD11c for dendritic cells). All data were analyzed using CellQuest software. Measurement of Serum Immunoglobulins To assess total levels of serum immunoglobulins, mice were bled under anesthesia from the auxiliary plexus, and serum was collected from blood after 30 min of resting at room temperature and 3 min centrifugation at 2000 rpm. Levels of isotype-specific Ig in sera were determined by ELISA. Cytokine Assays Cells isolated from spleen (erythrocyte depleted) and lymph nodes were cultured at 1 3 107 cells/ml with or without 4 mg/ml Concanavalin A (Con A). Supernatants were collected after 48 hr, and cytokine levels were measured by capture ELISA. For intracellular staining, primary cultures were stimulated with either ConA for 12 and 24 hr or OVA for 12 hr in the presence of 10 mg/ml brefeldin A. Cells were stained for flow cytometry analysis. Secondary response to OVA was measured by restimulation of primary cultures (kept without brefeldin) 12 hr after the first stimulation. Cells were incubated for another 12 hr in the presence of antigen and brefeldin A and subjected to flow cytometry analysis. Proliferation Assay Cultures contained erythrocyte-depleted spleen cells and lymph node cells from individual mice at 5 3 106 cells/ml were incubated in 96-well plates (Nunc, Naperville, Illinois) in complete RPMI with or without 100 mg/ml anti-CD3 monoclonal antibodies (BD-Pharmingen) for 72 hr. [3H thymidine] at 1 mCi/well was added for the last 12 hr of culture, and thymidine incorporation was measured. Statistical Analysis Results represent mean values 6 SD of a group (n = 7–8), and the statistical significance of differences was determined using ANOVA. In Vitro and In Vivo Survival Assays Nup96+/+ and Nup96+/2 MEFs were infected with VSV-GFP at MOIs of 0, 0.01, 0.1, 1, 5, and 10 for 6, 9, or 24 hr, and cell viability was followed by bright-field microscopy, GFP expression, and exclusion of 2 mM ethidium homodimer-1 (Molecular Probes) visualized on 5 randomly picked 1003 microscopic fields. Four hundred images were processed using the NIH Image J software. For in vivo assays, mice were infected intranasally with VSV at 105 plaque-forming units and were monitored for morbidity. Acknowledgments We thank V. Nussenzweig and M.C. Nussenzweig for productive discussions and G. Blobel, J. Glavy, and T. Boehmer for antibodies. We thank the Live Cell Imaging Facility at UTSW and H. Shio for support with EM samples. We thank H. Valentine, E. Casas, W. Zhu, E. Rabelo, L. Nathanson, and C. Arana for assistance. This work was supported by NIH (R01 GM067159-01 to B.M.A.F.); Florida Biomedical Research Grant (04TSP-02 project II to B.M.A.F.); American Cancer Society Institutional Grant (IRG98-277 to B.M.A.F.); Stanley J. Glaser Biomedical Research Grant (700393 to B.M.A.F.); CNPq, FAPEMIG grants to A.M.C.F., and NIH (AI28900 to D.E.L.). Received: September 5, 2005 Revised: January 10, 2006 Accepted: January 23, 2006 Published: March 21, 2006

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