Decreased SMG7 expression associates with lupus-risk variants and elevated antinuclear antibody production.

OBJECTIVES: Following up the systemic lupus erythematosus (SLE) genome-wide association studies (GWAS) identification of NMNAT2 at rs2022013, we fine-mapped its 150 kb flanking regions containing NMNAT2 and SMG7 in a 15 292 case-control multi-ancestry population and tested functions of identified variants. METHODS: We performed genotyping using custom array, imputation by IMPUTE 2.1.2 and allele specific functions using quantitative real-time PCR and luciferase reporter transfections. SLE peripheral blood mononuclear cells (PBMCs) were cultured with small interfering RNAs to measure antinuclear antibody (ANA) and cyto/chemokine levels in supernatants using ELISA. RESULTS: We confirmed association at NMNAT2 in European American (EA) and Amerindian/Hispanic ancestries, and identified independent signal at SMG7 tagged by rs2702178 in EA only (p=2.4×10-8, OR=1.23 (95% CI 1.14 to 1.32)). In complete linkage disequilibrium with rs2702178, rs2275675 in the promoter region robustly associated with SMG7 mRNA levels in multiple expression quantitative trait locus (eQTL) datasets. Its risk allele was dose-dependently associated with decreased SMG7 mRNA levels in PBMCs of 86 patients with SLE and 119 controls (p=1.1×10-3 and 6.8×10-8, respectively) and conferred reduced transcription activity in transfected HEK-293 (human embryonic kidney cell line) and Raji cells (p=0.0035 and 0.0037, respectively). As a critical component in the nonsense-mediated mRNA decay pathway, SMG7 could regulate autoantigens including ribonucleoprotein (RNP) and Smith (Sm). We showed SMG7 mRNA levels in PBMCs correlated inversely with ANA titres of patients with SLE (r=-0.31, p=0.01), and SMG7 knockdown increased levels of ANA IgG and chemokine (C-C motif) ligand 19 in SLE PBMCs (p=2.0×10-5 and 2.0×10-4, respectively). CONCLUSION: We confirmed NMNAT2 and identified independent SMG7 association with SLE. The inverse relationship between levels of the risk allele-associated SMG7 mRNAs and ANA suggested the novel contribution of mRNA surveillance pathway to SLE pathogenesis.

APOBEC3H polymorphisms and susceptibility to HIV-1 infection in an Indian population.

Human APOBEC3H (A3H) is a member of APOBEC cytidine deaminase family intensively constraining the HIV-1 replication. A3H is known to be polymorphic with different protein stability and anti-HIV-1 activity in vitro. We recently reported that A3H haplotypes composed of two functional polymorphisms, rs139292 (N15del) and rs139297 (G105R), were associated with the susceptibility to HIV-1 infection in Japanese. To confirm the association of A3H and HIV-1 infection in another ethnic group, a total of 241 HIV-1-infected Indian individuals and ethnic-matched 286 healthy controls were analyzed for the A3H polymorphisms. The frequency of 15del allele was high in the HIV-1-infected subjects as compared with the controls (0.477 vs 0.402, odds ratio (OR)=1.36, P=0.014). Haplotype analysis showed that the frequencies of 15del-105R was high (0.475 vs 0.400, OR=1.36, permutation P=0.037) in the HIV-1-infected subjects, confirming the association of A3H polymorphisms with the susceptibility to HIV-1 infection.

MicroRNA-3148 modulates allelic expression of toll-like receptor 7 variant associated with systemic lupus erythematosus.

We previously reported that the G allele of rs3853839 at 3'untranslated region (UTR) of Toll-like receptor 7 (TLR7) was associated with elevated transcript expression and increased risk for systemic lupus erythematosus (SLE) in 9,274 Eastern Asians [P = 6.5×10(-10), odds ratio (OR) (95%CI) = 1.27 (1.17-1.36)]. Here, we conducted trans-ancestral fine-mapping in 13,339 subjects including European Americans, African Americans, and Amerindian/Hispanics and confirmed rs3853839 as the only variant within the TLR7-TLR8 region exhibiting consistent and independent association with SLE (Pmeta = 7.5×10(-11), OR = 1.24 [1.18-1.34]). The risk G allele was associated with significantly increased levels of TLR7 mRNA and protein in peripheral blood mononuclear cells (PBMCs) and elevated luciferase activity of reporter gene in transfected cells. TLR7 3'UTR sequence bearing the non-risk C allele of rs3853839 matches a predicted binding site of microRNA-3148 (miR-3148), suggesting that this microRNA may regulate TLR7 expression. Indeed, miR-3148 levels were inversely correlated with TLR7 transcript levels in PBMCs from SLE patients and controls (R(2) = 0.255, P = 0.001). Overexpression of miR-3148 in HEK-293 cells led to significant dose-dependent decrease in luciferase activity for construct driven by TLR7 3'UTR segment bearing the C allele (P = 0.0003). Compared with the G-allele construct, the C-allele construct showed greater than two-fold reduction of luciferase activity in the presence of miR-3148. Reduced modulation by miR-3148 conferred slower degradation of the risk G-allele containing TLR7 transcripts, resulting in elevated levels of gene products. These data establish rs3853839 of TLR7 as a shared risk variant of SLE in 22,613 subjects of Asian, EA, AA, and Amerindian/Hispanic ancestries (Pmeta  = 2.0×10(-19), OR = 1.25 [1.20-1.32]), which confers allelic effect on transcript turnover via differential binding to the epigenetic factor miR-3148.

Preferential binding to Elk-1 by SLE-associated IL10 risk allele upregulates IL10 expression.

Immunoregulatory cytokine interleukin-10 (IL-10) is elevated in sera from patients with systemic lupus erythematosus (SLE) correlating with disease activity. The established association of IL10 with SLE and other autoimmune diseases led us to fine map causal variant(s) and to explore underlying mechanisms. We assessed 19 tag SNPs, covering the IL10 gene cluster including IL19, IL20 and IL24, for association with SLE in 15,533 case and control subjects from four ancestries. The previously reported IL10 variant, rs3024505 located at 1 kb downstream of IL10, exhibited the strongest association signal and was confirmed for association with SLE in European American (EA) (P = 2.7×10⁻8, OR = 1.30), but not in non-EA ancestries. SNP imputation conducted in EA dataset identified three additional SLE-associated SNPs tagged by rs3024505 (rs3122605, rs3024493 and rs3024495 located at 9.2 kb upstream, intron 3 and 4 of IL10, respectively), and SLE-risk alleles of these SNPs were dose-dependently associated with elevated levels of IL10 mRNA in PBMCs and circulating IL-10 protein in SLE patients and controls. Using nuclear extracts of peripheral blood cells from SLE patients for electrophoretic mobility shift assays, we identified specific binding of transcription factor Elk-1 to oligodeoxynucleotides containing the risk (G) allele of rs3122605, suggesting rs3122605 as the most likely causal variant regulating IL10 expression. Elk-1 is known to be activated by phosphorylation and nuclear localization to induce transcription. Of interest, phosphorylated Elk-1 (p-Elk-1) detected only in nuclear extracts of SLE PBMCs appeared to increase with disease activity. Co-expression levels of p-Elk-1 and IL-10 were elevated in SLE T, B cells and monocytes, associated with increased disease activity in SLE B cells, and were best downregulated by ERK inhibitor. Taken together, our data suggest that preferential binding of activated Elk-1 to the IL10 rs3122605-G allele upregulates IL10 expression and confers increased risk for SLE in European Americans.

Compensatory increase of left atrial external work to left ventricular dysfunction caused by afterload increase.

Afterload mismatch can cause acute decompensation leading to an occurrence of acute heart failure. We investigated how the left atrium (LA) and left ventricle (LV) react to acute increases in afterload using speckle tracking echocardiography (STE). LA strain and volume were obtained by STE in 10 dogs during banding of descending aorta (AoB). Simultaneously, LA pressure was measured by a micromanometer-tipped catheter. LA peak negative strain during LA contraction, strain change during LA relaxation (early reservoir strain), and that during LA dilatation (late reservoir strain) were obtained from LA longitudinal strain-volume curves. From pressure-strain curves, the areas of A-loop and V-loops were computed as the work during active contraction and relaxation (A-work) and that during passive filling and emptying (V-work). AoB increased LV systolic pressure (105 ± 15 vs. 163 ± 12 mmHg, P < 0.01) and mean LA pressure (3.8 ± 1.2 vs. 7.1 ± 2.0 mmHg, P < 0.01). LV global circumferential strain decreased (-18.8 ± 3.5 vs. -13.2 ± 3.5%, P < 0.01), but LV stroke volume was maintained (8.4 ± 2.3 vs. 9.6 ± 3.6 ml). LA peak negative strain (-2.9 ± 2.3 vs. -9.8 ± 4.0%, P < 0.01) and early reservoir strain (4.5 ± 2.1 vs. 7.7 ± 2.4%, P < 0.05) increased by AoB, but late reservoir strain did not change (8.9 ± 3.4 vs. 6.1 ± 3.4%). A-work significantly increased (3.2 ± 2.0 vs. 19.2 ± 15.1 mmHg %, P < 0.01), whereas V-work did not change (13.3 ± 7.1 vs. 13.1 ± 7.7 mmHg %). In conclusion, LA external work during active contraction and relaxation increased as compensation for LV dysfunction during aortic banding. Atrial dysfunction may lead failure of this mechanism and hemodynamic decompensation.

APOBEC3H polymorphisms associated with the susceptibility to HIV-1 infection and AIDS progression in Japanese.

Human APOBEC3H (A3H) is a member of APOBEC3 cytidine deaminase family that potently restricts HIV-1 replication. Because A3H is genetically divergent with different intracellular stability and anti-HIV-1 activity in vitro, we investigated a possible association of A3H with susceptibility to HIV-1 infection and disease progression in Japanese populations. A total of 191 HIV-1-infected individuals (HIV group), 93 long-term non-progressors to AIDS (LTNP group) and 421 healthy controls were genotyped for two functional APOBEC3H polymorphisms, rs139292 and rs139297. As compared with the controls, minor allele frequency (MAF) for rs139292 was high in the HIV group (MAF in cases vs. controls; 0.322 vs. 0.263, odds ratio (OR) = 1.33, 95% confidence interval (95% CI) = 1.02-1.74, p = 0.035) and low in the LTNP group (0.161 vs. 0.263, OR = 0.54, 95% CI = 0.36-0.82, p = 0.004, pc = 0.007), whereas the MAF for rs139297 was high in the HIV group (0.367 vs. 0.298, OR = 1.36, 95% CI = 1.07-1.76, p = 0.017, pc = 0.035). In addition, haplotype analyses revealed that the frequencies of A3H-hapC and -hapA were high (0.322 vs. 0.262, OR = 1.33, 95% CI = 1.02-1.74, p = 0.003) and low (0.634 vs. 0.697, OR = 0.75, 95 % CI = 0.58-0.97, p = 0.002), respectively, in the HIV group, whereas the frequencies of A3H-hapC and -hapB were low (0.161 vs. 0.262, OR = 0.54, 95% CI = 0.36-0.82, p = 0.00003) and high (0.097 vs. 0.040, OR = 2.55, 95% CI = 1.40-4.62, p = 0.000008), respectively, in the LTNP group, as compared with those in the controls. These observations suggest that the A3H with low anti-HIV-1 activity, A3H-hapC, is associated with the susceptibility to HIV-1 infection, whereas the A3H producing a stable protein, A3H-hapB, may confer a low risk of disease progression to AIDS.

CD2-mediated regulation of peripheral CD4(+) CD25(+) regulatory T-cell apoptosis accompanied by down-regulation of Bim.

Extensive studies on CD4(+)  CD25(+) regulatory T (Treg) cells suggest that they are important in regulating immune responses. However, mechanisms of peripheral Treg cell homeostasis are unknown. We found that stromal cells isolated from secondary lymphoid organs such as spleen and lymph nodes could support the survival of Treg cells. This was dependent on CD2 engagement and a direct interaction between Treg cells and stromal cells. In the presence of stromal cells, Bim, a pro-apoptotic factor, was partially decreased in Treg cells. This effect could be inhibited by anti-CD2 blocking antibodies, indicating that stimulation through CD2 on Treg cells regulates Bim expression, which may be relevant to Treg cell apoptosis. Therefore, Treg cell interactions with stromal cells through CD2 may be essential for Treg cell survival. Surprisingly, the expression of CD2 ligands on stromal cells was not detected. Hence, it is not clear how CD2 on Treg cells contributes to a direct interaction with the stromal cells and participates in survival support for Treg cells. Taken together, CD2 stimuli were mandatory for Treg cell survival with reduced Bim expression, but CD2 may not function as a direct receptor for molecules on stromal cells.

Differentiation stage-specific requirement in hypoxia-inducible factor-1alpha-regulated glycolytic pathway during murine B cell development in bone marrow.

Hypoxia-inducible factor (HIF)-1alpha plays a central role in oxygen homeostasis and energy supply by glycolysis in many cell types. We previously reported that an HIF-1alpha gene deficiency caused abnormal B cell development and autoimmunity. In this study we show that HIF-1alpha-enabled glycolysis during B cell development is required in a developmental stage-specific manner. Supporting this conclusion are observations that the glycolytic pathway in HIF-1alpha-deficient B220(+) bone marrow cells is much less functionally effective than in wild-type control cells. The expression of genes encoding the glucose transporters and the key glycolytic enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bishosphatase 3, was greatly reduced in HIF-1alpha-deficient cells. The compensatory adaptation to the defect of glycolysis was reflected in higher levels of expression of respiratory chain-related genes and TCA cycle-related genes in HIF-1alpha-deficient cells than in wild-type cells. In agreement with these findings, HIF-1alpha-deficient cells used pyruvate more efficiently than wild-type cells. The key role of HIF-1alpha-enabled glycolysis in bone marrow B cells was also demonstrated by glucose deprivation during in vitro bone marrow cell culture and by using a glycolysis inhibitor in the bone marrow cell culture. Taken together, these findings indicate that glucose dependency differs at different B cell developmental stages and that HIF-1alpha plays an important role in B cell development.

Identifying high-risk population of depression: association between metabolic syndrome and depression using a health checkup and claims database.

Depression and metabolic syndrome (MetS) are correlated, leading to an increased healthcare burden and decreased productivity. We aimed to investigate the association between MetS-related factors and depression using a health checkup and claims database. Individuals aged 18-75 years who underwent health examinations between 2014 and 2019 were enrolled in the study. Among 76,277 participants, "ever" and "incident" antidepressant users exhibited worse metabolic profiles and were more likely to be prescribed hypnotics and anxiolytics than "never" users. In a nested case-control study with a 1:10 ratio of incident users to controls, MetS was associated with incident antidepressant use (odds ratio, 1.53 [95% confidence interval 1.24-1.88]) adjusted for lifestyle information obtained from a self-administered questionnaire, medical history, and medications. Other metabolic traits also showed significant associations: body mass index (1.04 [1.02-1.06]), abdominal circumference per 10 cm (1.17 [1.08-1.27]), high blood pressure (1.17 [1.00-1.37]), glucose intolerance (1.29 [1.05-1.58]), and dyslipidemia (1.27 [1.08-1.51]). A bodyweight increase > 10 kg from age 20 years (1.46 [1.25-1.70]) was also significantly associated with incident antidepressant use. In conclusion, metabolic abnormalities were associated with incident antidepressant use and can be useful in identifying populations at high risk of depression.

TACI induces cIAP1-mediated ubiquitination of NIK by TRAF2 and TANK to limit non-canonical NF-kappaB signaling.

B-cell-activating factor of the TNF family (BAFF) is a critical factor for B-cell survival and maturation through non-canonical nuclear factor kappaB (NF-kappaB) pathway, a NF-kappaB inducing kinase (NIK)-dependent pathway for the processing of NF-kappaB2 p100 to generate p52. While BAFF acts primarily through BAFF receptor (BAFF-R), the transmembrane activator and CAML interactor (TACI), the other receptor for BAFF, is thought to serve as a negative regulator for B-cell responses. However, how TACI regulates NF-kappaB2 activity is largely unknown. In this study, we showed that constitutive activation of TACI signaling suppressed BAFF-R-mediated NF-kappaB2 p100 processing with the up-regulation of cellular inhibitors of apoptosis 1 (cIAP1) and TNF receptor associated factor (TRAF)-associated NF-kappaB activator (TANK). The ubiquitination of NIK by cIAP1 was inhibited by the expression of TRAF2 with physical binding to cIAP1. TANK deficiency by small interfering RNA (siRNA) impaired TACI-dependent inhibition of NF-kappaB2 p100 processing. TANK also inhibited TRAF2-mediated cIAP1 inactivation. Moreover, the recruitment of TRAF2 to TACI induced the ubiquitination of NIK. Taken together, the regulation of NIK by TACI through the interaction of TANK/TRAF2/cIAP1 plays a pivotal role in the suppression of non-canonical NF-kappaB signaling.