The triggering receptor expressed on myeloid cell 1 (TREM1) plays a critical role in development of chronic inflammatory disorders and the inflamed tumor microenvironment (TME) associated with most ...solid tumors. We examined whether loss of TREM1 signaling can abrogate the immunosuppressive TME and enhance cancer immunity. To investigate the therapeutic potential of TREM1 in cancer, we used mice deficient in Trem1 and developed a novel small molecule TREM1 inhibitor, VJDT. We demonstrated that genetic or pharmacological TREM1 silencing significantly delayed tumor growth in murine melanoma (B16F10) and fibrosarcoma (MCA205) models. Single-cell RNA-Seq combined with functional assays during TREM1 deficiency revealed decreased immunosuppressive capacity of myeloid-derived suppressor cells (MDSCs) accompanied by expansion in cytotoxic CD8+ T cells and increased PD-1 expression. Furthermore, TREM1 inhibition enhanced the antitumorigenic effect of anti-PD-1 treatment, in part, by limiting MDSC frequency and abrogating T cell exhaustion. In patient-derived melanoma xenograft tumors, treatment with VJDT downregulated key oncogenic signaling pathways involved in cell proliferation, migration, and survival. Our work highlights the role of TREM1 in cancer progression, both intrinsically expressed in cancer cells and extrinsically in the TME. Thus, targeting TREM1 to modify an immunosuppressive TME and improve efficacy of immune checkpoint therapy represents what we believe to be a promising therapeutic approach to cancer.
Chronic inflammation drives liver cancer pathogenesis, invasion, and metastasis. Liver Kupffer cells have crucial roles in mediating the inflammatory processes that promote liver cancer, but the ...mechanistic basis for their contributions are not fully understood. Here we show that expression of the proinflammatory myeloid cell surface receptor TREM-1 expressed by Kupffer cells is a crucial factor in the development and progression of liver cancer. Deletion of the murine homolog Trem1 in mice attenuated hepatocellular carcinogenesis triggered by diethylnitrosamine (DEN). Trem1 deficiency attenuated Kupffer cell activation by downregulating transcription and protein expression of interleukin (IL)-6, IL-1β, TNF, CCL2, and CXCL10. In addition, Trem1 ablation diminished activation of the p38, extracellular regulated kinase 1/2, JNK, mitogen-activated protein kinase, and NF-κB signaling pathways in Kupffer cells, resulting in diminished liver injury after DEN exposure. Adoptive transfer of wild-type Kupffer cells to Trem1-deficient mice complemented these defects and reversed unresponsiveness to DEN-induced liver injury and malignant development. Together, our findings offer causal evidence that TREM-1 is a pivotal determinant of Kupffer cell activation in liver carcinogenesis, deepening mechanistic insights into how chronic inflammation underpins the development and progression of liver cancer.
Heat shock protein 90 (Hsp90) and its co-chaperones promote cancer, and targeting Hsp90 holds promise for cancer treatment. Most of the efforts to harness this potential have focused on targeting the ...Hsp90 N-terminus ATP binding site. Although newer-generation inhibitors have shown improved efficacy in aggressive cancers, induction of the cellular heat shock response (HSR) by these inhibitors is thought to limit their clinical efficacy. Therefore, Hsp90 inhibitors with novel mechanisms of action and that do not trigger the HSR would be advantageous. Here, we investigated the mechanism by which capsaicin inhibits Hsp90. Through mutagenesis, chemical modifications, and proteomic studies, we show that capsaicin binds to the N-terminus of Hsp90 and inhibits its ATPase activity. Consequently, capsaicin and its analogs inhibit Hsp90 ATPase-dependent progesterone receptor reconstitution in vitro. Capsaicin did not induce the HSR, instead, it promoted the degradation of Hsp70 through the lysosome-autophagy pathway. Remarkably, capsaicin did not induce degradation of the constitutively expressed cognate Hsc70, indicating selectivity for Hsp70. Combined treatments of capsaicin and the Hsp90 inhibitor 17-AAG improved the anti-tumor efficacy of 17-AAG in cell culture and tridimensional tumor spheroid growth assays using breast and prostate cancer models. Consistent with this, in silico docking studies revealed that capsaicin binding to the ATP binding site of Hsp90 was distinct from classical N-terminus Hsp90 inhibitors, indicating a novel mechanism of action. Collectively, these findings support the use of capsaicin as a chemical scaffold to develop novel Hsp90 N-terminus inhibitors as well as its ability to be a potential cancer co-therapeutic.
The outcome of organ transplantation is largely dictated by selection of a well-matched donor, which results in less chance of graft rejection. An allogeneic immune response is the main immunological ...barrier for successful organ transplantation. Donor and recipient human leukocyte antigen (HLA) mismatching diminishes outcomes after solid organ transplantation. The current evaluation of HLA incompatibility does not provide information on the immunogenicity of individual HLA mismatches and impact of non-HLA-related alloantigens, especially
. Here we demonstrate a new method for analysis of alloimmune responsiveness between donor and recipient
by introducing a humanized mouse model. Using molecular, cellular, and genomic analyses, we demonstrated that a recipient's personalized humanized mouse provided the most sensitive assessment of allogeneic responsiveness to potential donors. In our study, HLA typing provided a better recipient-donor match for one donor among two related donors. In contrast, assessment of an allogeneic response by mixed lymphocyte reaction (MLR) was indistinguishable between these donors. We determined that, in the recipient's humanized mouse model, the donor selected by HLA typing induced the strongest allogeneic response with markedly increased allograft rejection markers, including activated cytotoxic Granzyme B-expressing CD8
T cells. Moreover, the same donor induced stronger upregulation of genes involved in the allograft rejection pathway as determined by transcriptome analysis of isolated human CD45
cells. Thus, the humanized mouse model determined the lowest degree of recipient-donor alloimmune response, allowing for better selection of donor and minimized immunological risk of allograft rejection in organ transplantation. In addition, this approach could be used to evaluate the level of alloresponse in allogeneic cell-based therapies that include cell products derived from pluripotent embryonic stem cells or adult stem cells, both undifferentiated and differentiated, all of which will produce allogeneic immune responses.
The expression of Ig-like transcript (ILT) inhibitory receptors is a characteristic of tolerogenic dendritic cells (DCs). However, the mechanisms of modulation of DCs via ILT receptors remain poorly ...defined. HLA-G is a preferential ligand for several ILTs. Recently, we demonstrated that triggering of ILT4 by HLA-G1 inhibits maturation of human monocyte-derived conventional DCs and murine DCs from ILT4 transgenic mice, resulting in diminished expression of MHC class II molecules, CD80 and CD86 costimulatory molecules, and prolongation of skin allograft survival. Different isoforms of HLA-G have diverse effects on the efficiency to induce ILT-mediated signaling. In this work, we show that HLA-G1 tetrameric complex and HLA-G5 dimer, but not HLA-G5 monomer, induce strong ILT-mediated signaling. We determined that the arrest of maturation of ILT4-positive DCs by HLA-G ligands involves the IL-6 signaling pathway and STAT3 activation. Ligation of ILT4 with HLA-G on DCs results in recruitment of SHP-1 and SHP-2 protein tyrosine phosphatases. We propose a model where SHP-2 and the IL-6-STAT3 signaling pathway play critical roles in the modulation of DC differentiation by ILT4 and HLA-G.
The non-classical Human leukocyte antigen G (HLA-G) differs from classical HLA class I molecules by its low genetic diversity, a tissue-restricted expression, the existence of seven isoforms, and ...immuno-inhibitory functions. Most of the known functions of HLA-G concern the membrane-bound HLA-G1 and soluble HLA-G5 isoforms, which present the typical structure of classical HLA class I molecule: a heavy chain of three globular domains α
1
–α
2
–α
3
non-covalently bound to β-2-microglobulin (B2M) and a peptide. Very little is known of the structural features and functions of other HLA-G isoforms or structural conformations other than B2M-associated HLA-G1 and HLA-G5. In the present work, we studied the capability of all isoforms to form homomultimers, and investigated whether they could bind to, and function through, the known HLA-G receptors LILRB1 and LILRB2. We report that all HLA-G isoforms may form homodimers, demonstrating for the first time the existence of HLA-G4 dimers. We also report that the HLA-G α
1
–α
3
structure, which constitutes the extracellular part of HLA-G2 and HLA-G6, binds the LILRB2 receptor but not LILRB1. This is the first report of a receptor for a truncated HLA-G isoform. Following up on this finding, we show that the α
1
–α
3
-Fc structure coated on agarose beads is tolerogenic and capable of prolonging the survival of skin allografts in B6-mice and in a LILRB2-transgenic mouse model. This study is the first proof of concept that truncated HLA-G isoforms could be used as therapeutic agents.
The human leukocyte antigen (HLA)‐G is a tolerogenic molecule, whose expression by allografts is associated with better acceptance. An increasing interest in producing HLA‐G as a clinical‐grade ...molecule for therapy use is impaired by its complexity and limited stability. Our purpose was to engineer simpler and more stable HLA‐G‐derived molecules than the full‐length HLA‐G trimolecular complex that are also tolerogenic, functional as soluble molecules, and compatible with good manufacturing practice (GMP) production conditions. We present two synthetic molecules: (α3‐L)x2 and (α1‐α3)x2 polypeptides. We show their capability to bind the HLA‐G receptor LILRB2 and their functions in vitro and in vivo. The (α1‐α3)x2 polypeptide proved to be a potent tolerogenic molecule in vivo: One treatment of skin allograft recipient mice with (α1‐α3)x2 was sufficient to significantly prolong graft survival, and four weekly treatments induced complete tolerance. Furthermore, (α1‐α3)x2 was active as a soluble molecule and capable of inhibiting the proliferation of tumor cell lines, as does the full length HLA‐G trimolecular complex. Thus, the synthetic (α1‐α3)x2 polypeptide is a stable and simpler alternative to the full‐length HLA‐G molecule. It can be produced under GMP conditions, it functions as a soluble molecule, and it is at least as tolerogenic as HLA‐G in vivo.—LeMaoult, J., Daouya, M., Wu, J., Loustau, M., Horuzsko, A., Carosella, E. D. Synthetic HLA‐G proteins for therapeutic use in transplantation. FASEB J. 27, 3643–3651 (2013). www.fasebj.org
Sickle cell disease (SCD) is a pathophysiological condition of chronic hemolysis, oxidative stress, and elevated inflammation. The transcription factor Nrf2 is a master regulator of oxidative stress. ...Here, we report that the FDA-approved oral agent simvastatin, an inhibitor of hydroxymethyl-glutaryl coenzyme A reductase, significantly activates the expression of Nrf2 and antioxidant enzymes. Simvastatin also induces fetal hemoglobin expression in SCD patient primary erythroid progenitors and a transgenic mouse model. Simvastatin alleviates SCD symptoms by decreasing hemoglobin S sickling, oxidative stress, and inflammatory stress in erythroblasts. Particularly, simvastatin increases cellular levels of cystine, the precursor for the biosynthesis of the antioxidant reduced glutathione, and decreases the iron content in SCD mouse spleen and liver tissues. Mechanistic studies suggest that simvastatin suppresses the expression of the critical histone methyltransferase enhancer of zeste homolog 2 to reduce both global and gene-specific histone H3 lysine 27 trimethylation. These chromatin structural changes promote the assembly of transcription complexes to fetal γ-globin and antioxidant gene regulatory regions in an antioxidant response element-dependent manner. In summary, our findings suggest that simvastatin activates fetal hemoglobin and antioxidant protein expression, modulates iron and cystine/reduced glutathione levels to improve the phenotype of SCD, and represents a therapeutic strategy for further development.
Endothelial cell‐selective adhesion molecule (ESAM) regulates inflammatory cell adhesion and transmigration and promotes angiogenesis. Here, we examined the role of ESAM in cardiac vascularization, ...inflammatory cell infiltration, and left ventricle (LV) diastolic function under basal and hemodynamic stress conditions. We employed mice with homozygous genetic deletion of ESAM (ESAM−/−) and also performed uninephrectomy and aldosterone infusion (UNX‐Aldo) to induce volume and pressure overload. Using echocardiography, we found that ESAM−/− mice display no change in systolic function. However, they develop LV diastolic dysfunction, as indicated by a significantly reduced E/A ratio (E = early, A = late mitral inflow peak velocities), increased E/e’ ratio, isovolumic relaxation time (IVRT), and E wave deceleration time. An unbiased automated tracing and 3D reconstruction of coronary vasculature revealed that ESAM−/− mice had reduced coronary vascular density. Arteries of ESAM−/− mice exhibited impaired endothelial sprouting and in cultured endothelial cells siRNA‐mediated ESAM knockdown reduced tube formation. Changes in ESAM−/− mice were accompanied by elevated myocardial inflammatory cytokine and myeloperoxidase‐positive neutrophil levels. Furthermore, UNX‐Aldo procedure in wild type mice induced LV diastolic dysfunction, which was accompanied by significantly increased serum ESAM levels. When compared to wild types, ESAM−/− mice with UNX‐Aldo displayed worsening of LV diastolic function, as indicated by increased IVRT and pulmonary edema. Thus, we propose that ESAM plays a mechanistic role in proper myocardial vascularization and the maintenance of LV diastolic function under basal and hemodynamic stress conditions.
Endothelial cell‐selective adhesion molecule (ESAM) regulates inflammatory cell adhesion and transmigration and promotes angiogenesis. We propose that animals deficient in ESAM develop left ventricular diastolic dysfunction due to reduced myocardial vascular density, hypoxia, and inflammation. Hemodynamic challenge lead to worsening of left ventricular diastolic dysfunction in ESAM knockout mice.
Abstract The expression of inhibitory immunoglobulin-like transcripts (ILTs) on dendritic cells (DCs) is a biomarker of tolerogenic DCs. In this article we discuss current knowledge on the function ...of ILTs and explore the molecular mechanisms involved in modulation of DCs via the inhibitory receptor and its natural ligand, rendering these cells tolerogenic. We propose a method to enhance targeting of inhibitory receptors on DCs using microparticles containing a preferential ligand, HLA-G, and monoclonal antibody against the pan-DCs marker CD11c. The double-coated microparticles increase the binding of ILT4 receptor and improve modulation of DCs in vitro and in vivo . This targeting concept can be used for regulation of specific immune responses to antigens in transplantation, autoimmunity, allergy, and cancer.
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