NQO1 and NQO2 Regulation of Humoral Immunity and Autoimmunity*

NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH:quinone oxidoreductase 2 (NQO2) are cytosolic enzymes that catalyze metabolic reduction of quinones and derivatives. NQO1-null and NQO2-null mice were generated that showed decreased lymphocytes in peripheral blood, myeloid hyperplasia, and increased sensitivity to skin carcinogenesis. In this report, we investigated the in vivo role of NQO1 and NQO2 in immune response and autoimmunity. Both NQO1-null and NQO2-null mice showed decreased B-cells in blood, lower germinal center response, altered B cell homing, and impaired primary and secondary immune responses. NQO1-null and NQO2-null mice also showed susceptibility to autoimmune disease as revealed by decreased apoptosis in thymocytes and pre-disposition to collagen-induced arthritis. Further experiments showed accumulation of NADH and NRH, cofactors for NQO1 and NQO2, indicating altered intracellular redox status. The studies also demonstrated decreased expression and lack of activation of immune-related factor NF-κB. Microarray analysis showed altered chemokines and chemokine receptors. These results suggest that the loss of NQO1 and NQO2 leads to altered intracellular redox status, decreased expression and activation of NF-κB, and altered chemokines. The results led to the conclusion that NQO1 and NQO2 are endogenous factors in the regulation of immune response and autoimmunity.

The roles of genetic factors in immune deficiency and autoimmune diseases have been recognized for decades (17). B cells play a key role in regulation of immune system. B cells produce antibodies, provide support to other mononuclear cells, and contribute directly to inflammatory pathways (18). Impaired B cell production, maturation, homing, and activation are known to lead to defective immune response (19). Dysfunctional immune response and impaired apoptosis in T cells have been implicated in many immunological abnormalities including autoimmune lymphoproliferative syndrome (18,20,21).
In this report, we investigated the in vivo role of NQO1 and NQO2 in immune response and autoimmunity. Both NQO1null mice as well as NQO2-null mice demonstrated lower B cells in peripheral blood, decreased germinal center response, altered B cell homing, and impaired antibody responses. NQO1-null and NQO2-null mice also showed increased susceptibility to autoimmune disease as revealed by decreased apoptosis in thymocyte and predisposition to collagen-induced arthritis. The loss of NQO1 and NQO2 led to accumulation of NADH and NRH that altered intracellular redox status. The studies also demonstrated decreased expression and lack of activation of NF-B and altered chemokines and chemokine receptors. These results suggest that the loss of NQO1 and NQO2 leads to altered intracellular redox status that results in decreased expression and lack of activation of NF-B. This leads to B-cell deficiency and alterations in the homing of B cells and impaired humoral immune response and autoimmunity. labeled anti-CD19, 2.5 l of PE-labeled anti-CD4, and 2.5 of l of CyChrom-labeled anti-CD8 antibodies gently vortexed and incubated on ice in the dark for 30 min. Red blood corpuscles were hemolyzed and fixed using Coulter Q-prep and analyzed using a Coulter EPICS XL-MCL flow cytometer. Femurs were cut at both ends. Bone marrow was flushed with sterile cold PBS. After two PBS washes, the cells were suspended in annexin binding buffer to a concentration of 10 ϫ 10 6 cells/ml. Spleen cells were suspended in cold PBS using the rough surface of glass slides. Red blood cells were lysed using red blood cell lysis buffer containing 15.5 mM NH4Cl, 1 mM KHCO3, and 0.001 mM EDTA. Cells were suspended in annexin binding buffer to a concentration of 10 ϫ 10 6 cells/ml. Thymocytes were obtained from the thymus and suspended in cold PBS using the rough surface of glass slides. Cells were then suspended in annexin binding buffer to a concentration of 10 ϫ 10 6 cells/ml. 100 l of cell suspensions (bone marrow, spleen, or thymus) was added to the appropriate antibodies (1 l of annexin V-FITC, 2.5 l of PE-labeled anti-CD19, 2.5 l of FITC-labeled anti-CD43, 2.5 l of FITC-labeled anti-CD25, 2.5 l of FITC-labeled anti-IgD, 2.5 l of FITC-labeled anti-CD19, 2.5 l of PE-labeled anti-CD4, and 2.5 l of CyChrom-labeled anti-CD8 antibodies). Samples were gently vortexed and incubated on ice, in the dark, for 30 min. Samples were then fixed in 4% paraformaldehyde and then analyzed using Coulter EPICS XL-MCL flow cytometer.
Thymidine Incorporation Assay of Proliferation in the Bone Marrow and Spleen Cells-The wild-type, NQO1-null, and NQO2-null mice were sacrificed, and their femurs and spleen were obtained. The bones were cut, and marrow was flushed out gently with RPMI 10% fetal bovine serum with antibiotics. Cell samples were cultured in triplicates in 96-well plates for 48 h. 1 Ci of [H 3 ]thymidine was added to each well. Twentyfour hours later, cells were harvested on a glass fiber filter mat using Tomtec Harvester 96. Incorporated thymidine was measured by scintillation counter. Cell suspensions were prepared from the spleens in RPMI 10% fetal bovine serum with antibiotics. Cell samples were cultured in triplicates in 96-well plates. 0.5 g of Con A was added to each well. Forty-eight hours later, 1 Ci of [H 3 ]thymidine was added to each well. Twenty-four hours later, cells were harvested, and incorporated thymidine was measured.
Evaluation of Germinal Center Response-Eight-week-old wild-type, NQO1-null, and NQO2-null mice were injected intraperitoneally with alum-precipitated 50 g of 4-hydroxy-3nitrophenyl acetate (NP) conjugated to chicken ␥-globulin (CGG). Twelve days after immunization, spleens were obtained. Each spleen was split in half. One half was frozen for histology (22). Sections were cut and then probed by immunohistochemistry using antibodies against germinal center B cells marker GL-7 by procedures as described (22). The other half was suspended in cold PBS using the rough surface of glass slides. Red blood cells were lysed using red blood cell lysis buffer containing 15.5 mM NH 4 Cl, 1 mM KHCO 3 , and 0.001 mM EDTA. Cells were suspended in cold PBS in 10 ϫ 10 6 cells/ml. 100 l of the spleen cell suspension was then added to 2.5 l of FITC-labeled anti-B220 and PE-labeled anti-GL-7. Samples were gently vortexed and incubated on ice, in the dark, for 30 min. Samples were then fixed in 4% paraformaldehyde and then analyzed using Coulter EPICS XL-MCL flow cytometer.
Primary and Secondary Immune Response Assessment-Eightweek-old wild-type, NQO1-null, and NQO2-null mice were injected intraperitoneally with 50 g of NP conjugated to CGG as described (22). Twelve days after immunization, spleen and bone marrow were obtained for primary immune response analysis. For another set of mice, 8 weeks after primary immunization, mice were injected intraperitoneally with 20 g of NP-CGG. Assays for serum immunoglobulin levels were performed at 0, 5, and 10 days after secondary immunization. On day 12, mice were sacrificed and analyzed for antibody-forming cells (AFC). NP-specific antibody-forming cells were quantitated by ELISPOT assay using nitrocellulose filters coated with NP-BSA 5:1 and 25:1. Labeled anti-IgM Ab and anti-IgG Ab were used to visualize NP-specific AFC.
Collagen-induced Arthritis Models-Eight-week-old wildtype, NQO1-null, and NQO2-null mice were immunized with chicken collagen II to induce arthritis as described (23). Incidences of arthritis were recorded. Clinical arthritis scores were calculated using a scale from 0 to 3 for each paw with a maximum score of 12 per mouse. Anti-collagen antibody titers were measured on days 21 and 42. On day 42, mice were euthanized. Sections in the paws were examined microscopically. NAD(P)H:NAD(P) and NRH:NR Ratio in Bone Marrow, Spleen, and Thymus-The following procedure was used to collect and process the tissue for determination of NAD(P): NAD(P)H and NRH:NR ratio because of the sensitivity of these molecules. The bone marrow, spleen, and thymus were surgically removed while the mice were under anesthesia to avoid changes in the levels of the pyridine nucleotides. The bone marrow was then instantly placed in liquid nitrogen (24). While frozen, the ends of the bone were cut using a surgical blade, and while the marrow was thawing, it was flushed by using the solution containing 200 mM KCN, 1 mM bathophenanthroline, and 60 mM KOH. The pyridines were extracted with chloroform and analyzed from these tissues by HPLC and procedures as described previously (4,25).
Electrophoretic Mobility Shift Assay-One million bone marrow cells were obtained from wild-type and NQO1-null mice and treated with 10 g/ml lipopolysaccharide (LPS). The nuclear extract was prepared, and an electrophoretic mobility shift assay was performed by procedures as described previously (26). The NF-B binding oligonucleotide sequence used was 5Ј-TTGTTACAAGGGACTTTCCGCTGGGGACTTTC-CAGGCAGGCGTGG-3Ј.
Microarray Analysis-We used Affymetrix GeneChip mouse expression set 430 and RNA from untreated mice bone marrow for microarray analysis. Three samples for each genotype (wild type, NQO1-null, and NQO2-null) were analyzed. Each sample included a pool of bone marrow cells from five mice. RNA samples were prepared from bone marrow cells using a Qiagen RNeasy kit. The excellent quality of the RNA samples was confirmed with an Agilent 2100 Bioanalyzer. Our core facility analyzed the samples and provided data to us. We used dChip 1.3 and GeneSpring software to analyze data. We categorized alterations in gene expression in several groups according to gene function using dChip 1.3. These categories included apoptotic genes such as p53, Bax, Bcl-2, caspase 2, caspase 3, caspase 8, apoptosis inhibitor 6, and C/EBP; interleukins, chemokines, and their receptors such as CXCR4, CCL9, CCR1, CXCL12, interferon ␥ receptor, interferon ␥-induced GTPase, interleukin 7, interleukin 10, and interleukin 10 receptor; and transcription regulation genes, DNA damage response genes, and DNA replication and metabolism genes. The results for chemokines and chemokine receptors are presented as fold increase or decrease.

Immunological Phenotype of NQO1-null and NQO2-null
Mice-Previously, we generated NQO1-null mice deficient in NQO1-and NQO2-null mice deficient in NQO2 (6, 27). An analysis of blood, bone marrow, and spleen from wild-type, NQO1-null, and NQO2-null mice was performed. Flow cytometry analysis of blood lymphocytes showed a decrease in the number of CD19ϩ B cells and an increase in CD4ϩ T cells (Fig.  1, A and B). However, NQO1-null and NQO2-null mice showed higher numbers of CD19ϩ cells in the bone marrow ( Fig. 1C; compare with A and B). To trace different stages of B cell development in the bone marrow, we analyzed bone marrow cells for CD19 and CD43 (for pro-B cells), CD19 and CD25 (for pre-B cells), and CD19 and IgD (for mature B cells). There was no difference between wild-type, NQO1-null, and NQO2null mice in the number of B cell progenitors, pro-B and pre-B cells (Fig. 1C). However, there was a significant increase in the Mice were euthanized, and bone marrow was collected from femurs. After two PBS washes, the cells were suspended in annexin binding buffer to a concentration of 10 ϫ 10 6 cells/ml. To cells in separate tubes, we added PE-labeled anti-CD19 antibody; annexin V-FITC and PE-labeled anti-CD19 antibody; FITClabeled anti-CD43 and PE-labeled anti-CD19 antibody, FITC-labeled anti-CD25 and PE-labeled anti-CD19 antibody, FITC-labeled anti-IgD and PE-labeled anti-CD19 antibody. Assays for the determination of B cells, apoptosis in B cells, and B cell development were essentially performed as described by the manufacturer and measured using a Coulter EPICS XL-MCL flow cytometer. E and F, flow cytometry analysis of the spleen. Spleens were obtained from mice after euthanization. Spleen cells were suspended in cold PBS. Red blood cells were lysed. Cells were suspended in annexin binding buffer to a concentration of 10 ϫ 10 6 cells/ml. 100 l of cell suspension was added to FITC-labeled anti-CD19, PE-labeled anti-CD4, and CyChrom-labeled anti-CD8 antibodies (E ) and to annexin V-FITC and PE-labeled anti-CD19 antibody (F ). Samples were gently vortexed and incubated on ice, in the dark, for 30 min. Assays for the determination of B cells, apoptosis in B cells, CD4ϩ T cells (helper), and CD8ϩ T cell (cytotoxic) were essentially performed as described by the manufacturer and measured using Coulter EPICS XL-MCL flow cytometer. G, proliferation of bone marrow and spleen cells. The mice were sacrificed, and their femurs and spleens were removed. Bone marrow and spleen cells were exposed to 1 Ci of [H 3 ]thymidine. Twenty-four hours later, cells were harvested, and incorporated thymidine was measured. OCTOBER 13, 2006 • VOLUME 281 • NUMBER 41 number of mature IgDϩ B cells in the bone marrow of NQO1null and NQO2-null mice (Fig. 1C). Apoptosis in bone marrow B cells was lower in NQO1-null and NQO2-null mice as compared with wild-type mice (Fig. 1D). Flow cytometry analysis of spleen lymphocytes showed no difference between wild-type, NQO1-null, and NQO2-null mice (Fig. 1E). In contrast to lower apoptosis in bone marrow B cells, the knock-out mice showed no difference in apoptosis of spleen B cells as compared with wild type (Fig. 1F).

NQO1 and NQO2 Regulate Immune Response and Autoimmunity
We then measured proliferation of bone marrow cells and spleen T cells using thymidine incorporation assay. The results are shown in Fig. 1G. NQO1-null bone marrow cells showed significantly higher proliferation rate than that of wild type ( p Ͻ 0.01). NQO2-null bone marrow does not proliferate significantly different from that of wild type. NQO1-null spleen cells showed significantly higher proliferation rate than that of wild type ( p Ͻ 0.001). NQO2-null spleen T cells proliferate slightly faster than that of wild type ( p Ͻ 0.05).
We observed that NQO1-null mice have bigger spleen than wild-type mice. We weighed the spleens of 10 wild-type, NQO1-null, and NQO2-null mice. NQO1-null mice spleen is slightly but significantly bigger than that of wild-type mice (wild type, 105 Ϯ 8, and NQO1-null, 134 Ϯ 10 mg; p Ͻ 0.05). No significant difference between NQO2-null and wild type in spleen weight was observed.
We analyzed the spleen cells from wild-type, NQO1-null, and NQO2-null mice for marginal zone B cells by flow cytometry after staining for marginal zone B cell markers CD23 and CD21 ( Fig. 2A). CD23ϩCD21ϩ marginal zone B cells showed a significant decrease in the spleen of NQO1-null mice ( Fig. 2A). CD23ϩCD21ϩ marginal zone B cells were also lower in NQO2-null mice as compared with wild type (Fig. 2A). In same experiment, CD21ϩ cells were found Spleens were obtained from wild-type, NQO1-null, and NQO2-null mice after euthanization. Spleen cells were suspended in cold PBS. Red blood cells were lysed. Cells were suspended in cold PBS to a concentration of 10 ϫ 10 6 cells/ml. 100 l of cell suspension was added to FITC-labeled anti-CD21 and PE-labeled anti-CD23 antibodies. Samples were gently vortexed and incubated on ice, in the dark, for 30 min. Assays for the determination of CD21ϩ and CD23ϩ splenic B cells were essentially performed as described by the manufacturer and measured using Coulter EPICS XL-MCL flow cytometer. B and C, germinal center (GC) response. Eight-weekold wild-type, NQO1-null, and NQO2-null mice were injected intraperitoneally with 50 g of NP conjugated to CGG. Twelve days after immunization, spleens were obtained. Germinal center response was evaluated by flow cytometry analysis for B cell marker B220 and germinal center marker Gl-7 and immunohistochemistry analysis using antibodies against germinal center marker GL-7.
increased in NQO1-null mice as compared with wild-type mice. NQO2-null mice also demonstrated an increase in CD21ϩ cells. However, the increase was significantly lower than NQO1-null mice. The significance of the increase in CD21ϩ cells remains unknown.
Humoral Immune Response in NQO1-null and NQO2-null Mice-To assess the humoral immune response in NQO1-null mice and NQO2-null mice, we started by evaluating the germinal center response. Eight-week-old wild-type, NQO1-null, and NQO2-null mice were injected intraperitoneally with 50 g of alum-precipitated NP conjugated to CGG (NP-CGG). Twelve days after immunization, spleen cells and tissue sections were analyzed for germinal center response. Both flow cytometry and immunohistochemistry analysis showed significantly lower germinal center response in NQO1-null mice ( p Ͻ 0.01) (Fig. 2, B and C). Some decrease, but not statistically significant, was observed in NQO2-null mice (Fig. 2, B and C).
For more thorough assessment of humoral immune response, we measured primary and secondary antigen-specific antibody response in wild-type, NQO1-null, and NQO2-null mice. Eight-FIGURE 3. Primary and secondary humoral immune response. A and C, primary humoral immune response. Eight-week-old wild-type, NQO1-null, and NQO2-null mice were injected intraperitoneally with NP conjugated to CGG. Twelve days after immunization, spleen and bone marrow were obtained for primary immune response analysis. NP-specific AFC were quantitated by ELISPOT assay using nitrocellulose filters coated with NP-BSA 5:1 and 25:1. Labeled anti-IgM Ab and anti-IgG Ab were used to visualize NP-specific AFC. B and D, secondary humoral immune response. Seven-week-old wild-type, NQO1-null, and NQO2-null mice were injected intraperitoneally with 50 g of NP conjugated to CGG. Eight weeks after primary immunization, mice were injected intraperitoneally with 20 g of NP-CGG. On day 12, mice were sacrificed and analyzed for AFC. NP-specific AFC were quantitated by ELISPOT assay using nitrocellulose filters coated with NP-BSA 5:1 and 25:1. Labeled anti-IgM Ab and anti-IgG Ab were used to visualize NP-specific AFC. . Serum analysis for antigen-specific IgG after primary and secondary immunization. Eight-week-old wild-type, NQO1-null, and NQO2null mice were injected intraperitoneally with 50 g of NP conjugated to CGG. Eight weeks after primary immunization, mice were injected intraperitoneally with 20 g of NP-CGG. Assays for serum immunoglobulin levels were performed at 0, 5, and 10 days after secondary immunization. OCTOBER 13, 2006 • VOLUME 281 • NUMBER 41

JOURNAL OF BIOLOGICAL CHEMISTRY 30921
week-old wild-type mice were immunized with NP-CGG as described earlier. Twelve days after immunization, spleen and bone marrow were obtained for primary immune response analysis. For another set of mice, 8 weeks after primary immunization, mice were injected intraperitoneally with 20 g of soluble NP-CGG. Twelve days later, mice were sacrificed and analyzed for secondary immune response. Assessment of immune response was done by measuring the number of AFC. Labeled anti-IgM antibody and anti-IgG1 antibody were used to visualize NP-specific AFC. Both NQO1-null and NQO2-null mice (especially NQO1-null) showed weaker primary and secondary immune response (Fig. 3). Serum levels of NP-specific IgG were measured on days 0, 5, and 10 after the secondary immunization. NP-specific IgG were lower in NQO1-null and NQO2-null mice (Fig. 4).
Autoimmunity in NQO1-null and NQO2-null Mice-Decreased apoptosis in the thymus and bone marrow cells of NQO1-null and NQO2-null mice and increased spleen T cell proliferation in NQO1-null mice pointed to the possibility of higher susceptibility to autoimmune disease. We used the collagen-induced arthritis model to assess the susceptibility of NQO1-null and NQO2-null mice to autoimmunity. We found that NQO1-null mice developed arthritis earlier and for a longer duration than wild-type mice (Fig. 5A). Arthritis in NQO1null mice was more severe than that in wild-type mice. This is reflected as a higher arthritis clinical score (Fig. 5B). There was no significant difference between NQO2-null and wild-type mice in the onset and severity of arthritis (Fig. 5, A and B). However, arthritis lasted longer in NQO2-null mice as compared with wild type (Fig. 5, A and B).

Alteration in Intracellular Redox Status, Decreased Expression, and Activation of NF-B and Altered
Chemokines and Chemokine Receptors-The analysis of bone marrow NADH, NAD, NRH, and NR showed a significant increase in NADH:NAD ratio in NQO1-null and NRH:NR ratio in NQO2-null mice (Fig. 6A). Similar analysis also showed a significant increase in NADPH:NADP ratio in NQO1-null mice as compared with wild-type mice (data not shown). We used electrophoretic mobility shift assay to investigate the expression and LPS activation of NF-B in wildtype, NQO1-null, and NQO2-null mice. The results are presented in Fig. 6B. Decreased NF-B binding to DNA was observed with bone marrow nuclear extract from NQO1null mice as compared with wild-type mice (compare lanes 3 and 5). LPS treatment demonstrated a significant increase in NF-B binding to DNA in wild-type mice (compare lanes 3  and 4). The LPS-mediated activation of NF-B binding was more or less not observed in NQO1-null mice (compare lanes 5 and 6). The decreased binding of NF-B and lack of LPS activation of NF-B binding to DNA was also observed in NQO2-null mice (data not shown). However, the magnitude of difference was lower in NQO2-null than in NQO1null mice. The results on the increase in NADH:NAD and NRH:NR ratios and lower NF-B binding and lack of LPS activation of NF-B binding to DNA were also observed in spleen and thymus of NQO1-null and NQO2-null mice (data not shown). Microarray analysis of bone marrow from untreated wild-type, NQO1-null, and NQO2-null mice were performed. The microarray analysis revealed alterations in chemokines and chemokine receptors associated with the loss of NQO1 and NQO2 in respective null mice (Fig. 6C). Specially noted were chemokine (CXC motif) ligand 12, receptor 4, and receptor 1. Interestingly, the alterations noted were of lower FIGURE 5. Collagen-induced arthritis model and apoptosis in thymocytes. A and B, collagen-induced arthritis. wild-type, NQO1-null, and NQO2-null mice were injected intradermally at the base of the tail with 200 g of chicken collagen II emulsified in complete Freund's adjuvant. Twenty-one days later, mice were injected with 100 g of collagen II in complete Freund's adjuvant. Mice were observed for signs of arthritis. Incidence of arthritis was recorded (A). Clinical arthritis scores were calculated using a scale from 0 to 3 for each paw with a maximum score of 12 per mouse (B). C and D, flow cytometry analysis of the thymus. Thymocytes were obtained from wild-type, NQO1-null, and NQO2-null mice after euthanization. Thymocytes were suspended in cold PBS. Cells were then suspended in annexin binding buffer to a concentration of 10 ϫ 10 6 cells/ml. 100 l of cell suspension was added to 2.5 l of PE-labeled anti-CD4 and 2.5 l of CyChrom-labeled anti-CD8 antibodies (C) and 1 l of annexin V-FITC (D). Samples were gently vortexed and incubated on ice, in the dark, for 30 min. CD4ϩCD8Ϫ T cells, CD8ϩCD4Ϫ T cell, CD4ϩCD8ϩ T cells, and apoptosis in thymocytes were measured using Coulter EPICS XL-MCL flow cytometer. magnitude in NQO2-null mice as compared with NQO1-null mice.

DISCUSSION
The experiments in this study, for the first time, establish a physiological role of NQO1 and NQO2 in control of immune response and autoimmunity. The NQO1-null and NQO2-null mice showed impaired humoral immune response. The magnitude of impairment in humoral immune response was of higher magnitude in NQO1-null mice than NQO2-null mice. Phenotypic analysis of NQO1-null and NQO2-null mice showed a decrease in the number of B cells in the blood and an increase in the bone marrow, whereas no change in B cell number was observed in the spleen. Further investigations revealed that mature B cells and not pro-and pre-B cells increased in bone marrow of NQO1-null and NQO2-null mice. Since the maturation of B cells to IgD positive mature B cells takes place in the peripheral lymphoid organs, spleen, and lymph nodes (not in the bone marrow), the increase in IgD positive mature B cells in the bone marrow most likely resulted due to the increased homing of mature B cells into the bone marrow. Microarray analysis of the bone marrow showed increased chemokine (CXC motif) receptor 4 (CXCr4) and chemokine (CXC motif) ligand 12 (CXCL12), 20% in NQO1-null and 5% in NQO2-null mice. CXCr4 and CXCL12 are the chemokine receptor and ligand involved in the homing of B cells to the bone marrow (28).
NQO1-null and NQO2-null mice (especially NQO1-null) showed weaker primary and secondary antibody response. This was demonstrated by the lower number of NP-specific AFC in the spleen and bone marrow and lower serum NP-specific IgG after primary and secondary immunization with NP-CGG. Germinal center response was also weaker, especially in NQO1-null mice. All these observations led to the conclusion that the loss of NQO1 or NQO2 (NQO1 more than NQO2) resulted in impaired humoral immune response, suggesting that NQO1 and NQO2 are significant endogenous factors in regulation and proper functioning of immune response.
T helper cells are major perpetrators in autoimmunity (29). The decreased apoptosis has been linked to autoimmunity (30). Mutations in genes that regulate apoptosis, such as Fas, FasL, caspase 10, and caspase 8, result in higher susceptibility to autoimmune diseases (20,21,31,33,34). Flow cytometric analysis of thymus showed lower apoptosis in NQO1-null and NQO2-null thymocytes as compared with wild-type mice. The thymus is a main place for T cell tolerance. T cell tolerance includes elimination of autoreactive T cells (negative selection), mostly by apoptosis (29). Lower apoptosis in the thymus of NQO1-null and NQO2-null mice might compromise T cell tolerance. This might have allowed autoreactive T cells to escape negative selection and thus increased susceptibility to develop autoimmune disease. These findings together pointed toward the possibility of increased susceptibility in NQO1-null or NQO2-null mice to develop autoimmunity. Indeed, NQO1-null mice developed arthritis earlier and for a longer duration than wild-type mice in collagen-induced arthritis model. NQO2-null mice did not show significant difference in autoimmunity from wild-type mice in the same experiment. The higher sensitivity of NQO1null mice to induce arthritis could be due to impaired T cell tolerance in the lymphoid organs as a result of decreased apoptosis and increased proliferation of T cells. This is supported by the lower apoptosis seen in the thymus and bone marrow cells of NQO1-null mice and the increased proliferation in bone marrow cells and splenic T cells. The decreased apoptosis and increased proliferation in the lymphoid organs of NQO2-null mice were milder than that in NQO1-null mice.
The above observations raised an interesting question regarding the mechanism of NQO1 and NQO2 regulation of immune response and autoimmunity. The loss of NQO1 and NQO2 led to alterations in intracellular redox status. This was due to accumulation of reduced NADH in NQO1-null mice and NRH in NQO2-null mice. Alterations in the redox status of the cells presumably changed transcription and/or modifica-FIGURE 6. NQO1 relationship to immune response and autoimmunity. A, NAD(P)H:NAD(P) ratio in bone marrow of wild-type, NQO1-null, and NQO2-null mice. Femurs were surgically removed, and pyridines were extracted with chloroform and analyzed by HPLC procedures as described under "Materials and Methods." The data are shown only for NADH/NAD. B, electrophoretic mobility shift assay. One million bone marrow cells were untreated or treated with 10 g/ml LPS for 30 min. Nuclear extracts were prepared, and NF-B binding was analyzed by electrophoretic mobility shift assay. Only the shifted bands are shown. C, microarray analysis. RNA from wild-type, NQO1-null, and NQO2-null mice bone marrow were used to perform microarray analysis. The differences in selected chemokines and receptors are listed as compared with wild-type mice.
tion of factors including the loss of expression and lack of LPS activation of NF-B and alterations in chemokines (including CXCr4 and CXCL12). The redox modulation of NF-B and chemokine/receptor is reported earlier (35). LPS has been shown to cause apoptosis in B cells by activating NF-B (36), CD4ϩCD8ϩ thymocytes, and lymphoid organs (32). Failure of activation of NF-B might have contributed to reduced apoptosis in thymocytes in NQO1-null and NQO2-null mice. The alterations in intracellular redox status combined with lower expression and lack of activation of NF-B might have altered the homing of B cells and reduced antibody responses. The changes in B cells were translated to decreased primary and secondary immune response. Decreased apoptosis and increased proliferation of thymocytes contributed to autoimmunity in NQO1-null mice.
The results on impaired immune response and autoimmunity in NQO1-null and NQO2-null mice are extremely significant and have major impact on human health. This is since 2-4% of human individuals are homozygous for C 3 T mutation in the NQO1 gene, leading to proline to serine substitution, and totally lack the NQO1 protein (10,11). In addition, greater than 20% individuals are heterozygous and carry one mutated NQO1 allele. These individuals lack 50% NQO1 protein. The NQO1 homozygous, and heterozygous mutant individuals are expected to have impaired immune response and are at risk for autoimmune diseases. This is the first study toward genotyping human individuals for lack of NQO1 and problems associated with impaired immune response and autoimmunity.
In conclusion, NQO1 and NQO2 are important endogenous factors in regulation of immune response and autoimmunity. The loss of NQO1 or NQO2 (especially NQO1) results in impaired humoral immune response and higher susceptibility to autoimmune diseases. The alterations in intracellular redox status due to the loss of NQO1 and NQO2 presumably led to changes in expression and induction of factors including NF-B, chemokines, and chemokine receptors. These changes resulted in altered B cell homing, reduced B cell response, and decreased apoptosis of thymocytes. These changes led to compromised immune response and autoimmunity. The detailed mechanisms of the role of NQO1 and NQO2 role in regulation of immune response and autoimmunity await future investigations.