Induction of Siglec-1 by Endotoxin Tolerance Suppresses the Innate Immune Response by Promoting TGF-β1 Production

Abstract

Sepsis is one of the leading causes of death worldwide. Although the prevailing theory for the sepsis syndrome is a condition of uncontrolled inflammation in response to infection, sepsis is increasingly being recognized as an immunosuppressive state known as endotoxin tolerance. We found sialylation of cell surface was significantly increased on LPS-induced tolerant cells; knockdown of Neu1 in macrophage cell line RAW 264.7 cells resulted in enhanced LPS-induced tolerance, whereas overexpression of Neu1 or treatment with sialidase abrogated LPS-induced tolerance, as defined by measuring TNF-␣ levels in the culture supernatants. We also found that the expression of Siglec-1 (a member of sialic acid-binding Ig (I)-like lectin family members, the predominant sialic acid-binding proteins on cell surface) was specifically up-regulated in endotoxin tolerant cells and the induction of Siglec-1 suppresses the innate immune response by promoting TGF-␤1 production. The enhanced TGF-␤1 production by Siglec-1 was significantly attenuated by spleen tyrosine kinase (Syk) inhibitor. Knockdown of siglec-1 in RAW 264.7 cells resulted in inhibiting the production of TGF-␤1 by ubiquitin-dependent degradation of Syk. Mechanistically, Siglec-1 associates with adaptor protein DNAX-activation protein of 12 kDa (DAP12) and transduces a signal to Syk to control the production of TGF-␤1 in endotoxin tolerance. Thus, Siglec-1 plays an important role in the development of endotoxin tolerance and targeted manipulation of this process could lead to a new therapeutic opportunity for patients with sepsis.Sepsis is generally defined as a systemic inflammatory response syndrome in response to infection. Sepsis can be potentially life threatening; of more than 1 million Americans who are diagnosed with severe sepsis every year, between 28 and 50% will die from this disease (1, 2). It is well-recognized that patients with sepsis are often immune suppressed, a state of reduced responsiveness to endotoxin known as endotoxin tolerance, deaths in this immunosuppressive phase are typically due to failure to control the secondary infections (3-5). The molecular mechanisms underlying this endotoxin tolerance phenomenon is poorly understood.Sialic acids are a family of nine-carbon acidic monosaccharides and are involved in immune response, such as host-pathogen recognition, migration, and antigen presentation. Mounting experimental evidence suggests that the presence of sialic acid residue act as a marker of self in the immune system, as such residues are absent from most microbes (6). Indeed, host cells can be lysed by immune cytotoxic effector mechanisms after extensive desialylation, an observation that demonstrates the significant role played by cell surface sialic acids in the selfrecognition process (7). In addition, normal human serum contains natural antibodies to sialidase-treated red blood cells (7-9), lymphocytes (10), and thymocytes (7). Recently, Meesmann et al. showed that desialylation acted as an "eat me" signal and caused an enhanced upt...

Siglec-E Negatively Regulates the Activation of TLR4 by Controlling Its Endocytosis

“…We found that expression of sialylation was significantly decreased on the cells over-expressing Neu1 compared with empty vector control cells (Fig. 2B) and in our recently published data (13). The efficiency of the E. coli induced TLR4 endocytosis was significantly decreased on the cells over-expressing Neu1 compared with cells expressing empty vector, indicating that Neu1 plays an important role in E. coli -induced TLR4 endocytosis in D2SC/1 cells (Fig.…”

“…We have demonstrated that Neu1 plays a critical role in regulating Siglec-TLR interaction and endotoxemia (26). In order to determine whether Neu1 contributes to the regulation of E. coli -induced TLR4 endocytosis, we established D2SC/1 cell clones stably over-expressing shRNA for Neu1 as described in our recently published work (13, 26), treated these cells with E. coli at 37°C for one hour and examined for TLR4 surface levels. As shown in Figure 2A, increased expression of sialylation was observed on the cells over-expressing shRNA for Neu1, compared with the cells expressing scrambled shRNA, however, silencing of Neu1 in D2SC/1 cells has little effect on E. coli -induced TLR4 endocytosis (Fig.…”

“…Increase in sialylation contributes to the tolerant phenotype in CD4 + T cells (12) and dendritic cells and macrophages (13), while desialylation plays a crucial role in the recognition process (10) and acts as an “eat me” signal in the clearance of apoptotic cells (15). Here, we found desialylation of cell surface inhibits endocytosis of TLR4 during E. coli infection, and our data showed that this is attributable to the action of Neu1 during E. coli infection.…”

“…Sialylation plays an important role in self-nonself discrimination and bacterial intake (10, 11). Increases of sialylation contribute to the tolerant phenotype in CD4 + T cells (12), dendritic cells, macrophages (13) and regulatory T cells (14); while desialylation acts as an “eat me” signal and promotes the clearance of apoptotic cells (15). The sialylation level of a cell is largely dependent on the activity of two enzymes; sialyltransferases, which are responsible for adding sialic acid residues to glycolipids or glycoproteins; and sialidases, which are responsible for removing sialic acid residues from glycolipids or glycoproteins.…”

Microglial-specific transcriptome changes following chronic alcohol consumption

“…Thus, future studies would benefit from investigating TGF-β signaling in the brain following ethanol consumption. Futhermore, although there haven’t been any studies thus far investigating the interaction between TGFβ and Siglec-H, there is evidence for other Siglecs regulating TGFβ signaling in immune cells (Takamiya et al, 2013; Wu et al, 2016) and the addition of TGF-β1 to microglial cultures upregulates Siglech expression. Thus, the interaction between Siglec-H and TGFβ in microglia is also worthy of investigation.…”