Granzyme B Induces IRF-3 Phosphorylation through a Perforin-Independent Proteolysis-Dependent Signaling Cascade without Inducing Cell Death.

Granzyme B (GrB) is an immune protease implicated in the pathogenesis of several human diseases. In the current model of GrB activity, perforin determines whether the downstream actions of GrB occur intracellularly or extracellularly, producing apoptotic cytotoxicity or nonapoptotic effects, respectively. In the current study, we demonstrate the existence of a broad range of GrB-dependent signaling activities that 1) do not require perforin, 2) occur intracellularly, and 3) for which cell death is not the dominant outcome. In the absence of perforin, we show that GrB enzymatic activity still induces substoichiometric activation of caspases, which through nonlethal DNA damage response signals then leads to activity-associated phosphorylation of IFN regulatory factor-3. These findings illustrate an unexpected potential interface between GrB and innate immunity separate from the traditional role of GrB in perforin-dependent GrB-mediated apoptosis that could have mechanistic implications for human disease.

Autoantibodies targeting LINE-1-encoded ORF1p are associated with systemic lupus erythematosus diagnosis but not disease activity.

OBJECTIVES: Long Interspersed Element 1 (LINE-1) is an endogenous retroelement that constitutes a significant portion of the human genome and has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). The LINE-1 RNA chaperone protein ORF1p was recently identified as an SLE autoantigen. Here we analyse ORF1p for qualities underlying SLE autoantigen status, compared anti-ORF1p antibodies to markers of SLE disease activity, and performed screening for antibodies against LINE-1 reverse transcriptase ORF2p. METHODS: ORF1p was examined in epithelial cell lines treated with cytotoxic lymphocyte granules and UV irradiation. Anti-ORF1p and anti-ORF2p antibodies were assayed by ELISA and analysed in two SLE cohorts. RESULTS: We found that ORF1p localises to cytoplasmic RNA-containing blebs in apoptotic cells, and is a substrate of the cytotoxic protease granzyme B (GrB). Anti-ORF1p antibodies were present in 4.2% of healthy controls, compared to 15.8% (p=0.0157) and 15.5% (p=0.036) of subjects in the two SLE cohorts. Anti-ORF1p antibodies were not associated with SLE disease activity nor peripheral blood markers of interferon (IFN) activation. Anti-ORF1p titres demonstrated stability over serial time points. Anti-ORF1p antibodies were not associated with anti-DNA, anti-RNP, or other SLE autoantibodies. There was no difference in anti-ORF2p ELISA results in controls versus SLE patients. CONCLUSIONS: LINE-1 ORF1p is a component of apoptotic blebs and a substrate for GrB. Anti-ORF1p antibodies are enriched in SLE subjects but are not associated with dynamic markers of disease activity. These data support a potential role for LINE-1 dysregulation in SLE pathogenesis.

Cryo-EM structure of the NLRC4CARD filament provides insights into how symmetric and asymmetric supramolecular structures drive inflammasome assembly.

Inflammasomes are supramolecular signaling platforms integral to innate immune defense against invading pathogens. The NOD-like receptor (NLR) family apoptosis inhibitory protein (NAIP)·NLR family caspase-recruiting domain (CARD) domain-containing 4 (NLRC4) inflammasome recognizes intracellular bacteria and induces the polymerization of the caspase-1 protease, which in turn executes maturation of interleukin-1ß (IL-1ß) and pyroptosis. Several high-resolution structures of the fully assembled NAIP·NLRC4 complex are available, but these structures do not resolve the architecture of the CARD filament in atomic detail. Here, we present the cryo-EM structure of the filament assembled by the CARD of human NLRC4 (NLRC4CARD) at 3.4 Å resolution. The structure revealed that the helical architecture of the NLRC4CARD filament is essentially identical to that of the downstream filament assembled by the CARD of caspase-1 (casp1CARD), but deviates from the split washer-like assembly of the NAIP·NLRC4 oligomer. Our results suggest that architectural complementarity is a major driver for the recognition between upstream and downstream CARD assemblies in inflammasomes. Furthermore, a Monte Carlo simulation of the NLRC4CARD filament assembly rationalized why an (un)decameric NLRC4 oligomer is optimal for assembling the helical base of the NLRC4CARD filament. Together, our results explain how symmetric and asymmetric supramolecular assemblies enable high-fidelity signaling in inflammasomes.

ANCA-Associated Vasculitis Pathogenesis: A Commentary.

PURPOSE OF REVIEW: The ANCA-associated vasculitides are a group of small vessel vasculitides characterized by autoantibodies recognizing the neutrophil cytoplasmic antigens PR3 and MPO. We examine the current clinical and molecular immunology understanding of ANCA-associated vasculitides and discuss the current needs in our understanding of the pathogenic mechanisms of these rare diseases. RECENT FINDINGS: The majority of efforts to understand the pathogenesis of these diseases have focused on dissecting neutrophil biology because the neutrophil is the primary expressor of ANCA autoantigens. However, a number of important genetic, clinical, and cellular biology observations suggest that attempts to understand the pathogenesis of ANCA vasculitides should move away from emphasis on the role of the neutrophil and instead re-focus on the potential role of other immune cell mediators. Whether or not neutrophils are the key determinant of ANCA-associated vasculitis pathogenesis should be revisited in detail. A neutrophil-centric view of the pathogenesis of these diseases cannot fully account for important genetic, clinical, and cellular biology observations that implicate important and under-appreciated roles for monocytes and T cells. Refocusing on these findings will likely lead to new discovery of novel therapeutic targets and the identification of clinically useful biomarkers for disease activity.

Identification of a ferrireductase required for efficient transferrin-dependent iron uptake in erythroid cells.

The reduction of iron is an essential step in the transferrin (Tf) cycle, which is the dominant pathway for iron uptake by red blood cell precursors. A deficiency in iron acquisition by red blood cells leads to hypochromic, microcytic anemia. Using a positional cloning strategy, we identified a gene, six-transmembrane epithelial antigen of the prostate 3 (Steap3), responsible for the iron deficiency anemia in the mouse mutant nm1054. Steap3 is expressed highly in hematopoietic tissues, colocalizes with the Tf cycle endosome and facilitates Tf-bound iron uptake. Steap3 shares homology with F(420)H(2):NADP(+) oxidoreductases found in archaea and bacteria, as well as with the yeast FRE family of metalloreductases. Overexpression of Steap3 stimulates the reduction of iron, and mice lacking Steap3 are deficient in erythroid ferrireductase activity. Taken together, these findings indicate that Steap3 is an endosomal ferrireductase required for efficient Tf-dependent iron uptake in erythroid cells.

Interferon and autoantigens: intersection in autoimmunity.

Interferon (IFN) is a key component of the innate immune response. For reasons that remain incompletely understood, the IFN system is upregulated in several rheumatic diseases, particularly those that feature autoantibody production, such as SLE, Sjögren's syndrome, myositis and systemic sclerosis. Interestingly, many of the autoantigens targeted in these diseases are components of the IFN system, representing IFN-stimulated genes (ISGs), pattern recognition receptors (PRRs), and modulators of the IFN response. In this review, we describe features of these IFN-linked proteins that may underlie their status as autoantigens. Note is also made of anti-IFN autoantibodies that have been described in immunodeficiency states.