Natural killer cell-mediated contact dermatitis-like reaction induced by treatment with TLR3 ligand poly(I:C).

INTRODUCTION: Poly(I:C) is recognised by endosomal Toll-like receptor 3 (TLR3) and activates cytotoxic CD8(+) lymphocytes and natural killer (NK) cells. It has been shown that the viral TLR3 agonist induces robust and long-lasting T-cell-mediated responses. In addition, TLR3 modulates the contact hypersensitivity reaction. OBJECTIVE: This study aimed to determine whether poly(I:C) injection can induce NK-mediated hapten reactivity in mice. METHODS: Mice were treated with poly(I:C), and their response to dinitrofluorobenzene hapten was measured by assessing ear swelling and serum interferon gamma (IFN-γ) production. Adoptive cell transfer and cell sorting were used to investigate the mechanism of the reaction, and the phenotype of poly(I:C)-activated liver NK cells was determined by flow cytometry analysis. RESULTS: The results showed that poly(I:C) administration increased ear swelling, serum IFN-γ levels and the response to hapten in both immunocompetent and T- and B-cell-deficient mice. Only liver poly(I:C)-activated DX5(+) NK cells were able to transfer reactivity to hapten into a naive recipient. Induction of liver NK cells after poly(I:C) administration was TLR3/TRIF- and IFN-γ-dependent, interleukin 12-independent, and not modulated by MyD88. CONCLUSION: This study provides new insights into how poly(I:C) stimulates NK-mediated reactivity to hapten and suggests that liver NK cells may modulate the immune response to non-pathogenic factors during viral infection.

Obesity aggravates contact hypersensitivity reaction in mice.

BACKGROUND: Obesity is associated with chronic, low-grade inflammation in tissues and predisposes to various complications, including inflammatory skin diseases. However, the link between obesity and contact hypersensitivity (CHS) is not fully understood. OBJECTIVES: We sought to determine the influence of obesity on T helper 1 (Th1)-mediated CHS. METHODS: The activity/phenotype/cytokine profile of the immune cells was tested in vivo and in vitro. Using quantitative polymerase chain reaction (qPCR) and fecal microbiota transplantation (FMT), we tested the role of a high-fat diet (HFD)-induced gut microbiota (GM) dysbiosis in increasing the effects of CHS. RESULTS: Exacerbated CHS correlates with an increased inflammation-inducing GM in obese mice. We showed a proinflammatory milieu in the subcutaneous adipose tissue of obese mice, accompanied by proinflammatory CD4+ T cells and dendritic cells in skin draining lymph nodes and spleen. Obese interleukin (IL)-17A-/-B6 mice are protected from CHS aggravation, suggesting the importance of IL-17A in CHS aggravation in obesity. CONCLUSIONS: Obesity creates a milieu that induces more potent CHS-effector cells but does not have effects on already activated CHS-effector cells. IL-17A is essential for the pathogenesis of enhanced CHS during obesity. Our study provides novel knowledge about antigen-specific responses in obesity, which may help with the improvement of existing treatment and/or in designing novel treatment for obesity-associated skin disorders.

Impact of a Graphene Oxide Reducing Agent on a Semi-Permeable Graphene/Reduced Graphene Oxide Forward Osmosis Membrane Filtration Efficiency.

Graphene has been considered as a material that may overcome the limitations of polymer semi-permeable membranes in water treatment technology. However, monolayer graphene still suffers from defects that cause leakage. Here, we report a method of sealing defects in graphene transferred onto porous polymer substrate via reduced graphene oxide (rGO). The influence of various reducing agents (e.g., vitamin C, hydrazine) on the properties of rGO was investigated by SEM, Raman, FTIR, and XRD. Subsequently, membranes based on graphene/reduced graphene oxide were tested in a forward osmosis system using sodium chloride (NaCl). The effect of the effectiveness of the reduction of graphene oxide, the type and number of attached groups, the change in the distance between the rGO flakes, and the structure of this material were examined in terms of filtration efficiency. As a result, semi-permeable centimetre-scale membranes with ion blocking efficiency of up to 90% and water flux of 20 mL h-1 m-2 bar-1 were proposed.

Diet-induced obesity aggravates NK cell-mediated contact hypersensitivity reaction in Rag1-/- mice.

BACKGROUND: Previous studies showed that natural killer (NK) cells mediate contact hypersensitivity (CHS) reaction. Many reports are showing that obesity promotes several inflammatory diseases. It was shown that diet-induced obesity (DIO) aggravates classical T cell-mediated CHS in mice. OBJECTIVES: To determine whether the high-fat diet (HFD)-induced obesity modulates antigen-specific NK cell-mediated response. METHODS: We evaluated the effect of DIO on NK cell-mediated CHS reaction using a model of dinitrofluorobenzene (DNFB)-induced CHS in Rag1-/- mice. RESULTS: Rag1-/- mice fed HFD for 8 but not for 4 weeks developed aggravated CHS reaction determined by ear swelling measurement when compared to animals kept on normal diet (ND) prior to DNFB sensitization. The obese Rag1-/- mice presented the adipose tissue inflammation. Furthermore, in vitro analysis showed that feeding with HFD significantly increases interferon γ (IFN-γ) and interleukin (IL)-12p70 and decreases adiponectin concentration in liver mononuclear cell (LMNC) culture supernatants. The flow cytometry analysis of LMNC revealed that HFD treatment prior to DNFB sensitization increases the percentage of NK1.1+ IFN-γ+ cell population and affects the development and maturation of NK1.1+ cells. CONCLUSIONS: In summary, current results suggest that the DIO significantly modulates the local and systemic inflammatory response, contributing to exacerbation of the CHS response mediated by liver NK cells.

Perinatal treatment of parents with the broad-spectrum antibiotic enrofloxacin aggravates contact sensitivity in adult offspring mice.

BACKGROUND: Antibiotics, while eliminating pathogens, also partially deplete commensal bacteria. Antibiotic-induced dysbiosis may contribute to the observed rise in "immune-mediated" diseases, including autoimmunity and allergy. The aim of this study is to investigate the impact of perinatal antibiotic treatment on T cell-mediated immune response in adult mice. METHODS: Oral treatment with broad-spectrum antibiotic enrofloxacin during gestation and breastfeeding or breastfeeding or gestation alone was used to evaluate whether antibiotic exposure early in life could modulate contact sensitivity (CS) in adult mice. RESULTS: Here, we demonstrated that enrofloxacin treatment during gestation and breastfeeding, but not during pregnancy or breastfeeding alone, aggravated CS reaction in adult mice measured by ear swelling. These data correlate with increased myeloperoxidase (MPO) activity in the ear extracts and elevated production of IL-6 and IL-17A by auricular lymph node cells (ELNC) and was not influenced by food consumption and body weight. In each dosing regimen, enrofloxacin treatment reduced the relative abundance of Enterococcus spp. but did not influence the relative abundances of Lactobacillus, Clostridium cluster XIVa, XIVab, I, Bacteroidetes, and segmented filamentous bacteria (SFB). However, prolonged enrofloxacin-treatment during both gestation and breastfeeding decreased the relative abundance of Clostridium cluster IV. CONCLUSION: These data show that long-term perinatal enrofloxacin treatment induces intestinal dysbiosis, characterized by decreased levels of anti-inflammatory Clostridium cluster IV, and alters T cell-dependent immune responses, enhancing CS reaction in adult mice.

1,2,3,4,6-Penta-O-galloyl-ß-d-glucose modulates perivascular inflammation and prevents vascular dysfunction in angiotensin II-induced hypertension.

BACKGROUND AND PURPOSE: Hypertension is a multifactorial disease, manifested by vascular dysfunction, increased superoxide production, and perivascular inflammation. In this study, we have hypothesized that 1,2,3,4,6-penta-O-galloyl-ß-d-glucose (PGG) would inhibit vascular inflammation and protect from vascular dysfunction in an experimental model of hypertension. EXPERIMENTAL APPROACH: PGG was administered to mice every 2 days at a dose of 10 mg·kg-1 i.p during 14 days of Ang II infusion. It was used at a final concentration of 20 µM for in vitro studies in cultured cells. KEY RESULTS: Ang II administration increased leukocyte and T-cell content in perivascular adipose tissue (pVAT), and administration of PGG significantly decreased total leukocyte and T-cell infiltration in pVAT. This effect was observed in relation to all T-cell subsets. PGG also decreased the content of T-cells bearing CD25, CCR5, and CD44 receptors and the expression of both monocyte chemoattractant protein 1 (CCL2) in aorta and RANTES (CCL5) in pVAT. PGG administration decreased the content of TNF+ and IFN-γ+ CD8 T-cells and IL-17A+ CD4+ and CD3+ CD4- CD8- cells. Importantly, these effects of PGG were associated with improved vascular function and decreased ROS production in the aortas of Ang II-infused animals independently of the BP increase. Mechanistically, PGG (20 µM) directly inhibited CD25 and CCR5 expression in cultured T-cells. It also decreased the content of IFN-γ+ CD8+ and CD3+ CD4- CD8- cells and IL-17A+ CD3+ CD4- CD8- cells. CONCLUSION AND IMPLICATION: PGG may constitute an interesting immunomodulating strategy in the regulation of vascular dysfunction and hypertension. LINKED ARTICLES: This article is part of a themed section on Immune Targets in Hypertension. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.12/issuetoc.

Cyclophosphamide-modified murine peritoneal macrophages induce CD4+ T contrasuppressor cells that protect contact sensitivity T effector cells from suppression.

BACKGROUND: Cyclophosphamide (CY) is one of the most widely used alkylating agents in the treatment of various cancers and some autoimmune diseases. Numerous reports suggest that CY exerts immunoregulatory effects. Animal studies have shown CY affects contact sensitivity (CS) response by depleting CD4+CD25+ T regulatory cells and CD8+ T suppressor (Ts) cells. In a mouse model of CS, we previously showed that in vivo treatment with CY shapes the immunogenic/immunoregulatory balance of peritoneal macrophages. The aim of the current study is to verify if macrophages (Mf) from CY-treated mice are indeed able to induce immunoregulatory cells that could protect from suppression. METHODS: Adoptive cell transfer of CS was used to examine immunomodulating properties of peritoneal Mf from CY-treated mice. Isolation of peritoneal Mf from animals that were (Mf-CY) or were not (Mf) treated with CY were cultured to identify cytokine repertoire. Further, we assessed spleen cell (SPLC) cytokine production following immunization with trinitrophenyl-conjugated Mf from donors treated (TNP-Mf-CY) or non-treated (TNP-Mf) with CY. RESULTS: In vitro experiments identified that Mf-CY produce more IL-6, TNF-α and TGF-ß than naïve Mf. Further, immunization with peritoneal TNP-Mf-CY induces CD4+ T contrasuppressor cells (Tcs) cells that protect CS-effector cells from suppression. Higher IL-17A secretion was observed from TNP-Mf-CY-treated mouse SPLC compared to SPLC from TNP-Mf injected mice suggesting that this cytokine might be important in mediating contrasuppression in this model. CONCLUSIONS: Our results show that in vivo treatment with CY influences mouse peritoneal Mf to induce CD4+ Tcs cells that protect CS-effector cells from suppressive signals of Ts cells.

Regulation of contact sensitivity in non-obese diabetic (NOD) mice by innate immunity.

BACKGROUND: Genetic background influences allergic immune responses to environmental stimuli. Non-obese diabetic (NOD) mice are highly susceptible to environmental stimuli. Little is known about the interaction of autoimmune genetic factors with innate immunity in allergies, especially skin hypersensitivity. OBJECTIVES: To study the interplay of innate immunity and autoimmune genetic factors in contact hypersensitivity (CHS) by using various innate immunity-deficient NOD mice. METHODS: Toll-like receptor (TLR) 2-deficient, TLR9-deficient and MyD88-deficient NOD mice were used to investigate CHS. The cellular mechanism was determined by flow cytometry in vitro and adoptive cell transfer in vivo. To investigate the role of MyD88 in dendritic cells (DCs) in CHS, we also used CD11cMyD88+ MyD88-/- NOD mice, in which MyD88 is expressed only in CD11c+ cells. RESULTS: We found that innate immunity negatively regulates CHS, as innate immunity-deficient NOD mice developed exacerbated CHS accompanied by increased numbers of skin-migrating CD11c+ DCs expressing higher levels of major histocompatibility complex II and CD80. Moreover, MyD88-/- NOD mice had increased numbers of CD11c+ CD207- CD103+ DCs and activated T effector cells in the skin-draining lymph nodes. Strikingly, re-expression of MyD88 in CD11c+ DCs (CD11cMyD88+ MyD88-/- NOD mice) restored hyper-CHS to a normal level in MyD88-/- NOD mice. CONCLUSION: Our results suggest that the autoimmune-prone NOD genetic background aggravates CHS regulated by innate immunity, through DCs and T effector cells.

Epicutaneous (EC) immunization with type II collagen (COLL II) induces CD4(+) CD8(+) T suppressor cells that protect from collagen-induced arthritis (CIA).

BACKGROUND: We have shown previously that epicutaneous (EC) immunization with protein antigen induces T suppressor cells that alleviate inflammatory response in contact hypersensitivity reactions, in an animal model of multiple sclerosis, and in TNBS-induced colitis. METHODS: DBA/1 mice were EC immunized with type II collagen (COLL II) spread over a gauze patch on days 0 and 4. On day 7, patches were removed and mice were intradermally (id) immunized with COLL II in CFA to induce collagen-induced arthritis (CIA). RESULTS: Our work shows that EC immunization with 100µg of COLL II prior to CIA induction reduces disease severity as determined by macroscopic evaluation. Reduced disease severity after EC immunization with COLL II correlates with milder histological changes found in joint sections. Experiments with the three non-cross-reacting antigens COLL II, ovalbumin (OVA) and myelin basic protein (MBP) showed that skin-induced suppression is antigen non-specific. Transfer experiments show that EC immunization with COLL II induces suppressor cells that belong to the population of CD4(+) CD8(+) double positive lymphocytes. Flow cytometry experiments showed increased percentage of CD4(+) CD8(+) RORγt(+) cells in axillary and inguinal lymph nodes isolated from mice patched with COLL II. CONCLUSION: Maneuver of EC immunization with a protein antigen that induces suppressor cells to inhibit inflammatory responses may become an attractive, noninvasive, needle-free therapeutic method for different clinical situations.

Oral treatment with enrofloxacin early in life promotes Th2-mediated immune response in mice.

BACKGROUND: Th2 lymphocytes play a crucial role in the development of allergy. These pathologies are caused by coordinated production of the cytokines IL-4, IL-5 and IL-13 that regulate the activity of eosinophils, basophils and B cells. According to the 'hygiene hypothesis', the reduced exposure to microorganisms favors allergy occurrence. The advances in medicine in the field of infection therapy promoted an increasing application of antibiotics which, apart from eliminating pathogens, also partially eliminate the microbiota. METHODS: Epicutaneous (EC) immunization with ovalbumin (OVA) followed by OVA challenge was used to study the influence of partial gut flora depletion by oral treatment with enrofloxacin on type-2 immune response. RESULTS: Current work describes the influence of enrofloxacin application on anti-OVA antibody production and cytokine synthesis in young and adult mice. Immune response in adult mice is less sensitive to modification of natural gut flora. We observed that enrofloxacin treatment of adult mice leads to significant decrease of anti-OVA IgG2a production while synthesis of anti-OVA IgE was not changed. The production of type-1 (IFN-γ), type-2 (IL-4, IL-5, IL-10, IL-13) and Th17-associated (IL-17A) cytokines was inhibited. On the other hand, treatment of young mice with enrofloxacin significantly upregulates the production of anti-OVA IgE and inhibits the secretion of anti-OVA IgG2a antibodies. Additionally, treatment with enrofloxacin early in life prior to OVA immunization results in increased production of type-2 (IL-4, IL-10 and IL-13) cytokines. CONCLUSION: Our results clearly indicate that the immune system is more vulnerable to decreased bacterial exposure early in life that may promote development of allergy.