Dijana Miljkovic

Association between Group 2 Innate Lymphoid Cells enrichment, nasal polyps and allergy in Chronic Rhinosinusitis

Group 2 innate lymphoid cells (ILC2s) were shown to be involved in the initiation and coordination of Th2‐type immune responses in allergic disease animal models. Recently, ILC2s enrichment was noted in chronic rhinosinusitis (CRS) patients; however, the role of ILC2s in coordinating the Th2 response in CRS remains to be elucidated. Here, we characterize the ILC2 compartment in CRS by investigating the correlations between ILC2s, Th2 cells and Th2 cytokines expression in CRS patients.

Opposing Effects of Omega-3 and Omega-6 Long Chain Polyunsaturated Fatty Acids on the Expression of Lipogenic Genes in Omental and Retroperitoneal Adipose Depots in the Rat

This study aimed to determine the effect of varying dietary intake of the major n-3 PUFA in human diets, α-linolenic acid (ALA; 18 : 3n-3), on expression of lipogenic genes in adipose tissue. Rats were fed diets containing from 0.095%en to 6.3%en ALA and a constant n-6 PUFA level for 3 weeks. Samples from distinct adipose depots (omental and retroperitoneal) were collected and mRNA expression of the pro-lipogenic transcription factors Sterol-Retinoid-Element-Binding-Protein1c (SREBP1c) and Peroxisome Proliferator Activated Receptor-γ (PPARγ), lipogenic enzymes Sterol-coenzyme Desaturase1 (SCD-1), Fatty Acid Synthase (FAS), lipoprotein lipase (LPL) and glycerol-3-phosphate dehydrogenase (G3PDH) and adipokines leptin and adiponectin determined by qRT-PCR. Increasing dietary ALA content resulted in altered expression of SREBP1c, FAS and G3PDH mRNA in both adipose depots. SREBP1c mRNA expression was related directly to n-6 PUFA concentrations (omental, r 2 = .71; P < .001; Retroperitoneal, r 2 = .20; P < .002), and inversely to n-3 PUFA concentrations (omental, r 2 = .59; P < .001; Retroperitoneal, r 2 = .19; P < .005) independent of diet. The relationship between total n-6 PUFA and SREBP1c mRNA expression persisted when the effects of n-3 PUFA were controlled for. Altering red blood cell concentrations of n-3 PUFA is thus associated with altered expression of lipogenic genes in a depot-specific manner and this effect is modulated by prevailing n-6 PUFA concentrations.

Maternal omega-3 supplementation increases fat mass in male and female rat offspring.

Adipogenesis and lipogenesis are highly sensitive to the nutritional environment in utero and in early postnatal life. Omega-3 long chain polyunsaturated fatty acids (LCPUFA) inhibit adipogenesis and lipogenesis in adult rats, however it is not known whether supplementing the maternal diet with omega-3 LCPUFA results in reduced fat deposition in the offspring. Female Albino Wistar rats were fed either a standard chow (Control, n = 10) or chow designed to provide ∼15 mg/kg/day of omega-3 LCPUFA, chiefly as docosahexaenoic acid (DHA), throughout pregnancy and lactation (Omega-3, n = 11) and all pups were weaned onto a commercial rat chow. Blood and tissues were collected from pups at 3 and 6 weeks of age and weights of visceral and subcutaneous fat depots recorded. The expression of adipogenic and lipogenic genes in the subcutaneous and visceral fat depots were determined using quantitative real time reverse transcription-PCR. Birth weight and postnatal growth were not different between groups. At 6 weeks of age, total percentage body fat was significantly increased in both male (5.09 ± 0.32% vs. 4.56 ± 0.2%, P < 0.04) and female (5.15 ± 0.37% vs. 3.89 ± 0.36%, P < 0.04) offspring of omega-3 dams compared to controls. The omega-3 LCPUFA content of erythrocyte phospholipids (as a% of total fatty acids) was higher in omega-3 offspring (6.7 ± 0.2% vs. 5.6 ± 0.2%, P < 0.001). There was no effect of maternal omega-3 LCPUFA supplementation on the expression of adipogenic or lipogenic genes in the offspring in either the visceral or subcutaneous fat depots. We have therefore established that an omega-3 rich environment during pregnancy and lactation in a rodent model increases fat accumulation in both male and female offspring, particularly in subcutaneous depots, but that this effect is not mediated via upregulation adipogenic/lipogenic gene transcription. These data suggest that maternal n−3 LCPUFA supplementation during pregnancy/lactation may not be an effective strategy for reducing fat deposition in the offspring.

CD4+ and CD8+ Regulatory T Cells in Chronic Rhinosinusitis Mucosa:

Background Chronic rhinosinusitis (CRS) mucosal inflammation is characterized by an accumulation of effector–memory T cells, but their immune regulatory potential has not been adequately examined. Coexpression of transcription factor, forkhead box P3 (Foxp3), an interleukin-2 receptor, CD25, in CD4+ and CD8+ T cells is linked with regulatory function in humans. The aim of this study was to investigate the regulatory ell (T phenotype of CD4+ (CD4Treg) and CD8+ (CD8Treg) T cells in peripheral blood (PB) and sinus mucosa of CRS patients. Methods Prospective study was performed involving 32 CRS with nasal polyp (CRSwNP), 14 CRS without nasal polyp (CRSsNP), and 8 control patients. Sinus and PB T lymphocytes were stained with CD3, CD4, CD8, CD25, and Foxp3 and analyzed using flow cytometry. Relevant clinical characteristics, sinus bacterial culture results, and eosinophilic mucus were examined. Results Sinus mucosa had a higher percentage of CD4Treg (CD3+CD4+CD25+Foxp3+) population compared with PB all patients. The percentage of PB CD4Treg and CD8Treg (CD3+CD8+CD25+Foxp3+) was not significantly different between the study groups. CRS mucosal tissue had a higher percentage of CD4Treg and activated T-helper cells than controls. There was no significant difference in PB and mucosal CD4Treg populations in CRS patients based on the presence of allergy, sinus culture results, or eosinophilic mucus. In controls creased mucosal CD4Treg correlated with coexisting allergy. Although overall CD4Treg numbers were higher, the regulatory potential of activated CD4+ T cells (CD4Treg/activated T-helper cell ratio) was significantly lower in CRS mucosa compared with controls. The CD8Treg subset was also significantly reduced in CRSwNP mucosa compared with controls. Conclusion A higher percentage of CD4Treg and activated T-helper cells in CRS mucosa suggests increased inflammation in CRS, independent of the presence of allergy, microbial culture results, or eosinophilic mucus. How, the decreased ratio of CD4Treg versus activated T-helper cells in CRS and reduced CD8Treg population in CRSwNPs indicates an inflammatory bias and the inability to control mucosal disease.BACKGROUND Chronic rhinosinusitis (CRS) mucosal inflammation is characterized by an accumulation of effector-memory T cells, but their immune regulatory potential has not been adequately examined. Coexpression of transcription factor, forkhead box P3 (Foxp3), and interleukin-2 receptor, CD25, in CD4+ and CD8+ T cells is linked with regulatory function in humans. The aim of this study was to investigate the regulatory T cell (Treg) phenotype of CD4+ (CD4Treg) and CD8+ (CD8Treg) T cells in peripheral blood (PB) and sinus mucosa of CRS patients. METHODS Prospective study was performed involving 32 CRS with nasal polyp (CRSwNP), 14 CRS without nasal polyp (CRSsNP), and 8 control patients. Sinus and PB T lymphocytes were stained with CD3, CD4, CD8, CD25, and Foxp3 and analyzed using flow cytometry. Relevant clinical characteristics, sinus bacterial culture results, and eosinophilic mucus were examined. RESULTS Sinus mucosa had a higher percentage of CD4Treg (CD3+CD4+CD25+Foxp3+) population compared with PB in all patients. The percentage of PB CD4Treg and CD8Treg (CD3+CD8+CD25+Foxp3+) was not significantly different between the study groups. CRS mucosal tissue had a higher percentage of CD4Treg and activated T-helper cells than controls. There was no significant difference in PB and mucosal CD4Treg populations in CRS patients based on the presence of allergy, sinus culture results, or eosinophilic mucus. In controls, increased mucosal CD4Treg correlated with coexisting allergy. Although overall CD4Treg numbers were higher, the regulatory potential of activated CD4+ T cells (CD4Treg/activated T-helper cell ratio) was significantly lower in CRS mucosa compared with controls. The CD8Treg subset was also significantly reduced in CRSwNP mucosa compared with controls. CONCLUSION A higher percentage of CD4Treg and activated T-helper cells in CRS mucosa suggests increased inflammation in CRS, independent of the presence of allergy, microbial culture results, or eosinophilic mucus. However, the decreased ratio of CD4Treg versus activated T-helper cells in CRS and reduced CD8Treg population in CRSwNPs indicates an inflammatory bias and the inability to control mucosal disease.

Accumulation of Effector Memory CD8+ T Cells in Nasal Polyps:

Background T lymphocytes are prevalent in sinus mucosa and are implicated in chronic rhinosinusitis (CRS) pathogenesis. However, the major T-cell subpopulations, helper (CD4+) and cytotoxic (CD8+), have not been adequately examined in CRS. This study was designed to characterize human sinus mucosa and peripheral blood (PB) CD4+ and CD8+ T cells and their level of differentiation in CRS with nasal polyps (NPs), CRS without NPs, and control patients. Methods A prospective study was performed. Percentages of CD4+ and CD8+ T cells and their levels of differentiation were analyzed in sinus mucosa and PB by flow cytometry. Cell populations were defined as naive, central memory, effector memory, and effector T cells using cell surface markers CD45RA, CD62L, and CD27. The influence of coexisting allergy, sinus eosinophilic mucus (EM), and culture results were examined. Results In all patients, sinus mucosa had a lower percentage of CD4+ and a higher percentage of CD8+ T cells compared with PB. However, CRS with NPs (n = 86) had a significantly higher percentage of mucosal CD8+ T cells compared with CRS without NPs (n = 40) in control (n = 13) patients (p < 0.0001). Effector memory T cells were increased in sinuses compared with PB in all patients; however, the percentage of effector memory CD8+ T cells was greatest in CRS with NP mucosa (p = 0.002). Surprisingly coexisting allergy or culture results did not influence the mucosal T-cell phenotype. CRS with NP patients with sinus EM had a significantly higher percentage of mucosal CD8+ T cells. Conclusion Sinus mucosa in CRS with NPs is characterized by a significant enrichment of CD8+ T cells and a relative deficiency of CD4+ T cells. The majority of NP CD8+ T cells had a terminally differentiated, mature, effector memory phenotype, which raises the question, whether these cells are pathogenic or appear as a consequence of inflammation, independent of the presence of allergy or positive microbial culture.

An in vivo safety and efficacy demonstration of a topical liposomal nitric oxide donor treatment for Staphylococcus aureus biofilm-associated rhinosinusitis

The burden of drug resistance emerges in the wake of chronic and repeated antibiotic use. This underpins the importance of discovering alternatives to current antibiotic regimens. In chronic rhinosinusitis (CRS), topical therapy such as nasal douches and steroid sprays is the mainstay of treatment. However, bacterial sinusitis such as those with Staphylococcus aureus biofilm infection point to more recalcitrant CRS subtypes, focusing research efforts into topical antimicrobial therapies. In the sinuses, both local mucosal and systemic effects must be considered in designing any new topical medication. Nitric oxide (NO), an endogenous antimicrobial agent, is found at extremely low levels in CRS sinuses and high levels in healthy sinuses. As a novel treatment modality, we have designed a liposomal formulation of an NO donor (LFNO) using isosorbide mononitrate, as a topical sinus wash in a sheep model of S. aureus biofilm rhinosinusitis. Heart rate (HR), blood pressure, mean arterial pressure (MAP), and histologic and ciliary analyses were assessed in the safety component. Efficacy was assessed by quantifying biofilm biomass post-treatment. LFNO-treated sheep had lesser inflammation (P = 0.02), and comparable ciliary preservation (P = 0.86) than the control group. A transient increase in HR and decrease in MAP were observed in the LFNO group (P < 0.05), but this was not accompanied by observable side effects. LFNO sheep had significantly lower biofilm biomass vs controls (P = 0.044). Our findings demonstrate the localized and systemic safety of LFNO in an animal model despite using high NO concentrations, thus warranting further investigation for its possible therapeutic role in CRS.

Long-Term Safety of Topical Bacteriophage Application to the Frontal Sinus Region

Background: Staphylococcus aureus biofilms contribute negatively to a number of chronic conditions, including chronic rhinosinusitis (CRS). With the inherent tolerance of biofilm-bound bacteria to antibiotics and the global problem of bacterial antibiotic resistance, the need to develop novel therapeutics is paramount. Phage therapy has previously shown promise in treating sinonasal S. aureus biofilms. Methods: This study investigates the long term (20 days) safety of topical sinonasal flushes with bacteriophage suspensions. The bacteriophage cocktail NOV012 against S. aureus selected for this work contains two highly characterized and different phages, P68 and K710. Host range was assessed against S. aureus strains isolated from CRS patients using agar spot tests. NOV012 was applied topically to the frontal sinus region of sheep, twice daily for 20 days. General sheep wellbeing, mucosal structural changes and inflammatory load were assessed to determine safety of NOV012 application. Results: NOV012 could lyse 52/61 (85%) of a panel of locally derived CRS clinical isolates. Application of NOV012 to the frontal sinuses of sheep for 20 days was found to be safe, with no observed inflammatory infiltration or tissue damage within the sinus mucosa. Conclusion: NOV012 cocktail appears safe to apply for extended periods to sheep sinuses and it could infect and lyse a wide range of S. aureus CRS clinical isolates. This indicates that phage therapy has strong potential as a treatment for chronic bacterial rhinosinusitis.

Gene expression differences in nitric oxide and reactive oxygen species regulation point to an altered innate immune response in chronic rhinosinusitis

The complex interplay between host, environment, and microbe in the etiopathogenesis of chronic rhinosinusitis (CRS) remains unclear. This study focuses on the host‐microbe interaction, specifically the regulation of nitric oxide (NO) and reactive oxygen species (ROS) against the pathogenic organism Staphylococcus aureus (S. aureus). NO and ROS play crucial roles in innate immunity and in the first‐line defense against microbial invasion.

T regulatory and Th17 cells in chronic rhinosinusitis with polyps

Chronic rhinosinusitis (CRS) is categorized into 2 types based on the absence (CRSsNP) and presence of nasal polyps (CRSwNP). Although CRSsNP patients lack nasal polyps, the mucosa may show variable degrees of polypoid change. This raises the question of whether or not the classification system is an over simplification and that CRSsNP and CRSwNP only represent 2 phenotypic extremes along a broader spectrum of immunologically different disease processes. To investigate this, adaptive and innate immune cells were compared in the different tissue types within CRSsNP and CRSwNP patients.

Discordant frequencies of tissue-resident and circulating CD180-negative B cells in chronic rhinosinusitis

The unconventional toll‐like receptor (TLR) CD180 is implicated in chronic inflammatory diseases; however, its role in chronic rhinosinusitis (CRS) has yet to be investigated. Here we study the expression of CD180, its homologue TLR4 and myeloid differentiation factor 1 (MD1) on mucosal and systemic immune cell populations in relation to serum immunoglobulin G (IgG) levels.