Oral administration of Manuka honey induces IFNγ-dependent resistance to tumor growth that correlates with beneficial modulation of gut microbiota composition.

Background: To investigate the potential of Manuka honey (MH) as an immunomodulatory agent in colorectal cancer (CRC) and dissect the underlying molecular and cellular mechanisms. Methods: MH was administered orally over a 4 week-period. The effect of MH treatment on microbiota composition was studied using 16S rRNA sequencing of fecal pellets collected before and after treatment. Pretreated mice were implanted with CRC cells and followed for tumor growth. Tumors and lymphoid organs were analyzed by flow cytometry (FACS), immunohistochemistry and qRT-PCR. Efficacy of MH was also assessed in a therapeutic setting, with oral treatment initiated after tumor implantation. We utilized IFNγ-deficient mice to determine the importance of interferon signaling in MH-induced immunomodulation. Results: Pretreatment with MH enhanced anti-tumor responses leading to suppression of tumor growth. Evidence for enhanced tumor immunogenicity included upregulated MHC class-II on intratumoral macrophages, enhanced MHC class-I expression on tumor cells and increased infiltration of effector T cells into the tumor microenvironment. Importantly, oral MH was also effective in retarding tumor growth when given therapeutically. Transcriptomic analysis of tumor tissue highlighted changes in the expression of various chemokines and inflammatory cytokines that drive the observed changes in tumor immunogenicity. The immunomodulatory capacity of MH was abrogated in IFNγ-deficient mice. Finally, bacterial 16S rRNA sequencing demonstrated that oral MH treatment induced unique changes in gut microbiota that may well underlie the IFN-dependent enhancement in tumor immunogenicity. Conclusion: Our findings highlight the immunostimulatory properties of MH and demonstrate its potential utilization in cancer prevention and treatment.

Characterization of immunomodulatory responses induced by manuka honey.

Manuka honey (MH) is known for its wound-healing, anti-microbial, anti-oxidant and anti-tumor properties. However, there is conflicting evidence regarding the role of MH in inflammatory responses, with some studies highlighting its pro-inflammatory capacity and others showing that it has a predominantly anti-inflammatory activity. The current study is aimed at characterizing the immunomodulatory capacity of MH using both in vitro and in vivo approaches, focusing on the underlying mechanisms. Treatment of RAW 264.7 macrophages with 1% MH (w/v) resulted in a significant increase in the gene expression (~26-fold) and secretion (~27-fold) of tumor necrosis factor-alpha (TNF-α). Similarly, an increase was observed in the gene expression of other inflammatory cytokines including interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS), as well as the chemokines; (C-X-C motif) ligand 2 (CXCL2) and (C-C) motif ligand 2 (CCL2). Using an in vivo model, intraperitoneal (i.p.) administration of MH in C57BL/6 mice elicited a peritoneal response characterized by a significant expansion in the number of peritoneal exudate cells (PECs), which was mainly due to a 35-fold increase in the recruitment of neutrophils. Importantly, this response was evident in toll-like receptor 4 (TLR4)-defective C3H/HeJ mice, indicating that the observed stimulatory effect occurs independently of TLR4 and unlikely to be mediated by any lipopolysaccharide (LPS) contaminant. MH administration also led to changes in the phenotypic expression and functional maturation of peritoneal macrophages, as evidenced by a shift towards the CD11blo F4/80lo phenotype and an increase in the expression of major histocompatibility complex (MHC) class II proteins. In contrast, the MH-initiated peritoneal response was largely abrogated in mice deficient in myeloid differentiation primary response 88 (MyD88) protein, a critical adaptor of most TLR signaling pathways. Thus, the current findings help to characterize the immunostimulatory properties of MH and their dependence on TLR signaling, and highlight the potential utility of MH as an immunomodulatory agent in a variety of disorders.

Attenuated Salmonella potentiate PD-L1 blockade immunotherapy in a preclinical model of colorectal cancer.

The use of immune checkpoint inhibitors to treat cancer resulted in unprecedented and durable clinical benefits. However, the response rate among patients remains rather modest. Previous work from our laboratory demonstrated the efficacy of using attenuated bacteria as immunomodulatory anti-cancer agents. The current study investigated the potential of utilizing a low dose of attenuated Salmonella typhimurium to enhance the efficacy of PD-L1 blockade in a relatively immunogenic model of colon cancer. The response of MC38 tumors to treatment with αPD-L1 monoclonal antibody (mAb) was variable, with only 30% of the mice being responsive. Combined treatment with αPD-L1 mAb and Salmonella resulted in 75% inhibition of tumor growth in 100% of animals. Mechanistically, the enhanced response correlated with a decrease in the percentage of tumor-associated granulocytic cells, upregulation in MHC class II expression by intratumoral monocytes and an increase in tumor infiltration by effector T cells. Collectively, these alterations resulted in improved anti-tumor effector responses and increased apoptosis within the tumor. Thus, our study demonstrates that a novel combination treatment utilizing attenuated Salmonella and αPD-L1 mAb could improve the outcome of immunotherapy in colorectal cancer.

CD40L modulates transcriptional signatures of neutrophils in the bone marrow associated with development and trafficking.

Neutrophils are produced in the BM in a process called granulopoiesis, in which progenitor cells sequentially develop into mature neutrophils. During the developmental process, which is finely regulated by distinct transcription factors, neutrophils acquire the ability to exit the BM, properly distribute throughout the body, and migrate to infection sites. Previous studies have demonstrated that CD40 ligand (CD40L) influences hematopoiesis and granulopoiesis. Here, we investigate the effect of CD40L on neutrophil development and trafficking by performing functional and transcriptome analyses. We found that CD40L signaling plays an essential role in the early stages of neutrophil generation and development in the BM. Moreover, CD40L modulates transcriptional signatures, indicating that this molecule enables neutrophils to traffic throughout the body and to migrate in response to inflammatory signals. Thus, our study provides insights into the complex relationships between CD40L signaling and granulopoiesis, and it suggests a potentially novel and nonredundant role of CD40L signaling in neutrophil development and function.

Integration of Salmonella into Combination Cancer Therapy.

Current modalities of cancer treatment have limitations related to poor target selectivity, resistance to treatment, and low response rates in patients. Accumulating evidence over the past few decades has demonstrated the capacity of several strains of bacteria to exert anti-tumor activities. Salmonella is the most extensively studied entity in bacterial-mediated cancer therapy, and has a good potential to induce direct tumor cell killing and manipulate the immune components of the tumor microenvironment in favor of tumor inhibition. In addition, Salmonella possesses some advantages over other approaches of cancer therapy, including high tumor specificity, deep tissue penetration, and engineering plasticity. These aspects underscore the potential of utilizing Salmonella in combination with other cancer therapeutics to improve treatment effectiveness. Herein, we describe the advantages that make Salmonella a good candidate for combination cancer therapy and summarize the findings of representative studies that aimed to investigate the therapeutic outcome of combination therapies involving Salmonella. We also highlight issues associated with their application in clinical use.

The Immunomodulatory Effects of Honey and Associated Flavonoids in Cancer.

Honey has exerted a high impact in the field of alternative medicine over many centuries. In addition to its wound healing, anti-microbial and antioxidant properties, several lines of evidence have highlighted the efficiency of honey and associated bioactive constituents as anti-tumor agents against a range of cancer types. Mechanistically, honey was shown to inhibit cancer cell growth through its pro-apoptotic, anti-proliferative and anti-metastatic effects. However, the potential of honey to regulate anti-tumor immune responses is relatively unexplored. A small number of in vitro and in vivo studies have demonstrated the ability of honey to modulate the immune system by inducing immunostimulatory as well as anti-inflammatory effects. In the present review, we summarize the findings from different studies that aimed to investigate the immunomodulatory properties of honey and its flavonoid components in relation to cancer. While these studies provide promising data, additional research is needed to further elucidate the immunomodulatory properties of honey, and to enable its utilization as an adjuvant therapy in cancer.

Ambrisentan, an endothelin receptor type A-selective antagonist, inhibits cancer cell migration, invasion, and metastasis.

Several studies reported a central role of the endothelin type A receptor (ETAR) in tumor progression leading to the formation of metastasis. Here, we investigated the in vitro and in vivo anti-tumor effects of the FDA-approved ETAR antagonist, Ambrisentan, which is currently used to treat patients with pulmonary arterial hypertension. In vitro, Ambrisentan inhibited both spontaneous and induced migration/invasion capacity of different tumor cells (COLO-357 metastatic pancreatic adenocarcinoma, OvCar3 ovarian carcinoma, MDA-MB-231 breast adenocarcinoma, and HL-60 promyelocytic leukemia). Whole transcriptome analysis using RNAseq indicated Ambrisentan's inhibitory effects on the whole transcriptome of resting and PAR2-activated COLO-357 cells, which tended to normalize to an unstimulated profile. Finally, in a pre-clinical murine model of metastatic breast cancer, treatment with Ambrisentan was effective in decreasing metastasis into the lungs and liver. Importantly, this was associated with a significant enhancement in animal survival. Taken together, our work suggests a new therapeutic application for Ambrisentan in the treatment of cancer metastasis.

Therapeutic and preventive properties of honey and its bioactive compounds in cancer: an evidence-based review.

Despite the much improved therapeutic approaches for cancer treatment that have been developed over the past 50 years, cancer remains a major cause of mortality globally. Considerable epidemiological and experimental evidence has demonstrated an association between ingestion of food and nutrients with either an increased risk for cancer or its prevention. There is rising interest in exploring agents derived from natural products for chemoprevention or for therapeutic purposes. Honey is rich in nutritional and non-nutritional bioactive compounds, as well as in natural antioxidants, and its potential beneficial function in human health is becoming more evident. A large number of studies have addressed the anti-cancer effects of different types of honey and their phenolic compounds using in vitro and in vivo cancer models. The reported findings affirm that honey is an agent able to modulate oxidative stress and has anti-proliferative, pro-apoptotic, anti-inflammatory, immune-modulatory and anti-metastatic properties. However, despite its reported anti-cancer activities, very few clinical studies have been undertaken. In the present review, we summarise the findings from different experimental approaches, including in vitro cell cultures, preclinical animal models and clinical studies, and provide an overview of the bioactive profile and bioavailability of the most commonly studied honey types, with special emphasis on the chemopreventive and therapeutic properties of honey and its major phenolic compounds in cancer. The implications of these findings as well as the future prospects of utilising honey to fight cancer will be discussed.

Inhibition of Tyrosine-Phosphorylated STAT3 in Human Breast and Lung Cancer Cells by Manuka Honey is Mediated by Selective Antagonism of the IL-6 Receptor.

Aberrantly high levels of tyrosine-phosphorylated signal transducer and activator of transcription 3 (p-STAT3) are found constitutively in ~50% of human lung and breast cancers, acting as an oncogenic transcription factor. We previously demonstrated that Manuka honey (MH) inhibits p-STAT3 in breast cancer cells, but the exact mechanism remained unknown. Herein, we show that MH-mediated inhibition of p-STAT3 in breast (MDA-MB-231) and lung (A549) cancer cell lines is accompanied by decreased levels of gp130 and p-JAK2, two upstream components of the IL-6 receptor (IL-6R) signaling pathway. Using an ELISA-based assay, we demonstrate that MH binds directly to IL-6Rα, significantly inhibiting (~60%) its binding to the IL-6 ligand. Importantly, no evidence of MH binding to two other cytokine receptors, IL-11Rα and IL-8R, was found. Moreover, MH did not alter the levels of tyrosine-phosphorylated or total Src family kinases, which are also constitutively activated in cancer cells, suggesting that signaling via other growth factor receptors is unaffected by MH. Binding of five major MH flavonoids (luteolin, quercetin, galangin, pinocembrin, and chrysin) was also tested, and all but pinocembrin could demonstrably bind IL-6Rα, partially (30-35%) blocking IL-6 binding at the highest concentration (50 µM) used. In agreement, each flavonoid inhibited p-STAT3 in a dose-dependent manner, with estimated IC50 values in the 3.5-70 µM range. Finally, docking analysis confirmed the capacity of each flavonoid to bind in an energetically favorable configuration to IL-6Rα at a site predicted to interfere with ligand binding. Taken together, our findings identify IL-6Rα as a direct target of MH and its flavonoids, highlighting IL-6R blockade as a mechanism for the anti-tumor activity of MH, as well as a viable therapeutic target in IL-6-dependent cancers.

CD40 ligand deficiency: treatment strategies and novel therapeutic perspectives.

INTRODUCTION: CD40 ligand (CD40L) deficiency or X-linked Hyper-IgM syndrome is a severe primary immunodeficiency caused by mutations in the CD40L gene. Despite currently available treatments, CD40L-deficient patients remain susceptible to life-threatening infections and have poor long term survival. Areas covered: Here, we discuss clinical and immunological characteristics of CD40L deficiency as well as current therapeutic strategies used for patient management. This review highlights that beyond B cell defects, patients' susceptibility to opportunistic pathogens might be due to impaired T cell and innate immune responses. In this context, we discuss how better knowledge of CD40L function and regulation may result in the development of new treatments. Expert opinion: Despite the introduction of hematopoietic stem-cell transplantation, immunoglobulin replacement, granulocyte colony-stimulating factor (G-CSF) administration, and prophylactic antibiotic therapies, life-threatening infections still cause high morbidity and mortality among CD40L-deficient patients. The reasons for this inadequate response to current therapies remains poorly understood, but recent reports suggest the involvement of CD40L-CD40 interaction in early stages of the innate immune system ontogeny. The development of novel gene therapeutic approaches and the use of redirected immunotherapies represent alternative treatment methods that could offer reduced morbidity and mortality rates for patients with CD40L deficiency.

GPCR-specific autoantibody signatures are associated with physiological and pathological immune homeostasis.

Autoantibodies have been associated with autoimmune diseases. However, studies have identified autoantibodies in healthy donors (HD) who do not develop autoimmune disorders. Here we provide evidence of a network of immunoglobulin G (IgG) autoantibodies targeting G protein-coupled receptors (GPCR) in HD compared to patients with systemic sclerosis, Alzheimer's disease, and ovarian cancer. Sex, age and pathological conditions affect autoantibody correlation and hierarchical clustering signatures, yet many of the correlations are shared across all groups, indicating alterations to homeostasis. Furthermore, we identify relationships between autoantibodies targeting structurally and functionally related molecules, such as vascular, neuronal or chemokine receptors. Finally, autoantibodies targeting the endothelin receptor type A (EDNRA) exhibit chemotactic activity, as demonstrated by neutrophil migration toward HD-IgG in an EDNRA-dependent manner and in the direction of IgG from EDNRA-immunized mice. Our data characterizing the in vivo signatures of anti-GPCR autoantibodies thus suggest that they are a physiological part of the immune system.

Attenuated Bacteria as Immunotherapeutic Tools for Cancer Treatment.

The use of attenuated bacteria as cancer therapeutic tools has garnered increasing scientific interest over the past 10 years. This is largely due to the development of bacterial strains that maintain good anti-tumor efficacy, but with reduced potential to cause toxicities to the host. Because of its ability to replicate in viable as well as necrotic tissue, cancer therapy using attenuated strains of facultative anaerobic bacteria, such as Salmonella, has several advantages over standard treatment modalities, including chemotherapy and radiotherapy. Despite some findings suggesting that it may operate through a direct cytotoxic effect against cancer cells, there is accumulating evidence demonstrating that bacterial therapy acts by modulating cells of the immune system to counter the growth of the tumor. Herein, we review the experimental evidence underlying the success of bacterial immunotherapy against cancer and highlight the cellular and molecular alterations in the peripheral immune system and within the tumor microenvironment that have been reported following different forms of bacterial therapy. Our improved understanding of these mechanisms should greatly aid in the translational application of bacterial therapy to cancer patients.

CD40 ligand deficiency causes functional defects of peripheral neutrophils that are improved by exogenous IFN-γ.

BACKGROUND: Patients with X-linked hyper-IgM syndrome caused by CD40 ligand (CD40L) deficiency often present with episodic, cyclic, or chronic neutropenia, suggesting abnormal neutrophil development in the absence of CD40L-CD40 interaction. However, even when not neutropenic and despite immunoglobulin replacement therapy, CD40L-deficient patients are susceptible to life-threatening infections caused by opportunistic pathogens, suggesting impaired phagocyte function and the need for novel therapeutic approaches. OBJECTIVES: We sought to analyze whether peripheral neutrophils from CD40L-deficient patients display functional defects and to explore the in vitro effects of recombinant human IFN-γ (rhIFN-γ) on neutrophil function. METHODS: We investigated the microbicidal activity, respiratory burst, and transcriptome profile of neutrophils from CD40L-deficient patients. In addition, we evaluated whether the lack of CD40L in mice also affects neutrophil function. RESULTS: Neutrophils from CD40L-deficient patients exhibited defective respiratory burst and microbicidal activity, which were improved in vitro by rhIFN-γ but not soluble CD40L. Moreover, neutrophils from patients showed reduced CD16 protein expression and a dysregulated transcriptome suggestive of impaired differentiation. Similar to CD40L-deficient patients, CD40L knockout mice were found to have impaired neutrophil responses. In parallel, we demonstrated that soluble CD40L induces the promyelocytic cell line HL-60 to proliferate and mature by regulating the expression of genes of the same Gene Ontology categories (eg, cell differentiation) when compared with those dysregulated in peripheral blood neutrophils from CD40L-deficient patients. CONCLUSION: Our data suggest a nonredundant role of CD40L-CD40 interaction in neutrophil development and function that could be improved in vitro by rhIFN-γ, indicating a potential novel therapeutic application for this cytokine.

The IL-6/STAT3 Signaling Pathway Is an Early Target of Manuka Honey-Induced Suppression of Human Breast Cancer Cells.

There is renewed interest in the potential use of natural compounds in cancer therapy. Previously, we demonstrated the anti-tumor properties of manuka honey (MH) against several cancers. However, the underlying mechanism and molecular targets of this activity remain unknown. For this study, the early targets of MH and its modulatory effects on proliferation, invasiveness, and angiogenic potential were investigated using two human breast cancer cell lines, the triple-negative MDA-MB-231 cells and estrogen receptor-positive MCF-7 cells, and the non-neoplastic breast epithelial MCF-10A cell line. Exposure to MH at concentrations of 0.3-1.25% (w/v) induced a dose-dependent inhibition of the proliferation of MDA-MB-231 and MCF-7, but not MCF-10A, cells. This inhibition was independent of the sugar content of MH as a solution containing equivalent concentrations of its three major sugars failed to inhibit cell proliferation. At higher concentrations (>2.5%), MH was found to be generally deleterious to the growth of all three cell lines. MH induced apoptosis of MDA-MB-231 cells through activation of caspases 8, 9, 6, and 3/7 and this correlated with a loss of Bcl-2 and increased Bax protein expression in MH-treated cells. Incubation with MH induced a time-dependent translocation of cytochrome c from mitochondria to the cytosol and Bax translocation from the cytosol into the mitochondria. MH also induced apoptosis of MCF-7 cells via the activation of caspases 9 and 6. Low concentrations of MH (0.03-1.25% w/v) induced a rapid reduction in tyrosine-phosphorylated STAT3 (pY-STAT3) in MDA-MB-231 and MCF-7 cells. Maximum inhibition of pY-STAT3 was observed at 1 h with a loss of >80% and coincided with decreased interleukin-6 (IL-6) production. Moreover, MH inhibited the migration and invasion of MDA-MB-231 cells as well as the angiogenic capacity of human umbilical vein endothelial cells. Our findings identify multiple functional pathways affected by MH in human breast cancer and highlight the IL-6/STAT3 signaling pathway as one of the earliest potential targets in this process.

Deficiency of mitochondrial modulator MCJ promotes chemoresistance in breast cancer.

Despite major advances in early detection and prognosis, chemotherapy resistance is a major hurdle in the battle against breast cancer. Identifying predictive markers and understanding the mechanisms are key steps to overcoming chemoresistance. Methylation-controlled J protein (MCJ, also known as DNAJC15) is a negative regulator of mitochondrial respiration and has been associated with chemotherapeutic drug sensitivity in cancer cell lines. Here we show, in a retrospective study of a large cohort of breast cancer patients, that low MCJ expression in breast tumors predicts high risk of relapse in patients treated with chemotherapy; however, MCJ expression does not correlate with response to endocrine therapy. In a prospective study in breast cancer patients undergoing neoadjuvant therapy, low MCJ expression also correlates with poor clinical response to chemotherapy and decreased disease-free survival. Using MCJ-deficient mice, we demonstrate that lack of MCJ is sufficient to induce mammary tumor chemoresistance in vivo. Thus, loss of expression of this endogenous mitochondrial modulator in breast cancer promotes the development of chemoresistance.

Acute systemic exposure to silver-based nanoparticles induces hepatotoxicity and NLRP3-dependent inflammation.

Nanoparticles (NPs) are increasingly being commercialized for use in biomedicine. NP toxicity following acute or chronic exposure has been described, but mechanistic insight into this process remains incomplete. Recent evidence from in vitro studies suggested a role for NLRP3 in NP cytotoxicity. In this study, we investigated the effect of systemic administration of composite inorganic NP, consisting of Ag:Cu:B (dose range 1-20 mg/kg), on the early acute (4-24 h post-exposure) and late phase response (96 h post-exposure) in normal and NLRP3-deficient mice. Our findings indicate that systemic exposure (≥2 mg/kg) was associated with acute liver injury due to preferential accumulation of NP in this organ and resulted in elevated AST, ALT and LDH levels. Moreover, within 24 h of NP administration, there was a dose-dependent increase in intraperitoneal neutrophil recruitment and upregulation in gene expression of several proinflammatory mediators, including TNF-α, IL-1ß and S100A9. Histological analysis of liver tissue revealed evidence of dose-dependent hepatocyte necrosis, increase in sinusoidal Kupffer cells, lobular granulomas and foci of abscess formation which were most pronounced at 24 h following NP administration. NP deposition in the liver led to a significant upregulation in gene expression of S100A9, an endogenous danger signal recognition molecule of phagocytes, IL-1ß and IL-6. The extent of proinflammatory cytokine activation and hepatotoxicity was significantly attenuated in mice deficient in the NLRP3 inflammasome, demonstrating the critical role of this innate immune system recognition receptor in the response to NP.

A missense mutation in TFRC, encoding transferrin receptor 1, causes combined immunodeficiency.

Patients with a combined immunodeficiency characterized by normal numbers but impaired function of T and B cells had a homozygous p.Tyr20His substitution in transferrin receptor 1 (TfR1), encoded by TFRC. The substitution disrupts the TfR1 internalization motif, resulting in defective receptor endocytosis and markedly increased TfR1 expression on the cell surface. Iron citrate rescued the lymphocyte defects, and expression of wild-type but not mutant TfR1 rescued impaired transferrin uptake in patient-derived fibroblasts. Tfrc(Y20H/Y20H) mice recapitulated the immunological defects of patients. Despite the critical role of TfR1 in erythrocyte development and function, patients had only mild anemia and only slightly increased TfR1 expression in erythroid precursors. We show that STEAP3, a metalloreductase expressed in erythroblasts, associates with TfR1 and partially rescues transferrin uptake in patient-derived fibroblasts, suggesting that STEAP3 may provide an accessory TfR1 endocytosis signal that spares patients from severe anemia. These findings demonstrate the importance of TfR1 in adaptive immunity.

Phenotypic alterations, clinical impact and therapeutic potential of regulatory T cells in cancer.

INTRODUCTION: Regulatory T cells (Tregs) have been characterised in different cancers. They accumulate in peripheral blood and tumour microenvironments where they suppress tumour-specific immune responses, enabling tumours to develop without challenge. This tumour immune evasion represents a major obstacle to successful cancer therapies. Whilst Tregs are generally divided into thymic-derived and peripherally induced, Tregs exhibit a wide spectrum of phenotypes and functional capacity dependent on microenvironment. This phenotypic diversity is also reflected in tumour-infiltrating Treg (TI Treg) populations, which may explain the variable impact of Treg accumulation on prognosis in different cancers. Identifying TI Treg subsets is critical to understand TI Treg biology and for developing effective immunotherapies. AREAS COVERED: This review discusses current and potential markers, and the modulation of these markers in cancer. In addition, we systematically review the clinical impact of Tregs in cancer and their potential as a therapeutic target, with a focus on TI Tregs. EXPERT OPINION: TI Tregs represent dynamic and diverse subsets that are key in promoting tumour progression through their suppressive activities. Targeting specific TI Treg subpopulations and functional TI Treg markers represents a feasible therapeutic strategy that might allow reestablishment of antitumour immune responses without affecting physiological immune regulation.

Salmonella-mediated tumor regression involves targeting of tumor myeloid suppressor cells causing a shift to M1-like phenotype and reduction in suppressive capacity.

The effectiveness of attenuated Salmonella in inhibiting tumor growth has been demonstrated in many therapeutic models, but the precise mechanisms remain incompletely understood. In this study, we show that the anti-tumor capacity of Salmonella depends on a functional MyD88-TLR pathway and is independent of adaptive immune responses. Since myeloid suppressor cells play a critical role in tumor growth, we investigated the consequences of Salmonella treatment on myeloid cell recruitment, phenotypic characteristics, and functional activation in spleen and tumor tissue of B16.F1 melanoma-bearing mice. Salmonella treatment led to increased accumulation of splenic and intratumoral CD11b(+)Gr-1(+) myeloid cells, exhibiting significantly increased expression of various activation markers such as MHC class II, costimulatory molecules, and Sca-1/Ly6A proteins. Gene expression analysis showed that Salmonella treatment induced expression of iNOS, arginase-1 (ARG1), and IFN-γ in the spleen, but down-regulated IL-4 and TGF-ß. Within the tumor, expression of iNOS, IFN-γ, and S100A9 was markedly increased, but ARG1, IL-4, TGF-ß, and VEGF were inhibited. Functionally, splenic CD11b(+) cells maintained their suppressive capacity following Salmonella treatment, but intratumoral myeloid cells had significantly reduced suppressive capacity. Our findings demonstrate that administration of attenuated Salmonella leads to phenotypic and functional maturation of intratumoral myeloid cells making them less suppressive and hence enhancing the host's anti-tumor immune response. Modalities that inhibit myeloid suppressor cells may be useful adjuncts in cancer immunotherapy.