Ali A. Abdul-Sater

Inflammasome-dependent Caspase-1 Activation in Cervical Epithelial Cells Stimulates Growth of the Intracellular Pathogen Chlamydia trachomatis

Inflammasomes have been extensively characterized in monocytes and macrophages, but not in epithelial cells, which are the preferred host cells for many pathogens. Here we show that cervical epithelial cells express a functional inflammasome. Infection of the cells by Chlamydia trachomatis leads to activation of caspase-1, through a process requiring the NOD-like receptor family member NLRP3 and the inflammasome adaptor protein ASC. Secretion of newly synthesized virulence proteins from the chlamydial vacuole through a type III secretion apparatus results in efflux of K+ through glibenclamide-sensitive K+ channels, which in turn stimulates production of reactive oxygen species. Elevated levels of reactive oxygen species are responsible for NLRP3-dependent caspase-1 activation in the infected cells. In monocytes and macrophages, caspase-1 is involved in processing and secretion of pro-inflammatory cytokines such as interleukin-1β. However, in epithelial cells, which are not known to secrete large quantities of interleukin-1β, caspase-1 has been shown previously to enhance lipid metabolism. Here we show that, in cervical epithelial cells, caspase-1 activation is required for optimal growth of the intracellular chlamydiae.

Enhancement of Reactive Oxygen Species Production and Chlamydial Infection by the Mitochondrial Nod-like Family Member NLRX1

Chlamydia trachomatis infections cause severe and irreversible damage that can lead to infertility and blindness in both males and females. Following infection of epithelial cells, Chlamydia induces production of reactive oxygen species (ROS). Unconventionally, Chlamydiae use ROS to their advantage by activating caspase-1, which contributes to chlamydial growth. NLRX1, a member of the Nod-like receptor family that translocates to the mitochondria, can augment ROS production from the mitochondria following Shigella flexneri infections. However, in general, ROS can also be produced by membrane-bound NADPH oxidases. Given the importance of ROS-induced caspase-1 activation in growth of the chlamydial vacuole, we investigated the sources of ROS production in epithelial cells following infection with C. trachomatis. In this study, we provide evidence that basal levels of ROS are generated during chlamydial infection by NADPH oxidase, but ROS levels, regardless of their source, are enhanced by an NLRX1-dependent mechanism. Significantly, the presence of NLRX1 is required for optimal chlamydial growth.

Chlamydial infection of monocytes stimulates IL-1β secretion through activation of the NLRP3 inflammasome

Chlamydia trachomatis infections represent the leading cause of bacterial sexually-transmitted disease in the United States and can cause serious tissue damage leading to infertility and ectopic pregnancies in women. Inflammation and hence the innate immune response to chlamydial infection contributes significantly to tissue damage, particularly by secreting proinflammatory cytokines such as interleukin (IL)-1beta from monocytes, macrophages and dendritic cells. Here we demonstrate that C. trachomatis or Chlamydia muridarum infection of a monocytic cell line leads to caspase-1 activation and IL-1beta secretion through a process requiring the NLRP3 inflammasome. Thus, secretion of IL-1beta decreased significantly when cells were depleted of NLRP3 or treated with the anti-inflammatory inhibitors parthenolide or Bay 11-7082, which inhibit inflammasomes and the transcription factor NF-kappaB. As for other infections causing NRLP3 inflammasome assembly, caspase-1 activation in monocytes is triggered by potassium efflux and reactive oxygen species production. However, anti-oxidants inhibited IL-1beta secretion only partially. Atypically for a bacterial infection, caspase-1 activation during chlamydial infection also involves partially the spleen tyrosine kinase (Syk), which is usually associated with a pathogen recognition receptor for fungal pathogens. Secretion of IL-1beta during infection by many bacteria requires both microbial products from the pathogen and an exogenous danger signal, but chlamydial infection provides both the pathogen-associated molecular patterns and danger signals necessary for IL-1beta synthesis and its secretion from human monocytes. Use of inhibitors that target the inflammasome in animals should therefore dampen inflammation during chlamydial infection.

Different STAT Transcription Complexes Drive Early and Delayed Responses to Type I IFNs

IFNs, which transduce pivotal signals through Stat1 and Stat2, effectively suppress the replication of Legionella pneumophila in primary murine macrophages. Although the ability of IFN-γ to impede L. pneumophila growth is fully dependent on Stat1, IFN-αβ unexpectedly suppresses L. pneumophila growth in both Stat1- and Stat2-deficient macrophages. New studies demonstrating that the robust response to IFN-αβ is lost in Stat1-Stat2 double-knockout macrophages suggest that Stat1 and Stat2 are functionally redundant in their ability to direct an innate response toward L. pneumophila. Because the ability of IFN-αβ to signal through Stat1-dependent complexes (i.e., Stat1-Stat1 and Stat1-Stat2 dimers) has been well characterized, the current studies focus on how Stat2 is able to direct a potent response to IFN-αβ in the absence of Stat1. These studies reveal that IFN-αβ is able to drive the formation of a Stat2 and IFN regulatory factor 9 complex that drives the expression of a subset of IFN-stimulated genes, but with substantially delayed kinetics. These observations raise the possibility that this pathway evolved in response to microbes that have devised strategies to subvert Stat1-dependent responses.

The signaling adaptor TRAF1 negatively regulates Toll-like receptor signaling and this underlies its role in rheumatic disease

TRAF1 is a signaling adaptor known for its role in tumor necrosis factor receptor-induced cell survival. Here we show that monocytes from healthy human subjects with a rheumatoid arthritis–associated single-nucleotide polymorphism (SNP) in the TRAF1 gene express less TRAF1 protein but greater amounts of inflammatory cytokines in response to lipopolysaccharide (LPS). The TRAF1 MATH domain binds directly to three components of the linear ubiquitination (LUBAC) complex, SHARPIN, HOIP and HOIL-1, to interfere with the recruitment and linear ubiquitination of NEMO. This results in decreased NF-κB activation and cytokine production, independently of tumor necrosis factor. Consistent with this, Traf1−/− mice show increased susceptibility to LPS-induced septic shock. These findings reveal an unexpected role for TRAF1 in negatively regulating Toll-like receptor signaling, providing a mechanistic explanation for the increased inflammation seen with a disease-associated TRAF1 SNP.

Dichotomous Expression of TNF Superfamily Ligands on Antigen-Presenting Cells Controls Post-priming Anti-viral CD4+ T Cell Immunity

SUMMARY T cell antigen‐presenting cell (APC) interactions early during chronic viral infection are crucial for determining viral set point and disease outcome, but how and when different APC subtypes contribute to these outcomes is unclear. The TNF receptor superfamily (TNFRSF) member GITR is important for CD4+ T cell accumulation and control of chronic lymphocytic choriomeningitis virus (LCMV). We found that type I interferon (IFN‐I) induced TNFSF ligands GITRL, 4–1BBL, OX40L, and CD70 predominantly on monocyte‐derived APCs and CD80 and CD86 predominantly on classical dendritic cells (cDCs). Mice with hypofunctional GITRL in Lyz2+ cells had decreased LCMV‐specific CD4+ T cell accumulation and increased viral load. GITR signals in CD4+ T cells occurred after priming to upregulate OX40, CD25, and chemokine receptor CX3CR1. Thus IFN‐I (signal 3) induced a post‐priming checkpoint (signal 4) for CD4+ T cell accumulation, revealing a division of labor between cDCs and monocyte‐derived APCs in regulating T cell expansion. HIGHLIGHTSType I IFN coordinately induces several TNF family ligands, including GITRL, on APCsTNF family ligands are largely expressed on monocyte‐derived APCs, not classical DCsGITRL on Lyz2+ APCs contributes to CD4+ T cell accumulation and viral controlGITR‐dependent signals occur after priming to upregulate CD25, OX40, and CX3CR1 T cell‐APC interactions modulate outcomes in chronic viral infections, but the division of labor between APC subtypes is unclear. Chang et al. reveal a dichotomy in costimulatory molecule expression whereby CD80 and CD86 are expressed mainly on classical DCs, and monocyte‐derived APCs provide TNF family ligands for the post‐priming accumulation of CD4+ T cells.

Statins Modulate Cyclooxygenase‐2 and Microsomal Prostaglandin E Synthase‐1 in Human Hepatic Myofibroblasts

Statins have been shown to exert anti‐inflammatory and anti‐fibrogenic properties in the liver. In the present study, we explored the mechanisms underlying anti‐fibrogenic effects of statins in isolated hepatic myofibroblasts and focused on cyclooxyegnase‐2, a major anti‐proliferative pathway in these cells. We show that simvastatin and fluvastatin inhibit thymidine incorporation in hMF in a dose‐dependent manner. Pretreatment of cells with NS398, a COX‐2 inhibitor, partially blunted this effect. cAMP levels, essential to the inhibition of hMF proliferation, were increased by statins and inhibited by non‐steroidal anti‐inflammatory drugs. Since statins modify prenylation of some important proteins in gene expression, we investigated the targets involved using selective inhibitors of prenyltransferases. Inhibition of geranylgeranylation resulted in the induction of COX‐2 and mPGES‐1. Using gel retardation assays, we further demonstrated that statins potentially activated the NFκB and CRE/E‐box binding for COX‐2 promoter and the binding of GC‐rich regions and GATA for mPGES‐1. Together these data demonstrate that statin limit hepatic myofibroblasts proliferation via a COX‐2 and mPGES‐1 dependent pathway. These data suggest that statin‐dependent increase of prostaglandin in hMF contributes to its anti‐fibrogenic effect. J. Cell. Biochem. 117: 1176–1186, 2016.

Interferon Regulation of the Innate Response to Bacteria

Characterization of potent antiviral response to Interferons (IFNs) led to identification of the JAK-STAT signaling cascade almost two decades ago. More recently, studies have begun to explore how IFNs participate in the innate response to bacterial infections. This includes the activation of classic antibacterial responses, like expression on inducible nitric oxidase (iNOS) and GTPases, as well as the induction of autophagy. Not surprisingly, studies on its anti-bacterial activity of IFNs have provided important new insights into IFN biology and JAK-STAT signaling.