Kristi L. Williams

NLRP3 inflammasome induces chemotactic immune cell migration to the CNS in experimental autoimmune encephalomyelitis

The NLRP3 inflammasome is a multiprotein complex consisting of three kinds of proteins, NLRP3, ASC, and pro-caspase-1, and plays a role in sensing pathogens and danger signals in the innate immune system. The NLRP3 inflammasome is thought to be involved in the development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). However, the mechanism by which the NLRP3 inflammasome induces EAE is not clear. In this study, we found that the NLRP3 inflammasome played a critical role in inducing T-helper cell migration into the CNS. To gain migratory ability, CD4+ T cells need to be primed by NLRP3 inflammasome-sufficient antigen-presenting cells to up-regulate chemotaxis-related proteins, such as osteopontin, CCR2, and CXCR6. In the presence of the NLRP3 inflammasome, dendritic cells and macrophages also induce chemotactic ability and up-regulate chemotaxis-related proteins, such as α4β1 integrin, CCL7, CCL8, and CXCL16. On the other hand, reduced Th17 cell population size in immunized Nlrp3−/− and Asc−/− mice is not a determinative factor for their resistance to EAE. As currently applied in clinical interventions of MS, targeting immune cell migration molecules may be an effective approach in treating MS accompanied by NLRP3 inflammasome activation.

Interferon-β Therapy Against EAE Is Effective Only When Development of the Disease Depends on the NLRP3 Inflammasome

Characterization of an animal model may explain why not all patients with multiple sclerosis respond to interferon-β. Inflammasome Dependency Determines Therapy? Multiple sclerosis (MS) is an inflammatory autoimmune disease in which the myelin sheath surrounding axons is destroyed by cells of the immune system. MS and experimental autoimmune encephalitis (EAE), an animal model of MS, can be ameliorated by interferon-β (IFN-β); however, IFN-β is not effective in all cases. Inoue et al. determined a mechanism by which IFN-β decreases the severity of EAE in mice by inhibiting the activity of the NLRP3 inflammasome. However, the authors also characterized a form of EAE that was independent of NLRP3 and was refractory to IFN-β. Given other reports that have suggested the involvement of inflammasomes in MS, it will be important to investigate whether patients who fail to respond to IFN-β have inflammasome-independent disease. Interferon-β (IFN-β) is widely used to treat multiple sclerosis (MS), and its efficacy was demonstrated in the setting of experimental autoimmune encephalomyelitis (EAE), an animal model of MS; however, IFN-β is not effective in treating all cases of MS. Here, we demonstrate that signaling by IFNAR (the shared receptor for IFN-α and IFN-β) on macrophages inhibits activation of Rac1 and the generation of reactive oxygen species (ROS) through suppressor of cytokine signaling 1 (SOCS1). The inhibition of Rac1 activation and ROS generation suppressed the activity of the Nod-like receptor (NLR) family, pyrin domain–containing 3 (NLRP3) inflammasome, which resulted in attenuated EAE pathogenicity. We further found that two subsets of EAE could be defined on the basis of their dependency on the NLRP3 inflammasome and that IFN-β was not an effective therapy when EAE was induced in an NLRP3 inflammasome–independent fashion. Thus, our study demonstrates a previously uncharacterized signaling pathway that is involved in the suppression of EAE by IFN-β and characterizes NLRP3-independent EAE, which cannot be treated with IFN-β.

ATP binding by monarch-1/NLRP12 is critical for its inhibitory function

ABSTRACT The recently discovered nucleotide binding domain-leucine rich repeat (NLR) gene family is conserved from plants to mammals, and several members are associated with human autoinflammatory or immunodeficiency disorders. This family is defined by a central nucleotide binding domain that contains the highly conserved Walker A and Walker B motifs. Although the nucleotide binding domain is a defining feature of this family, it has not been extensively studied in its purified form. In this report, we show that purified Monarch-1/NLRP12, an NLR protein that negatively regulates NF-κB signaling, specifically binds ATP and exhibits ATP hydrolysis activity. Intact Walker A/B motifs are required for this activity. These motifs are also required for Monarch-1 to undergo self-oligomerization, Toll-like receptor- or CD40L-activated association with NF-κB-inducing kinase (NIK) and interleukin-1 receptor-associated kinase 1 (IRAK-1), degradation of NIK, and inhibition of IRAK-1 phosphorylation. The stable expression of a Walker A/B mutant in THP-1 monocytes results in increased production of proinflammatory cytokines and chemokines to an extent comparable to that in cells in which Monarch-1 is silenced via short hairpin RNA. The results of this study are consistent with a model wherein ATP binding regulates the anti-inflammatory activity of Monarch-1.

Cutting Edge: Monarch-1: A Pyrin/Nucleotide-Binding Domain/Leucine-Rich Repeat Protein That Controls Classical and Nonclassical MHC Class I Genes

Proteins containing a limited number of N-terminal motifs followed by nucleotide-binding domain and leucine-rich repeat regions are emerging as important regulators for immunity. A search of human genome scaffold databases has identified a large family of known and unknown genes, which we have recently called the CATERPILLER (caspase recruitment domain, transcription enhancer, r(purine)-binding, pyrin, lots of leucine repeats) gene family. This work describes the characterization of a new member, Monarch-1. Monarch-1 has four different splice forms due to the differential splicing of leucine-rich repeat motifs. It is expressed in cells of myeloid-monocytic origin. Affymetrix microarrays and small interfering RNA were used to elucidate the downstream effects of Monarch-1 expression in cells including those of myeloid-monocytic origin. These analyses show that Monarch-1 enhances nonclassical and classical MHC class I expression at the level of the promoter, RNA, and protein expression.

Characterization of murine BATF: a negative regulator of activator protein-1 activity in the thymus.

BATF belongs to the AP‐1/ATF superfamily of transcription factors and forms heterodimers with Jun proteins to bind AP‐1 consensus DNA. Unlike Fos/Jun heterodimers which stimulate gene transcription, BATF/Jun heterodimers are transcriptionally inert and inhibit biological processes that are associated with the overstimulation of AP‐1 activity. Here, we describe the murine BATF cDNA and genomic clones and map the BATF locus to chromosome 12 D2‐3. Using in situ hybridization of BATF mRNA, we show that BATF gene expression is highly restricted, with the most prominent signals detected in the thymus. BATF mRNA levels are regulated differentially during discrete stages of T cell development and are up‐regulated following activation of T cells in the periphery. To demonstrate the impact of BATF on AP‐1 activity in vivo, AP‐1 luciferase reporter mice were crossed to transgenic mice overexpressing BATF exclusively inthymic T cells. Results show that elevated levels of BATF protein correlate with reduced transactivation by AP‐1. Since the differential regulation of AP‐1 activity is linked to key transitions in the developing immune system, our observations support a critical role for BATF in determining the overall level of AP‐1 activity, and thus AP‐1 target gene expression, in specific T cell subtypes.

The role of surfactant protein A in bleomycin-induced acute lung injury.

RATIONALE Surfactant protein A (SP-A) is a collectin family member that has multiple immunomodulatory roles in lung host defense. SP-A levels are altered in the bronchoalveolar lavage (BAL) fluid and serum of patients with acute lung injury and acute respiratory distress syndrome, suggesting the importance of SP-A in the pathogenesis of acute lung injury. OBJECTIVES Investigate the role of SP-A in the murine model of noninfectious lung injury induced by bleomycin treatment. METHODS Wild-type (WT) or SP-A deficient (SP-A(-/-)) mice were challenged with bleomycin, and various indices of lung injury were analyzed. MEASUREMENTS AND MAIN RESULTS On challenge with bleomycin, SP-A(-/-) mice had a decreased survival rate as compared with WT mice. SP-A(-/-) mice had a higher degree of neutrophil-dominant cell recruitment and the expression of the inflammatory cytokines in BAL fluid than did WT mice. In addition, SP-A(-/-) mice had increased lung edema as assessed by the increased levels of intravenously injected Evans blue dye leaking into the lungs. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and active caspase-3 staining suggested the increased apoptosis in the lung sections from SP-A(-/-) mice challenged with bleomycin. SP-A also specifically reduced bleomycin-induced apoptosis in mouse lung epithelial 12 cells in vitro. Moreover, intratracheal administration of exogenous SP-A rescued the phenotype of SP-A(-/-) mice in vivo. CONCLUSIONS These data suggest that SP-A plays important roles in modulating inflammation, apoptosis, and epithelial integrity in the lung in response to acute noninfectious challenges.

BATF Transgenic Mice Reveal a Role for Activator Protein-1 in NKT Cell Development

The importance of regulated AP-1 activity during T cell development was assessed using transgenic mice overexpressing BATF, a basic leucine zipper transcription factor and an AP-1 inhibitor. BATF transgenic animals possess normal thymic cellularity and all major T cell subsets, but show impaired thymocyte proliferation in vitro and no induction of IL-2, IL-4, IL-5, IL-10, and IL-13 expression. Since NKT cells are largely responsible for cytokine production in the thymus, this population was examined by detection of the Vα14-Jα281 TCR, flow cytometry of NK1.1+ TCRβ+ cells, and analysis of cytokine production by heat-stable Aglow thymocytes and peripheral NKT cells stimulated in vivo. Results show a severe under-representation of NKT cells in BATF transgenic animals, providing the first evidence that the precise control of AP-1-mediated transcription is critical for the proper emergence of thymus-derived NKT cells in the mouse.

The inflammasome regulatory pathway and infections: Role in pathophysiology and clinical implications

The innate immune system serves to generate immediate host defenses against pathogens. Advance in the mechanism of innate immunity has provided new insights into host-pathogen microbial interactions. The cytosolic multi-protein complex called the inflammasome, which regulates the caspase-1 dependent processing of inflammatory cytokines IL-1β and IL-18, is critical for the innate defense against pathogens. We summarize the current knowledge regarding the regulatory functions of the inflammasome in the pathogenesis of infections by various microbes (e.g., bacteria, fungi, viruses, and protozoa), and discuss its potential application in a clinical setting. Understanding of the unique role of the inflammasome signaling pathway in initiating and regulating inflammation is pivotal for the development of innovative approaches to optimize management of these infections.

Blimp-1/PRDM1 Mediates Transcriptional Suppression of the NLR Gene NLRP12/Monarch-1

NLR (nucleotide-binding domain, leucine-rich repeat) proteins are intracellular regulators of host defense and immunity. One NLR gene, NLRP12 (NLR family, pyrin domain containing 12)/Monarch-1, has emerged as an important inhibitor of inflammatory gene expression in human myeloid cells. This is supported by genetic analysis linking the loss of a functional NLRP12 protein to hereditary periodic fever. NLRP12 transcription is diminished by specific TLR stimulation and myeloid cell maturation, consistent with its role as a negative regulator of inflammation. The NLRP12 promoter contains a novel Blimp-1 (B lymphocyte-induced maturation protein-1)/PRDM1 (PR domain-containing 1, with ZNF domain) binding site, and Blimp-1 reduces NLRP12 promoter activity, expression, and histone 3 acetylation. Blimp-1 associates with the endogenous NLRP12 promoter in a TLR-inducible manner and mediates the down-regulation of NLRP12 expression by TLR agonists. As expected, the expression of NLRP12 and Blimp-1 is inversely correlated. Analysis of Blimp-1−/− murine myeloid cells provides physiologic evidence that Blimp-1 reduces NLRP12 gene expression during cell differentiation. This demonstrates a novel role for Blimp-1 in the regulation of an NLR gene.

Direct manipulation of activator protein-1 controls thymocyte proliferation in vitro.

B cell activating transcription factor (BATF) belongs to the activator protein‐1 (AP‐1) superfamily of basic leucine zipper transcription factors and forms heterodimers with Jun that possess minimal transcriptional activity. Mice carrying a p56lckHA‐BATF transgene were created to observe the effects of constitutive expression of this well‐characterized AP‐1 inhibitor on T cell proliferation. Consistent with the role of AP‐1 in promoting the proliferation of many cell types, BATF‐transgenic thymocytes proliferate poorly in vitro when stimulated with anti‐CD3ϵ and anti‐CD28 antibodies or with Concanavalin A. However, when BATF‐transgenic thymocytes were stimulated using a standard treatment of PMA and ionomycin, proliferation is normal. The responsiveness to PMA and ionomycin can be attributed to the dramatic disappearance of the hemagglutinin antigen (HA)‐tagged BATF protein which is a PKC‐dependent process caused by the down‐regulation of the p56lck proximal promoter coupled with the rapid turnover of the HA‐BATF protein. These studies describe conditions of T cell stimulation that negatively influence transcription of the widely used p56lck proximal promoter expression cassette. In addition, the unique circumstances of this regulation were exploited to demonstrate that inhibition of AP‐1 activity by BATF exerts a direct, and reversible, effect on T cell proliferation in vitro.