Lori Broderick

From NAFLD to NASH to cirrhosis—new insights into disease mechanisms

NAFLD has evolved as a serious public health problem in the USA and around the world. In fact, NASH—the most serious form of NAFLD—is predicted to become the leading cause of liver transplantation in the USA by the year 2020. The pathogenesis of NAFLD and NASH, in particular the mechanisms responsible for liver injury and fibrosis, is the result of a complex interplay between host and environmental factors, and is at the centre of intense investigation. In this Review, we focus on recently uncovered aspects of the genetic, biochemical, immunological and molecular events that are responsible for the development and progression of this highly prevalent and potentially serious disease. These studies bring new insight into this complex disorder and have led to the development of novel therapeutic and diagnostic strategies that might enable a personalized approach in the management of this disease.

The inflammasomes and autoinflammatory syndromes.

Inflammation, a vital response of the immune system to infection and damage to tissues, can be initiated by various germline-encoded innate immune-signaling receptors. Among these, the inflammasomes are critical for activation of the potent proinflammatory interleukin-1 cytokine family. Additionally, inflammasomes can trigger and maintain inflammatory responses aimed toward excess nutrients and the numerous danger signals that appear in a variety of chronic inflammatory diseases. We discuss our understanding of how inflammasomes assemble to trigger caspase-1 activation and subsequent cytokine release, describe how genetic mutations in inflammasome-related genes lead to autoinflammatory syndromes, and review the contribution of inflammasome activation to various pathologies arising from metabolic dysfunction. Insights into the mechanisms that govern inflammasome activation will help in the development of novel therapeutic strategies, not only for managing genetic diseases associated with overactive inflammasomes, but also for treating common metabolic diseases for which effective therapies are currently lacking.

Characterization of Human Lung Tumor-Associated Fibroblasts and Their Ability to Modulate the Activation of Tumor-Associated T Cells

The tumor microenvironment of human non-small cell lung cancer (NSCLC) is composed largely of stromal cells, including fibroblasts, yet these cells have been the focus of few studies. In this study, we established stromal cell cultures from primary NSCLC through isolation of adherent cells. Characterization of these cells by flow cytometry demonstrated a population which expressed a human fibroblast-specific 112-kDa surface molecule, Thy1, α-smooth muscle actin, and fibroblast activation protein, but failed to express CD45 and CD11b, a phenotype consistent with that of an activated myofibroblast. A subset of the tumor-associated fibroblasts (TAF) was found to express B7H1 (PD-L1) and B7DC (PD-L2) constitutively, and this expression was up-regulated by IFN-γ. Production of cytokines and chemokines, including IFN-γ, monokine induced by IFN-γ, IFN-γ-inducible protein-10, RANTES, and TGF-β1 was also demonstrated in these cells. Together, these characteristics provide multiple opportunities for the TAF to influence cellular interactions within the tumor microenvironment. To evaluate the ability of TAF to modulate tumor-associated T cell (TAT) activation, we conducted coculture experiments between autologous TAF and TAT. In five of eight tumors, TAF elicited a contact-dependent enhancement of TAT activation, even in the presence of a TGF-β1-mediated suppressive effect. In the three other tumors, TAF had a net suppressive effect upon TAT activation, and, in one of these cases, blockade of B7H1 or B7DC was able to completely abrogate the TAF-mediated suppression. We conclude that TAF in human NSCLC are functionally and phenotypically heterogeneous and provide multiple complex regulatory signals that have the potential to enhance or suppress TAT function in the tumor microenvironment.

Inflammasome activation leads to Caspase-1–dependent mitochondrial damage and block of mitophagy

Significance Sensors of the innate immune system trigger the induction of inflammatory responses upon infection and cellular stress. One such sensor is the inflammasome pathway, which is best described for its role in the production of the inflammatory cytokines IL-1β and IL-18. Other effector functions of the inflammasome pathway remain less well defined, and here we show that activation of this pathway leads to induction of mitochondrial damage and dismantling of the organelle. We link such mitochondrial damage to another inflammasome-regulated process called pyroptosis, which is a proinflammatory form of cell death. In summary, we characterize the role of mitochondrial damage during activation of the inflammasome pathway, of relevance to host defense and other physiological and pathophysiological settings. Inflammasomes are intracellular sensors that couple detection of pathogens and cellular stress to activation of Caspase-1, and consequent IL-1β and IL-18 maturation and pyroptotic cell death. Here, we show that the absent in melanoma 2 (AIM2) and nucleotide-binding oligomerization domain-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasomes trigger Caspase-1–dependent mitochondrial damage. Caspase-1 activates multiple pathways to precipitate mitochondrial disassembly, resulting in mitochondrial reactive oxygen species (ROS) production, dissipation of mitochondrial membrane potential, mitochondrial permeabilization, and fragmentation of the mitochondrial network. Moreover, Caspase-1 inhibits mitophagy to amplify mitochondrial damage, mediated in part by cleavage of the key mitophagy regulator Parkin. In the absence of Parkin activity, increased mitochondrial damage augments pyroptosis, as indicated by enhanced plasma membrane permeabilization and release of danger-associated molecular patterns (DAMPs). Therefore, like other initiator caspases, Caspase-1 activation by inflammasomes results in mitochondrial damage.

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.

Human CD4+ Effector Memory T Cells Persisting in the Microenvironment of Lung Cancer Xenografts Are Activated by Local Delivery of IL-12 to Proliferate, Produce IFN-γ, and Eradicate Tumor Cells

The implantation of small pieces of human primary lung tumor biopsy tissue into SCID mice results in a viable s.c. xenograft in which the tissue architecture, including tumor-associated leukocytes, tumor cells, and stromal cells, is preserved in a functional state. By monitoring changes in tumor volume, gene expression patterns, cell depletion analysis, and the use of function-blocking Abs, we previously established in this xenograft model that exogenous IL-12 mobilizes human tumor-associated leukocytes to kill tumor cells in situ by indirect mechanisms that are dependent upon IFN-γ. In this study immunohistochemistry and FACS characterize the early cellular events in the tumor microenvironment induced by IL-12. By 5 days post-IL-12 treatment, the constitutively present human CD45+ leukocytes have expanded and infiltrated into tumor-rich areas of the xenograft. Two weeks post-treatment, there is expansion of the human leukocytes and complete effacement of the tumor compared with tumor progression and gradual loss of most human leukocytes in control-treated xenografts. Immunohistochemical analyses reveal that the responding human leukocytes are primarily activated or memory T cells, with smaller populations of B cells, macrophages, plasma cells, and plasmacytoid dendritic cells capable of producing IFN-α. The predominant cell population was also characterized by FACS and was shown to have a phenotype consistent with a CD4+ effector memory T cell. We conclude that quiescent CD4+ effector memory T cells are present within the tumor microenvironment of human lung tumors and can be reactivated by the local and sustained release of IL-12 to proliferate and secrete IFN-γ, leading to tumor cell eradication.

Membrane-Associated TGF-β1 Inhibits Human Memory T Cell Signaling in Malignant and Nonmalignant Inflammatory Microenvironments

TGF-β1 is present on cells derived from the microenvironment of human lung tumors and nonmalignant inflammatory tissues. We establish that this cell-associated cytokine mediates hyporesponsiveness of the memory T cells in these microenvironments in situ by blocking TCR signaling. T cells derived from these tissues failed to translocate NF-κB to the nucleus in response to CD3 + CD28 cross-linking. This nonresponsiveness was reversed by an anti-TGF-β1-neutralizing Ab. Refractoriness of the memory T cells to TCR activation was also reversed by the removal of TGF-β1 by briefly pulsing the cells in a low pH buffer. Addition of exogenous TGF-β1 to eluted T cells re-established their nonresponsive state. Neither TGF-β1, anti-TGF-β1 Ab, nor low pH affected TCR signaling potential of peripheral blood T cells. We conclude that TGF-β1 mediates a physiologically relevant regulatory mechanism, selective for memory T cells present in the tumor microenvironment and nonmalignant chronic inflammatory tissues.

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.

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

Mutations of Complement Factor I and Potential Mechanisms of Neuroinflammation in Acute Hemorrhagic Leukoencephalitis

PurposeAcute Hemorrhagic Leukoencephalitis (AHLE) is a rare demyelinating disorder of acute onset, rapid deterioration and significant morbidity and mortality. Most often described as a post-infectious complication of an upper respiratory illness, its precise pathophysiology remains unclear. We describe two pediatric patients with AHLE with partial complement factor I (FI) deficiency whose successful treatment included the interleukin-1 (IL-1) receptor antagonist, anakinra, implicating a role for FI and IL-1 in this disorder.MethodsExtensive clinical workup of two patients presenting with AHLE revealed complement abnormalities, specifically related to the alternative pathway and its regulator, FI. Aggressive management with steroids, immunoglobulin, and anakinra ultimately led to improvement of clinical status and near return to neurologic baseline in both patients. Genetic sequencing of the FI coding regions of the patients and their families was performed. In vitro protein expression studies and immunohistochemistry of fixed brain tissue was used to investigate pathogenic mechanisms.ResultsTwo novel mutations in FI were identified in our patients, which result in failure to secrete FI. Immunohistochemical evaluation of brain tissue demonstrated positive staining for C3, membrane attack complex (MAC) and IL-1.ConclusionsWe propose AHLE is an unreported, rare phenotype for partial FI deficiency. The upregulation of C3, MAC and IL-1 with subsequent demyelination support a pathologic role for complement activation in AHLE, and suggest anakinra as an important adjunctive therapy in this disease.