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Female Bias in Systemic Lupus Erythematosus is Associated with the Differential Expression of X-Linked Toll-Like Receptor 8.

McDonald G, Cabal N, Vannier A, Umiker B, Yin RH, Orjalo AV, Johansson HE, Han JH, Imanishi-Kari T - Front Immunol (2015)

Bottom Line: Here, we show the results of our investigation into the role of Tlr8 expression in SLE pathogenesis in 564Igi females.Bone marrow-derived macrophages from 564Igi females have a significant increase in Tlr8 expression compared to male-derived cells, and RNA fluorescence in situ hybridization data suggest that Tlr8 may escape X-inactivation in female-derived macrophages.These results propose a model by which females may be more susceptible to SLE pathogenesis due to inefficient inactivation of Tlr8.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Physiology and Pathobiology, Tufts University , Boston, MA , USA.

ABSTRACT
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of anti-nuclear antibodies. SLE is one of many autoimmune disorders that have a strong gender bias, with 70-90% of SLE patients being female. Several explanations have been postulated to account for the severity of autoimmune diseases in females, including hormonal, microbiota, and gene dosage differences. X-linked toll-like receptors (TLRs) have recently been implicated in disease progression in females. Our previous studies using the 564Igi mouse model of SLE on a Tlr7 and Tlr9 double knockout background showed that the presence of Tlr8 on both X chromosomes was required for the production of IgG autoantibodies, Ifn-I expression and granulopoiesis in females. Here, we show the results of our investigation into the role of Tlr8 expression in SLE pathogenesis in 564Igi females. Female mice have an increase in serum pathogenic anti-RNA IgG2a and IgG2b autoantibodies. 564Igi mice have also been shown to have an increase in neutrophils in vivo, which are major contributors to Ifn-α expression. Here, we show that neutrophils from C57BL/6 mice express Ifn-α in response to 564 immune complexes and TLR8 activation. Bone marrow-derived macrophages from 564Igi females have a significant increase in Tlr8 expression compared to male-derived cells, and RNA fluorescence in situ hybridization data suggest that Tlr8 may escape X-inactivation in female-derived macrophages. These results propose a model by which females may be more susceptible to SLE pathogenesis due to inefficient inactivation of Tlr8.

No MeSH data available.


Related in: MedlinePlus

BMDM from 564Igi females have increased transcript levels of Ifn-α6, Tlr7, and Tlr8 compared to BMDM from male 564Igi mice. (A) Whole bone marrow cell suspensions from female and male mice were grown in cell culture media to select for the growth of macrophages (20% FCS, 25% L-292 cell culture supernatant, 10 U/mL penicillin/streptomycin, RPMI). Shown is a representative plot showing the percentage of neutrophils, monocytes, and macrophages after culturing compared to whole bone marrow and RAW 264.7 macrophage cell line. (B–D) After 7 days in culture, RNA was isolated from the cells, converted to cDNA, and (B)Ifn-α6, (C)Tlr7, and (D)Tlr8 expression was measured by RT-qPCR. Expression was normalized to β-actin and compared to Ifn-α6, Tlr7, and Tlr8 expression in the RAW 264.7 macrophage cell line. Shown is the average ± SEM fold-increase in expression two mice per group, tested in two independent experiments, each done in triplicate.
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Figure 4: BMDM from 564Igi females have increased transcript levels of Ifn-α6, Tlr7, and Tlr8 compared to BMDM from male 564Igi mice. (A) Whole bone marrow cell suspensions from female and male mice were grown in cell culture media to select for the growth of macrophages (20% FCS, 25% L-292 cell culture supernatant, 10 U/mL penicillin/streptomycin, RPMI). Shown is a representative plot showing the percentage of neutrophils, monocytes, and macrophages after culturing compared to whole bone marrow and RAW 264.7 macrophage cell line. (B–D) After 7 days in culture, RNA was isolated from the cells, converted to cDNA, and (B)Ifn-α6, (C)Tlr7, and (D)Tlr8 expression was measured by RT-qPCR. Expression was normalized to β-actin and compared to Ifn-α6, Tlr7, and Tlr8 expression in the RAW 264.7 macrophage cell line. Shown is the average ± SEM fold-increase in expression two mice per group, tested in two independent experiments, each done in triplicate.

Mentions: As described above, 564Igi females have increased IgG autoantibodies compared to males (Figures 1A,B). Autoantibody production in 564Igi mice is mediated by TLR7 and TLR8 (15, 20). Therefore, the increase in pathogenic IgG autoantibodies in 564Igi females may be the result of differential expression of X-linked Tlr7 and/or Tlr8. To examine the expression of Tlr7 and Tlr8, we used RT-qPCR and RNA FISH. Neutrophils were not used in these experiments because neutrophils did not survive the culturing process necessary for RNA-FISH. However, we have previously shown that neutrophils from 564Igi mice over-express Tlr7 and Tlr8 compared to C57BL/6 mice at both the mRNA and protein level (15). Here, we use BMDM from male and female C57BL/6 and 564Igi mice because monocytes and macrophages were also shown to produce IFN-I in 564Igi mice (30). BMDM were cultured from whole bone marrow cell suspensions and analyzed by flow cytometry for phenotypic similarity to the RAW264.7 mouse macrophage cell line (Figure 4A). RAW264.7 was subsequently used for normalization in qRT-PCR analyses. BMDM from both C57BL/6 and 564Igi male and female mice express Ifn-α6 (Figure 4B), similar to the primary neutrophils and monocytes/macrophages found in 564Igi mice (30). However, BMDM from female 564Igi mice express significantly more Ifn-α6 than BMDM from male 564Igi mice and C57BL/6 female mice, suggesting that BMDM may significantly contribute to IFN-I production in vivo. The phenotypic similarities between BMDM and primary neutrophils/macrophages from 564Igi mice support our use of these cells in subsequent experiments. BMDM from female 564Igi mice also over-express Tlr7 and Tlr8 compared to male 564Igi-derived and C57BL/6-derived BMDM (Figures 4C,D). These results are similar to what has been published for primary neutrophils from 564Igi mice, which over-express Tlr7 and Tlr8 compared to neutrophils from C57BL/6 mice by both qRT-PCR and Western blot (15). Furthermore, the fold-increase in Tlr8 expression in female BMDM is several orders of magnitude higher than the fold-increase in Tlr7 expression. Although both Tlr7 and Tlr8 are located on the X chromosome, it appears that Tlr8 may be more significantly dysregulated than Tlr7 and may play a larger role in facilitating SLE pathogenesis in females.


Female Bias in Systemic Lupus Erythematosus is Associated with the Differential Expression of X-Linked Toll-Like Receptor 8.

McDonald G, Cabal N, Vannier A, Umiker B, Yin RH, Orjalo AV, Johansson HE, Han JH, Imanishi-Kari T - Front Immunol (2015)

BMDM from 564Igi females have increased transcript levels of Ifn-α6, Tlr7, and Tlr8 compared to BMDM from male 564Igi mice. (A) Whole bone marrow cell suspensions from female and male mice were grown in cell culture media to select for the growth of macrophages (20% FCS, 25% L-292 cell culture supernatant, 10 U/mL penicillin/streptomycin, RPMI). Shown is a representative plot showing the percentage of neutrophils, monocytes, and macrophages after culturing compared to whole bone marrow and RAW 264.7 macrophage cell line. (B–D) After 7 days in culture, RNA was isolated from the cells, converted to cDNA, and (B)Ifn-α6, (C)Tlr7, and (D)Tlr8 expression was measured by RT-qPCR. Expression was normalized to β-actin and compared to Ifn-α6, Tlr7, and Tlr8 expression in the RAW 264.7 macrophage cell line. Shown is the average ± SEM fold-increase in expression two mice per group, tested in two independent experiments, each done in triplicate.
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Figure 4: BMDM from 564Igi females have increased transcript levels of Ifn-α6, Tlr7, and Tlr8 compared to BMDM from male 564Igi mice. (A) Whole bone marrow cell suspensions from female and male mice were grown in cell culture media to select for the growth of macrophages (20% FCS, 25% L-292 cell culture supernatant, 10 U/mL penicillin/streptomycin, RPMI). Shown is a representative plot showing the percentage of neutrophils, monocytes, and macrophages after culturing compared to whole bone marrow and RAW 264.7 macrophage cell line. (B–D) After 7 days in culture, RNA was isolated from the cells, converted to cDNA, and (B)Ifn-α6, (C)Tlr7, and (D)Tlr8 expression was measured by RT-qPCR. Expression was normalized to β-actin and compared to Ifn-α6, Tlr7, and Tlr8 expression in the RAW 264.7 macrophage cell line. Shown is the average ± SEM fold-increase in expression two mice per group, tested in two independent experiments, each done in triplicate.
Mentions: As described above, 564Igi females have increased IgG autoantibodies compared to males (Figures 1A,B). Autoantibody production in 564Igi mice is mediated by TLR7 and TLR8 (15, 20). Therefore, the increase in pathogenic IgG autoantibodies in 564Igi females may be the result of differential expression of X-linked Tlr7 and/or Tlr8. To examine the expression of Tlr7 and Tlr8, we used RT-qPCR and RNA FISH. Neutrophils were not used in these experiments because neutrophils did not survive the culturing process necessary for RNA-FISH. However, we have previously shown that neutrophils from 564Igi mice over-express Tlr7 and Tlr8 compared to C57BL/6 mice at both the mRNA and protein level (15). Here, we use BMDM from male and female C57BL/6 and 564Igi mice because monocytes and macrophages were also shown to produce IFN-I in 564Igi mice (30). BMDM were cultured from whole bone marrow cell suspensions and analyzed by flow cytometry for phenotypic similarity to the RAW264.7 mouse macrophage cell line (Figure 4A). RAW264.7 was subsequently used for normalization in qRT-PCR analyses. BMDM from both C57BL/6 and 564Igi male and female mice express Ifn-α6 (Figure 4B), similar to the primary neutrophils and monocytes/macrophages found in 564Igi mice (30). However, BMDM from female 564Igi mice express significantly more Ifn-α6 than BMDM from male 564Igi mice and C57BL/6 female mice, suggesting that BMDM may significantly contribute to IFN-I production in vivo. The phenotypic similarities between BMDM and primary neutrophils/macrophages from 564Igi mice support our use of these cells in subsequent experiments. BMDM from female 564Igi mice also over-express Tlr7 and Tlr8 compared to male 564Igi-derived and C57BL/6-derived BMDM (Figures 4C,D). These results are similar to what has been published for primary neutrophils from 564Igi mice, which over-express Tlr7 and Tlr8 compared to neutrophils from C57BL/6 mice by both qRT-PCR and Western blot (15). Furthermore, the fold-increase in Tlr8 expression in female BMDM is several orders of magnitude higher than the fold-increase in Tlr7 expression. Although both Tlr7 and Tlr8 are located on the X chromosome, it appears that Tlr8 may be more significantly dysregulated than Tlr7 and may play a larger role in facilitating SLE pathogenesis in females.

Bottom Line: Here, we show the results of our investigation into the role of Tlr8 expression in SLE pathogenesis in 564Igi females.Bone marrow-derived macrophages from 564Igi females have a significant increase in Tlr8 expression compared to male-derived cells, and RNA fluorescence in situ hybridization data suggest that Tlr8 may escape X-inactivation in female-derived macrophages.These results propose a model by which females may be more susceptible to SLE pathogenesis due to inefficient inactivation of Tlr8.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Physiology and Pathobiology, Tufts University , Boston, MA , USA.

ABSTRACT
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of anti-nuclear antibodies. SLE is one of many autoimmune disorders that have a strong gender bias, with 70-90% of SLE patients being female. Several explanations have been postulated to account for the severity of autoimmune diseases in females, including hormonal, microbiota, and gene dosage differences. X-linked toll-like receptors (TLRs) have recently been implicated in disease progression in females. Our previous studies using the 564Igi mouse model of SLE on a Tlr7 and Tlr9 double knockout background showed that the presence of Tlr8 on both X chromosomes was required for the production of IgG autoantibodies, Ifn-I expression and granulopoiesis in females. Here, we show the results of our investigation into the role of Tlr8 expression in SLE pathogenesis in 564Igi females. Female mice have an increase in serum pathogenic anti-RNA IgG2a and IgG2b autoantibodies. 564Igi mice have also been shown to have an increase in neutrophils in vivo, which are major contributors to Ifn-α expression. Here, we show that neutrophils from C57BL/6 mice express Ifn-α in response to 564 immune complexes and TLR8 activation. Bone marrow-derived macrophages from 564Igi females have a significant increase in Tlr8 expression compared to male-derived cells, and RNA fluorescence in situ hybridization data suggest that Tlr8 may escape X-inactivation in female-derived macrophages. These results propose a model by which females may be more susceptible to SLE pathogenesis due to inefficient inactivation of Tlr8.

No MeSH data available.


Related in: MedlinePlus