Jennifer K. Mulligan

Vitamin D3 correlates inversely with systemic dendritic cell numbers and bone erosion in chronic rhinosinusitis with nasal polyps and allergic fungal rhinosinusitis

Vitamin D3 (VD3) is a steroid hormone that regulates bone health and numerous aspects of immune function and may play a role in respiratory health. We hypothesized that T helper type 2 (Th2) disorders, chronic rhinosinusitis with nasal polyps (CRSwNP) and allergic fungal rhinosinusitis (AFRS) would have VD3 deficiencies, resulting in increased mature dendritic cells (DCs) and bone erosion. We conducted a retrospective study examining VD3 levels in patients with AFRS (n = 14), CRSwNP (n = 9), chronic rhinosinusitis without nasal polyps (CRSsNP) (n = 20) and cerebrospinal fluid leak repair (non‐diseased controls) (n = 14) at time of surgery. Circulating immune cell levels were determined by immunostaining and flow cytometric analysis. Plasma VD3 and immune regulatory factors (granulocyte–macrophage colony‐stimulating factor and prostaglandin E2) were measured by enzyme‐linked immunosorbent assay. It was observed that CRSwNP and AFRS demonstrated increased circulating DCs, while chronic rhinosinusitis without nasal polyps displayed increased circulating macrophages. CRSwNP and AFRS were to found to have insufficient levels of VD3 which correlated inversely with circulating numbers of mature DCs, DC regulatory factors and bone erosion. CRSsNP displayed no change in circulating DC numbers or VD3 status compared to control, but did display increased numbers of circulating macrophages that was independent of VD3 status. Lastly, VD3 deficiency was associated with more severe bone erosion. Taken together, these results suggest support a role for VD3 as a key player in the immunopathology of CRSwNP and AFRS.

Cytokine correlation between sinus tissue and nasal secretions among chronic rhinosinusitis and controls.

Compare cytokine levels in sinus tissue to sinus secretions from controls and chronic rhinosinusitis patients.

Increased presence of dendritic cells and dendritic cell chemokines in the sinus mucosa of chronic rhinosinusitis with nasal polyps and allergic fungal rhinosinusitis

The aim of this study was to determine if there is a link between local dendritic cells (DCs) and various subtypes of chronic rhinosinusitis (CRS): CRS with nasal polyposis (CRSwNP), CRS without nasal polyposis (CRSsNP), and allergic fungal rhinosinusitis (AFRS). Once DC presence was established we considered possible mechanisms for DC recruitment to the sinuses.

Secretion of vascular endothelial growth factor by oral squamous cell carcinoma cells skews endothelial cells to suppress T-cell functions

Patients with oral squamous cell carcinoma (OSCC) have severe defects in antitumor immune function. Endothelial cells are potential regulators of immune cell function and have therefore been examined to determine their role in tumor-induced immune suppression. The present studies demonstrated that supernatants from endothelial cells exposed to OSCC-conditioned media (endo(OSCC-sup)) exhibited elevated levels of the immune suppressive products prostaglandin E(2) (PGE(2)) and vascular endothelial growth factor (VEGF) compared with supernatants from endothelial cells treated with medium alone (endo(medium)) or with keratinocyte-conditioned medium (endo(ker-sup)). Antibody neutralization of OSCC-derived VEGF prevented tumor-conditioned media from inducing endothelial cells to increase production of PGE(2)and VEGF. Furthermore, treatment of T-cells with supernatants from endo(OSCC-sup) resulted in diminished T-cell proliferation and decreased interferon-gamma (IFN-gamma) production compared with T-cells treated with medium or supernatants from endo(medium) or endo(ker-sup) controls. T-cell levels of granzyme B and perforin were reduced after treatment with supernatant from endo(OSCC-sup) compared with control treatments. The addition of VEGF neutralizing antibody to the OSCC-conditioned medium prevented endothelial cells from being skewed to downregulate T-cell proliferation and production of IFN-gamma, perforin, and granzyme B. Taken together, these studies provide support for the use of VEGF-targeting therapies as an immunotherapeutic agent to block induction of immune suppressive endothelial cells in patients with OSCC.

Vitamin D3 deficiency increases sinus mucosa dendritic cells in pediatric chronic rhinosinusitis with nasal polyps.

Objective Dendritic cells are professional antigen presenting cells, capable of initiating Th1 or Th2 responses, and have been implicated in the pathogenesis of a number of diseases, including sinusitis. Vitamin D3 is a steroid hormone that acts on dendritic cells in a manner similar to corticosteroids. Investigators examined whether children with allergic fungal rhinosinusitis (AFRS) or chronic rhinosinusitis with nasal polyposis (CRSwNP) were vitamin D3 deficient and the relationship of vitamin D3 deficiency to dendritic cell infiltrate in the sinus mucosa. Setting Tertiary care university hospital. Study Design Retrospective, controlled study using samples collected from pediatric patients seen from August 2009 to July 2011. Subjects and Methods Plasma levels of 25-hydroxy vitamin D3 were measured by enzyme-linked immunosorbent assay in children (≤18 years old) with AFRS, CRSwNP, or CRS without nasal polyposis (CRSsNP) and in controls undergoing surgery for adenotonsillar hypertrophy. Vitamin D3 levels were confirmed using clinical diagnostic methods for those with CRSwNP or AFRS. Tissue samples were immunohistochemically stained for the dendritic cell marker CD209 and the costimulatory molecules CD80 and CD86. Results There was no difference in mean vitamin D3 levels between control and CRSsNP, whereas mean CRSwNP and AFRS levels were both well below the minimum recommended level of 30 ng/mL and significantly lower than control and CRSsNP levels. CD209+ dendritic cells inversely correlated with vitamin D3 but not costimulatory molecule expression. Conclusions These studies identify that children with CRSwNP or AFRS are vitamin D3 deficient, which may be linked to increased dendritic cell infiltrate. These results suggest a role for vitamin D3 as a key player in the immunopathology of pediatric CRSwNP.

Tumor Secretion of VEGF Induces Endothelial Cells to Suppress T cell Functions Through the Production of PGE2

Endothelial cells are potent regulators of immune cell functions and have therefore been examined to determine their role in tumor-induced immune suppression. Previous studies by our laboratory showed that exposure to Lewis lung carcinoma (LLC)-secreted products induced endothelial cells to suppress T-cell functions in vitro. The current studies examined in vitro and in vivo the mechanism by which tumors induce the formation of suppressor endothelial cells and the means by which suppressor endothelial cells disrupt T-cell functions. In vitro studies demonstrated that inhibition of tumor-derived VEGF with neutralizing antibodies or treatment of endothelial cells with the VEGF receptor tyrosine kinase inhibitor, SU5416, prevented endothelial cells from being induced to suppress T-cell functions. Treatment of tumor-bearing mice with SU5416 blocked the development of endothelial cells that are suppressive to CD4+ and CD8+ T-cell functions. We next examined the role of suppressor endothelial cell-derived PGE2 in the inhibition of T-cell functions. Abrogation of endothelial cell PGE2 production in vitro with indomethacin prevented tumor-conditioned media from stimulating endothelial cell production of immune inhibitory activity toward T-cell functions. Similar treatment of endothelial cells from lungs of tumor-bearing mice blocked their capacity to produce T-cell-inhibitory mediators. These studies demonstrate that tumor-derived VEGF induces endothelial cells to upregulate production of PGE2 which, in turn, leads to suppression of T-cell functions.

Tumors induce the formation of suppressor endothelial cells in vivo.

Patients with solid tumors have defects in immune effector cells, which have been associated with a poorer prognosis. Previous studies by our laboratory have shown that exposure to Lewis lung carcinoma (LLC)-secreted products induces the formation of suppressor endothelial cells in vitro. The current studies examined if tumors could induce the formation of suppressor endothelial cells in vivo. Endothelial cells were immunomagnetically isolated from the lungs of tumor-bearing mice or normal controls and examined for their ability to modulate NK cell, T-cell and macrophage functions. Compared to normal controls, supernatants from endothelial cells isolated from tumor-bearing lungs had elevated secretion of PGE2, IL-6, IL-10 and VEGF. Conditioned media from endothelial cells isolated from normal lungs increased CD8+ T-cell IFN-γ and CD4+ T-cell IL-2 production in response to anti-CD3 stimulation, while media conditioned by endothelial cells from tumor-bearing lungs had a diminished stimulatory capacity. Examination of NK cell functions showed that supernatants from endothelial cells isolated from normal lungs were potent activators of NK cells, as indicated by their secretion of TNF-α and IFN-γ. Endothelial cells isolated from tumor-bearing lungs had a significantly diminished capacity to activate NK cells. Finally, supernatants from endothelial cells of tumor-bearing lungs diminished macrophage phagocytosis compared to either treatment with supernatants of normal endothelial cells or treatment with media alone. The results of these studies demonstrate that tumors induce the formation of suppressor endothelial cells in vivo and provide support for the role of endothelial cells in tumor-induced immune suppression.

Impact of vitamin D deficiency upon clinical presentation in nasal polyposis

The objective of this work was to determine if specific chronic rhinosinusitis with nasal polyps (CRSwNP) populations are at risk for vitamin D3 (VD3) deficiency and if VD3 levels correlate with radiographic measures of disease severity or eosinophilia.

Tumors skew endothelial cells to disrupt NK cell, T-cell and macrophage functions.

IntroductionPatients and mice with solid tumors, such as Lewis lung carcinoma (LLC), have defects in functions of immune effector cells. Endothelial cells, a component of the tumor vasculature, are potential regulators of immune cell functions. Therefore, these studies examined the impact of exposure to LLC tumor on the ability of endothelial cells to modulate immune cell functions.Materials and methodsEndothelial cells were pre-treated with LLC tumor-conditioned medium (EndoT-sup) for 24 h. Control endothelial cells that were exposed to medium (EndoMedia) or epithelial cell-conditioned medium (EndoEpi-sup). After the initial 24 h incubation, endothelial cells were washed and fresh media was added. Cells were allowed to incubate for an additional 24 h. Supernatants from EndoMedia, EndoEpi-sup or EndoT-sup were collected and assayed for immune modulatory products and for immune modulatory activity.ResultsSupernatant from EndoT-sup contained increased levels of PGE2, IL-6 and VEGF as compared to EndoMedia and EndoEpi-sup controls. NK cell activity, as measured by TNF-α and IFN-γ secretion, was increased following exposure to media conditioned by EndoMedia and EndoEpi-sup. Exposure of NK cells to supernatants of EndoT-sup, also increases TNF-α and IFN-γ secretion, but to a lesser extent than by EndoMedia and EndoEpi-sup. Examination of macrophage functions demonstrated that supernatant from EndoT-sup decreased microbead phagocytosis and increased production of the immune suppressive mediators, IL-10 and PGE2. Lastly, T-cell responses to stimulation with anti-CD3 in the presence of supernatants from EndoT-sup were examined. IFN-γ production by CD8+ T-cells was reduced after exposure to EndoT-sup-conditioned medium, as compared to cells treatments with medium or control conditioned medium. Production of IFN-γ by CD4+ T-cells exposed to EndoT-sup was not altered.ConclusionsTaken together, these studies demonstrate that tumors skew endothelial cells to disrupt NK cell, T-cell and macrophages functions, and represents a novel mechanism of tumor-induced immune suppression.

Mucous Cytokine Levels in Chronic Rhinosinusitis-Associated Olfactory Loss

IMPORTANCE Olfactory loss is a frequent symptom of patients with chronic rhinosinusitis (CRS), but our understanding of how inflammatory cytokines affect olfaction is limited. OBJECTIVES To examine whether inflammatory cytokines are present in the olfactory cleft and whether they correlate with objective olfaction. DESIGN, SETTING, AND PARTICIPANTS In this cross-sectional study, patients with CRS underwent quantitative olfactory testing using the Sniffin Sticks test to calculate a composite threshold discrimination identification (TDI) score from October 21, 2013, to November 12, 2015. Nasal mucus was collected using a sponge placed in the olfactory cleft for 5 minutes, and Cytometric Bead Array was used to measure secreted immunomodulatory products. Correlations between TDI score and secreted mediators were then calculated. Data analysis was performed from October 15, 2015, to December 17, 2015. MAIN OUTCOMES AND MEASURES Composite TDI scores and mean secreted mediator levels in mucus from the olfactory cleft. RESULTS Thirty-four patients were enrolled (mean [SD] age, 57.3 [15.7] years; female, 21 [61.8%]; white, 26 [76.5]). The TDI scores were lower in patients with CRS with nasal polyps (CRSwNP) than in patients with CRS without nasal polyps (CRSsNP) (difference, 8.7; 95% CI, 2.5-15.0; P = .007). Interleukin (IL) 5 levels were inversely correlated with TDI scores in patients with CRSwNP and those with CRSsNP (mean [SE] β estimate, -46.56 [15.11]; P = .005), whereas IL-6, IL-7, and vascular endothelial growth factor A were positively correlated with TDI scores only in the CRSwNP cohort. Subscale olfactory TDI scores followed similar correlations to composite TDI scores. CONCLUSIONS AND RELEVANCE In this study, inflammatory cytokines were found in mucus collected from the olfactory cleft. Levels of IL-5, in addition to other cytokines, were associated with objective olfactory function. Further inquiry is needed to establish the source of mucous cytokines and establish whether they play a causal role in olfactory loss.