Integrins are critical for the migration and function of leukocytes in inflammation. However, the interaction between integrin alpha(M) (CD11b), which has high expression in monocytes and macrophages, and Toll-like receptor (TLR)-triggered innate immunity remains unclear. Here we report that CD11b deficiency enhanced TLR-mediated responses in macrophages, rendering mice more susceptible to endotoxin shock and Escherichia coli-caused sepsis. CD11b was activated by TLR-triggered phosphatidylinositol 3-OH kinase (PI(3)K) and the effector RapL and fed back to inhibit TLR signaling by activating the tyrosine kinases Src and Syk. Syk interacted with and induced tyrosine phosphorylation of MyD88 and TRIF, which led to degradation of these adaptor molecules by the E3 ubiquitin ligase Cbl-b. Thus, TLR-triggered, active CD11b integrin engages in crosstalk with the MyD88 and TRIF pathways and subsequently inhibits TLR signaling in innate immune responses.

RIG-I is a critical RNA virus sensor that serves to initiate antiviral innate immunity. However, posttranslational regulation of RIG-I signaling remains to be fully understood. We report here that RNA viruses, but not DNA viruses or bacteria, specifically upregulate lectin family member Siglecg expression in macrophages by RIG-I- or NF-κB-dependent mechanisms. Siglec-G-induced recruitment of SHP2 and the E3 ubiquitin ligase c-Cbl to RIG-I leads to RIG-I degradation via K48-linked ubiquitination at Lys813 by c-Cbl. By increasing type I interferon production, targeted inactivation of Siglecg protects mice against lethal RNA virus infection. Taken together, our data reveal a negative feedback loop of RIG-I signaling and identify a Siglec-G-mediated immune evasion pathway exploited by RNA viruses with implication in antiviral applications. These findings also provide insights into the functions and crosstalk of Siglec-G, a known adaptive response regulator, in innate immunity.

Toll-like receptor 4 (TLR4) initiates both myeloid differentiation factor 88 (MyD88)-dependent and Toll/interleukin (IL)-1R domain-containing adapter, inducing interferon (IFN)-␤-dependent signaling, leading to production of proinflammatory mediators and type I interferon (IFN) to eliminate pathogens. However, uncontrolled TLR4 activation may contribute to pathogenesis of autoimmune and inflammatory diseases. TLR4 is transported from the plasma membrane to the endosome for ubiqutination and to the lysosome for degradation, and downregulation of TLR4 expression or promotion of TLR4 degradation are important ways for negative regulation of TLR4 signaling. We previously identified a lysosome-associated small guanosine triphosphatase (GTPase) Rab7b that may be involved in lysosomal trafficking and degradation of proteins. Here we demonstrate that Rab7b can negatively regulate lipopolysaccharide (LPS)-induced production of tumor necrosis factor (TNF)-␣, IL-6, nitric oxide, and IFN-␤, and potentiate LPS-induced activation of mitogen-activated protein kinase, nuclear factor B, and IFN regulatory factor 3 signaling pathways in macrophages by promoting the degradation of TLR4. Rab7b is localized in LAMP-1-positive subcellular compartments and colocalized with TLR4 after LPS treatment and can decrease the protein level of TLR4. Our findings suggest that Rab7b is a negative regulator of TLR4 signaling, potentially by promoting the translocation of TLR4 into lysosomes for degradation. IntroductionToll-like receptors (TLRs) play a critical role in innate immunity by recognizing structurally conserved bacterial and viral components termed pathogen-associated molecular patterns. [1][2][3] TLRs activate signaling through the Toll/IL-1R (TIR) domain found in the cytoplasmic tails of these proteins, which in turn triggers the binding of the adaptor protein myeloid differentiation factor 88 (MyD88) to the TIR domain, allowing for interaction and phosphorylation of IL-1R-associated kinases and subsequent activation of tumor necrosis factor receptor (TNF)-associated factor 6 (TRAF-6), leading to the activation of nuclear factor B (NF-B) and mitogen-activated protein kinase (MAPK) pathways and the induction of proinflammatory cytokines. [1][2][3] However, recent studies have identified molecules involved in the MyD88-independent signaling for TLR3 and TLR4, including the TIR domain-containing adaptor protein (TIRAP/MAL), as a second adaptor instead of MyD88 for activation of 4,5 and the TIR domain-containing adapter inducing interferon (IFN)-␤ (TRIF) as a critical MyD88-independent adaptor used by TLR3 and TLR4 to regulate type I IFN production. 6,7 The signaling receptor for lipopolysaccharide (LPS) is TLR4/MD-2, which receives LPS from CD14. LPS delivered by CD14 to TLR4/MD-2 initiates a signaling cascade through the TIR domain containing adaptors MyD88, TIRAP/ MAL, TRIF, and TRAM (TRIF-related adaptor molecule), which eventually leads to activation of MAPK (including extracellular signal-regulated kinase [ERK], c-Jun N-terminal k...

T raditionally, heat shock proteins (HSP) 5 are regarded as chaperones assisting protein folding and translocation. However, HSP can also serve as cytokines that can stimulate dendritic cells (DC) and macrophages to produce proinflammatory cytokines and chemokines (1-5). More importantly, HSP derived from tumor cells are capable of chaperoning tumor Ags to DC and then cross-presenting the Ags to T cells (6). HSP70 proteins, including the constitutively expressed cognate HSP70 (HSC70 or HSP73), the stress-inducible HSP70 (HSP70i or HSP72), and the mitochondrial HSP70 (HSP75), constitute the most conserved class of all HSP. Previous reports have shown that HSP can be released from various cells via passive (e.g., HSP released during cell injury conditions, such as surgery, excessive exercise, and necrosis) and active (e.g., translocation of HSP to plasma membrane and subsequent secretion) pathways (3-5). However, the roles of HSP70 proteins released from tumor cells in the induction of antitumor immunity and the underlying mechanisms have not been fully elucidated.Hyperthermia (HT) has been reported to enhance the immunogenecity of cancer cells concomitantly with expression of HSP (7,8). Recent reports demonstrate that heat stress (HS) can induce the cell surface expression and the release of HSP70, HSP90, and gp96 (glucose-regulated protein 94; Grp94) (1-5). However, the underlying mechanisms that local HT can initiate antitumor immunity via released HSP and via subsequent activation of DC still lack direct evidence and thus need to be further investigated.During the investigations of local HT (42-43°C)-elicited antitumor immunity, we find that infiltration of DC and T cells within heat-stressed tumor is significantly increased. We thus hypothesize that chemokines, induced by HS, may be involved in the initiation of HT-elicited antitumor immunity by chemoattraction and activation of DC. Our studies show that HSP70 proteins simultaneously released by tumor cells can serve as autocrine and paracrine cytokines inducing the production of various chemokines by tumor cells and the activation of DC via TLR4 signaling pathway. Our data provide direct evidence for the important roles of releasable HSP in the initiation of antitumor immunity during local HT.

Background: The molecular mechanisms of endotoxin tolerance remain not well elucidated. Results: IRG1, up-regulated by LPS and during sepsis, can feedback suppress the Toll-like receptor-triggered inflammatory response by increasing A20 expression via reactive oxygen species (ROS) in LPS-tolerized macrophages. Conclusion: Inducible IRG1 promotes endotoxin tolerance by increasing A20 expression through ROS. Significance: Providing new molecular mechanisms regulating hypoinflammation of sepsis and endotoxin tolerance.