Jessica Doerner

TNF-like weak inducer of apoptosis promotes blood brain barrier disruption and increases neuronal cell death in MRL/lpr mice

Neuropsychiatric disease is one of the most common manifestations of human systemic lupus erythematosus, but the mechanisms remain poorly understood. In human brain microvascular endothelial cells in vitro, TNF-like weak inducer of apoptosis (TWEAK) decreases tight junction ZO-1 expression and increases the permeability of monolayer cell cultures. Furthermore, knockout (KO) of the TWEAK receptor, Fn14, in the MRL/lpr lupus mouse strain markedly attenuates neuropsychiatric disease, as demonstrated by significant reductions in depressive-like behavior and improved cognitive function. The purpose of the present study was to determine the mechanisms by which TWEAK signaling is instrumental in the pathogenesis of neuropsychiatric lupus (NPSLE). Evaluating brain sections of MRL/lpr Fn14WT and Fn14KO mice, we found that Fn14KO mice displayed significantly decreased cellular infiltrates in the choroid plexus. To evaluate the integrity of the blood brain barrier (BBB) in MRL/lpr mice, Western blot for fibronectin, qPCR for iNOS, and immunohistochemical staining for VCAM-1/ICAM-1 were performed. We found preserved BBB permeability in MRL/lpr Fn14KO mice, attributable to reduced brain expression of VCAM-1/ICAM-1 and iNOS. Additionally, administration of Fc-TWEAK intravenously directly increased the leakage of a tracer (dextran-FITC) into brain tissue. Furthermore, MRL/lpr Fn14KO mice displayed reduced antibody (IgG) and complement (C3, C6, and C4a) deposition in the brain. Finally, we found that MRL/lpr Fn14KO mice manifested reduced neuron degeneration and hippocampal gliosis. Our studies indicate that TWEAK/Fn14 interactions play an important role in the pathogenesis of NPSLE by increasing the accumulation of inflammatory cells in the choroid plexus, disrupting BBB integrity, and increasing neuronal damage, suggesting a novel target for therapy in this disease.

TWEAK/Fn14 Signaling Involvement in the Pathogenesis of Cutaneous Disease in the MRL/lpr Model of Spontaneous Lupus

TNF-like weak inducer of apoptosis (TWEAK, TNFSF12) and its sole receptor Fn14, belonging to the TNF ligand and receptor superfamilies respectively, are involved in cell survival and cytokine production. The role of TWEAK/Fn14 interactions in the pathogenesis of cutaneous lupus has not been explored. TWEAK treatment of murine PAM212 keratinocytes stimulated the secretion of RANTES via Fn14, and promoted apoptosis. Parthenolide, but not wortmanin or the MAPK inhibitor PD98059, significantly decreased production of RANTES, indicating that this effect of TWEAK is mediated via NF-κB signaling. Ultraviolet-B irradiation significantly upregulated the expression of Fn14 on keratinocytes in vitro and in vivo, and increased RANTES production. MRL/lpr Fn14 knockout lupus mice were compared with MRL/lpr Fn14 wild-type mice to evaluate for any possible differences in the severity of cutaneous lesions and the presence of infiltrating immune cells. MRL/lpr Fn14 knockout mice had markedly attenuated cutaneous disease as compared to their Fn14 wild-type littermates, as evidenced by the well maintained architecture of the skin and significantly decreased skin infiltration of T cells and macrophages. Our data strongly implicate TWEAK/Fn14 signaling in the pathogenesis of the cutaneous manifestations in the MRL/lpr model of spontaneous lupus, and suggest a possible target for therapeutic intervention.

T cells targeting a neuronal paraneoplastic antigen mediate tumor rejection and trigger CNS autoimmunity with humoral activation.

Paraneoplastic neurologic diseases (PND) involving immune responses directed toward intracellular antigens are poorly understood. Here, we examine immunity to the PND antigen Nova2, which is expressed exclusively in central nervous system (CNS) neurons. We hypothesized that ectopic expression of neuronal antigen in the periphery could incite PND. In our C57BL/6 mouse model, CNS antigen expression limits antigen‐specific CD4+ and CD8+ T‐cell expansion. Chimera experiments demonstrate that this tolerance is mediated by antigen expression in nonhematopoietic cells. CNS antigen expression does not limit tumor rejection by adoptively transferred transgenic T cells but does limit the generation of a memory population that can be expanded upon secondary challenge in vivo. Despite mediating cancer rejection, adoptively transferred transgenic T cells do not lead to paraneoplastic neuronal targeting. Preliminary experiments suggest an additional requirement for humoral activation to induce CNS autoimmunity. This work provides evidence that the requirements for cancer immunity and neuronal autoimmunity are uncoupled. Since humoral immunity was not required for tumor rejection, B‐cell targeting therapy, such as rituximab, may be a rational treatment option for PND that does not hamper tumor immunity.

CSF-1R inhibition attenuates renal and neuropsychiatric disease in murine lupus.

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease that can affect multiple end organs. Kidney and brain are two of the organs most commonly involved in SLE. Past studies have suggested the importance of macrophages in the pathogenesis of lupus nephritis (LN). Furthermore, as the immune effectors of the brain, microglia have been implicated in pathways leading to neuropsychiatric SLE (NPSLE). We depleted macrophages and microglia using GW2580, a small colony stimulating factor-1 receptor (CSF-1R) kinase inhibitor, in MRL-lpr/lpr (MRL/lpr) mice, a classic murine lupus model that displays features of both LN and NPSLE. Treatment was initiated before the onset of disease, and mice were followed for the development of LN and neurobehavioral dysfunction throughout the study. Treatment with GW2580 significantly ameliorated kidney disease, as evidenced by decreased proteinuria, BUN, and improved renal histopathology, despite equivalent levels of IgG and C3 deposition in the kidneys of treated and control mice. We were able to confirm macrophage depletion within the kidney via IBA-1 staining. Furthermore, we observed specific improvement in the depression-like behavioral deficit of MRL/lpr mice with GW2580 treatment. Circulating antibody and autoantibody levels were, however, not affected. These results provide additional support for the role of macrophages as a potentially valuable therapeutic target in SLE. Inhibiting CSF-1 receptor signaling would be more targeted than current immunosuppressive therapies, and may hold promise for the treatment of renal and neuropsychiatric end organ disease manifestations.

B cell and/or autoantibody deficiency do not prevent neuropsychiatric disease in murine systemic lupus erythematosus

BackgroundNeuropsychiatric lupus (NPSLE) can be one of the earliest clinical manifestations in human lupus. However, its mechanisms are not fully understood. In lupus, a compromised blood-brain barrier may allow for the passage of circulating autoantibodies into the brain, where they can induce neuropsychiatric abnormalities including depression-like behavior and cognitive abnormalities. The purpose of this study was to determine the role of B cells and/or autoantibodies in the pathogenesis of murine NPSLE.MethodsWe evaluated neuropsychiatric manifestations, brain pathology, and cytokine expression in constitutively (JhD/MRL/lpr) and conditionally (hCD20-DTA/MRL/lpr, inducible by tamoxifen) B cell-depleted mice as compared to MRL/lpr lupus mice.ResultsWe found that autoantibody levels were negligible (JhD/MRL/lpr) or significantly reduced (hCD20-DTA/MRL/lpr) in the serum and cerebrospinal fluid, respectively. Nevertheless, both JhD/MRL/lpr and hCD20-DTA/MRL/lpr mice showed profound depression-like behavior, which was no different from MRL/lpr mice. Cognitive deficits were also observed in both JhD/MRL/lpr and hCD20-DTA/MRL/lpr mice, similar to those exhibited by MRL/lpr mice. Furthermore, although some differences were dependent on the timing of depletion, central features of NPSLE in the MRL/lpr strain including increased blood-brain barrier permeability, brain cell apoptosis, and upregulated cytokine expression persisted in B cell-deficient and B cell-depleted mice.ConclusionsOur study surprisingly found that B cells and/or autoantibodies are not required for key features of neuropsychiatric disease in murine NPSLE.

Intracerebroventricular administration of TNF-like weak inducer of apoptosis induces depression-like behavior and cognitive dysfunction in non-autoimmune mice.

Fn14, the sole known signaling receptor for the TNF family member TWEAK, is inducibly expressed in the central nervous system (CNS) in endothelial cells, astrocytes, microglia, and neurons. There is increasing recognition of the importance of the TWEAK/Fn14 pathway in autoimmune neurologic conditions, including experimental autoimmune encephalomyelitis and neuropsychiatric lupus. Previously, we had found that Fn14 knockout lupus-prone MRL/lpr mice display significantly attenuated neuropsychiatric manifestations. To investigate whether this improvement in disease is secondary to inhibition of TWEAK/Fn14 signaling within the CNS or the periphery, and determine whether TWEAK-mediated neuropsychiatric effects are strain dependent, we performed intracerebroventricular (ICV) injection of Fc-TWEAK or an isotype matched control protein to C57Bl6/J non-autoimmune mice. We found that Fc-TWEAK injected C57Bl6/J mice developed significant depression-like behavior and cognitive dysfunction. Inflammatory mediators associated with lupus brain disease, including CCL2, C3, and iNOS, were significantly elevated in the brains of Fc-TWEAK treated mice. Furthermore, Fc-TWEAK directly increased blood brain barrier (BBB) permeability, as demonstrated by increased IgG deposition in the brain and reduced aquaporin-4 expression. Finally, Fc-TWEAK increased apoptotic cell death in the cortex and hippocampus. In conclusion, TWEAK can contribute to lupus-associated neurobehavioral deficits including depression and cognitive dysfunction by acting within the CNS to enhance production of inflammatory mediators, promote disruption of the BBB, and induce apoptosis in resident brain cells. Our study provides further support that the TWEAK/Fn14 signaling pathway may be a potential therapeutic target for inflammatory diseases involving the CNS.

B7x/B7-H4 modulates the adaptive immune response and ameliorates renal injury in antibody-mediated nephritis

Kidney disease is one of the leading causes of death in patients with lupus and other autoimmune diseases affecting the kidney, and is associated with deposition of antibodies as well as infiltration of T lymphocytes and macrophages, which are responsible for initiation and/or exacerbation of inflammation and tissue injury. Current treatment options have relatively limited efficacy; therefore, novel targets need to be explored. The co‐inhibitory molecule, B7x, a new member of the B7 family expressed predominantly by non‐lymphoid tissues, has been shown to inhibit the proliferation, activation and functional responses of CD4 and CD8 T cells. In this study, we found that B7x was expressed by intrinsic renal cells, and was up‐regulated upon stimulation with inflammatory triggers. After passive administration of antibodies against glomerular antigens, B7x−/− mice developed severe renal injury accompanied by a robust adaptive immune response and kidney up‐regulation of inflammatory mediators, as well as local infiltration of T cells and macrophages. Furthermore, macrophages in the spleen of B7x−/− mice were polarized to an inflammatory phenotype. Finally, treatment with B7x‐immunoglobulin (Ig) in this nephritis model decreased kidney damage and reduced local inflammation. We propose that B7x can modulate kidney damage in autoimmune diseases including lupus nephritis and anti‐glomerular basement membrane disease. Thus, B7x mimetics may be a novel therapeutic option for treatment of immune‐mediated kidney disease.

Highly selective inhibition of Bruton’s tyrosine kinase attenuates skin and brain disease in murine lupus

BackgroundSystemic lupus erythematosus (SLE) is a systemic autoimmune disease that affects different end organs, including skin and brain. We and others have previously shown the importance of macrophages in the pathogenesis of cutaneous and neuropsychiatric lupus. Additionally, autoantibodies produced by autoreactive B cells are thought to play a role in both the skin and central nervous system pathologies associated with SLE.MethodsWe used a novel inhibitor of Bruton’s tyrosine kinase (BTK), BI-BTK-1, to target both macrophage and B cell function in the MRL-lpr/lpr murine model of SLE, and examined the effect of treatment on skin and brain disease.ResultsWe found that treatment with BI-BTK-1 significantly attenuated the lupus associated cutaneous and neuropsychiatric disease phenotypes in MRL/lpr mice. Specifically, BI-BTK-1 treated mice had fewer macroscopic and microscopic skin lesions, reduced cutaneous cellular infiltration, and diminished inflammatory cytokine expression compared to control mice. BTK inhibition also significantly improved cognitive function, and decreased accumulation of T cells, B cells, and macrophages within the central nervous system, specifically the choroid plexus.ConclusionsDirected therapies may improve the response rate in lupus-driven target organ involvement, and decrease the dangerous side effects associated with global immunosuppression. Overall, our results suggest that inhibition of BTK may be a promising therapeutic option for cutaneous and neuropsychiatric disease associated with SLE.

TWEAK is not elevated in patients with newly diagnosed inflammatory bowel disease

Abstract Objective: Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) may be involved in the pathogenesis of inflammatory bowel disease. The aim was to investigate if TWEAK may reflect disease activity in inflammatory bowel disease. Materials and methods: In this cohort study, 139 consecutive patients with newly diagnosed and previously untreated inflammatory bowel disease – 95 with ulcerative colitis (UC) and 44 with Crohn’s disease (CD) – underwent colonoscopy. Disease activity was assessed by the Mayo score and the Mayo endoscopic score (MES) for UC, or the Simple Endoscopic Score (SES) for CD. Serum C-reactive protein (CRP) and fecal calprotectin were measured in IBD patients, as were plasma TWEAK levels in patients and 85 healthy subjects. Associations between TWEAK levels and disease activity markers were explored. Results: In the total IBD group, the median (interquartile range) TWEAK level was 430 pg/ml (109–6570), in UC 502 pg/ml (109–4547) and in CD patients 352 pg/ml (101–9179), respectively. Healthy subjects had a median (IQR) TWEAK of 307 pg/ml (63–3492). There were no significant differences in TWEAK levels between the total IBD group and healthy control subjects, nor between UC and CD, or between UC/CD and healthy subjects. Furthermore, we found no significant associations between Mayo scores, MES-UC, SES-CD, CRP, and fecal calprotectin with plasma TWEAK levels. Conclusions: Plasma TWEAK levels do not reflect disease activity or the grade of inflammation in patients with newly diagnosed inflammatory bowel disease. NCT01551563.

OP0164 Blockade of Immune Complex-Mediated Glomerulonephritis by Highly Selective Inhibition of Bruton's Tyrosine Kinase

Background Lupus nephritis (LN) causes significant morbidity and mortality in SLE patients. Autoantibody-containing immune complexes (ICs) can activate complement cascades and Fc receptors on resident and infiltrating cells within the kidney, thus promoting inflammation. Brutons tyrosine kinase (BTK) is a tyrosine kinase important for B cell development, Fc receptor signaling, and macrophage polarization. Objectives To assess the role of BTK in the pathogenesis of nephritis in an inducible model of LN, and to evaluate the therapeutic potential of BTK inhibition. Methods A novel, highly selective, and potent (mouse whole blood CD69 IC50=13±2 nM) BTK inhibitor, BI-BTK-1 (Boehringer Ingelheim), was tested in female 129 sv/J mice (10 weeks of age) injected with nephrotoxic serum (NTS), an experimental model which closely mimics LN. Mice pre-immunized with rabbit IgG (Day 0) were administered NTS containing rabbit anti-mouse glomerular antibodies (Day 5), inducing a severe IC-mediated crescentic glomerulonephritis. Mice that did not receive the NTS transfer were used as healthy controls. Mice were treated once daily with vehicle alone or BI-BTK-1 (0.3–10 mg/kg, n=16/group), before the transfer of NTS as a prophylactic assessment, or after the transfer of NTS as a therapeutic assessment. Results NTS-challenged mice treated with BI-BTK-1 exhibited a statistically significant, dose responsive protection from kidney disease. Compared to vehicle treated mice, NTS-challenged mice treated with 10 mg/kg BI-BTK-1 had significantly less proteinuria (1220 mg/dl vs 10 mg/dl, respectively, p<0.0005), serum creatinine (0.74 mg/dl vs 0.48 mg/dl, respectively, p<0.03), and BUN (82 mg/dl vs 25 mg/dl, respectively, p<0.03) at the end of study (Day 11). Histology assessment confirmed marked renal protection in the BI-BTK-1 treatment groups. BI-BTK-1 treatment resulted in decreased recruitment of inflammatory monocytes from the splenic reservoir, and a decrease in infiltrating IBA-1+ cells as well as C3 deposition within the kidney. RNA-seq revealed that more than 500 genes were modulated by BTK treatment, with further analysis highlighting many of the modulated pathways being related to inflammation and glomerular injury in LN. Serum profiling and selective kidney gene expression analyses by Q-PCR revealed that BTK inhibition was associated with a significant decrease in the levels of key LN-relevant inflammatory cytokines and chemokines. Importantly, delayed treatment with 3 mg/kg of BI-BTK-1in mice with established proteinuria was able to reverse proteinuria and improve renal histopathology. Conclusions Our results further confirm the important role for BTK activation in the pathogenesis of immune complex-mediated nephritis, and highlight BTK as a valuable therapeutic target. Taken together with previously published studies and our preliminary results in spontaneous lupus models, these findings further strengthen the rationale for selective BTK inhibition as a promising approach to the treatment of LN. Disclosure of Interest S. Chalmers: None declared, J. Doerner: None declared, T. Bosanac Employee of: Boehringer Ingelheim, S. Khalil Employee of: Boehringer Ingelheim, D. Smith Employee of: Boehringer Ingelheim, C. Harcken Employee of: Boehringer Ingelheim, J. Dimock Employee of: Boehringer Ingelheim, E. Der: None declared, L. Herlitz: None declared, D. Webb Employee of: Boehringer Ingelheim, E. Seccareccia Employee of: Boehringer Ingelheim, D. Feng Employee of: Boehringer Ingelheim, J. Fine Employee of: Boehringer Ingelheim, M. Ramanujam Employee of: Boehringer Ingelheim, E. Klein Employee of: Boehringer Ingelheim, C. Putterman Grant/research support from: Boehringer Ingelheim