Matthew D. McGeough

Inflammasome-mediated disease animal models reveal roles for innate but not adaptive immunity.

NLRP3 nucleates the inflammasome, a protein complex responsible for cleavage of prointerleukin-1beta (IL-1beta) to its active form. Mutations in the NLRP3 gene cause the autoinflammatory disease spectrum cryopyrin-associated periodic syndromes (CAPS). The central role of IL-1beta in CAPS is supported by the response to IL-1-targeted therapy. We developed two Nlrp3 mutant knockin mouse strains to model CAPS to examine the role of other inflammatory mediators and adaptive immune responses in an innate immune-driven disease. These mice had systemic inflammation and poor growth, similar to some human CAPS patients, and demonstrated early mortality, primarily mediated by myeloid cells. Mating these mutant mice to various gene mutant backgrounds showed that the mouse disease phenotype required an intact inflammasome, was only partially dependent on IL-1beta, and was independent of T cells. These data suggest that CAPS are true inflammasome-mediated diseases and provide insight for more common inflammatory disorders.

NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice

Inflammasome activation plays a central role in the development of drug‐induced and obesity‐associated liver disease. However, the sources and mechanisms of inflammasome‐mediated liver damage remain poorly understood. Our aim was to investigate the effect of NLRP3 inflammasome activation on the liver using novel mouse models. We generated global and myeloid cell‐specific conditional mutant Nlrp3 knock‐in mice expressing the D301N Nlrp3 mutation (ortholog of D303N in human NLRP3), resulting in a hyperactive NLRP3. To study the presence and significance of NLRP3‐initiated pyroptotic cell death, we separated hepatocytes from nonparenchymal cells and developed a novel flow‐cytometry–based (fluorescence‐activated cell sorting; FACS) strategy to detect and quantify pyroptosis in vivo based on detection of active caspase 1 (Casp1)‐ and propidium iodide (PI)‐positive cells. Liver inflammation was quantified histologically by FACS and gene expression analysis. Liver fibrosis was assessed by Sirius Red staining and quantitative polymerase chain reaction for markers of hepatic stellate cell (HSC) activation. NLRP3 activation resulted in shortened survival, poor growth, and severe liver inflammation; characterized by neutrophilic infiltration and HSC activation with collagen deposition in the liver. These changes were partially attenuated by treatment with anakinra, an interleukin‐1 receptor antagonist. Notably, hepatocytes from global Nlrp3‐mutant mice showed marked hepatocyte pyroptotic cell death, with more than a 5‐fold increase in active Casp1/PI double‐positive cells. Myeloid cell‐restricted mutant NLRP3 activation resulted in a less‐severe liver phenotype in the absence of detectable pyroptotic hepatocyte cell death. Conclusions: Our data demonstrate that global and, to a lesser extent, myeloid‐specific NLRP3 inflammasome activation results in severe liver inflammation and fibrosis while identifying hepatocyte pyroptotic cell death as a novel mechanism of NLRP3‐mediated liver damage. (Hepatology 2014;59:898–910)

Divergence of IL-1, IL-18, and cell death in NLRP3 inflammasomopathies

The inflammasome is a cytoplasmic multiprotein complex that promotes proinflammatory cytokine maturation in response to host- and pathogen-derived signals. Missense mutations in cryopyrin (NLRP3) result in a hyperactive inflammasome that drives overproduction of the proinflammatory cytokines IL-1β and IL-18, leading to the cryopyrin-associated periodic syndromes (CAPS) disease spectrum. Mouse lines harboring CAPS-associated mutations in Nlrp3 have elevated levels of IL-1β and IL-18 and closely mimic human disease. To examine the role of inflammasome-driven IL-18 in murine CAPS, we bred Nlrp3 mutations onto an Il18r-null background. Deletion of Il18r resulted in partial phenotypic rescue that abolished skin and visceral disease in young mice and normalized serum cytokines to a greater extent than breeding to Il1r-null mice. Significant systemic inflammation developed in aging Nlrp3 mutant Il18r-null mice, indicating that IL-1 and IL-18 drive pathology at different stages of the disease process. Ongoing inflammation in double-cytokine knockout CAPS mice implicated a role for caspase-1-mediated pyroptosis and confirmed that CAPS is inflammasome dependent. Our results have important implications for patients with CAPS and residual disease, emphasizing the need to explore other NLRP3-mediated pathways and the potential for inflammasome-targeted therapy.

ORMDL3 transgenic mice have increased airway remodeling and airway responsiveness characteristic of asthma

Orosomucoid-like (ORMDL)3 has been strongly linked with asthma in genetic association studies. Because allergen challenge induces lung ORMDL3 expression in wild-type mice, we have generated human ORMDL3 zona pellucida 3 Cre (hORMDL3zp3-Cre) mice that overexpress human ORMDL3 universally to investigate the role of ORMDL3 in regulating airway inflammation and remodeling. These hORMDL3zp3-Cre mice have significantly increased levels of airway remodeling, including increased airway smooth muscle, subepithelial fibrosis, and mucus. hORMDL3zp3-Cre mice had spontaneously increased airway responsiveness to methacholine compared to wild-type mice. This increased airway remodeling was associated with selective activation of the unfolded protein response pathway transcription factor ATF6 (but not Ire1 or PERK). The ATF6 target gene SERCA2b, implicated in airway remodeling in asthma, was strongly induced in the lungs of hORMDL3zp3-Cre mice. Additionally, increased levels of expression of genes associated with airway remodeling (TGF-β1, ADAM8) were detected in airway epithelium of these mice. Increased levels of airway remodeling preceded increased levels of airway inflammation in hORMDL3zp3-Cre mice. hORMDL3zp3-Cre mice had increased levels of IgE, with no change in levels of IgG, IgM, and IgA. These studies provide evidence that ORMDL3 plays an important role in vivo in airway remodeling potentially through ATF6 target genes such as SERCA2b and/or through ATF6-independent genes (TGF-β1, ADAM8).

Cutting Edge: IL-6 Is a Marker of Inflammation with No Direct Role in Inflammasome-Mediated Mouse Models

IL-6 is a known downstream target of IL-1β and is consistently increased in serum from patients with NLRP3 inflammasome-mediated conditions. Therefore, IL-6 could be a therapeutic target in the treatment of IL-1β–provoked inflammation. IL-6 was increased in serum with accompanying neutrophilia in tissues of an inducible mouse model of Muckle–Wells syndrome. However, an IL-6–null background failed to provide phenotypic rescue and did not significantly impact inflammatory cytokine levels. In a second model of IL-1β–driven inflammation, NLRP3 activation by monosodium urate crystals similarly increased IL-6. Consistent with our Muckle–Wells syndrome model, ablation of IL-6 did not impact an acute neutrophilic response in this in vivo evaluation of gouty arthritis. Taken together, our results indicate that IL-6 is a reliable marker of inflammation, with no direct role in inflammasome-mediated disease.

Independent roles of the priming and the triggering of the NLRP3 inflammasome in the heart

AIMS The NLRP3 inflammasome is activated in the ischaemic heart promoting caspase-1 activation, inflammation, and cell death. Ischaemic injury establishes both a priming signal (transcription of inflammasome components) and a trigger (NLRP3 activation). Whether NLRP3 activation, without priming, induces cardiac dysfunction and/or failure is unknown. The aim of this study was to assess the independent and complementary roles of the priming and the triggering signals in the heart, in the absence of ischaemia or myocardial injury. METHODS AND RESULTS We used mice with mutant NLRP3 (constitutively active), NLRP3-A350V, under the control of tamoxifen-driven expression of the Cre recombinase (Nlrp3-A350V/CreT mice). The mice were treated for 10 days with tamoxifen before measuring the activity of caspase-1, the effector enzyme in the inflammasome. Tamoxifen treatment induced the inflammasome in the spleen but not in the heart, despite expression of the mutant NLRP3-A350V. The components of the inflammasome were significantly less expressed in the heart compared with the spleen. Subclinical low-dose lipopolysaccharide (LPS; 2 mg/kg) in Nlrp3-A350V/CreT mice induced the expression of the components of the inflammasome (priming), measured using real-time PCR and western blot, leading to the formation of an active inflammasome (caspase-1 activation) in the heart and LV systolic dysfunction while low-dose LPS was insufficient to induce LV systolic dysfunction in wild-type mice (all P < 0.01 for mutant vs. wild-type mice). CONCLUSION The signalling pathway governing the inflammasome formation in the heart requires a priming signal in order for an active NLRP3 to induce caspase-1 activation and LV dysfunction.

NLRP3 mediates osteolysis through inflammation-dependent and -independent mechanisms

Activating‐mutations in NOD‐like receptor (NLR) family, pyrin domain‐containing 3 (NLRP3) cause neonatal‐onset multisystem inflammatory disease. However, the ontogeny of skeletal anomalies in this disorder is poorly understood. Mice globally expressing the D301N mutation in Nlrp3 (D303N in human) model the human phenotype, including systemic inflammation and skeletal deformities. To gain insights into the skeletal manifestations, we generated mice in which the expression of D301N Nlrp3 (Nlrp3D301N) is restricted to myeloid cells. These mice exhibit systemic inflammation and severe osteopenia (~60% lower bone mass) similar to mice globally expressing the knock‐in mutation, consistent with the paradigm of innate immune‐driven cryopyrinopathies. Because systemic inflammation may indirectly affect bone homeostasis, we engineered mice in which Nlrp3D301N is expressed specifically in osteoclasts, the cells that resorb bone. These mice also develop ~50% lower bone mass due to increased osteolysis, but there is no systemic inflammation and no change in osteoclast number. Mechanistically, aside from its role in IL‐1β maturation, Nlrp3D301N expression enhances osteoclast bone resorbing ability through reorganization of actin cytoskeleton while promoting the degradation of poly(ADP‐ribose) polymerase 1, an inhibitor of osteoclastogenesis. Thus, NLRP3 inflammasome activation is not restricted to the production of proinflammatory mediators but also leads to cytokine‐autonomous responses.—Qu, C., Bonar, S. L., Hickman‐Brecks, C. L., Abu‐Amer, S., McGeough, M. D., Peña, C. A., Broderick, L., Yang, C., Grimston, S., K., Kading, J., Abu‐Amer, Y., Novack, D. V., Hoffman, H. M., Civitelli, R., Mbalaviele, G. NLRP3 mediates osteolysis through inflammation‐dependent and ‐independent mechanisms. FASEB J. 29, 1269‐1279 (2015). www.fasebj.org

Functional consequences of EpCam mutation in mice and men

Congenital tufting enteropathy (CTE) is a severe diarrheal disease of infancy characterized by villous changes and epithelial tufts. We previously identified mutations in epithelial cell adhesion molecule (EpCAM) as the cause of CTE. We developed an in vivo mouse model of CTE based on EpCAM mutations found in patients with the aim to further elucidate the in vivo role of EpCAM and allow for a direct comparison to human CTE. Using Cre-LoxP recombination technology, we generated a construct lacking exon 4 in Epcam. Epcam(Δ4/Δ4) mice and CTE patient intestinal tissue integrity was analyzed by histology using both light immunohistochemistry and electron microscopy. Epcam(Δ4/Δ4) mice demonstrate neonatal lethality and growth retardation with pathological features, including epithelial tufts, enterocyte crowding, altered desmosomes, and intercellular gaps, similar to human CTE patients. Mutant EpCAM protein is present at low levels and is mislocalized in the intestine of Epcam(Δ4/Δ4) mice and CTE patients. Deletion of exon 4 was found to decrease expression of both EpCAM and claudin-7 causing a loss of colocalization, functionally disrupting the EpCAM/claudin-7 complex, a finding for the first time confirmed in CTE patients. Furthermore, compared with unaffected mice, mutation of Epcam leads to enhanced permeability and intestinal cell migration, uncovering underlying disease mechanisms.

NLRP3 inflammasome driven liver injury and fibrosis: Roles of IL-17 and TNF in mice.

The NLRP3 inflammasome, a caspase‐1 activation platform, plays a key role in the modulation of liver inflammation and fibrosis. Here, we tested the hypothesis that interleukin 17 (IL‐17) and tumor necrosis factor (TNF) are key cytokines involved in amplifying and perpetuating the liver damage and fibrosis resulting from NLRP3 activation. To address this hypothesis, gain‐of‐function Nlrp3A350V knock‐in mice were bred onto il17a and Tnf knockout backgrounds allowing for constitutive Nlrp3 activation in myeloid derived cells in mice deficient in IL‐17 or TNF. Livers of Nlrp3A350V knock‐in mice exhibited severe liver inflammatory changes characterized by infiltration with neutrophils, increased expression of chemokine (C‐X‐C motif) ligand (CXCL) 1 and CXCL2 chemokines, activated inflammatory macrophages, and elevated levels of IL‐17 and TNF. Mutants with ablation of il17a signal showed fewer neutrophils when compared to intact Nlrp3A350V mutants, but still significant inflammatory changes when compared to the nonmutant il17a knockout littermates. The severe inflammatory changes associated with mutant Nlrp3 were almost completely rescued by Tnf knockout in association with a marked decrease in circulating IL‐1β levels. Intact Nlrp3A350Vmutants showed changes in liver fibrosis, as evidenced by morphometric quantitation of Sirius Red staining and increased mRNA levels of profibrotic genes, including connective tissue growth factor and tissue inhibitor of matrix metalloproteinase 1. Il17a lacking mutants exhibited amelioration of the aforementioned fibrosis, whereas Tnf‐deficient mutants showed no signs of fibrosis when compared to littermate controls. Conclusion: Our study uncovers key roles for TNF and, to a lesser extent, IL‐17 as mediators of liver inflammation and fibrosis induced by constitutive NLRP3 inflammasome activation in myeloid‐derived cells. These findings may lead to therapeutic strategies aimed at halting the progression of liver injury and fibrogenesis in various liver pathogeneses driven by NLRP3 activation. (Hepatology 2018;67:736‐749).

Cutting Edge: Targeting Epithelial ORMDL3 Increases, Rather than Reduces, Airway Responsiveness and Is Associated with Increased Sphingosine-1-Phosphate

In this study, we used cre-lox techniques to generate mice selectively deficient in ORMDL3 in airway epithelium (Ormdl3Δ2-3/Δ2-3/CC10) to simulate an inhaled therapy that effectively inhibited ORMDL3 expression in the airway. In contrast to the anticipated reduction in airway hyperresponsiveness (AHR), OVA allergen–challenged Ormdl3Δ2-3/Δ2-3/CC10 mice had a significant increase in AHR compared with wild-type mice. Levels of airway inflammation, mucus, fibrosis, and airway smooth muscle were no different in Ormdl3Δ2-3/Δ2-3/CC10 and wild-type mice. However, levels of sphingosine-1-phosphate (S1P) were significantly increased in Ormdl3Δ2-3/Δ2-3/CC10 mice as well as in airway epithelial cells in which ORMDL3 was inhibited with small interfering RNA. Incubation of S1P with airway smooth muscle cells significantly increased contractility. Overall, Ormdl3Δ2-3/Δ2-3/CC10 mice exhibit increased allergen-induced AHR independent of inflammation and associated with increased S1P generation. These studies raise concerns for inhaled therapies that selectively and effectively inhibit ORMDL3 in airway epithelium in asthma.