Epicutaneous immunization with TNP-Ig antigen induces CD11c+IL-10+ dendritic cells that promote suppression of Th1-mediated contact hypersensitivity in humanized HLA-DR4 transgenic mice.

The contact hypersensitivity response (CHS) is a mouse model of allergic contact dermatitis in humans. The reaction is classified as type IV hypersensitivity and underlies many autoimmune disorders. Experiments employing the CHS model in wild-type mice showed that the protein antigen applied to the skin in the form of a gauze patch one week before the induction of Th1-dependent CHS was an effective strategy to reduce the inflammatory response in the skin. The approach of epicutaneous (EC) immunization also effectively suppressed the inflammatory response in various mouse models of autoimmune diseases. To evaluate the potential of EC immunization to suppress T cell-dependent immune response in humans, we used HLA-DR4 tg mice, which express the human DRB1*0401 allele and lack all endogenous mouse MHC class II genes. Our data show that EC immunization with TNP-conjugated protein antigen followed by induction of CHS to trinitrochlorobenzene (TNCB), effectively suppressed the CHS response as described by ear swelling, MPO activity in ear extracts, and the number of TCRß+CD4+IFN-γ+ CHS T-effector cells in auxiliary and inguinal lymph nodes (ALN) and spleen (SPL) of HLA-DR4 tg mice. EC-induced suppression increases the frequency of CD11c+IL-10+ DCs in SPL. Their immunoregulatory role was confirmed by s.c. immunization with TNP-CD11c+DCs prior to CHS elicitation and induction. Our data in HLA-DR4 tg mice show that EC protein immunization induces IL-10-producing DCs, which suppress the development of CD4+IFN-γ+ T cell-dependent CHS, implying that EC protein immunization could be of therapeutic importance for T cell-mediated diseases in humans.

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.

Myeloperoxidase Inhibition Ameliorates Plaque Psoriasis in Mice.

Plaque psoriasis is a common inflammatory condition of the skin characterized by red, flaking lesions. Current therapies for plaque psoriasis target many facets of the autoimmune response, but there is an incomplete understanding of how oxidative damage produced by enzymes such as myeloperoxidase contributes to skin pathology. In this study, we used the Aldara (Imiquimod) cream model of plaque psoriasis in mice to assess myeloperoxidase inhibition for treating psoriatic skin lesions. To assess skin inflammation severity, an innovative mouse psoriasis scoring system was developed. We found that myeloperoxidase inhibition ameliorated psoriasis severity when administered either systemically or topically. The findings of this study support the role of oxidative damage in plaque psoriasis pathology and present potential new therapeutic avenues for further exploration.

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.

Neutrophil-Derived Myeloperoxidase Facilitates Both the Induction and Elicitation Phases of Contact Hypersensitivity.

Background: Allergic contact dermatitis (ACD) is a common skin disorder affecting an estimated 15-20% of the general population. The mouse model of ACD is contact hypersensitivity (CHS), which consists of two phases: induction and elicitation. Although neutrophils are required for both CHS disease phases their mechanisms of action are poorly understood. Neutrophils release myeloperoxidase (MPO) that through oxidation of biomolecules leads to cellular damage. Objectives: This study investigated mechanisms whereby MPO contributes to CHS pathogenesis. Methods: CHS was induced in mice using oxazolone (OX) as the initiating hapten applied to the skin. After 7 days, CHS was elicited by application of OX to the ear and disease severity was measured by ear thickness and vascular permeability in the ear. The role of MPO in the two phases of CHS was determined utilizing MPO-deficient mice and a specific MPO inhibitor. Results: During the CHS induction phase MPO-deficiency lead to a reduction in IL-1ß production in the skin and a subsequent reduction in migratory dendritic cells (DC) and effector T cells in the draining lymph node. During the elicitation phase, inhibition of MPO significantly reduced both ear swelling and vascular permeability. Conclusion: MPO plays dual roles in CHS pathogenesis. In the initiation phase MPO promotes IL-1ß production in the skin and activation of migratory DC that promote effector T cell priming. In the elicitation phase MPO drives vascular permeability contributing to inflammation. These results indicate that MPO it could be a potential therapeutic target for the treatment of ACD in humans.

Oral treatment with enrofloxacin creates anti-inflammatory environment that supports induction of tolerogenic dendritic cells.

BACKGROUND: Oral enrofloxacin treatment altered the gut microbiome promoting anti-inflammatory bacteria. The dysbiosis promotes regulatory cell induction in the intestines and in the periphery, which suppresses contact sensitivity. Bacterial-derived signals promote regulatory cell induction both directly and indirectly by influencing the phenotype of dendritic cells (DC). METHODS: Oral treatment with broad-spectrum antibiotic enrofloxacin was used to evaluate how gut flora perturbation shapes the immune response in the gut and the periphery. RESULTS: Enrofloxacin-induced dysbiosis creates an anti-inflammatory environment characterized by increased IL-10 concentration in the gut lumen and tissues. The production of IFN-γ and IL-17A did not change. Oral enrofloxacin treatment skewed the profile of the immune response towards an anti-inflammatory phenotype locally in small intestinal Peyer's Patches (PP) and systematically in the spleen (SPL). Enrofloxacin administration changed immune response in PP by increasing TGF-ß secretion from an increased percentage of TGF-ß-producing. In the SPL, enrofloxacin treatment increased the secretion of TGF-ß and IL-10 and decreased the secretion of IL-17A and IFN-γ. The shift in cytokine profile correlated with a higher percentage of latency-associated peptide and IL-10-producing cells and a decreased percentage of IFN-γ-producing T cells. This anti-inflammatory immune response in the PP and SPL promoted a higher frequency of tolerogenic DC. CONCLUSION: Our data indicate that two-week enrofloxacin treatment induces dysbiosis, skews immune response towards an anti-inflammatory phenotype, and elevates secretion of TGF-ß and IL-10 in the intestines and periphery. Additionally, we observed higher frequencies of tolerogenic DC, characterized by CD11b and IL-10 expression, which are known inducers of Treg cells.

Antibiotics and autoimmune and allergy diseases: Causative factor or treatment?

The newborn infant emerges from an almost sterile environment into a world of bacteria. Bacteria colonize the infant's skin, lungs, and, of most importance, the gut. The process of bacterial colonization is coordinated, and each body niche acquires a unique composition of bacteria. In the gut, most bacteria belong to the Firmicutes and Bacteroidetes phyla, while Actinobacteria and Proteobacteria are far less abundant. Some of these bacteria possess strong immunoregulatory properties. Bacterial colonization is essential to skew the newborn's immune response away from the allergy-favoring Type-2 response towards a Type-1 immune response, which is essential for pathogen elimination. Imbalance between Type 1 and Type 2 responses, however, can promote autoimmunity. In addition, the microbiota shapes immune responses in adults. Autoimmune and allergic diseases are commonly associated with an altered composition of resident bacteria, which is known as dysbiosis. Perhaps the most common cause of disruption and alteration of the bacterial colonization of newborns is the use of antibiotics. It is not known whether the dysbiosis precedes or is the consequence of allergic and autoimmune disorders, and whether antibiotics can be a trigger for these disorders, depending on the type of antibiotic used and the maturity of immune system. In this review, we discuss the development of the microbiota in different body niches and their immunomodulatory potential. We evaluate the impact of antibiotics, both in mice and in humans, on microbial communities and how that may impact the development and manifestation of diseases through all stages of life: the prenatal period, childhood, and adulthood.

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.

Myeloperoxidase: A new player in autoimmunity.

Myeloperoxidase (MPO) is the most toxic enzyme found in the azurophilic granules of neutrophils. MPO utilizes H2O2 to generate hypochlorous acid (HClO) and other reactive moieties, which kill pathogens during infections. In contrast, in the setting of sterile inflammation, MPO and MPO-derived oxidants are thought to be pathogenic, promoting inflammation and causing tissue damage. In contrast, evidence also exists that MPO can limit the extent of immune responses. Elevated MPO levels and activity are observed in a number of autoimmune diseases including in the central nervous system (CNS) of multiple sclerosis (MS) and the joints of rheumatoid arthritis (RA) patients. A pathogenic role for MPO in driving autoimmune inflammation was demonstrated using mouse models. Mechanisms whereby MPO is thought to contribute to disease pathogenesis include tuning of adaptive immune responses and/or the induction of vascular permeability.

Broad spectrum antibiotic enrofloxacin modulates contact sensitivity through gut microbiota in a murine model.

BACKGROUND: Medical advances in the field of infection therapy have led to an increasing use of antibiotics, which, apart from eliminating pathogens, also partially eliminate naturally existing commensal bacteria. It has become increasingly clear that less exposure to microbiota early in life may contribute to the observed rise in "immune-mediated" diseases, including autoimmunity and allergy. OBJECTIVE: We sought to test whether the change of gut microbiota with the broad spectrum antibiotic enrofloxacin will modulate contact sensitivity (CS) in mice. METHODS: Natural gut microbiota were modified by oral treatment with enrofloxacin prior to sensitization with trinitrophenyl chloride followed by CS testing. Finally, adoptive cell transfers were performed to characterize the regulatory cells that are induced by microbiota modification. RESULTS: Oral treatment with enrofloxacin suppresses CS and production of anti-trinitrophenyl chloride IgG1 antibodies. Adoptive transfer experiments show that antibiotic administration favors induction of regulatory cells that suppress CS. Flow cytometry and adoptive transfer of purified cells show that antibiotic-induced suppression of CS is mediated by TCR αß+CD4+CD25+FoxP3+ Treg, CD19+B220+CD5+ IL-10+, IL-10+ Tr1, and IL-10+ TCR γδ+ cells. Treatment with the antibiotic induces dysbiosis characterized by increased proportion of Clostridium coccoides (cluster XIVa), C coccoides-Eubacterium rectale (cluster XIVab), Bacteroidetes, and Bifidobacterium spp, but decreased segmented filamentous bacteria. Transfer of antibiotic-modified gut microbiota inhibits CS, but this response can be restored through oral transfer of control gut bacteria to antibiotic-treated animals. CONCLUSIONS: Oral treatment with a broad spectrum antibiotic modifies gut microbiota composition and promotes anti-inflammatory response, suggesting that manipulation of gut microbiota can be a powerful tool to modulate the course of CS.

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.

Epicutaneous immunization with TNP-Ig and Zymosan induces TCRαß+ CD4+ contrasuppressor cells that reverse skin-induced suppression via IL-17A.

BACKGROUND: Our previous work showed that epicutaneous (EC) immunization with protein antigen e.g. TNP-conjugated mouse immunoglobulin (TNP-Ig) in the form of a patch prior to hapten sensitization inhibits Th1-mediated contact hypersensitivity (CHS) in mice. We also found that suppression of CHS was mediated by TCRαß+ CD4+ CD8+ T suppressor cells producing TGF-ß. The aim of this study was to investigate the role of innate immunity in the suppression of CHS. METHODS: Mice were immunized by applying gauze patches containing protein antigen alone or in the presence of zymosan, and were then tested for the CHS response. Adoptive cell transfer experiments were used to study the mechanisms involved in the reversal of skin-induced suppression. The influence of EC immunization on cytokine production by lymph node cells was measured by ELISA. RESULTS: We found that EC immunization with TNP-Ig and zymosan before trinitrophenyl chloride sensitization reverses skin-induced suppression, demonstrated in vivo and in vitro. The reversal of skin-induced suppression was transferable by antigen-specific TCRαß+ CD4+ T contrasuppressor cells. Furthermore, we showed that the contrasuppression was IL-17A-dependent and TLR2- and MyD88-independent. CONCLUSIONS: Our work strongly suggests that EC immunization with protein antigen and zymosan reverses skin-induced suppression and that this approach may be a potential tool to increase the immunogenicity of weakly immunogenic antigens.

Epicutaneous immunization with protein antigen TNP-Ig and NOD2 ligand muramyl dipeptide (MDP) reverses skin-induced suppression of contact hypersensitivity.

BACKGROUND: Epicutaneous (EC) immunization offers a new method of a needle-free and self-administrable immunization by using a topically applied patch to deliver vaccine. We have previously shown that EC immunization with hapten-conjugated protein antigen TNP-Ig prior to hapten sensitization inhibits Th1-mediated contact hypersensitivity (CHS) in mice. Our further work showed that EC immunization with TNP-Ig and Toll-like receptor (TLR) ligands prior to hapten sensitization reverses skin-induced suppression. METHODS: Animal model of contact hypersensitivity was used to study reversal of skin-induced suppression. RESULTS: Current work showed that EC immunization with protein antigen TNP-Ig and MDP NOD2 agonist - muramyldipeptide (L isoform) reverses skin-induced suppression of CHS. On the other hand L18-MDP NOD2 agonist - muramyldipeptide with a C18 fatty acid chain and MDP control - negative control for MDP - muramyldipeptide (D isoform, inactive) did not reverse skin-induced suppression. "Transfer in" experiment showed that reversal of skin-induced suppression can be adoptively transferred with lymphoid cells isolated from donors EC treated with TNP-Ig and MDP NOD2 agonist. Moreover, experiment employing two non-cross-reacting antigens TNP-Ig and OX-Ig proved that reversal of skin-induced suppression is antigen specific. Additionally, lymph node cells isolated from mice EC immunized with TNP-Ig and MDP NOD2 agonist produced increased level of IFN-γ suggesting that this cytokine might be involved in reversal of skin-induced suppression. CONCLUSION: This work shows that EC immunization with protein antigen plus NOD2 ligand MDP may be a potential tool to increase the immunogenicity of weekly immunogenic antigens.

GammadeltaT cells positively regulate contact sensitivity (CS) reaction via modulation of INF-gamma, IL-12 and TNF-alpha production.

The gammadeltaT cells were identified as positive as well as negative regulators of immune responses. They take part in pathogen clearance, modulation of innate and adaptive immunity as well as in healing and tissue maintenance. The course of many pathological conditions such as collagen induced arthritis (CIA), experimental autoimmune encephalomyelitis (EAE) and airway hyperresponsiveness is positively regulated by gammadeltaT cells. It was shown previously that contact sensitivity (CS), an example of antigen-specific cell-mediated immune response, is also positively regulated by gammadeltaT cells. The current work confirmed the regulatory function of gammadeltaT cells in CS response as their depletion with anti-TCRdelta monoclonal antibody and complement significantly decreased adoptive transfer of the CS reaction. In vitro study showed that removal of gammadeltaT cells with magnetic beads significantly decreased the production of the proinflammatory cytokines IFN-gamma, IL-12 and TNF-alpha. Reconstitution of gammadeltaT-depleted cells with gammadeltaT-enriched cells restored cytokine production, proving the reversibility of the investigated process. In summary, gammadeltaT cells positively regulate the CS reaction via modulation of proinflammatory cytokine production.

[Influence of natural gut flora on immune response]. / Wplyw naturalnej flory jelitowej na odpowiedz immunologiczna.

Our intestines are habitat for trillions of microorganisms such as bacteria, viruses and eukaryotes, known as microbiota. They are indispensable for our well-being due to their metabolic activities. Microbiota digests complex plant polysaccharides, which are normally unprocessed by humans; as well it retrieves other essential nutrients. It is well established that microbiota is crucial for proper development of intestinal as well systemic immune compartments. Recent results indicate that composition of natural gut flora is responsible for shaping of immune response. Alerted bacterial profile, known as dysbiosis precedes development of allergy in children. Many autoimmune conditions are associated with shift in intestinal bacterial profile. Apart of direct association between gut flora and systemic immune compartment little is known about the mechanisms by which microbiota exerts its immunoregulatory function. At the moment several bacterial strains as well some bacterial products were recognized as immunomodulators. This review describes the composition of normal gut flora as well disease-associated microbiota. It deals with unique mechanisms, found in GALT, that favor induction of tolerance towards orally administrated antigens as well discriminate between commensal and pathogens to minimize induction of inflammatory response. Further, the review tries to establish the connection between microbiota and systemic immune response. Finally the factors that modulate the composition of our gut flora are described.