Autoimmune Mice Research Articles

Autoimmune disease mouse model exhibits pulmonary arterial hypertension.

BackgroundPulmonary arterial hypertension is often associated with connective tissue disease. Although there are some animal models of pulmonary hypertension, an autoimmune disease-based model has not yet been reported. MRL/lpr mice, which have hypergammaglobulinemia, produce various autoimmune antibodies, and develop vasculitis and nephritis spontaneously. However, little is known about pulmonary circulation in these mice. In the present study, we examined the pulmonary arterial pressure in MRL/lpr mice.Methods and resultsWe used female MRL/lpr mice aged between 12 and 14 weeks. Fluorescent immunostaining showed that there was no deposition of immunoglobulin or C3 in the lung tissue of the MRL/lpr mice. Elevation of interferon-γ and interleukin-6 was recognized in the lung tissue of the MRL/lpr mice. Right ventricular systolic pressure, Fulton index and the ratio of right ventricular weight to body weight in the MRL/lpr mice were significantly higher than those in wild type mice with same background (C57BL/6). The medial smooth muscle area and the proportion of muscularized vessels in the lung tissue of the MRL/lpr mice were larger than those of the C57BL/6 mice. Western blot analysis demonstrated markedly elevated levels of prepro-endothelin-1 and survivin as well as decreased endothelial nitric oxide synthase phosphorylation in the lung tissue of the MRL/lpr mice. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling assay showed the resistance against apoptosis of pulmonary arterial smooth muscle cells in the MRL/lpr mice.ConclusionWe showed that MRL/lpr mice were complicated with pulmonary hypertension. MRL/lpr mice appeared to be a useful model for studying the mechanism of pulmonary hypertension associated with connective tissue diseases.

Quantitative Characterization of Autoimmune Uveoretinitis in an Experimental Mouse Model.

To accurately evaluate the autoimmune inflammation, we aim to develop three quantitative measurements to monitor the inflammatory changes in the retina: retinal-choroidal thickness, major retinal vessel diameter, and electroretinography amplitudes. During a 21-day experimental period, eyes were examined by confocal scanning laser ophthalmoscopy, optical coherence tomography, fundus fluorescein angiography, and electroretinography in living mice, which were then subsequently killed for histologic assessments. On day 21 postimmunization, inflammation was observed both in vivo and in vitro. Fold change of retinal-choroidal thickness and major retinal vessel diameter in experimental autoimmune uveoretinitis mice were significantly greater than controls (P < 0.001). Both scotopic and photopic electroretinography amplitudes were significantly attenuated when compared with control mice (P < 0.01). Our results showed that these three quantifiable indicators provided an objective and accurate evaluation of autoimmune inflammation, which are in good correlations with the reported clinical and histopathologic scoring systems (P < 0.05). These three indicators will be useful to detect the small but significant differences in the severity of experimental autoimmune uveoretinitis for future longitudinally therapeutic studies.

Blocking IL-10 signalling at the time of immunization does not increase unwanted side effects in mice

BackgroundCancer therapeutic vaccine induced cytotoxic T cell (CTL) responses are pivotal for the killing of tumour cells. Blocking interleukin 10 (IL-10) signalling at the time of immunization increases vaccine induced CTL responses and improves prevention of tumour growth in animal models compared to immunization without an IL-10 signalling blockade. Therefore, this immunization strategy may have potential to curtail cancer in a clinical setting. However, IL-10 deficiency leads to autoimmune disease in the gut. Blocking IL-10 at the time of immunization may result in unwanted side effects, especially immune-pathological diseases in the intestine.MethodsWe investigated whether blocking IL-10 at the time of immunization results in intestinal inflammation responses in a mouse TC-1 tumour model and in a NOD autoimmune disease prone mouse model.ResultsWe now show that blocking IL-10 at the time of immunization increases IL-10 production by CD4+ T cells in the spleen and draining lymph nodes, and does not result in blood cell infiltration to the intestines leading to intestinal pathological changes. Moreover, immunization with papillomavirus like particles combined with simultaneously blocking IL-10 signalling does not increase the incidence of autoimmune disease in Non-obese diabetic (NOD) mice.ConclusionsOur results indicate that immunization with an IL-10 inhibitor may facilitate the generation of safe, effective therapeutic vaccines against chronic viral infection and cancer.

Expression and significance of estrogen receptor in bladder of experimental autoimmune encephalomyelitis mouse model

Objective To study the changes of estrogen receptor (ER) expression in neurogenic bladder caused by multiple sclerosis (MS) and its significance by establishing an experimental autoimmune encephalomyelitis (EAE) model in mice. Methods The EAE models were induced by an antigen emulsion mixture that was well mixed with mog35-55 and complete Freund’s adjuvant. Twenty female C57Bl/6 mice were randomly divided into experimental group and control group. The clinical symptoms of mice were observed and recorded after immunization. Twenty-third days after immunization, micturition frequency and mean void weight were observed within 4 h. The spinal cord as well as the bladder was collected and the bladder was weighed. The pathological changes were observed by hematoxylin-eosin (HE) staining and Luxol fast blue (LFB) staining. The expression of ER in bladder of EAE mice was detected by Western blotting. Results The clinical score of neurological function in EAE mice increased, and typical MS symptoms appeared. The weight of the control group and experimental group was (23.0±1.1) g and (16.5±1.5) g respectively. As compared with the control group, the weight of the mice in the model group was obviously reduced. The average number of 4H urination in model group (8.7±1.4) was significantly greter than in the control group (4.2±1.1, P=0.001). The urine volume in the model group [(0.22±0.11) mg] was significantly less than that in the control group [(0.34±0.07) mg, P=0.008]. In the model group, the bladder weight ratio [(0.26±0.09)‰] was significantly higher than that in the model group [(0.11±0.03)‰, P=0.001]. HE-LFB staining showed obvious inflammatory cell infiltration and demyelination in the spinal cord of EAE model mice, and Western blotting showed that the relative expression of ER in the bladder of model group (0.38±0.08) was significantly lower than that in the control group (0.71±0.19, P=0.001). Conclusion Mog35-55 can successfully induce the EAE mice model. The EAE mice model can well simulate the MS neurogenic bladder symptoms. ER and estrogen may be the important factors in bladder dysfunction caused by multiple sclerosis. Key words: Estrogen receptor; Experimental autoimmune encephalomyelitis; Neurogenic bladder

Featured Article: Serum [Met5]-enkephalin levels are reduced in multiple sclerosis and restored by low-dose naltrexone.

Low-dose naltrexone is a widely used off-label therapeutic prescribed for a variety of immune-related disorders. The mechanism underlying low-dose naltrexone's efficacy for fatigue, Crohn's disease, fibromyalgia, and multiple sclerosis is, in part, intermittent blockade of opioid receptors followed by upregulation of endogenous opioids. Short, intermittent blockade by naltrexone specifically blocks the opioid growth factor receptor resulting in biofeedback events that increase production of the endogenous opioid growth factor (OGF) (chemically termed [Met5]-enkephalin) facilitating interactions between opioid growth factor and opioid growth factor receptor that ultimately, result in inhibited cell proliferation. Preclinical studies have reported that enkephalin levels are deficient in animal models of experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. Our hypothesis is that serum enkephalin levels are diminished in humans with multiple sclerosis and experimental autoimmune encephalomyelitis mice, and that change in serum opioid growth factor levels may serve as a reasonable candidate biomarker for the onset of experimental autoimmune encephalomyelitis and response to therapy. To address this, we designed a two-part study to measure endogenous opioids in multiple sclerosis patients, and to investigate the temporal pattern of decline in serum enkephalin concentrations in mice with chronic progressive experimental autoimmune encephalomyelitis and treated with low-dose naltrexone. For comparison, we investigated whether low-dose naltrexone exposure in normal mice also resulted in altered enkephalin levels. In both animal models, we monitored tactile and heat sensitivity, as well as differential white blood cell counts as indicators of inflammation. Serum [Met5]-enkephalin levels were lower in humans with multiple sclerosis relative to non-multiple sclerosis patients, and low-dose naltrexone restored their levels. In experimental autoimmune encephalomyelitis mice, [Met5]-enkephalin levels were depressed prior to the appearance of clinical disease, and were restored with low-dose naltrexone treatment. Low-dose naltrexone therapy had no effect on serum [Met5]-enkephalin or β-endorphin in normal mice. Thus, [Met5]-enkephalin (i.e. opioid growth factor) may be a reasonable candidate biomarker for multiple sclerosis, and may signal new pathways for treatment of autoimmune disorders. Impact statement This report presents human and animal data identifying a novel biomarker for the onset and progression of multiple sclerosis (MS). Humans diagnosed with MS have reduced serum levels of OGF (i.e. [Met5]-enkephalin) relative to non-MS neurologic patients, and low-dose naltrexone (LDN) therapy restored their enkephalin levels. Serum OGF levels were reduced in mice immunized with MOG35-55 prior to any clinical behavioral sign of experimental autoimmune encephalomyelitis, and LDN therapy restored their serum OGF levels. β-endorphin concentrations were not altered by LDN in humans or mice. Thus, blood levels of OGF may serve as a new, selective biomarker for the progression of MS, as well as response to therapy.

The therapeutic effects of Yongdamsagan-tang on autoimmune hepatitis models

Autoimmune hepatitis (AIH) is an immunity disorder that is the result of antibodies in the liver tissue of the patient that are attacked by activated immune cells due to an unknown cause. In this study, we aimed to investigate the anti-inflammatory effect of Yongdamsagan-tang (YST) extracts and confirm effects on autoimmune hepatitis models as the therapeutic agent using the YST extracted by various solvents. YST, a mixture of 11 herbal extracts, is known in traditional Korean medicine as a widely used treatment for inflammatory diseases. We proposed the AIH-condition in vitro model by the addition of recombinant IL-17A and then observed several markers linked to AIH symptoms, including an increase of IL-6 expression, lipid accumulation, and fibrosis. In AIH-condition hepatic cell model, YST reduced IL-6 expression and lipid accumulation caused by treatment of IL-17 combination in hepatocyte cells. Also, YST blocked several activated fibrosis factors including transforming growth factor-β (TGF- β1), collagen type 1 (Col-α1(I)), and α-smooth muscle actin (α-SMA) in liver stellate cells. Furthermore, pretreatment with YST protected hepatic damage and reduces histological injury by suppressing apoptosis mediator and inflammatory cytokines expression in concanavalin A (Con A)-induced autoimmune hepatitis mice model. The findings here improve our understanding of YST extracted by 80% ethanol, suggesting that YST can be used as a therapeutic treatment for AIH.

Adoptive transfer of autoimmune splenic dendritic cells to lupus-prone mice triggers a B lymphocyte humoral response

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by increased autoantibody production that leads to multiple tissue injuries. Dendritic cells (DCs) are important orchestrators of immune responses and key components in fine-tuning the balance between tolerance and immunity. However, their role in autoimmune disorders such as SLE remains uncertain. We analyzed the contribution of DCs in triggering SLE by adoptively transferring splenic DCs from aged autoimmune [NZB×NZW]F1 (BWF1) mice to young healthy BWF1 mice. We observed that the transfer of DCs from autoimmune mice to pre-autoimmune mice induced high autoantibody titers in the serum of recipient mice. Moreover, autoimmune DCs from aged BWF1 mice were crucial for the expansion and differentiation of plasmablasts and CD5+ B cells or B1-like cells in the peripheral blood, and spleen of recipient BWF1 mice, a phenomenon that is observed in autoimmune BWF1 mice. On the other hand, DCs from aged BWF1 mice participated in the expansion and differentiation of DCs and IFN-γ-producing T cells. These results reveal that DCs from autoimmune BWF1 mice exhibit functional and phenotypic characteristics that allow them to trigger B cell hyperactivation, as well as DC and T cell expansion and differentiation, thereby promoting an exacerbated humoral response in lupus-prone mice.

Protective and therapeutic role of 2-carba-cyclic phosphatidic acid in demyelinating disease

BackgroundMultiple sclerosis is a neuroinflammatory demyelinating and neurodegenerative disease of the central nervous system characterized by recurrent and progressive demyelination/remyelination cycles, neuroinflammation, oligodendrocyte loss, demyelination, and axonal degeneration. Cyclic phosphatidic acid (cPA) is a natural phospholipid mediator with a unique cyclic phosphate ring structure at the sn-2 and sn-3 positions of the glycerol backbone. We reported earlier that cPA elicits a neurotrophin-like action and protects hippocampal neurons from ischemia-induced delayed neuronal death. We designed, chemically synthesized, and metabolically stabilized derivatives of cPA: 2-carba-cPA (2ccPA), a synthesized compound in which one of the phosphate oxygen molecules is replaced with a methylene group at the sn-2 position. In the present study, we investigated whether 2ccPA exerts protective effects in oligodendrocytes and suppresses pathology in the two most common mouse models of multiple sclerosis.MethodsTo evaluate whether 2ccPA has potential beneficial effects on the pathology of multiple sclerosis, we investigated the effects of 2ccPA on oligodendrocyte cell death in vitro and administrated 2ccPA to mouse models of experimental autoimmune encephalomyelitis (EAE) and cuprizone-induced demyelination.ResultsWe demonstrated that 2ccPA suppressed the CoCl2-induced increase in the Bax/Bcl-2 protein expression ratio and phosphorylation levels of p38MAPK and JNK protein. 2ccPA treatment reduced cuprizone-induced demyelination, microglial activation, NLRP3 inflammasome, and motor dysfunction. Furthermore, 2ccPA treatment reduced autoreactive T cells and macrophages, spinal cord injury, and pathological scores in EAE, the autoimmune multiple sclerosis mouse model.ConclusionsWe demonstrated that 2ccPA protected oligodendrocytes via suppression of the mitochondrial apoptosis pathway. Also, we found beneficial effects of 2ccPA in the multiperiod of cuprizone-induced demyelination and the pathology of EAE. These data indicate that 2ccPA may be a promising compound for the development of new drugs to treat demyelinating disease and ameliorate the symptoms of multiple sclerosis.

Associations of Autoimmunity, Immunodeficiency, Lymphomagenesis, and Gut Microbiota in Mice with Knockins for a Pathogenic Autoantibody

A number of mouse strains transgenic for B-cell receptors specific for nucleic acids or other autoantigens have been generated to understand how autoreactive B cells are regulated in normal and autoimmune mice. Previous studies of nonautoimmune C57BL/6 mice heterozygous for both the IgH and IgL knockins of the polyreactive autoantibody, 564, produced high levels of autoantibodies in a largely Toll-like receptor 7-dependent manner. Herein, we describe studies of mice homozygous for the knockins that also expressed high levels of autoantibodies but, unlike the heterozygotes, exhibited a high incidence of mature B-cell lymphomas and enhanced susceptibility to bacterial infections. Microarray analyses and serological studies suggested that lymphomagenesis might be related to chronic B-cell activation promoted by IL-21. Strikingly, mice treated continuously with antibiotic-supplemented water did not develop lymphomas or abscesses and exhibited less autoimmunity. This mouse model may help us understand the reasons for enhanced susceptibility to lymphoma development exhibited by humans with a variety of autoimmune diseases, such as Sjögren syndrome, systemic lupus erythematosus, and highly active rheumatoid arthritis.

Breaching peripheral tolerance promotes the production of HIV-1-neutralizing antibodies.

A subset of characterized HIV-1 broadly neutralizing antibodies (bnAbs) are polyreactive with additional specificities for self-antigens and it has been proposed immunological tolerance may present a barrier to their participation in protective humoral immunity. We address this hypothesis by immunizing autoimmune-prone mice with HIV-1 Envelope (Env) and characterizing the primary antibody response for HIV-1 neutralization. We find autoimmune mice generate neutralizing antibody responses to tier 2 HIV-1 strains with alum treatment alone in the absence of Env. Importantly, experimentally breaching immunological tolerance in wild-type mice also leads to the production of tier 2 HIV-1-neutralizing antibodies, which increase in breadth and potency following Env immunization. In both genetically prone and experimentally induced mouse models of autoimmunity, increased serum levels of IgM anti-histone H2A autoantibodies significantly correlated with tier 2 HIV-1 neutralization, and anti-H2A antibody clones were found to neutralize HIV-1. These data demonstrate that breaching peripheral tolerance permits a cross-reactive HIV-1 autoantibody response able to neutralize HIV-1.

Netrin‐1 and multiple sclerosis: a new biomarker for neuroinflammation?

Netrin-1, an axon guidance protein, reduces serum levels of pro-inflammatory mediators and stabilizes the blood-brain barrier limiting the entrance of immune cells into the central nervous system. The aim was to investigate its presence in the experimental autoimmune encephalomyelitis (EAE) model and in multiple sclerosis (MS) patients with and without clinical activity. Netrin-1 levels were evaluated in EAE mouse tissues. Afterwards, serum netrin-1 was cross-sectionally quantified in 90 patients with different MS phenotypes and 30 control subjects. An additional group of 10 relapsing-remitting MS (RRMS) patients was longitudinally evaluated throughout a relapse (RRMSr) with an interval of 60 days. Tumour necrosis factor α (TNFα), a reference inflammatory cytokine, and netrin-1 were quantified by enzyme-linked immunosorbent assay. Experimental autoimmune encephalomyelitis mice showed significantly lower netrin-1 levels and higher TNFα amounts in sera, spinal cord and cerebella than healthy control mice. MS patients showed significantly lower serum netrin-1 levels than controls (511.62 ± 209.30 and 748.32 ± 103.24 pg/ml, respectively; P ≤ 0.005). The lowest protein levels were found in RRMSr, remaining significantly lower throughout the relapse. TNFα serum concentrations were higher in MS patients compared to controls, and negatively correlated with netrin-1 levels (r = -0.3734, P ≤ 0.0001). Netrin-1 decreased in EAE and in MS patients, mainly during relapse, suggesting an anti-inflammatory role of netrin-1. Further research should be performed in a larger cohort of patients to validate netrin-1 as a biomarker of MS inflammatory activity.

Chinese medicine Ginseng and Astragalus granules ameliorate autoimmune diabetes by upregulating both CD4+FoxP3+ and CD8+CD122+PD1+ regulatory T cells.

Type 1 diabetes mellitus (T1DM) is an autoimmune disease mainly mediated by effector T cells that are activated by autoantigen, thereby resulting in the destruction of pancreatic islets and deficiency of insulin. Cyclosporine is widely used as an immunosuppressant that suppresses autoimmunity in clinic. However, continuous treatments with conventional immunosuppressive drugs may cause severe side effects. Therefore it is important to seek alternative medicine. Chinese medicine Ginseng and Astragalus granule (GAG) was used to successfully treat type 2 diabetes mellitus in clinic in China. Here we found that GAG ameliorated T1DM in autoimmune NOD mice by increasing the level of insulin and reducing the level of blood glucose. Treatments with both GAG and CsA further decreased the blood glucose level. Moreover, GAG increased both CD4+FoxP3+ and CD8+CD122+PD-1+ Treg numbers in both spleens and lymph nodes of NOD mice. In particular, GAG could reverse a decline in CD4+FoxP3+ Tregs resulted from CsA treatments. The percentage of effector/memory CD8+ T cells (CD44highCD62Llow) was significantly reduced by GAG, especially in the presence of low-doses of CsA. Histopathology also showed that GAG attenuated cellular infiltration and lowered CD3+ T cell numbers around and in islets. Thus, we demonstrated that GAG ameliorated autoimmune T1DM by upregulating both CD4+FoxP3+ and CD8+CD122+PD-1+ Tregs while GAG synergized with CsA to further suppress autoimmunity and T1DM by reversing the decline in CD4+FoxP3+ Tregs resulted from CsA treatments. This study may have important clinical implications for the treatment of T1DM using traditional Chinese medicine.

IFN-β-induced reactive oxygen species and mitochondrial damage contribute to muscle impairment and inflammation maintenance in dermatomyositis

Dermatomyositis (DM) is an autoimmune disease associated with enhanced type I interferon (IFN) signalling in skeletal muscle, but the mechanisms underlying muscle dysfunction and inflammation perpetuation remain unknown. Transcriptomic analysis of early untreated DM muscles revealed that the main cluster of down-regulated genes was mitochondria-related. Histochemical, electron microscopy, and in situ oxygraphy analysis showed mitochondrial abnormalities, including increased reactive oxygen species (ROS) production and decreased respiration, which was correlated with low exercise capacities and a type I IFN signature. Moreover, IFN-β induced ROS production in human myotubes was found to contribute to mitochondrial malfunctions. Importantly, the ROS scavenger N-acetyl cysteine (NAC) prevented mitochondrial dysfunctions, type I IFN-stimulated transcript levels, inflammatory cell infiltrate, and muscle weakness in an experimental autoimmune myositis mouse model. Thus, these data highlight a central role of mitochondria and ROS in DM. Mitochondrial dysfunctions, mediated by IFN-β induced-ROS, contribute to poor exercise capacity. In addition, mitochondrial dysfunctions increase ROS production that drive type I IFN-inducible gene expression and muscle inflammation, and may thus self-sustain the disease. Given that current DM treatments only induce partial recovery and expose to serious adverse events (including muscular toxicity), protecting mitochondria from dysfunctions may open new therapeutic avenues for DM.

Clinical and Histopathological Amelioration of Experimental Autoimmune Encephalomyelitis by AAV Vectors Expressing a Soluble Interleukin-23 Receptor

The role of the T helper (Th)17 pathway has been clearly demonstrated in the onset and progression of autoimmune diseases, where interleukin (IL)-23 is a key molecule in maintaining the response mediated by Th17 cells. As a consequence, recent strategies based on blocking the interaction between IL-23 and its receptor (IL-23R), for example the anti-p19 antibody tildrakizumab, have been developed to regulate the Th17 pathway from the initial stages of the disease. Here, a soluble (s)IL-23R cDNA was cloned in expression plasmids and viral vectors. The clinical efficacy of sIL-23R was evaluated in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis mice intravenously injected with a single dose of adeno-associated virus AAV8–sIL-23R vectors. Cytokine secretion was determined by multiplex assay, while histopathological analysis of the central nervous system was performed to study demyelination, inflammatory infiltration, and microglia and astroglia activation. We observed that administration of adeno-associated vector 8 encoding sIL-23R was associated with a significant disease improvement, including delay in the onset of the clinical signs; slower progress of the disease; interference with IL-23-mediated signal transducer and activator of transcription response by inhibiting of signal transducer and activator of transcription 3 phosphorylation; reduced demyelination and infiltration in the central nervous system; and lower astrocyte and microglia activation. Our results suggest that the use of vectors carrying sIL-23R to block the IL-23/IL-23R interaction may be a new therapeutic strategy for the treatment of multiple sclerosis.

Inactivation of the KSRP gene modifies collagen antibody induced arthritis

The KH type splicing regulatory protein (KSRP) is a nucleic acid binding protein, which negatively regulates the stability and/or translatability of many mRNA species encoding immune-relevant proteins. As KSRP is expressed in immune cells including T and B cells, neutrophils, macrophages and dendritic cells, we wanted to analyze its importance for the development of autoimmune diseases.We chose collagen antibody-induced arthritis (CAIA) as an appropriate autoimmune disease mouse model in which neutrophils and macrophages constitute the main effector cell populations. We compared arthritis induction in wild type (WT) and KSRP−/− mice and paws were taken for histological sections and qPCR analysis. Furthermore, we determined the frequencies of spleen immune cells by flow cytometry. Cytokine levels in spleen cell supernatants were determined by cytometric bead array analyses (CBA).After CAIA induction we unexpectedly observed in WT animals much stronger swelling of the paws than in KSRP−/− mice. In accordance, histological staining of paw sections of KSRP−/− animals revealed much lower frequencies of infiltrating immune cells in the joints compared to WT animals. Furthermore, CAIA-treatment resulted in reduced expression of several inflammatory factors (like CXCL-1, iNOS, TNF-α and S100A8) as well as immune cell marker genes (e.g. LFA-1, CD68, Ly6G) in the joints of KSRP−/− mice.Spleen cells of KSRP−/− mice showed lower frequencies of myeloid cells. On cytokine level IFN-γ production was increased in spleen cells of KSRP−/− mice compared to WT samples.These data surprisingly suggest that the absence of KSRP protects against the induction of inflammatory arthritis.

Multiple extrinsic signals are required for the enrichment of dual-reactive B cells in autoimmune mice

Abstract Dual-reactive B cells expressing two different immunoglobulin heavy or light chains are present in both healthy humans and mice. However, a subset of systemic lupus erythematosus patients are enriched for dual-reactive B cells. To study dual-reactive B cells in an autoimmune background we used congenic mice bearing a gene targeted human Cκ allele which allowed for the identification and tracking of dual-κ B cells within a polyclonal B cell repertoire. We previously showed that dual-κ B cells positively correlated with disease progression and were enriched in the plasmablast and memory B cell compartments of lupus-prone MRL/lpr mice. Recent studies showed that T cells play a major role in the enrichment of dual-κ B cells, as MRL/lpr mice deficient in the IL-21 receptor had a significant reduction in total dual-κ B cells. In addition, IL-21 signaling was required for the maximal differentiation of dual-κ B cells into plasmablasts and was essential for the development of dual-κ memory B cells. Dual-κ B cells were shown to significantly upregulate antigen presenting molecules such as MHCII, CD80, and CD86. While dual-κ B cells are enriched for polyreactive B cells, immunization with a T-dependent antigen elicited a similar response between single and dual-κ B cells. However, the addition of TLR9 stimulation amplified the antigen-specific response exclusively in dual-k B cells. Furthermore, in vivo EdU incorporation studies showed that dual-k B cells have a higher proliferation rate than single-κ B cells in the absence of exogenous stimuli. Overall, elucidating the diverse molecular pathways promoting the enrichment of dual-reactive B cells in autoimmunity will allow for the selective targeting of these cells in autoimmune patients.

Inhibition of BAFF binding to its receptor, BR3, by low molecular weight compounds results in suppression of activation of monocytes and B cells in vitro and in vivo model of autoimmune diseases.

Abstract Background and Purpose We have demonstrated that the elevated expression of BAFF receptor (BR3) on monocytes is involved in the pathogenesis of primary Sjogren’s syndrome and BR3 is a therapeutic target to treat pSS. In our previous study, we have discovered two pyrrolopryimidine derivatives, BIK12 and BIK13, which inhibit binding of BAFF to BR3, using our original high-throughput screening system. In this study, we investigated inhibitory effects of these compounds on BAFF signaling pathways in monocytes and B cells in vitro. In addition, we analyzed the effects of the compounds on serum level of an anti-dsDNA antibody in autoimmune disease model mice. Methods Peripheral monocytes were stimulated with soluble BAFF (sBAFF) and cultured in vitro with or without peripheral B cells in the presence of BIK-12 or BIK-13. IL-6 production by monocytes and IgG production by B cells were measured by ELISA. MRL/lpr and NZBWF1 mice were treated with these compounds three times a week at a dose of 1.0 or 0.2 mg/kg for 6 months. The titer of an anti-ds DNA antibody was measured by ELISA. Results and Discussion sBAFF-induced IL-6 production by peripheral monocytes and IgG production by B cells co-cultured with sBAFF-stimulated peripheral monocytes were significantly suppressed by BIK12 and BIK13 in a dose dependent manner. Interestingly, the titer of an anti-dsDNA antibody in both MRL/lpr and NZBWF1 mice received BIK13 was significantly lower than control mice after 16 weeks’ treatment, indicating that the compound was also efficacious in vivo.

Model specific role of B cell-intrinsic IFNαR signaling in promoting spontaneous germinal centers and autoimmunity

Abstract In systemic lupus erythematosus (SLE), germinal centers can form spontaneously (Spt-GCs) in the absence of foreign antigen and drive high-affinity autoantibody (autoAb) production. Overexpression of Type 1 interferon (IFNα) accelerates autoimmunity and alters B cell selection in certain SLE mouse models, but in another model, B cell intrinsic IFNαR signaling is shown to be dispensable for autoimmune GC response and autoimmunity. Using B6.Sle1b autoimmune mice derived from the NZM2410 SLE mouse model, we show here that IFNαR signaling is not required for Spt-GC formation but is important for elevated Spt-GC response and autoreactive B cell selection into AFCs. IFNα receptor1 deficient B6.Sle1b mice (B6.Sle1b.IFNαR1−/−) have lower Spt-GC and Tfh responses, reduced CD86 expression on B cells and diminished autoAb titers as compared to B6.Sle1b mice. We also observe lower Ca2+ flux in B cells deficient in IFNα receptor compared to control B cells upon BCR cross-linking in vitro. Consistently, we find that B cell-intrinsic IFNαR signaling promotes differentiation of Sle1b-expressing DNA-reactive B cells into GC B cells. RNA sequencing studies show that B6.Sle1b and B6.Sle1b.IFNαR1−/− GC B cells have distinct transcriptional profiles that are associated with proliferation, cell survival, and antigen presentation pathways. Compared to B6.Sle1b control, B6.Sle1b.IFNαR1−/− B cells fail to form stable conjugates with T cells in vitro and GC B cells express reduced levels of ICOS-ligand in vivo, suggesting B6.Sle1b.IFNαR1−/− B cells may not acquire adequate T cell help. These data reveal novel information about the model specific role of B cell-intrinsic IFNαR signaling in the regulation of Spt-GCs and autoimmunity.

B Cell Tolerance to Deiminated Histones in BALB/c, C57BL/6, and Autoimmune-Prone Mouse Strains.

Deimination, a posttranslational modification of arginine to citrulline carried out by peptidylarginine deiminases, may compromise tolerance of self-antigens. Patients with connective tissue autoimmunity, particularly rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), or Felty’s syndrome, present with autoantibodies to deiminated histones (dH), which thus form a category of antibodies to citrullinated protein antigens (ACPA). In general, ACPA are a sensitive diagnostic for RA and may form in response to the release of nuclear chromatin (DNA plus dH) from granulocytes, usually referred to as neutrophil extracellular traps. The aim of this study was to examine spontaneously autoimmune mice for autoantibodies and T cell responses to dH. We compared IgG binding to deiminated and non-deiminated histones (nH) by ELISA and Western blotting in spontaneously autoimmune strains of (NZB × NZW) F1 and NZM2410 together with their derivative congenic strains, C57BL/6.Sle1 and C57BL/6.Sle1.Sle3, which display profound autoreactivity against nuclear self-antigens. The splenocyte proliferation against the two antigens was determined in the spontaneously autoimmune (NZB × NZW) F1 strain from which other autoimmune strains used in the study were derived. Immunizations with dH and nH were attempted in BALB/c mice to assess their splenocyte response. Splenocytes from BALB/c mice and from autoimmune mice at the time of conversion to autoimmunity proliferated strongly in response to dH, yet serum IgG from autoimmune (NZB × NZW) F1, NZM2410, and C57BL/6.Sle1.Sle3 mice displayed a remarkable bias against binding to dH. At the time of seroconversion, the antibodies already exhibited preference for nH, and only nH were recovered from circulating immune complexes. Analysis of histone deimination showed constitutive deimination in thymic extracts from C57BL/6 and C57BL/6.Sle1.Sle2.Sle3 triply congenic mice and in spleens of autoimmune triply congenic mice. Our study demonstrates that tolerance mechanisms against dH are intact in BALB/c and C57BL/6 mice and continue to be effective in mice with overt autoimmunity to nH. We conclude that, in contrast to human RA and SLE patients, where we frequently observe autoantibodies against dH, autoimmune mice maintain strong tolerance mechanisms to prevent the development of autoantibodies to dH.

The Function of FK506-Binding Protein 13 in Protein Quality Control Protects Plasma Cells from Endoplasmic Reticulum Stress-Associated Apoptosis.

Plasma cells (PCs) are exposed to intense endoplasmic reticulum (ER) stress imposed by enormous rates of immunoglobulin (Ig) synthesis and secretion. Therefore, protein homeostasis is crucial for the survival of PCs, but its molecular mechanism remains largely unknown. Here, we found marked overexpression of FK506-binding protein 13 (FKBP13) in long-lived PCs from autoimmune mice and investigated its function using a plasmacytoma cell line secreting IgA. FKBP13 expression was induced largely in the lumen of ER in response to treatment with an ER stressor tunicamycin or overexpression of an adaptive unfolded protein response (UPR) protein X-box binding protein 1 (XBP1). Silencing of FKBP13 expression led to induction of molecules involved in the terminal UPR and ER stress-associated apoptosis. FKBP13 interacted with Ig, facilitated its ubiquitination, and lowered the extent of ER stress. FKBP13 overexpression caused a significant reduction in secreted IgA in plasmacytoma cells, and FKBP13 knockdown exerted an opposite effect. Rapamycin interfered with the interaction between FKBP13 and IgA and enhanced the amount of secreted IgA. Importantly, the level of FKBP13 was inversely correlated with the amount of secreted antibody in long-lived PCs from autoimmune mice. These results suggest that FKBP13 is a marker of long-lived PCs and a component of XBP1-dependent ER protein homeostasis. FKBP13 is likely to act as a molecular chaperone that delivers misfolded ER clients, including Ig, to ER-associated degradation, so reducing proteotoxic stress on the PC. Our data reveal a novel cytoprotective role for FKBP13 in long-lived PCs occurring at the expense of antibody production.