Kirsten Bruderek

Myeloid‐derived suppressor cells in the peripheral blood of cancer patients contain a subset of immature neutrophils with impaired migratory properties

In tumor‐bearing mice, immunosuppressive granulocytic and monocytic MDSC have been identified. The identity and function of MDSC in cancer patients are less clear and need further characterization. We analyzed the peripheral blood of 103 patients with HNC, lung cancer, or cancers of bladder and ureter. Based on sedimentation properties in density gradients, a subset of LD‐PMN was identified and analyzed. LD‐PMN were expanded in the peripheral blood of cancer patients, suppressed proliferation, and IFN‐γ production of polyclonally stimulated T cells and thus, qualify as human MDSC. Immunophenotyping and morphological analysis revealed the accumulation of immature PMN in the MDSC fraction. Neutrophilic MDSC showed altered surface marker expression, prolonged survival, and impaired effector functions when compared with conventional, mature PMN of regular density. MDSC displayed markedly reduced chemotaxis toward tumor‐conditioned medium and lacked expression of chemokine receptors CXCR1 and CXCR2, which are normally required for PMN extravasation from the bloodstream and subsequent tissue infiltration. Collectively, our data suggest the accumulation and persistence of long‐lived, immature granulocytic MDSC with T cell‐suppressive function and impaired migratory properties in the peripheral blood of cancer patients.

Polymorphonuclear granulocytes in human head and neck cancer: enhanced inflammatory activity, modulation by cancer cells and expansion in advanced disease.

The progression of epithelial cancer is associated with an intense immunological interaction between the tumor cells and immune cells of the host. However, little is known about the interaction between tumor cells and polymorphonuclear granulocytes (PMNs) in patients with head and neck squamous cell carcinoma (HNSCC). In our study, we investigated systemic PMN‐related alterations in HNSCC, the role of tumor‐infiltrating PMNs and their modulation by the tumor microenvironment. We assessed the infiltration of HNSCC tissue by PMNs (retrospectively) and systemic PMN‐related alterations in blood values (prospectively) in HNSCC patients (n = 99 and 114, respectively) and control subjects (n = 41). PMN recruitment, apoptosis and inflammatory activity were investigated in an in vitro system of peripheral blood PMNs and a human HNSCC cell line (FaDu). HNSCC tissue exhibited considerable infiltration by PMNs, and strong infiltration was associated with poorer survival in advanced disease. PMN count, neutrophil‐to‐lymphocyte ratio and serum concentrations of CXCL8 (interleukin‐8), CCL4 (MIP‐1β) and CCL5 (RANTES) were significantly higher in the peripheral blood of HNSCC patients than in that of controls. In vitro, HNSCC‐conditioned medium inhibited apoptosis of PMNs, increased chemokinesis and chemotaxis of PMNs, induced release of lactoferrin and matrix metalloproteinase 9 by PMNs and enhanced the secretion of CCL4 by PMN. Our findings demonstrate alterations in PMN biology in HNSCC patients. In vitro, tumor‐derived factors modulate cellular functions of PMNs and increase their inflammatory activity. Thus, the interaction between HNSCC and PMNs may contribute to host‐mediated changes in the tumor microenvironment.

Tissue-resident mesenchymal stem cells attract peripheral blood neutrophils and enhance their inflammatory activity in response to microbial challenge.

Human MSCs may respond to TLR ligation, and recent research has suggested that many tissues contain tissue‐specific MSCs, possibly located in periendothelial and perivascular regions. At present, the functional consequences of these findings are unclear. We hypothesized that tissue‐specific MSCs could play an instructional role during early phases of bacterial challenge. To investigate this hypothesis further, we set up a coculture system of glandular MSCs and peripheral blood neutrophils so that we could analyze the cellular interactions of these cells in response to LPS challenge. We found that stimulation with bacterial endotoxin induced chemokine receptor expression and mobility of MSCs. Activated MSCs secreted large amounts of inflammatory cytokines and recruited neutrophils in an IL‐8‐ and MIF‐dependent manner. Recruited and activated neutrophils showed a prolonged lifespan, an increased expression of inflammatory chemokines, and an enhanced responsiveness toward subsequent challenge with LPS. Our findings demonstrate a complex, functional interaction between tissue‐resident MSCs and peripheral blood neutrophils upon bacterial challenge and suggest a role for MSCs in the early phases of pathogen challenge, when classical immune cells have not been recruited yet.

Tumor-derived macrophage migration inhibitory factor modulates the biology of head and neck cancer cells via neutrophil activation

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that has been reported to enhance the aggressiveness and metastatic potential of tumor cells. However, the mechanisms through which MIF influences tumor development and progression are not understood. The objectives of our study were to assess the effects of tumor‐derived MIF on neutrophils in head and neck cancer (HNC) and to identify possible feedback effects on tumor cells. To this end, we used an in vitro system to model the interaction between human HNC cells and neutrophils. In addition, we analyzed expression of MIF in tissues from HNC patients in relation to neutrophilic infiltration and clinical parameters. Our results show that human HNC is infiltrated by neutrophils proportional to the levels of tumoral MIF. Strong MIF expression by the tumor is associated with higher lymph node metastasis and reduced survival in HNC patients. In vitro, MIF modulated functions of human neutrophils by inducing chemokine CXC motif receptor 2(CXCR2)‐dependent chemotaxis, enhancing neutrophil survival and promoting release of chemokine C‐C Motif Ligand 4 (CCL4) and matrix metalloprotease 9(MMP9). Further, neutrophils activated with tumor‐derived MIF enhanced migratory properties of HNC cells. In conclusion, our data indicate that the effects of tumor‐derived MIF on neutrophils represent an additional mechanism by which MIF might contribute to tumor progression.

Failure to detect production of IL-10 by activated human neutrophils.

To the Editor: We read with great interest the paper by De Santo et al.1 published in the November 2010 issue of Nature Immunology, which has received wide attention because of its potential implications for inflammation and immunotherapy. The authors show that the acute-phase protein serum amyloid A1 (SAA-1) induces substantial secretion of the immunosuppressive cytokine interleukin 10 (IL-10) by human neutrophils (up to 100–400 ng/ml) and that invariant natural killer T cells (iNKT cells) are able to diminish this. The authors conclude that harnessing iNKT cells might “be useful therapeutically by decreasing the frequency of immunosuppressive neutrophils and restoring tumor-specific immune responses” in patients with melanoma and other patients with high plasma SAA-1 concentrations1. Intimate crosstalk of neutrophils and monocytes with another population of ‘unconventional’ human T cells, Vg9Vd2 gd T cells, has been demonstrated2. Of relevance for immunotherapy, Vg9Vd2 T cells are much more abundant in human blood than are iNKT cells and can readily be activated by aminobisphosphonate drugs such as zoledronate (Zometa). Small-scale trials have shown promising results for therapy targeting Vg9Vd2 T cells to treat metastatic disease in patients with multiple myeloma, prostate cancer or breast cancer3. Thus, we were keen to ‘translate’ the described interaction of iNKT cells with neutrophils to our own experimental system2,4 and to test whether activated Vg9Vd2 T cells likewise inhibits IL-10-producing neutrophils. Unexpectedly, we found that highly purified, obtained by negative selection human neutrophils (Supplementary Fig. 1) did not secrete IL-10 (Fig. 1a) or express IL-10 mRNA (Fig. 1b) in response to recombinant SAA-1 at the same concentrations used by De Santo et al.1 Notably, and in contrast to De Santo et al., we also did not detect IL-10 in the culture supernatants of neutrophils stimulated with either Salmonella abortus equi lipopolysaccharide (LPS) or ultrapure Escherichia coli LPS (Fig. 1 and Supplementary Fig. 2). The failure to detect IL-10 production by human neutrophils in response to SAA-1 or LPS was reproducible in four independent laboratories at Cardiff (UK), Verona (Italy), Milan (Italy) and Essen (Germany) and was independent of the isolation procedure, the method used to lyse red blood cells, the culture dishes used, cell numbers and medium supplements (Supplementary Methods). The lack of IL-10 in neutrophil culture supernatants was consistent with the absence of both IL-10 mRNA (Fig. 1b) and IL-10 protein (data not shown) in total lysates of LPS-stimulated neutrophils, which confirmed published data5,6. Finally, human neutrophils did not secrete IL-10 even when incubated with other Toll-like receptor agonists (poly(I:C), Pam3CSK4 or R-848), cytokines (IFN-g, TNF, GM-CSF or G-CSF), curdlan, neutrophil-activating protein derived from Helicobacter pylori, the chemoattractant fMLF or insoluble immunocomplexes (Supplementary Fig. 2 and data not shown). In their study, De Santo et al. positively select cells on the basis of CD11b expression and define them as >95% CD11b+CD15+ (ref. 1), compared with the value of over 98–99% achieved in our laboratories2,4,7,8 (Supplementary Methods). Of note, both peripheral neutrophils and monocytes expressed CD11b. Although neutrophils were distinctly CD15+, monocytes had intermediate surface expression of CD15 (Supplementary Fig. 1). Contaminating monocytes may thus give rise to false-positive data in whole-population analyses by enzyme-linked immunosorbent assay (ELISA), quantitative RT-PCR or immunoblot analysis. When we added

Peripheral Blood Neutrophil Granulocytes from Patients with Head and Neck Squamous Cell Carcinoma Functionally Differ from Their Counterparts in Healthy Donors

Solid tumors such as head and neck squamous cell carcinoma (HNSCC) display an intense interaction between tumoral factors and the immune system. Functional modulation of tumor-infiltrating and peripheral blood immune cells plays an important role during tumor progression. In this pilot study we compared biological functions of polymorphonuclear granulocytes (PMN) from the peripheral blood of HNSCC patients and healthy subjects. PMN were simultaneously isolated from the peripheral blood of HNSCC patients and healthy donors for functional analysis (apoptosis, production of reactive oxygen species (ROS), cytokine release and immunophenotyping). PMN from HNSCC patients showed a significantly lower inducible production of ROS (P = 0.02) and reduced spontaneous apoptosis (P = 0.008) compared with PMN from healthy donors. Under standard culture conditions, there was no significant difference regarding the release of inflammatory cytokines between PMN from HNSCC patients and PMN from healthy donors. Confirming previous observations, serum concentrations of PMN-related cytokines were significantly higher in the peripheral blood of HNSCC patients than in that of controls. Importantly, immunophenotyping revealed an increased number of immature PMN in PMN fractions isolated from HNSCC patients. Peripheral blood PMN from HNSCC patients and healthy donors show distinct functional differences. The presence of increased numbers of immature stages of PMN in HNSCC patients may partly contribute to the changes observed. After recruitment to and infiltration of the tumor, PMN may be further modulated in the local tumor microenvironment. This pilot study justifies functional analyses of myeloid cells in larger cohorts of patients with HNSCC.

Granulocytic myeloid-derived suppressor cells are cryosensitive and their frequency does not correlate with serum concentrations of colony-stimulating factors in head and neck cancer

Granulocytic myeloid-derived suppressor cells (MDSC) are a MDSC subset expanded in various cancer types. As many clinical studies rely on the use of stored collections of frozen blood samples, we first tested the influence of freezing/thawing procedures on immunophenotyping and enumeration of granulocytic MDSC (G-MDSC). To identify factors involved in expansion of human G-MDSC, we then analyzed correlations between G-MDSC frequencies, clinical parameters and granulocyte-related factors in the peripheral blood of head and neck cancer patients. HLA-DR, CD14, CD33 and CD66b allowed a clear discrimination of G-MDSC from monocytic MDSC and immature myeloid cells. MDSC subsets were sensitive to cryopreservation with immature G-MDSC showing the highest sensitivity. G-MDSC frequencies were increased in advanced disease stage and associated with the level of CCL4 and CXCL8, but not with colony-stimulating factors, IL-6, S100A8/9, CXCL1 and other cytokines. Our results indicate that the frequency of MDSC, in particular G–MDSC, may be underestimated in retrospective clinical analyses using frozen blood samples. Increased G-MDSC frequencies correlate with advanced disease and increased concentrations of CXCL8, but, unexpectedly, not with growth factors (such as granulocyte colony-stimulating factor), IL-6 and CXCL1. Our data suggest that CXCL8 promotes accumulation of G-MDSC in cancer patients independent of classical colony-stimulating factors.

Cutting Edge: An Inactive Chromatin Configuration at the IL-10 Locus in Human Neutrophils

To identify the molecular basis of IL-10 expression in human phagocytes, we evaluated the chromatin modification status at their IL-10 genomic locus. We analyzed posttranslational modifications of histones associated with genes that are active, repressed, or poised for transcriptional activation, including H3K4me3, H4Ac, H3K27Ac, and H3K4me1 marks. Differently from autologous IL-10–producing monocytes, none of the marks under evaluation was detected at the IL-10 locus of resting or activated neutrophils from healthy subjects or melanoma patients. By contrast, increased H3K4me3, H4Ac, H3K4me1, and H3K27Ac levels were detected at syntenic regions of the IL-10 locus in mouse neutrophils. Altogether, data demonstrate that human neutrophils, differently from either monocytes or mouse neutrophils, cannot switch on the IL-10 gene because its locus is in an inactive state, likely reflecting a neutrophil-specific developmental outcome. Implicitly, data also definitively settle a currently unsolved issue on the capacity of human neutrophils to produce IL-10.

Low adiponectin, high levels of apoptosis and increased peripheral blood neutrophil activity in healthy obese subjects.

Objective: Growing evidence supports a link between obesity and inflammation. Current research is focused on the role of adipokines such as adiponectin and immune cells, especially macrophages, in adipose tissue. Our aim was to examine the role of inflammation not in tissue but in the peripheral blood of healthy overweight and obese subjects. We especially investigated the role of neutrophils and their possible regulation by adiponectin. Methods: In healthy normal-weight, overweight, and obese human subjects (n = 32) the peripheral blood concentrations of adipokines, satiety hormones, apoptosis markers, and cytokines as well as the blood count were related to inflammation and neutrophils, at 3 independent days of examination. The response of neutrophils to stimulation by adiponectin was also investigated in vitro. Results: In obese and by tendency already in overweight subjects, inflammation was increased showing a higher neutrophil-to-lymphocyte ratio, elevated high-sensitivity C-reactive protein, increased chemokines (CXCL8, CCL3, CCL5), increased apoptosis markers (M30 and M65), and changes in hormone levels in the peripheral blood. LPS- and fMLP-induced production of CXCL8 by neutrophils was elevated in overweight and obese subjects. High plasma levels of adiponectin were associated with reduced CXCL8 production in peripheral blood neutrophils. In vitro, production of CXCL8 by neutrophils was inhibited by adiponectin. Conclusion: Reduced adiponectin and enhanced apoptosis may occur already in the peripheral blood of healthy overweight subjects. This process seems to further enhance neutrophil activity in overweight and obese.

Mesenchymal stem cells augment the anti-bacterial activity of neutrophil granulocytes.

Background Mesenchymal stem cells (MSCs) participate in the regulation of inflammation and innate immunity, for example by responding to pathogen-derived signals and by regulating the function of innate immune cells. MSCs from the bone-marrow and peripheral tissues share common basic cell-biological functions. However, it is unknown whether these MSCs exhibit different responses to microbial challenge and whether this response subsequently modulates the regulation of inflammatory cells by MSCs. Methodology/Principal Findings We isolated MSCs from human bone-marrow (bmMSCs) and human salivary gland (pgMSCs). Expression levels of TLR4 and LPS-responsive molecules were determined by flow cytometry and quantitative PCR. Cytokine release was determined by ELISA. The effect of supernatants from unstimulated and LPS-stimulated MSCs on recruitment, cytokine secretion, bacterial clearance and oxidative burst of polymorphonuclear neutrophil granulocytes (PMN) was tested in vitro. Despite minor quantitative differences, bmMSCs and pgMSCs showed a similar cell biological response to bacterial endotoxin. Both types of MSCs augmented anti-microbial functions of PMNs LPS stimulation, particularly of bmMSCs, further augmented MSC-mediated activation of PMN. Conclusions/Significance This study suggests that MSCs may contribute to the resolution of infection and inflammation by promoting the anti-microbial activity of PMNs. This property is exerted by MSCs derived from both the bone-marrow and peripheral glandular tissue.