NF-κB-inducing kinase maintains T cell metabolic fitness in antitumor immunity.

Metabolic reprograming toward aerobic glycolysis is a pivotal mechanism shaping immune responses. Here we show that deficiency in NF-κB-inducing kinase (NIK) impairs glycolysis induction, rendering CD8+ effector T cells hypofunctional in the tumor microenvironment. Conversely, ectopic expression of NIK promotes CD8+ T cell metabolism and effector function, thereby profoundly enhancing antitumor immunity and improving the efficacy of T cell adoptive therapy. NIK regulates T cell metabolism via a NF-κB-independent mechanism that involves stabilization of hexokinase 2 (HK2), a rate-limiting enzyme of the glycolytic pathway. NIK prevents autophagic degradation of HK2 through controlling cellular reactive oxygen species levels, which in turn involves modulation of glucose-6-phosphate dehydrogenase (G6PD), an enzyme that mediates production of the antioxidant NADPH. We show that the G6PD-NADPH redox system is important for HK2 stability and metabolism in activated T cells. These findings establish NIK as a pivotal regulator of T cell metabolism and highlight a post-translational mechanism of metabolic regulation.

NIK signaling axis regulates dendritic cell function in intestinal immunity and homeostasis.

Dendritic cells (DCs) play an integral role in regulating mucosal immunity and homeostasis, but the signaling network mediating this function of DCs is poorly defined. We identified the noncanonical NF-κB-inducing kinase (NIK) as a crucial mediator of mucosal DC function. DC-specific NIK deletion impaired intestinal immunoglobulin A (IgA) secretion and microbiota homeostasis, rendering mice sensitive to an intestinal pathogen, Citrobacter rodentium. DC-specific NIK was required for expression of the IgA transporter polymeric immunoglobulin receptor (pIgR) in intestinal epithelial cells, which in turn relied on the cytokine IL-17 produced by TH17 cells and innate lymphoid cells (ILCs). NIK-activated noncanonical NF-κB induced expression of IL-23 in DCs, contributing to the maintenance of TH17 cells and type 3 ILCs. Consistent with the dual functions of IL-23 and IL-17 in mucosal immunity and inflammation, NIK deficiency also ameliorated colitis induction. Thus, our data suggest a pivotal role for the NIK signaling axis in regulating DC functions in intestinal immunity and homeostasis.

Intestinal Epithelial TBK1 Prevents Differentiation of T-helper 17 Cells and Tumorigenesis in Mice.

BACKGROUND & AIMS: Intestinal epithelial cells (IECs) regulate intestinal immune cells, particularly development of T-helper 17 (Th17) cells. Deregulation of this process leads to intestinal inflammation and tumorigenesis, via unknown mechanisms. TANK-binding kinase 1 (TBK1) is expressed by IECs and cells in the innate immune system. We studied the functions of TBK1 in the intestinal immune response and tumorigenesis in mice. METHODS: We performed studies of wild-type mice, mice with conditional disruption of Tbk1 (Tbk1IEC-KO), Tbk1IEC-KO mice crossed with ApcMin/+ mice, and Mt-/- mice crossed with ApcMin/+ mice. Some mice were given intraperitoneal injections of a neutralizing antibody against interleukin 17 (IL17) or IL1ß. Intestine tissues were collected from mice and analyzed by histology, for numbers of adenomas and Th17 cells, and expression of inflammatory cytokines by real-time PCR. IECs were isolated from wild-type and Tbk1IEC-KO mice, stimulated with lipopolysaccharide, co-cultured for with bone marrow-derived macrophages, and analyzed by RNA sequencing and biochemical analyses. RESULTS: Compared to ApcMin/+Tbk1WT mice, ApcMin/+Tbk1IEC-KO mice had significant increases in number and size of intestinal polyps, and significantly more Th17 cells in lamina propria. Administration of an antibody against IL17 reduced the number of intestinal polyps in ApcMin/+Tbk1IEC-KO mice to that observed in ApcMin/+Tbk1WT mice. In culture, TBK1-deficient IECs promoted expression of IL1ß by macrophages, which induced differentiation of naïve CD4+ T cells into Th17 cells. RNA sequencing analysis revealed that the TBK1-deficient IECs had increased expression of metallothionein 1 (MT1), an immune regulator that promotes intestinal inflammation. Intestine tissues from ApcMin/+Mt-/- mice had significant fewer Th17 cells than ApcMin/+Mt+/+ mice, and a significantly lower number of polyps. Analyses of colorectal tumors in the Cancer Genome Atlas found colorectal tumors with high levels of MT1 and IL17 mRNAs to be associated with reduced survival times of patients. CONCLUSIONS: Expression of TBK1 by IECs suppresses expression of MT1 and prevents expression of IL1ß by macrophages and differentiation of Th17 cells, to prevent inflammation and tumorigenesis. Strategies to block this pathway might be developed for colorectal tumorigenesis.

RKIP and TBK1 form a positive feedback loop to promote type I interferon production in innate immunity.

TANK-binding kinase 1 (TBK1) activation is a central event in type I interferon production in anti-virus innate immunity. However, the regulatory mechanism underlying TBK1 activation remains unclear. Here we report that Raf kinase inhibitory protein (RKIP) is essential for TBK1 activation and type I interferon production triggered by viral infection. Upon viral infection, RKIP is phosphorylated at serine 109 (S109) by TBK1. Phosphorylation of RKIP enhances its interaction with TBK1 and in turn promotes TBK1 autophosphorylation. Mutation of RKIP S109 to alanine abrogates the interaction between RKIP and TBK1, and the anti-viral function of RKIP RKIP deficiency inhibits intracellular double-stranded RNA- or DNA-induced type I interferon production. Consistently, RKIP deficiency renders the mice more susceptible to vesicular stomatitis virus (VSV) and herpes simplex virus (HSV) infections. This study reveals a previously unrecognized positive feedback loop between RKIP and TBK1 that is essential for type I interferon production in anti-viral innate immunity.

Raf Kinase Inhibitor Protein Preferentially Promotes TLR3-Triggered Signaling and Inflammation.

Raf kinase inhibitor protein (RKIP) protects against host immunological responses in nematodes and Drosophila Whether RKIP functions in innate immune responses in mammals remains unknown. In this article, we report that RKIP preferentially regulates the TLR3-mediated immune response in macrophages. RKIP deficiency or silencing significantly decreases polyinosinic:polycytidylic acid [Poly(I:C)]-induced IFN-ß, IL-6, and TNF-α production without affecting the counterpart induced by LPS or CpG. Compared with their wild-type counterparts, RKIP-deficient mice produce less IFN-ß, IL-6, and TNF-α in serum and display decreased lethality upon peritoneal Poly(I:C) plus d-galactosamine injection. Mechanistically, RKIP interacts with TBK1 and promotes the Poly(I:C)-induced TANK-binding kinase 1/IRF3 activation. Simultaneously, RKIP enhances the Poly(I:C)-induced interaction between TGF-ß-activated kinase 1 and MAPK kinase 3 (MKK3), thus promoting MKK3/6 and p38 activation. We further demonstrated that Poly(I:C) treatment, but not LPS treatment, induces RKIP phosphorylation at S109. This action is required for RKIP to promote TANK-binding kinase 1 activation, as well as the interaction between TGF-ß-activated kinase 1 and MKK3, which lead to activation of the downstream IRF3 and p38, respectively. Therefore, RKIP acts as a positive-feedback regulator of the TLR3-induced inflammatory response and may be a potential therapeutic target for inflammatory disease.

Phosphatase holoenzyme PP1/GADD34 negatively regulates TLR response by inhibiting TAK1 serine 412 phosphorylation.

The molecular mechanisms that fine tune TLRs responses need to be fully elucidated. Protein phosphatase-1 (PP1) has been shown to be important in cell death and differentiation. However, the roles of PP1 in TLR-triggered immune response remain unclear. In this study, we demonstrate that PP1 inhibits the activation of the MAPK and NF-κB pathway and the production of TNF-α, IL-6 in macrophages triggered by TLR3, TLR4, and TLR9 in a phosphatase-dependent manner. Conversely, PP1 knockdown increases TLRs-triggered signaling and proinflammatory cytokine production. Tautomycetin, a specific inhibitor of PP1, aggravates LPS-induced endotoxin shock in mice. We further demonstrate that PP1 negatively regulates TLR-triggered signaling by targeting TGF-ß-activated kinase 1 (TAK1) serine 412 (Ser412) phosphorylation, which is required for activation of TAK1-mediated IL-1R and TLR signaling. Mutation of TAK1 Serine 412 to alanine (S412A) significantly inhibits TLR/IL-1R-triggered NF-κB and MAPK activation and induction of proinflammatory cytokines in macrophage and murine embryonic fibroblast cells. DNA damage-inducible protein 34 (GADD34) specifies PP1 to dephosphorylate TAK1 at Ser412. GADD34 depletion abolished the interaction between TAK1 and PP1, and it relieved PP1 overexpression-induced inhibition of TLRs signaling and proinflammatory cytokine production. In addition, knockdown of GADD34 significantly promotes TLR-induced TAK1 Ser412 phosphorylation, downstream NF-κB and MAPK activation, and proinflammatory cytokine production. Therefore, PP1, as a physiologic inhibitor, together with its regulatory subunit GADD34, tightly controls TLR-induced TAK1 Ser412 phosphorylation, preventing excessive activation of TLRs and protecting the host from overwhelmed inflammatory immune responses.

Transforming growth factor (TGF)-ß-activated kinase 1 (TAK1) activation requires phosphorylation of serine 412 by protein kinase A catalytic subunit α (PKACα) and X-linked protein kinase (PRKX).

TGF-ß-activated kinase 1 (TAK1) is a key kinase in mediating Toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R) signaling. Although TAK1 activation involves the phosphorylation of Thr-184 and Thr-187 residues at the activation loop, the molecular mechanism underlying the complete activation of TAK1 remains elusive. In this work, we show that the Thr-187 phosphorylation of TAK1 is regulated by its C-terminal coiled-coil domain-mediated dimerization in an autophosphorylation manner. Importantly, we find that TAK1 activation in mediating downstream signaling requires an additional phosphorylation at Ser-412, which is critical for TAK1 response to proinflammatory stimuli, such as TNF-α, LPS, and IL-1ß. In vitro kinase and shRNA-based knockdown assays reveal that TAK1 Ser-412 phosphorylation is regulated by cAMP-dependent protein kinase catalytic subunit α (PKACα) and X-linked protein kinase (PRKX), which is essential for proper signaling and proinflammatory cytokine induction by TLR/IL-1R activation. Morpholino-based in vivo knockdown and rescue studies show that the corresponding site Ser-391 in zebrafish TAK1 plays a conserved role in NF-κB activation. Collectively, our data unravel a previously unknown mechanism involving TAK1 phosphorylation mediated by PKACα and PRKX that contributes to innate immune signaling.

Inhibition of STAT6 with Antisense Oligonucleotides Enhances the Systemic Antitumor Effects of Radiotherapy and Anti-PD-1 in Metastatic Non-Small Cell Lung Cancer.

Diverse factors contribute to the limited clinical response to radiotherapy (RT) and immunotherapy in metastatic non-small cell lung cancer (NSCLC), among which is the ability of these tumors to recruit a retinue of suppressive immune cells-such as M2 tumor-associated macrophages (TAM)-thereby establishing an immunosuppressive tumor microenvironment that contributes to tumor progression and radio resistance. M2 TAMs are activated by the STAT6 signaling pathway. Therefore, we targeted STAT6 using an antisense oligonucleotide (ASO) along with hypofractionated RT (hRT; 3 fractions of 12 Gy each) to primary tumors in three bilateral murine NSCLC models (Lewis lung carcinoma, 344SQ-parental, and anti-PD-1-resistant 344SQ lung adenocarcinomas). We found that STAT6 ASO plus hRT slowed growth of both primary and abscopal tumors, decreased lung metastases, and extended survival. Interrogating the mechanism of action showed reduced M2 macrophage tumor infiltration, enhanced TH1 polarization, improved T-cell and macrophage function, and decreased TGFß levels. The addition of anti-PD-1 further enhanced systemic antitumor responses. These results provide a preclinical rationale for the pursuit of an alternative therapeutic approach for patients with immune-resistant NSCLC.

Dapl1 controls NFATc2 activation to regulate CD8+ T cell exhaustion and responses in chronic infection and cancer.

CD8+ T cells are central mediators of immune responses against infections and cancer. Here we identified Dapl1 as a crucial regulator of CD8+ T cell responses to cancer and infections. Dapl1 deficiency promotes the expansion of tumour-infiltrating effector memory-like CD8+ T cells and prevents their functional exhaustion, coupled with increased antitumour immunity and improved efficacy of adoptive T cell therapy. Dapl1 controls activation of NFATc2, a transcription factor required for the effector function of CD8+ T cells. Although NFATc2 mediates induction of the immune checkpoint receptor Tim3, competent NFATc2 activation prevents functional exhaustion of CD8+ T cells. Interestingly, exhausted CD8+ T cells display attenuated NFATc2 activation due to Tim3-mediated feedback inhibition; Dapl1 deletion rescues NFATc2 activation and thereby prevents dysfunction of exhausted CD8+ T cells in chronic infection and cancer. These findings establish Dapl1 as a crucial regulator of CD8+ T cell immunity and a potential target for cancer immunotherapy.

Stk24 protects against obesity-associated metabolic disorders by disrupting the NLRP3 inflammasome.

Adipose tissue macrophages (ATMs) regulate the occurrence of obesity and its related diseases. Here, we found that serine/threonine protein kinase 24 (Stk24) expression is downregulated significantly in ATMs in obese subjects or obese subjects with type 2 diabetes and mice fed a high-fat diet (HFD). We further identified that glucolipotoxicity downregulated Stk24 expression in ATMs. Stk24-deficient mice develop severe HFD-induced metabolic disorders and insulin insensitivity. Mechanistically, Stk24 intervenes in NLRP3 inflammasome assembly in ATMs by associating directly with NLRP3, decreasing interleukin-1ß (IL-1ß) secretion. Accordingly, Stk24 deficiency in the hematopoietic system promotes NLRP3 inflammasome activation, which contributes to exacerbation of metabolic disorders. Intriguingly, Stk24 expression correlates negatively with body mass index (BMI) and the levels of glucose, cholesterol, triglycerides, and low-density lipoprotein in human subjects. These findings provide insights into the function and clinical implications of Stk24 in obesity-mediated metabolic disorders.

Pulsed Radiation Therapy to Improve Systemic Control of Metastatic Cancer.

Radiation therapy (RT) is emerging as an interventional modality in the cancer-immunity cycle, augmenting the activation of an adaptive immune response against tumors. RT, particularly in combination with immunotherapy, can enhance immune memory effects and shape the tumor-directed T-cell populations. However, a single cycle of RT delivered to a limited number of polymetastatic lesions is rarely sufficient to achieve systemic control. We hypothesize that several rounds of RT, akin to several rounds of immunotherapeutic drugs, is likely to provide greater clinical benefit to patients with metastatic disease. We propose that the repeated exposure to tumor antigens released by "pulsed-RT" (i.e., treating 2-4 tumor lesions with 3 irradiation cycles given one month apart) may amplify the adaptive immune response by expanding the tumor-specific T-cell receptor repertoire, the production of high-affinity tumor antibodies, and the generation of memory lymphocytes and thereby improve immune control of systemic disease.

Microglia promote autoimmune inflammation via the noncanonical NF-κB pathway.

Microglia have been implicated in neuroinflammatory diseases, including multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). We demonstrate that microglia mediate EAE disease progression via a mechanism relying on the noncanonical nuclear factor kB (NF-κB) pathway. Microglia-specific deletion of the noncanonical NF-κB-inducing kinase (NIK) impairs EAE disease progression. Although microglial NIK is dispensable for the initial phase of T cell infiltration into the central nervous system (CNS) and EAE disease onset, it is critical for the subsequent CNS recruitment of inflammatory T cells and monocytes. Our data suggest that following their initial CNS infiltration, T cells activate the microglial noncanonical NF-κB pathway, which synergizes with the T cell-derived cytokine granulocyte-macrophage colony-stimulating factor to induce expression of chemokines involved in the second-wave of T cell recruitment and disease progression. These findings highlight a mechanism of microglial function that is dependent on NIK signaling and required for EAE disease progression.

TRAF2 regulates T cell immunity by maintaining a Tpl2-ERK survival signaling axis in effector and memory CD8 T cells.

Generation and maintenance of antigen-specific effector and memory T cells are central events in immune responses against infections. We show that TNF receptor-associated factor 2 (TRAF2) maintains a survival signaling axis in effector and memory CD8 T cells required for immune responses against infections. This signaling axis involves activation of Tpl2 and its downstream kinase ERK by NF-κB-inducing kinase (NIK) and degradation of the proapoptotic factor Bim. NIK mediates Tpl2 activation by stimulating the phosphorylation and degradation of the Tpl2 inhibitor p105. Interestingly, while NIK is required for Tpl2-ERK signaling under normal conditions, uncontrolled NIK activation due to loss of its negative regulator, TRAF2, causes constitutive degradation of p105 and Tpl2, leading to severe defects in ERK activation and effector/memory CD8 T cell survival. Thus, TRAF2 controls a previously unappreciated signaling axis mediating effector/memory CD8 T cell survival and protective immunity.

Addition of TLR9 agonist immunotherapy to radiation improves systemic antitumor activity.

Radiotherapy (RT) has been used to control tumors by physically damaging DNA and inducing apoptosis; it also promotes antitumor immune responses via neoantigens release and augmenting immune-oncology agents to elicit systemic response. Tumor regression after RT can recruit inflammatory cells, such as tumor-associated macrophages and CD11b+ myeloid cell populations, a major subset of which may actually be immunosuppressive. However, these inflammatory cells also express Toll-like receptors (TLRs) that can be stimulated to reverse suppressive characteristics and promote systemic antitumor outcomes. Here, we investigated the effects of adding CMP-001, a CpG-A oligodeoxynucleotide TLR9 agonist delivered in a virus-like particle (VLP), to RT in two murine models (344SQ metastatic lung adenocarcinoma and CT26 colon carcinoma). High-dose RT (12Gy x 3 fractions) significantly increased the percentages of plasmacytoid dendritic cells within the tumor islets 3- and 5-days post-RT; adding CMP-001 after RT also enhanced adaptive immunity by increasing the proportion of CD4+ and CD8+ T cells. RT plus CMP-001-mediated activation of the immune system led to significant inhibition of tumor growth at both primary and abscopal tumor sites, thereby suggesting a new combinatorial treatment strategy for systemic disease.

Role of Mitochondria in Cancer Immune Evasion and Potential Therapeutic Approaches.

The role of mitochondria in cancer formation and progression has been studied extensively, but much remains to be understood about this complex relationship. Mitochondria regulate many processes that are known to be altered in cancer cells, from metabolism to oxidative stress to apoptosis. Here, we review the evolving understanding of the role of mitochondria in cancer cells, and highlight key evidence supporting the role of mitochondria in cancer immune evasion and the effects of mitochondria-targeted antitumor therapy. Also considered is how knowledge of the role of mitochondria in cancer can be used to design and improve cancer therapies, particularly immunotherapy and radiation therapy. We further offer critical insights into the mechanisms by which mitochondria influence tumor immune responses, not only in cancer cells but also in immune cells. Given the central role of mitochondria in the complex interactions between cancer and the immune system, high priority should be placed on developing rational strategies to address mitochondria as potential targets in future preclinical and clinical studies. We believe that targeting mitochondria may provide additional opportunities in the development of novel antitumor therapeutics.

NDR2 promotes the antiviral immune response via facilitating TRIM25-mediated RIG-I activation in macrophages.

Retinoic acid-inducible gene I (RIG-I), a pivotal cytosolic sensor, recognizes viral RNAs to initiate antiviral innate immunity. However, posttranslational regulation of RIG-I signaling is not well understood. We report here that nuclear Dbf2-related kinase 2 (NDR2) functions as a crucial positive regulator of the RIG-I-mediated antiviral immune response. Overexpression of NDR2 or its kinase-inactive mutants potentiates RNA virus-induced production of type I interferons and proinflammatory cytokines and dampens viral replication. NDR2 conditional knockout mice (Lysm+NDR2f/f) show an impaired antiviral immune response. Mechanistically, NDR2 directly associates with RIG-I and TRIM25, thus facilitating the RIG-I/TRIM25 complex and enhancing the TRIM25-mediated K63-linked polyubiquitination of RIG-I, which is required for the RIG-I-mediated antiviral immune response. Furthermore, NDR2 expression is notably down-regulated in peripheral blood from respiratory syncytial virus-infected patients and in virus-infected macrophages. Collectively, these findings provide insights into the function of NDR2 in antiviral immunity and its related clinical significance.

Lymphatic endothelial cells regulate B-cell homing to lymph nodes via a NIK-dependent mechanism.

B cells home to the lymph nodes (LNs) via high endothelial venules (HEVs) under the guidance of chemokines, particularly CXCL13. However, as CXCL13 is not directly made in HEVs, the molecular mechanism mediating B-cell homing to LNs has remained unclear. We show here that nuclear factor (NF)-κB-inducing kinase (NIK), a kinase mediating activation of the noncanonical NF-κB pathway, functions in lymphatic endothelial cells (LECs) to regulate B-cell homing to LNs. LEC-conditional deletion of NIK in mice did not affect the integrity or global function of lymphatic vessels but caused a severe reduction in the frequency of B cells in LNs. The LEC-specific NIK deficiency did not affect the survival of B cells or the frequency of B cells in the spleen. B-cell adoptive transfer studies revealed that the LEC-specific NIK deletion impairs the ability of LNs to recruit B cells. We further show that NIK mediates expression of the chemokines CXCL13 and CCL19 in LECs. Although CCL19 is also expressed in blood endothelial cells (BECs), CXCL13 is not produced in BECs. These results suggest that NIK regulates naive B-cell homing to LNs via mediating production of the B-cell homing chemokine CXCL13 in LECs.