Macrophage differentiation and polarization via phosphatidylinositol 3-kinase/Akt-ERK signaling pathway conferred by serum amyloid P component

Macrophage differentiation and polarization is influenced by, and act on, many processes associated with autoimmunity. However, the molecular mechanisms underlying macrophage polarization in systemic lupus erythematosus (SLE) remain largely debated. We previously demonstrated that macrophage M2b polarization conferred by activated lymphocyte-derived (ALD)-DNA immunization could initiate and propagate murine lupus nephritis. Serum amyloid P component (SAP), a conserved acute-phase protein in mice, has been reported to bind to DNA and modulate immune responses. In this study, murine SAP was shown to promote macrophage-mediated ALD-DNA uptake through binding to ALD-DNA (SAP/ALD-DNA). Moreover, macrophage phenotypic switch from a proinflammatory M2b phenotype induced by ALD-DNA alone to an anti-inflammatory M2a phenotype stimulated with SAP/ALD-DNA were found because of PI3K/Akt-ERK signaling activation. Both in vivo SAP supplements and adoptive transfer of ex vivo programmed M2a macrophages induced by SAP/ALD-DNA into SLE mice could efficiently alleviate lupus nephritis. Importantly, increased IL-10 secretion, accompanied by anti-inflammatory effect exerted by M2a macrophages, was found to predominantly impede macrophage M2b polarization. Furthermore, neutralization of IL-10 notably reduced the suppressive effect of M2a macrophages. Our results demonstrate that binding of SAP to ALD-DNA could switch macrophage phenotypic polarization from proinflammatory M2b to anti-inflammatory M2a via PI3K/Akt-ERK signaling activation, thus exerting protective and therapeutic interventions on murine lupus nephritis. These data provide a possible molecular mechanism responsible for modulation of macrophage polarization in the context of lupus nephritis and open a new potential therapeutic avenue for SLE.     

Mannose-Binding Lectin Blunts Macrophage Polarization and Ameliorates Lupus Nephritis

Background

Deficiency in clearance of self nuclear antigens, including DNA, is the hallmark of systemic lupus erythematosus (SLE), a chronic autoimmnue disease characterized by the production of various autoantibodies, immune complex deposition and severe organ damage. Our previous studies revealed that administration of syngeneic BALB/c mice with activated lymphocyte-derived DNA (ALD-DNA) could induce SLE disease. Mannose-binding lectin (MBL), a secreted pattern recognition receptor with binding activity to DNA, has been proved to be a modulator of inflammation, but whether MBL takes responsibility for DNA clearance, modulates the DNA-mediated immune responses, and is involved in the development of DNA-induced SLE disease remain poorly understood.

Methodology/Principal Findings

The levels of serum MBL significantly decreased in lupus mice induced by ALD-DNA and were negatively correlated with SLE disease. MBL blunted macrophage M2b polarization by inhibiting the MAPK and NF-κB signaling while enhancing the activation of CREB. Furthermore, MBL suppressed the ability of ALD-DNA–stimulated macrophages to polarize T cells toward Th1 cells and Th17 cells. Importantly, MBL supplement in vivo could ameliorate lupus nephritis.

Conclusion/Significance

These results suggest MBL supplement could alleviate SLE disease and might imply a potential therapeutic strategy for DNA-induced SLE, which would further our understanding of the protective role of MBL in SLE disease.     

Dynamic expression of microRNAs in M2b polarized macrophages associated with systemic lupus erythematosus

Macrophage polarization contributes to the initiation and perpetuation of systemic lupus erythematosus (SLE). Our previous study demonstrated that M2b polarized macrophages induced by activated lymphocyte-derived DNA (ALD-DNA) have a crucial function in the initiation and progress of SLE disease. Accumulated data suggest that microRNAs (miRNAs) serve as critical regulators to control macrophage polarization. To investigate miRNA regulation during macrophage M2b polarization of SLE, miRNA microarrays of murine bone marrow derived macrophages (BMDMs) were performed following stimulation with ALD-DNA for 6 and 36 h. Over 11% of the 1111 analyzed miRNAs appeared differentially expressed during ALD-DNA triggered macrophage M2b polarization. Cluster analysis revealed certain patterns in miRNA expression that are closely linked to ALD-DNA induced macrophage M2b polarization. Analysis of the network structure showed that the predicted functions of the differentially regulated miRNAs at 6 h are significantly associated with inflammatory response and disease. Differentially regulated miRNAs identified at 36 h were determined to be significantly related to cell proliferation by biological network analysis. In this study, dynamic miRNA expression patterns and network analysis are described for the first time during ALD-DNA induced macrophage M2b polarization. The data not only provide a better understanding of miRNA-mediated macrophage polarization but also demonstrate the future therapeutic potential of targeting miRNAs in SLE patients.     

Amelioration of lupus nephritis by serum amyloid P component gene therapy with distinct mechanisms varied from different stage of the disease

Background

Our previous study revealed that administration of syngeneic female BALB/c mice with excessive self activated lymphocyte-derived DNA (ALD-DNA) could induce systemic lupus erythematosus (SLE) disease, indicating that overload of self-DNA might exceed normal clearance ability and comprise the major source of autoantigens in lupus mice. Serum amyloid P component (SAP), an acute-phase serum protein with binding reactivity to DNA in mice, was proved to promote the clearance of free DNA and prevent mice against self-antigen induced autoimmune response. It is reasonable to hypothesize that SAP treatment might contribute to alleviation of SLE disease, whereas its role in ALD-DNA-induced lupus nephritis is not fully understood.

Methodology/Principal Findings

The ratios of SAP to DNA significantly decreased and were negatively correlated with the titers of anti-dsDNA antibodies in ALD-DNA-induced lupus mice, indicating SAP was relatively insufficient in lupus mice. Herein a pcDNA3-SAP plasmid (pSAP) was genetically constructed and intramuscularly injected into BALB/c mice. It was found that SAP protein purified from the serum of pSAP-treated mice bound efficiently to ALD-DNA and inhibited ALD-DNA-mediated innate immune response in vitro. Treatment of ALD-DNA-induced lupus mice with pSAP in the early stage of SLE disease with the onset of proteinuria reversed lupus nephritis via decreasing anti-dsDNA autoantibody production and immune complex (IC) deposition. Further administration of pSAP in the late stage of SLE disease that had established lupus nephritis alleviated proteinuria and ameliorated lupus nephritis. This therapeutic effect of SAP was not only attributable to the decreased levels of anti-dsDNA autoantibodies, but also associated with the decreased infiltration of lymphocytes and the reduced production of inflammatory markers.

Conclusion/Significance

These results suggest that SAP administration could effectively alleviated lupus nephritis via modulating anti-dsDNA antibody production and the inflammation followed IC deposition, and SAP-based intervening strategy may provide new approaches for treating SLE disease.     

HMGB1 protein promotes glomerular mesangial matrix deposition via TLR2 in lupus nephritis

Lupus nephritis (LN) is the most common complication of systemic lupus erythematosus. Patients with LN mostly die of sclerosing glomerulonephritis and renal failure. The inhibition of glomerular mesangial matrix deposition is an efficient method to restrict the progress of renal injury. By recognizing and binding extracellular and intracellular ligands, Toll-like receptor 2 (TLR2) contributes to the pathogenesis of most immune diseases. However, the relationship between TLR2 and LN is still unknown. Our previous studies confirmed that high-mobility group box 1 (HMGB1), an important ligand of TLR2, promotes the progression of LN by inducing the proliferation of glomerular mesangial cells. However, whether or not HMGB1 participates in the pathogenesis of glomerular mesangial matrix deposition in LN remains unknown. In this study, we observed the upregulated expression of TLR2 in the glomeruli of LN patients and MRL/lpr mice. The inhibition of either TLR2 or HMGB1 inhibited the release of fibronectin and the activation of the MyD88/NF-κB pathway in mesangial cells cultured with LN plasma. In addition, both TLR2- and HMGB1-deficient mice showed reduced 24 hr urine protein levels and improved glomerular histological changes and sclerosis levels. These results indicate that TLR2 regulates glomerular mesangial matrix deposition in LN through the activation of the MyD88/NF-κB pathway by binding to HMGB1.     

Urine levels of HMGB1 in Systemic Lupus Erythematosus patients with and without renal manifestations

Introduction

Lupus nephritis (LN) is a severe and frequent manifestation of systemic lupus erythematosus (SLE). Its pathogenesis has not been fully elucidated but immune complexes are considered to contribute to the inflammatory pathology in LN. High Mobility Group Box 1 (HMGB1) is a nuclear non-histone protein which is secreted from different types of cells during activation and/or cell death and may act as a pro-inflammatory mediator, alone or as part of DNA-containing immune complexes in SLE. Urinary excretion of HMGB1 might reflect renal inflammatory injury. To assess whether urinary HMGB1 reflects renal inflammation we determined serum levels of HMGB1 simultaneously with its urinary levels in SLE patients with and without LN in comparison to healthy controls (HC). We also analyzed urinary HMGB1 levels in relation with clinical and serological disease activity.

Methods

The study population consisted of 69 SLE patients and 17 HC. Twenty-one patients had biopsy proven active LN, 15 patients had a history of LN without current activity, and 33 patients had non-renal SLE. Serum and urine levels of HMGB1 were both measured by western blotting. Clinical and serological parameters were assessed according to routine procedures. In 17 patients with active LN a parallel analysis was performed on the expression of HMGB1 in renal biopsies.

Results

Serum and urinary levels of HMGB1 were significantly increased in patients with active LN compared to patients without active LN and HC. Similarly, renal tissue of active LN patients showed strong expression of HMGB1 at cytoplasmic and extracellular sites suggesting active release of HMGB1. Serum and urinary levels in patients without active LN were also significantly higher compared to HC. Urinary HMGB1 levels correlated with SLEDAI, and showed a negative correlation with complement C3 and C4.

Conclusion

Levels of HMGB1 in urine of SLE patients, in particular in those with active LN, are increased and correlate with SLEDAI scores. Renal tissue of LN patients shows increased release of nuclear HMGB1 compared to control renal tissue. HMGB1, although at lower levels, is, however, also present in the urine of patients without active LN. These data suggest that urinary HMGB1 might reflect both local renal inflammation as well as systemic inflammation.     

Self DNA from Lymphocytes That Have Undergone Activation-Induced Cell Death Enhances Murine B Cell Proliferation and Antibody Production

Systemic lupus erythematosus (SLE) is characterized by prominent autoinflammatory tissue damage associated with impaired removal of dying cells and DNA. Self DNA-containing immune complexes are able to activate both innate and adaptive immune responses and play an important role in the maintenance and exacerbation of autoimmunity in SLE. In this study, we used DNA from lymphocytes that have undergone activation-induced cell death (ALD-DNA) and analyzed its role on the activation and differentiation of B cells from normal BALB/c mice as well as lupus-prone MRL^+/+ and MRL/lpr mice. We found that ALD-DNA directly increased the expression of costimulatory molecules and the survival of naïve B cells in vitro. Although ALD-DNA alone had little effect on the proliferation of naïve B cells, it enhanced LPS-activated B cell proliferation in vitro and in vivo. In addition, ALD-DNA increased plasma cell numbers and IgG production in LPS-stimulated cultures of naïve B cells, in part via enhancing IL-6 production. Importantly, B cells from lupus mice were hyperresponsive to ALD-DNA and/or LPS relative to normal control B cells in terminal plasma cell differentiation, as evidenced by increases in CD138⁺ cell numbers, IgM production, and mRNA levels of B lymphocyte-induced maturation protein-1 (Blimp-1) and the X-box binding protein 1 (XBP1). Furthermore, ALD-DNA enhanced CD40-activated naïve B cell proliferation. Collectively, these data indicate that self DNA can serve as a DAMP (damage-associated molecular pattern) that cooperates with signals from both innate and adaptive immunity to promote polyclonal B cell activation, a common characteristic of autoimmune diseases.     

Decrease in circulating DNA, IL-10 and BAFF levels in newly-diagnosed SLE patients after corticosteroid and chloroquine treatment

Arsenal of pattern-recognition receptors alongside antibody production machinery make B cells vulnerable to autoimmune response if an autoantigen elicits both pathways in a self-sustained fashion. Systemic lupus erythematosus is an autoimmune disease characterized by autoantibodies to DNA, RNA and related structures. Murine studies demonstrated autoreactive B cell activation upon TLR9 stimulation with DNA-containing immune complexes. This activation could be abolished with chloroquine, a drug used in SLE treatment that also blocks TLR9 signaling. We investigated whether chloroquine modulates TLR9 expression, circulating DNA levels and B cell-related cytokines in newly discovered, untreated SLE patients. TLR9 was measured in peripheral blood B cells by flow cytometry, serum DNA by real-time PCR, and IL-10 and BAFF by ELISA before treatment, after 3weeks on corticosteroids, and 3months after introduction of chloroquine. We found that circulating DNA is higher in SLE patients than in controls in every time-point and decreases significantly after chloroquine treatment. Untreated patients had higher serum IL-10 than controls or patients on corticosteroids. Also, corticosteroids decreased and chloroquine completely abolished CpG-mediated CD86 upregulation on B cells and IL-10 secretion in PBMC culture. Providing the TLR9 pathway activation demonstrates its importance in pathogenesis of human SLE, this data supports continuation of chloroquine in SLE treatment protocol. In addition, observed modulation of cytokine and DNA levels after immunomodulatory treatment prompts for inclusion of untreated patients in studies of human immune disorders.     

circLRP6 regulates high glucose-induced proliferation,oxidative stress,ECM accumulation,and inflammation in mesangial cells

Aberrant regulation in mesangial cell proliferation, extracellular matrix (ECM) accumulation, oxidative stress, and inflammation under hyperglycemic condition contributes significantly to the occurrence and development of diabetic nephropathy (DN). However, the mechanisms underlying the hyperglycemia-induced dysregulations have not been clearly elucidated. Here, we reported that high mobility group box 1 (HMGB1) was highly elevated in high glucose (HG)-treated mesangial cells, and induced the phosphorylation, nuclear translocation, and DNA binding activity of NF-κB via toll-like receptor 4 (TLR4). Function assays showed that inhibition of HMGB1 mitigated HG-induced proliferation, oxidative stress, ECM accumulation, and inflammation in mesangial cells via TLR4/NF-κB pathway. Increasing evidence has shown that circRNA, a large class of noncoding RNAs, functions by binding with miRNAs and terminating regulation of their target genes. We further investigated whether HMGB1 is involved in circRNA–miRNA–mRNA regulatory network. First, HMGB1 was identified and confirmed to be the target of miR-205, and miR-205 played a protective role against HG-induced cell injure via targeting HMGB1. Then circLRP6 was found to be upregulated in HG-treated mesangial cells, and regulate HG-induced mesangial cell injure via sponging miR-205. Besides, overexpression of miR-205 or knockdown of circLRP6 inhibited the NF-κB signaling pathway. Collectively, these data suggest that circLRP6 regulates HG-induced proliferation, oxidative stress, ECM accumulation, and inflammation in mesangial cells via sponging miR-205, upregulating HMGB1 and activating TLR4/NF-κB pathway. These findings provide a better understanding for the pathogenesis of DN.     

Hemorrhagic shock induces NAD(P)H oxidase activation in neutrophils: role of HMGB1-TLR4 signaling

Hemorrhagic shock/resuscitation (HS/R)-induced generation of reactive oxygen species (ROS) plays an important role in posthemorrhage inflammation and tissue injury. We have recently reported that HS/R-activated neutrophils (PMN), through release of ROS, serve an important signaling function in mediating alveolar macrophage priming and lung inflammation. PMN NAD(P)H oxidase has been thought to be an important source of ROS following HS/R. TLR4 sits at the interface of microbial and sterile inflammation by mediating responses to both bacterial endotoxin and multiple endogenous ligands, including high-mobility group box 1 (HMGB1). Recent studies have implicated HMGB1 as an early mediator of inflammation after HS/R and organ ischemia/reperfusion. In the present study, we tested the hypothesis that HS/R activates NAD(P)H oxidase in PMN through HMGB1/TLR4 signaling. We demonstrated that HS/R induced PMN NAD(P)H oxidase activation, in the form of phosphorylation of p47phox subunit of NAD(P)H oxidase, in wild-type mice; this induction was significantly diminished in TLR4-mutant C3H/HeJ mice. HMGB1 levels in lungs, liver, and serum were increased as early as 2 h after HS/R. Neutralizing Ab to HMGB1 prevented HS/R-induced phosphorylation of p47phox in PMN. In addition, in vitro stimulation of PMN with recombinant HMGB1 caused TLR4-dependent activation of NAD(P)H oxidase as well as increased ROS production through both MyD88-IRAK4-p38 MAPK and MyD88-IRAK4-Akt signaling pathways. Thus, PMN NAD(P)H oxidase activation, induced by HS/R and as mediated by HMGB1/TLR4 signaling, is an important mechanism responsible for PMN-mediated inflammation and organ injury after hemorrhage.     

Novel Autoantibodies Related to Cell Death and DNA Repair Pathways in Systemic Lupus Erythematosus

Systemic lupus erythematosus(SLE) is a complex autoimmune syndrome characterized by various co-existing autoantibodies(auto Abs) in patients' blood.However,the full spectrum of auto Abs in SLE has not been comprehensively elucidated.In this study,a commercial platform bearing 9400 antigens(Proto Array) was used to identify auto Abs that were significantly elevated in the sera of SLE patients.By comparing the auto Ab profiles of SLE patients with those of healthy controls,we identified 437 Ig G and 1213 Ig M auto Abs that the expression levels were significantly increased in SLE(P 0.05).Use of the Proto Array platform uncovered over 300 novel auto Abs targeting a broad range of nuclear,cytoplasmic,and membrane antigens.Molecular interaction network analysis revealed that the antigens targeted by the auto Abs were most significantly enriched in cell death,cell cycle,and DNA repair pathways.A group of auto Abs associated with cell apoptosis and DNA repair function,including those targeting APEX1,AURKA,POLB,AGO1,HMGB1,IFIT5,MAPKAPK3,PADI4,RGS3,SRP19,UBE2 S,and VRK1,were further validated by ELISA and Western blot in a larger cohort.In addition,the levels of auto Abs against APEX1,HMGB1,VRK1,AURKA,PADI4,and SRP19 were positively correlated with the level of anti-ds DNA in SLE patients.Comprehensive auto Ab screening has identified novel auto Abs,which may shed light on potential pathogenic pathways leading to lupus.     

Recent Developments in the Role of High-Mobility Group Box 1 in Systemic Lupus Erythematosus

High-mobility group box 1 (HMGB1) is an important molecule for several nuclear processes. Recently, HMGB1 has gained much attention as a damage-associated molecular pattern (DAMP) and has been implicated in the pathogenesis of several (auto)-immune diseases, in particular, systemic lupus erythematosus (SLE). A main pathogenic feature in SLE is the accumulation of apoptotic cells. Since HMGB1 is released from apoptotic cells it has been hypothesized that HMGB1 might fuel the inflammatory processes, as seen in this disease, and play a fundamental role in the pathogenesis. In this review, we discuss evidence in support of the theory that HMGB1 is an important mediator in SLE and may be considered a new autoantigen.     

Physiologic Concentrations of HMGB1 Have No Impact on Cytokine-Mediated Eosinophil Survival or Chemotaxis in Response to Eotaxin-2 (CCL24)

HMGB1 is an alarmin that can stimulate the innate immune system alone or in a complex with other inflammatory mediators. Given the recent interest in HMGB1 with respect to the pathogenesis of eosinophil-associated disorders, including asthmatic inflammation and chronic rhinosinusitis, we have explored the role of this mediator and in promoting eosinophil activation. HMGB1 receptors RAGE and TLR4 but not TLR2 were detected on freshly isolated human eosinophils from healthy donors. Physiologic and relevant pathophysiologic levels of biologically-active HMGB1 had no effect on survival of human eosinophils alone or in combination with pro-survival cytokines IL-5, IL-3, or GM-CSF, and increasing concentrations of HMGB1 had no impact on surface expression of RAGE, TLR2 or TLR4. Similarly, HMGB1 did not elicit chemotaxis of human eosinophils alone and had no effect in combination with the eosinophil chemotactic agent, eotaxin-2 (CCL24). However, surface expression of TLR2 and TLR4 increased in response to cell stress, notably on eosinophils that remain viable after 48 hours without IL-5. As such, HMGB1 signaling on eosinophils may be substantially more detailed, and may involve complex immunostimulatory pathways other than or in addition to those evaluated here.     

The HIV Protease Inhibitor Saquinavir Inhibits HMGB1-Driven Inflammation by Targeting the Interaction of Cathepsin V with TLR4/MyD88

Extracellular high-mobility group box 1 (HMGB1) (disulfide form), via activation of toll-like receptor 4 (TLR4)-dependent signaling, is a strong driver of pathologic inflammation in both acute and chronic conditions. Identification of selective inhibitors of HMGB1-TLR4 signaling could offer novel therapies that selectively target proximal endogenous activators of inflammation. A cell-based screening strategy led us to identify first generation HIV-protease inhibitors (PI) as potential inhibitors of HMGB1-TLR4 driven cytokine production. Here we report that the first-generation HIV-PI saquinavir (SQV), as well as a newly identified mammalian protease inhibitor STO33438 (334), potently block disulfide HMGB1-induced TLR4 activation, as assayed by the production of TNF-α by human monocyte-derived macrophages (THP-1). We further report on the identification of mammalian cathepsin V, a protease, as a novel target of these inhibitors. Cellular as well as recombinant protein studies show that the mechanism of action involves a direct interaction between cathepsin V with TLR4 and its adaptor protein MyD88. Treatment with SQV, 334 or the known cathepsin inhibitor SID26681509 (SID) significantly improved survival in murine models of sepsis and reduced liver damage following warm liver ischemia/reperfusion (I/R) models, both characterized by strong HMGB1-TLR4 driven pathology. The current study demonstrates a novel role for cathepsin V in TLR4 signaling and implicates cathepsin V as a novel target for first-generation HIV-PI compounds. The identification of cathepsin V as a target to block HMGB1-TLR4-driven inflammation could allow for a rapid transition of the discovery from the bench to the bedside. Disulfide HMGB1 drives pathologic inflammation in many models by activating signaling through TLR4. Cell-based screening identified the mammalian protease cathepsin V as a novel therapeutic target to inhibit TLR4-mediated inflammation induced by extracellular HMGB1 (disulfide form). We identified two protease inhibitors (PIs) that block cathepsin V and thereby inhibit disulfide HMGB1-induced TLR4 activation: saquinavir (SQV), a first-generation PI targeting viral HIV protease and STO33438 (334), targeting mammalian proteases. We discovered that cathepsin V binds TLR4 under basal and HMGB1-stimulated conditions, but dissociates in the presence of SQV over time. Thus cathepsin V is a novel target for first-generation HIV PIs and represents a potential therapeutic target of pathologic inflammation.     

Role of HMGB1 signaling in the inflammatory process in diabetic retinopathy

High mobility group box 1 (HMGB1) is a key player in retinal inflammation. HMGB1 is a danger associated protein pattern receptor which can sense high glucose as a stressor. Increased HMGB1 levels have been found in patients with late stage diabetic retinopathy. HMGB1 can bind toll-like receptor 4 (TLR4) and the receptor for advanced glycation end-products (RAGE), leading to increased inflammation commonly through nuclear factor kappa beta (NFkB). Because diabetic patients have been found to have increased HMGB1 and RAGE levels, as well as polymorphisms of TLR4, a number of investigations have focused on inhibition of these pathways in the diabetic retina. Work in diabetic animal models and cell culture have demonstrated a number of factors that can inhibit HMGB1/TLR4/RAGE signaling. This regulation offers potential new avenues for therapeutic development. This review is focused on HMGB1 signaling and downstream pathways leading to inflammation in the diabetic retina.     

High Mobility Group Box-1 Promotes Inflammation-Induced Lymphangiogenesis via Toll-Like Receptor 4-Dependent Signalling Pathway

Lymphangiogenesis in inflammation has received considerable attention in recent years. Administration of modulating lymphangiogenesis provides more possibilities of treating inflammation-associated diseases. However, the main mediators and factors governing inflammation-induced lymphangiogenesis (ILA) are yet to be defined. Here, we explored the role of HMGB1-TLR4 signalling pathway in modulating inflammation-induced lymphangiogenesis and its underlying mechanisms using an ILA mouse model and 2 cell lines. Our results show that HMGB1 promoted VEGF-C-induced HDLECs proliferation in a dose-dependent manner and TLR4 mediates HMGB1-induced LECs proliferation and tube formation in vitro. And in vivo, rHMGB1 treatment significantly promoted ILA, and the promoting effects was inhibited notably when HMGB1-TLR4 was blocked. HMGB1-associated ILA is primarily dependent on TLR4 but not on TLR2. In mechanisms, the recruitment and activation of CD11b+ cells are important cellular mechanisms in HMGB1-TLR4 associated ILA, and multiple key pro-lymphangiogenesis molecules mediates HMGB1-TLR4 associated ILA, including VEGF-C/VEGFR3, inflammatory factors IL-1β and TNF-α, MMP-2 and MMP-9 and NF-κB p65. In conclusion, HMGB1-associated ILA is primarily dependent on TLR4, and CD11b+ cells and multiple molecular mechanisms mediate HMGB1-TLR4 associated ILA. Furthermore, the ILA can be effectively modulated by HMGB1-TLR4 signalling. Consequently, administration of modulating ILA through HMGB1-TLR4 pathway may provide us more possibilities of treating inflammation and lymphangiogenesis associated diseases.