Grass carp (Ctenopharyngodon idellus) SHP2 suppresses IFN I expression via decreasing the phosphorylation of GSK3ß in a non-contact manner.

As a tyrosine phosphatase, Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) serves as an inhibitor in PI3K-Akt pathway. In mammals, SHP2 can phosphorylate GSK3ß at Y216 site to control the expression of IFN. So far, the multiple functions of SHP2 have been reported in mammals. However, little is known about fish SHP2. In this study, we cloned and identified a grass carp (Ctenopharyngodon idellus) SHP2 gene (CiSHP2, MT373151). SHP2 is conserved among different vertebrates by amino acid sequences alignment and the phylogenetic tree analysis. CiSHP2 shared the closest homology with Danio rerio SHP2. Simultaneously, SHP2 was also tested in grass carp tissues and CIK (C. idellus kidney) cells. We found that it responded to poly I:C stimulation. CiSHP2 was located in the cytoplasm just as the same as those of mammals. Interestingly, it inhibited the phosphorylation level of GSK3ß in a non-contact manner. Meanwhile CiGSK3ß interacted with and directly phosphorylated CiTBK1. In addition, we found that CiSHP2 also reduced the phosphorylation level of CiTBK1 by CiGSK3ß, and then it depressed the expression of IFN I via GSK3ß-TBK1 axis. These results suggested that CiSHP2 was involved in CiGSK3ß and CiTBK1 activity but not regulated their transcriptional level. At the same time, we also found that CiSHP2 also influenced the activity of CiIRF3. Therefore, fish SHP2 inhibited IFN I expression through blocking GSK3ß-TBK1 signal axis.

SHP2 blockade enhances anti-tumor immunity via tumor cell intrinsic and extrinsic mechanisms.

SHP2 is a ubiquitous tyrosine phosphatase involved in regulating both tumor and immune cell signaling. In this study, we discovered a novel immune modulatory function of SHP2. Targeting this protein with allosteric SHP2 inhibitors promoted anti-tumor immunity, including enhancing T cell cytotoxic function and immune-mediated tumor regression. Knockout of SHP2 using CRISPR/Cas9 gene editing showed that targeting SHP2 in cancer cells contributes to this immune response. Inhibition of SHP2 activity augmented tumor intrinsic IFNγ signaling resulting in enhanced chemoattractant cytokine release and cytotoxic T cell recruitment, as well as increased expression of MHC Class I and PD-L1 on the cancer cell surface. Furthermore, SHP2 inhibition diminished the differentiation and inhibitory function of immune suppressive myeloid cells in the tumor microenvironment. SHP2 inhibition enhanced responses to anti-PD-1 blockade in syngeneic mouse models. Overall, our study reveals novel functions of SHP2 in tumor immunity and proposes that targeting SHP2 is a promising strategy for cancer immunotherapy.

Protein Tyrosine Phosphatase SHP2 Suppresses Host Innate Immunity against Influenza A Virus by Regulating EGFR-Mediated Signaling.

Influenza A virus (IAV) is a highly contagious pathogen, causing acute respiratory illnesses in human beings and animals and frequently giving rise to epidemic outbreaks. Evasion by IAV of host immunity facilitates viral replication and spread, which can be initiated through various mechanisms, including epidermal growth factor receptor (EGFR) activation. However, how EGFR mediates the suppression of antiviral systems remains unclear. Here, we examined host innate immune responses and their relevant signaling to EGFR upon IAV infection. IAV was found to induce the phosphorylation of EGFR and extracellular signal-regulated kinase (ERK) at an early stage of infection. Inhibition of EGFR or ERK suppressed the viral replication but increased the expression of type I and type III interferons (IFNs) and interferon-stimulated genes (ISGs), supporting the idea that IAV escapes from antiviral innate immunity by activating EGFR/ERK signaling. Meanwhile, IAV infection also induced the activation of Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2). Pharmacological inhibition or small interfering RNA (siRNA)-based silencing of SHP2 enhanced the IFN-dependent antiviral activity and reduced virion production. Furthermore, knockdown of SHP2 attenuated the EGFR-mediated ERK phosphorylation triggered by viral infection or EGF stimulation. Conversely, ectopic expression of constitutively active SHP2 noticeably promoted ERK activation and viral replication, concomitant with diminished immune function. Altogether, the results indicate that SHP2 is crucial for IAV-induced activation of the EGFR/ERK pathway to suppress host antiviral responses.IMPORTANCE Viral immune evasion is the most important strategy whereby viruses evolve for their survival. This work shows that influenza A virus (IAV) suppressed the antiviral innate immunity through downregulation of IFNs and ISGs by activating EGFR/ERK signaling. Meanwhile, IAV also induced the activation of protein tyrosine phosphatase SHP2, which was found to be responsible for modulating the EGFR-mediated ERK activity and subsequent antiviral effectiveness both in vitro and in vivo The results suggest that SHP2 is a key signal transducer between EGFR and ERK and plays a crucial role in suppressing host innate immunity during IAV infection. The finding enhances our understanding of influenza immune evasion and provides a new therapeutic approach to viral infection.

SHP2 inhibition diminishes KRASG12C cycling and promotes tumor microenvironment remodeling.

KRAS is the most frequently mutated human oncogene, and KRAS inhibition has been a longtime goal. Recently, inhibitors were developed that bind KRASG12C-GDP and react with Cys-12 (G12C-Is). Using new affinity reagents to monitor KRASG12C activation and inhibitor engagement, we found that an SHP2 inhibitor (SHP2-I) increases KRAS-GDP occupancy, enhancing G12C-I efficacy. The SHP2-I abrogated RTK feedback signaling and adaptive resistance to G12C-Is in vitro, in xenografts, and in syngeneic KRASG12C-mutant pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC). SHP2-I/G12C-I combination evoked favorable but tumor site-specific changes in the immune microenvironment, decreasing myeloid suppressor cells, increasing CD8+ T cells, and sensitizing tumors to PD-1 blockade. Experiments using cells expressing inhibitor-resistant SHP2 showed that SHP2 inhibition in PDAC cells is required for PDAC regression and remodeling of the immune microenvironment but revealed direct inhibitory effects on tumor angiogenesis and vascularity. Our results demonstrate that SHP2-I/G12C-I combinations confer a substantial survival benefit in PDAC and NSCLC and identify additional potential combination strategies.

ROS1 Fusion Mediates Immunogenicity by Upregulation of PD-L1 After the Activation of ROS1-SHP2 Signaling Pathway in Non-Small Cell Lung Cancer.

The drug resistance of first-line crizotinib therapy for ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) fusion non-small cell lung cancer (NSCLC) is inevitable. Whether the administration of immune checkpoint inhibitor (ICI) therapy is suitable for ROS 1 fusion NSCLCs or after the development of crizotinib resistance is still unknown. In this study, five different crizotinib resistant concentration cell lines (HCC78CR1-5) from primary sensitive HCC78 cells were cultured. Ba/F3 cells expressing crizotinib sensitive ROS1 fusion and crizotinib resistant ROS1-G2032R mutation were used to explore the relationship between ROS1 fusion, ROS1-G2032R mutation and programmed death-ligand 1 (PD-L1) expression and the clinical potential of anti-PD-L1 ICI therapy. The signaling pathway net was compared between HCC78 and HCC78CR1-5 cells using RNA sequencing. Anti- PD-L1 ICI therapy was performed on mouse xenograft models with Ba/F3 ROS1 fusion or ROS1-G2032R mutation. HCC78CR1-5 showed more immunogenicity than HCC78 in immune-related pathways. The PD-L1 expression level was remarkably higher in HCC78CR1-5 with ROS1 fusion upregulation than HCC78 primary cell. Furthermore, the expression of PD-L1 was down-regulated by RNA interference with ROS1 siRNAs and up-regulated lower in Ba/F3 ROS1-G2032R resistant mutation than ROS1 fusion. Western blotting analysis showed the ROS1-SHP2 signaling pathway activation in HCC78CR1-5 cells, Ba/F3 ROS1 fusion and ROS1-G2032R resistant mutation. Mouse xenograft models with Ba/F3 ROS1 fusion showed more CD3+PD-1+ T cells both in blood and tissue, and more sensitivity than the cells with Ba/F3 ROS1-G2032R resistant mutation after anti-PD-L1 therapy. Our findings indicate that PD-L1 upregulation depends on ROS1 fusion more than ROS1-G2032R mutation. We share our insights of NSCLCs treatment management into the use of anti-PD-L1 ICI therapy in ROS1 fusion and not in ROS1-G2032R resistant mutation.

Identification, recombinant expression and immunological study of Lja-SHP2 in Lampetra japonica.

The protein tyrosine phosphatase SHP2 of higher vertebrates, encoded by ptpn11 gene, catalyzes the dephosphorylation of tyrosine phosphorylation site, and plays regulatory roles in various signaling pathways by cooperating with other protein tyrosine kinase. Previous studies have shown that SHP2 plays an important role in the activation and signal transduction of T and B cells in higher vertebrates. To study the role of a SHP2 homologous molecule of lampreys (Lja-SHP2) in immune response, we cloned and expressed the open reading frame sequence of Lja-SHP2 gene in prokaryotic expression vector pET-32a. The recombinant protein was successfully expressed in E. coli and the rabbit-derived polyclonal antibody was prepared. Lampetra japonica were immunized with mixed bacteria, and the mRNA and protein of Lja-SHP2 in immune-related cells and tissues were detected by real-time quantitative PCR and Western blotting after immunization. The Lja-SHP2 mRNA and protein were not significantly affected in leukocytes and supraneural myeloid bodies, but up-regulated significantly in gill tissues (P<0.05) after challenged by mixed bacteria, which indicated that Lja-SHP2 mainly participates in the immune response of gill tissues after mixed bacteria stimulation. To further investigate whether Lja-SHP2 level was affected in three lymphocyte subsets, the B-cell mitogen lipopolysaccharide (LPS) and T-cell mitogen phytohaemagglutinin (PHA) were employed to boost the immune response in L. japonica. LPS immune stimulation increased Lja-SHP2 in leucocytes significantly compared with the control group, and but had a marginal effect on Lja-SHP2 expression in gills and supraneural myeloid bodies. PHA immune stimulation could up-regulate Lja-SHP2 level in leukocytes, gill tissues and supraneural myeloid bodies. The change of Lja-SHP2 was especially dramatical in leukocytes, which was about 2.5 times higher than that in the control group, suggesting that Lja-SHP2 is involved in the lamprey immune response mediated by PHA. Consistent with the previous finding that PHA could induce the activation of VLRA+ lymphocytes, our results showed that Lja-SHP2 might be included in the immune response of VLRA+ lymphocytes mediated by PHA in gills. This research will benefit exploring the functions of Lja-SHP2 in the immune response of lamprey and will provide clues for understanding the phylogenesis of SHP2 molecular family, and its roles in the early occurrence and evolution of adaptive immune system in higher vertebrates.

The immune checkpoint receptor associated phosphatases SHP-1 and SHP-2 are not required for γδT17 cell development, activation, or skin inflammation.

IL-17-producing gamma delta (γδT17) cells are innate lymphocytes critical for antibacterial protection at barrier surfaces such as the skin but also highly pathogenic during inflammation. It is therefore important to understand the cellular and molecular mechanisms that could counter-balance overt γδT17 cell activation. Immune checkpoint receptors (ICRs) deliver inhibitory signals to activated lymphocytes and have been implicated as negative regulators of mouse γδT17 cells. In this report, we investigated the cytokine signals that induce ICR expression on γδT17 cells and studied the in vivo role of the Src-homology-2 phosphatases 1 and 2 (SHP-1 and SHP-2) in the context of γδT17-induced psoriasis. We found that surface expression of ICRs can be induced by cytokines; however, SHP-1 or SHP-2 could not inhibit γδT17 responses. In this regard, conditional deletion of SHP-1, SHP-2, or both did no impact γδT17 cell development, expansion, cytokine production, or skin pathology.

Targeting SHP2 as a promising strategy for cancer immunotherapy.

Src homology-2-containing protein tyrosine phosphatase 2 (SHP2) is a major phosphatase involved in several cellular processes. In recent years, SHP2 has been the focus of significant attention in human diseases, particular in cancer. Several studies have shown that SHP2 plays an important role in regulating immune cell functions in tumor microenvironment. A few clinical trials conducted using SHP2 allosteric inhibitors have shown remarkable anti-tumor benefits and good safety profiles. This review focuses on the current understanding of the regulation of SHP2 and highlights the vital roles of SHP2 in T lymphocytes, macrophages and cancer cells. It also summarizes the current development of SHP2 inhibitors as a promising strategy for cancer immunotherapy.

SHP2 deficiency promotes Staphylococcus aureus pneumonia following influenza infection.

OBJECTIVES: Secondary bacterial pneumonia is common following influenza infection. However, it remains unclear about the underlying molecular mechanisms. MATERIALS AND METHODS: We established a mouse model of post-influenza S aureus pneumonia using conditional Shp2 knockout mice (LysMCre/+ :Shp2flox/flox ). The survival, bacterial clearance, pulmonary histology, phenotype of macrophages, and expression of type I interferons and chemokines were assessed between SHP2 deletion and control mice (Shp2flox/flox ). We infused additional KC and MIP-2 to examine the reconstitution of antibacterial immune response in LysMCre/+ :Shp2flox/flox mice. The effect of SHP2 on signal molecules including MAPKs (JNK, p38 and Erk1/2), NF-κB p65 and IRF3 was further detected. RESULTS: LysMCre/+ :Shp2flox/flox mice displayed impaired antibacterial immunity and high mortality compared with control mice in post-influenza S aureus pneumonia. The attenuated antibacterial ability was associated with the induction of type I interferon and suppression of chemo-attractants KC and MIP-2, which reduced the infiltration of neutrophils into the lung upon secondary bacterial invasion. In additional, Shp2 knockout mice displayed enhanced polarization to alternatively activated macrophages (M2 phenotype). Further in vitro analyses consistently demonstrated that SHP2-deficient macrophages were skewed towards an M2 phenotype and had a decreased antibacterial capacity. Moreover, SHP2 modulated the inflammatory response to secondary bacterial infection via interfering with NF-κB and IRF3 signalling in macrophages. CONCLUSIONS: Our findings reveal that the SHP2 expression enhances the host immune response and prompts bacterial clearance in post-influenza S aureus pneumonia.

Immune and genomic correlates of response to anti-PD-1 immunotherapy in glioblastoma.

Immune checkpoint inhibitors have been successful across several tumor types; however, their efficacy has been uncommon and unpredictable in glioblastomas (GBM), where <10% of patients show long-term responses. To understand the molecular determinants of immunotherapeutic response in GBM, we longitudinally profiled 66 patients, including 17 long-term responders, during standard therapy and after treatment with PD-1 inhibitors (nivolumab or pembrolizumab). Genomic and transcriptomic analysis revealed a significant enrichment of PTEN mutations associated with immunosuppressive expression signatures in non-responders, and an enrichment of MAPK pathway alterations (PTPN11, BRAF) in responders. Responsive tumors were also associated with branched patterns of evolution from the elimination of neoepitopes as well as with differences in T cell clonal diversity and tumor microenvironment profiles. Our study shows that clinical response to anti-PD-1 immunotherapy in GBM is associated with specific molecular alterations, immune expression signatures, and immune infiltration that reflect the tumor's clonal evolution during treatment.

Phosphatase Shp2 exacerbates intestinal inflammation by disrupting macrophage responsiveness to interleukin-10.

Inflammatory cytokines produced by activated macrophages largely contribute to the pathological signs of inflammatory bowel disease (IBD). Interleukin-10 (IL-10) is the predominant anti-inflammatory cytokine in the intestine, and its therapeutic efficacy for IBD has been clinically tested. Nevertheless, how the function of IL-10 is regulated in the intestinal microenvironment remains unknown, which largely hinders the further development of IL-10-based therapeutic strategies. Here, we found that the expression of phosphatase Shp2 was increased in colonic macrophages and blood monocytes from IBD patients compared with those from healthy controls. Shp2 deficiency in macrophages protects mice from colitis and colitis-driven colon cancer. Mechanistically, Shp2 disrupts IL-10-STAT3 signaling and its dependent anti-inflammatory response in human and mouse macrophages. Furthermore, a Shp2-inducing role of TNF-α is unveiled in our study. Collectively, our work identifies Shp2 as a detrimental factor for intestinal immune homeostasis and hopefully will be helpful in the future exploitation of IL-10 immunotherapy for IBD.

Shp-2 Is Dispensable for Establishing T Cell Exhaustion and for PD-1 Signaling In Vivo.

In chronic infection and cancer, T cells acquire a dysfunctional state characterized by the expression of inhibitory receptors. In vitro studies implicated the phosphatase Shp-2 downstream of these receptors, including PD-1. However, whether Shp-2 is responsible in vivo for such dysfunctional responses remains elusive. To address this, we generated T cell-specific Shp-2-deficient mice. These mice did not show differences in controlling chronic viral infections. In this context, Shp-2-deleted CD8+ T lymphocytes expanded moderately better but were less polyfunctional than control cells. Mice with Shp-2-deficient T cells also showed no significant improvement in controlling immunogenic tumors and responded similarly to controls to α-PD-1 treatment. We therefore showed that Shp-2 is dispensable in T cells for globally establishing exhaustion and for PD-1 signaling in vivo. These results reveal the existence of redundant mechanisms downstream of inhibitory receptors and represent the foundation for defining these relevant molecular events.

Interleukin 17 regulates SHP-2 and IL-17RA/STAT-3 dependent Cyr61, IL-23 and GM-CSF expression and RANKL mediated osteoclastogenesis by fibroblast-like synoviocytes in rheumatoid arthritis.

Interleukin (IL)-17 predominately produced by the Th17 cells, plays a crucial role in the fibroblast-like synoviocytes (FLS) mediated disease process of rheumatoid arthritis (RA). IL-17 exerts its pathogenic effects in RA-FLS by IL-17/IL-17RA/STAT-3 signaling. Recent studies have shown that RA-FLS produces SHP-2, Cyr61, IL-23, GM-CSF and RANKL which results in worsening of the disease. However, whether IL-17/IL-17RA/STAT-3 signaling regulates SHP-2, Cyr61, IL-23, GM-CSF and RANKL expressions in RA-FLS remains unknown. In this study, IL-17 treatment dramatically induced the production of Cyr61, IL-23 and GM-CSF in FLS isolated from adjuvant induced arthritis (AA) rats. Conversely, IL-17 mediated production of Cyr61, IL-23 and GM-CSF was abrogated by knockdown of IL-17RA using a small interfering RNA or blockade of STAT-3 activation with S3I-201 in AA-FLS. Interestingly, IL-17 treatment noticeably increased the expression of IL-17RA and SHP-2 in AA-FLS. However, silencing of IL-17RA reversed the effect of IL-17 on the expression of IL-17RA and SHP-2 in AA-FLS. In addition, an increased number of TRAP-positive multinucleated cells were observed in a coculture system consisting of IL-17 treated AA-FLS and rat bone marrow derived monocytes/macrophages. Further, mechanistically we found that IL-17 upregulated RANKL expression in AA-FLS that was dependent on the IL-17/IL-17RA/STAT-3 signaling cascade. Knockdown of IL-17RA or inhibition of STAT-3 activation decreased the IL- 17 induced RANKL expression by AA-FLS and their osteoclastogenic potential. Taken together, our findings demonstrate that IL-17 regulates SHP-2 expression and IL-17RA/STAT-3 dependent production of Cyr61, IL-23, GM-CSF and RANKL in AA-FLS and may reveal a new insight into the pathogenesis of RA.

E3 ligase FBXW7 is critical for RIG-I stabilization during antiviral responses.

Viruses can escape from host recognition by degradation of RIG-I or interference with the RIG-I signalling to establish persistent infections. However, the mechanisms by which host cells stabilize RIG-I protein for avoiding its degradation are largely unknown. We report here that, upon virus infection, the E3 ubiquitin ligase FBXW7 translocates from the nucleus into the cytoplasm and stabilizes RIG-I. FBXW7 interacts with SHP2 and mediates the degradation and ubiquitination of SHP2, thus disrupting the SHP2/c-Cbl complex, which mediates RIG-I degradation. When infected with VSV or influenza A virus, FBXW7 conditional knockout mice (Lysm+FBXW7f/f) show impaired antiviral immunity. FBXW7-deficient macrophages have decreased RIG-I protein levels and type-I interferon signalling. Furthermore, PBMCs from RSV-infected children have reduced FBXW7 mRNA levels. Our results identify FBXW7 as an important interacting partner for RIG-I. These findings provide insights into the function of FBXW7 in antiviral immunity and its related clinical significance.

Myelodysplastic and myeloproliferative disorders of childhood.

Myelodysplastic syndrome (MDS) and myeloproliferative disorders are rare in children; they are divided into low-grade MDS (refractory cytopenia of childhood [RCC]), advanced MDS (refractory anemia with excess blasts in transformation), and juvenile myelomonocytic leukemia (JMML), each with different characteristics and management strategies. Underlying genetic predisposition is recognized in an increasing number of patients. Germ line GATA2 mutation is found in 70% of adolescents with MDS and monosomy 7. It is challenging to distinguish RCC from aplastic anemia, inherited bone marrow failure, and reactive conditions. RCC is often hypoplastic and may respond to immunosuppressive therapy. In case of immunosuppressive therapy failure, hypercellular RCC, or RCC with monosomy 7, hematopoietic stem cell transplantation (HSCT) using reduced-intensity conditioning regimens is indicated. Almost all patients with refractory anemia with excess blasts are candidates for HSCT; children age 12 years or older have a higher risk of treatment-related death, and the conditioning regimens should be adjusted accordingly. Unraveling the genetics of JMML has demonstrated that JMML in patients with germ line PTPN11 and CBL mutations often regresses spontaneously, and therapy is seldom indicated. Conversely, patients with JMML and neurofibromatosis type 1, somatic PTPN11, KRAS, and most of those with NRAS mutations have a rapidly progressive disease, and early HSCT is indicated. The risk of relapse after HSCT is high, and prophylaxis for graft-versus-host disease and monitoring should be adapted to this risk.

Inhibition of SHP2 ameliorates the pathogenesis of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a devastating multisystemic autoimmune disorder. However, the molecular mechanisms underlying its pathogenesis remain elusive. Some patients with Noonan syndrome, a congenital disorder predominantly caused by gain-of-function mutations in the protein tyrosine phosphatase SH2 domain-containing PTP (SHP2), have been shown to develop SLE, suggesting a functional correlation between phosphatase activity and systemic autoimmunity. To test this directly, we measured SHP2 activity in spleen lysates isolated from lupus-prone MRL/lpr mice and found it was markedly increased compared with that in control mice. Similar increases in SHP2 activity were seen in peripheral blood mononuclear cells isolated from lupus patients relative to healthy patients. To determine whether SHP2 alters autoimmunity and related immunopathology, we treated MRL/lpr mice with an SHP2 inhibitor and found increased life span, suppressed crescentic glomerulonephritis, reduced spleen size, and diminished skin lesions. SHP2 inhibition also reduced numbers of double-negative T cells, normalized ERK/MAPK signaling, and decreased production of IFN-γ and IL-17A/F, 2 cytokines involved in SLE-associated organ damage. Moreover, in cultured human lupus T cells, SHP2 inhibition reduced proliferation and decreased production of IFN-γ and IL-17A/F, further implicating SHP2 in lupus-associated immunopathology. Taken together, these data identify SHP2 as a critical regulator of SLE pathogenesis and suggest targeting of its activity as a potent treatment for lupus patients.

The adhesion molecule PECAM-1 enhances the TGF-ß-mediated inhibition of T cell function.

Transforming growth factor-ß (TGF-ß) is an immunosuppressive cytokine that inhibits the proinflammatory functions of T cells, and it is a major factor in abrogating T cell activity against tumors. Canonical TGF-ß signaling results in the activation of Smad proteins, which are transcription factors that regulate target gene expression. We found that the cell surface molecule platelet endothelial cell adhesion molecule-1 (PECAM-1) facilitated noncanonical (Smad-independent) TGF-ß signaling in T cells. Subcutaneously injected tumor cells that are dependent on TGF-ß-mediated suppression of immunity for growth grew more slowly in PECAM-1(-/-) mice than in their wild-type counterparts. T cells isolated from PECAM-1(-/-) mice demonstrated relative insensitivity to the TGF-ß-dependent inhibition of interferon-γ (IFN-γ) production, granzyme B synthesis, and cellular proliferation. Similarly, human T cells lacking PECAM-1 demonstrated decreased sensitivity to TGF-ß in a manner that was partially restored by reexpression of PECAM-1. Co-incubation of T cells with TGF-ß and a T cell-activating antibody resulted in PECAM-1 phosphorylation on an immunoreceptor tyrosine-based inhibitory motif (ITIM) and the recruitment of the inhibitory Src homology 2 (SH2) domain-containing tyrosine phosphatase-2 (SHP-2). Such conditions also induced the colocalization of PECAM-1 with the TGF-ß receptor complex as identified by coimmunoprecipitation, confocal microscopy, and proximity ligation assays. These studies indicate a role for PECAM-1 in enhancing the inhibitory functions of TGF-ß in T cells and suggest that therapeutic targeting of the PECAM-1-TGF-ß inhibitory axis represents a means to overcome TGF-ß-dependent immunosuppression within the tumor microenvironment.

Role of Aspergillus fumigatus in Triggering Protease-Activated Receptor-2 in Airway Epithelial Cells and Skewing the Cells toward a T-helper 2 Bias.

Aspergillus fumigatus (AF) infection and sensitization are common and promote Th2 disease in individuals with asthma. Innate immune responses of bronchial epithelial cells are now known to play a key role in determination of T cell responses upon encounter with inhaled pathogens. We have recently shown that extracts of AF suppress JAK-STAT signaling in epithelial cells and thus may promote Th2 bias. To elucidate the impact of AF on human bronchial epithelial cells, we tested the hypothesis that AF can modulate the response of airway epithelial cells to favor a Th2 response and explored the molecular mechanism of the effect. Primary normal human bronchial epithelial (NHBE) cells were treated with AF extract or fractionated AF extract before stimulation with poly I:C or infection with human rhinovirus serotype 16 (HRV16). Expression of CXCL10 mRNA (real-time RT-PCR) and protein (ELISA) were measured as markers of IFN-mediated epithelial Th1-biased responses. Western blot was performed to evaluate expression of IFN regulatory factor-3 (IRF-3), NF-κB, and tyrosine-protein phosphatase nonreceptor type 11 (PTPN11), which are other markers of Th1 skewing. Knockdown experiments for protease-activated receptor-2 (PAR-2) and PTPN11 were performed to analyze the role of PAR-2 in the mechanism of suppression by AF. AF and a high-molecular-weight fraction of AF extract (HMW-AF; > 50 kD) profoundly suppressed poly I:C- and HRV16-induced expression of both CXCL10 mRNA and protein from NHBE cells via a mechanism that relied upon PAR-2 activation. Both AF extract and a specific PAR-2 activator (AC-55541) suppressed the poly I:C activation of phospho-IRF-3 without affecting activation of NF-κB. Furthermore, HMW-AF extract enhanced the expression of PTPN11, a phosphatase known to inhibit IFN signaling, and concurrently suppressed poly I:C-induced expression of both CXCL10 mRNA and protein from NHBE cells. These results show that exposure of bronchial epithelial cells to AF extract suppressed poly I:C and HRV16 signaling via a mechanism shown to involve activation of PAR-2 and PTPN11. This action of AF may promote viral disease exacerbations and may skew epithelial cells to promote Th2 inflammation in allergic airway disorders mediated or exacerbated by AF, such as asthma and chronic rhinosinusitis.

Helicobacter pylori infection activates Src homology-2 domain-containing phosphatase 2 to suppress IFN-γ signaling.

Helicobacter pylori infection not only induces gastric inflammation but also increases the risk of gastric tumorigenesis. IFN-γ has antimicrobial effects; however, H. pylori infection elevates IFN-γ-mediated gastric inflammation and may suppress IFN-γ signaling as a strategy to avoid immune destruction through an as-yet-unknown mechanism. This study was aimed at investigating the mechanism of H. pylori-induced IFN-γ resistance. Postinfection of viable H. pylori decreased IFN-γ-activated signal transducers and activators of transcription 1 and IFN regulatory factor 1 not only in human gastric epithelial MKN45 and AZ-521 but also in human monocytic U937 cells. H. pylori caused an increase in the C-terminal tyrosine phosphorylation of Src homology-2 domain-containing phosphatase (SHP) 2. Pharmacologically and genetically inhibiting SHP2 reversed H. pylori-induced IFN-γ resistance. In contrast to a clinically isolated H. pylori strain HP238, the cytotoxin-associated gene A (CagA) isogenic mutant strain HP238(CagAm) failed to induce IFN-γ resistance, indicating that CagA regulates this effect. Notably, HP238 and HP238(CagAm) differently caused SHP2 phosphorylation; however, imaging and biochemical analyses demonstrated CagA-mediated membrane-associated binding with phosphorylated SHP2. CagA-independent generation of reactive oxygen species (ROS) contributed to H. pylori-induced SHP2 phosphorylation; however, ROS/SHP2 mediated IFN-γ resistance in a CagA-regulated manner. This finding not only provides an alternative mechanism for how CagA and ROS coregulate SHP2 activation but may also explain their roles in H. pylori-induced IFN-γ resistance.