IL-36α Enhances Host Defense against Pseudomonas aeruginosa Keratitis in C57BL/6 Mouse Corneas.

The IL-36 cytokines are known to play various roles in mediating the immune response to infection in a tissue- and pathogen-dependent manner. The present study seeks to investigate the role of IL-36R signaling in C57BL/6 mouse corneas in response to Pseudomonas aeruginosa infection. IL-36α-/-, IL-36γ-/-, and IL-36R-/- mice had significantly more severe keratitis than wild-type mice. At six hours postinfection, IL-36α pretreatment augmented P. aeruginosa-induced expression of IL-1Ra, IL-36γ, LCN2, and S100A8/A9. At one day postinfection, exogenous IL-36α suppressed, whereas IL-36α deficiency promoted, the expression of IL-1ß. At three days postinfection, exogenous IL-36α suppressed Th1 but promoted Th2 immune response. IL-36α stimulated the infiltration of IL-22-expressing immune cells, and IL-22 neutralization resulted in more severe keratitis. IL-36α alone stimulated dendritic cell infiltration in B6 mouse corneas. Taken together, our study suggests that IL-36R signaling plays a protective role in the pathogenesis of P. aeruginosa keratitis by promoting the innate immune defense, Th2, and/or Th22/IL-22 immune responses. Exogenous IL-36α might be a potential therapy for improving the outcome of P. aeruginosa keratitis.

A New Understanding of the Role of IL-1 in Age-Related Intervertebral Disc Degeneration in a Murine Model.

Increased cytokine expression, in particular interleukin-1ß (IL-1ß), is considered a hallmark of intervertebral disc degeneration. However, the causative relationship between IL-1 and age-dependent degeneration has not been established. To investigate the role of IL-1 in driving age-related disc degeneration, we studied the spine phenotype of global IL-1α/ß double knockout (IL-1KO) mice at 12 and 20 months. Multiplex ELISA analysis of blood revealed significant reductions in the concentrations of IFN-γ, IL-5, IL-15, TNF-α, IP-10, and a trend of reduced concentrations of IL-10, macrophage inflammatory protein 1α (MIP-1α), keratinocyte chemoattractant/human growth-regulated oncogene (KC/GRO), and IL-6. However, the circulating level of MIP-2, a neutrophil chemoattractant, was increased in the IL-1KO. The alterations in systemic cytokine levels coincided with altered bone morphology-IL-1KO mice exhibited significantly thicker caudal cortical bone at 12 and 20 months. Despite these systemic inflammatory and bony changes, IL-1 deletion only minimally affected disc health. Both wild-type (WT) and IL-1KO mice showed age-dependent disc degeneration. Unexpectedly, rather than protecting the animals from degeneration, the aging phenotype was more pronounced in IL-1KO animals: knockout mice evidenced significantly more degenerative changes in the annulus fibrosis (AF) together with alterations in collagen type and maturity. At 20 months, there were no changes in nucleus pulposus (NP) extracellular matrix composition or cellular marker expression; however, the IL-1KO NP cells occupied a smaller proportion of the NP compartment that those of WT controls. Taken together, these results show that IL-1 deletion altered the systemic inflammatory environment and vertebral bone morphology. However, instead of protecting discs from age-related disc degeneration, global IL-1 deletion amplified the degenerative phenotype. © 2019 American Society for Bone and Mineral Research.

Overlapping Roles for Interleukin-36 Cytokines in Protective Host Defense against Murine Legionella pneumophila Pneumonia.

Legionella pneumophila causes life-threatening pneumonia culminating in acute lung injury. Innate and adaptive cytokines play an important role in host defense against L. pneumophila infection. Interleukin-36 (IL-36) cytokines are recently described members of the larger IL-1 cytokine family known to exert potent inflammatory effects. In this study, we elucidated the role for IL-36 cytokines in experimental pneumonia caused by L. pneumophila Intratracheal (i.t.) administration of L. pneumophila induced the upregulation of both IL-36α and IL-36γ mRNA and protein production in the lung. Compared to the findings for L. pneumophila-infected wild-type (WT) mice, the i.t. administration of L. pneumophila to IL-36 receptor-deficient (IL-36R-/-) mice resulted in increased mortality, a delay in lung bacterial clearance, increased L. pneumophila dissemination to extrapulmonary organs, and impaired glucose homeostasis. Impaired lung bacterial clearance in IL-36R-/- mice was associated with a significantly reduced accumulation of inflammatory cells and the decreased production of proinflammatory cytokines and chemokines. Ex vivo, reduced expression of costimulatory molecules and impaired M1 polarization were observed in alveolar macrophages isolated from infected IL-36R-/- mice compared to macrophages from WT mice. While L. pneumophila-induced mortality in IL-36α- or IL-36γ-deficient mice was not different from that in WT animals, antibody-mediated neutralization of IL-36γ in IL-36α-/- mice resulted in mortality similar to that observed in IL-36R-/- mice, indicating redundant and overlapping roles for these cytokines in experimental murine L. pneumophila pneumonia.

IL-37 affects the occurrence and development of endometriosis by regulating the biological behavior of endometrial stromal cells through multiple signaling pathways.

Endometriosis (EMs) is a chronic inflammatory condition. Interleukin (IL)-37 is a member of the IL-1 family and an anti-inflammatory cytokine. This study aimed to evaluate the possible role of IL-37 in the EMs pathogenesis. We investigated the in vivo effect of IL-37 on EMs by injection with recombinant human IL-37 (rhIL-37) into EMs mice. Furthermore, we evaluated the in vitro effects of IL-37 on proliferation, adhesion, migration and invasiveness of endometrial stromal cells (ESCs), and explored whether Wnt/ß-catenin and mitogen-activated protein kinase (MAPK) pathways were involved in this process. In cultured ESCs, IL-37 overexpression significantly suppressed both protein and mRNA expression of the inflammation-associated cytokines, including IL-1ß, IL-6, IL-10 and tumor necrosis factor (TNF-α). Furthermore, IL-37 overexpression significantly inhibited ESCs proliferation, adhesion, migration, invasion and the activity of matrix metalloproteinase (MMP)-2 and MMP-9. In contrast, knockdown of IL-37 exerted the opposite effects. Importantly, the IL-37-mediated action in ESCs was through inactivation of Wnt/ß-catenin, p38 MAPK, extracellular signal-related kinases MAPK and c-Jun N-terminal kinase MAPK pathways. Moreover, EMs mice treated with rhIL-37 showed the decreased endometriotic-like lesion size and lesion weight, lower expression of IL-1ß, IL-6, IL-10, TNF-α, vascular endothelial growth factor (VEGF), soluble intercellular adhesion molecule-I (ICAM-I) and MMP-2/9 activity in peritoneal fluid compared with the wide type (WT) EMs mice. These findings suggest that IL-37 suppresses cell proliferation, adhesion, migration and invasion of human ESCs through multiple signaling pathways, thereby affecting the occurrence and development of EMs.

Role of the inflammasome-related cytokines Il-1 and Il-18 during infection with murine coronavirus.

The inflammasome, a cytosolic protein complex that mediates the processing and secretion of pro-inflammatory cytokines, is one of the first responders during viral infection. The cytokines secreted following inflammasome activation, which include IL-1 and IL-18, regulate cells of both the innate and adaptive immune system, guiding the subsequent immune responses. In this study, we used murine coronavirus, mouse hepatitis virus (MHV), infection of the central nervous system and liver to assess of the role of the inflammasome and its related cytokines on pathogenesis and host defense during viral infection. Mice lacking all inflammasome signaling due to the absence of caspase-1 and -11 were more vulnerable to infection, with poor survival and elevated viral replication compared to wild-type mice. Mice lacking IL-1 signaling experienced elevated viral replication but similar survival compared to wild-type controls. In the absence of IL-18, mice had elevated viral replication and poor survival, and this protective effect of IL-18 was found to be due to promotion of interferon gamma production in αß T cells. These data suggest that inflammasome signaling is largely protective during murine coronavirus infection, in large part due to the pro-inflammatory effects of IL-18.

Interleukin-36ß provides protection against HSV-1 infection, but does not modulate initiation of adaptive immune responses.

Interleukin-36 (IL-36) represents three cytokines, IL-36α, IL-36ß and IL-36γ, which bind to the same receptor, IL-1RL2; however, their physiological function(s) remain poorly understood. Here, the role of IL-36 in immunity against HSV-1 was examined using the flank skin infection mouse model. Expression analyses revealed increased levels of IL-36α and IL-36ß mRNA in infected skin, while constitutive IL-36γ levels remained largely unchanged. In human keratinocytes, IL-36α mRNA was induced by HSV-1, while IL-1ß and TNFα increased all three IL-36 mRNAs. The dominant alternative splice variant of human IL-36ß mRNA was isoform 2, which is the ortholog of the known mouse IL-36ß mRNA. Mice deficient in IL-36ß, but not IL-36α or IL-36γ, succumbed more frequently to HSV-1 infection than wild type mice. Furthermore, IL-36ß-/- mice developed larger zosteriform skin lesions along infected neurons. Levels of HSV-1 specific antibodies, CD8+ cells and IFNγ-producing CD4+ cells were statistically equal in wild type and IL-36ß-/- mice, suggesting similar initiation of adaptive immunity in the two strains. This correlated with the time at which HSV-1 genome and mRNA levels in primary skin lesions started to decline in both wild type and IL-36ß-/- mice. Our data indicate that IL-36ß has previously unrecognized functions protective against HSV-1 infection.

Role of Interleukin 36γ in Host Defense Against Tuberculosis.

Tuberculosis remains a major killer worldwide, not the least because of our incomplete knowledge of protective and pathogenic immune mechanism. The roles of the interleukin 1 (IL-1) and interleukin 18 pathways in host defense are well established, as are their regulation through the inflammasome complex. In contrast, the regulation of interleukin 36γ (IL-36γ), a recently described member of the IL-1 family, and its immunological relevance in host defense remain largely unknown. Here we show that Mycobacterium tuberculosis infection of macrophages induces IL-36γ production in a 2-stage-regulated fashion. In the first stage, microbial ligands trigger host Toll-like receptor and MyD88-dependent pathways, leading to IL-36γ secretion. In the second stage, endogenous IL-1ß and interleukin 18 further amplify IL-36γ synthesis. The relevance of this cytokine in the control of M. tuberculosis is demonstrated by IL-36γ-induced antimicrobial peptides and IL-36 receptor-dependent restriction of M. tuberculosis growth. Thus, we provide first insight into the induction and regulation of the proinflammatory cytokine IL-36γ during tuberculosis.

Gastric Metaplasia Induced by Helicobacter pylori Is Associated with Enhanced SOX9 Expression via Interleukin-1 Signaling.

Histopathological changes of the gastric mucosa after Helicobacter pylori infection, such as atrophy, metaplasia, and dysplasia, are considered to be precursors of gastric cancer, yet the mechanisms of histological progression are unknown. The aim of this study was to analyze the histopathological features of the gastric mucosa in mice infected with H. pylori strain PMSS1 in relation to gastric stem cell marker expression. C57BL/6J mice infected with PMSS1 were examined for histopathological changes, levels of proinflammatory cytokines, and expression of stem cell markers. Histopathological gastritis scores, such as atrophy and metaplasia, and levels of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß), were increased after PMSS1 infection. Expression levels of the cell proliferation and stem cell markers CD44 and SOX9 were also significantly increased in PMSS1-infected mice. Importantly, almost all metaplastic cells induced by PMSS1 infection expressed SOX9. When IL-1 receptor (IL-1R) knockout mice were infected with PMSS1, metaplastic changes and expression levels of stem cell markers were significantly decreased compared with those in wild-type (WT) mice. In conclusion, H. pylori infection induced the expression of cytokines and stem cell markers and histopathological metaplasia in the mouse gastric mucosa. SOX9 expression, in particular, was strongly associated with metaplastic changes, and these changes were dependent on IL-1 signaling. The results suggested the importance of SOX9 in gastric carcinogenesis.

Interleukin-driven insulin-like growth factor promotes prostatic inflammatory hyperplasia.

Prostatic inflammation is of considerable importance to urologic research because of its association with benign prostatic hyperplasia and prostate cancer. However, the mechanisms by which inflammation leads to proliferation and growth remain obscure. Here, we show that insulin-like growth factors (IGFs), previously known as critical developmental growth factors during prostate organogenesis, are induced by inflammation as part of the proliferative recovery to inflammation. Using genetic models and in vivo IGF receptor blockade, we demonstrate that the hyperplastic response to inflammation depends on interleukin-1-driven IGF signaling. We show that human prostatic hyperplasia is associated with IGF pathway activation specifically localized to foci of inflammation. This demonstrates that mechanisms of inflammation-induced epithelial proliferation and hyperplasia involve the induction of developmental growth factors, further establishing a link between inflammatory and developmental signals and providing a mechanistic basis for the management of proliferative diseases by IGF pathway modulation.

Activation of microglia induces symptoms of Parkinson's disease in wild-type, but not in IL-1 knockout mice.

BACKGROUND: Parkinson's disease (PD) is an age-related progressive neurodegenerative disorder caused by selective loss of dopaminergic neurons from the substantia nigra (SN) to the striatum. The initial factor that triggers neurodegeneration is unknown; however, inflammation has been demonstrated to be significantly involved in the progression of PD. The present study was designed to investigate the role of the pro-inflammatory cytokine interleukin-1 (IL-1) in the activation of microglia and the decline of motor function using IL-1 knockout (KO) mice. METHODS: Lipopolysaccharide (LPS) was stereotaxically injected into the SN of mice brains as a single dose or a daily dose for 5 days (5 mg/2 ml/injection, bilaterally). Animal behavior was assessed with the rotarod test at 2 hr and 8, 15 and 22 days after the final LPS injection. RESULTS: LPS treatment induced the activation of microglia, as demonstrated by production of IL-1ß and tumor necrosis factor (TNF) α as well as a change in microglial morphology. The number of cells immunoreactive for 4-hydroxynonenal (4HNE) and nitrotyrosine (NT), which are markers for oxidative insults, increased in the SN, and impairment of motor function was observed after the subacute LPS treatment. Cell death and aggregation of α-synuclein were observed 21 and 30 days after the final LPS injection, respectively. Behavioral deficits were observed in wild-type and TNFα KO mice, but IL-1 KO mice behaved normally. Tyrosine hydroxylase (TH) gene expression was attenuated by LPS treatment in wild-type and TNFα KO mice but not in IL-1 KO mice. CONCLUSIONS: The subacute injection of LPS into the SN induces PD-like pathogenesis and symptoms in mice that mimic the progressive changes of PD including the aggregation of α-synuclein. LPS-induced dysfunction of motor performance was accompanied by the reduced gene expression of TH. These findings suggest that activation of microglia by LPS causes functional changes such as dopaminergic neuron attenuation in an IL-1-dependent manner, resulting in PD-like behavioral impairment.

Role of interleukin-1/interleukin-1 receptor antagonist family of cytokines in long-term continuous glucose monitoring in vivo.

BACKGROUND: Glucose-sensor-induced tissue reactions (e.g., inflammation and wound healing) are known to negatively impact sensor function in vivo. The roles of cytokine networks in controlling these tissue reactions (i.e., sensor biofouling) is not understood. In the present study, we investigated the role of interleukin-1 receptor antagonist (IL-1Ra), a key anti-inflammatory antagonist of the proinflammatory interleukin-1 cytokines [i.e. interleukin-1 (IL-1) alpha and IL-1 beta] in controlling continuous glucose monitoring (CGM). METHODS: To investigate the role of IL-1Ra in long-term CGM in vivo, we compared CGM in transgenic mice that overexpress IL-1Ra [interleukin-1 receptor antagonist overexpresser (IL-1Ra~OE), B6.Cg-Tg(IL1rn)1Dih/J] or are deficient in IL-1Ra [interleukin-1 receptor antagonist knockout (IL-1Ra~KO), B6.129S-IL1rn(tm1Dih)/J] with mice that have normal levels of IL-1Ra (C57BL/6) over a 28-day time period. RESULTS: Mean absolute relative difference (MARD) analysis of CGM results among the mice of varying IL-1Ra levels demonstrated that during the first 21 days, IL-1~KO mice had the greatest tissue inflammation and the poorest sensor performance (i.e., higher MARD values) when compared with normal or IL-1Ra~OE mice. By 28 days post-sensor implantation, the inflammatory reactions had subsided and were replaced by varying degrees of fibrosis. CONCLUSIONS: These data support our hypothesis on the importance of the IL-1 family of agonists and antagonists in controlling tissue reactions and sensor function in vivo. These data also suggest that local delivery of IL-1Ra genes or recombinant proteins (anakinra) or other IL-1 antagonists such as antibodies or soluble IL-1 receptors would suppress sensor-induced tissue reactions and likely enhance glucose sensor function by inhibiting inflammation and wound healing at sensor implantation sites.

Hepatic macrophages but not dendritic cells contribute to liver fibrosis by promoting the survival of activated hepatic stellate cells in mice.

UNLABELLED: Although it is well established that hepatic macrophages play a crucial role in the development of liver fibrosis, the underlying mechanisms remain largely elusive. Moreover, it is not known whether other mononuclear phagocytes such as dendritic cells (DCs) contribute to hepatic stellate cell (HSC) activation and liver fibrosis. We show for the first time that hepatic macrophages enhance myofibroblast survival in a nuclear factor kappa B (NF-κB)-dependent manner and thereby promote liver fibrosis. Microarray and pathway analysis revealed no induction of HSC activation pathways by hepatic macrophages but a profound activation of the NF-κB pathway in HSCs. Conversely, depletion of mononuclear phagocytes during fibrogenesis in vivo resulted in suppressed NF-κB activation in HSCs. Macrophage-induced activation of NF-κB in HSCs in vitro and in vivo was mediated by interleukin (IL)-1 and tumor necrosis factor (TNF). Notably, IL-1 and TNF did not promote HSC activation but promoted survival of activated HSCs in vitro and in vivo and thereby increased liver fibrosis, as demonstrated by neutralization in coculture experiments and genetic ablation of IL-1 and TNF receptor in vivo. Coculture and in vivo ablation experiments revealed only a minor contribution to NF-κB activation in HSCs by DCs, and no contribution of DCs to liver fibrosis development, respectively. CONCLUSION: Promotion of NF-κB-dependent myofibroblast survival by macrophages but not DCs provides a novel link between inflammation and fibrosis.

Interleukin-1 participates in the classical and alternative activation of microglia/macrophages after spinal cord injury.

BACKGROUND: Microglia and macrophages (MG/MΦ) have a diverse range of functions depending on unique cytokine stimuli, and contribute to neural cell death, repair, and remodeling during central nervous system diseases. While IL-1 has been shown to exacerbate inflammation, it has also been recognized to enhance neuroregeneration. We determined the activating phenotype of MG/MΦ and the impact of IL-1 in an in vivo spinal cord injury (SCI) model of IL-1 knock-out (KO) mice. Moreover, we demonstrated the contribution of IL-1 to both the classical and alternative activation of MG in vitro using an adult MG primary culture. METHODS: SCI was induced by transection of the spinal cord between the T9 and T10 vertebra in wild-type and IL-1 KO mice. Locomotor activity was monitored and lesion size was determined for 14 days. TNFα and Ym1 levels were monitored to determine the MG/MΦ activating phenotype. Primary cultures of MG were produced from adult mice, and were exposed to IFNγ or IL-4 with and without IL-1ß. Moreover, cultures were exposed to IL-4 and/or IL-13 in the presence and absence of IL-1ß. RESULTS: The locomotor activity and lesion area of IL-1 KO mice improved significantly after SCI compared with wild-type mice. TNFα production was significantly suppressed in IL-1 KO mice. Also, Ym1, an alternative activating MG/MΦ marker, did not increase in IL-1 KO mice, suggesting that IL-1 contributes to both the classical and alternative activation of MG/MΦ. We treated primary MG cultures with IFNγ or IL-4 in the presence and absence of IL-1ß. Increased nitric oxide and TNFα was present in the culture media and increased inducible NO synthase was detected in cell suspensions following co-treatment with IFNγ and IL-1ß. Expression of the alternative activation markers Ym1 and arginase-1 was increased after exposure to IL-4 and further increased after co-treatment with IL-4 and IL-1ß. The phenotype was not observed after exposure of cells to IL-13. CONCLUSIONS: We demonstrate here in in vivo experiments that IL-1 suppressed SCI in a process mediated by the reduction of inflammatory responses. Moreover, we suggest that IL-1 participates in both the classical and alternative activation of MG in in vivo and in vitro systems.

Involvement of interleukin-1 in lead nitrate-induced hypercholesterolemia in mice.

Hepatic 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and cholesterol 7α-hydroxylase (Cyp7a1) are rate-limiting enzymes for cholesterol biosynthesis and catabolism, respectively. Involvement of inflammatory cytokines, particularly interleukin-1 (IL-1), in alterations of HMGR and Cyp7a1 gene expression during development of lead nitrate (LN)-induced hypercholesterolemia was examined in IL-1α/ß-knockout (IL-1-KO) and wild-type (WT) mice. Lead nitrate treatment of WT mice led to not only a marked downregulation of the Cyp7a1 gene at 6-12 h, but also a significant upregulation of the HMGR gene at 12 h. However, such changes were not observed at significant levels in IL-1-KO mice, although a slight, transient downregulation of the Cyp7a1 gene and a minimal upregulation of the HMGR gene occurred at 6 h and 24 h, respectively. Consequently, LN treatment led to development of hypercholesterolemia at 24 h in WT mice, but not in IL-1-KO mice. Furthermore, in WT mice, significant LN-mediated increases were observed at 3-6 h in hepatic IL-1 levels, which can modulate gene expression of Cyp7a1 and HMGR. These findings indicate that, in mice, LN-mediated increases in hepatic IL-1 levels contribute, at least in part, to altered expressions of Cyp7a1 and HMGR genes, and eventually to hypercholesterolemia development.

VSV oncolytic virotherapy in the B16 model depends upon intact MyD88 signaling.

We show here, for the first time to our knowledge, that the antitumor therapy of oncolytic vesicular stomatitis virus (VSV) in the B16ova model depends upon signaling through myeloid differentiation primary response gene 88 (MyD88) in host cells. VSV-mediated therapy of B16ova tumors was abolished in MyD88(-/-) mice despite generation of antigen-specific T cell responses similar to those in immune-competent mice. Mice defective in only toll-like receptor 4 (TLR4), TLR7, or interleukin 1 (IL-1) signaling retained VSV-induced therapy, suggesting that multiple, redundant pathways of innate immune activation by the virus contribute to antitumor immune reactivity. Lack of MyD88 signaling was associated with decreased expression of proinflammatory cytokines and neutrophil infiltration in response to intratumoral virus, as well as decreased infiltration of draining lymph nodes (LN) with plasmacytoid dendritic cells (pDCs) (CD11b(-)GR1(+)B220(+)) and myeloid-derived suppressor cells (CD11b(+)GR1(+)F4/80(+)). MyD88 signaling in response to VSV was also closely associated with a type I interferon (IFN) response. This inhibited virus replication within the tumor but also protected the host from viral dissemination from the tumor. Therefore, the innate immune response to oncolytic viruses can be, simultaneously, protherapeutic, antioncolytic, and systemically protective. These paradoxically conflicting roles need to be carefully considered in future strategies designed to improve the efficacy of oncolytic virotherapy.

IL-33 is a crucial amplifier of innate rather than acquired immunity.

IL-33, a member of the IL-1-related cytokines, is considered to be a proallergic cytokine that is especially involved in Th2-type immune responses. Moreover, like IL-1α, IL-33 has been suggested to act as an "alarmin" that amplifies immune responses during tissue injury. In contrast to IL-1, however, the precise roles of IL-33 in those settings are poorly understood. Using IL-1- and IL-33-deficient mice, we found that IL-1, but not IL-33, played a substantial role in induction of T cell-mediated type IV hypersensitivity such as contact and delayed-type hypersensitivity and autoimmune diseases such as experimental autoimmune encephalomyelitis. Most notably, however, IL-33 was important for innate-type mucosal immunity in the lungs and gut. That is, IL-33 was essential for manifestation of T cell-independent protease allergen-induced airway inflammation as well as OVA-induced allergic topical airway inflammation, without affecting acquisition of antigen-specific memory T cells. IL-33 was significantly involved in the development of dextran-induced colitis accompanied by T cell-independent epithelial cell damage, but not in streptozocin-induced diabetes or Con A-induced hepatitis characterized by T cell-mediated apoptotic tissue destruction. In addition, IL-33-deficient mice showed a substantially diminished LPS-induced systemic inflammatory response. These observations indicate that IL-33 is a crucial amplifier of mucosal and systemic innate, rather than acquired, immune responses.

NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals.

The inflammatory nature of atherosclerosis is well established but the agent(s) that incite inflammation in the artery wall remain largely unknown. Germ-free animals are susceptible to atherosclerosis, suggesting that endogenous substances initiate the inflammation. Mature atherosclerotic lesions contain macroscopic deposits of cholesterol crystals in the necrotic core, but their appearance late in atherogenesis had been thought to disqualify them as primary inflammatory stimuli. However, using a new microscopic technique, we revealed that minute cholesterol crystals are present in early diet-induced atherosclerotic lesions and that their appearance in mice coincides with the first appearance of inflammatory cells. Other crystalline substances can induce inflammation by stimulating the caspase-1-activating NLRP3 (NALP3 or cryopyrin) inflammasome, which results in cleavage and secretion of interleukin (IL)-1 family cytokines. Here we show that cholesterol crystals activate the NLRP3 inflammasome in phagocytes in vitro in a process that involves phagolysosomal damage. Similarly, when injected intraperitoneally, cholesterol crystals induce acute inflammation, which is impaired in mice deficient in components of the NLRP3 inflammasome, cathepsin B, cathepsin L or IL-1 molecules. Moreover, when mice deficient in low-density lipoprotein receptor (LDLR) were bone-marrow transplanted with NLRP3-deficient, ASC (also known as PYCARD)-deficient or IL-1alpha/beta-deficient bone marrow and fed on a high-cholesterol diet, they had markedly decreased early atherosclerosis and inflammasome-dependent IL-18 levels. Minimally modified LDL can lead to cholesterol crystallization concomitant with NLRP3 inflammasome priming and activation in macrophages. Although there is the possibility that oxidized LDL activates the NLRP3 inflammasome in vivo, our results demonstrate that crystalline cholesterol acts as an endogenous danger signal and its deposition in arteries or elsewhere is an early cause rather than a late consequence of inflammation. These findings provide new insights into the pathogenesis of atherosclerosis and indicate new potential molecular targets for the therapy of this disease.

Promotion of arthritis and allergy in mice by aminoglycoglycerophospholipid, a membrane antigen specific to Mycoplasma fermentans.

Mycoplasmas, which lack a cell wall and are the smallest self-replicating bacteria, have been linked to some chronic diseases, such as AIDS, rheumatoid arthritis (RA), and oncogenic transformation of cells. Their membrane components (lipoproteins and glycolipids) have been identified as possible causative factors in such diseases. Glycoglycerophospholipid (GGPL)-III, a unique phosphocholine-containing aminoglycoglycerophospholipid, is a major specific antigen of Mycoplasma fermentans, and has been detected in 38% of RA patients. Unlike those of lipoproteins, which induce inflammation via Toll-like receptor 2 (TLR2), the pathologic effects of GGPL-III are poorly understood. RA and metal allergies are chronic inflammatory diseases in which autoantigens have been implicated. Here, we examined the effects of chemically synthesized GGPL-III in murine arthritis and allergy models. GGPL-III alone exhibited little inflammatory effect, but promoted both collagen-induced arthritis and nickel (Ni) allergy, although less powerfully than Escherichia coli lipopolysaccharide. The augmenting effect of GGPL-III on Ni allergy was present in mice deficient in either T cells or active TLR4, but it was markedly weaker in mice deficient in macrophages, interleukin-1, or the histamine-forming enzyme histidine decarboxylase than in their control strains. These results suggest that GGPL-III may play roles in some types of chronic diseases via the innate immune system.

Identification of the cellular sensor that stimulates the inflammatory response to sterile cell death.

Cell death provokes a robust inflammatory response. We have previously shown that this response is dependent on IL-1alpha. In this study, we investigate the cellular mechanism used by a host to sense cell death, produce IL-1alpha and also the role of IL-1beta in this response. In almost all cases examined, the IL-1 that stimulated the death-induced inflammatory response came from the host rather than the cell that was dying. In these situations, host bone marrow-derived cells were the key source of the IL-1alpha that was required for the inflammatory response. Conditional cellular depletion and reconstitution in CD11b promoter-driven diphtheria toxin receptor transgenic mice revealed that host macrophages played an essential role in the generation of the inflammatory response and were the source of the required IL-1alpha. In addition, we found a role for IL-1beta in the death-induced inflammatory response and that this cytokine was generated by both bone marrow-derived and radioresistant host cells. The one exception to these findings was that when dendritic cells were injected into mice, they provided a portion of the IL-1 that stimulated inflammation, and this was observed whether the dendritic cells were live or necrotic. Together, these findings demonstrate that macrophages play a key role as the primary sentinels that are required to sense and report cell death in ways that initiate the inflammatory response. One key way they accomplish this important task is by producing IL-1alpha that is needed to initiate the inflammatory response.