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In this study, the role of NF-κB1 was examined during toxoplasmosis. While wildtype BALB/c mice generated protective responses, NF-κB1−/− mice developed Toxoplasmic encephalitis, characterized by increased parasite burden and necrosis in the brain. Susceptibility was primarily associated with a local decrease in the number of CD8+ T cells and IFN-γ production, while accessory cell function appeared intact in NF-κB1−/− mice. Consistent with these findings, T cell transfer studies revealed that NF-κB1−/− T cells provided SCID mice less protection than wildtype T cells. These results demonstrate an intrinsic role for NF-κB1 in T cell-mediated immunity to T. gondii.
Many aspects of the immune response are dependent on the NF-κB family of transcription factors: RelA, RelB, c-Rel, NF-κB1, and NF-κB2. NF-κB1 (p50) is processed from the p105 protein and regulated by inhibitor of NF-κB (IκB) proteins. Stimuli, including TLR ligands, TNF-α, or T cell receptor engagement lead to the nuclear localization of NF-κB1 hetero- or homodimers which regulate gene expression (Beinke and Ley, 2004; Mason et al., 2004a; Zhang and Ghosh, 2001). The importance of NF-κB1 in the generation of protective immunity and autoimmunity has been demonstrated using mice deficient in this subunit. While NF-κB1−/− mice develop normally, they have several immune defects, including decreased T and B cell proliferation, antibody production, and macrophage expression of IL-10 (Artis et al., 2003; Beinke and Ley, 2004; Cao et al., 2006; Corn et al., 2003; Mason et al., 2004a; Sha et al., 1995; Snapper et al., 1996; Sriskantharajah et al., 2009). Initial studies demonstrated that NF-κB1−/− mice are more susceptible to the bacterial pathogens Listeria monocytogenes and Streptococcus pneumoniae, but were more resistant to encephalomyocarditis virus, due to increased apoptosis of infected cells (Schwarz et al., 1998; Sha et al., 1995). Later, cell-specific roles for NF-κB1 in the generation of protective immune responses to parasites were identified. The failure of NF-κB1−/− mice to control Leishmania major was attributed to defects in CD4+ T cell proliferation, which was associated with a reduced protective Th1 cell response (Artis et al., 2003). On the other hand, NF-κB1 was shown to be essential for dendritic cell (DC)-mediated induction of Th2 responses (Artis et al., 2005). Additionally, NF-κB1−/− mice develop severe colitis in response to Trichuris muris infection, characterized by increased IFN-γ production (Artis et al., 2002). Consistent with the identified defects in the generation of CD4+ T cells responses, NF-κB1−/− mice fail to generate autoreactive T cells and do not develop experimental autoimmune encephalomyeletis (Hilliard et al., 1999). Taken together, these studies demonstrate that the absence of NF-κB1 differentially impacts the generation of a variety of immune responses.
T. gondii is a protozoan parasite that infects approximately one-quarter of the U.S. population and roughly one-third of the world population. Infection can be acquired by ingestion of oocysts, consumption of undercooked meat, and congenitally. Despite the development of protective immunity, parasites persist in a latent cyst form in multiple tissues, most prominently the brain. In immuno-competent individuals, the chronic phase is asymptomatic, but persons with acquired immune-deficiencies in T cell function develop Toxoplasmic encephalitis when parasites reactivate within the brain (Hill et al., 2005; Hunter and Remington, 1994; Luft and Remington, 1992).
Consistent with the role of NF-κB in the recognition and control of infections, several pathogens interfere with this signaling pathway in order to subvert the host immune response (Tato and Hunter, 2002). Relevant to these studies, multiple groups have described the ability of T. gondii to disrupt NF-κB activation, by preventing the phosphorylation and nuclear translocation of RelA to the nucleus of infected macrophages (Butcher et al., 2001; Shapira et al., 2005; Shapira et al., 2002). Overall, the inhibition of NF-κB signaling by T. gondii leads to reduced capacity of infected cells to produce proinflammatory cytokines required for resistance to infection with this organism (Butcher et al., 2001).
Although this parasite interferes with immune responses generated through NF-κB, several studies from this laboratory have identified essential roles of NF-κB subunits in the immune response to T. gondii. Resistance to T. gondii is generated by innate recognition of the parasite leading to production of IL-12 which stimulates NK and T cells to secrete IFN-γ (Gazzinelli et al., 1994; Liu et al., 2006; Scanga et al., 2002; Scharton-Kersten et al., 1995). IFN-γ-mediated effector responses are required for clearance of the parasite during the acute phase of infection (Lieberman et al., 2004; Scharton-Kersten et al., 1996; Taylor et al., 2004). Once infection progresses to the chronic phase, current models suggest that IFN-γ and CD4+ and CD8+ T cells are required to limit parasite replication in the brain (Gazzinelli et al., 1992; Suzuki et al., 1989). The NF-κB family has been implicated in all of these processes and, not surprisingly, this infection leads to global activation of NF-κB (Shapira et al., 2002). Previous work from this laboratory identified roles for NF-κB2, c-Rel, and RelB as well as a T cell-intrinsic requirement for NF-κB in resistance T. gondii (Caamano et al., 1999; Caamano et al., 2000; Mason et al., 2002; Mason et al., 2004b; Tato et al., 2003). Moreover, NF-κB1 has been implicated in the regulation of NK cell proliferation and IFN-γ production during Toxoplasmosis (Tato et al., 2006). The studies presented here demonstrate that while NF-κB1-deficient mice are able to control the early phase of infection, a T cell-intrinsic role for NF-κB1 is essential for long-term resistance to the parasite.
NF-κB1−/− mice were originally provided by Rodrigo Bravo at Bristol-Myers Squibb Pharmaceutical Research Institute (Princeton, NJ) and bred in University Laboratory Animal Resources facilities at the University of Pennsylvania. Mice were crossed onto the BALB/c background for greater than nine generations, bred as heterozygotes, and genotyped by PCR. Age and sex-matched knockout mice were used in experiments with wildtype littermates serving as controls. BALB/c SCID and CBA mice were purchased from Taconic Farms, Inc. (Germantown, NY). All procedures were performed in accordance to the guidelines of the University of Pennsylvania Institutional Animal Care and Use Committee. For infections, the Me49 strain T. gondii parasites were isolated from chronically infected CBA mice. Brains were removed and homogenized by serial needle passage in PBS. Parasite cysts were enumerated by light microscopy. Mice were infected with 20 cysts i.p.
To measure the amount of parasite DNA in the brain and liver, real-time PCR was utilized as previously described (Wilson et al., 2005). Briefly, approximately 50 mg of tissue was digested and DNA was purified using the High Pure PCR template preparation kit (Roche, Mannheim, Germany). Real-time PCR specific for the Toxoplasma B1 repeat region was used to quantify the amount of parasite DNA from 500 ng of DNA purified from tissue (Wilson et al., 2005). For the analysis of gene expression, brain tissue was placed in Trizol (Invitrogen, Carlsbad, CA) and mRNA was extracted as instructed by the manufacturer. Purified RNA was treated with DNAse I to eliminate any contamination with genomic DNA (Promega, Madison, WI). cDNA was generated using Superscript II reverse transcriptase (Invitrogen). Real-time PCR was performed using primers for iNOS and normalized to HPRT (Qiagen, Germantown, MD). Samples, standard amounts of purified T. gondii DNA, or cDNA were amplified using Power SYBR® Green PCR Master Mix and a 7500 Fast Real-Time PCR System. Analysis was performed with system software v1.3.1 (Applied Biosystems, Warrington, UK).
For histological examination of brain tissue, the organ was excised and placed in 4% buffered formalin. Tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Sections were examined by light microscopy on a Nikon Eclipse E600 scope and images captured using a Nikon DS-Fi1 camera run by NIS Elements software (Nikon, Melville, NY). For immunohistochemistry, organs were embedded in OCT and flash frozen. Six μm sections were cut using a Microm cryostat. Sections were fixed with a solution of 75% acetone and 25% ethanol. Anti-CD4 (5 μg/ml), anti-CD8 (5 μg/ml) (eBioscience, San Diego, CA), anti-I/A I/E (BD Pharmingen), anti-iNOS (10 μg/ml), anti-Ki67 (3.25 μg/ml) (Abcam, Cambridge, MA), anti-Me49 (1:4000), and anti-GFAP (10 μg/ml) (Invitrogen) antibodies were used. Anti-rabbit Alexa 488 (Invitrogen) and anti-rat Cy3 (Jackson Immunoresearch, West Grove, PA) were used as secondary antibodies for fluorescence staining. DAPI (Invitrogen) was used to visualize nuclei. To detect the expression of iNOS, a biotinylated goat anti-rabbit antibody was used, followed by Vectastain ABC reagent (Vector Laboratories, Burlingame, CA). DAB was used as an HRP substrate that created a brown precipitate and slides were counterstained with hematoxylin. Images were captured using light microscopy or standard fluorescence microscopy using a Nikon Eclipse E600 microscope (Melville, NY) equipped with a Photometrics Cool Snap EZ CCD camera (Tucson, AZ). Nikon NIS Elements software was used to capture and overlay images. The percentage of Ki67+ cells was determined by scoring at least 150 T cells from images from two separate experiments.
Spleens were dissected, dissociated, and subjected to hypotonic red blood cell lysis to generate a cell suspension. The remaining cells were enumerated and used in subsequent assays. Brain mononuclear cells (BMNCs) were isolated as previously described (Wilson et al., 2005). Briefly, perfused brains were cut into small pieces, passed through an 18 gauge needle, and digested with collagenase/dispase and DNase (Roche) for 90 minutes. Following the digestion, the cells were washed and strained through a 70 μm filter. Subsequently, cells were resuspended in 60% percoll, overlayed with 30% percoll, and centrifuged at room temperature for 25 minutes at 2000 rpm. BMNCs were collected from the interface, washed, and enumerated. For T cell recall responses, splenocytes were plated at 500,000 cells/well and BMNCs at 200,000 cells/well. Cells were stimulated with medium alone, 25μg/ml soluble Toxoplasma antigen (STAg), or 1 μg/ml soluble anti-CD3 antibody (BD Pharmingen) for 48 hours at 37° C with 5% CO2. IFN-γ and IL-12p40 were measured by ELISA from the culture supernatant or serum, as previously described (Caamano et al., 1999; Sander et al., 1989). For flow cytometry, cells were washed with FACs buffer (1X PBS, 0.2% BSA, and 2mM EDTA) and incubated in Fc block (0.1 μg/ml 24G2 antibody) for 15 minutes prior to surface staining with CD4-FITC, CD8-PerCp Cy5.5, CD44-APC, and CD62L-APC Alexa 750 (eBioscience). Dendritic cells were identified as CD3−, NK1.1−, CD19−, CD45+, and CD11c+ (all antibodies from eBioscience) and stained with anti-Kd-PE or anti-I/A-PE (BD) to assess MHC class I and II expression, respectively. For intracellular cytokine staining, 5 × 105 splenocytes and 2 × 105 BMNCs were plated in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal calf serum, 1% sodium pyruvate, 1% non-essential amino acids, 0.1% β-mercaptoethanol, 100 U of penicillin/ml and 100 μg/ml of streptomycin (Gibco, Invitrogen Incorporation, Grand Island, NY). Cells were stimulated with PMA (50 ng/ml) and ionomycin (500ng/ml) in the presence of brefeldin A (10μg/ml) for four hours at 37° C with 5% CO2. Cells were washed, incubated with Fc block, and surfaced stained with anti-CD4-FITC and anti-CD8-PerCp Cy5.5 (eBioscience). The cells were fixed with 2% paraformaldehyde (Electron Microscopy Science, Hatfield, PA) for 10 minutes. For intracellular staining, the cells were permeabilized with 0.1% saponin (Sigma, St. Louis, MO) in FACS buffer and stained with anti-IFN-γ-PE (eBioscience). All flow cytometry was performed on a FACsCanto using FACsDIVA 6.0 software (BD Biosciences, San Jose, CA). Analysis was performed using FloJo software (Treestar Inc., Ashland, OR).
T cells were purified from the spleens and lymph nodes of wildtype BALB/c mice and NF-κB1−/− mice using a T cell enrichment column (R&D Systems, Minneapolis, MN). The cells were determined to be greater than 90% pure by flow cytometric analysis (data not shown). 2 × 106 T cells in PBS were transferred i.p. to BALB/c-SCID mice.
Significance was determined using student’s t-test. p ≤ 0.05 was considered significant. Analysis was performed using Prism software (GraphPad Software, Inc.).
To determine the role of the NF-κB subunit, NF-κB1, in the immune response to T. gondii, wildtype BALB/c and NF-κB1-deficient mice were injected i.p. with 20 cysts of the Me49 strain. Whereas the wildtype BALB/c were resistant to infection, the knockout mice succumbed approximately 3–4 weeks post-infection (Figure 1A). Quantification of parasite DNA at day 22 post-infection revealed a two-log increase in parasite burden in the brain and liver of the NF-κB1−/− mice (Figure 1B). Histological analysis indicated that the most prominent pathology was evident in the brain, with only limited indicators of pathology in the liver. In NF-κB1−/− mice, extensive regions of parasite replication, necrosis, and some inflammatory infiltrate were present in the brain (Figure 1C). While a significant difference in parasite burden was observed during the chronic phase, clearance of parasites from the peritoneal cavity on day 10 post-infection was evident in wildtype and knockout mice (data not shown). These results indicate that NF-κB1-deficient mice survive the acute phase of infection, but succumb as a consequence of the presence of large numbers of parasites in the brain.
Multiple studies have implicated astrocytes in the local control of T. gondii in the CNS (Wilson and Hunter, 2004). Therefore, the function of astrocytes in the absence of NF-κB1 was addressed. As an indicator of astrocyte activation in vivo, GFAP expression was assessed by immunohistochemistry. Widespread astrocyte activation was observed in NF-κB1−/− and wildtype mice (Figure 2A). In addition, when primary astrocyte and macrophage cultures were treated with IFN-γ and TNF-α and infected with T. gondii, NF-κB1−/− cells were able to control the parasites as well as wildtype cells (Supplemental Figure 1). These results suggest that NF-κB1 deficiency does not appreciably affect the ability of astrocytes or macrophages to respond to infection in vivo or control parasite replication in vitro.
Several NF-κB-regulated molecules are induced in the brain during chronic T. gondii infection, including inducible nitric oxide synthase (iNOS), MHC class I, and MHC class II, which are all required for resistance to T. gondii (Gazzinelli et al., 1993; Scharton-Kersten et al., 1997; Schluter et al., 1999). Levels of these molecules were measured in the brains of wildtype and NF-κB1−/− mice on day 22 post-infection. Immunohistochemical staining of brain sections identified numerous regions of iNOS production associated with parasite replication (Figure 2B). The marked increase in parasite burden in the NF-κB1−/− mice correlated with increased iNOS staining in comparison to wildtype mice. The relative amount of iNOS transcript was also elevated in the brains of NF-κB1−/− mice in comparison to wildtype mice (Figure 2C). Following infection, the expression levels of MHC class I and II molecules increase on antigen presenting cells (APCs). Using flow cytometry, MHC class I and II expression was measured on microglia, macrophages, and dendritic cells from the spleen and CNS to assess the activation status of APCs. While MHC class I and II levels were decreased on dendritic cells (DCs) in the brain (Figure 2D), expression was equivalent on macrophages and microglia from the CNS and splenic DCs and macrophages (data not shown). Immunohistochemical staining demonstrated the association of MHC class II positive cells near regions of parasite replication in wildtype and knockout mice (Figure 2E). While MHC class I and II expression were largely intact and higher levels of iNOS were present, NF-κB1−/− mice fail to limit parasite replication. Expression of IFN-γ-induced GTPases required for parasite control were also assessed by real-time PCR. During chronic infection, higher levels of ifi47 and igtp were measured in the knockout mice versus wildtype mice (Supplemental Figure 2). Thus, it is likely that additional NF-κB1-dependent immune responses are altered in the knockout mice, which ultimately leads to increased susceptibility to T. gondii.
The control of T. gondii in the CNS is dependent on local T cell production of IFN-γ, leading to the activation of anti-parasitic effector mechanisms (Gazzinelli et al., 1992; Suzuki et al., 1988). Given a role for NF-κB1 in the regulation of T cell responses (Beinke and Ley, 2004), studies were performed to assess if the absence of this transcription factor affects the local T cell response. Therefore, T cells in the CNS were examined on day 22 post-infection by immunohistochemistry. These studies revealed that, as in wildtype mice, CD4+ and CD8+ T cells from NF-κB1−/− mice were observed in close proximity to parasites (Figure 3A and 3B). Since NF-κB1 affects T cell proliferation, brain sections from wildtype and knockout mice were stained with antibodies to Ki67, a marker used to detect cells in active cell cycle (Barber et al., 2006; Scholzen and Gerdes, 2000). Approximately 30% of T cells in the brains of wildtype mice were Ki-67+. The majority of CD4+ (56%) and CD8+ (62%) T cells in the brains of NF-κB1−/− mice were Ki-67+ (Figure 3C), although the overall number of T cells was decreased (Figure 4A). Thus, despite having a higher percentage of T cells in the cell cycle, NF-κB1−/− mice appeared to have fewer T cells in the brain. Based on histology, T cell recruitment to the CNS and activation of cell cycle are not defective in NF-κB1−/− mice, but survival or proliferation deficits may lead to reduced T cell populations during chronic infection.
To quantify the number and activation status of T cells following infection, T cells were isolated and analyzed by flow cytometry during the acute (day 10 post-infection) and chronic (day 22 post-infection) stage of disease. Early during infection there were no differences found in CD4+ T cell number or activation status (CD44hi and CD62Llo). In addition, there was a trend toward fewer total and activated CD8+ T cells isolated from the spleens of NF-κB1−/− mice, but no significant differences were detected (results not shown). Later, during chronic infection, more dramatic defects in the T cell compartment were revealed. While the total number of CD4+ T cells in the spleen and brain were not dramatically altered at this timepoint, there was a significant reduction in the number of CD8+ T cells in the spleen (Figure 4A, p=0.0475) and brain (Figure 4B, p=0.0108). Additionally, there were significant decreases in activated CD4+ and CD8+ T cells in the spleen by percentage (Figure 4C) and number (Figure 4D). The most striking difference in activated T cell numbers in the CNS was in the CD8+ T cell compartment (Figure 4E, p=0.01). These results confirmed previous observations that NF-κB1−/− mice have a defective T cell response in the brain, predominantly in the CD8+ compartment and also revealed a CD4+ T cell defect in the spleen.
Control of T. gondii during the acute and chronic stages of infection relies on a strong Th1 response. Thus, the ability of the NF-κB1 knockout mice to mount a Th1 response during the acute (day 10) and chronic (day 22) stage was examined. Serum levels of IL-12p40 and IFN-γ were measured by ELISA on day 10 post-infection. NF-κB1−/− mice displayed increased levels of IL-12 p40 and decreased levels of IFN-γ (Figure 5A and 5B). These results suggest that the defect in IFN-γ production during the acute phase was not caused by a reduction in IL-12 production by innate immune cells.
As an additional readout of T cell function, splenic T cell recall responses to soluble Toxoplasma antigen (STAg) and anti-CD3 were measured on day 10 and 22 post-infection. Following a 48-hour incubation with antigen, cells from the knockout mice produced less IFN-γ in response to STAg than wildtype mice (Figure 5C and 5D). IFN-γ production was also assessed by intracellular cytokine staining. Cells from the spleen and brain isolated on day 22 post-infection were stimulated with PMA and ionomycin for 4 hours ex vivo in the presence of brefeldin A. Flow cytometric analysis revealed that T cells from the knockout mice were capable of making IFN-γ; in fact, a higher percentage of T cells were producing cytokine in comparison to wildtype mice (Figure 6A). Despite this, the numbers of cytokine-producing CD8+ T cells were diminished in spleens and significantly reduced in the brains (p=0.0076) of NF-κB1−/− mice, while the number of CD4+ T cells producing IFN-γ was comparable to wildtype mice (Figure 6B and 6C). These data suggest that T cells from NF-κB1−/− mice are capable of producing IFN-γ, but the severe Toxoplasmic encephalitis in NF-κB1−/− mice is associated with a marked defect in the number of CD8+ T cells producing this cytokine in the brain.
The studies described above identify a major role for NF-κB1 in the regulation of T cell-mediated responses to T. gondii, but it was unclear whether this was a secondary consequence of reduced APC function or was T cell intrinsic. To directly distinguish this, a transfer model was utilized. Cells isolated from the spleen and lymph nodes of uninfected wildtype and NF-κB1−/− mice were enriched for T cells and 2 × 106 T cells were transferred i.p. to SCID mice one day prior to infection with T. gondii. SCID mice that were not given T cells succumbed to infection prior to day 20, while SCID mice that received T cells from WT mice survived for approximately 40 days. Mice that received NF-κB1 deficient T cells demonstrated an intermediate phenotype, where the majority of the mice survived 25 days post-infection (Figure 6D). On day 10 post-transfer, IFN-γ levels were measured in the serum of mice receiving T cell transfer. Recipients of NF-κB1−/− T cells had less systemic IFN-γ in comparison to mice receiving wildtype T cells (Figure 6E). This defect in cytokine production may underlie the increased susceptibility of SCID mice receiving NF-κB1 deficient T cells. Together these data identify a T cell-intrinsic role for NF-κB1.
The results presented here establish a role for NF-κB1 in T cell-mediated control of T. gondii in the CNS. Given the ubiquitous role of NF-κB transcription factors in the immune response, it was surprising that NF-κB1 deficient mice survived acute infection. Several immune parameters remained intact or were enhanced in NF-κB1−/− mice, and thus may contribute to the early control of the parasite. For example, it has been previously reported that NK cells from NF-κB1−/− mice infected with T. gondii are hyper-proliferative and produce more IFN-γ than their wildtype counterparts (Tato et al., 2006). In addition, the data presented here indicate increased levels of IL-12 are found in the serum of NF-κB1−/− mice. Previous reports have demonstrated that NF-κB1 homodimers and NF-κB1 dimers with RelA and c-Rel are capable of binding the iL-12p40 promoter (Sanjabi et al., 2000). Given the role of NF-κB1 homodimers as transcriptional repressors (May and Ghosh, 1997), the results presented here are consistent with a negative regulatory role of NF-κB1 on IL-12p40 production. Nevertheless, despite enhanced innate responses in the absence of NF-κB1, the knockout mice ultimately fail to control parasite replication in the brain. Due to the role of NF-κB in autoimmune disease and cancer, NF-κB inhibitors have been developed and are currently in clinical trials (Karin et al., 2004). Based on the findings of this study, reactivation of latent T. gondii in the CNS may be a negative side effect of such therapies.
The enhanced parasite replication and associated areas of necrosis in the brains of NF-κB1−/− mice are reminiscent of the phenotypes observed in mice that lack iNOS or TNF-α signaling (Deckert-Schluter et al., 1998; Gazzinelli et al., 1993; Scharton-Kersten et al., 1997; Yap et al., 1998). Since TNF-α signaling is required for the optimal induction of IFN-γ-mediated control of intracellular T. gondii (Sibley et al., 1991), cell-intrinsic NF-κB1 may be required in for parasite control in infected macrophages and astrocytes. However, the use of in vitro studies, where exogenous TNF-α and IFN-γ was provided to cells infected with T. gondii, did not reveal an inability of NF-κB1 deficient cells to control parasite replication. Moreover, astrocyte activation in infected knockout mice, assessed by expression of GFAP, appeared to be equivalent to wildtype mice. Consistent with these results, more iNOS was detected in the brains of NF-κB1−/− mice in comparison to wildtype mice. In addition, MHC class I and II expression was not decreased on NF-κB1−/− macrophages isolated from the brain. Taken together, these results suggested that the increased susceptibility of NF-κB1−/− mice to TE was not due to an intrinsic defect in the effector functions of two commonly infected cell types, macrophages and astroyctes.
The ability of T cells, particularly CD8+ T cells, to produce IFN-γ is an essential element of the immune response necessary for the local control of T. gondii in the CNS (Gazzinelli et al., 1992; Suzuki et al., 1989; Suzuki et al., 1988). Characterization of the T cell response in chronically infected NF-κB1−/− mice revealed a marked decrease in the number of activated and cytokine-producing CD8+ T cells in the spleen and brain. When assessing T cells in the brain by immunohistochemistry, it was apparent that NF-κB1−/− T cells were able to traffic to the CNS to regions of parasite replication and expressed Ki67, a marker of active cell cycle. Consistent with the idea that accessory cell functions are intact in NF-κB1−/− mice, T cells were activated during acute infection and there was no significant decrease in CD8+ T cells in the knockout mice at this stage of disease. However, T cell transfer to SCID mice confirmed that NF-κB1−/− T cells were not able to provide as much protection as wildtype T cells. Together, these results support the idea that the increased susceptibility of NF-κB1−/− mice to TE is associated with an intrinsic role for NF-κB1 in the regulation of T cell functions.
An important question these studies raised is how NF-κB1 influences the CD8+ T cell response. Given the prominent role of NF-κB in T cell proliferation and survival, one possible explanation is that the absence of NF-κB1 these processes are affected, leading to the loss of parasite-specific T cells (Beinke and Ley, 2004; Li and Verma, 2002; Mason et al., 2004a). Based on the histological analysis of proliferation using the marker Ki67, more cells in the brains of NF-κB1−/− mice were in active cell cycle in comparison to T cells in brains of wildtype mice. These results suggest that NF-κB1 may impact cell survival. Alternatively, the role of NF-κB1 may not be intrinsic to the CD8+ T cells, as altered CD4+ T cell responses could impact CD8+ T cells. This piece of data is analogous to the situation in AIDS patients that develop TE, where a loss of CD4+ T cells is associated with a decrease in CD8+ T cell numbers and function (Hunter and Remington, 1994; Luft and Remington, 1988; Luft and Remington, 1992). In support of this hypothesis, previous studies addressing the role of NF-κB1 during parasitic infections have described defects in CD4+ T cells. For example, NF-κB1−/− CD4+ T cells were shown to have proliferative defects in response to L. major infection and polyclonal stimulation in vitro (Artis et al., 2003). In addition, mice lacking the ability to process the NF-κB1 precursor protein, p105, have T cell-intrinsic defects in CD4+ T cell proliferation following stimulation with anti-CD3 and anti-CD28, which was not attributed to apoptosis but rather lower IL-2 production and a failure to enter S-phase (Sriskantharajah et al., 2009). In support of this idea, it appears that the CD4+ cell responses induced early during infection in NF-κB1−/− mice were intact, but there was a significant decrease in activated CD4+ T cells in the spleen but not the brain during TE. However, these studies do not dissect the impact of NF-κB1 on the individual role of CD4+ and CD8+ T cells. Additional experiments will be required to gain an understanding of whether NF-κB1 has a role in promoting CD4+ T cell functions that are required to either generate or maintain parasite-specific CD8+ T cells.
Previous work from this laboratory has addressed the individual roles of NF-κB2, c-Rel, RelB, and IκB-α degradation during T. gondii infection (Caamano et al., 1999; Caamano et al., 2000; Hilliard et al., 2002; Mason et al., 2004b; Tato et al., 2003). These studies have shown that the absence of a single family member or the inability to degrade IκB-α causes unique immune defects and that the remaining family members fail to completely compensate for the deficiency. For example, RelB−/− mice do not survive the acute phase of infection, while NF-κB2−/− and c-Rel−/− mice succumbed to TE. In each of these knockout strains aberrant T cell function and reduced production of IFN-γ was associated with increased susceptibility, similar to what was observed with NF-κB1−/− mice. While many previous studies have identified a critical role for NF-κB1 in the control of CD4+ T cell mediated immunity to a variety of parasitic infections, this study provides the first indication that NF-κB1 impacts the CD8+ T cell population. One difference between the current study and those previously published on the role of NF-κB in resistance to infection is the genetic background of the mice. Previous studies have utilized mice on the C57BL/6 background, while the mice in this study are on the BALB/c background. Since resistance to T. gondii in BALB/c mice is critically dependent on CD8+ T cell responses generated by antigens presented by Ld, it is perhaps not that surprising that defects in the CD8+ T cell population were identified in NF-κB1−/− mice on this genetic background (Blanchard et al., 2008). Nevertheless, the results presented in this study identify a previously unrecognized T cell-intrinsic role for NF-κB1 in the control of T. gondii in the CNS. These studies not only identify a role for this transcription factor in the generation of CD8+ T cell population but also complete the assessment of the role of individual NF-κB family members in the immune response to T. gondii.
Supported by research grants from the National Institutes of Health AI-41158 (C.A.H), AI-42334 (CAH), T32-AI-055400 (T.H.H.), AI-081478 (T.H.H), T32-AI-07532 (E.D.T) and the State of Pennsylvania.
The authors would like to thank Shileen Bynum and Qun Fang for their contributions to this work.
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