Type 2 dendritic cells mediate control of cytotoxic T cell resistant tumors.

Type 2 DCs (DC2s) comprise the majority of conventional DCs within most tumors; however, little is known about their ability to initiate and sustain antitumor immunity, as most studies have focused on antigen cross-presenting DC1s. Here, we report that DC2 infiltration identified by analysis of multiple human cancer data sets showed a significant correlation with survival across multiple human cancers, with the benefit being seen in tumors resistant to cytotoxic T cell control. Characterization of DC subtype infiltration into an immunotherapy-resistant model of breast cancer revealed that impairment of DC1s through 2 unique models resulted in enhanced DC2 functionality and improved tumor control. BATF3 deficiency depleted intratumoral DC1s, which led to increased DC2 lymph node migration and CD4+ T cell activation. Enhancing DC2 stimulatory potential by genetic deletion of Hsp90b1 (encoding molecular chaperon GP96) led to a similar enhancement of T cell immunity and improved survival in a spontaneous breast cancer model. These data highlight the therapeutic and prognostic potential of DC2s within checkpoint blockade-resistant tumors.

Pro-Survival Lipid Sphingosine-1-Phosphate Metabolically Programs T Cells to Limit Anti-tumor Activity.

Sphingosine 1-phosphate (S1P), a bioactive lysophospholipid generated by sphingosine kinase 1 (SphK1), regulates lymphocyte egress into circulation via S1P receptor 1 (S1PR1) signaling, and it controls the differentiation of regulatory T cells (Tregs) and T helper-17 cells. However, the mechanisms by which receptor-independent SphK1-mediated intracellular S1P levels modulate T cell functionality remains unknown. We show here that SphK1-deficient T cells maintain central memory phenotype and exhibit higher mitochondrial respiration and reduced differentiation to Tregs. Mechanistically, we discovered a direct correlation between SphK1-generated S1P and lipid transcription factor PPARγ (peroxisome proliferator-activated receptor gamma) activity, which in turn regulates lipolysis in T cells. Genetic and pharmacologic inhibition of SphK1 improved metabolic fitness and anti-tumor activity of T cells against murine melanoma. Further, inhibition of SphK1 and PD1 together led to improved control of melanoma. Overall, these data highlight the clinical potential of limiting SphK1/S1P signaling for enhancing anti-tumor-adoptive T cell therapy.

Hornerin promotes tumor progression and is associated with poor prognosis in hepatocellular carcinoma.

BACKGROUND: The function of hornerin (HRNR), a member of the S100 protein family, is poorly clarified in the development of human tumors. The role of HRNR in hepatocellular carcinoma (HCC) progression is investigated in the study. METHODS: The expression levels of HRNR were assessed in tumor samples from a cohort of 271 HCC patients. The effect of HRNR on proliferation, colony formation and invasion of tumor cells was examined. We further determined the role of HRNR in tumor growth in vivo by using xenograft HCC tumor models. The possible mechanism of the HRNR promotion of HCC progression was explored. RESULTS: We found that HRNR was overexpressed in HCC tissues. The high expression of HRNR in HCCs was significantly associated with vascular invasion, poor tumor differentiation, and advanced TNM stage. The disease-free survival (DFS) and overall survival (OS) of HCC patients with high HRNR expression were poorer than those in the low HRNR expression group. HRNR expression was an independent risk factor linked to both poor DFS (HR = 2.209, 95% CI = 1.627-2.998,P <  0.001) and OS (HR = 2.459,95% CI = 1.736-3.484, P <  0.001). In addition, the knockdown of HRNR by shRNAs significantly inhibited the proliferation, colony formation, migration and invasion of HCC tumor cells. HRNR silencing led to the decreased phosphorylation of AKT signaling. Notably, tumor growth was markedly inhibited by HRNR silencing in a xenograft model of HCC. CONCLUSIONS: HRNR promotes tumor progression and is correlated with a poor HCC prognosis. HRNR may contribute to HCC progression via the regulation of the AKT pathway.

Inflammasomes and Cancer: The Dynamic Role of the Inflammasome in Tumor Development.

Chronic Inflammation in tumor microenvironments is not only associated with various stages of tumor development, but also has significant impacts on tumor immunity and immunotherapy. Inflammasome are an important innate immune pathway critical for the production of active IL-1ß and interleukin 18, as well as the induction of pyroptosis. Although extensive studies have demonstrated that inflammasomes play a vital role in infectious and autoimmune diseases, their role in tumor progression remains elusive. Multiple studies using a colitis-associated colon cancer model show that inflammasome components provide protection against the development of colon cancer. However, very recent studies demonstrate that inflammasomes promote tumor progression in skin and breast cancer. These results indicate that inflammasomes can promote and suppress tumor development depending on types of tumors, specific inflammasomes involved, and downstream effector molecules. The complicated role of inflammasomes raises new opportunities and challenges to manipulate inflammasome pathways in the treatment of cancer.

ER Stress-induced Inflammasome Activation Contributes to Hepatic Inflammation and Steatosis.

Endoplasmic reticulum (ER) stress functions as a protein folding and quality control mechanism to maintain cell homeostasis. Emerging evidence indicates that ER stress is also involved in metabolic and inflammatory diseases. However, the link between ER stress and inflammation remains not well characterized. In this study, we have demonstrated that ER stress-induced inflammasome activation plays a critical role in the pathogenesis of hepatic steatosis. By utilizing genetic and pharmacological agent-induced hepatic steatosis animal models, we found that hepatic steatosis was associated with inflammasome activation and ER stress. Our results show that caspase-1 ablation alleviated liver inflammation and injury. Liver tissues from caspase-1 KO mice had significantly reduced production of IL-1ß under ER stress conditions. We also found that ER stress promoted inflammasome activation and IL-1ß processing in both hepatocytes and Kupffer cells/macrophages. Moreover, lack of caspase-1 ameliorated cell death or pyropoptosis of hepatocytes induced by ER stress. Taken together, our findings suggest that ER stress-induced inflammasome activation and IL-1ß production generate a positive feedback loop to amplify inflammatory response, eventually leading to liver steatosis and injury.

Targeting inflammasome/IL-1 pathways for cancer immunotherapy.

The inflammatory microenvironment has been shown to play important roles in various stages of tumor development including initiation, growth, and metastasis. The inflammasome is a critical innate immune pathway for the production of active IL-1ß, a potent inflammatory cytokine. Although inflammasomes are essential for host defense against pathogens and contribute to autoimmune diseases, their role in tumor progression remains controversial. Here, our results demonstrate that the inflammasome and IL-1ß pathway promoted tumor growth and metastasis in animal and human breast cancer models. We found that tumor progression was associated with the activation of inflammasome and elevated levels of IL-1ß at primary and metastatic sites. Mice deficient for inflammasome components exhibited significantly reduced tumor growth and lung metastasis. Furthermore, inflammasome activation promoted the infiltration of myeloid cells such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) into tumor microenvironments. Importantly, blocking IL-1R with IL-1R antagonist (IL-Ra) inhibited tumor growth and metastasis accompanied by decreased myeloid cell accumulation. Our results suggest that targeting the inflammasome/IL-1 pathway in tumor microenvironments may provide a novel approach for the treatment of cancer.

IL-10 Modulates Th17 Pathogenicity during Autoimmune Diseases.

The immune system is essential for host defense against pathogen infections; however dysregulated immune response may lead to inflammatory or autoimmune diseases. Elevated activation of both innate immune cells and T cells such as Th17 cells are linked to many autoimmune diseases, including Multiple Sclerosis (MS), arthritis and inflammatory bowel disease (IBD). To keep immune homeostasis, the immune system develops a number of negative feedback mechanisms, such as the production of anti-inflammatory cytokine IL-10, to dampen excessive production of inflammatory cytokines and uncontrolled activation of immune cells. Our recent studies uncover a novel immunoregulatory function of interferon (IFN) pathways on the innate and antigen-specific immune response. Our results show that IFNα/ß induced IL-10 production from macrophages and Th17 cells, which in turn negatively regulated Th17 function in autoimmune diseases such as Experimental Allergic Encephalomyelitis (EAE), an animal model of human MS. In a chronic colitis model resembling human IBD, we also found that IL-10 inhibited inflammasome/IL-1 pathway, and the pathogenicity of Th17 cells, leading to reduced chronic intestinal inflammation. Results from our and other studies further suggest that IL-10 produced by both macrophages and regulatory T cells may shift Th17 into more regulatory phenotypes, leading to reduced inflammatory response.

Endoplasmic reticulum stress in hepatic steatosis and inflammatory bowel diseases.

As an adaptive response to the overloading with misfolded proteins in the endoplasmic reticulum (ER), ER stress plays critical roles in maintaining protein homeostasis in the secretory pathway to avoid damage to the host. Such a conserved mechanism is accomplished through three well-orchestrated pathways known collectively as unfolded protein response (UPR). Persistent and pathological ER stress has been implicated in a variety of diseases in metabolic, inflammatory, and malignant conditions. Furthermore, ER stress is directly linked with inflammation through UPR pathways, which modulate transcriptional programs to induce the expression of inflammatory genes. Importantly, the inflammation induced by ER stress is directly responsible for the pathogenesis of metabolic and inflammatory diseases. In this review, we will discuss the potential signaling pathways connecting ER stress with inflammation. We will also depict the interplay between ER stress and inflammation in the pathogenesis of hepatic steatosis, inflammatory bowel diseases and colitis-associated colon cancer.

The molecular chaperone gp96/GRP94 interacts with Toll-like receptors and integrins via its C-terminal hydrophobic domain.

The structural basis for molecular chaperones to discern misfolded proteins has long been an enigma. As the endoplasmic reticulum paralogue of the cytosolic HSP90, gp96 (GRP94, HSP90b1) is an essential molecular chaperone for Toll-like receptors (TLRs) and integrins. However, little is known about its client-binding domain (CBD). Herein, we provide genetic and biochemical evidence to definitively demonstrate that a C-terminal loop structure, formed by residues 652-678, is the critical region of CBD for both TLRs and integrins. Deletion of this region affects neither the intrinsic ATPase activity nor the overall conformation of gp96. However, without it, the chaperoning function of gp96 collapses. We also find a critical Met pair (Met(658)-Met(662)) for the folding of integrins but not TLRs. Moreover, we find that the TLR binding to gp96 is also dependent on the C-terminal dimerization domain but not the N-terminal ATP-binding pocket of gp96. Our study has unveiled surprisingly the exquisite specificity of gp96 in substrate binding and suggests a manipulation of its CBD as an alternative strategy for targeted therapy of a variety of diseases.

Type I IFN promotes IL-10 production from T cells to suppress Th17 cells and Th17-associated autoimmune inflammation.

Whereas the immune system is essential for host defense against pathogen infection or endogenous danger signals, dysregulated innate and adaptive immune cells may facilitate harmful inflammatory or autoimmune responses. In the CNS, chronic inflammation plays an important role in the pathogenesis of neurodegenerative diseases such as multiple sclerosis (MS). Our previous study has demonstrated a critical role for the type I IFN induction and signaling pathways in constraining Th17-mediated experimental autoimmune encephalomyelitis (EAE), an animal model of human MS. However, it remains unknown if self-reactive Th17 cells can be reprogrammed to have less encephalitogenic activities or even have regulatory effects through modulation of innate pathways. In this study, we investigated the direct effects of type I IFN on Th17 cells. Our data show that IFNß treatment of T cells cultured under Th17 polarizing conditions resulted in reduced production of IL-17, but increased production of IL-10. We also found that IFNß induced IL-10 production by antigen specific T cells derived from immunized mice. Furthermore, IFNß treatment could suppress the encephalitogenic activity of myelin-specific T cells, and ameliorate clinical symptoms of EAE in an adoptive transfer model. Together, results from this study suggest that IFNß may induce antigen-specific T cells to produce IL-10, which in turn negatively regulate Th17-mediate inflammatory and autoimmune response.

Toll-like receptor 3-mediated suppression of TRAMP prostate cancer shows the critical role of type I interferons in tumor immune surveillance.

Inflammation has increasingly been recognized as a critical component influencing tumor growth. Recent reports have revealed conflicting evidence for the role of Toll-like receptors (TLR) in modulating tumorigenesis. In our study, we implicate TLR3 in mediating immune surveillance with increased growth of implanted transgenic adenocarcinoma of the mouse prostate (TRAMP) tumors in TLR3(-/-) compared with TLR3(+/+) mice. Activation of TLR3 by polyinosinic-polycytidylic acid (polyI:C) leads to induction of multiple inflammatory pathways, including NF-kappaB, mitogen-activated protein kinases, and interferon (IFN) regulatory factors. We explored the potential of TLR3 stimulation in prostate cancer immunotherapy and showed that treatment with polyI:C can strongly suppress both s.c. implanted TRAMP tumors in syngenic mice as well as orthotopic prostate cancers in TRAMP C57Bl6 x FvB F1 Tg(+/-) transgenic mice. Treated tumors remained well differentiated to moderately differentiated with increased infiltration of T lymphocytes and natural killer (NK) cells compared with poorly differentiated adenocarcinoma observed in untreated tumors. Like TLR3(-/-) mice, IFN-alpha receptor 1 (IFNAR1)(-/-) mice exhibited reduced tumor surveillance and impaired tumor suppression following polyI:C treatment. We observed that type I IFN-dependent induction of cytokines was responsible for NK activation, with depletion of NK cells leading to increased tumor growth as well as expansion of CD4(+)CD25(+)Foxp3(+) T regulatory lymphocytes. Our study therefore delineates the importance of IFNAR-dependent functions in TLR3-mediated tumor suppression and supports the use of TLR3 agonists for prostate cancer immune-based therapies.

Macrophage differentiation from embryoid bodies derived from human embryonic stem cells.

Human embryonic stem cells can differentiate into CD34+ hematopoietic progenitors by co-culture on murine feeders such as OP9 and S17. These CD34+ progenitors can be further differentiated into several cells of the hematopoietic lineage including macrophages. However, co-culture on murine feeders is time consuming and involves extensive manipulations. Furthermore, CD45 expression is low on hematopoietic cultures derived from stromal co-cultures. In this study we describe a novel and highly efficient system of generating differentiated macrophages from hematopoietic progenitors generated from embryoid body cultures of human embryonic stem cells. The hematopoietic progenitors generated from these embryoid bodies express higher numbers of CD45+ cells and are able to differentiate to macrophages when cultured in presence of cytokines. Using this system we were able to generate higher yields of CD14+ macrophages compared to traditional stromal cell culture methods. The embryoid body derived macrophages are phagocytic, respond to Toll-like receptor stimulation and express phenotypic markers of mature macrophages. Importantly, the embryoid body system generates hematopoietic progenitors suitable for clinical use by eliminating the need for murine feeder cells. Furthermore, this system is amenable to genetic manipulation and may thus be used to study important mechanisms of macrophage differentiation and function.

PLP2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type I interferon production.

Infections by coronaviruses such as severe acute respiratory syndrome (SARS) coronavirus (SCoV) and mouse hepatitis virus A59 (MHV-A59) result in very little type I interferon (IFN) production by host cells, which is potentially responsible for the rapid viral growth and severe immunopathology associated with SARS. However, the molecular mechanisms for the low IFN production in cells infected with coronaviruses remain unclear. Here, we provide evidence that Papain-like protease domain 2 (PLP2), a catalytic domain of the nonstructural protein 3 (nsp3) of MHV-A59, can bind to IRF3, cause its deubiquitination and prevent its nuclear translocation. As a consequence, co-expression of PLP2 strongly inhibits CARDIF-, TBK1- and IRF3-mediated IFNbeta reporter activities. In addition, we show that wild-type PLP2 but not the mutant PLP2 lacking the deubiquitinase (DUB) activity can reduce IFN induction and promote viral growth in cells infected with VSV. Thus, our study uncovered a viral DUB which coronaviruses may use to escape from the host innate antiviral responses.

The type I IFN induction pathway constrains Th17-mediated autoimmune inflammation in mice.

IFN-beta, a type I IFN, is widely used for the treatment of MS. However, the mechanisms behind its therapeutic efficacy are not well understood. Using a murine model of MS, EAE, we demonstrate that the Th17-mediated development of autoimmune disease is constrained by Toll-IL-1 receptor domain-containing adaptor inducing IFN-beta-dependent (TRIF-dependent) type I IFN production and its downstream signaling pathway. Mice with defects in TRIF or type I IFN receptor (IFNAR) developed more severe EAE. Notably, these mice exhibited marked CNS inflammation, as manifested by increased IL-17 production. In addition, IFNAR-dependent signaling events were essential for negatively regulating Th17 development. Finally, IFN-beta-mediated IL-27 production by innate immune cells was critical for the immunoregulatory role of IFN-beta in the CNS autoimmune disease. Together, our findings not only may provide a molecular mechanism for the clinical benefits of IFN-beta in MS but also demonstrate a regulatory role for type I IFN induction and its downstream signaling pathways in limiting Th17 development and autoimmune inflammation.

IRF3-dependent type I interferon response in B cells regulates CpG-mediated antibody production.

Hypomethylated CpG oligonucleotides (CpG) are not only potent adjuvants for enhancing adaptive immune responses but may also play a critical role in the development of autoimmune diseases such as Rheumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE). Here we provide evidence that, in addition to dendritic cells, murine B lymphocytes also exhibit a type I IFN response to CpG-B. Unlike dendritic cells, B cell-mediated type I IFN induction depended on the transcription factor IRF3, but similar to dendritic cells this pathway was independent of the IRF3 kinase TBK1. Utilizing type I IFN receptor-deficient mice, we were able to demonstrate that this IFN pathway enhanced Syndecan-1 expression and IgM production and was required for IgG2a production following CpG-B stimulation. Overall, our findings identify a unique IFN pathway in B cells that may play a central role in mediating B cell biology in response to CpG, potentially implicating this pathway in autoantibody production and the pathogenesis of certain autoimmune diseases.

LILRA2 activation inhibits dendritic cell differentiation and antigen presentation to T cells.

The differentiation of monocytes into dendritic cells (DC) is a key mechanism by which the innate immune system instructs the adaptive T cell response. In this study, we investigated whether leukocyte Ig-like receptor A2 (LILRA2) regulates DC differentiation by using leprosy as a model. LILRA2 protein expression was increased in the lesions of the progressive, lepromatous form vs the self-limited, tuberculoid form of leprosy. Double immunolabeling revealed LILRA2 expression on CD14+, CD68+ monocytes/macrophages. Activation of LILRA2 on peripheral blood monocytes impaired GM-CSF induced differentiation into immature DC, as evidenced by reduced expression of DC markers (MHC class II, CD1b, CD40, and CD206), but not macrophage markers (CD209 and CD14). Furthermore, LILRA2 activation abrogated Ag presentation to both CD1b- and MHC class II-restricted, Mycobacterium leprae-reactive T cells derived from leprosy patients, while cytokine profiles of LILRA2-activated monocytes demonstrated an increase in TNF-alpha, IL-6, IL-8, IL-12, and IL-10, but little effect on TGF-beta. Therefore, LILRA2 activation, by altering GM-CSF-induced monocyte differentiation into immature DC, provides a mechanism for down-regulating the ability of the innate immune system to activate the adaptive T cell response while promoting an inflammatory response.

Cutting edge: involvement of the type I IFN production and signaling pathway in lipopolysaccharide-induced IL-10 production.

Macrophages respond to LPS by the rapid activation of proinflammatory cytokines that serve to initiate host defense against microbial invasion. To prevent injury to the host from excess production of these cytokines, IL-10 is up-regulated to feedback inhibit the proinflammatory response. However, the molecular events responsible for LPS-induced up-regulation of IL-10 remain to be elucidated. In this study, we provide evidence that production of and signaling by type I IFN is required for LPS-induced IL-10 up-regulation. In addition, we demonstrate that defect in type I IFN production and signaling results in a trend toward LPS-mediated superinduction of proinflammatory genes and cytokines in bone marrow-derived macrophages. Our findings suggest a novel anti-inflammatory role for the type I IFN production and signaling pathway in regulating LPS response in bone marrow-derived macrophages.

Modulation of the interferon antiviral response by the TBK1/IKKi adaptor protein TANK.

Induction of type I interferons can be triggered by viral components through Toll-like receptors or intracellular viral receptors such as retinoic acid-inducible gene I. Here, we demonstrate that the TRAF (tumor necrosis factor receptor-associated factor) family member-associated NF-kappaB activator (TANK) plays an important role in interferon induction through both retinoic acid-inducible gene I- and Toll-like receptor-dependent pathways. TANK forms complexes with both upstream signal mediators, such as Cardif/MAVS/IPS-1/VISA, TRIF (Toll-interleukin-1 receptor domain-containing adaptor inducing interferon-beta), and TRAF3 and downstream mediators TANK-binding kinase 1, inducible IkappaB kinase, and interferon regulatory factor 3. In addition, it synergizes with these signaling components in interferon induction. Specific knockdown of TANK results in reduced type I interferon production, increased viral titers, and enhanced cell sensitivity to viral infection. Thus, TANK may be a critical adaptor that regulates the assembly of the TANK-binding kinase 1-inducible IkappaB kinase complex with upstream signaling molecules in multiple antiviral pathways.

Critical role of TRAF3 in the Toll-like receptor-dependent and -independent antiviral response.

Type I interferon (IFN) production is a critical component of the innate defence against viral infections. Viral products induce strong type I IFN responses through the activation of Toll-like receptors (TLRs) and intracellular cytoplasmic receptors such as protein kinase R (PKR). Here we demonstrate that cells lacking TRAF3, a member of the TNF receptor-associated factor family, are defective in type I IFN responses activated by several different TLRs. Furthermore, we show that TRAF3 associates with the TLR adaptors TRIF and IRAK1, as well as downstream IRF3/7 kinases TBK1 and IKK-epsilon, suggesting that TRAF3 serves as a critical link between TLR adaptors and downstream regulatory kinases important for IRF activation. In addition to TLR stimulation, we also show that TRAF3-deficient fibroblasts are defective in their type I IFN response to direct infection with vesicular stomatitis virus, indicating that TRAF3 is also an important component of TLR-independent viral recognition pathways. Our data demonstrate that TRAF3 is a major regulator of type I IFN production and the innate antiviral response.

Phosphorylation of the CARMA1 linker controls NF-kappaB activation.

PKC isoforms and CARMA1 play crucial roles in immunoreceptor-dependent NF-kappaB activation. We tested whether PKC-dependent phosphorylation of CARMA1 directly regulates this signaling cascade. B cell antigen receptor (BCR) engagement led to the progressive recruitment of CARMA1 into lipid rafts and to the association of CARMA1 with, and phosphorylation by, PKCbeta. Furthermore, PKCbeta interacted with the serine-rich CARMA1 linker, and both PKCbeta and PKCtheta phosphorylated identical serine residues (S564, S649, and S657) within this linker. Mutation of two of these sites ablated the functional activity of CARMA1. In contrast, deletion of the linker resulted in constitutive, receptor- and PKC-independent NF-kappaB activation. Together, our data support a model whereby CARMA1 phosphorylation controls NF-kappaB activation by triggering a shift from an inactive to an active CARMA1 conformer. This PKC-dependent switch regulates accessibility of the CARD and CC domains and controls assembly and full activation of the membrane-associated IkappaB kinase (IKK) signalosome.