Intermediate monocytes induced by IFN-γ inhibit cancer metastasis by promoting NK cell activation through FOXO1 and interleukin-27.

BACKGROUND: Circulating monocytes are functionally heterogeneous and can be divided into classical (CMo), intermediate (IMo), and non-CMo/patrolling monocyte (PMo) subsets. CMo can differentiate into PMo through IMo. PMos have been shown to inhibit cancer metastasis but the role of IMo is unclear. To date, no strategy has been developed to inhibit cancer metastasis through enhancing PMo/IMo differentiation. METHODS: We screened multiple inflammatory cytokines/chemokines activity of modulating PMo/IMo associated cell markers expression using human monocyte in vitro culture system. We tested our candidate cytokine activity in vivo using multiple mice models. We identified critical key factors and cytokines for our candidate cytokine activity by using gene-knockout mice and neutralization antibodies. RESULTS: We identified IFN-γ as a candidate inflammatory cytokine in the regulation of human IMo/PMo marker expression. Our in vivo data demonstrated that IMo expansion was induced by short-term (3 days) IFN-γ treatment through increasing CMo-IMo differentiation and blocking IMo-PMo differentiation. The IMo induced by IFN-γ (IFN-IMo), but not IFN-γ activated CMo (IFN-CMo), inhibited cancer metastasis by 90%. Surprizing, the effect of IFN-γ is greater in PMo deficiency mice, indicating the effect of IFN-IMo is not mediated through further differentiation into PMo. We also found that IFN-IMos induced by short-term IFN-γ treatment robustly boosted NK cell expansion for threefold and promoted NK differentiation and function through IL-27 and CXCL9. Furthermore, we identified that FOXO1, a key molecule controlling cellular energy metabolism, mediated the effect of IFN-γ induced IL-27 expression, and that NR4A1, a key molecule controlling PMo differentiation and inhibiting cancer metastasis, inhibited the pro-NK cell and anti-metastasis activity of IFN-IMo by suppressing CXCL9 expression. CONCLUSIONS: We have discovered the antimetastasis and pro-NK cell activity of IFN-IMo, identified FOXO1 as a key molecule for IFN-γ driven monocyte differentiation and function, and found NR4A1 as an inhibitory molecule for IFN-IMo activity. Our study has not only shown novel mechanisms for a classical antitumor cytokine but also provided potential target for developing superior monocytic cell therapy against cancer metastasis.

IL-27 Mediates Pro-Inflammatory Effects via the ERK Signaling Pathway During Preterm Labor.

Preterm labor (PTL) is a multifactorial syndrome that results in birth prior to 37 weeks of gestation. However, the specific molecular mechanisms underlying this condition have yet to be elucidated. Previous research demonstrated that the abnormal expression of IL-27, and its receptors, played a role in the pathophysiology of preterm labor. In the present study, we established a Lipopolysaccharide (LPS)-stimulated, infection-induced, preterm mouse model based on wild-type C57BL/6 mice and WSX-1-/-C57BL/6 mice. WSX-1 knockdown led to a significant delay in birth by 11.32 ± 2.157h. In addition, compared with wild-type C57B/6 mice, the expression levels of IFN-γ, IL-1ß, IL-6, TNF-α, and CXCL10, in the fetal membrane and myometrium of WSX-1-/-mice were significantly lower, particularly in the myometrium. We also confirmed similar pro-inflammatory effects arising from IL-27 in human amniotic cell line (WISH) and human myometrial smooth muscle cell line (HMSMC). Once stimulated by LPS, the pro-inflammatory action exhibited a synergistic effect and appeared to be time-dependent. Finally, we demonstrated that LY3214996, an inhibitor of the ERK pathway, significantly inhibited the pro-inflammatory effect mediated by IL-27. Overall, our data confirmed that the inflammatory effect mediated by the IL-27/IFN-r/ERK axis is involved in preterm labor. Our findings, therefore, provide an enhancement in our etiological understanding of the mechanisms underlying PTL.

ENTPD1 (CD39) Expression Inhibits UVR-Induced DNA Damage Repair through Purinergic Signaling and Is Associated with Metastasis in Human Cutaneous Squamous Cell Carcinoma.

UVR and immunosuppression are major risk factors for cutaneous squamous cell carcinoma (cSCC). Regulatory T cells promote cSCC carcinogenesis, and in other solid tumors, infiltrating regulatory T cells and CD8+ T cells express ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1) (also known as CD39), an ectoenzyme that catalyzes the rate-limiting step in converting extracellular adenosine triphosphate (ATP) to extracellular adenosine (ADO). We previously showed that extracellular purine nucleotides influence DNA damage repair. In this study, we investigate whether DNA damage repair is modulated through purinergic signaling in cSCC. We found increased ENTPD1 expression on T cells within cSCCs when compared with the expression on T cells from blood or nonlesional skin, and accordingly, concentrations of derivative extracellular adenosine diphosphate (ADP), adenosine monophosphate (AMP), and ADO are increased in tumors compared with those in normal skin. Importantly, ENTPD1 expression is significantly higher in human cSCCs that metastasize than in those that are nonmetastatic. We also identify in a mouse model that ENTPD1 expression is induced by UVR in an IL-27-dependent manner. Finally, increased extracellular ADO is shown to downregulate the expression of NAP1L2, a nucleosome assembly protein we show to be important for DNA damage repair secondary to UVR. Together, these data suggest a role for ENTPD1 expression on skin-resident T cells to regulate DNA damage repair through purinergic signaling to promote skin carcinogenesis and metastasis.

Interleukin-17D Aggravates Sepsis by Inhibiting Macrophage Phagocytosis.

OBJECTIVES: Interleukin-17D has been shown to participate in the control of viral infections and cancer. Here we hypothesized that interleukin-17D may play a potential role in sepsis. DESIGN: Prospective randomized animal investigation and in vitro human blood studies. SETTING: Research laboratory from a university hospital. SUBJECTS: Female C57BL/6J mice, sepsis patients by Sepsis-3 definitions, ICU patient controls, and healthy individuals. INTERVENTIONS: Serum concentrations of interleukin-17D were measured and analyzed in human sepsis patients, patient controls, and healthy individuals. The contribution of interleukin-17D to sepsis-related survival, bacterial burden, and organ injury was assessed in a murine model of cecal ligation and puncture-induced polymicrobial sepsis by the use of anti-interleukin-17D antibody and recombinant interleukin-17D protein. The effects of interleukin-17D on bacterial phagocytosis by macrophages were also investigated using in vitro cell models. MEASUREMENTS AND MAIN RESULTS: On the day of ICU admission (day 0), septic patients had significantly higher serum concentrations of interleukin-17D than patient controls and healthy individuals. Serum interleukin-17D levels remained significantly elevated in septic patients from ICU admission to day 3 and correlated with Sequential (Sepsis-related) Organ Failure Assessment scores and documented bacteremia on day 0. Furthermore, nonsurvivors of septic patients displayed significantly higher interleukin-17D levels compared with survivors of septic patients on days 0 and 1 of ICU admission. In animal models of sepsis, treatment with anti-interleukin-17D antibody protected mice from cecal ligation and puncture-induced severe sepsis, which was associated with improved bacterial clearance and organ injury. Conversely, administration of recombinant interleukin-17D protein aggravated cecal ligation and puncture-induced nonsevere sepsis. Furthermore, we found that interleukin-17D impaired bacterial phagocytosis by macrophages. Phagocytosis inhibition by interleukin-17D involved its ability to down-regulate the activation of nuclear factor-κB signaling pathway in macrophages upon bacterial infection. CONCLUSIONS: This study indicates a previously undescribed role of interleukin-17D in sepsis and identifies a new target for antisepsis treatment.

Interleukin-27 enhances TNF-α-mediated activation of human coronary artery endothelial cells.

Immune cells and inflammatory mediators play important roles in the development of atherosclerotic vascular inflammation. IL-27 is a member of the IL-6/IL-12 family that can promote Th1 responses and augment the release of inflammatory mediators from human mast cells and monocytes. However, the direct effect of IL-27 on human coronary artery endothelial cells was unclear. In this study, the effects of IL-27 and TNF-α on the cell surface expression of adhesion molecules, inflammatory cytokines, and chemokines were investigated. Results showed that IL-27 alone could significantly promote the release of chemokine CXCL10. However, IL-27 could further significantly enhance the TNF-α-mediated upregulation of adhesion molecules ICAM-1 and VCAM-1, inflammatory cytokine IL-6, as well as chemokines CCL5 and CXCL10 from human coronary artery endothelial cells. The release of IL-6, CCL5, and CXCL10 were significantly suppressed by specific signaling molecule inhibitors, implying that the induction of these mediators from the human coronary artery endothelial cells could be differentially regulated by the c-Jun N-terminal kinase, p38 mitogen-activated protein kinase, and nuclear factor-κB pathways. These results provided new insights into the effect of IL-27 on the TNF-α mediated activation of human coronary artery endothelial cells in atherosclerotic vascular inflammation.

The immunobiology of interleukin-27.

Interleukin-27 (IL-27) is a cytokine with strikingly diverse influences on the immune response. Although it was initially linked with the development of Th1 responses, it is now recognized as a potent antagonist of different classes of inflammation through its ability to directly modify CD4(+) and CD8(+) T cell effector functions, to induce IL-10, and to promote specialized T regulatory cell responses. Although this aspect of IL-27 biology has provided insights into how the immune system prevents hyperactivity in the setting of infectious and autoimmune inflammation, in vaccination and cancer models the stimulatory effects of IL-27 on CD8(+) T cell function appear prominent. Additionally, associations between IL-27 and antibody-mediated disease have led to an interest in defining the impact of IL-27 on innate immunity and humoral responses in different disease states. The maturation of this literature has been accompanied by attempts to translate these findings from experimental models into human diseases and by efforts to define where IL-27 might represent a viable therapeutic target.

Dendritic cell subsets require cis-activation for cytotoxic CD8 T-cell induction.

Dendritic cells (DCs) are required for the induction of cytotoxic T cells (CTL). In most tissues, including the lung, the resident DCs fall into two types expressing the integrin markers CD103 and CD11b. The current supposition is that DC function is predetermined by lineage, designating the CD103(+) DC as the major cross-presenting DC able to induce CTL. Here we show that Poly I:C (TLR3 agonist) or R848 (TLR7 agonist) do not activate all endogenous DCs. CD11b(+) DCs can orchestrate a CTL response in vivo in the presence of a TLR7 agonist but not a TLR3 agonist, whereas CD103(+) DCs require ligation of TLR3 for this purpose. This selectivity does not extend to antigen cross-presentation for T-cell proliferation but is required for induction of cytotoxicity. Thus, we demonstrate that the ability of DCs to induce functional CTLs is specific to the nature of the pathogen-associated molecular pattern (PAMP) encountered by endogenous DC.

IL-27 controls sepsis-induced impairment of lung antibacterial host defence.

BACKGROUND: Interleukin 27 (IL-27) is an important cytokine regulating host immune responses. However, its role in sepsis-induced immunosuppression remains unclear. AIM: To investigate the role of IL-27 in modulating sepsis-induced immunosuppression using a murine model of caecal ligation and puncture (CLP)-induced sepsis followed by secondary challenge with Pseudomonas aeruginosa. METHODS: CLP or sham surgery was performed in wild-type (WT) and IL-27 receptor (IL-27R)/WSX-1 knockout (KO) mice, and then mice were infected with intratracheal P aeruginosa. RESULTS: IL-27 was upregulated in patients with sepsis and septic mice. Following sepsis and secondary intrapulmonary bacterial challenge, IL-27R KO mice had higher survival rates and improved bacterial clearance from lung and blood compared with WT mice, which was associated with early increased pulmonary cytokine/chemokine production, as well as enhanced neutrophil recruitment to airspaces. Neutralisation of IL-27 in septic mice significantly improved survival and clearance of bacteria from the lungs of septic mice infected with P aeruginosa, and direct application of recombinant IL-27 could increase susceptibility to P aeruginosa infection. The resistance of septic IL-27R KO mice to secondary P aeruginosa infection was abrogated by depletion of alveolar macrophages (AMs) and neutrophils. AMs from septic IL-27R KO mice had higher bacterial uptake and killing capacities, enhanced cytokine/chemokine production, and increased expression of costimulatory molecules compared with those from WT mice, while neutrophils from septic IL-27R KO mice had increased bacterial killing ability and higher expression of adhesion molecule Mac-1 compared with WT neutrophils. CONCLUSIONS: IL-27 is an important mediator of sepsis-induced impairment of lung antibacterial host defence.

The suppressive effect of IL-27 on encephalitogenic Th17 cells induced by multiwalled carbon nanotubes reduces the severity of experimental autoimmune encephalomyelitis.

BACKGROUND: Both Th1 and Th17 cells specific for neuroantigen are described as encephalitogenic in the experimental autoimmune encephalomyelitis (EAE) model. AIM: The proposal of this study was to investigate how carbon nanotubes internalized by antigen-presenting cells (APCs) affect the development of encephalitogenic CD4(+) T cells. METHODS: Therefore, we stimulated encephalitogenic T cells in the presence or not of multiwalled carbon nanotube (MWCNT). After the incubation, we analyzed the expression profile of the encephalitogenic T cells and their capacity to induce EAE. RESULTS: Encephalitogenic CD4(+) T cells cultured with APCs that were previously incubated with MWCNTs do not express IL-17. The adoptive transfer of these cells causes less severe EAE than the transfer of both Th1 and Th17 cells that are not incubated with MWCNTs. These results suggest that the increased IL-27 level produced by the APCs incubated with the carbon nanotubes inhibits the development of Th17 cells. This observation is confirmed by the concomitant reduction in the level of RORγt, which is a transcription factor essential for the development of Th17 cells. Moreover, the incubation of encephalitogenic T cells devoid of Th17 cells with neutralizing anti-IL-27 antibodies restored the production of IL-17. CONCLUSION: This finding confirms the suppressive effect of IL-27 on encephalitogenic Th17 cells. The results presented suggest that the stimulation of APCs with carbon nanoparticles prior to neuroantigen presentation affects the development of the Th17 subset of encephalitogenic CD4(+) T lymphocytes and results in less severe EAE.

[Toward therapeutic application of IL-10-producing regulatory T cells].

IL-10 is an anti-inflammatory cytokine with an important role in preventing inflammatory and autoimmune responses. IL-10 is also important for suppressive function of inducible regulatory T (iTreg) cells, several types of which were reported in succession. Type1 regulatory T (Tr1) cell is a representative of IL-10-prroducing regulatory T cells. Although specific surface markers or origin of Tr1 cells have not fully been clarified yet, IL-27 was recently reported to induce IL-10 production in T cells and be an inducer of Tr1 cells. We previously reported that CD4(+)CD25(-)lymphocyte activation gene (LAG-3)(+) Treg (LAG3(+) Treg) is one of the peripherally inducible Tregs and functions as an immune-regulator through IL-10 production. We found that the expression level of Egr-2, a transcription factor required for T cell anergy induction, is elevated in LAG3(+) Treg and that forced expression of Egr-2 induces LAG-3 expression and IL-10 production. Egr-2 has been suggested to be a key player of regulatory function in LAG3(+) Treg. In this study, we review Tr1 cells and the mechanism of IL-10 induction by IL-27 in T cells. Also, we introduce LAG3(+) Treg and discuss the therapeutic potential of these regulatory T cells.