Ablation of SigH+ pDCs in B6.Nba2 mice prevents lupus-like disease development only if started before disease is fully established.

Systemic lupus erythematosus is characterized by hyper-activation of the immune system, multi-organ inflammation, and end-organ damage. Type I interferons (IFN-I) have been strongly implicated a role in disease etiology as has the main IFN-I-producing cell subset, the plasmacytoid dendritic cell (pDC). The B6.Nba2 mouse model develops a lupus-like disease characterized by elevated IFN-I levels and pDC pathogenicity. We have previously shown that pDC ablation prior to disease development in B6.Nba2 mice effectively prevents disease; however, it remains unclear if a similar protection can be seen if pDC ablation is initiated during later disease stages. This is important as Systemic lupus erythematosus patients are rarely diagnosed until disease is well-established and thus preventative treatment is unlikely to take place. Here we show that ablation of pDCs in the B6.Nba2 mouse model must be initiated early in order to effectively block disease development and that sustained reduction in pDC numbers is necessary for sustained effects. Finally, targeting of pDCs have been hypothesized to affect immunity towards infectious agents, in particular virus and intracellular bacteria. We show here that pDC ablation in B6.Nba2 mice does not affect the anti-viral response to encephalomyocarditic virus or a model T-dependent antigen. In summary, pDC ablation does not affect general immunity, but needs to happen early and be sustained to prevent lupus-like disease development in B6.Nba2 mice.

Spontaneous CD4+ T Cell Activation and Differentiation in Lupus-Prone B6.Nba2 Mice Is IFNAR-Independent.

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by dysregulated T and B lymphocytes. Type I interferons (IFN-I) have been shown to play important pathogenic roles in both SLE patients and mouse models of lupus. Recent studies have shown that B cell intrinsic responses to IFN-I are enough to drive B cell differentiation into autoantibody-secreting memory B cells and plasma cells, although lower levels of residual auto-reactive cells remain present. We speculated that IFN-I stimulation of T cells would similarly drive specific T-cell associated lupus phenotypes including the upregulation of T follicular helper cells and Th17, thereby affecting autoantibody production and the development of glomerulonephritis. Using the B6.Nba2 mouse model of lupus, we evaluated disease parameters in T cell specific IFN-I receptor (IFNAR)-deficient mice (cKO). Surprisingly, all measured CD4+ T cell abnormalities and associated intra-splenic cytokine levels (IFNγ, IL-6, IL-10, IL-17, IL-21) were unchanged and thus independent of IFN-I. In contrast B6.Nba2 cKO mice displayed reduced levels of effector CD8+ T cells and increased levels of Foxp3+ CD8+ regulatory T cells, suggesting that IFN-I induced signaling specifically affecting CD8+ T cells. These data suggest a role for both pathogenic and immunosuppressive CD8+ T cells in Nba2-driven autoimmunity, providing a model to further evaluate the role of these cell subsets during lupus-like disease development in vivo.

Lupus-prone B6.Nba2 male and female mice display anti-DWEYS reactivity and a neuropsychiatric phenotype.

OBJECTIVE: Neuropsychiatric lupus (NPSLE), a manifestation of the autoimmune disease systemic lupus erythematosus (SLE), is characterized by psychiatric symptoms including anxiety and depression and upregulated autoantibodies. The B6.Nba2 spontaneous mouse model develops SLE, but has not previously been tested for NPSLE. METHODS: We investigated the NPSLE phenotype in male and female B6.Nba2 mice (n = 12 each) and age- and sex-matched B6 controls (n = 10 each) via behavioral assessments for anxiety, depression, and memory deficits. Serum anti-dsDNA, anti-nRNP, anti-DWEYS peptide reactive IgG autoantibody levels and soluble TWEAK levels were determined by ELISA. Hippocampal regions were stained for activated microglia and neurons. RESULTS: Both male and female B6.Nba2 mice showed elevated anti-dsDNA IgG, anti-nRNP IgG and anti-DWEYS reactive antibodies, elevated serum soluble TWEAK levels, and a strong anxiety and depression phenotype (p < 0.05-0.0001). Male B6.Nba2 mice developed this phenotype at a slightly older age than females. Female B6.Nba2 mice displayed reduced numbers of neurons in the hippocampal region compared to female B6 controls (p < 0.05). CONCLUSION: The B6.Nba2 mouse model recapitulates many known NPSLE phenotypes, making it a promising model to investigate the development of NPSLE in the context of SLE.

Immunological effects of vitamin D and their relations to autoimmunity.

Vitamin D deficiency is an established risk factor for many autoimmune diseases and the anti-inflammatory properties of vitamin D underscore its potential therapeutic value for these diseases. However, results of vitamin D3 supplementation clinical trials have been varied. To understand the clinical heterogeneity, we reviewed the pre-clinical data on vitamin D activity in four common autoimmune diseases: multiple sclerosis (MS), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and inflammatory bowel disease (IBD), in which patients are commonly maintained on oral vitamin D3 supplementation. In contrast, many pre-clinical studies utilize other methods of manipulation (i.e. genetic, injection). Given the many actions of vitamin D3 and data supporting a vitamin D-independent role of the Vitamin D receptor (VDR), a more detailed mechanistic understanding of vitamin D3 activity is needed to properly translate pre-clinical findings into the clinic. Therefore, we assessed studies based on route of vitamin D3 administration, and identified where discrepancies in results exist and where more research is needed to establish the benefit of vitamin D supplementation.

Differential Internalization Rates and Postendocytic Sorting of the Norepinephrine and Dopamine Transporters Are Controlled by Structural Elements in the N Termini.

The norepinephrine transporter (NET) mediates reuptake of synaptically released norepinephrine in central and peripheral noradrenergic neurons. The molecular processes governing availability of NET in the plasma membrane are poorly understood. Here we use the fluorescent cocaine analogue JHC 1-64, as well as several other approaches, to investigate the trafficking itinerary of NET in live noradrenergic neurons. Confocal imaging revealed extensive constitutive internalization of JHC 1-64-labeled NET in the neuronal somata, proximal extensions and presynaptic boutons. Phorbol 12-myristate 13-acetate increased intracellular accumulation of JHC 1-64-labeled NET and caused a parallel reduction in uptake capacity. Internalized NET strongly colocalized with the "long loop" recycling marker Rab11, whereas less overlap was seen with the "short loop" recycling marker Rab4 and the late endosomal marker Rab7. Moreover, mitigating Rab11 function by overexpression of dominant negative Rab11 impaired NET function. Sorting of NET to the Rab11 recycling compartment was further supported by confocal imaging and reversible biotinylation experiments in transfected differentiated CATH.a cells. In contrast to NET, the dopamine transporter displayed markedly less constitutive internalization and limited sorting to the Rab11 recycling compartment in the differentiated CATH.a cells. Exchange of domains between the two homologous transporters revealed that this difference was determined by non-conserved structural elements in the intracellular N terminus. We conclude that NET displays a distinct trafficking itinerary characterized by continuous shuffling between the plasma membrane and the Rab11 recycling compartment and that the functional integrity of the Rab11 compartment is critical for maintaining proper presynaptic NET function.

Gr1+ cells suppress T-dependent antibody responses in (NZB x NZW)F1 male mice through inhibition of T follicular helper cells and germinal center formation.

Systemic lupus erythematosus is an autoimmune disease characterized by elevated production of autoreactive Abs. The disease has a much higher prevalence in women than in men. Although testosterone has been shown to be protective in the disease, and estrogens exacerbating, the discrepancy in prevalence between men and women is still not well understood and the mechanism behind it is unknown. We have recently described that male (New Zealand black [NZB] × New Zealand white [NZW])F1 mice have higher levels of Gr1(+)CD11b(+) cells, and that these cells suppress autoantibody production in vivo. In this article, we extend our findings to show that similarly to humans, female lupus-prone (NZB × NZW)F1 mice also respond with stronger Ab responses to thymus-dependent Ag immunization than male littermates. Furthermore, the presence or absence of Gr1-expressing cells not only control Ag-specific Ab responses in male, but not female, (NZB × NZW)F1 mice, but also significantly alter the activation and differentiation of CD4(+) T cells in vitro and in vivo. In particular, we found that Gr1(+) cells from male (NZB × NZW)F1 mice suppress the differentiation and effector function of CXCR5(+)PD-1(+) T follicular helper cells, thereby controlling germinal center formation and plasma cell differentiation. This new finding strongly supports efforts to develop new drugs that target myeloid cell subsets in a number of T and B cell-mediated diseases with a female predominance.

Suppressive effects of androgens on the immune system.

Sex-based disparities in immune responses are well known phenomena. The two most important factors accounting for the sex-bias in immunity are genetics and sex hormones. Effects of female sex hormones, estrogen and progesterone are well established, however the role of testosterone is not completely understood. Evidence from unrelated studies points to an immunosuppressive role of testosterone on different components of the immune system, but the underlying molecular mechanisms remains unknown. In this review we evaluate the effect of testosterone on key cellular components of innate and adaptive immunity. Specifically, we highlight the importance of testosterone in down-regulating the systemic immune response by cell type specific effects in the context of immunological disorders. Further studies are required to elucidate the molecular mechanisms of testosterone-induced immunosuppression, leading the way to the identification of novel therapeutic targets for immune disorders.

Spontaneous loss of tolerance of autoreactive B cells in Act1-deficient rheumatoid factor transgenic mice.

Self-reactive B cells in BALB/c AM14 transgenic (Tg) rheumatoid factor mice are not subject to central or peripheral tolerization. Instead, they remain at a stage of "clonal ignorance"; that is, they do not proliferate and differentiate into Ab-forming cells. However, the immunoregulatory mechanisms that prevent autoantibody production in these mice remain unclear. In this study, we show that crossing AM14 Tg mice to a mouse strain deficient in Act1, a molecule involved in the regulation of BAFF-R and CD40-signaling in B cells, results in spontaneous activation of AM14 Tg B cells and production of AM14-specific Abs. Three- to 5-mo-old AM14 Tg Act1(-/-) mice showed significant expansion of AM14 Tg B cells, including a 2- to 3-fold increase in the spleen and cervical lymph nodes compared with AM14 Tg Act1(+/+) mice. Furthermore, in the presence of endogenous self-Ag (IgH(a) congenic background), AM14 Tg Act1(-/-) B cells were spontaneously activated and differentiated into Ab-forming cells. In contrast with previous studies using AM14 Tg MLR.Fas(lpr) mice, we found that a significant number of AM14 Tg cells AM14 Tg Act1(-/-) mice displayed phenotypic characteristics of germinal center B cells. Anti-CD40L treatment significantly limited the expansion and activation of AM14 Tg Act1(-/-) B cells, suggesting that CD40L-mediated signals are required for the retention of these cells. Our results support the important role of Act1 in the regulation of self-reactive B cells and reveal how Act1 functions to prevent the production of autoantibodies.

Gr-1(high) CD11b+ cells suppress B cell differentiation and lupus-like disease in lupus-prone male mice.

OBJECTIVE: Systemic lupus erythematosus (SLE) develops much more readily in females than in males. Previous research has focused primarily on identifying mechanisms pertinent to the pathology in females. The aim of the current study was to delineate active protective mechanisms in males. We present evidence of a new male-associated mechanism of protection against the development of lupus-like disease in lupus-prone (NZB × NZW)F1 mice. METHODS: We identified previously uncharacterized cellular and functional differences in myeloid cells between male and female (NZB × NZW)F1 mice, with the use of flow cytometry, confocal imaging, in vivo antibody-mediated depletion, and in vitro cell coculture assays. RESULTS: A population of Gr-1(high) Ly-6G+CD11b+ myeloid cells was found to be constitutively increased in male (NZB × NZW)F1 mice as compared with female mice and was regulated by testosterone. The cells were located adjacent to spleen B cell follicles in vivo and were found to directly inhibit cytokine-induced differentiation of naive B cells into antibody-secreting cells in vitro. Most notably, treatment with anti-Gr-1-depleting antibodies increased the spontaneous production of antinuclear autoantibodies in male (NZB × NZW)F1 mice, while a similar approach in female mice had no effect on disease development. CONCLUSION: Male lupus-prone (NZB × NZW)F1 mice harbor elevated levels of a population of myeloid cells with pronounced immunosuppressive capacities that specifically target B cells and the production of antibodies in vivo. We suggest that these cells represent a male-driven inhibitory mechanism involved in the control of B cell pathogenesis, delaying (or preventing) lupus-like disease development in otherwise genetically predisposed male (NZB × NZW)F1 mice.

SLC6 neurotransmitter transporters: structure, function, and regulation.

The neurotransmitter transporters (NTTs) belonging to the solute carrier 6 (SLC6) gene family (also referred to as the neurotransmitter-sodium-symporter family or Na(+)/Cl(-)-dependent transporters) comprise a group of nine sodium- and chloride-dependent plasma membrane transporters for the monoamine neurotransmitters serotonin (5-hydroxytryptamine), dopamine, and norepinephrine, and the amino acid neurotransmitters GABA and glycine. The SLC6 NTTs are widely expressed in the mammalian brain and play an essential role in regulating neurotransmitter signaling and homeostasis by mediating uptake of released neurotransmitters from the extracellular space into neurons and glial cells. The transporters are targets for a wide range of therapeutic drugs used in treatment of psychiatric diseases, including major depression, anxiety disorders, attention deficit hyperactivity disorder and epilepsy. Furthermore, psychostimulants such as cocaine and amphetamines have the SLC6 NTTs as primary targets. Beginning with the determination of a high-resolution structure of a prokaryotic homolog of the mammalian SLC6 transporters in 2005, the understanding of the molecular structure, function, and pharmacology of these proteins has advanced rapidly. Furthermore, intensive efforts have been directed toward understanding the molecular and cellular mechanisms involved in regulation of the activity of this important class of transporters, leading to new methodological developments and important insights. This review provides an update of these advances and their implications for the current understanding of the SLC6 NTTs.

Control of dermatomyositis skin disease activity lags behind control of muscles disease activity during the early treatment stages of classic dermatomyositis: A retrospective, single-centre study.

This study aimed to retrospectively identify differences in relative control of cutaneous and muscular disease activity in adult dermatomyositis (DM) patients at a single, tertiary care centre following initiation of diagnosis and treatment. Our results demonstrated a significantly lower complete treatment response rate of skin disease compared to muscular disease at 6-months and persistent but not statistically significant lower skin disease response at 12 months. These results suggest DM skin disease activity may be more refractory to treatment than muscle disease activity, especially in the early disease phase.

Intracellular and circulating neuronal antinuclear antibodies in human epilepsy.

There are overwhelming data supporting the inflammatory origin of some epilepsies (e.g., Rasmussen's encephalitis and limbic encephalitis). Inflammatory epilepsies with an autoimmune component are characterized by autoantibodies against membrane-bound, intracellular or secreted proteins (e.g., voltage gated potassium channels). Comparably, little is known regarding autoantibodies targeting nuclear antigen. We tested the hypothesis that in addition to known epilepsy-related autoantigens, the human brain tissue and serum from patients with epilepsy contain autoantibodies recognizing nuclear targets. We also determined the specific nuclear proteins acting as autoantigen in patients with epilepsy. Brain tissue samples were obtained from patients undergoing brain resections to treat refractory seizures, from the brain with arteriovenous malformations or from post-mortem multiple sclerosis brain. Patients with epilepsy had no known history of autoimmune disease and were not diagnosed with autoimmune epilepsy. Tissue was processed for immunohistochemical staining. We also obtained subcellular fractions to extract intracellular IgGs. After separating nuclear antibody-antigen complexes, the purified autoantigen was analyzed by mass spectrometry. Western blots using autoantigen or total histones were probed to detect the presence of antinuclear antibodies in the serum of patients with epilepsy. Additionally, HEp-2 assays and antinuclear antibody ELISA were used to detect the staining pattern and specific presence of antinuclear antibodies in the serum of patients with epilepsy. Brain regions from patients with epilepsy characterized by blood-brain barrier disruption (visualized by extravasated albumin) contained extravasated IgGs. Intracellular antibodies were found in epilepsy (n=13/13) but not in multiple sclerosis brain (n=4/4). In the brain from patients with epilepsy, neurons displayed higher levels of nuclear IgGs compared to glia. IgG colocalized with extravasated albumin. All subcellular fractions from brain resections of patients with epilepsy contained extravasated IgGs (n=10/10), but epileptogenic cortex, where seizures originated from, displayed the highest levels of chromatin-bound IgGs. In the nuclear IgG pool, anti-histone autoantibodies were identified by two independent immunodetection methods. HEp-2 assay and ELISA confirmed the presence of anti-histone (n=5/8) and anti-chromatin antibodies in the serum from patients with epilepsy. We developed a multi-step approach to unmask autoantigens in the brain and sera of patients with epilepsy. This approach revealed antigen-bound antinuclear antibodies in neurons and free antinuclear IgGs in the serum of patients with epilepsy. Conditions with blood-brain barrier disruption but not seizures, were characterized by extravasated but not chromatin-bound IgGs. Our results show that the pool of intracellular IgG in the brain of patients with epilepsy consists of nucleus-specific autoantibodies targeting chromatin and histones. Seizures may be the trigger of neuronal uptake of antinuclear antibodies.

Lack of T cells in Act1-deficient mice results in elevated IgM-specific autoantibodies but reduced lupus-like disease.

Act1 is a negative regulator of B-cell activation factor of the TNF family (BAFF) and CD40L-induced signaling. BALB/C mice lacking Act1 develop systemic autoimmunity resembling systemic lupus erythematosus (SLE) and Sjögren's syndrome (SjS). SLE and SjS are characterized by anti-nuclear IgG autoantibody (ANA-IgG) production and inflammation of peripheral tissues. As autoantibody production can occur in a T-cell dependent or T-cell independent manner, we investigated the role of T-cell help during Act1-mediated autoimmunity. Act1-deficiency was bred onto C57Bl/6 (B6.Act1(-/-) ) mice and B6.TCRß(-/-) TCRδ(-/-) Act1(-/-) (TKO) mice were generated. While TCRß/δ-sufficient B6.Act1(-/-) mice developed splenomegaly and lymphadenopathy, hypergammaglobulinemia, elevated levels of ANA-IgG, and kidney pathology, TKO mice failed to develop any such signs of disease. Neither B6.Act1(-/-) nor TKO mice developed SjS-like disease, suggesting that epigenetic interactions on the BALB/C background are responsible for this phenotype in BALB/C.Act1(-/-) mice. Interestingly, BAFF-driven transitional B-cell abnormalities, previously reported in BALB/C.Act1(-/-) mice, were intact in B6.Act1(-/-) mice and largely independent of T cells. In conclusion, T cells are necessary for the development of SLE-like disease in B6.Act1(-/-) mice, but not BAFF-driven transitional B-cell differentiation.

Brain-derived neurotrophic factor (BDNF) enhances GABA transport by modulating the trafficking of GABA transporter-1 (GAT-1) from the plasma membrane of rat cortical astrocytes.

The γ-aminobutyric acid (GABA) transporters (GATs) are located in the plasma membrane of neurons and astrocytes and are responsible for termination of GABAergic transmission. It has previously been shown that brain derived neurotrophic factor (BDNF) modulates GAT-1-mediated GABA transport in nerve terminals and neuronal cultures. We now report that BDNF enhances GAT-1-mediated GABA transport in cultured astrocytes, an effect mostly due to an increase in the V(max) kinetic constant. This action involves the truncated form of the TrkB receptor (TrkB-t) coupled to a non-classic PLC-γ/PKC-δ and ERK/MAPK pathway and requires active adenosine A(2A) receptors. Transport through GAT-3 is not affected by BDNF. To elucidate if BDNF affects trafficking of GAT-1 in astrocytes, we generated and infected astrocytes with a functional mutant of the rat GAT-1 (rGAT-1) in which the hemagglutinin (HA) epitope was incorporated into the second extracellular loop. An increase in plasma membrane of HA-rGAT-1 as well as of rGAT-1 was observed when both HA-GAT-1-transduced astrocytes and rGAT-1-overexpressing astrocytes were treated with BDNF. The effect of BDNF results from inhibition of dynamin/clathrin-dependent constitutive internalization of GAT-1 rather than from facilitation of the monensin-sensitive recycling of GAT-1 molecules back to the plasma membrane. We therefore conclude that BDNF enhances the time span of GAT-1 molecules at the plasma membrane of astrocytes. BDNF may thus play an active role in the clearance of GABA from synaptic and extrasynaptic sites and in this way influence neuronal excitability.

The adaptor molecule Act1 regulates BAFF responsiveness and self-reactive B cell selection during transitional B cell maturation.

The transitional stage is a key check-point for elimination of autoreactive B cells in the periphery. This selection process requires fine regulation of signals received through BCR and B cell activating factor (BAFF) receptor. We previously identified the adaptor molecule Act1 as a negative regulator of BAFF-mediated signaling. Deficiency of Act1 in mice results in peripheral B cell hyperplasia and development of autoimmunity. In this study, we demonstrate that Act1 plays a critical role in the regulation of transitional B cell survival and maturation. We found that the ratio of late-transitional (T2) to early-transitional (T1) cells was increased in spleens from Act1-deficient mice. Moreover, BAFF stimulation induced better T1 cell survival and promoted more efficient maturation of T1 cells into T2 cells ex vivo in the absence of Act1. BAFF stimulation induced higher levels of the anti-apoptotic Bcl-2 member Mc1-l in Act1-deficient T1 cells than in wild-type control cells, suggesting that Mcl-1 might be one of the key effector molecules for BAFF-mediated survival of the Act1-deficient transitional B cells. Importantly, costimulation with BAFF was able to rescue Act1-deficient T1 cells from BCR-induced apoptosis more effectively than Act1-sufficient T1 B cells. Finally, by using hen egg lysozyme double transgenic mice, we demonstrated that Act1 deficiency can promote the maturation of Ag-specific autoreactive B cells. Taken together, our results suggest that the transitional stage is a critical point of action of Act1 in the elimination of autoreactive B cells and in the regulation of peripheral B cell homeostasis.

Development of murine lupus involves the combined genetic contribution of the SLAM and FcgammaR intervals within the Nba2 autoimmune susceptibility locus.

Autoantibodies are of central importance in the pathogenesis of Ab-mediated autoimmune disorders. The murine lupus susceptibility locus Nba2 on chromosome 1 and the syntenic human locus are associated with a loss of immune tolerance that leads to antinuclear Ab production. To identify gene intervals within Nba2 that control the development of autoantibody-producing B cells and to determine the cellular components through which Nba2 genes accomplish this, we generated congenic mice expressing various Nba2 intervals where genes for the FcgammaR, SLAM, and IFN-inducible families are encoded. Analysis of congenic strains demonstrated that the FcgammaR and SLAM intervals independently controlled the severity of autoantibody production and renal disease, yet are both required for lupus susceptibility. Deregulated homeostasis of terminally differentiated B cells was found to be controlled by the FcgammaR interval where FcgammaRIIb-mediated apoptosis of germinal center B cells and plasma cells was impaired. Increased numbers of activated plasmacytoid dendritic cells that were distinctly CD19+ and promoted plasma cell differentiation via the proinflammatory cytokines IL-10 and IFNalpha were linked to the SLAM interval. These findings suggest that SLAM and FcgammaR intervals act cooperatively to influence the clinical course of disease through supporting the differentiation and survival of autoantibody-producing cells.

Lack of Association Between Sex Hormones, MDSCs, LDGs and pDCs in Males and Females With Systemic Lupus Erythematosus.

Plasmacytoid dendritic cells (pDCs) and low-density granulocytes (LDGs) are interferon-alpha producing cells that create a pro-inflammatory response in Systemic Lupus Erythematosus (SLE) leading to auto antibody production and organ damage. Both pDCs and LDGs have been shown to be dysfunctional in patients with active SLE. Myeloid-derived suppressor cells (MDSCs) have the capacity to control T and B cell activation and differentiation, and have recently been identified as cells of interest in SLE as well. While not fully understood, previous studies have suggested that pDCs are regulated in part by both X chromosome inactivation and estradiol. Whether sex chromosomes or sex hormones regulate MDSCs and LDGs remain to be determined. We aimed to explore the relative role of sex and sex hormones on pDC, MDSC and LDG frequency and function in SLE patients. We recruited patients with SLE as defined by ACR or SLICC classification criteria and healthy controls in conjunction with the Cleveland Clinic Lupus Cohort and Clinical Research Unit. We analyzed serum sex hormone levels by ELISA, and frequencies of pDCs, MDSCs, and LDGs among PBMCs and serum cytokine levels by flow cytometry. PBMCs were further analyzed for expression of genes involved in or induced by toll-like receptor (TLR)7 or TLR9 stimulation. In all SLE patients, the serum estradiol/testosterone ratio and levels of granulocytic MDSCs and LDGs were increased, while levels of pDCs were decreased. Furthermore, pDCs from active SLE patients expressed lower levels of TLR7 and TLR9 and showed diminished production of TLR9-induced IFNα and TNFα as compared to healthy controls. LDGs from healthy controls and SLE patients expressed very low levels of TLR7 and TLR9 and largely failed to respond to TLR9 stimulation. Thus, regardless of sex and sex-hormone levels, frequencies of pDCs, MDSCs and LDGs, TLR7 and TLR9 expression, and TLR9-driven cytokine production were similarly altered in male and female SLE patients.

The N terminus of monoamine transporters is a lever required for the action of amphetamines.

The serotonin transporter (SERT) terminates neurotransmission by removing serotonin from the synaptic cleft. In addition, it is the site of action of antidepressants (which block the transporter) and of amphetamines (which induce substrate efflux). We explored the functional importance of the N terminus in mediating the action of amphetamines by focusing initially on the highly conserved threonine residue at position 81, a candidate site for phosphorylation by protein kinase C. Molecular dynamics simulations of the wild type SERT, compared with its mutations SERT(T81A) and SERT(T81D), suggested structural changes in the inner vestibule indicative of an opening of the inner vestibule. Predictions from this model (e.g. the preferential accumulation of SERT(T81A) in the inward conformation, its reduced turnover number, and a larger distance between its N and C termini) were verified. Most importantly, SERT(T81A) (and the homologous mutations in noradrenaline and dopamine) failed to support amphetamine-induced efflux, and this was not remedied by aspartate at this position. Amphetamine-induced currents through SERT(T81A) were comparable with those through the wild type transporter. Both abundant Na(+) entry and accumulation of SERT(T81A) in the inward facing conformation ought to favor amphetamine-induced efflux. Thus, we surmised that the N terminus must play a direct role in driving the transporter into a state that supports amphetamine-induced efflux. This hypothesis was verified by truncating the first 64 amino acids and by tethering the N terminus to an additional transmembrane helix. Either modification abolished amphetamine-induced efflux. We therefore conclude that the N terminus of monoamine transporters acts as a lever that sustains reverse transport.

S100a9 Protects Male Lupus-Prone NZBWF1 Mice From Disease Development.

Systemic lupus erythematosus (SLE) is an autoimmune disorder disproportionally affecting women. A similar sex difference exists in the murine New Zealand Black/White hybrid model (NZBWF1) of SLE with all females, but only 30-40% of males, developing disease within the first year of life. Myeloid-derived suppressor cells (MDSCs) are prominent in NZBWF1 males and while depletion of these cells in males, but not females, promotes disease development, the mechanism of suppression remains unknown. S100a9, expressed by neutrophils and MDSCs, has previously been shown to exert immunosuppressive functions in cancer and inflammation. Here we investigated if S100a9 exerts immunosuppressive functions in NZBWF1 male and female mice. S100a9+/+, S100a9+/- and S100a9-/- NZBWF1 mice were followed for disease development for up to 8 months of age. Serum autoantibody levels, splenomegaly, lymphocyte activation, glomerulonephritis and proteinuria were measured longitudinally or at the time of harvest. In accordance with an immunosuppressive function of MDSCs in male mice, S100a9-deficient male NZBWF1 mice developed accelerated autoimmunity as indicated by increased numbers of differentiated effector B and T cells, elevated serum autoantibody levels, increased immune-complex deposition and renal inflammation, and accelerated development of proteinuria. In contrast, female mice showed either no response to S100a9-deficiency or even a slight reduction in disease symptoms. Furthermore, male, but not female, S100a9-/- NZBWF1 mice displayed an elevated type I interferon-induced gene signature, suggesting that S100a9 may dampen a pathogenic type I interferon signal in male mice. Taken together, S100a9 exerts an immunosuppressive function in male NZBWF1 mice effectively moderating lupus-like disease development via inhibition of type I interferon production, lymphocyte activation, autoantibody production and the development of renal disease.