Constitutive expression of interleukin-27 diminishes proinflammatory cytokine production without impairing effector function of engineered T cells

Immunomodulatory cytokines can alter the tumor microenvironment and promote tumor eradication. Interleukin (IL)-27 is a pleiotropic cytokine that has potential to augment anti-tumor immunity while also facilitating anti-myeloma activity. We engineered human T cells to express a recombinant single-chain (sc)IL-27 and a synthetic antigen receptor targeting the myeloma antigen, B-cell maturation antigen, and evaluated the anti-tumor function of T cells bearing scIL-27 in vitro and in vivo. We discovered that T cells bearing scIL-27 sustained anti-tumor immunity and cytotoxicity yet manifested a profound reduction in pro-inflammatory cytokines granulocyte-macrophage colony-stimulating factor and tumor necrosis factor alpha. IL-27–expressing T cells therefore present a potential avenue to avert treatment-related toxicities commonly associated with engineered T-cell therapy due to the reduced pro-inflammatory cytokine profile.     

The role of IL-27 in susceptibility to post-influenza Staphylococcus aureus pneumonia

Background

Influenza is a common respiratory virus and Staphylococcus aureus frequently causes secondary pneumonia during influenza infection, leading to increased morbidity and mortality. Influenza has been found to attenuate subsequent Type 17 immunity, enhancing susceptibility to secondary bacterial infections. IL-27 is known to inhibit Type 17 immunity, suggesting a potential critical role for IL-27 in viral and bacterial co-infection.

Methods

A murine model of influenza and Staphylococcus aureus infection was used to mimic human viral, bacterial co-infection. C57BL/6 wild-type, IL-27 receptor α knock-out, and IL-10 knock-out mice were infected with Influenza H1N1 (A/PR/8/34) or vehicle for 6 days followed by challenge with Staphylococcus aureus or vehicle for 24 hours. Lung inflammation, bacterial burden, gene expression, and cytokine production were determined.

Results

IL-27 receptor α knock-out mice challenged with influenza A had increased morbidity compared to controls, but no change in viral burden. IL-27 receptor α knock-out mice infected with influenza displayed significantly decreased IL-10 production compared to wild-type. IL-27 receptor α knock-out mice co-infected with influenza and S. aureus had improved bacterial clearance compared to wild-type controls. Importantly, there were significantly increased Type 17 responses and decreased IL-10 production in IL-27 receptor α knock-out mice. Dual infected IL-10−/− mice had significantly less bacterial burden compared to dual infected WT mice.

Conclusions

These data reveal that IL-27 regulates enhanced susceptibility to S. aureus pneumonia following influenza infection, potentially through the induction of IL-10 and suppression of IL-17.     

Interleukin-27 Induces a STAT1/3- and NF-κB-dependent Proinflammatory Cytokine Profile in Human Monocytes

IL-27 is a heterodimeric cytokine bridging innate and adaptive immunity by playing a role in the activation of naive T cells and in development of Th1 cells. Additionally, recent evidence supports a role for IL-27 in the activation of monocytic cells. Both pro-inflammatory and anti-inflammatory activities have been attributed to IL-27; however, the role played by IL-27 in the activation of human monocytic cells in terms of cytokine production has not been well described. Our results show that IL-27 is a strong inducer of proinflammatory cytokine and chemokine expression, including enhancement of IL-6, IP-10, MIP-1α, MIP-1β, and TNF-α expression in human primary monocytes. Furthermore, we observed that IL-27-induced cytokine and chemokine production was mediated by STAT1, STAT3, and NF-κB activation. Understanding how IL-27 exerts its effects on monocytic cells will identify important molecular mechanisms in the regulation of immune responses, particularly in the modulation of monocyte activation.     

Evaluating the potential of IL-27 as a novel therapeutic agent in HIV-1 infection

Interleukin 27 (IL-27) is an immunomodulatory cytokine with important roles in both the innate and adaptive immune systems. In the last five years, the addition of exogenous IL-27 to primary cell cultures has been demonstrated to decrease HIV-1 replication in a number of cell types including peripheral blood mononuclear cells (PBMCs), CD4+ T cells, macrophages and dendritic cells. These in vitro findings suggest that IL-27 may have therapeutic value in the setting of HIV-1 infection. In this review, we describe the current knowledge of the biology of IL-27, its effects primarily on HIV-1 replication but also in other viral infections and explore its potential role as a therapeutic cytokine for the treatment of patients with HIV-1 infection.     

IL-27 affects helper T cell responses via regulation of PGE2 production by macrophages

IL-27 is a heterodimeric cytokine that regulates both innate and adaptive immunity. The immunosuppressive effect of IL-27 largely depends on induction of IL-10-producing Tr1 cells. To date, however, effects of IL-27 on regulation of immune responses via mediators other than cytokines remain poorly understood. To address this issue, we examined immunoregulatory effects of conditional medium of bone marrow-derived macrophages (BMDMs) from WSX-1 (IL-27Rα)-deficient mice and found enhanced IFN-γ and IL-17A secretion by CD4⁺ T cells as compared with that of control BMDMs. We then found that PGE₂ production and COX-2 expression by BMDMs from WSX-1-deficient mice was increased compared to control macrophages in response to LPS. The enhanced production of IFN-γ and IL-17A was abolished by EP2 and EP4 antagonists, demonstrating PGE₂ was responsible for enhanced cytokine production. Murine WSX-1-expressing Raw264.7 cells (mWSX-1-Raw264.7) showed phosphorylation of both STAT1 and STAT3 in response to IL-27 and produced less amounts of PGE₂ and COX-2 compared to parental RAW264.7 cells. STAT1 knockdown in parental RAW264.7 cells and STAT1-deficiency in BMDMs showed higher COX-2 expression than their respective control cells. Collectively, our result indicated that IL-27/WSX-1 regulated PGE₂ secretion via STAT1–COX-2 pathway in macrophages and affected helper T cell response in a PGE₂-mediated fashion.     

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.     

The biology of IL-12: coordinating innate and adaptive immune responses

Cytokines play critical roles in regulating all aspects of immune responses, including lymphoid development, homeostasis, differentiation, tolerance and memory. Interleukin (IL)-12 is especially important because its expression during infection regulates innate responses and determines the type and duration of adaptive immune response. IL-12 induces interferon-gamma (IFN-gamma) production by NK, T cells, dendritic cells (DC), and macrophages. IL-12 also promotes the differentiation of na?ve CD4+ T cells into T helper 1 (Th1) cells that produce IFN-gamma and aid in cell-mediated immunity. As IL-12 is induced by microbial products and regulates the development of adaptive immune cells, IL-12 plays a central role in coordinating innate and adaptive immunity. IL-12 and the recently identified cytokines, IL-23 and IL-27, define a family of related cytokines that induce IFN-gamma production and promote T cell expansion and proliferation.     

Trichomonas vaginalis Lipophosphoglycan Exploits Binding to Galectin-1 and -3 to Modulate Epithelial Immunity

Trichomoniasis is the most common non-viral sexually transmitted infection caused by the vaginotropic extracellular protozoan parasite Trichomonas vaginalis. The infection is recurrent, with no lasting immunity, often asymptomatic, and linked to pregnancy complications and risk of viral infection. The molecular mechanisms of immune evasion by the parasite are poorly understood. We demonstrate that galectin-1 and -3 are expressed by the human cervical and vaginal epithelial cells and act as pathogen-recognition receptors for the ceramide phosphoinositol glycan core (CPI-GC) of the dominant surface protozoan lipophosphoglycan (LPG). We used an in vitro model with siRNA galectin knockdown epithelial clones, recombinant galectins, clinical Trichomonas isolates, and mutant protozoan derivatives to dissect the function of galectin-1 and -3 in the context of Trichomonas infection. Galectin-1 suppressed chemokines that facilitate recruitment of phagocytes, which can eliminate extracellular protozoa (IL-8) or bridge innate to adaptive immunity (MIP-3α and RANTES (regulated on activation normal T cell expressed and secreted)). Silencing galectin-1 increased and adding exogenous galectin-1 suppressed chemokine responses to Trichomonas or CPI-GC/LPG. In contrast, silencing galectin-3 reduced IL-8 response to LPG. Live Trichomonas depleted the extracellular levels of galectin-3. Clinical isolates and mutant Trichomonas CPI-GC that had reduced affinity to galectin-3 but maintained affinity to galectin-1 suppressed chemokine expression. Thus via CPI-GC binding, Trichomonas is capable of regulating galectin bioavailability and function to the benefit of its parasitic survival. These findings suggest novel approaches to control trichomoniasis and warrant further studies of galectin-binding diversity among clinical isolates as a possible source for symptom disparity in parasitic infections.     

Kinetics of IL-23 and IL-12 Secretion in Response to Toxoplasma gondii Antigens from THP-1 Monocytic Cells

IL-23 and IL-12 are structurally similar and critical for the generation of efficient cellular immune responses. Toxoplasma gondii induces a strong cell-mediated immune response. However, little is known about IL-23 secretion profiles in T. gondii-infected immune cells in connection with IL-12. We compared the patterns of IL-23 and IL-12 production by THP-1 human monocytic cells in response to stimulation with live or heat-killed T. gondii tachyzoites, or with equivalent quantities of either T. gondii excretory/secretory proteins (ESP) or soluble tachyzoite antigen (STAg). IL-23 and IL-12 were significantly increased from 6 hr after stimulation with T. gondii antigens, and their secretions were increased with parasite dose-dependent manner. IL-23 concentrations were significantly higher than those of IL-12 at the same multiplicity of infection. IL-23 secretion induced by live parasites was significantly higher than that by heat-killed parasites, ESP, or STAg, whereas IL-12 secretion by live parasite was similar to those of ESP or STAg. However, the lowest levels of both cytokines were at stimulation with heat-killed parasites. These data indicate that IL-23 secretion patterns by stimulation with various kinds of T. gondii antigens at THP-1 monocytic cells are similar to those of IL-12, even though the levels of IL-23 induction were significantly higher than those of IL-12. The detailed kinetics induced by each T. gondii antigen were different from each other.     

Cutting edge: adaptive versus innate receptor signals selectively control the pool sizes of murine IFN-γ- or IL-17-producing γδ T cells upon infection

γδ T lymphocytes are commonly viewed as embracing properties of both adaptive and innate immunity. Contributing to this is their responsiveness to pathogen products, either with or without the involvement of the TCR and its coreceptors. This study clarifies this paradoxical behavior by showing that these two modes of responsiveness are the properties of two discrete sets of murine lymphoid γδ T cells. Thus, MyD88 deficiency severely impaired the response to malaria infection of CD27((-)), IL-17A-producing γδ T cells, but not of IFN-γ-producing γδ cells. Instead, the latter compartment was severely contracted by ablating CD27, which synergizes with TCRγδ in the induction of antiapoptotic mediators and cell cycle-promoting genes in CD27((+)), IFN-γ-secreting γδ T cells. Hence, innate versus adaptive receptors differentially control the peripheral pool sizes of discrete proinflammatory γδ T cell subsets during immune responses to infection.     

Antimicrobial decapeptide KSL-W attenuates Candida albicans virulence by modulating its effects on Toll-like receptor, human β-defensin, and cytokine expression by engineered human oral mucosa

We investigated the toxicity of synthetic antimicrobial decapeptide KSL-W on normal human gingival epithelial cell cultures, its effect on Candida albicans adhesion and growth, and the activation of epithelial cell innate immunity. Our results indicate that KSL-W had no toxic effect on cell adhesion or growth, suggesting its safe use with human cells. Pre-treating C. albicans with KSL-W attenuated the yeast's virulence as demonstrated by its reduced adhesion and growth on engineered human oral mucosa epithelium and the subsequent decreased expression of some innate defense molecules by targeted epithelial cells. Indeed, the expression of Toll-like receptors and human β-defensins was reduced in tissues infected with KSL-W-treated Candida. Proinflammtory cytokine secretion (IL-1β and IL-6) by the epithelial cells was also regulated by KSL-W in a manner similar to that of antifungal molecule amphotericin B. These findings therefore show that KSL-W is safe for use with human cells and is able to attenuate Candida virulence by modulating its effects on host innate immunity. This study proposes the potential application of KSL-W peptide as an alternative antifungal agent.     

Enhancement of phagocytosis and cytotoxicity in macrophages by tumor-derived IL-18 stimulation

Inoculation of mice with the murine NFSA cell line caused the formation of large tumors with necrotic tumor cores. FACS analysis revealed accumulations of CD11b⁺ cells in the tumors. Microarray analysis indicated that the NFSA cells expressed a high level of the pro-inflammatory factor interleukin-18 (il-18), which is known to play a critical role in macrophages. However, little is known about the physiological function of IL-18-stimulated macrophages. Here, we provide direct evidence that IL-18 enhances the phagocytosis of RAW264 cells and peritoneal macrophages, accompanied by the increased expression of tumor necrosis factor (tnf-α), interleukin-6 (il-6) and inducible nitric oxide synthase (Nos2). IL-18-stimulated RAW264 cells showed an enhanced cytotoxicity to endothelial F-2 cells via direct cell-to-cell interaction and the secretion of soluble mediators. Taken together, our results demonstrate that tumor-derived IL-18 plays an important role in the phagocytosis of macrophages and that IL-18-stimulated macrophages may damage tumor endothelial cells. [BMB Reports 2014; 47(5): 286-291]     

IL-33 facilitates rapid expulsion of the parasitic nematode Strongyloides ratti from the intestine via ILC2- and IL-9-driven mast cell activation

Parasitic helminths are sensed by the immune system via tissue-derived alarmins that promote the initiation of the appropriate type 2 immune responses. Here we establish the nuclear alarmin cytokine IL-33 as a non-redundant trigger of specifically IL-9-driven and mast cell-mediated immunity to the intestinal parasite Strongyloides ratti. Blockade of endogenous IL-33 using a helminth-derived IL-33 inhibitor elevated intestinal parasite burdens in the context of reduced mast cell activation while stabilization of endogenous IL-33 or application of recombinant IL-33 reciprocally reduced intestinal parasite burdens and increased mast cell activation. Using gene-deficient mice, we show that application of IL-33 triggered rapid mast cell-mediated expulsion of parasites directly in the intestine, independent of the adaptive immune system, basophils, eosinophils or Gr-1⁺ cells but dependent on functional IL-9 receptor and innate lymphoid cells (ILC). Thereby we connect the described axis of IL-33-mediated ILC2 expansion to the rapid initiation of IL-9-mediated and mast cell-driven intestinal anti-helminth immunity.     

NK cell intrinsic regulation of MIP-1α by granzyme M

Granzymes are generally recognized for their capacity to induce various pathways of perforin-dependent target cell death. Within this serine protease family, Granzyme M (GrzM) is unique owing to its preferential expression in innate effectors such as natural killer (NK) cells. During Listeria monocytogenes infection, we observed markedly reduced secretion of macrophage inflammatory protein-1 alpha (MIP-1α) in livers of GrzM-deficient mice, which resulted in significantly impaired NK cell recruitment. Direct stimulation with IL-12 and IL-15 demonstrated that GrzM was required for maximal secretion of active MIP-1α. This effect was not due to reduced protein induction but resulted from heightened intracellular accumulation of MIP-1α, with reduced release. These results demonstrate that GrzM is a critical mediator of innate immunity that can regulate chemotactic networks and has an important role in the initiation of immune responses and pathogen control.     

Leishmania mexicana: participation of NF-kappaB in the differential production of IL-12 in dendritic cells and monocytes induced by lipophosphoglycan (LPG)

Dendritic cells (DC) and macrophages (Mφ) are well known as important effectors of the innate immune system and their ability to produce IL-12 indicates that they possess the potential of directing acquired immunity toward a Th1-biased response. Interestingly, the intracellular parasite Leishmania has been shown to selectively suppress Mφ IL-12 production and are DC the principal source of this cytokine. The molecular details of this phenomenon remain enigmatic. In the present study we examined the effect of Leishmania mexicana lipophosphoglycan (LPG) on the production of IL-12, TNF-α, and IL-10 and nuclear translocation of NF-κB. The results show that LPG induced more IL-12 in human DC than in monocytes. This difference was due in part to nuclear translocation of NF-κB, since LPG induced more translocation in DC than in monocytes. These results suggest that Leishmania LPG impairs nuclear translocation of NF-κB in monocytes with the subsequent decrease in IL-12 production.     

Invariant NKT cells amplify the innate immune response to lipopolysaccharide

NKT cells are thought of as a bridge between innate and adaptive immunity. In this study, we demonstrate that mouse NKT cells are activated in response to Escherichia coli LPS, and produce IFN-gamma, but not IL-4, although activation through their TCR typically induces both IL-4 and IFN-gamma production. IFN-gamma production by NKT cells is dependent on LPS-induced IL-12 and IL-18 from APC. LPS induced IFN-gamma production by NKT cells does not require CD1d-mediated presentation of an endogenous Ag and exposure to a combination of IL-12 and IL-18 is sufficient to activate them. In mice that are deficient for NKT cells, innate immune cells are activated less efficiently in response to LPS, resulting in the reduced production of TNF and IFN-gamma. We propose that in addition to acting as a bridge to adaptive immunity, NKT cells act as an early amplification step in the innate immune response and that the rapid and complete initiation of this innate response depends on the early production of IFN-gamma by NKT cells.     

Identification of IFN-γ-producing innate B cells

Although B cells play important roles in the humoral immune response and the regulation of adaptive immunity, B cell subpopulations with unique phenotypes, particularly those with non-classical immune functions, should be further investigated. By challenging mice with Listeria monocytogenes, Escherichia coli, vesicular stomatitis virus and Toll-like receptor ligands, we identified an inducible CD11a^hiFcγRIII^hi B cell subpopulation that is significantly expanded and produces high levels of IFN-γ during the early stage of the immune response. This subpopulation of B cells can promote macrophage activation via generating IFN-γ, thereby facilitating the innate immune response against intracellular bacterial infection. As this new subpopulation is of B cell origin and exhibits the phenotypic characteristics of B cells, we designated these cells as IFN-γ-producing innate B cells. Dendritic cells were essential for the inducible generation of these innate B cells from the follicular B cells via CD40L-CD40 ligation. Increased Bruton''s tyrosine kinase activation was found to be responsible for the increased activation of non-canonical NF-κB pathway in these innate B cells after CD40 ligation, with the consequent induction of additional IFN-γ production. The identification of this new population of innate B cells may contribute to a better understanding of B cell functions in anti-infection immune responses and immune regulation.     

Interleukin-12 augments a Th1-type immune response manifested as lymphocyte proliferation and interferon gamma production in Leishmania infantum-infected dogs

The dog is the major reservoir for human visceral leishmaniasis caused by Leishmania infantum. Interleukin-12 is considered to have an essential role in the development of both innate and adaptive immunity to Leishmania spp. and other intracellular pathogens. This study focused on the influence of IL-12 in experimental and natural canine visceral leishmaniasis. Responses of peripheral blood mononuclear cells to IL-12, interleukin-10 and Leishmania soluble antigen were evaluated in L. infantum experimentally infected oligosymptomatic beagles, uninfected beagles, naturally infected polysymptomatic dogs, and their matched uninfected controls. Leishmania soluble antigen induced strong peripheral blood mononuclear cells proliferation both in experimentally infected dogs (median stimulation index [SI]=15.01), and in naturally infected dogs (SI=8.86), but not by cells from the control groups. IL-12 addition further enhanced cell proliferation in naturally (SI=14.95), but not in experimentally infected animals. Peripheral blood mononuclear cells from experimentally infected dogs were able to produce significant amounts of IFN-gamma (3.39 ng/ml) upon LSA stimulation, but no such production was detected in cells from naturally infected or control animals. Interestingly, addition of IL-12 reversed the inhibitory effect of LSA on IFN-gamma production by cells from polysymptomatic naturally infected dogs and the uninfected beagles (4.84 and 7.45 ng/ml, respectively), and further increased IFN-gamma production by peripheral blood mononuclear cells from experimentally infected oligosymptomatic dogs (29.28 ng/ml). IFN-gamma mRNA expression correlated well with IFN-gamma production. Addition of IL-10 to Leishmania soluble antigen stimulated peripheral blood mononuclear cells inhibited proliferation and IFN-gamma production in experimentally infected dogs. Thus, the ability of IL-12 to augment IFN-gamma production by peripheral blood mononuclear cells from dogs with experimental or natural symptomatic canine visceral leishmaniasis makes it a good candidate for cytokine therapy in dogs that are refractory to current therapy.