Susann Schönefeldt

Antiapoptotic Mcl-1 is critical for the survival and niche-filling capacity of Foxp3 + regulatory T cells

Foxp3+ regulatory T (Treg) cells are a crucial immunosuppressive population of CD4+ T cells, yet the homeostatic processes and survival programs that maintain the Treg cell pool are poorly understood. Here we report that peripheral Treg cells markedly alter their proliferative and apoptotic rates to rapidly restore numerical deficit through an interleukin 2–dependent and costimulation-dependent process. By contrast, excess Treg cells are removed by attrition, dependent on the Bim-initiated Bak- and Bax-dependent intrinsic apoptotic pathway. The antiapoptotic proteins Bcl-xL and Bcl-2 were dispensable for survival of Treg cells, whereas Mcl-1 was critical for survival of Treg cells, and the loss of this antiapoptotic protein caused fatal autoimmunity. Together, these data define the active processes by which Treg cells maintain homeostasis via critical survival pathways.

Rapamycin increases survival in ALS mice lacking mature lymphocytes

BackgroundAmyotrophic Lateral Sclerosis (ALS) is a devastating progressive neurodegenerative disease. Disease pathophysiology is complex and not yet fully understood, but is proposed to include the accumulation of misfolded proteins, as aggregates are present in spinal cords from ALS patients and in ALS model organisms. Increasing autophagy is hypothesized to be protective in ALS as it removes these aggregates. Rapamycin is frequently used to increase autophagy, but is also a potent immune suppressor. To properly assess the role of rapamycin-induced autophagy, the immune suppressive role of rapamycin should be negated.FindingsAutophagy is increased in the spinal cord of ALS mice. Dietary supplementation of rapamycin increases autophagy, but does not increase the survival of mutant SOD1 mice. To measure the effect of rapamycin in ALS independent of immunosuppression, we tested the effect of rapamycin in ALS mice deficient of mature lymphocytes. Our results show that rapamycin moderately increases the survival of these ALS mice deficient of mature lymphocytes.ConclusionsRapamycin could suppress protective immune responses while enhancing protective autophagy reactions during the ALS disease process. While these opposing effects can cancel each other out, the use of immunodeficient mice allows segregation of effects. Our results indicate that maximal therapeutic benefit may be achieved through the use of compounds that enhance autophagy without causing immune suppression.

The microRNA-29 Family Dictates the Balance Between Homeostatic and Pathological Glucose Handling in Diabetes and Obesity

The microRNA-29 (miR-29) family is among the most abundantly expressed microRNA in the pancreas and liver. Here, we investigated the function of miR-29 in glucose regulation using miR-29a/b-1 (miR-29a)-deficient mice and newly generated miR-29b-2/c (miR-29c)-deficient mice. We observed multiple independent functions of the miR-29 family, which can be segregated into a hierarchical physiologic regulation of glucose handling. miR-29a, and not miR-29c, was observed to be a positive regulator of insulin secretion in vivo, with dysregulation of the exocytotic machinery sensitizing β-cells to overt diabetes after unfolded protein stress. By contrast, in the liver both miR-29a and miR-29c were important negative regulators of insulin signaling via phosphatidylinositol 3-kinase regulation. Global or hepatic insufficiency of miR-29 potently inhibited obesity and prevented the onset of diet-induced insulin resistance. These results demonstrate strong regulatory functions for the miR-29 family in obesity and diabetes, culminating in a hierarchical and dose-dependent effect on premature lethality.

Loss of T cell microRNA provides systemic protection against autoimmune pathology in mice.

With an increasing number of studies demonstrating alterations in T cell microRNA expression during autoimmune disease, modulation of the T cell microRNA network is considered a potential therapeutic strategy. Due to the complex and often opposing interactions of individual microRNA, prioritization of therapeutic targets first requires dissecting the dominant effects of the T cell microRNA network. Initial results utilizing a unidirectional screen suggested that the tolerogenic functions were dominant, with spontaneous colitis resulting from T cell-specific excision of Dicer. Here we performed a bidirectional screen for microRNA function by removing Dicer from the T cells of both wildtype mice and Transforming Growth Factor β (TGFβ) receptor-deficient mice. This allowed the impact of microRNA loss on T cell activation, effector T cell differentiation and autoimmune pathology to be systematically assessed. This bidirectional screen revealed a dominant immunogenic function for T cell microRNA, with potent suppression of T cell activation, IFNγ production and autoimmune pathology in all targeted organs except the colon, where Dicer-dependent microRNA demonstrated a dominant tolerogenic function. These results reverse the original conclusions of microRNA function in T cells by revealing a systemic pro-autoimmune function.

Type 1 Diabetes in NOD Mice Unaffected by Mast Cell Deficiency

Mast cells have been invoked as important players in immune responses associated with autoimmune diseases. Based on in vitro studies, or in vivo through the use of Kit mutant mice, mast cells have been suggested to play immunological roles in direct antigen presentation to both CD4+ and CD8+ T cells, in the regulation of T-cell and dendritic cell migration to lymph nodes, and in Th1 versus Th2 polarization, all of which could significantly impact the immune response against self-antigens in autoimmune disease, including type 1 diabetes (T1D). Until now, the role of mast cells in the onset and incidence of T1D has only been indirectly tested through the use of low-specificity mast cell inhibitors and activators, and published studies reported contrasting results. Our three laboratories have generated independently two strains of mast cell–deficient nonobese diabetic (NOD) mice, NOD.Cpa3Cre/+ (Heidelberg) and NOD.KitW-sh/W-sh (Leuven and Boston), to address the effects of mast cell deficiency on the development of T1D in the NOD strain. Our collective data demonstrate that both incidence and progression of T1D in NOD mice are independent of mast cells. Moreover, analysis of pancreatic lymph node cells indicated that lack of mast cells has no discernible effect on the autoimmune response, which involves both innate and adaptive immune components. Our results demonstrate that mast cells are not involved in T1D in the NOD strain, making their role in this process nonessential and excluding them as potential therapeutic targets.

Beta-2 microglobulin is important for disease progression in a murine model for amyotrophic lateral sclerosis

Beta-2 microglobulin (β2m) is an essential component of the major histocompatibility complex (MHC) class I proteins and in the nervous system β2m is predominantly expressed in motor neurons. As β2m can promote nerve regeneration, we investigated its potential role in amyotrophic lateral sclerosis (ALS) by investigating its expression level as well as the effect of genetically removing β2m on the disease process in mutant superoxide dismutase 1 (SOD1G93A) mice, a model of ALS. We observed a strong upregulation of β2m in motor neurons during the disease process and ubiquitous removal of β2m dramatically shortens the disease duration indicating that β2m plays an essential and positive role during the disease process. We hypothesize that β2m contributes to plasticity that is essential for muscle reinnervation. Absence of this plasticity will lead to faster muscle denervation and counteracting this process could be a relevant therapeutic target.

C-kit is important for SOD1G93A mouse survival independent of mast cells

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease leading to progressive and lethal paralysis. The disease process is multi-factorial and is characterized by selective motor neuron degeneration. Previous work demonstrated that the local concentration of various growth factors can influence motor neuron survival and disease progression. A potential role for c-kit, a growth factor receptor present in the spinal cord, in ALS is unknown. To dissect the role of c-kit in ALS we interbred SOD1(G93A) mice with kit(w-sh/w-sh) mice, which have a 70% decrease in c-kit expression in the spinal cord. kit(w-sh/w-sh) SOD1(G93A) mice have a reduced survival compared to SOD1(G93A) mice, while the amount of motor neurons at end stage is similar. By means of grip strength and nerve conductance analysis we show that kit(w-sh/w-sh) mice have diminished strength and slightly impaired compound muscle action potential latency, although the number of neurons is similar across genotypes. Decreasing kit gene expression in SOD1(G93A) mice is detrimental and our results imply that this effect is independent of mast cells, as tested by ketotifen administration. To conclude, our data expand on the protective role of growth factors in ALS, as decreasing c-kit by approximately 70% is detrimental in SOD1(G93A) mice.

Cytotoxic T-lymphocyte-associated protein 4-Ig effectively controls immune activation and inflammatory disease in a novel murine model of leaky severe combined immunodeficiency

Background Severe combined immunodeficiency can be caused by loss‐of‐function mutations in genes involved in the DNA recombination machinery, such as recombination‐activating gene 1 (RAG1), RAG2, or DNA cross‐link repair 1C (DCLRE1C). Defective DNA recombination causes a developmental block in T and B cells, resulting in high susceptibility to infections. Hypomorphic mutations in the same genes can also give rise to a partial loss of T cells in a spectrum including leaky severe combined immunodeficiency (LS) and Omenn syndrome (OS). These patients not only experience life‐threatening infections because of immunodeficiency but also experience inflammatory/autoimmune conditions caused by the presence of autoreactive T cells. Objective We sought to develop a preclinical model that fully recapitulates the symptoms of patients with LS/OS, including a model for testing therapeutic intervention. Methods We generated a novel mutant mouse (Dclre1cleaky) that develops a LS phenotype. Mice were monitored for diseases, and immune phenotype and immune function were evaluated by using flow cytometry, ELISA, and histology. Results Dclre1cleaky mice present with a complete blockade of B‐cell differentiation, with a leaky block in T‐cell differentiation resulting in an oligoclonal T‐cell receptor repertoire and enhanced cytokine secretion. Dclre1cleaky mice also had inflammatory symptoms, including wasting, dermatitis, colitis, hypereosinophilia, and high IgE levels. Development of a preclinical murine model for LS allowed testing of potential treatment, with administration of cytotoxic T‐lymphocyte‐associated protein 4‐Ig reducing disease symptoms and immunologic disturbance, resulting in increased survival. Conclusion These data suggest that cytotoxic T‐lymphocyte‐associated protein 4‐Ig should be evaluated as a potential treatment of inflammatory symptoms in patients with LS and those with OS.

Transcriptional upregulation of myelin components in spontaneous myelin basic protein-deficient mice

Myelin is essential for efficient signal transduction in the nervous system comprising of multiple proteins. The intricacies of the regulation of the formation of myelin, and its components, are not fully understood. Here, we describe the characterization of a novel myelin basic protein (Mbp) mutant mouse, mbp(jive), which spontaneously occurred in our mouse colony. These mice displayed the onset of a shaking gait before 3 weeks of age and seizure onset before 2 months of age. Due to a progressive increase of seizure intensity, mbp(jive) mice experienced premature lethality at around 3 months of age. Mbp mRNA transcript or protein was undetectable and, accordingly, genetic analysis demonstrated a homozygous loss of exons 3 to 6 of Mbp. Peripheral nerve conductance was mostly unimpaired. Additionally, we observed grave structural changes in white matter predominant structures were detected by T1, T2 and diffusion weighted magnetic resonance imaging. We additionally observed that Mbp-deficiency results in an upregulation of Qkl, Mag and Cnp, suggestive of a regulatory feedback mechanism whereby compensatory increases in Qkl have downstream effects on Mag and Cnp. Further research will clarify the role and specifications of this myelin feedback loop, as well as determine its potential role in therapeutic strategies for demyelinating disorders.