Ruoxi Yuan

Alteration of Lysosome Fusion and Low-grade Inflammation Mediated by Super-low-dose Endotoxin

Background: Super-low-dose endotoxemia contributes to cell stress and low-grade inflammation. Results: Super-low-dose LPS removed Tollip from late endosomes/lysosomes and blocked lysosome fusion with endosomes or autophagosomes. Tollip knock-out mice had impaired wound healing. Conclusion: Super-low-dose LPS leads to cell stress through clearing Tollip and blocking lysosome fusion. Significance: Our data reveal molecular dynamics of innate immunity regulation. Subclinical super-low-dose endotoxin LPS is a risk factor for the establishment of low-grade inflammation during the pathogenesis and progression of chronic diseases. However, the underlying mechanisms are not well understood. At the cellular level, a disruption of lysosome fusion with endosomes or autophagosomes may contribute to the potentiation of low-grade inflammation. In this study, we identified that subclinical super-low-dose endotoxin LPS can potently inhibit the process of endosome acidification and lysosome fusion with endosomes or autophagosomes in primary macrophages. Super-low-dose LPS induced the inhibitory phosphorylation of VPS34, thus leading to the disruption of endosome-lysosome fusion. This effect may depend upon the clearance and relocation of Tollip in macrophages by super-low-dose LPS. Consistent with this notion, Tollip-deficient macrophages had constitutively elevated levels of VPS34 inhibitory phosphorylation and constitutive disruption of endosome-lysosome fusion. By employing a skin excision wound-healing model, we observed that Tollip-deficient mice had significantly elevated levels of cell stress and reduced wound repair. This study reveals a novel mechanism responsible for the modulation of endosome-lysosome fusion and low-grade inflammation in innate macrophages.

The persistence of low-grade inflammatory monocytes contributes to aggravated atherosclerosis

Sustained low-grade inflammation mediated by non-resolving inflammatory monocytes has long been suspected in the pathogenesis of atherosclerosis; however, the molecular mechanisms responsible for the sustainment of non-resolving inflammatory monocytes during atherosclerosis are poorly understood. Here we observe that subclinical endotoxemia, often seen in humans with chronic inflammation, aggravates murine atherosclerosis through programming monocytes into a non-resolving inflammatory state with elevated Ly6C, CCR5, MCP-1 and reduced SR-B1. The sustainment of inflammatory monocytes is due to the disruption of homeostatic tolerance through the elevation of miR-24 and reduction of the key negative-feedback regulator IRAK-M. miR-24 reduces the levels of Smad4 required for the expression of IRAK-M and also downregulates key lipid-processing molecule SR-B1. IRAK-M deficiency in turn leads to elevated miR-24 levels, sustains disruption of monocyte homeostasis and aggravates atherosclerosis. Our data define an integrated feedback circuit in monocytes and its disruption may lead to non-resolving low-grade inflammation conducive to atherosclerosis.

Super-low dose endotoxin pre-conditioning exacerbates sepsis mortality.

Sepsis mortality varies dramatically in individuals of variable immune conditions, with poorly defined mechanisms. This phenomenon complements the hypothesis that innate immunity may adopt rudimentary memory, as demonstrated in vitro with endotoxin priming and tolerance in cultured monocytes. However, previous in vivo studies only examined the protective effect of endotoxin tolerance in the context of sepsis. In sharp contrast, we report herein that pre-conditioning with super-low or low dose endotoxin lipopolysaccharide (LPS) cause strikingly opposite survival outcomes. Mice pre-conditioned with super-low dose LPS experienced severe tissue damage, inflammation, increased bacterial load in circulation, and elevated mortality when they were subjected to cecal-ligation and puncture (CLP). This is in contrast to the well-reported protective phenomenon with CLP mice pre-conditioned with low dose LPS. Mechanistically, we demonstrated that super-low and low dose LPS differentially modulate the formation of neutrophil extracellular trap (NET) in neutrophils. Instead of increased ERK activation and NET formation in neutrophils pre-conditioned with low dose LPS, we observed significantly reduced ERK activation and compromised NET generation in neutrophils pre-conditioned with super-low dose LPS. Collectively, our findings reveal a mechanism potentially responsible for the dynamic programming of innate immunity in vivo as it relates to sepsis risks.

Low-grade inflammatory polarization of monocytes impairs wound healing.

Impaired wound healing often accompanies low‐grade inflammatory conditions, during which circulating levels of subclinical super‐low‐dose endotoxin may persist. Low‐grade inflammatory monocyte polarization may occur during chronic inflammation and deter effective wound repair. However, little is understood about the potential mechanisms of monocyte polarization by sustained insult of subclinical super‐low‐dose endotoxin. We observed that super‐low‐dose endotoxin preferentially programmes a low‐grade inflammatory monocyte state in vitro and in vivo, as represented by the elevated population of CD11b+ Ly6Chigh monocytes and sustained expression of CCR5. Mechanistically, super‐low‐dose endotoxin caused cellular stress, altered lysosome function and increased the transcription factor IRF5. TUDCA, a potent inhibitor of cellular stress, effectively blocked monocyte polarization and improved wound healing in mice injected with super‐low‐dose endotoxin. Our data revealed the polarization of low‐grade inflammatory monocytes by sustained endotoxin challenge, its underlying mechanisms and a potential intervention strategy. Copyright

Tollip Deficiency Alters Atherosclerosis and Steatosis by Disrupting Lipophagy

Background Compromised lipophagy with unknown mechanisms may be critically involved in the intracellular accumulation of lipids, contributing to elevated atherosclerosis and liver steatosis. We hypothesize that toll‐interacting protein (Tollip), a key innate immune molecule involved in the fusion of autolysosome, may play a significant role in lipophagy and modulate lipid accumulation during the pathogenesis of atherosclerosis and liver steatosis. Methods and Results By comparing mice fed with either a Western high‐fat diet or a regular chow diet, we observed that both atherosclerosis and liver steatosis were aggravated in apolipoprotein E–deficient (ApoE−/−)/Tollip−/− mice as compared with ApoE−/− mice. Through electron microscopy analyses, we observed compromised fusion of lipid droplets with lysosomes within aortic macrophages as well as liver hepatocytes from ApoE−/−/Tollip−/− mice as compared with ApoE−/− mice. As a molecular indicator for disrupted lysosome fusion, the levels of p62 were significantly elevated in aortic and liver tissues from ApoE−/−/Tollip−/− mice. Molecules involved in facilitating lipophagy completion such as Ras‐related protein 7 and gamma‐aminobutyric acid receptor‐associated protein were reduced in ApoE−/−/Tollip−/− mice as compared with ApoE−/− mice. Intriguingly, ApoE−/−/Tollip−/− mice had reduced circulating levels of inflammatory cytokines such as tumor necrosis factor‐α and increased levels of transforming growth factor‐β. The reduced inflammation due to Tollip deficiency is consistent with a stable atherosclerotic plaque phenotype with increased levels of plaque collagen and smooth muscle cells in ApoE−/−/Tollip−/− mice. Conclusions Tollip deficiency selectively leads to enlarged yet stable atherosclerotic plaques, increased circulating lipids, liver steatosis, and reduced inflammation. Compromised lipophagy and reduced expression of inflammatory mediators due to Tollip deficiency may be the underlying causes. Our data suggest that lipid accumulation and inflammation may be intertwined yet independent processes during the progression of atherosclerosis and steatosis.

Deficiency in Toll-interacting protein (Tollip) skews inflamed yet incompetent innate leukocytes in vivo during DSS-induced septic colitis

Functionally compromised neutrophils contribute to adverse clinical outcomes in patients with severe inflammation and injury such as colitis and sepsis. However, the ontogeny of dysfunctional neutrophil during septic colitis remain poorly understood. We report that the dysfunctional neutrophil may be derived by the suppression of Toll-interacting-protein (Tollip). We observed that Tollip deficient neutrophils had compromised migratory capacity toward bacterial product fMLF due to reduced activity of AKT and reduction of FPR2, reduced potential to generate bacterial-killing neutrophil extra-cellular trap (NET), and compromised bacterial killing activity. On the other hand, Tollip deficient neutrophils had elevated levels of CCR5, responsible for their homing to sterile inflamed tissues. The inflamed and incompetent neutrophil phenotype was also observed in vivo in Tollip deficient mice subjected to DSS-induced colitis. We observed that TUDCA, a compound capable of restoring Tollip cellular function, can potently alleviate the severity of DSS-induced colitis. In humans, we observed significantly reduced Tollip levels in peripheral blood collected from human colitis patients as compared to blood samples from healthy donors. Collectively, our data reveal a novel mechanism in Tollip alteration that underlies the inflamed and incompetent polarization of neutrophils leading to severe outcomes of colitis.

Dynamic modulation of innate immunity programming and memory.

Recent progress harkens back to the old theme of immune memory, except this time in the area of innate immunity, to which traditional paradigm only prescribes a rudimentary first-line defense function with no memory. However, both in vitro and in vivo studies reveal that innate leukocytes may adopt distinct activation states such as priming, tolerance, and exhaustion, depending upon the history of prior challenges. The dynamic programming and potential memory of innate leukocytes may have far-reaching consequences in health and disease. This review aims to provide some salient features of innate programing and memory, patho-physiological consequences, underlying mechanisms, and current pressing issues.

Molecular Mechanisms That Underlie the Dynamic Adaptation of Innate Monocyte Memory to Varying Stimulant Strength of TLR Ligands

In adaptation to rising stimulant strength, innate monocytes can be dynamically programed to preferentially express either pro- or anti-inflammatory mediators. Such dynamic innate adaptation or programing may bear profound relevance in host health and disease. However, molecular mechanisms that govern innate adaptation to varying strength of stimulants are not well understood. Using lipopolysaccharide (LPS), the model stimulant of toll-like-receptor 4 (TLR4), we reported that the expressions of pro-inflammatory mediators are preferentially sustained in monocytes adapted by lower doses of LPS, and suppressed/tolerized in monocytes adapted by higher doses of LPS. Mechanistically, monocytes adapted by super-low dose LPS exhibited higher levels of transcription factor, interferon regulatory factor 5 (IRF5), and reduced levels of transcriptional modulator B lymphocyte-induced maturation protein-1 (Blimp-1). Intriguingly, the inflammatory monocyte adaptation by super-low dose LPS is dependent upon TRAM/TRIF but not MyD88. Similar to LPS, we also observed biphasic inflammatory adaptation and tolerance in monocytes challenged with varying dosages of TLR7 agonist. In sharp contrast, rising doses of TLR3 agonist preferentially caused inflammatory adaptation without inducing tolerance. At the molecular level, the differential regulation of IRF5 and Blimp-1 coincides with unique monocyte adaptation dynamics by TLR4/7 and TLR3 agonists. Our study provides novel clue toward the understanding of monocyte adaptation and memory toward distinct TLR ligands.

Tissue-resident dendritic cells and diseases involving dendritic cell malfunction.

Dendritic cells (DCs) control immune responses and are central to the development of immune memory and tolerance. DCs initiate and orchestrate immune responses in a manner that depends on signals they receive from microbes and cellular environment. Although DCs consist mainly of bone marrow-derived and resident populations, a third tissue-derived population resides the spleen and lymph nodes (LNs), different subsets of tissue-derived DCs have been identified in the blood, spleen, lymph nodes, skin, lung, liver, gut and kidney to maintain the tolerance and control immune responses. Tissue-resident DCs express different receptors for microbe-associated molecular patterns (MAMPs) and damage-associated molecular patterns (DAMPs), which were activated to promote the production of pro- or anti-inflammatory cytokines. Malfunction of DCs contributes to diseases such as autoimmunity, allergy, and cancer. It is therefore important to update the knowledge about resident DC subsets and diseases associated with DC malfunction.

Subclinical-Dose Endotoxin Sustains Low-Grade Inflammation and Exacerbates Steatohepatitis in High-Fat Diet–Fed Mice

Subclinical circulating bacterial endotoxin LPS has been implicated as an important cofactor in the development and progression of nonalcoholic steatohepatitis, but the underlying mechanisms remain unclear. In this study, we demonstrated that 4-wk injection with superlow-dose LPS significantly promoted neutrophil infiltration and accelerated nonalcoholic steatohepatitis progression, including exacerbated macrovesicular steatosis, inflammation, and hepatocyte ballooning in high-fat diet–fed apolipoprotein E knockout mice. This effect could sustain for a month after stoppage of LPS injection. LPS also significantly increased numbers of apoptotic nuclei in hepatocytes and expressions of proapoptotic regulators. Moreover, LPS sustained the low-grade activation of p38 MAPK and inhibited the expression of the upstream MAPK phosphatase 7. By applying selective inhibitors, we demonstrated that the activation of p38 MAPKs is required for neutrophil migration induced by superlow-dose LPS in vitro. Together, these data suggest that superlow-dose LPS may sustain the low-grade activation of p38 MAPKs and neutrophil infiltration, leading to the exacerbation of steatohepatitis.