Bernadette Marrero

An activating NLRC4 inflammasome mutation causes autoinflammation with recurrent macrophage activation syndrome

Inflammasomes are innate immune sensors that respond to pathogen- and damage-associated signals with caspase-1 activation, interleukin (IL)-1β and IL-18 secretion, and macrophage pyroptosis. The discovery that dominant gain-of-function mutations in NLRP3 cause the cryopyrin-associated periodic syndromes (CAPS) and trigger spontaneous inflammasome activation and IL-1β oversecretion led to successful treatment with IL-1–blocking agents. Herein we report a de novo missense mutation (c.1009A>T, encoding p.Thr337Ser) affecting the nucleotide-binding domain of the inflammasome component NLRC4 that causes early-onset recurrent fever flares and macrophage activation syndrome (MAS). Functional analyses demonstrated spontaneous inflammasome formation and production of the inflammasome-dependent cytokines IL-1β and IL-18, with the latter exceeding the levels seen in CAPS. The NLRC4 mutation caused constitutive caspase-1 cleavage in cells transduced with mutant NLRC4 and increased production of IL-18 in both patient-derived and mutant NLRC4–transduced macrophages. Thus, we describe a new monoallelic inflammasome defect that expands the monogenic autoinflammatory disease spectrum to include MAS and suggests new targets for therapy.

Additive loss-of-function proteasome subunit mutations in CANDLE/PRAAS patients promote type I IFN production

Autosomal recessive mutations in proteasome subunit β 8 (PSMB8), which encodes the inducible proteasome subunit β5i, cause the immune-dysregulatory disease chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE), which is classified as a proteasome-associated autoinflammatory syndrome (PRAAS). Here, we identified 8 mutations in 4 proteasome genes, PSMA3 (encodes α7), PSMB4 (encodes β7), PSMB9 (encodes β1i), and proteasome maturation protein (POMP), that have not been previously associated with disease and 1 mutation in PSMB8 that has not been previously reported. One patient was compound heterozygous for PSMB4 mutations, 6 patients from 4 families were heterozygous for a missense mutation in 1 inducible proteasome subunit and a mutation in a constitutive proteasome subunit, and 1 patient was heterozygous for a POMP mutation, thus establishing a digenic and autosomal dominant inheritance pattern of PRAAS. Function evaluation revealed that these mutations variably affect transcription, protein expression, protein folding, proteasome assembly, and, ultimately, proteasome activity. Moreover, defects in proteasome formation and function were recapitulated by siRNA-mediated knockdown of the respective subunits in primary fibroblasts from healthy individuals. Patient-isolated hematopoietic and nonhematopoietic cells exhibited a strong IFN gene-expression signature, irrespective of genotype. Additionally, chemical proteasome inhibition or progressive depletion of proteasome subunit gene transcription with siRNA induced transcription of type I IFN genes in healthy control cells. Our results provide further insight into CANDLE genetics and link global proteasome dysfunction to increased type I IFN production.

STAT3 Protein Interacts with Class O Forkhead Transcription Factors in the Cytoplasm and Regulates Nuclear/Cytoplasmic Localization of FoxO1 and FoxO3a Proteins in CD4+ T Cells

Background: Molecular signals that control how long activated lymphocytes remain activated are unknown. Results: Unphosphorylated STAT3 interacts with and sequesters pFoxO1/pFoxO3a in cytoplasm whereas pSTAT3 terminates TCR activation by inducing nuclear localization of FoxO1/FoxO3a and p27Kip1 expression. Conclusion: STAT3/FoxO are gatekeepers that determine whether T cells remain quiescent or proliferate. Significance: STAT3 is convergence point for mechanisms that regulate cellular quiescence and lymphocyte activation. An important feature of the adaptive immune response is its remarkable capacity to regulate the duration of inflammatory responses, and effector T cells have been shown to limit excessive immune responses by producing anti-inflammatory cytokines such as IL-10 and IL-27. However, how anti-inflammatory cytokines mediate their suppressive activities is not well understood. In this study, we show that STAT3 contributes to mechanisms that control the duration of T cell proliferation by regulating the subcellular location of FoxO1 and FoxO3a, two Class O Forkhead transcription factors that mediate lymphocyte quiescence and inhibit T cell activation. We show that active FoxO1 and FoxO3a reside exclusively in the nucleus of naïve T cells whereas inactive pFoxO1 and pFoxO3a were most abundant in activated T cells and sequestered in their cytoplasm in association with unphosphorylated STAT3 (U-STAT3) and 14-3-3. We further show that FoxO1/FoxO3a rapidly relocalized into the nucleus in response to pSTAT3 activation by IL-6 or IL-10, and the accumulation of FoxO1/FoxO3a in their nuclei coincided with increased expression of p27Kip1 and p21WAF1. STAT3 inhibitors completely abrogated cytokine-induced translocation of FoxO1/FoxO3a into the nucleus. In naïve or resting STAT3-deficient T cells, expression of pFoxO1/pFoxO3a was predominantly in the cytoplasm and correlated with defects in p27Kip1 and p21WAF1 expression, suggesting requirement of STAT3 for importation or retention of FoxO in the nucleus and attenuation of lymphocyte proliferation. Taken together, these results suggest that U-STAT3 collaborates with 14-3-3 to sequester pFoxO1/pFoxO3a in cytoplasm and thus prolong T cell activation, whereas pSTAT3 activation by anti-inflammatory cytokines would curtail the duration of TCR activation and re-establish lymphocyte quiescence by inducing nuclear localization of FoxO1/FoxO3a and FoxO-mediated expression of growth-inhibitory proteins.

BAFF Suppresses IL-15 Expression in B Cells

Clinical trials have shown that BAFF inhibitors do not reduce memory B cell levels but can reduce the number of mature B cells. It remains uncertain whether BAFF affects memory-maintaining cytokines such as IL-15. We found that BAFF suppressed IL-15 expression in B cells from lupus-like or experimental allergic encephalomyelitis mice. When BAFF was blocked with atacicept-IgG, IL-15 expression was upregulated in lupus-like or experimental allergic encephalomyelitis mice. Finally, we showed that BAFF suppressed IL-15 expression in transitional 2 B cells by reducing Foxo1 expression and inducing Foxo1 phosphorylation. This study suggests that BAFF suppresses IL-15 expression in autoimmune diseases, and this opens up the possible opportunity for the clinical application of BAFF- and IL-15–specific therapeutic agents.

C5a Regulates IL-12 + DC Migration to Induce Pathogenic Th1 and Th17 Cells in Sepsis

Objective It is well known that complement system C5a is excessively activated during the onset of sepsis. However, it is unclear whether C5a can regulate dentritic cells (DCs) to stimulate adaptive immune cells such as Th1 and Th17 in sepsis. Methods Sepsis was induced by cecal ligation and puncture (CLP). CLP-induced sepsis was treated with anti-C5a or IL-12. IL-12+DC, IFNγ+Th1, and IL-17+Th17 cells were analyzed by flow cytometry. IL-12 was measured by ELISA. Results Our studies here showed that C5a induced IL-12+DC cell migration from the peritoneal cavity to peripheral blood and lymph nodes. Furthermore, IL-12+DC cells induced the expansion of pathogenic IFNγ+Th1 and IL-17+Th17 cells in peripheral blood and lymph nodes. Moreover, IL-12, secreted by DC cells in the peritoneal cavity, is an important factor that prevents the development of sepsis. Conclusion Our data suggests that C5a regulates IL-12+DC cell migration to induce pathogenic Th1 and Th17 cells in sepsis.

Interleukin 27 Induces the Expression of Complement Factor H (CFH) in the Retina

Complement factor H (CFH) is a central regulator of the complement system and has been implicated in the etiology of age-related macular degeneration (AMD), a leading cause of blindness in the elderly. In view of previous studies showing that reduced expression of CFH in the retina is a risk factor for developing AMD, there is significant interest in understanding how CFH expression is regulated in the retina. In this study, we have shown that the anti-inflammatory cytokine, IL-27, induced CFH expression in mouse retinal cells and human retinal pigmented epithelial cells (RPE) through STAT1-mediated up-regulation of Interferon Regulatory Factor-1 (IRF-1) and IRF-8. We further show that cells in the ganglion and inner-nuclear layers of the retina constitutively express IRF-1 and IRF-8 and enhanced CFH expression in the retina during ocular inflammation correlated with significant increase in the expression of IRF-1, IRF-8 and IL-27 (IL-27p28 and Ebi3). Our data thus reveal a novel role of IL-27 in regulating complement activation through up-regulation of CFH and suggest that defects in IL-27 signaling or expression may contribute to the reduction of CFH expression in the retina of patients with AMD.

BAFF maintains T-cell survival by inducing OPN expression in B cells

Dysregulation of T-cell survival and apoptosis is the common cause of autoimmune diseases such as multiple sclerosis (MS). However, the factors inducing imbalance of T-cell survival and apoptosis in MS remains unclear. Here, we show that the resistance to apoptosis was associated with high levels of B-cell activating factor (BAFF). Blockade of BAFF with TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor)-IgG significantly reduced T-cell survival in myelin oligodendroglia glycoprotein (MOG)-induced chronic experimental allergic encephalitis (EAE). Furthermore, BAFF induced anti-apoptotic molecule Bcl2 expression in T cells by up-regulating osteopontin (OPN) secretion from B cells. BAFF mainly induced OPN expression in splenic CD21(-)CD23(+) B cells via a NF-kB dependent signaling pathway. In addition, we found that BAFF and OPN levels were increased in MS patients similar to the results obtained from our mice research. The study suggests that BAFF regulates T-cell survival by inducing OPN secretion in B cells in autoimmune diseases.

Combination of TACI-IgG and anti-IL-15 treats murine lupus by reducing mature and memory B cells.

Clinical trials suggest that BAFF inhibitors such as atacicept (TACI-IgG) and belimumab (anti-BAFF antibody) could not reduce memory B-cell numbers, although they reduced the numbers of CD20(+) naïve B cells and activated B cells. In the present study, we explored the way to reduce memory B-cell numbers. First, we used TACI-IgG to treat murine lupus. We found that TACI-IgG was effective in reducing mature B cell numbers. Accordingly it controlled the level of the anti-dsDNA antibody in lupus-like mice. In addition, TACI-IgG up-regulated memory B cells in murine lupus. Furthermore, we found that TACI-IgG up-regulated IL-15 expression in lupus-like mice. Thus, the combination of TACI-IgG and anti-IL-15 antibodies was explored to understand their effects on the treatment of murine lupus. Compared to treatments with TACI-IgG or anti-IL-15 alone, the combination of TACI-IgG and anti-IL-15 antibodies efficiently ameliorated murine lupus phenotypes. The study provides hints for the clinical application of BAFF- and IL-15-specific therapeutic agents.

Erratum: Additive loss-of-function proteasome subunit mutations in CANDLE/PRAAS patients promote type i IFN production (Journal of Clinical Investigation (2015) 125:11 (4196-4211) DOI: 10.1172/JCI81260)

Autosomal recessive mutations in proteasome subunit b 8 (PSMB8), which encodes the inducible proteasome subunit b5i, cause the immune-dysregulatory disease chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE), which is classified as a proteasome-associated autoinflammatory syndrome (PRAAS). Here, we identified 8 mutations in 4 proteasome genes, PSMA3 (encodes a7), PSMB4 (encodes b7), PSMB9 (encodes b1i), and proteasome maturation protein (POMP), that have not been previously associated with disease and 1 mutation in PSMB8 that has not been previously reported. One patient was compound heterozygous for PSMB4 mutations, 6 patients from 4 families were heterozygous for a missense mutation in 1 inducible proteasome subunit and a mutation in a constitutive proteasome subunit, and 1 patient was heterozygous for a POMP mutation, thus establishing a digenic and autosomal dominant inheritance pattern of PRAAS. Function evaluation revealed that these mutations variably affect transcription, protein expression, protein folding, proteasome assembly, and, ultimately, proteasome activity. Moreover, defects in proteasome formation and function were recapitulated by siRNA-mediated knockdown of the respective subunits in primary fibroblasts from healthy individuals. Patient-isolated hematopoietic and nonhematopoietic cells exhibited a strong IFN gene-expression signature, irrespective of genotype. Additionally, chemical [...] Research Article Immunology

A157: Macrophage Activation Syndrome‐like Illness Due to an Activating Mutation in NLRC4

Macrophage Activation Syndrome (MAS) is a life‐threatening systemic inflammatory disorder of unknown etiology. While MAS has no known genetic basis, clinical similarity with a genetic disorder of impaired cytotoxicity known as primary Hemophagocytic Lymphohistiocytosis (HLH) has suggested shared pathogenesis. In contrast, other investigations have suggested innate immune dysregulation drives MAS.