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.

Colon cancer cells acquire immune regulatory molecules from tumor-infiltrating lymphocytes by trogocytosis.

Cancer cells can develop an immunosuppressive tumor microenvironment to control tumor-infiltrating lymphocytes. The underlying mechanisms still remain unclear. Here, we report that mouse and human colon cancer cells acquire lymphocyte membrane proteins including cellular markers such as CD4 and CD45. We observed cell populations harboring both a tumor-specific marker and CD4 in the tumor microenvironment. Sorted cells from these populations were capable of forming organoids, identifying them as cancer cells. Live imaging analysis revealed that lymphocyte membrane proteins were transferred to cancer cells via trogocytosis. As a result of the transfer in vivo, cancer cells also acquired immune regulatory surface proteins such as CTLA4 and Tim3, which suppress activation of immune cells [T. L. Walunas et al, Immunity 1, 405-413 (1994) and L. Monney et al., Nature 415, 536-541 (2002)]. RNA sequencing analysis of ex vivo-cocultured splenocytes with trogocytic cancer cells showed reductions in Th1 activation and natural killer cell signaling pathways compared with the nontrogocytic control. Cancer cell trogocytosis was confirmed in the patient-derived xenograft models of colorectal cancer and head and neck cancer. These findings suggest that cancer cells utilize membrane proteins expressed in lymphocytes, which in turn contribute to the development of the immunosuppressive tumor microenvironment.

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.

Functional Diversity of Myeloid-Derived Suppressor Cells: The Multitasking Hydra of Cancer.

Myeloid-derived suppressor cells (MDSCs) are immature suppressive cells found in tumors and immunological niches. In this article, we highlight the ability of MDSCs to promote IL-17-producing T cells (Th17) and regulatory T cells in addition to suppressing cytotoxic T cells in different tumor models. These interactions between MDSCs and T cells support tumor growth because IL-17 is tumorigenic in many cancer types and regulatory T cells suppress antitumor T cells. Besides T cells, MDSCs promote regulatory B cells and suppress overall B cell function; however, tumor-evoked regulatory B cells also regulate MDSC function, suggesting cross-regulation between MDSCs and B cells. These multiple functions shed light on how MDSCs dysregulate several arms of host immune response. Moreover, MDSCs promote tumor cell survival and angiogenesis to support tumors. Therefore, the multifunctional feature of MDSCs make them attractive immunotherapeutic targets.

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.

Intranuclear delivery of the transcription modulation domain of Tbet-improved lupus nephritis in (NZB/NZW) F1 lupus-prone mice.

Excessive expression of Tbet and IFNγ is evidence of systemic lupus erythematosus (SLE) in lupus patients. In this study, the nucleus-transducible form of Transcription Modulation Domain (TMD) of Tbet (ntTbet-TMD), which is a fusion protein between Protein Transduction Domain Hph-1 (Hph-1-PTD) and the TMD of Tbet comprising DNA binding domain and isotype-specific domain, was generated to inhibit Tbet-mediated transcription in the interactomic manner. ntTbet-TMD was effectively delivered into the nucleus of the cells and specifically inhibited Tbet-mediated transcription without influencing the differentiation of other T cell subsets and signaling events for T cell activation. The severity of nephritis was significantly reduced by ntTbet-TMD as effectively as methylprednisolone in lupus-prone mice. The number of Th1, Th2 or Th17 cells and the secretion of their cytokines substantially decreased in the spleen and kidney of lupus-prone mice by ntTbet-TMD treatment. In contrast to methylprednisolone, the marked increase of Treg cells and the secretion of their immunosuppressive cytokine were detected in the spleen of (NZB/NZW) F1 mice treated with ntTbet-TMD. Thus, ntTbet-TMD can improve nephritis in lupus-prone mice by modulating the overall proinflammatory microenvironment and rebalancing T cell subsets, leading to new immune therapeutics for Th1-mediated autoimmune diseases.

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.

RORγt-specific transcriptional interactomic inhibition suppresses autoimmunity associated with TH17 cells.

The nuclear hormone receptor retinoic acid-related orphan receptor gamma t (RORγt) is a transcription factor (TF) specific to TH17 cells that produce interleukin (IL)-17 and have been implicated in a wide range of autoimmunity. Here, we developed a novel therapeutic strategy to modulate the functions of RORγt using cell-transducible form of transcription modulation domain of RORγt (tRORγt-TMD), which can be delivered effectively into the nucleus of cells and into the central nerve system (CNS). tRORγt-TMD specifically inhibited TH17-related cytokines induced by RORγt, thereby suppressing the differentiation of naïve T cells into TH17, but not into TH1, TH2, or Treg cells. tRORγt-TMD injected into experimental autoimmune encephalomyelitis (EAE) animal model can be delivered effectively in the splenic CD4(+) T cells and spinal cord-infiltrating CD4(+) T cells, and suppress the functions of TH17 cells. The clinical severity and incidence of EAE were ameliorated by tRORγt-TMD in preventive and therapeutic manner, and significant reduction of both infiltrating CD4(+) IL-17(+) T cells and inflammatory cells into the CNS was observed. As a result, the number of spinal cord demyelination was also reduced after tRORγt-TMD treatment. With the same proof of concept, tTbet-TMD specifically blocking TH1 differentiation improved the clinical incidence of rheumatoid arthritis (RA). Therefore, tRORγt-TMD and tTbet-TMD can be novel therapeutic reagents with the natural specificity for the treatment of inflammatory diseases associated with TH17 or TH1. This strategy can be applied to treat various diseases where a specific transcription factor has a key role in pathogenesis.

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.

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.

Sex-Based Selectivity of PPARγ Regulation in Th1, Th2, and Th17 Differentiation.

Peroxisome proliferator-activated receptor gamma (PPARγ) has recently been recognized to regulate adaptive immunity through Th17 differentiation, Treg functions, and TFH responses. However, its role in adaptive immunity and autoimmune disease is still not clear, possibly due to sexual differences. Here, we investigated in vitro treatment study with the PPARγ agonist pioglitazone to compare Th1, Th2, and Th17 differentiation in male and female mouse splenic T cells. Pioglitazone treatment significantly inhibited various effector T cell differentiations including Th1, Th2, and Th17 cells from female naïve T cells, but it selectively reduced IL-17 production in male Th17 differentiation. Interestingly, pioglitazone and estradiol (E2) co-treatment of T cells in males inhibited differentiation of Th1, Th2, and Th17 cells, suggesting a mechanism for the greater sensitivity of PPARγ to ligand treatment in the regulation of effector T cell differentiation in females. Collectively, these results demonstrate that PPARγ selectively inhibits Th17 differentiation only in male T cells and modulates Th1, Th2, and Th17 differentiation in female T cells based on different level of estrogen exposure. Accordingly, PPARγ could be an important immune regulator of sexual differences in adaptive immunity.

Intranuclear interactomic inhibition of FoxP3 suppresses functions of Treg cells.

Regulatory T cells (Treg cells) are crucial for the maintenance of immunological tolerance, and it has been reported that Treg cells are enriched within the tumor micro-environment for immune evasion due to their immunosuppressive functions. To inhibit Treg cells functions, FoxP3, a lineage-specific transcription factor responsible for the differentiation and functions of Treg cells, was functionally targeted by a nucleus-transducible (nt) form of various FoxP3 functional subdomains. These nt modified domains can be delivered into the nucleus effectively and work as interactomic inhibitors via disruption of the endogenous FoxP3-mediated transcription complex. Among these domains, nt-FoxP3-FKH (Forkhead DNA binding domain) is most effective at restoring NFAT activity suppressed by FoxP3, and inhibiting the binding of endogenous FKH-containing proteins to FKH DNA binding sequences without influencing the viability and activation of T cells. The suppressive functions of TGF-ß-induced iTreg cells and thymus-derived tTreg cells were substantially blocked by nt-FoxP3-FKH, accompanied with down-regulation of CTLA-4 surface expression and IL-10 secretion of Treg cells. In addition, nt-FoxP3-FKH upregulated the expression of IL-2 and IFN-γ in Treg cells. Therefore, nt-FoxP3-FKH has the potential to be a novel therapeutic agent to modulate the immune-evasive tumor environment created by Treg cells without the need for genetic modifications.

Renal cancer cells acquire immune surface protein through trogocytosis and horizontal gene transfer.

Trogocytosis is an underappreciated phenomenon that shapes the immune microenvironment surrounding many types of solid tumors. The consequences of membrane-bound proteins being deposited from a donor immune cell to a recipient cancer cell via trogocytosis are still unclear. Here, we report that human clear cell renal carcinoma tumors stably express the lymphoid markers CD45, CD56, CD14, and CD16. Flow cytometry performed on fresh kidney tumors revealed consistent CD45 expression on tumor cells, as well as varying levels of the other markers mentioned previously. These results were consistent with our immunofluorescent analysis, which also revealed colocalization of lymphoid markers with carbonic anhydrase 9 (CAIX), a standard kidney tumor marker. RNA analysis showed a significant upregulation of genes typically associated with immune cells in tumor cells following trogocytosis. Finally, we show evidence of chromosomal DNA being transferred from immune cells to tumor cells during trogocytosis. This horizontal gene transfer has transcriptional consequences in the recipient tumor cell, resulting in a fusion phenotype that expressed both immune and cancer specific proteins. This work demonstrates a novel mechanism by which tumor cell protein expression is altered through the acquisition of surface membrane fragments and genomic DNA from infiltrating lymphocytes. These results alter the way in which we understand tumor-immune cell interactions and may reveal new insights into the mechanisms by which tumors develop. Additionally, further studies into trogocytosis will help push the field towards the next generation of immunotherapies and biomarkers for treating renal cell carcinoma and other types of cancers. SIGNIFICANCE STATEMENT: We have identified trogocytosis as a mechanism by which human clear cell renal carcinoma tumors acquire lymphocyte surface protein expression from tumor infiltrating immune cells. In addition to the transfer of membrane fragments, we have provided evidence to show that genomic DNA is transferred from a normal immune cell to a tumor cell during trogocytosis. This process alters the transcriptome of cancer cells such that they express significantly more mRNA for immune proteins such as the lymphocyte marker CD45 compared to tumor cells that have not undergone trogocytosis. This study provides an in-depth analysis of the interactions between cancer cells and tumor infiltrating lymphocytes, and how these interactions alter the development of human tumors.

Triple Negative Breast Cancer Cells Acquire Lymphocyte Proteins and Genomic DNA During Trogocytosis with T Cells.

Trogocytosis is the process by which a recipient cell siphons small membrane fragments and proteins from a donor cell and may be utilized by cancer cells to avoid immune detection. We observed lymphocyte specific protein expressed by TNBC cells via immunofluorescence imaging of patient samples. Image analysis of CD45RA expression, a T cell specific protein, revealed that all stages of TNBCs express CD45RA. Flow cytometry revealed TNBC cells trogocytose CD45 protein from T cells. We also showed that the acquisition of these lymphoid markers is contact dependent. Confocal and super-resolution imaging further revealed CD45 + spherical structures containing T cell genomic DNA inside TNBC cells after co-culture. Trogocytosis between T cells and TNBC cells altered cancer cell gene expression. Our results revealed that CD45 is obtained by TNBC cells from T cells via trogocytosis and that TNBC cells express CD45 intracellularly and on the membrane. Teaser: TNBC cells acquire small spherical structures from T cells containing lymphocyte-specific membrane proteins and genomic DNA.

Metastasis of colon cancer requires Dickkopf-2 to generate cancer cells with Paneth cell properties.

Metastasis is the leading cause of cancer-related mortality. Paneth cells provide stem cell niche factors in homeostatic conditions, but the underlying mechanisms of cancer stem cell niche development are unclear. Here we report that Dickkopf-2 (DKK2) is essential for the generation of cancer cells with Paneth cell properties during colon cancer metastasis. Splenic injection of Dkk2-knockout (KO) cancer organoids into C57BL/6 mice resulted in a significant reduction of liver metastases. Transcriptome analysis showed reduction of Paneth cell markers such as lysozymes in KO organoids. Single cell RNA sequencing analyses of murine metastasized colon cancer cells and patient samples identified the presence of lysozyme positive cells with Paneth cell properties including enhanced glycolysis. Further analyses of transcriptome and chromatin accessibility suggested Hepatocyte nuclear factor 4-alpha (HNF4A) as a downstream target of DKK2. Chromatin immunoprecipitation followed by sequencing analysis revealed that HNF4A binds to the promoter region of Sox9, a well-known transcription factor for Paneth cell differentiation. In the liver metastatic foci, DKK2 knockout rescued HNF4A protein levels followed by reduction of lysozyme positive cancer cells. Taken together, DKK2-mediated reduction of HNF4A protein promotes the generation of lysozyme positive cancer cells with Paneth cell properties in the metastasized colon cancers.

Neuroprotection against neuroblastoma cell death induced by depletion of mitochondrial glutathione.

Mitochondrial glutathione pool is vital in protecting cells against oxidative stress as the majority of the cellular reactive oxygen species are generated in mitochondria. Oxidative stress is implicated as a causative factor in neuronal death in neurodegenerative disorders. We hypothesized that depletion of mitochondrial glutathione leads to mitochondrial dysfunction and apoptotic death of SK-N-SH (human neuroblastoma) cells and investigated the neuroprotective strategies against GSH depletion. SK-N-SH cells were treated with two distinct inhibitors of glutathione metabolism: L-buthionine-(S, R)-sulfoximine (BSO) and ethacrynic acid (EA). EA treatment caused depletion of both the total and mitochondrial glutathione (while BSO had no effect on mitochondrial glutathione), enhanced rotenone-induced ROS production, and reduced the viability of SK-N-SH cells. Glutathione depletion by BSO or EA demonstrated positive features of mitochondria-mediated apoptosis in neuroblastoma cell death. Prevention of apoptosis by Bcl2 overexpression or use of antioxidant ebselen did not confer neuroprotection. Co-culture with U-87 (human glioblastoma) cells protected SK-N-SH cells from the cell death. Our data suggest that depletion of mitochondrial glutathione leads to mitochondrial dysfunction and apoptosis. The study indicates that preventing mitochondrial glutathione depletion could become a novel strategy for the development of neuroprotective therapeutics in neurodegenerative disorders.

Reperfusion injury intensifies the adaptive human T cell alloresponse in a human-mouse chimeric artery model.

OBJECTIVE: Perioperative nonimmune injuries to an allograft can decrease graft survival. We have developed a model for studying this process using human materials. METHODS AND RESULTS: Human artery segments were transplanted as infrarenal aortic interposition grafts into an immunodeficient mouse host, allowed to "heal in" for 30 days, and then retransplanted into a second mouse host. To induce a reperfusion injury, the healed-in artery segments were incubated for 3 hours under hypoxic conditions ex vivo before retransplantation. To induce immunologic rejection, the animals receiving the retransplanted artery segment were adoptively transferred with human peripheral blood mononuclear cells or purified T cells from a donor allogeneic to the artery 1 week before surgery. To compare rejection of injured versus healthy tissues, these manipulations were combined. Results were analyzed ex vivo by histology, morphometry, immunohistochemistry, and mRNA quantitation or in vivo by ultrasound. Our results showed that reperfusion injury, which otherwise heals with minimal sequelae, intensifies the degree of allogeneic T cell-mediated injury to human artery segments. CONCLUSIONS: We developed a new human-mouse chimeric model demonstrating interactions of reperfusion injury and alloimmunity using human cells and tissues that may be adapted to study other forms of nonimmune injury and other types of adaptive immune responses.

Ablation of IL-17A abrogates progression of spontaneous intestinal tumorigenesis.

The intrinsic role of endogenous IL-17A in spontaneous intestinal tumorigenesis has not been addressed previously to our knowledge. Ablation of IL-17A significantly reduced tumor development in mice bearing a heterozygote mutation in the adenomatous polyposis coli (APC) gene (Apc(Min/+) mice). There was also a decrease in inflammatory cytokines and proinflammatory mediators, reduced infiltration of lymphocytes including T cells, and preservation of intestinal architecture and the presence of APC protein in intestinal epithelial cells. Interestingly, IL-17A ablation also corrected immunological abnormalities such as splenomegaly and thymic atrophy in Apc(Min/+) mice. CD4 T cells from Apc(Min/+) mice showed hyperproliferative potential in vitro and in vivo and increased levels of IL-17A and IL-10. The effector CD4 T cells from Apc(Min/+) mice were more resistant to regulatory T cell-mediated suppression. Finally, these CD4 T cells induced colitis in immunodeficient mice upon adoptive transfer, whereas the ablation of IL-17A in CD4 T cells in Apc(Min/+) mice completely abolished this pathogenic potential in vivo. Taken together, our results show that CD4 T cell-derived IL-17A promotes spontaneous intestinal tumorigenesis with altered functions of CD4 T cells in Apc(Min/+) mice.

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.

Leishmania major-derived lipophosphoglycan influences the host's early immune response by inducing platelet activation and DKK1 production via TLR1/2.

Background: Platelets are rapidly deployed to infection sites and respond to pathogenic molecules via pattern recognition receptors (TLR, NLRP). Dickkopf1 (DKK1) is a quintessential Wnt antagonist produced by a variety of cell types including platelets, endothelial cells, and is known to modulate pro-inflammatory responses in infectious diseases and cancer. Moreover, DKK1 is critical for forming leukocyte-platelet aggregates and induction of type 2 cell-mediated immune responses. Our previous publication showed activated platelets release DKK1 following Leishmania major recognition. Results: Here we probed the role of the key surface virulence glycoconjugate lipophosphoglycan (LPG), on DKK1 production using null mutants deficient in LPG synthesis (Δlpg1- and Δlpg2-). Leishmania-induced DKK1 production was reduced to control levels in the absence of LPG in both mutants and was restored upon re-expression of the cognate LPG1 or LPG2 genes. Furthermore, the formation of leukocyte-platelet aggregates was dependent on LPG. LPG mediated platelet activation and DKK1 production occurs through TLR1/2. Conclusion: Thus, LPG is a key virulence factor that induces DKK1 production from activated platelets, and the circulating DKK1 promotes Th2 cell polarization. This suggests that LPG-activated platelets can drive innate and adaptive immune responses to Leishmania infection.