Teresa K. Tarrant

An Endogenous TNF-α Antagonist Induced by Splice-switching Oligonucleotides Reduces Inflammation in Hepatitis and Arthritis Mouse Models

Tumor necrosis factor-α (TNF-α) is a key mediator of inflammatory diseases, including rheumatoid arthritis (RA), and anti-TNF-α drugs such as etanercept are effective treatments. Splice-switching oligonucleotides (SSOs) are a new class of drugs designed to induce therapeutically favorable splice variants of targeted genes. In this work, we used locked nucleic acid (LNA)-based SSOs to modulate splicing of TNF receptor 2 (TNFR2) pre-mRNA. The SSO induced skipping of TNFR2 exon 7, which codes the transmembrane domain (TM), switching endogenous expression from the membrane-bound, functional form to a soluble, secreted form (Δ7TNFR2). This decoy receptor protein accumulated in the circulation of treated mice, antagonized TNF-α, and altered disease in two mouse models: TNF-α-induced hepatitis and collagen-induced arthritis (CIA). This is the first report of upregulation of the endogenous, circulating TNF-α antagonist by oligonucleotide-induced splicing modulation.Tumor necrosis factor-alpha (TNF-alpha) is a key mediator of inflammatory diseases, including rheumatoid arthritis (RA), and anti-TNF-alpha drugs such as etanercept are effective treatments. Splice-switching oligonucleotides (SSOs) are a new class of drugs designed to induce therapeutically favorable splice variants of targeted genes. In this work, we used locked nucleic acid (LNA)-based SSOs to modulate splicing of TNF receptor 2 (TNFR2) pre-mRNA. The SSO induced skipping of TNFR2 exon 7, which codes the transmembrane domain (TM), switching endogenous expression from the membrane-bound, functional form to a soluble, secreted form (Delta7TNFR2). This decoy receptor protein accumulated in the circulation of treated mice, antagonized TNF-alpha, and altered disease in two mouse models: TNF-alpha-induced hepatitis and collagen-induced arthritis (CIA). This is the first report of upregulation of the endogenous, circulating TNF-alpha antagonist by oligonucleotide-induced splicing modulation.

Autoimmunity in immunodeficiency.

Primary immunodeficiencies (PID) comprise a diverse group of clinical disorders with varied genetic defects. Paradoxically, a substantial proportion of PID patients develop autoimmune phenomena in addition to having increased susceptibility to infections from their impaired immunity. Although much of our understanding comes from data gathered through experimental models, there are several well-characterized PID that have improved our knowledge of the pathways that drive autoimmunity. The goals of this review will be to discuss these immunodeficiencies and to review the literature with respect to the proposed mechanisms for autoimmunity within each put forth to date.

Autoimmune Lymphoproliferative Syndrome: an Update and Review of the Literature

Autoimmune lymphoproliferative syndrome (ALPS) is characterized by immune dysregulation due to a defect in lymphocyte apoptosis. The clinical manifestations may be noted in multiple family members and include lymphadenopathy, splenomegaly, increased risk of lymphoma, and autoimmune disease, which typically involves hematopoietic cell lines manifesting as multilineage cytopenias. Since the disease was first characterized in the early 1990s, there have been many advances in the diagnosis and management of this syndrome. The inherited genetic defect of many ALPS patients has involved (FAS) pathway signaling proteins, but there remain those patients who carry undefined genetic defects. Despite ALPS having historically been considered a primary immune defect presenting in early childhood, adult onset presentation is increasingly becoming recognized and more so in genetically undefined patients and those with somatic FAS mutations. Thus, future research may identify novel pathways and/or regulatory proteins important in lymphocyte activation and apoptosis.

A desensitization protocol for the mAb cetuximab.

could be somehow regarded as a disease-specific biomarker: this fact also suggests that HVA, which is a pure IgE-mediated disease, differs in its immunologic regulation from other types of allergic diseases. Daniele Saverino, BS Anna Maria Riccio, BS Anthi Rogkakou, MD Marcello Bagnasco, MD Patrizia Bonadonna, MD Rita Simone, BS Alessandra Chiappori, BS Cinzia Gamalero, BS Giovanni Passalacqua, MD Giorgio Walter Canonica, MD Giampaola Pesce, BS From the Human Anatomy Section, Department of Experimental Medicine, University of Genoa, Genoa, Italy; Allergy and Respiratory Diseases, Department of Internal Medicine, University of Genoa, Genoa, Italy; Medical and Radiomethabolic Therapy, University of Genoa, Genoa, Italy; Allergy Service, Verona General Hospital, Verona, Italy; and Internal Medicine, Department of Clinical and Experimental Medicine, University of Verona, Verona, Italy. E-mail: passalacqua@unige.it. Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest.

Enhanced Th17-Cell Responses Render CCR2-Deficient Mice More Susceptible for Autoimmune Arthritis

CCR2 is considered a proinflammatory mediator in many inflammatory diseases such as rheumatoid arthritis. However, mice lacking CCR2 develop exacerbated collagen-induced arthritis. To explore the underlying mechanism, we investigated whether autoimmune-associated Th17 cells were involved in the pathogenesis of the severe phenotype of autoimmune arthritis. We found that Th17 cells were expanded approximately 3-fold in the draining lymph nodes of immunized CCR2−/− mice compared to WT controls (p = 0.017), whereas the number of Th1 cells and regulatory T cells are similar between these two groups of mice. Consistently, levels of the Th17 cell cytokine IL-17A and Th17 cell-associated cytokines, IL-6 and IL-1β were approximately 2–6-fold elevated in the serum and 22–28-fold increased in the arthritic joints in CCR2−/− mice compared to WT mice (p = 0.04, 0.0004, and 0.01 for IL-17, IL-6, and IL-1β, respectively, in the serum and p = 0.009, 0.02, and 0.02 in the joints). Furthermore, type II collagen-specific antibodies were significantly increased, which was accompanied by B cell and neutrophil expansion in CCR2−/− mice. Finally, treatment with an anti-IL-17A antibody modestly reduced the disease severity in CCR2−/− mice. Therefore, we conclude that while we detect markedly enhanced Th17-cell responses in collagen-induced arthritis in CCR2-deficient mice and IL-17A blockade does have an ameliorating effect, factors additional to Th17 cells and IL-17A also contribute to the severe autoimmune arthritis seen in CCR2 deficiency. CCR2 may have a protective role in the pathogenesis of autoimmune arthritis. Our data that monocytes were missing from the spleen while remained abundant in the bone marrow and joints of immunized CCR2−/− mice suggest that there is a potential link between CCR2-expressing monocytes and Th17 cells during autoimmunity.

Granulocyte chemotaxis and disease expression are differentially regulated by GRK subtype in an acute inflammatory arthritis model (K/BxN)

OBJECTIVE Chemokine receptors are G-protein coupled receptors (GPCRs) phosphorylated by G-protein receptor kinases (GRKs) after ligand-mediated activation. We hypothesized that GRK subtypes differentially regulate granulocyte chemotaxis and clinical disease expression in the K/BxN model. METHODS Clinical, histologic, and cytokine responses in GRK6-/-, GRK5-/-, GRK2+/-, and wildtype mice were evaluated using K/BxN serum transfer. Granulocyte chemotaxis was analyzed by transendothelial migration assays. RESULTS Both GRK6-/- and GRK2+/- mice had increased arthritis disease severity (p<0.001); whereas GRK5-/- was not different from controls. Acute weight loss was enhanced in GRK6-/- and GRK2+/- mice (p<0.001, days 3-10). However, GRK6-/- mice uniquely had more weight loss (>10%), elevated serum IL-6, and enhanced migration toward LTB4 and C5a in vitro. CONCLUSIONS GRK6 and -2, but not GRK5, are involved in the pathogenesis of acute arthritis in the K/BxN model. In particular, GRK6 may dampen inflammatory responses by regulating granulocyte trafficking toward chemoattractants.

Decreased Th17 and antigen‐specific humoral responses in CX3CR1‐deficient mice in the collagen‐induced arthritis model

OBJECTIVE CX(3) CR1 is a chemokine receptor that uniquely binds to its ligand fractalkine (CX(3) CL1) and has been shown to be important in inflammatory arthritis responses, largely due to its effects on cellular migration. This study was undertaken to test the hypothesis that genetic deficiency of CX(3) CR1 is protective in the chronic inflammatory arthritis model collagen-induced arthritis (CIA). Because CX(3) CR1 is expressed on T cells and antigen-presenting cells, we also examined adaptive immune functions in this model. METHODS Autoantibody formation, clinical, histologic, T cell proliferative, and cytokine responses were evaluated in wild-type and CX(3) CR1-deficient DBA/1J mice after immunization with heterologous type II collagen (CII). RESULTS CX(3) CR1(-/-) mice had an ∼30% reduction in arthritis severity compared to wild-type mice, as determined by 2 independent measures, paw swelling (P < 0.01) and clinical disease score (P < 0.0001). Additionally, compared to wild-type mice, CX(3) CR1(-/-) mice had an ∼50% decrease in anti-CII autoantibody formation (P < 0.05), decreased Th17 intraarticular cytokine expression (P < 0.01 for interleukin-17 [IL-17] and P < 0.001 for IL-23), and decreased total numbers of Th17 cells in inflamed joints (P < 0.05). CONCLUSION Our findings indicate that CX(3) CR1 deficiency is protective in inflammatory arthritis and may have effects that extend beyond migration that involve adaptive immune responses in autoimmune disease.

G-protein signaling modulator-3, a gene linked to autoimmune diseases, regulates monocyte function and its deficiency protects from inflammatory arthritis

Polymorphism at the GPSM3 gene locus is inversely associated with four systemic autoimmune diseases, including rheumatoid arthritis and ankylosing spondylitis. G-protein signaling modulator-3 (GPSM3) expression is most pronounced in myeloid cells, in which it targets heterotrimeric G-protein Gαi subunits of chemokine receptors, critical to immune function. To begin to explore the regulatory role of GPSM3 in monocytes, human THP-1 and primary mouse myeloid cells were cultured under stimulus conditions; GPSM3 was found by immunoblotting to be expressed at highest levels in the mature monocyte. To evaluate the effects of GPSM3 deficiency on a myeloid-dependent autoimmune disease, collagen antibody-induced arthritis (CAIA) was induced in Gpsm3-/- and control mice, which were then analyzed for clinical score, paw swelling, intra-articular proinflammatory markers, and histopathology. Mice lacking GPSM3 were protected from CAIA, and expression of monocyte-representative pro-inflammatory chemokine receptors and cytokines in paws of Gpsm3-/- mice were decreased. Flow cytometry, apoptosis, and transwell chemotaxis experiments were conducted to further characterize the effect of GPSM3 deficiency on survival and chemokine responsiveness of monocytes. GPSM3-deficient myeloid cells had reduced migration ex vivo to CCL2, CX3CL1, and chemerin and enhanced apoptosis in vitro. Our results suggest that GPSM3 is an important regulator of monocyte function involving mechanisms of differentiation, survival, and chemotaxis, and deficiency in GPSM3 expression is protective in acute inflammatory arthritis.

G protein-coupled receptor kinase-3-deficient mice exhibit WHIM syndrome features and attenuated inflammatory responses

Chemokine receptor interactions coordinate leukocyte migration in inflammation. Chemokine receptors are GPCRs that when activated, are phosphorylated by GRKs to turn off G protein‐mediated signaling yet recruit additional signaling machinery. Recently, GRK3 was identified as a negative regulator of CXCL12/CXCR4 signaling that is defective in human WHIM syndrome. Here, we report that GRK3−/− mice exhibit numerous features of human WHIM, such as impaired CXCL12‐mediated desensitization, enhanced CXCR4 signaling to ERK activation, altered granulocyte migration, and a mild myelokathexis. Moreover, GRK3−/− protects mice from two acute models of inflammatory arthritis (K/BxN serum transfer and CAIA). In these granulocyte‐dependent disease models, protection of GRK3−/− mice is mediated by retention of cells in the marrow, fewer circulating granulocytes in the peripheral blood, and reduced granulocytes in the joints during active inflammation. In contrast to WHIM, GRK3−/− mice have minimal hypogammaglobulinemia and a peripheral leukocytosis with increased lymphocytes and absent neutropenia. Thus, we conclude that the loss of GRK3‐mediated regulation of CXCL12/CXCR4 signaling contributes to some, but not all, of the complete WHIM phenotype and that GRK3 inhibition may be beneficial in the treatment of inflammatory arthritis.

S100A9 is not essential for disease expression in an acute (K/BxN) or chronic (CIA) model of inflammatory arthritis

Objective: S100A8 (calgranulin A, MRP8) and S100A9 (calgranulin B, MRP14) are calcium-binding proteins highly expressed by activated myeloid cells and thought to be involved in the pathogenesis of inflammatory diseases. Circulating levels of S100A8/S100A9 are elevated in both human and experimental models of autoimmune disease, including rheumatoid arthritis (RA). Methods: Mice deficient in S100A9 (S100A9 − /−) and wild-type controls were immunized using standard techniques for the K/BxN serum transfer or the collagen-induced arthritis (CIA) model. Results: S100A9 − /− animals, with defective expression of both S100A8 and S100A9 proteins, had similar arthritis and histopathology to that of wild-type controls in both mouse models. Conclusion: S100A8 and S100A9 are not essential for disease expression in either the K/BxN serum transfer or the CIA model of inflammatory arthritis.