Injury by Inflammasome
Inflammasomes form within the cytoplasm in response to infection or other intracellular changes and can activate caspase-1, which promotes maturation and release of the inflammatory cytokines IL-1β and IL-18. Activated caspase-1 has also been associated with a form of induced cell death known as pyroptosis. Kovarova et al. (p. 2006) characterized a role for the NLRP1b-containing inflammasome in response to anthrax lethal toxin (LT) using Nlrp1b−/− mice. Macrophages from Nlrp1b−/− mice were resistant to cell death upon exposure to LT, and further analysis in vitro demonstrated that LT induced NLRP1 inflammasome formation, caspase-1 activation, and IL-1β secretion. In contrast, the microbial ligand peptidoglycan or its derivative, muramyl dipeptide, required inflammasome formation with NLRP3, but not NLRP1, for IL-1β release. Nlrp1b−/− mice were protected from acute lung injury and death following intratracheal treatment with LT and showed few if any signs of morbidity and lung damage relative to control mice. Furthermore, LT-induced pyroptosis of alveolar macrophages was dependent on NLRP1b expression and caspase-1 activity. Together these observations show that NLRP1 inflammasome formation in response to LT causes pyroptosis, which is thought to contribute to escalated inflammation, devastating lung injury, and death.
Microbial sepsis arises in part from excessive host inflammation, which can be triggered by innate immune responses to pathogen-associated molecules like LPS. Leukocyte mono-Ig–like receptor 5 (LMIR5) is an activating receptor on myeloid cells, and Yamanishi et al. (p. 1773) now show that a soluble form of LMIR5 (sLMIR5) augments LPS-induced sepsis and death. LPS stimulation of the myeloid cell line RAW264.7 caused decreases in surface expression of LMIR5 due to extracellular release of sLMIR5 into the culture supernatant. Analysis of LPS-treated primary myeloid cells identified neutrophils as the primary source of sLMIR5, which was generated by proteolytic cleavage of surface LMIR5. Neutrophils were also identified as the major source of sLMIR5 in mice treated i.p. with LPS. I.p. injection of mice with LMIR5-Fc, a sLMIR5 surrogate, induced a significant increase in IL-6 and TNF-α in serum and peritoneal lavage fluid relative to injection with control Fc. This proinflammatory response was triggered by an interaction between LMIR5-Fc and an unknown ligand on peritoneal macrophages. LMIR5−/− mice injected i.p. with LPS or subjected to abdominal sepsis produced lower levels of proinflammatory cytokines and were protected from death compared with wild-type mice. Overall, these observations indicate that sLMIR5 from neutrophils may contribute to lethal inflammatory responses associated with sepsis.
PHD3’s Part in Inflammation
Hypoxic conditions stimulate macrophage proinflammatory activity. The prolyl hydroxylase domain-containing enzymes (PHD1, -2, and -3) activate the hypoxia-inducible factors (HIF)-1α and -2α, and each have additional, distinct functions. Two articles in this issue address the involvement of PHD3 in macrophage activity during inflammation. In mouse models of sepsis, Kiss et al. (p. 1955) found that mice deficient in PHD3, but not PHD1 or PHD2, suffered strikingly higher mortality than wild-type littermates. This heightened susceptibility to sepsis in PHD3−/− mice was accompanied by increased levels of plasma proinflammatory cytokines and multiorgan infiltration by macrophages and was dependent on the absence of PHD3 in myeloid cells. Compared with wild-type macrophages, PHD3−/− macrophages demonstrated enhanced proinflammatory cytokine production, migration, and phagocytic activity in response to LPS stimulation. PHD3 deficiency stimulated maturation of monocytes into macrophages and enhanced their LPS-induced polarization to the M1 (proinflammatory) phenotype, relative to wild-type cells. Finally, LPS treatment of PHD3−/− macrophages, compared with control cells, caused increases in NF-κB activation and HIF-1α protein stabilization, which were required for much of the excessive inflammatory activity observed in the absence of PHD3. Taken together, these data reveal a role for PHD3 in the regulation of macrophage inflammatory activity in polymicrobial sepsis.
In the second article, Escribese et al. (p. 1946) investigated PHD3 expression in human macrophages. Hypoxia is one of many factors that can alter macrophage polarization, and the involvement of PHD enzymes in HIF regulation prompted the authors to examine these enzymes in macrophages. PHD3 was upregulated at both the mRNA and protein levels in proinflammatory M1 macrophages but not in M2 macrophages. In vivo, PHD3 expression was detected at a high level within inflammatory environments and in a subset of alveolar macrophages under homeostatic conditions. Tumor-associated macrophages expressed PHD3 heterogeneously, such that it was found only on cells lacking anti-inflammatory M2 markers. Activin A, which is produced by M1 macrophages, promoted expression of the gene that encodes PHD3, EGLN3. Interestingly, hypoxia did not affect EGLN3 expression in M1 macrophages yet significantly upregulated this gene in M2 macrophages in an activin A-dependent manner. It remains to be seen whether the PHD3 expressed on human macrophages has a function similar to or distinct from that observed in mouse macrophages. However, these studies agree that PHD3 expression in macrophages is involved in the inflammatory response in both mice and humans.
Resisting Insulin Resistance
Obesity-induced insulin resistance is a major factor in the development of type 2 diabetes and is associated with proinflammatory macrophage infiltration of insulin target tissues. The G protein-coupled receptor GPR105 is activated by UDP-glucose and can regulate leukocyte chemotaxis, leading Xu et al. (p. 1992) to hypothesize that GPR105 might play a role in insulin resistance in high-fat diet (HFD)-induced obesity. Compared with wild-type littermates, HFD-fed GPR105-deficient mice were protected from systemic insulin resistance and showed improved insulin sensitivity in the liver, skeletal muscle, and adipose tissue. HFD-fed GPR105−/− mice also demonstrated reduced liver inflammation and lipid accumulation, relative to wild-type mice. However, significant differences from wild-type were not observed in macrophage infiltration or inflammatory gene expression in adipose tissue of GPR105−/− mice. Recruitment of GPR105−/− macrophages to the liver was significantly lower than that of wild-type macrophages, and UDP-glucose, which was upregulated in HFD-fed mice, could serve as a chemoattractant for wild-type but not GPR105−/− macrophages. Transplantation of GPR105−/− bone marrow into irradiated wild-type recipients confirmed that immune cells were the source of improved glucose tolerance in GPR105−/− mice. This study reveals a role for GPR105 in the development of insulin resistance in diet-induced obesity and encourages investigation of this receptor as a potential therapeutic target.
Protection Allows Presentation
The proteasome is an important cytosolic endoprotease that cleaves peptides for MHC class I presentation. The β1, β2, and β5 constitutive catalytic subunits are replaced during an immune response by the inducible subunits LMP2, MECL-1, and LMP7, respectively, to form the immunoproteasome. Previous data suggested that the structural properties but not the catalytic activities of the inducible subunits can be important for epitope generation. Basler et al. (p. 1868) analyzed the presentation of the HY Ag-derived epitope UTY246–254 to address the mechanism responsible for this somewhat counterintuitive observation. Presentation of the UTY246–254 epitope required the presence of LMP2 and LMP7. However, neither a selective inhibitor of LMP2 nor a catalytically inactive LMP2 mutant affected UTY246–254 presentation. Processing of the UTY protein by a proteasome containing the constitutive subunit β1 destroyed the UTY246–254 epitope, and this epitope could be rescued by a selective β1 inhibitor or overexpression of a catalytically inactive β1 mutant. Presentation of the LMP7-dependent influenza matrix M1 58–66 epitope was similarly unaffected by a specific LMP7 inhibitor but was enhanced by a β5 inhibitor. These data suggest that LMP7 protects matrix M1 58–66 from cleavage by β5 and LMP2 protects UTY246–254 from cleavage by β2, proposing a novel mechanistic basis for the importance of the inducible subunits of the immunoproteasome.
MicroRNAs (miRNAs) can regulate gene expression through targeted degradation of mRNAs. A recent study in mice deficient in the Dicer enzyme, which is required for miRNA generation, indicated that miRNAs may influence Th1 differentiation. Smith et al. (p. 1567) identified a role for miR-29b as a negative regulator of Th1 polarization. miR-29b was detected as a potential Th1-related miRNA by an in silico screen for miRNAs that target mRNA transcripts of the genes encoding T-bet and IFN-γ. Luciferase reporter gene expression assays confirmed that miR-29b directly targeted regions in the 3′-UTRs of both mRNAs. Mice generated with a deletion of the miR-29ab1 genomic cluster defined this cluster as the primary source of miR-29b in activated T cells. Polyclonal stimulation of miR-29ab–deficient CD4+ T cells induced significantly greater T-bet and IFN-γ expression relative to wild-type CD4+ T cells. IFN-γ treatment of polyclonally stimulated CD4+ T cells induced significantly greater miR-29b expression relative to polyclonal stimulation alone, which was linked to IFN-γ–induced STAT1 activity. Interestingly, miR-29ab–deficient mice were protected from severe experimental autoimmune encephalomyelitis (EAE). Relative to healthy controls, miR-29b levels were significantly higher in memory CD4+ T cells from multiple sclerosis (MS) patients but decreased during reactivation of resting memory CD4+ T cells. Together these results suggest that miR-29b regulates T-bet and IFN-γ expression, thus influencing Th1 polarization, and may become dysregulated during EAE and MS.