Cancer-associated fibroblast-derived Dickkopf-1 suppresses NK cell cytotoxicity in breast cancer.

Breast cancer is poorly immunogenic, hence able to evade T cell recognition and respond poorly to immune checkpoint blockade. Breast cancer cells can also evade NK cell-mediated immune surveillance, but the mechanism remains enigmatic. Dickkopf-1 (DKK1) is a Wnt/b-catenin inhibitor, whose levels are increased in breast cancer patients and correlate with reduced overall survival. DKK1 is expressed by cancer-associated fibroblasts (CAFs) in orthotopic breast tumors and patient samples, and at higher levels by bone cells. While bone-derived DKK1 contributes to the systemic elevation of DKK1 in tumor-bearing mice, CAFs represent the primary source of DKK1 at the tumor site. Systemic or bone-specific DKK1 targeting reduces primary tumor growth. Intriguingly, specific deletion of CAF-derived DKK1 also limits breast cancer progression, regardless of its elevated levels in circulation and in the bone. DKK1 does not support tumor proliferation directly but rather suppresses the activation and tumoricidal activity of NK cells. Importantly, increased DKK1 levels and reduced number of cytotoxic NK cells are detected in breast cancer patients with progressive bone metastases compared to those with stable disease. Our findings indicate that DKK1 creates a tumor-supporting environment through the suppression of NK cells in breast cancer.

Tmem178 Negatively Regulates IL-1ß Production Through Inhibition of the NLRP3 Inflammasome.

OBJECTIVE: Inflammasomes modulate the release of bioactive interleukin (IL)-1ß. Excessive IL-1ß levels are detected in patients with systemic juvenile idiopathic arthritis (sJIA) and cytokine storm syndrome (CSS) with mutated and unmutated inflammasome components, raising questions on the mechanisms of IL-1ß regulation in these disorders. METHODS: To investigate how the NLRP3 inflammasome is modulated in sJIA, we focused on Transmembrane protein 178 (Tmem178), a negative regulator of calcium levels in macrophages, and measured IL-1ß and caspase-1 activation in wild-type (WT) and Tmem178-/- macrophages after calcium chelators, silencing of Stim1, a component of store-operated calcium entry (SOCE), or by expressing a Tmem178 mutant lacking the Stromal Interaction Molecule 1 (Stim1) binding site. Mitochondrial function in both genotypes was assessed by measuring oxidative respiration, mitochondrial reactive oxygen species (mtROS), and mitochondrial damage. CSS development was analyzed in Perforin-/- /Tmem178-/- mice infected with lymphocytic choriomeningitis virus (LCMV) in which inflammasome or IL-1ß signaling was pharmacologically inhibited. Human TMEM178 and IL1B transcripts were analyzed in data sets of whole blood and peripheral blood monocytes from healthy controls and patients with active sJIA. RESULTS: TMEM178 levels are reduced in whole blood and monocytes from patients with sJIA while IL1B levels are increased. Accordingly, Tmem178-/- macrophages produce elevated IL-1ß compared with WT cells. The elevated intracellular calcium levels after SOCE activation in Tmem178-/- macrophages induce mitochondrial damage, release mtROS, and ultimately promote NLRP3 inflammasome activation. In vivo, inhibition of inflammasome or IL-1ß neutralization prolongs Tmem178-/- mouse survival in LCMV-induced CSS. CONCLUSION: Down-regulation of TMEM178 levels may represent a marker of disease activity and help identify patients who could benefit from inflammasome targeting.

Tmem178 negatively regulates IL-1ß production through inhibition of the NLRP3 inflammasome.

Objective: Inflammasomes modulate the release of bioactive IL-1ß. Excessive IL-1ß levels are detected in patients with systemic juvenile idiopathic arthritis (sJIA) and cytokine storm syndrome (CSS) with mutated and unmutated inflammasome components, raising questions on the mechanisms of IL-1ß regulation in these disorders. Methods: To investigate how the NLRP3 inflammasome is modulated in sJIA, we focused on Tmem178, a negative regulator of calcium levels in macrophages, and measured IL-1ß and caspase-1 activation in wild-type (WT) and Tmem178 -/- macrophages following calcium chelators, silencing of Stim1, a component of store-operated calcium entry (SOCE), or by expressing a Tmem178 mutant lacking Stim1 binding site. Mitochondrial function in both genotypes was assessed by measuring oxidative respiration, mitochondrial reactive oxygen species (mtROS), and mitochondrial damage. CSS development was analyzed in Perforin -/- /Tmem178 -/- mice infected with LCMV in which inflammasome or IL-1 signaling was pharmacologically inhibited. Human TMEM178 and IL-1B transcripts were analyzed in a dataset of peripheral blood monocytes from healthy controls and active sJIA patients. Results: TMEM178 levels are reduced in monocytes from sJIA patients while IL-1B show increased levels. Accordingly, Tmem178 -/- macrophages produce elevated IL-1ß compared to WT cells. The elevated intracellular calcium levels following SOCE activation in Tmem178 -/- macrophages induce mitochondrial damage, release mtROS, and ultimately, promote NLRP3 inflammasome activation. In vivo , inhibition of inflammasome or IL-1 neutralization prolongs Tmem178 -/- mouse survival to LCMV-induced CSS. Conclusion: Downregulation of Tmem178 levels may represent a new biomarker to identify sJIA/CSS patients that could benefit from receiving drugs targeting inflammasome signaling.

The microbiome restrains melanoma bone growth by promoting intestinal NK and Th1 cell homing to bone.

Bone metastases are frequent complications of malignant melanoma leading to reduced quality of life and significant morbidity. Regulation of immune cells by the gut microbiome influences cancer progression, but the role of the microbiome in tumor growth in bone is unknown. Using intracardiac or intratibial injections of B16-F10 melanoma cells into mice, we showed that gut microbiome depletion by broad-spectrum antibiotics accelerated intraosseous tumor growth and osteolysis. Microbiome depletion blunted melanoma-induced expansion of intestinal NK cells and Th1 cells and their migration from the gut to tumor-bearing bones. Demonstrating the functional relevance of immune cell trafficking from the gut to the bone marrow (BM) in bone metastasis, blockade of S1P-mediated intestinal egress of NK and Th1 cells, or inhibition of their CXCR3/CXCL9-mediated influx into the BM, prevented the expansion of BM NK and Th1 cells and accelerated tumor growth and osteolysis. Using a mouse model, this study revealed mechanisms of microbiota-mediated gut-bone crosstalk that are relevant to the immunological restraint of melanoma metastasis and tumor growth in bone. Microbiome modifications induced by antibiotics might have negative clinical consequences in patients with melanoma.

Conditional loss of IKKα in Osterix + cells has no effect on bone but leads to age-related loss of peripheral fat.

NF-κB has been reported to both promote and inhibit bone formation. To explore its role in osteolineage cells, we conditionally deleted IKKα, an upstream kinase required for non-canonical NF-κB activation, using Osterix (Osx)-Cre. Surprisingly, we found no effect on either cancellous or cortical bone, even following mechanical loading. However, we noted that IKKα conditional knockout (cKO) mice began to lose body weight after 6 months of age with severe reductions in fat mass and lower adipocyte size in geriatric animals. qPCR analysis of adipogenic markers in fat pads of cKO mice indicated no difference in early differentiation, but instead markedly lower leptin with age. We challenged young mice with a high fat diet finding that cKO mice gained less weight and showed improved glucose metabolism. Low levels of recombination at the IKKα locus were detected in fat pads isolated from old cKO mice. To determine whether recombination occurs in adipocytes, we examined fat pads in Osx-Cre;TdT reporter mice; these showed increasing Osx-Cre-mediated expression in peripheral adipocytes from 6 weeks to 18 months. Since Osx-Cre drives recombination in peripheral adipocytes with age, we conclude that fat loss in cKO mice is most likely caused by progressive deficits of IKKα in adipocytes.

Effective Treatment of Established Bone Metastases Can Be Achieved by Combinatorial Osteoclast Blockade and Depletion of Granulocytic Subsets.

Osteoclast (OC) blockade has been successful in reducing tumor growth in bone in preclinical settings, but antiresorptive drugs, such as zoledronic acid (ZA), fail to improve the overall survival rate of patients with bone metastasis despite ameliorating skeletal complications. To address this unmet clinical need, we interrogated what other cells modulated tumor growth in bone in addition to OCs. Because myeloid-derived suppressor cells (MDSC)-heterogeneous populations expressing CD11b, Ly6C, and Ly6G markers-originate in the bone marrow and promote tumor progression, we hypothesized that their accumulation hinders ZA antitumor effects. By using a murine model of bone metastasis insensitive to OC blockade, we assessed the antitumor effect of MDSC depletion using anti-Gr1 in mice bearing skeletal lung [Lewis lung carcinoma (LLC)], melanoma (B16-F10), and mammary (4T1) tumors. Differently from soft tissue tumors, anti-Gr1 did not reduce bone metastases and led to the paradoxical accumulation of bone marrow-resident CD11b+Ly6CintLy6Gint cells that differentiated into OCs when cultured in vitro Anti-Gr1-mediated depletion of Ly6G+ granulocytic MDSCs combined with ZA-induced OC blockade reduced growth of established skeletal metastases compared with each agent alone. CD15+ granulocytic populations were increased in patients with breast cancer with progressive bone disease after antiresorptive treatment compared with those with stable bone disease. We provide evidence that antiresorptive therapies fail to reduce bone metastases in the presence of elevated granulocytic populations and that effective treatment of established skeletal metastases requires combinatorial depletion of granulocytes and OC blockade.

Constitutive activation of NF-κB inducing kinase (NIK) in the mesenchymal lineage using Osterix (Sp7)- or Fibroblast-specific protein 1 (S100a4)-Cre drives spontaneous soft tissue sarcoma.

Aberrant NF-κB signaling fuels tumor growth in multiple human cancer types including both hematologic and solid malignancies. Chronic elevated alternative NF-κB signaling can be modeled in transgenic mice upon activation of a conditional NF-κB-inducing kinase (NIK) allele lacking the regulatory TRAF3 binding domain (NT3). Here, we report that expression of NT3 in the mesenchymal lineage with Osterix (Osx/Sp7)-Cre or Fibroblast-Specific Protein 1 (FSP1)-Cre caused subcutaneous, soft tissue tumors. These tumors displayed significantly shorter latency and a greater multiple incidence rate in Fsp1-Cre;NT3 compared to Osx-Cre;NT3 mice, regardless of sex. Histological assessment revealed poorly differentiated solid tumors with some spindled patterns, as well as robust RelB immunostaining, confirming activation of alternative NF-κB. Even though NT3 expression also occurs in the osteolineage in Osx-Cre;NT3 mice, we observed no bony lesions. The staining profiles and pattern of Cre expression in the two lines pointed to a mesenchymal tumor origin. Immunohistochemistry revealed that these tumors stain strongly for alpha-smooth muscle actin (αSMA), although vimentin staining was uniform only in Osx-Cre;NT3 tumors. Negative CD45 and S100 immunostains precluded hematopoietic and melanocytic origins, respectively, while positive staining for cytokeratin 19 (CK19), typically associated with epithelia, was found in subpopulations of both tumors. Principal component, differential expression, and gene ontology analyses revealed that NT3 tumors are distinct from normal mesenchymal tissues and are enriched for NF-κB related biological processes. We conclude that constitutive activation of the alternative NF-κB pathway in the mesenchymal lineage drives spontaneous sarcoma and provides a novel mouse model for NF-κB related sarcomas.

Multi-tissue single-cell analysis deconstructs the complex programs of mouse natural killer and type 1 innate lymphoid cells in tissues and circulation.

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1s) are heterogenous innate lymphocytes broadly defined in mice as Lin-NK1.1+NKp46+ cells that express the transcription factor T-BET and produce interferon-γ. The ILC1 definition primarily stems from studies on liver and small intestinal populations. However, NK1.1+NKp46+ cells in the salivary glands, uterus, adipose, and other tissues exhibit nonuniform programs that differ from those of liver or intestinal ILC1s or NK cells. Here, we performed single-cell RNA sequencing on murine NK1.1+NKp46+ cells from blood, spleen, various tissues, and solid tumors. We identified gene expression programs of tissue-specific ILC1s, tissue-specific NK cells, and non-tissue-specific populations in blood, spleen, and other tissues largely corresponding to circulating cells. Moreover, we found that circulating NK cell programs were reshaped in tumor-bearing mice. Core programs of circulating and tumor NK cells paralleled conserved human NK cells signatures, advancing our understanding of the human NK-ILC1 spectrum.

Osteolineage depletion of mitofusin2 enhances cortical bone formation in female mice.

Mitochondria are essential organelles that form highly complex, interconnected dynamic networks inside cells. The GTPase mitofusin 2 (MFN2) is a highly conserved outer mitochondrial membrane protein involved in the regulation of mitochondrial morphology, which can affect various metabolic and signaling functions. The role of mitochondria in bone formation remains unclear. Since MFN2 levels increase during osteoblast (OB) differentiation, we investigated the role of MFN2 in the osteolineage by crossing mice bearing floxed Mfn2 alleles with those bearing Prx-cre to generate cohorts of conditional knock out (cKO) animals. By ex vivo microCT, cKO female mice, but not males, display an increase in cortical thickness at 8, 18, and 30 weeks, compared to wild-type (WT) littermate controls. However, the cortical anabolic response to mechanical loading was not different between genotypes. To address how Mfn2 deficiency affects OB differentiation, bone marrow-derived mesenchymal stromal cells (MSCs) from both wild-type and cKO mice were cultured in osteogenic media with different levels of ß-glycerophosphate. cKO MSCs show increased mineralization and expression of multiple markers of OB differentiation only at the lower dose. Interestingly, despite showing the expected mitochondrial rounding and fragmentation due to loss of MFN2, cKO MSCs have an increase in oxygen consumption during the first 7 days of OB differentiation. Thus, in the early phases of osteogenesis, MFN2 restrains oxygen consumption thereby limiting differentiation and cortical bone accrual during homeostasis in vivo.

Osterix-Cre marks distinct subsets of CD45- and CD45+ stromal populations in extra-skeletal tumors with pro-tumorigenic characteristics.

Cancer-associated fibroblasts (CAFs) are a heterogeneous population of mesenchymal cells supporting tumor progression, whose origin remains to be fully elucidated. Osterix (Osx) is a marker of osteogenic differentiation, expressed in skeletal progenitor stem cells and bone-forming osteoblasts. We report Osx expression in CAFs and by using Osx-cre;TdTomato reporter mice we confirm the presence and pro-tumorigenic function of TdTOSX+ cells in extra-skeletal tumors. Surprisingly, only a minority of TdTOSX+ cells expresses fibroblast and osteogenic markers. The majority of TdTOSX+ cells express the hematopoietic marker CD45, have a genetic and phenotypic profile resembling that of tumor infiltrating myeloid and lymphoid populations, but with higher expression of lymphocytic immune suppressive genes. We find Osx transcript and Osx protein expression early during hematopoiesis, in subsets of hematopoietic stem cells and multipotent progenitor populations. Our results indicate that Osx marks distinct tumor promoting CD45- and CD45+ populations and challenge the dogma that Osx is expressed exclusively in cells of mesenchymal origin.

TREM2 Modulation Remodels the Tumor Myeloid Landscape Enhancing Anti-PD-1 Immunotherapy.

Checkpoint immunotherapy unleashes T cell control of tumors, but is undermined by immunosuppressive myeloid cells. TREM2 is a myeloid receptor that transmits intracellular signals that sustain microglial responses during Alzheimer's disease. TREM2 is also expressed by tumor-infiltrating macrophages. Here, we found that Trem2-/- mice are more resistant to growth of various cancers than wild-type mice and are more responsive to anti-PD-1 immunotherapy. Furthermore, treatment with anti-TREM2 mAb curbed tumor growth and fostered regression when combined with anti-PD-1. scRNA-seq revealed that both TREM2 deletion and anti-TREM2 are associated with scant MRC1+ and CX3CR1+ macrophages in the tumor infiltrate, paralleled by expansion of myeloid subsets expressing immunostimulatory molecules that promote improved T cell responses. TREM2 was expressed in tumor macrophages in over 200 human cancer cases and inversely correlated with prolonged survival for two types of cancer. Thus, TREM2 might be targeted to modify tumor myeloid infiltrates and augment checkpoint immunotherapy.

The tethering function of mitofusin2 controls osteoclast differentiation by modulating the Ca2+-NFATc1 axis.

Dynamic regulation of the mitochondrial network by mitofusins (MFNs) modulates energy production, cell survival, and many intracellular signaling events, including calcium handling. However, the relative importance of specific mitochondrial functions and their dependence on MFNs vary greatly among cell types. Osteoclasts have many mitochondria, and increased mitochondrial biogenesis and oxidative phosphorylation enhance bone resorption, but little is known about the mitochondrial network or MFNs in osteoclasts. Because expression of each MFN isoform increases with osteoclastogenesis, we conditionally deleted MFN1 and MFN2 (double conditional KO (dcKO)) in murine osteoclast precursors, finding that this increased bone mass in young female mice and abolished osteoclast precursor differentiation into mature osteoclasts in vitro Defective osteoclastogenesis was reversed by overexpression of MFN2 but not MFN1; therefore, we generated mice lacking only MFN2 in osteoclasts. MFN2-deficient female mice had increased bone mass at 1 year and resistance to Receptor Activator of NF-κB Ligand (RANKL)-induced osteolysis at 8 weeks. To explore whether MFN-mediated tethering or mitophagy is important for osteoclastogenesis, we overexpressed MFN2 variants defective in either function in dcKO precursors and found that, although mitophagy was dispensable for differentiation, tethering was required. Because the master osteoclastogenic transcriptional regulator nuclear factor of activated T cells 1 (NFATc1) is calcium-regulated, we assessed calcium release from the endoplasmic reticulum and store-operated calcium entry and found that the latter was blunted in dcKO cells. Restored osteoclast differentiation by expression of intact MFN2 or the mitophagy-defective variant was associated with normalization of store-operated calcium entry and NFATc1 levels, indicating that MFN2 controls mitochondrion-endoplasmic reticulum tethering in osteoclasts.

Therapy-Induced Senescence Drives Bone Loss.

Chemotherapy is important for cancer treatment, however, toxicities limit its use. While great strides have been made to ameliorate the acute toxicities induced by chemotherapy, long-term comorbidities including bone loss remain a significant problem. Chemotherapy-driven estrogen loss is postulated to drive bone loss, but significant data suggests the existence of an estrogen-independent mechanism of bone loss. Using clinically relevant mouse models, we showed that senescence and its senescence-associated secretory phenotype (SASP) contribute to chemotherapy-induced bone loss that can be rescued by depleting senescent cells. Chemotherapy-induced SASP could be limited by targeting the p38MAPK-MK2 pathway, which resulted in preservation of bone integrity in chemotherapy-treated mice. These results transform our understanding of chemotherapy-induced bone loss by identifying senescent cells as major drivers of bone loss and the p38MAPK-MK2 axis as a putative therapeutic target that can preserve bone and improve a cancer survivor's quality of life. SIGNIFICANCE: Senescence drives chemotherapy-induced bone loss that is rescued by p38MAPK or MK2 inhibitors. These findings may lead to treatments for therapy-induced bone loss, significantly increasing quality of life for cancer survivors.

Diacylglycerol Kinase ζ Regulates Macrophage Responses in Juvenile Arthritis and Cytokine Storm Syndrome Mouse Models.

Dysregulation of monocyte and macrophage responses are often observed in children with systemic juvenile idiopathic arthritis (sJIA) and cytokine storm syndrome (CSS), a potentially fatal complication of chronic rheumatic diseases. Both conditions are associated with activation of TLR signaling in monocyte and macrophage lineage cells, leading to overwhelming inflammatory responses. Despite the importance of TLR engagement in activating proinflammatory macrophages, relatively little is known about activation of intrinsic negative regulatory pathways to attenuate excessive inflammatory responses. In this study, we demonstrate that loss of diacylglycerol (DAG) kinase (Dgk) ζ, an enzyme which converts DAG into phosphatidic acid, limits inflammatory cytokine production in an arthritic mouse model dependent on TLR2 signaling and in a CSS mouse model dependent on TLR9 signaling. In vitro, Dgkζ deficiency results in reduced production of TNF-α, IL-6, and IL-1ß and in limited M1 macrophage polarization. Mechanistically, Dgkζ deficiency decreases STAT1 and STAT3 phosphorylation. Moreover, Dgkζ levels are increased in macrophages derived from mice with CSS or exposed to plasma from sJIA patients with active disease. Our data suggest that Dgkζ induction in arthritic conditions perpetuates systemic inflammatory responses mediated by macrophages and highlight a potential role of Dgkζ-DAG/phosphatidic acid axis as a modulator of inflammatory cytokine production in sJIA and CSS.

Importance of the Conserved Carboxyl-Terminal CNOT1 Binding Domain to Tristetraprolin Activity In Vivo.

Tristetraprolin (TTP) is an anti-inflammatory protein that modulates the stability of certain cytokine/chemokine mRNAs. After initial high-affinity binding to AU-rich elements in 3' untranslated regions of target mRNAs, mediated through its tandem zinc finger (TZF) domain, TTP promotes the deadenylation and ultimate decay of target transcripts. These transcripts and their encoded proteins accumulate abnormally in TTP knockout (KO) mice, leading to a severe inflammatory syndrome. To assess the importance of the highly conserved C-terminal CNOT1 binding domain (CNBD) of TTP to the TTP deficiency phenotype in mice, we created a mouse model in which TTP lacked its CNBD. CNBD deletion mice exhibited a less severe phenotype than the complete TTP KO mice. In macrophages, the stabilization of target transcripts seen in KO mice was partially normalized in the CNBD deletion mice. In cell-free experiments, recombinant TTP lacking its CNBD could still activate target mRNA deadenylation by purified recombinant Schizosaccharomyces pombe CCR4/NOT complexes, although to a lesser extent than full-length TTP. Thus, TTP lacking its CNBD can still act to promote target mRNA instability in vitro and in vivo These data have implications for TTP family members throughout the eukarya, since species from all four kingdoms contain proteins with linked TZF and CNOT1 binding domains.

Tmem178 negatively regulates store-operated calcium entry in myeloid cells via association with STIM1.

Store-operated calcium entry (SOCE) modulates cytosolic calcium in multiple cells. Endoplasmic reticulum (ER)-localized STIM1 and plasma membrane (PM)-localized ORAI1 are two main components of SOCE. STIM1:ORAI1 association requires STIM1 oligomerization, its re-distribution to ER-PM junctions, and puncta formation. However, little is known about the negative regulation of these steps to prevent calcium overload. Here, we identified Tmem178 as a negative modulator of STIM1 puncta formation in myeloid cells. Using site-directed mutagenesis, co-immunoprecipitation assays and FRET imaging, we determined that Tmem178:STIM1 association occurs via their transmembrane motifs. Mutants that increase Tmem178:STIM1 association reduce STIM1 puncta formation, SOCE activation, impair inflammatory cytokine production in macrophages and osteoclastogenesis. Mutants that reduce Tmem178:STIM1 association reverse these effects. Furthermore, exposure to plasma from arthritic patients decreases Tmem178 expression, enhances SOCE activation and cytoplasmic calcium. In conclusion, Tmem178 modulates the rate-limiting step of STIM1 puncta formation and therefore controls SOCE in inflammatory conditions.

Plcγ2/Tmem178 dependent pathway in myeloid cells modulates the pathogenesis of cytokine storm syndrome.

Cytokine storm syndrome (CSS) is a life-threatening condition characterized by excessive activation of T cells and uncontrolled inflammation, mostly described in patients with familial hemophagocytic lymphohistiocytosis and certain systemic auto-inflammatory diseases, such as systemic juvenile idiopathic arthritis (sJIA). Defects in T cell cytotoxicity as a mechanism for uncontrolled inflammation following viral infections fail to represent the whole spectrum of CSS. Evidence implicates dysregulated innate immune responses, especially activation of monocytes and macrophages, in patients with CSS. However, the direct contribution of monocytes/macrophages to CSS development and the signaling pathways involved in their activation have not been formally investigated. We find that depletion of monocytes/macrophages during early stages of CSS development, by clodronate-liposomes or neutralizing anti-CSF1 antibody, reduces mortality and inflammatory cytokine levels in two CSS mouse models, one dependent on T cells and the second induced by repeated TLR9 stimulation. We further demonstrate that activation of Plcγ2 in myeloid cells controls CSS development by driving macrophage pro-inflammatory responses. Intriguingly, the Plcγ2 downstream effector Tmem178, a negative modulator of calcium levels, acts in a negative feedback loop to restrain inflammatory cytokine production. Genetic deletion of Tmem178 leads to pro-inflammatory macrophage polarization in vitro and more severe CSS in vivo. Importantly, Tmem178 levels are reduced in macrophages from mice with CSS and after exposure to plasma from sJIA patients with active disease. Our data identify a novel Plcγ2/Tmem178 axis as a modulator of inflammatory cytokine production by monocytes/macrophages. We also find that loss of Tmem178 accentuates the pro-inflammatory responses in CSS.

Breast and pancreatic cancer interrupt IRF8-dependent dendritic cell development to overcome immune surveillance.

Tumors employ multiple mechanisms to evade immune surveillance. One mechanism is tumor-induced myelopoiesis, whereby the expansion of immunosuppressive myeloid cells can impair tumor immunity. As myeloid cells and conventional dendritic cells (cDCs) are derived from the same progenitors, we postulated that myelopoiesis might impact cDC development. The cDC subset, cDC1, which includes human CD141+ DCs and mouse CD103+ DCs, supports anti-tumor immunity by stimulating CD8+ T-cell responses. Here, to understand how cDC1 development changes during tumor progression, we investigated cDC bone marrow progenitors. We found localized breast and pancreatic cancers induce systemic decreases in cDC1s and their progenitors. Mechanistically, tumor-produced granulocyte-stimulating factor downregulates interferon regulatory factor-8 in cDC progenitors, and thus results in reduced cDC1 development. Tumor-induced reductions in cDC1 development impair anti-tumor CD8+ T-cell responses and correlate with poor patient outcomes. These data suggest immune surveillance can be impaired by tumor-induced alterations in cDC development.

A Knock-In Tristetraprolin (TTP) Zinc Finger Point Mutation in Mice: Comparison with Complete TTP Deficiency.

Tristetraprolin (TTP) is a tandem CCCH zinc finger protein that can bind to AU-rich element-containing mRNAs and promote their decay. TTP knockout mice develop a severe inflammatory syndrome, largely due to excess tumor necrosis factor (TNF), whose mRNA is a direct target of TTP binding and destabilization. TTP's RNA binding activity and its ability to promote mRNA decay are lost when one of the zinc-coordinating residues of either zinc finger is mutated. To address several long-standing questions about TTP activity in intact animals, we developed a knock-in mouse with a cysteine-to-arginine mutation within the first zinc finger. Homozygous knock-in mice developed a severe inflammatory syndrome that was essentially identical to that of complete TTP deficiency, suggesting that TTP's critical anti-inflammatory role in mammalian physiology is secondary to its ability to bind RNA. In addition, there was no evidence for a "dominant-negative" effect of the mutant allele in heterozygotes, as suggested by previous experiments. Finally, mRNA decay experiments in mutant macrophages demonstrated that TTP can regulate the stability of its own mRNA, albeit to a minor extent. These studies suggest that RNA binding is an essential first step in the physiological activities of members of this protein family.

Phospholipase Cγ1 (PLCγ1) Controls Osteoclast Numbers via Colony-stimulating Factor 1 (CSF-1)-dependent Diacylglycerol/ß-Catenin/CyclinD1 Pathway.

Phospholipases Cγ (PLCγ) 1 and 2 are a class of highly homologous enzymes modulating a variety of cellular pathways through production of inositol 1,4,5-trisphosphate and diacylglycerol (DAG). Our previous studies demonstrated the importance of PLCγ2 in osteoclast (OC) differentiation by modulating inositol 1,4,5-trisphosphate-mediated calcium oscillations and the up-regulation of the transcription factor NFATc1. Surprisingly, despite being expressed throughout osteoclastogenesis, PLCγ1 did not compensate for PLCγ2 deficiency. Because both isoforms are activated during osteoclastogenesis, it is plausible that PLCγ1 modulates OC development independently of PLCγ2. Here, we utilized PLCγ1-specific shRNAs to delete PLCγ1 in OC precursors derived from wild type (WT) mice. Differently from PLCγ2, we found that PLCγ1 shRNA significantly suppresses OC differentiation by limiting colony-stimulating factor 1 (CSF-1)-dependent proliferation and ß-catenin/cyclinD1 levels. Confirming the specificity toward CSF-1 signaling, PLCγ1 is recruited to the CSF-1 receptor following exposure to the cytokine. To understand how PLCγ1 controls cell proliferation, we turned to its downstream effector, DAG. By utilizing cells lacking the DAG kinase ζ, which have increased DAG levels, we demonstrate that DAG modulates CSF-1-dependent proliferation and ß-catenin/cyclinD1 levels in OC precursors. Most importantly, the proliferation and osteoclastogenesis defects observed in the absence of PLCγ1 are normalized in PLCγ1/DAG kinase ζ double null cells. Taken together, our study shows that PLCγ1 controls OC numbers via a CSF-1-dependent DAG/ß-catenin/cyclinD1 pathway.