Low-density lipoprotein receptor-related protein 6 ablation in macrophages differentially inhibits lung injury-mediated inflammation and metastasis.

Low-density lipoprotein receptor-related protein 6 (LRP6) is a receptor protein for Wnt ligands. Yet, their role in immune cell regulation remains elusive. Here we demonstrated that genetic deletion of LRP6 in macrophages using LysM-cre Lrp6fl/fl (Lrp6MKO) mice showed differential inhibition of inflammation in the bleomycin (BLM)-induced lung injury model and B16F10 melanoma lung metastasis model. Lrp6MKO mice showed normal immune cell populations in the lung and circulating blood in homeostatic conditions. In the BLM-induced lung injury model, Lrp6MKO mice showed a decreased number of monocyte-derived alveolar macrophages, reduced collagen deposition and alpha-smooth muscle actin (αSMA) protein levels in the lung. In B16F10 lung metastasis model, Lrp6MKO mice reduced lung tumor foci. Monocytic and granulocytic-derived myeloid-derived suppressor cells (M-MDSCs and G-MDSCs) were increased in the lung. In G-MDSCs, hypoxia-inducible factor 1α (HIF1α)+ PDL1+ population was markedly decreased but not in M-MDSCs. Taken together, our results show that the role of LRP6 in macrophages is differential depending on the inflammation microenvironment in the lung.

Dickkopf1 Promotes Pulmonary Fibrosis upon Bleomycin-Induced Lung Injury.

Orchestration of inflammation and tissue repair processes is critical to maintaining homeostasis upon tissue injury. Tissue fibrosis is a pathological process characterized by aberrant accumulation of extracellular matrix proteins, such as collagen, upon injury. Dickkopf1 (DKK1) is a quintessential Wnt antagonist. The role of DKK1 in bleomycin (BLM)-induced lung injury and fibrosis model remains elusive. This study shows that BLM-induced lung injury markedly elevated DKK1 protein expressions in the lungs in mice, consistent with human pulmonary fibrosis patient lung tissues. The elevated DKK1 levels coincided with immune cell infiltration and collagen deposition. Notably, the reduced expression of DKK1 in Dkk1 hypomorphic doubleridge (Dkk1d/d) mice abrogated BLM-induced lung inflammation and fibrosis. Immune cell infiltration, collagen deposition, expression of profibrotic cytokine transforming growth factor ß1 (TGF-ß1), and extracellular matrix protein-producing myofibroblast marker α-smooth muscle actin (α-SMA) were reduced in Dkk1d/d mice. Consistent with these results, local DKK1 antibody administration after BLM-induced lung injury substantially decreased lung inflammation and fibrosis phenotypes. Taken together, these results demonstrate that DKK1 is a proinflammatory and profibrotic ligand that promotes inflammation and fibrosis upon BLM-induced lung injury, placing it as an attractive molecular target for dysregulated pulmonary inflammation and tissue repair.

Thrombocyte-derived Dickkopf1 promotes macrophage polarization in the Bleomycin-induced lung injury model.

Immune responses are crucial to maintaining tissue homeostasis upon tissue injury. Upon various types of challenges, macrophages play a central role in regulating inflammation and tissue repair processes. While an immunomodulatory role of Wnt antagonist Dickkopf1 (DKK1) has been implicated, the role of Wnt antagonist DKK1 in regulating macrophage polarization in inflammation and the tissue repair process remains elusive. Here we found that DKK1 induces gene expression profiles to promote inflammation and tissue repair in macrophages. Importantly, DKK1 induced various genes, including inflammation and tissue repair, via JNK (c-jun N-terminal kinase) in macrophages. Furthermore, DKK1 potentiated IL-13-mediated macrophage polarization and activation. The co-inhibition of JNK and STAT6 markedly decreased gene expressions relevant to inflammation and fibrosis by DKK1 and IL-13. Interestingly, thrombocyte-specific deletion of DKK1 in mice reduced collagen deposition and decreased Arg1, CD206, HIF1α, and IL1ß protein expressions in monocyte-derived alveolar macrophages in the acute sterile bleomycin (BLM)-induced lung injury model. These data suggested that thrombocytes communicate with macrophages via DKK1 to orchestrate inflammation and repair in this model. Taken together, our study demonstrates DKK1's role as an important regulatory ligand for macrophage polarization in the injury-induced inflammation and repair process in the lung.

Macrophages and Wnts in Tissue Injury and Repair.

Macrophages are important players in the immune system that sense various tissue challenges and trigger inflammation. Tissue injuries are followed by inflammation, which is tightly coordinated with tissue repair processes. Dysregulation of these processes leads to chronic inflammation or tissue fibrosis. Wnt ligands are present both in homeostatic and pathological conditions. However, their roles and mechanisms regulating inflammation and tissue repair are being investigated. Here we aim to provide an overview of overarching themes regarding Wnt and macrophages by reviewing the previous literature. We aim to gain future insights into how tissue inflammation, repair, regeneration, and fibrosis events are regulated by macrophages.

Dickkopf proteins in pathological inflammatory diseases.

The human body encounters various challenges. Tissue repair and regeneration processes are augmented after tissue injury to reinstate tissue homeostasis. The Wnt pathway plays a crucial role in tissue repair since it induces target genes required for cell proliferation and differentiation. Since tissue injury causes inflammatory immune responses, it has become increasingly clear that the Wnt ligands can function as immunomodulators while critical for tissue homeostasis. The Wnt pathway and Wnt ligands have been studied extensively in cancer biology and developmental biology. While the Wnt ligands are being studied actively, how the Wnt antagonists and their regulatory mechanisms can modulate immune responses during chronic pathological inflammation remain elusive. This review summarizes DKK family proteins as immunomodulators, aiming to provide an overarching picture for tissue injury and repair. To this end, we first review the Wnt pathway components and DKK family proteins. Next, we will review DKK family proteins (DKK1, 2, and 3) as a new class of immunomodulatory protein in cancer and other chronic inflammatory diseases. Taken together, DKK family proteins and their immunomodulatory functions in chronic inflammatory disorders provide novel insights to understand immune diseases and make them attractive molecular targets for therapeutic intervention.

Dickkopf1: An Immunomodulator in Tissue Injury, Inflammation, and Repair.

Upon injury, inflammation and repair processes are orchestrated to maintain tissue homeostasis. The Wnt ligands play essential roles in cell differentiation and proliferation for tissue repair and regeneration. It is increasingly clear that Wnt ligands play crucial immune-modulatory roles in inflammatory diseases. It is predicted that comprehensive research regarding the cross-talk between nonimmune and immune cells in tissue injury and repair will flourish. The Wnt system and immune system interaction will be critical to understanding tissue injury, inflammation, and repair. In this study, we will first introduce the Wnt system and review the role of the Wnt system in tissue regeneration and repair. We will review the previous literature regarding how the Wnt ligands regulate the immune system. Next, we will discuss the current and future perspectives of Wnt ligands to target cancer and other immunological diseases. Finally, we will discuss the quintessential Wnt antagonist Dickkopf1 as an immunomodulatory ligand.

Overexpression of ERAP2N in Human Trophoblast Cells Promotes Cell Death.

The genes involved in implantation and placentation are tightly regulated to ensure a healthy pregnancy. The endoplasmic reticulum aminopeptidase 2 (ERAP2) gene is associated with preeclampsia (PE). Our studies have determined that an isoform of ERAP2-arginine (N), expressed in trophoblast cells (TC), significantly activates immune cells, and ERAP2N-expressing TCs are preferentially killed by both cytotoxic T lymphocytes (CTLs) and Natural Killer cells (NKCs). To understand the cause of this phenomenon, we surveyed differentially expressed genes (DEGs) between ERAP2N expressing and non-expressing TCs. Our RNAseq data revealed 581 total DEGs between the two groups. 289 genes were up-regulated, and 292 genes were down-regulated. Interestingly, most of the down-regulated genes of significance were pro-survival genes that play a crucial role in cell survival (LDHA, EGLN1, HLA-C, ITGB5, WNT7A, FN1). However, the down-regulation of these genes in ERAP2N-expressing TCs translates into a propensity for cell death. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that 64 DEGs were significantly enriched in nine pathways, including "Protein processing in endoplasmic reticulum" and "Antigen processing and presentation", suggesting that the genes may be associated with peptide processes involved in immune recognition during the reproductive cycle.

Dickkopf1: An immunomodulatory ligand and Wnt antagonist in pathological inflammation.

The Wnt signaling pathway plays essential roles in tissue or organ homeostasis by regulating cell proliferation and differentiation. Upon tissue or organ injury, inflammation is coupled with tissue repair and regeneration process. The canonical Wnt signaling transduction pathway is crucial for cell proliferation, cell differentiation, and tissue regeneration. Dickkopf1 (DKK1) is a quintessential Wnt antagonist that inhibits the Wnt-mediated tissue repair process. Recent studies reported increased levels of DKK1 in many diseases such as cancer, infection, and musculoskeletal diseases. In many cases, the role of DKK1 has been identified as a pro-inflammatory ligand and the expression levels are associated with poor disease outcomes. A variety of cell types including platelets, endothelial cells, and cancer cells secrete DKK1 upon stimuli. This puts DKK1 in a unique place to view immune responses from multicellular interactions in tissue injury and repair process. In this review, we discuss recent efforts to address the underlying mechanism regarding the pro-inflammatory role of DKK1 in cancer, bone diseases, and other inflammatory diseases.

Canonical and Non-Canonical Wnt Signaling in Immune Cells.

Cell differentiation, proliferation, and death are vital for immune homeostasis. Wnt signaling plays essential roles in processes across species. The roles of Wnt signaling proteins and Wnt ligands have been studied in the past, but the context-dependent mechanisms and functions of these pathways in immune responses remain unclear. Recent findings regarding the role of Wnt ligands and Wnt signaling in immune cells and their immunomodulatory mechanisms suggest that Wnt ligands and signaling are significant in regulating immune responses. We introduce recent key findings and future perspectives on Wnt ligands and their signaling pathways in immune cells as well as the immunological roles and functions of Wnt antagonists.

Therapeutic Potential of Gene-Modified Regulatory T Cells: From Bench to Bedside.

Regulatory T cells (Tregs) are an important subset of adaptive immune cells and control immune reactions for maintaining homeostasis. Tregs are generated upon their encounter with self or non-self-antigen and mediate tolerance or suppress aberrant immune responses. A high level of specificity of Tregs to recognize antigen(s) suggested their instrumental potential to treat various inflammatory diseases. This review will first introduce seminal basic research findings in the field of Tregs over the last two decades pertinent to therapeutic approaches in progress. We will then discuss the previous approaches to use Tregs for therapeutic purposes and the more recent development of gene-modification approaches. The suppressive function of Tregs has been studied intensively in clinical settings, including cancer, autoimmunity, and allotransplantation. In cancer, Tregs are often aberrantly increased in their number, and their suppressor function inhibits mounting of effective antitumor immune responses. We will examine potential approaches of using gene-modified Tregs to treat cancer. In autoimmunity and allotransplantation, chronic inflammation due to inherent genetic defects in the immune system or mismatch between organ donor and recipient results in dysfunction of Tregs, leading to inflammatory diseases or rejection, respectively. Since the recognition of antigen is a central part in Treg function and their therapeutic use, the modulation of T cell receptor specificity will be discussed. Finally, we will focus on future novel strategies employing the therapeutic potential of Tregs using gene modification to broaden our perspective.

Membrane-bound Dickkopf-1 in Foxp3+ regulatory T cells suppresses T-cell-mediated autoimmune colitis.

Induction of tolerance is a key mechanism to maintain or to restore immunological homeostasis. Here we show that Foxp3+ regulatory T (Treg) cells use Dickkopf-1 (DKK-1) to regulate T-cell-mediated tolerance in the T-cell-mediated autoimmune colitis model. Treg cells from DKK-1 hypomorphic doubleridge mice failed to control CD4+ T-cell proliferation, resulting in CD4 T-cell-mediated autoimmune colitis. Thymus-derived Treg cells showed a robust expression of DKK-1 but not in naive or effector CD4 T cells. DKK-1 expression in Foxp3+ Treg cells was further increased upon T-cell receptor stimulation in vitro and in vivo. Interestingly, Foxp3+ Treg cells expressed DKK-1 in the cell membrane and the functional inhibition of DKK-1 using DKK-1 monoclonal antibody abrogated the suppressor function of Foxp3+ Treg cells. DKK-1 expression was dependent on de novo protein synthesis and regulated by the mitogen-activated protein kinase pathway but not by the canonical Wnt pathway. Taken together, our results highlight membrane-bound DKK-1 as a novel Treg-derived mediator to maintain immunological tolerance in T-cell-mediated autoimmune colitis.

The Wnt Antagonist Dickkopf-1 Promotes Pathological Type 2 Cell-Mediated Inflammation.

Exposure to a plethora of environmental challenges commonly triggers pathological type 2 cell-mediated inflammation. Here we report the pathological role of the Wnt antagonist Dickkopf-1 (Dkk-1) upon allergen challenge or non-healing parasitic infection. The increased circulating amounts of Dkk-1 polarized T cells to T helper 2 (Th2) cells, stimulating a marked simultaneous induction of the transcription factors c-Maf and Gata-3, mediated by the kinases p38 MAPK and SGK-1, resulting in Th2 cell cytokine production. Circulating Dkk-1 was primarily from platelets, and the increase of Dkk-1 resulted in formation of leukocyte-platelet aggregates (LPA) that facilitated leukocyte infiltration to the affected tissue. Functional inhibition of Dkk-1 impaired Th2 cell cytokine production and leukocyte infiltration, protecting mice from house dust mite (HDM)-induced asthma or Leishmania major infection. These results highlight that Dkk-1 from thrombocytes is an important regulator of leukocyte infiltration and polarization of immune responses in pathological type 2 cell-mediated inflammation.

Spontaneous Intestinal Tumorigenesis in Apc (/Min+) Mice Requires Altered T Cell Development with IL-17A.

The control of inflammatory diseases requires functional regulatory T cells (Tregs) with significant Gata-3 expression. Here we address the inhibitory role of Tregs on intestinal tumorigenesis in the Apc (/Min+) mouse model that resembles human familial adenomatous polyposis (FAP). Apc (/Min+) mice had a markedly increased frequency of Foxp3+ Tregs and yet decreased Gata-3 expression in the lamina propria. To address the role of heterozygous Apc gene mutation in Tregs, we generated Foxp3-Cre, Apc (flox/+) mice. Tregs from these mice effectively inhibited tumorigenesis comparable to wild type Tregs after adoptive transfer into Apc (/Min+) mice, demonstrating that the heterozygous Apc gene mutation in Tregs does not induce the loss of control over tumor microenvironment. Adoptive transfer of in vitro generated Apc (/Min+) iTregs (inducible Tregs) failed to inhibit intestinal tumorigenesis, suggesting that naïve CD4 T cells generated from Apc (/Min+) mice thymus were impaired. We also showed that adoptively transferred IL-17A-deficient Apc (/Min+) Tregs inhibited tumor growth, suggesting that IL-17A was critical to impair the tumor regression function of Apc (/Min+) Tregs. Taken together, our results suggest that both T cell development in a functional thymus and IL-17A control the ability of Treg to inhibit intestinal tumorigenesis in Apc (/Min+) mice.

The immunotherapeutic role of regulatory T cells in Leishmania (Viannia) panamensis infection.

Leishmania (Viannia) parasites are etiological agents of cutaneous leishmaniasis in the New World. Infection is characterized by a mixed Th1/Th2 inflammatory response, which contributes to disease pathology. However, the role of regulatory T cells (Tregs) in Leishmania (Viannia) disease pathogenesis is unclear. Using the mouse model of chronic L. (V.) panamensis infection, we examined the hypothesis that Treg functionality contributes to control of pathogenesis. Upon infection, Tregs (CD4(+)Foxp3(+)) presented with a dysregulated phenotype, in that they produced IFN-γ, expressed Tbet, and had a reduced ability to suppress T cell proliferation in vitro. Targeted ablation of Tregs resulted in enlarged lesions, increased parasite load, and enhanced production of IL-17 and IFN-γ, with no change in IL-10 and IL-13 levels. This indicated that an increased inflammatory response was commensurate with disease exacerbation and that the remaining impaired Tregs were important in regulation of disease pathology. Conversely, adoptive transfer of Tregs from naive mice halted disease progression, lowered parasite burden, and reduced cytokine production (IL-10, IL-13, IL-17, IFN-γ). Because Tregs appeared to be important for controlling infection, we hypothesized that their expansion could be used as an immunotherapeutic treatment approach. As a proof of principle, chronically infected mice were treated with rIL-2/anti-IL-2 Ab complex to expand Tregs. Treatment transitorily increased the numbers and percentage of Tregs (draining lymph node, spleen), which resulted in reduced cytokine responses, ameliorated lesions, and reduced parasite load (10(5)-fold). Thus, immunotherapy targeting Tregs could provide an alternate treatment strategy for leishmaniasis caused by Leishmania (Viannia) parasites.

Interplay between DNA repair and inflammation, and the link to cancer.

DNA damage and repair are linked to cancer. DNA damage that is induced endogenously or from exogenous sources has the potential to result in mutations and genomic instability if not properly repaired, eventually leading to cancer. Inflammation is also linked to cancer. Reactive oxygen and nitrogen species (RONs) produced by inflammatory cells at sites of infection can induce DNA damage. RONs can also amplify inflammatory responses, leading to increased DNA damage. Here, we focus on the links between DNA damage, repair, and inflammation, as they relate to cancer. We examine the interplay between chronic inflammation, DNA damage and repair and review recent findings in this rapidly emerging field, including the links between DNA damage and the innate immune system, and the roles of inflammation in altering the microbiome, which subsequently leads to the induction of DNA damage in the colon. Mouse models of defective DNA repair and inflammatory control are extensively reviewed, including treatment of mouse models with pathogens, which leads to DNA damage. The roles of microRNAs in regulating inflammation and DNA repair are discussed. Importantly, DNA repair and inflammation are linked in many important ways, and in some cases balance each other to maintain homeostasis. The failure to repair DNA damage or to control inflammatory responses has the potential to lead to cancer.

Alleviation of rheumatoid arthritis by cell-transducible methotrexate upon transcutaneous delivery.

Rheumatoid arthritis (RA) is a systemic autoimmune disease that is initiated and maintained by various inflammatory/immune cells and their cytokines, leading to cartilage degradation and bone erosion. Despite its potent therapeutic efficacy on RA, the oral administration of methotrexate (MTX) provokes serious adverse systemic complications, thus necessitating the local application of MTX. Here, we show that transcutaneous MTX (TC-MTX) can efficiently penetrate joint skin ex vivo and in vivo, and that TC-MTX can significantly improve the various inflammatory symptoms associated with RA. Further, TC-MTX preserved the joint-structures in mice with collagen-induced arthritis (CIA), which was also confirmed by three-dimensional micro-computed tomography scan. TC-MTX markedly decreased the secretion of inflammatory cytokines both in the serum and in inflamed joints of CIA mice. Further, its therapeutic potential is comparable to that of etanercept, a biological agent that block tumor necrosis factor (TNF)-α. Importantly, the systemic cytotoxicity of TC-MTX was not detected. Thus, TC-MTX can be a new therapeutic modality for RA patients without systemic complications.

IL-17F deficiency inhibits small intestinal tumorigenesis in ApcMin/+ mice.

IL-17 plays an important role in gut homeostasis. However, the role of IL-17F in intestinal tumorigenesis has not been addressed. Here we demonstrate that ablation of IL-17F significantly inhibits spontaneous intestinal tumorigenesis in the small intestine of Apc(Min/+) mice. IL-17F ablation decreased IL-1ß and Cox-2 expression as well as IL-17 receptor C (IL-17RC) expression, which were increased in tumors from Apc(Min/+) mice. Lack of IL-17F did not reverse the splenomegaly but partially restored thymic atrophy, suggesting a local effect of IL-17F in the intestine. IL-17F deficient Apc(Min/+) mice showed a significant decrease in immune cell infiltration in the lamina propria. Interestingly, the expression of IL-17A from CD4 T cells in the lamina propria remains unchanged in the absence of IL-17F. Collectively, our results suggest the proinflammatory and essential role of IL-17F to develop spontaneous intestinal tumorigenesis in Apc(Min/+) mice in the presence of IL-17A.

Neuropilin-1 attenuates autoreactivity in experimental autoimmune encephalomyelitis.

Neuropilin-1 (Nrp1) is a cell surface molecule originally identified for its role in neuronal development. Recently, Nrp1 has been implicated in several aspects of immune function including maintenance of the immune synapse and development of regulatory T (T(reg)) cells. In this study, we provide evidence for a central role of Nrp1 in the regulation of CD4 T-cell immune responses in experimental autoimmune encephalitis (EAE). EAE serves as an animal model for the central nervous system (CNS) inflammatory disorder multiple sclerosis (MS). EAE is mediated primarily by CD4(+) T cells that migrate to the CNS and mount an inflammatory attack against myelin components, resulting in CNS pathology. Using a tissue-specific deletion system, we observed that the lack of Nrp1 on CD4(+) T cells results in increased EAE severity. These conditional knockout mice exhibit preferential T(H)-17 lineage commitment and decreased T(reg)-cell functionality. Conversely, CD4(+) T cells expressing Nrp1 suppress effector T-cell proliferation and cytokine production both in vivo and in vitro independent of T(reg) cells. Nrp1-mediated suppression can be inhibited by TGF-ß blockade but not by IL-10 blockade. These results suggest that Nrp1 is essential for proper maintenance of peripheral tolerance and its absence can result in unchecked autoreactive responses, leading to diseases like EAE and potentially MS.

Cell-permeable Foxp3 protein alleviates autoimmune disease associated with inflammatory bowel disease and allergic airway inflammation.

Foxp3 is a key transcription factor for differentiation and function of regulatory T (Treg) cells that is critical for maintaining immunological self-tolerance. Therefore, increasing Treg function by Foxp3 transduction to regulate an inflammatory immune response is an important goal for the treatment of autoimmune and allergic diseases. Here we have generated a cell-permeable Foxp3 protein by fusion with the unique human HHph-1-PTD (protein transduction domain), examined its regulatory function in T cells, and characterized its therapeutic effect in autoimmune and allergic disease models. HHph-1-Foxp3 was rapidly and effectively transduced into cells within 30 min and conferred suppressor function to CD4(+)CD25(-) T cells as well as directly inhibiting T-cell activation and proliferation. Systemic delivery of HHph-1 Foxp3 remarkably inhibited the autoimmune symptoms of scurfy mice and the development of colitis induced by scurfy or wild-type CD4 T cells. Moreover, intranasal delivery of HHph-1-Foxp3 strongly suppressed ovalbumin-induced allergic airway inflammation. These results demonstrate the clinical potential of the cell-permeable recombinant HHph-1-Foxp3 protein in autoimmune and hypersensitive allergic diseases.