Kathleen M. Azzam

Glucocorticoids sensitize the innate immune system through regulation of the NLRP3 inflammasome

Background: The ability of glucocorticoids to regulate the expression and function of the NOD-like receptors is unknown. Results: Glucocorticoids enhance the expression and function of NLRP3, promoting the secretion of IL-1β in response to ATP. Conclusion: Glucocorticoids sensitize the innate immune system to specific stimuli. Significance: This work demonstrates a novel, proinflammatory role for glucocorticoids, enhancing the activation of the innate immune system in response to danger signals. Glucocorticoids have long been recognized as powerful anti-inflammatory compounds that are one of the most widely prescribed classes of drugs in the world. However, their role in the regulation of innate immunity is not well understood. We sought to examine the effects of glucocorticoids on the NOD-like receptors (NLRs), a central component of the inflammasome and innate immunity. Surprisingly, we show that glucocorticoids induce both NLRP3 messenger RNA and protein, which is a critical component of the inflammasome. The glucocorticoid-dependent induction of NLRP3 sensitizes the cells to extracellular ATP and significantly enhances the ATP-mediated release of proinflammatory molecules, including mature IL-1β, TNF-α, and IL-6. This effect was specific for glucocorticoids and dependent on the glucocorticoid receptor. These studies demonstrate a novel role for glucocorticoids in sensitizing the initial inflammatory response by the innate immune system.

p53 Integrates host defense and cell fate during bacterial pneumonia.

p53 deletion augments neutrophil-mediated bacterial clearance in the lung at the expense of tissue homeostasis, leading to increased mortality.

Apolipoproteins and apolipoprotein mimetic peptides modulate phagocyte trafficking through chemotactic activity

Background: The anti-inflammatory properties of apolipoproteins are incompletely defined. Results: Apolipoprotein A-I and E mimetic peptides suppress CXCR2-dependent neutrophil migration in vivo. Mimetic L-37pA itself induces formyl peptide receptor-2-dependent chemotaxis. Conclusion: Apolipoprotein mimetics display complex structure-activity relationships to multiple chemotactic receptors. Significance: Apolipoproteins and their mimetics regulate leukocyte migration. The plasma lipoprotein-associated apolipoproteins (apo) A-I and apoE have well described anti-inflammatory actions in the cardiovascular system, and mimetic peptides that retain these properties have been designed as therapeutics. The anti-inflammatory mechanisms of apolipoprotein mimetics, however, are incompletely defined. Whether circulating apolipoproteins and their mimetics regulate innate immune responses at mucosal surfaces, sites where transvascular emigration of leukocytes is required during inflammation, remains unclear. Herein, we report that Apoai−/− and Apoe−/− mice display enhanced recruitment of neutrophils to the airspace in response to both inhaled lipopolysaccharide and direct airway inoculation with CXCL1. Conversely, treatment with apoA-I (L-4F) or apoE (COG1410) mimetic peptides reduces airway neutrophilia. We identify suppression of CXCR2-directed chemotaxis as a mechanism underlying the apolipoprotein effect. Pursuing the possibility that L-4F might suppress chemotaxis through heterologous desensitization, we confirmed that L-4F itself induces chemotaxis of human PMNs and monocytes. L-4F, however, fails to induce a calcium flux. Further exploring structure-function relationships, we studied the alternate apoA-I mimetic L-37pA, a bihelical analog of L-4F with two Leu-Phe substitutions. We find that L-37pA induces calcium and chemotaxis through formyl peptide receptor (FPR)2/ALX, whereas its D-stereoisomer (i.e. D-37pA) blocks L-37pA signaling and induces chemotaxis but not calcium flux through an unidentified receptor. Taken together, apolipoprotein mimetic peptides are novel chemotactic agents that possess complex structure-activity relationships to multiple receptors, displaying anti-inflammatory efficacy against innate immune responses in the airway.

p53 integrates host defense and cell fate during bacterial pneumonia

Cancer and infection are predominant causes of human mortality and derive, respectively, from inadequate genomic and host defenses against environmental agents. The transcription factor p53 plays a central role in human tumor suppression. Despite its expression in immune cells and broad responsiveness to stressors, it is virtually unknown whether p53 regulates host defense against infection. We report that the lungs of naive p53 / mice display genome-wide induction of NF-B response element–enriched proinflammatory genes, suggestive of type 1 immune priming. p53-null and p53 inhibitor–treated mice clear Gram-negative and -positive bacteria more effectively than controls after intrapulmonary infection. This is caused, at least in part, by cytokines produced by an expanded population of apoptosis-resistant, TLR-hyperresponsive alveolar macrophages that enhance airway neutrophilia. p53 / neutrophils, in turn, display heightened phagocytosis, Nox-dependent

MicroRNA-33 Regulates the Innate Immune Response via ATP Binding Cassette Transporter-mediated Remodeling of Membrane Microdomains

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression by promoting degradation and/or repressing translation of specific target mRNAs. Several miRNAs have been identified that regulate the amplitude of the innate immune response by directly targeting Toll-like receptor (TLR) pathway members and/or cytokines. miR-33a and miR-33b (the latter present in primates but absent in rodents and lower species) are located in introns of the sterol regulatory element-binding protein (SREBP)-encoding genes and control cholesterol/lipid homeostasis in concert with their host gene products. These miRNAs regulate macrophage cholesterol by targeting the lipid efflux transporters ATP binding cassette (ABC)A1 and ABCG1. We and others have previously reported that Abca1−/− and Abcg1−/− macrophages have increased TLR proinflammatory responses due to augmented lipid raft cholesterol. Given this, we hypothesized that miR-33 would augment TLR signaling in macrophages via a raft cholesterol-dependent mechanism. Herein, we report that multiple TLR ligands down-regulate miR-33 in murine macrophages. In the case of lipopolysaccharide, this is a delayed, Toll/interleukin-1 receptor (TIR) domain-containing adapter-inducing interferon-β-dependent response that also down-regulates Srebf-2, the host gene for miR-33. miR-33 augments macrophage lipid rafts and enhances proinflammatory cytokine induction and NF-κB activation by LPS. This occurs through an ABCA1- and ABCG1-dependent mechanism and is reversible by interventions upon raft cholesterol and by ABC transporter-inducing liver X receptor agonists. Taken together, these findings extend the purview of miR-33, identifying it as an indirect regulator of innate immunity that mediates bidirectional cross-talk between lipid homeostasis and inflammation.

Proteomic Analysis of ABCA1-Null Macrophages Reveals a Role for Stomatin-Like Protein-2 in Raft Composition and Toll-Like Receptor Signaling

Lipid raft membrane microdomains organize signaling by many prototypical receptors, including the Toll-like receptors (TLRs) of the innate immune system. Raft-localization of proteins is widely thought to be regulated by raft cholesterol levels, but this is largely on the basis of studies that have manipulated cell cholesterol using crude and poorly specific chemical tools, such as β-cyclodextrins. To date, there has been no proteome-scale investigation of whether endogenous regulators of intracellular cholesterol trafficking, such as the ATP binding cassette (ABC)A1 lipid efflux transporter, regulate targeting of proteins to rafts. Abca1−/− macrophages have cholesterol-laden rafts that have been reported to contain increased levels of select proteins, including TLR4, the lipopolysaccharide receptor. Here, using quantitative proteomic profiling, we identified 383 proteins in raft isolates from Abca1+/+ and Abca1−/− macrophages. ABCA1 deletion induced wide-ranging changes to the raft proteome. Remarkably, many of these changes were similar to those seen in Abca1+/+ macrophages after lipopolysaccharide exposure. Stomatin-like protein (SLP)-2, a member of the stomatin-prohibitin-flotillin-HflK/C family of membrane scaffolding proteins, was robustly and specifically increased in Abca1−/− rafts. Pursuing SLP-2 function, we found that rafts of SLP-2-silenced macrophages had markedly abnormal composition. SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux but reduced macrophage responsiveness to multiple TLR ligands. This was associated with reduced raft levels of the TLR co-receptor, CD14, and defective lipopolysaccharide-induced recruitment of the common TLR adaptor, MyD88, to rafts. Taken together, we show that the lipid transporter ABCA1 regulates the protein repertoire of rafts and identify SLP-2 as an ABCA1-dependent regulator of raft composition and of the innate immune response.

Key role for scavenger receptor B-I in the integrative physiology of host defense during bacterial pneumonia

Scavenger receptor B-I (SR-BI) is a multirecognition receptor that regulates cholesterol trafficking and cardiovascular inflammation. Although it is expressed by neutrophils (PMNs) and lung-resident cells, no role for SR-BI has been defined in pulmonary immunity. Herein, we report that, compared with SR-BI+/+ counterparts, SR-BI−/− mice suffer markedly increased mortality during bacterial pneumonia associated with higher bacterial burden in the lung and blood, deficient induction of the stress glucocorticoid corticosterone, higher serum cytokines, and increased organ injury. SR-BI−/− mice had significantly increased PMN recruitment and cytokine production in the infected airspace. This was associated with defective hematopoietic cell-dependent clearance of lipopolysaccharide from the airspace and increased cytokine production by SR-BI−/− macrophages. Corticosterone replacement normalized alveolar neutrophilia but not alveolar cytokines, bacterial burden, or mortality, suggesting that adrenal insufficiency derepresses PMN trafficking to the SR-BI−/− airway in a cytokine-independent manner. Despite enhanced alveolar neutrophilia, SR-BI−/− mice displayed impaired phagocytic killing. Bone marrow chimeras revealed this defect to be independent of the dyslipidemia and adrenal insufficiency of SR-BI−/− mice. During infection, SR-BI−/− PMNs displayed deficient oxidant production and CD11b externalization, and increased surface L-selectin, suggesting defective activation. Taken together, SR-BI coordinates several steps in the integrated neutrophilic host defense response to pneumonia.

Irgm1 coordinately regulates autoimmunity and host defense at select mucosal surfaces

The pathogenesis of primary Sjogrens syndrome (SS), an autoimmune disease that targets the mucosa of exocrine tissues, is poorly understood. Although several mouse models have been developed that display features of SS, most of these are within the larger context of a lupus-like presentation. Immunity-related GTPase family M protein 1 (Irgm1) is an interferon-inducible cytoplasmic GTPase that is reported to regulate autophagy and mitochondrial homeostasis. Here, we report that naive Irgm1-/- mice display lymphocytic infiltration of multiple mucosal tissues including the lung in a manner reminiscent of SS, together with IgA class-predominant autoantibodies including anti-Ro and anti-La. This phenotype persists in the germ-free state, but is abolished by deletion of Irgm3. Irgm1-/- mice have increased local production in the lung of TECP15-idiotype IgA, a natural antibody with dual reactivity against host and pneumococcal phosphorylcholine. Associated with this, Irgm1-/- mice display enhanced opsonization and clearance of Streptococcus pneumoniae from the lung and increased survival from pneumococcal pneumonia. Taken together, our results identify Irgm1 as a master regulator of mucosal immunity that dually modulates evolutionarily conserved self- and other-directed immune responses at the interface of host with environment.