High-power polarization-maintaining LP11-mode fiber laser based on long-period fiber grating for precise welding.

An LP11-mode output all-fiber laser was presented, utilizing long-period fiber gratings (LPFGs) and polarization-maintaining optical fiber (PMF). The LPFG was designed and fabricated, achieving a 90.56% efficiency in LP01 to LP11 mode conversion. Furthermore, the transmission stability of LP11-mode in the PMF was also explored, with the spatial mode overlap ratio exceeding 0.95. Ultimately, the high-power polarization-maintaining (PM) fiber laser, capable of the LP11 mode output, was constructed, with the output power of 600 W and the beam quality M2 of 2.84. During the process of welding a thick Al-plate, the LP11 fiber laser exhibits a notable 1.88 times greater depth of fusion compared to the commercial single-mode fiber laser, when operating at the laser welding head speed of 100 mm/s. For applications demanding non-circular symmetric high-order modes, this research holds substantial potential for widespread adoption within the field of industrial processing.

SARS-COV-2 protein NSP9 promotes cytokine production by targeting TBK1.

SARS-COV-2 infection-induced excessive or uncontrolled cytokine storm may cause injury of host tissue or even death. However, the mechanism by which SARS-COV-2 causes the cytokine storm is unknown. Here, we demonstrated that SARS-COV-2 protein NSP9 promoted cytokine production by interacting with and activating TANK-binding kinase-1 (TBK1). With an rVSV-NSP9 virus infection model, we discovered that an NSP9-induced cytokine storm exacerbated tissue damage and death in mice. Mechanistically, NSP9 promoted the K63-linked ubiquitination and phosphorylation of TBK1, which induced the activation and translocation of IRF3, thereby increasing downstream cytokine production. Moreover, the E3 ubiquitin ligase Midline 1 (MID1) facilitated the K48-linked ubiquitination and degradation of NSP9, whereas virus infection inhibited the interaction between MID1 and NSP9, thereby inhibiting NSP9 degradation. Additionally, we identified Lys59 of NSP9 as a critical ubiquitin site involved in the degradation. These findings elucidate a previously unknown mechanism by which a SARS-COV-2 protein promotes cytokine storm and identifies a novel target for COVID-19 treatment.

Glutamylation of an HIV-1 protein inhibits the immune response by hijacking STING.

Cyclic GMP-AMP synthase (cGAS) recognizes Y-form cDNA of human immunodeficiency virus type 1 (HIV-1) and initiates antiviral immune response through cGAS-stimulator of interferon genes (STING)-TBK1-IRF3-type I interferon (IFN-I) signalingcascade. Here, we report that the HIV-1 p6 protein suppresses HIV-1-stimulated expression of IFN-I and promotes immune evasion. Mechanistically, the glutamylated p6 at residue Glu6 inhibits the interaction between STING and tripartite motif protein 32 (TRIM32) or autocrine motility factor receptor (AMFR). This subsequently suppresses the K27- and K63-linked polyubiquitination of STING at K337, therefore inhibiting STING activation, whereas mutation of the Glu6 residue partially reverses the inhibitory effect. However, CoCl2, an agonist of cytosolic carboxypeptidases (CCPs), counteracts the glutamylation of p6 at the Glu6 residue and inhibits HIV-1 immune evasion. These findings reveal a mechanism through which an HIV-1 protein mediates immune evasion and provides a therapeutic drug candidate to treat HIV-1 infection.

Extracellular fibrinogen-binding protein released by intracellular Staphylococcus aureus suppresses host immunity by targeting TRAF3.

Many pathogens secrete effectors to hijack intracellular signaling regulators in host immune cells to promote pathogenesis. However, the pathogenesis of Staphylococcus aureus secretory effectors within host cells is unclear. Here, we report that Staphylococcus aureus secretes extracellular fibrinogen-binding protein (Efb) into the cytoplasm of macrophages to suppress host immunity. Mechanistically, RING finger protein 114, a host E3 ligase, mediates K27-linked ubiquitination of Efb at lysine 71, which facilitates the recruitment of tumor necrosis factor receptor associated factor (TRAF) 3. The binding of Efb to TRAF3 disrupts the formation of the TRAF3/TRAF2/cIAP1 (cellular-inhibitor-of-apoptosis-1) complex, which mediates K48-ubiquitination of TRAF3 to promote degradation, resulting in suppression of the inflammatory signaling cascade. Additionally, the Efb K71R mutant loses the ability to inhibit inflammation and exhibits decreased pathogenicity. Therefore, our findings identify an unrecognized mechanism of Staphylococcus aureus to suppress host defense, which may be a promising target for developing effective anti-Staphylococcus aureus immunomodulators.

HIV-1 Vif suppresses antiviral immunity by targeting STING.

HIV-1 infection-induced cGAS-STING-TBK1-IRF3 signaling activates innate immunity to produce type I interferon (IFN). The HIV-1 nonstructural protein viral infectivity factor (Vif) is essential in HIV-1 replication, as it degrades the host restriction factor APOBEC3G. However, whether and how it regulates the host immune response remains to be determined. In this study, we found that Vif inhibited the production of type I IFN to promote immune evasion. HIV-1 infection induced the activation of the host tyrosine kinase FRK, which subsequently phosphorylated the immunoreceptor tyrosine-based inhibitory motif (ITIM) of Vif and enhanced the interaction between Vif and the cellular tyrosine phosphatase SHP-1 to inhibit type I IFN. Mechanistically, the association of Vif with SHP-1 facilitated SHP-1 recruitment to STING and inhibited the K63-linked ubiquitination of STING at Lys337 by dephosphorylating STING at Tyr162. However, the FRK inhibitor D-65495 counteracted the phosphorylation of Vif to block the immune evasion of HIV-1 and antagonize infection. These findings reveal a previously unknown mechanism through which HIV-1 evades antiviral immunity via the ITIM-containing protein to inhibit the posttranslational modification of STING. These results provide a molecular basis for the development of new therapeutic strategies to treat HIV-1 infection.

CCRL2 promotes antitumor T-cell immunity via amplifying TLR4-mediated immunostimulatory macrophage activation.

Macrophages are the key regulator of T-cell responses depending on their activation state. C-C motif chemokine receptor-like 2 (CCRL2), a nonsignaling atypical receptor originally cloned from LPS-activated macrophages, has recently been shown to regulate immune responses under several inflammatory conditions. However, whether CCRL2 influences macrophage function and regulates tumor immunity remains unknown. Here, we found that tumoral CCRL2 expression is a predictive indicator of robust antitumor T-cell responses in human cancers. CCRL2 is selectively expressed in tumor-associated macrophages (TAM) with immunostimulatory phenotype in humans and mice. Conditioned media from tumor cells could induce CCRL2 expression in macrophages primarily via TLR4, which is negated by immunosuppressive factors. Ccrl2-/- mice exhibit accelerated melanoma growth and impaired antitumor immunity characterized by significant reductions in immunostimulatory macrophages and T-cell responses in tumor. Depletion of CD8+ T cells or macrophages eliminates the difference in tumor growth between WT and Ccrl2-/- mice. Moreover, CCRL2 deficiency impairs immunogenic activation of macrophages, resulting in attenuated antitumor T-cell responses and aggravated tumor growth in a coinjection tumor model. Mechanically, CCRL2 interacts with TLR4 on the cell surface to retain membrane TLR4 expression and further enhance its downstream Myd88-NF-κB inflammatory signaling in macrophages. Similarly, Tlr4-/- mice exhibit reduced CCRL2 expression in TAM and accelerated melanoma growth. Collectively, our study reveals a functional role of CCRL2 in activating immunostimulatory macrophages, thereby potentiating antitumor T-cell response and tumor rejection, and suggests CCLR2 as a potential biomarker candidate and therapeutic target for cancer immunotherapy.

Helicobacter pylori CagA Interacts with SHP-1 to Suppress the Immune Response by Targeting TRAF6 for K63-Linked Ubiquitination.

Helicobacter pylori is the major etiological agent for most gastric cancer. CagA has been reported to be an important virulence factor of H. pylori, but its effect on the immune response is not yet clear. In this study, wild-type C57BL/6 mice and Ptpn6me-v/me-v mice were randomly assigned for infection with H. pylori We demonstrated that CagA suppressed H. pylori-stimulated expression of proinflammatory cytokines in vivo. Besides, we infected mouse peritoneal macrophages RAW264.7 and AGS with H. pylori Our results showed that CagA suppressed expression of proinflammatory cytokines through inhibiting the MAPKs and NF-κB pathways activation in vitro. Mechanistically, we found that CagA interacted with the host cellular tyrosine phosphatase SHP-1, which facilitated the recruitment of SHP-1 to TRAF6 and inhibited the K63-linked ubiquitination of TRAF6, which obstructed the transmission of signal downstream. Taken together, these findings reveal a previously unknown mechanism by which CagA negatively regulates the posttranslational modification of TRAF6 in innate antibacterial immune response and provide molecular basis for new therapeutics to treat microbial infection.

ß-arrestin 2 as an activator of cGAS-STING signaling and target of viral immune evasion.

Virus infection may induce excessive interferon (IFN) responses that can lead to host tissue injury or even death. ß-arrestin 2 regulates multiple cellular events through the G protein-coupled receptor (GPCR) signaling pathways. Here we demonstrate that ß-arrestin 2 also promotes virus-induced production of IFN-ß and clearance of viruses in macrophages. ß-arrestin 2 interacts with cyclic GMP-AMP synthase (cGAS) and increases the binding of dsDNA to cGAS to enhance cyclic GMP-AMP (cGAMP) production and the downstream stimulator of interferon genes (STING) and innate immune responses. Mechanistically, deacetylation of ß-arrestin 2 at Lys171 facilitates the activation of the cGAS-STING signaling and the production of IFN-ß. In vitro, viral infection induces the degradation of ß-arrestin 2 to facilitate immune evasion, while a ß-blocker, carvedilol, rescues ß-arrestin 2 expression to maintain the antiviral immune response. Our results thus identify a viral immune-evasion pathway via the degradation of ß-arrestin 2, and also hint that carvedilol, approved for treating heart failure, can potentially be repurposed as an antiviral drug candidate.

SHP-1 suppresses the antiviral innate immune response by targeting TRAF3.

Type I interferons play a pivotal role in innate immune response to virus infection. The protein tyrosine phosphatase SHP-1 was reported to function as a negative regulator of inflammatory cytokine production by inhibiting activation of NF-κB and MAPKs during bacterial infection, however, the role of SHP-1 in regulating type I interferons remains unknown. Here, we demonstrated that knockout or knockdown of SHP-1 in macrophages promoted both HSV-1- and VSV-induced antiviral immune response. Conversely, overexpression of SHP-1 in L929 cells suppressed the HSV-1- and VSV-induced immune response; suppression was directly dependent on phosphatase activity. We identified a direct interaction between SHP-1 and TRAF3; the association between these two proteins resulted in diminished recruitment of CK1ε to TRAF3 and inhibited its K63-linked ubiquitination; SHP-1 inhibited K63-linked ubiquitination of TRAF3 by promoting dephosphorylation at Tyr116 and Tyr446. Taken together, our results identify SHP-1 as a negative regulator of antiviral immunity and suggest that SHP-1 may be a target for intervention in acute virus infection.

ß-arrestin 2 quenches TLR signaling to facilitate the immune evasion of EPEC.

The protein translocated intimin receptor (Tir) from enteropathogenic Escherichia coli shares sequence similarity with the host cellular immunoreceptor tyrosine-based inhibition motifs (ITIMs). The ITIMs of Tir are required for Tir-mediated immune inhibition and evasion of host immune responses. However, the underlying molecular mechanism by which Tir regulates immune inhibition remains unclear. Here we demonstrated that ß-arrestin 2, which is involved in the G-protein-coupled receptor (GPCR) signal pathway, interacted with Tir in an ITIM-dependent manner. For the molecular mechanism, we found that ß-arrestin 2 enhanced the recruitment of SHP-1 to Tir. The recruited SHP-1 inhibited K63-linked ubiquitination of TRAF6 by dephosphorylating TRAF6 at Tyr288, and inhibited K63-linked ubiquitination and phosphorylation of TAK1 by dephosphorylating TAK1 at Tyr206, which cut off the downstream signal transduction and subsequent cytokine production. Moreover, the inhibitory effect of Tir on immune responses was diminished in ß-arrestin 2-deficient mice and macrophages. These findings suggest that ß-arrestin 2 is a key regulator in Tir-mediated immune evasion, which could serve as a new therapeutic target for bacterial infectious diseases.

Enterohemorrhagic Escherichia coli Tir inhibits TAK1 activation and mediates immune evasion.

Many pathogens infect hosts through various immune evasion strategies. However, the molecular mechanisms by which pathogen proteins modulate and evade the host immune response remain unclear. Enterohemorrhagic Escherichia coli (EHEC) is a pathological strain that can induce mitogen-activated protein (MAP) kinase (Erk, Jnk and p38 MAPK) and NF-κB pathway activation and proinflammatory cytokine production, which then causes diarrheal diseases such as hemorrhagic colitis and hemolytic uremic syndrome. Transforming growth factor ß-activated kinase-1 (TAK1) is a key regulator involved in distinct innate immune signalling pathways. Here we report that EHEC translocated intimin receptor (Tir) protein inhibits the expression of EHEC-induced proinflammatory cytokines by interacting with the host tyrosine phosphatase SHP-1, which is dependent on the phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs). Mechanistically, the association of EHEC Tir with SHP-1 facilitated the recruitment of SHP-1 to TAK1 and inhibited TAK1 phosphorylation, which then negatively regulated K63-linked polyubiquitination of TAK1 and downstream signal transduction. Taken together, these results suggest that EHEC Tir negatively regulates proinflammatory responses by inhibiting the activation of TAK1, which is essential for immune evasion and could be a potential target for the treatment of bacterial infection.

PLCß2 negatively regulates the inflammatory response to virus infection by inhibiting phosphoinositide-mediated activation of TAK1.

Excessive or uncontrolled release of proinflammatory cytokines caused by severe viral infections often results in host tissue injury or even death. Phospholipase C (PLC)s degrade phosphatidylinositol-4, 5-bisphosphate (PI(4,5)P2) lipids and regulate multiple cellular events. Here, we report that PLCß2 inhibits the virus-induced expression of pro-inflammatory cytokines by interacting with and inhibiting transforming growth factor-ß-activated kinase 1 (TAK1) activation. Mechanistically, PI(4,5)P2 lipids directly interact with TAK1 at W241 and N245, and promote its activation. Impairing of PI(4,5)P2's binding affinity or mutation of PIP2-binding sites on TAK1 abolish its activation and the subsequent production of pro-inflammatory cytokines. Moreover, PLCß2-deficient mice exhibit increased expression of proinflammatory cytokines and a higher frequency of death in response to virus infection, while the PLCß2 activator, m-3M3FBS, protects mice from severe Coxsackie virus A 16 (CVA16) infection. Thus, our findings suggest that PLCß2 negatively regulates virus-induced pro-inflammatory responses by inhibiting phosphoinositide-mediated activation of TAK1.

Nuclear cGAS suppresses DNA repair and promotes tumorigenesis.

Accurate repair of DNA double-stranded breaks by homologous recombination preserves genome integrity and inhibits tumorigenesis. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that activates innate immunity by initiating the STING-IRF3-type I IFN signalling cascade1,2. Recognition of ruptured micronuclei by cGAS links genome instability to the innate immune response3,4, but the potential involvement of cGAS in DNA repair remains unknown. Here we demonstrate that cGAS inhibits homologous recombination in mouse and human models. DNA damage induces nuclear translocation of cGAS in a manner that is dependent on importin-α, and the phosphorylation of cGAS at tyrosine 215-mediated by B-lymphoid tyrosine kinase-facilitates the cytosolic retention of cGAS. In the nucleus, cGAS is recruited to double-stranded breaks and interacts with PARP1 via poly(ADP-ribose). The cGAS-PARP1 interaction impedes the formation of the PARP1-Timeless complex, and thereby suppresses homologous recombination. We show that knockdown of cGAS suppresses DNA damage and inhibits tumour growth both in vitro and in vivo. We conclude that nuclear cGAS suppresses homologous-recombination-mediated repair and promotes tumour growth, and that cGAS therefore represents a potential target for cancer prevention and therapy.

Mechanisms of fibronectin-binding protein A (FnBPA110-263) vaccine efficacy in Staphylococcus aureus sepsis versus skin infection.

Increasing rates of life-threatening infections and decreasing susceptibility to antibiotics urge an effective vaccine targeting Staphylococcus aureus. Here we investigate the role of cellular immunity in FnBPA110-263 mediated protection in Staphylococcus aureus infection. This study revealed FnBPA110-263 broadly protected mice from seven FnBPA isotypes strains in the sepsis model. FnBPA110-263 immunized B-cell deficient mice were protected against lethal challenge, while T-cell deficient mice were not. Reconstituting mice with FnBPA110-263 specific CD4+ T-cells conferred antigen specific protection. In vitro assays indicated that isolated FnBPA110-263 specific splenocytes from immunized mice produced abundant IL-17A. IL-17A deficient mice were not protected from a lethal challenge by FnBPA110-263 vaccination. Moreover, neutralizing IL-17A, but not IFN-γ,reverses FnBPA110-263-induced protective efficacy in sepsis and skin infection model. These findings suggest that IL-17A producing Th17 cells play an essential role in FnBPA110-263 vaccine-mediated defense against S. aureus sepsis and skin infection in mice.

HIF1α-dependent glycolysis promotes macrophage functional activities in protecting against bacterial and fungal infection.

Macrophages are important innate immune defense system cells in the fight against bacterial and fungal pathogenic infections. They exhibit significant plasticity, particularly with their ability to undergo functional differentiation. Additionally, HIF1α is critically involved in the functional differentiation of macrophages during inflammation. However, the role of macrophage HIF1α in protecting against different pathogenic infections remains unclear. In this study, we investigated and compared the roles of HIF1α in different macrophage functional effects of bacterial and fungal infections in vitro and in vivo. We found that bacterial and fungal infections produced similar effects on macrophage functional differentiation. HIF1α deficiency inhibited pro-inflammatory macrophage functional activities when cells were stimulated with LPS or curdlan in vitro or when mice were infected with L. monocytogenes or C. albicans in vivo, thus decreasing pro-inflammatory TNFα and IL-6 secretion associated with pathogenic microorganism survival. Alteration of glycolytic pathway activation was required for the functional differentiation of pro-inflammatory macrophages in protecting against bacterial and fungal infections. Thus, the HIF1α-dependent glycolytic pathway is essential for pro-inflammatory macrophage functional differentiation in protecting against bacterial and fungal infections.

Notch4 Negatively Regulates the Inflammatory Response to Mycobacterium tuberculosis Infection by Inhibiting TAK1 Activation.

Tuberculosis, caused by Mycobacterium tuberculosis infection, remains a global threat to human health, but knowledge of the molecular mechanisms underlying the pathogenesis of tuberculosis is still limited. Although Notch4, a member of the Notch receptor family, is involved in the initiation of mammary tumors, its function in M. tuberculosis infection remains unclear. In this study, we found that Notch4-deficient mice were more resistant to M. tuberculosis infection, with a much lower bacterial burden and fewer pathological changes in the lungs. Notch4 inhibited M. tuberculosis-induced production of proinflammatory cytokines by interaction with TAK1 and inhibition of its activation. Furthermore, we found that Notch intracellular domain 4 prevented TRAF6 autoubiquitination and suppressed TRAF6-mediated TAK1 polyubiquitination. Finally, Notch inhibitors made mice more resistant to M. tuberculosis infection. These results suggest that Notch4 is a negative regulator of M. tuberculosis-induced inflammatory response, and treatment with a Notch inhibitor could serve as a new therapeutic strategy for tuberculosis.

Posttranslational Modification Control of Inflammatory Signaling.

Inflammation is usually the defensive reaction of the immune system to the invasion of pathogen and the exogenous objects. The activation of inflammation helps our body to eliminate pathogenic microbe, virus, and parasite harming our health, while under many circumstances inflammation is the direct cause of the pathological damage in tissues and dysfunction of organs. The posttranslational modification (PTM) of the inflammatory pathways, such as TLR pathways, RLR pathways, NLR pathway, intracellular DNA sensors, intracellular RNA sensors, and inflammasomes, is crucial in the regulation of these signaling trails. Ubiquitination, phosphorylation, polyubiquitination, methylation, and acetylation are the main forms of the PTM, and they respectively play different roles in signaling regulation. The effects of the PTM range from the production of pro-inflammatory factors and the interaction between adaptors and receptors to cell translocation in response to the infectious or other dangerous factors. In this chapter, we will have an overview of the different ways of the posttranslational modifications in different inflammatory signaling pathways and their essential roles in regulation of inflammation.

Acyloxyacyl hydrolase promotes the resolution of lipopolysaccharide-induced acute lung injury.

Pulmonary infection is the most common risk factor for acute lung injury (ALI). Innate immune responses induced by Microbe-Associated Molecular Pattern (MAMP) molecules are essential for lung defense but can lead to tissue injury. Little is known about how MAMP molecules are degraded in the lung or how MAMP degradation/inactivation helps prevent or ameliorate the harmful inflammation that produces ALI. Acyloxyacyl hydrolase (AOAH) is a host lipase that inactivates Gram-negative bacterial endotoxin (lipopolysaccharide, or LPS). We report here that alveolar macrophages increase AOAH expression upon exposure to LPS and that Aoah+/+ mice recover more rapidly than do Aoah-/- mice from ALI induced by nasally instilled LPS or Klebsiella pneumoniae. Aoah-/- mouse lungs had more prolonged leukocyte infiltration, greater pro- and anti-inflammatory cytokine expression, and longer-lasting alveolar barrier damage. We also describe evidence that the persistently bioactive LPS in Aoah-/- alveoli can stimulate alveolar macrophages directly and epithelial cells indirectly to produce chemoattractants that recruit neutrophils to the lung and may prevent their clearance. Distinct from the prolonged tolerance observed in LPS-exposed Aoah-/- peritoneal macrophages, alveolar macrophages that lacked AOAH maintained or increased their responses to bioactive LPS and sustained inflammation. Inactivation of LPS by AOAH is a previously unappreciated mechanism for promoting resolution of pulmonary inflammation/injury induced by Gram-negative bacterial infection.

Modulation of host signaling in the inflammatory response by enteropathogenic Escherichia coli virulence proteins.

To successfully infect host cells and evade the host immune response, a type III secretion system (T3SS) is commonly used by enteric bacterial pathogens such as enteropathogenic Escherichia coli (EPEC). Recent findings have revealed that various effectors are injected into host cells through the T3SS and exert an inhibitory effect on inflammatory signaling pathways, subverting the immune responses to these pathogens. Here we review recent studies aimed at addressing the modulation of several important inflammatory signaling pathways modulated by EPEC effector proteins, such as the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, which provides insight into the unfinished work in this unexplored field and helps to identify novel positions in inflammatory signaling networks for EPEC effectors.Cellular & Molecular Immunology advance online publication, 31 October 2016; doi:10.1038/cmi.2016.52.

High power all-fiber amplifier with different seed power injection.

We report a high power all-fiber amplifier with suitable seed power injected by an all-fiber laser. Different seed powers were injected into the all-fiber amplifier during our amplification experiments, and we found the stimulated Raman scattering (SRS) threshold was inversely proportional to the injected seed power. More than 3 kW signal light with good beam quality (M2 = 1.28) has been obtained with a suitable seed power injected, and the slope efficiency of the all-fiber amplifier was about 84.4%.