Cell-mediated cytotoxicity within CSF and brain parenchyma in spinal muscular atrophy unaltered by nusinersen treatment.

5q-associated spinal muscular atrophy (SMA) is a motoneuron disease caused by mutations in the survival motor neuron 1 (SMN1) gene. Adaptive immunity may contribute to SMA as described in other motoneuron diseases, yet mechanisms remain elusive. Nusinersen, an antisense treatment, enhances SMN2 expression, benefiting SMA patients. Here we have longitudinally investigated SMA and nusinersen effects on local immune responses in the cerebrospinal fluid (CSF) - a surrogate of central nervous system parenchyma. Single-cell transcriptomics (SMA: N = 9 versus Control: N = 9) reveal NK cell and CD8+ T cell expansions in untreated SMA CSF, exhibiting activation and degranulation markers. Spatial transcriptomics coupled with multiplex immunohistochemistry elucidate cytotoxicity near chromatolytic motoneurons (N = 4). Post-nusinersen treatment, CSF shows unaltered protein/transcriptional profiles. These findings underscore cytotoxicity's role in SMA pathogenesis and propose it as a therapeutic target. Our study illuminates cell-mediated cytotoxicity as shared features across motoneuron diseases, suggesting broader implications.

The Receptor for Advanced Glycation Endproducts (RAGE) Contributes to Severe Inflammatory Liver Injury in Mice.

Background: The receptor for advanced glycation end products (RAGE) is a multiligand receptor involved in a number of processes and disorders. While it is known that RAGE-signaling can contribute to toxic liver damage and fibrosis, its role in acute inflammatory liver injury and septic multiorgan failure is yet undefined. We examined RAGE in lipopolysaccharide (LPS) induced acute liver injury of D-galN sensitized mice as a classical model for tumor necrosis factor alpha (TNF-α) dependent inflammatory organ damage. Methods: Mice (Rage-/- and C57BL/6) were intraperitoneally injected with D-galN (300 mg/kg) and LPS (10 µg/kg). Animals were monitored clinically, and cytokines, damage associated molecular pattern molecules (DAMPs) as well as liver enzymes were determined in serum. Liver histology, hepatic cytokines as well as RAGE mRNA expression were analyzed. Cellular activation and functionality were evaluated by flow cytometry both in bone marrow- and liver-derived cells. Results: Genetic deficiency of RAGE significantly reduced the mortality of mice exposed to LPS/D-galN. Hepatocyte damage markers were reduced in Rage-/- mice, and liver histopathology was less severe. Rage-/- mice produced less pro-inflammatory cytokines and DAMPs in serum and liver. While immune cell functions appeared normal, TNF-α production by hepatocytes was reduced in Rage-/- mice. Conclusions: We found that RAGE deletion attenuated the expression of pro-inflammatory cytokines and DAMPs in hepatocytes without affecting cellular immune functions in the LPS/D-galN model of murine liver injury. Our data highlight the importance of tissue-specific RAGE-signaling also in acute inflammatory liver stress contributing to sepsis and multiorgan failure.

Activation-dependent cell death of human monocytes is a novel mechanism of fine-tuning inflammation and autoimmunity.

In patients with juvenile idiopathic arthritis (JIA), increased release of IFN-γ and GM-CSF in cells infiltrating synovial tissue can be a potent driver of monocyte activation. Given the fundamental role of monocyte activation in remodeling the early phases of inflammatory responses, here we analyze the GM-CSF/IFN-γ induced activity of human monocytes in such a situation in vitro and in vivo. Monocytes from healthy donors were isolated and stimulated with GM-CSF ± IFN-γ. Monocyte activation and death were analyzed by flow cytometry, immunofluorescence microscopy, ELISA, and qPCR. T-cell GM-CSF/IFN-γ expression and monocyte function were determined in synovial fluid and peripheral blood from 15 patients with active JIA and 21 healthy controls. Simultaneous treatment with GM-CSF and IFN-γ induces cell death of monocytes. This cell death is partly cathepsin B-associated and has morphological characteristics of necrosis. Monocytes responding to costimulation with strong proinflammatory activities are consequently eliminated. Monocytes surviving this form of hyperactivation retain normal cytokine production. Cathepsin B activity is increased in monocytes isolated from synovial fluid from patients with active arthritis. Our data suggest GM-CSF/IFN-γ induced cell death of monocytes as a novel mechanism to eliminate overactivated monocytes, thereby potentially balancing inflammation and autoimmunity in JIA.

Reprogramming of monocytes by GM-CSF contributes to regulatory immune functions during intestinal inflammation.

Human and murine studies showed that GM-CSF exerts beneficial effects in intestinal inflammation. To explore whether GM-CSF mediates its effects via monocytes, we analyzed effects of GM-CSF on monocytes in vitro and assessed the immunomodulatory potential of GM-CSF-activated monocytes (GMaMs) in vivo. We used microarray technology and functional assays to characterize GMaMs in vitro and used a mouse model of colitis to study GMaM functions in vivo. GM-CSF activates monocytes to increase adherence, migration, chemotaxis, and oxidative burst in vitro, and primes monocyte response to secondary microbial stimuli. In addition, GMaMs accelerate epithelial healing in vitro. Most important, in a mouse model of experimental T cell-induced colitis, GMaMs show therapeutic activity and protect mice from colitis. This is accompanied by increased production of IL-4, IL-10, and IL-13, and decreased production of IFN-γ in lamina propria mononuclear cells in vivo. Confirming this finding, GMaMs attract T cells and shape their differentiation toward Th2 by upregulating IL-4, IL-10, and IL-13 in T cells in vitro. Beneficial effects of GM-CSF in Crohn's disease may possibly be mediated through reprogramming of monocytes to simultaneously improved bacterial clearance and induction of wound healing, as well as regulation of adaptive immunity to limit excessive inflammation.

Granulocyte Macrophage Colony-Stimulating Factor-Activated CD39+/CD73+ Murine Monocytes Modulate Intestinal Inflammation via Induction of Regulatory T Cells.

BACKGROUND & AIMS: Granulocyte macrophage colony-stimulating factor (GM-CSF) treatment induces clinical response in patients with active Crohn's disease. To explore whether monocytes mediate GM-CSF effects in vivo, we used a mouse model of chronic colitis induced by dextran sulfate sodium (DSS). METHODS: Murine bone marrow-derived monocytes were activated with GM-CSF in vitro, and gene expression, phenotype, and function of GM-CSF-activated monocytes (GMaM) were analyzed. Therapeutic effects of GMaM were assessed in a model of chronic colitis induced by repeated cycles of DSS. Monocytes were administered intravenously and their immunomodulatory functions were evaluated in vivo by clinical monitoring, histology, endoscopy, immunohistochemistry, and expression of inflammatory markers in the colon. The distribution of injected monocytes in the intestine was measured by in vivo imaging. RESULTS: GMaM expressed significantly higher levels of anti-inflammatory molecules. Production of reactive oxygen species was also increased while phagocytosis and adherence were decreased. GMaM up-regulated CD39 and CD73, which allows the conversion of adenosine triphosphate into adenosine and coincided with the induction of Foxp3+ (forkhead-box-protein P3 positive) regulatory T cells (Treg) in cocultures of GMaM and naive T cells. In chronic DSS-induced colitis, adoptive transfer of GMaM led to significant clinical improvement, as demonstrated by reduced weight loss, inflammatory infiltration, ulceration, and colon shrinkage. As GMaM migrated faster and persisted longer in the inflamed intestine compared with control monocytes, their presence induced Treg generation in vivo. CONCLUSIONS: GM-CSF leads to specific monocyte activation that modulates experimental colitis via mechanisms that include the induction of Treg. We demonstrate a possible mechanism of Treg induction through CD39 and CD73 expression on monocytes.

The sympathetic nervous system modulates CD4(+)Foxp3(+) regulatory T cells via noradrenaline-dependent apoptosis in a murine model of lymphoproliferative disease.

The sympathetic nervous system (SNS) plays a crucial role in the course and development of autoimmune disease in Fas-deficient lpr/lpr mice. As regulatory T cells (Tregs) are considered important modulators of autoimmune processes, we analyzed the interaction between the SNS and Tregs in this murine model of lymphoproliferative disease. We found that the percentage of Tregs among CD4(+) T cells is increased in the spleen, lymph nodes, and thymus of lpr/lpr mice as compared to age-matched C57Bl/6J (B6) mice. Furthermore, noradrenaline (NA), the main sympathetic neurotransmitter, induced apoptosis in B6- and lpr/lpr-derived Tregs. NA also reduced the frequency of Foxp3(+) cells and Foxp3 mRNA expression via ß2-adrenoceptor (ß2-AR)-mediated mechanisms in a concentration and time-dependent manner. Destruction of peripheral sympathetic nerves by 6-hydroxydopamine significantly increased the percentage of Tregs in B6 control mice to an extent comparable to aged-matched lpr/lpr mice. The concentration of splenic NA negatively correlated with the frequency of CD4(+)Foxp3(+) Tregs. Additionally, 60days after sympathectomy, a partial recovery of NA concentrations led to Treg percentages comparable to those of intact, vehicle-treated controls. Immunohistochemical analysis of the spleen revealed localization of single Foxp3(+) Tregs in proximity to NA-producing nerve fibers, providing an interface between Tregs and the SNS. Taken together, our data suggest a relation between the degree of splenic sympathetic innervation and the size of the Treg compartment. While there are few examples of endogenous substances capable of affecting Tregs, our results provide a possible explanation of how the magnitude of the Treg compartment in the spleen can be regulated by the SNS.

Amygdaloid signature of peripheral immune activation by bacterial lipopolysaccharide or staphylococcal enterotoxin B.

Activated immune cells produce soluble mediators that not only coordinate local and systemic immune responses but also act on the brain to initiate behavioral, neuroendocrine and metabolic adaptations. Earlier studies have shown that the amygdala, a group of nuclei located in the medial temporal lobe, is engaged in the central processing of afferent signals from the peripheral immune system. Here, we compared amygdaloid responses to lipopolysaccharide (LPS) and staphylococcal enterotoxin B (SEB), two prototypic bacterial products that elicit distinct immune responses. Intraperitoneal administration of LPS (0.1 mg/kg) or SEB (1 mg/kg) in adult rats induced substantial increases in amygdaloid neuronal activity as measured by intracerebral electroencephalography and c-fos gene expression. Amygdaloid neuronal activation was accompanied by an increase in anxiety-related behavior in the elevated plus-maze test. However, only treatment with LPS, but not SEB, enhanced amygdaloid IL-1ß and TNF-α mRNA expression. This supports the view of the immune system as a sensory organ that recognizes invading pathogens and rapidly relays this information to the brain, independent of the nature of the immune response induced. The observation that neuronal and behavioral responses to peripheral immune challenges are not necessarily accompanied by increased brain cytokine expression suggests that cytokines are not the only factors driving sickness-related responses in the CNS.

Stimulation of ß2-adrenergic receptors inhibits calcineurin activity in CD4(+) T cells via PKA-AKAP interaction.

The sympathetic nervous system (SNS) is able to modulate immune functions via adrenoceptor-dependent mechanisms. Activation of ß2-adrenergic receptors (AR) on CD4(+) T lymphocytes has been shown to inhibit Th1-cytokine production and cell proliferation. Here, we investigated the role of the calcium/calmodulin-dependent protein phosphatase calcineurin (CaN), a key element of the T cell receptor (TCR)-signaling pathway, in ß2-AR-mediated suppression of T cell function. Purified rat splenic CD4(+) T cells were stimulated with anti-CD3/anti-CD28 in presence or absence of the ß2-AR agonist terbutaline (TERB). Treatment with TERB induced a dose-dependent inhibition of cellular CaN activity, along with a reduction in IL-2 and IFN-γ production, and T cell proliferation. Co-administration of the ß-AR antagonist nadolol abolished these effects. Blockade of the cAMP-dependent protein kinase A (PKA) with the inhibitor H-89 completely prevented TERB-induced CaN inhibition. However, a receptor-independent rise in the second messenger cAMP was not sufficient to suppress CaN activity. Disruption of the interaction between PKA and A-kinase anchoring protein (AKAP) by the inhibitor peptide St-Ht31 fully blocked TERB-induced CaN inhibition, demonstrating that PKA-AKAP interaction is essential for the ß2-AR-mediated CaN inhibition. Taken together, this study provides evidence for a link between the ß2-AR and TCR signaling pathways since expression of IL-2 and IFN-γ in activated T cells largely depends on dephosphorylation of the transcription factor NFAT by CaN, and identifies a novel intracellular mechanism that can lead to downregulation of T cell function after SNS activation.