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.

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.

TRIF deficiency protects non-obese diabetic mice from type 1 diabetes by modulating the gut microbiota and dendritic cells.

The incidence of type 1 diabetes (T1D) is determined by both genetic and environmental factors. In recent years, the gut microbiota have been identified to be an important environmental factor that could modify diabetes susceptibility. We have previously shown that Myeloid differentiation primary response gene 88 (MyD88), a major adaptor protein downstream of most innate immune Toll-like receptor (TLR) signaling, is important for mediating diabetes susceptibility in the non-obese diabetic (NOD) mouse model of human T1D. Here we report the role of TIR-domain-containing adapter-inducing interferon-ß (TRIF) in T1D development, as TRIF is an important adaptor protein downstream of TLR3 and TLR4 signaling. We found that TRIF-deficient (TRIF-/-) NOD mice were protected from development of diabetes, but only when housed with TRIF-deficient (TRIF-/-) NOD mice. When housed with TRIF-sufficient wild type (WT, i.e., TRIF+/+) NOD mice, the mice developed diabetes. We further investigated the gut microbiota as a potential cause for the altered diabetes development. Interestingly, TRIF-/-NOD mice had a different microbiota composition compared to WT NOD mice, only if they were housed with TRIF-/-NOD mice. However, the composition of gut microbiota in the TRIF-/-NOD mice was indistinguishable from WT NOD mice, if they were housed with WT NOD mice. The difference in the gut microbiota in TRIF-/-NOD mice, due to cohousing, accorded with the diabetes development in TRIF-/-NOD mice. Comparing the gut microbiota in TRIF-/- and WT NOD mice, we identified changes in percentage of Sutterella, Rikenella and Turicibacter species. Moreover, bacteria from WT NOD mice induced significantly stronger inflammatory immune responses in vitro compared to those from TRIF-/-NOD mice. Further immunological analysis revealed impaired function of dendritic cells and reduced T cell activation and proliferation in TRIF-/-NOD mice. Our data show that TRIF-deficiency protects NOD mice from diabetes development through alteration of the gut microbiota and reduced immune cell activation; however, that protection is over-ridden upon exposure to WT NOD bacteria. Therefore exposure to different microbiota can modify disease susceptibility determined by genetic factors related to innate immunity.

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.

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.

Epicutaneous immunization with ovalbumin and CpG induces TH1/TH17 cytokines, which regulate IgE and IgG2a production.

BACKGROUND: Subcutaneous allergen-specific immunotherapy is a standard route for the immunotherapy of allergic diseases. It modulates the course of allergy and can generate long-term remission. However, subcutaneous allergen-specific immunotherapy can also induce anaphylaxis in some patients, and therefore additional routes of administration should be investigated to improve the safety and tolerability of immunotherapy. OBJECTIVE: We sought to determine whether epicutaneous treatment with antigen in the presence of a Toll-like receptor 9 agonist can suppress TH2-mediated responses in an antigen-specific manner. METHODS: Epicutaneous immunization was performed by applying a skin patch soaked with ovalbumin (OVA) plus CpG, and its suppressor activity was determined by using the mouse model of atopic dermatitis. Finally, adoptive cell transfers were implemented to characterize the regulatory cells that are induced by epicutaneous immunization. RESULTS: Epicutaneous immunization with OVA and CpG reduces the production of OVA-specific IgE and increases the synthesis of OVA-specific IgG2a antibodies in an antigen-specific manner. Moreover, eosinophil peroxidase activity in the skin and production of IL-4, IL-5, IL-10, and IL-13 are suppressed. The observed reduction of IgE synthesis is transferable with T-cell receptor (TCR) αß(+)CD4(+)CD25(-) cells, whereas IgG2a production is dependent on both TCRαß(+) and TCRγδ(+) T cells. Further experiments show that the described phenomenon is myeloid differentiation primary response 88, IFN-γ, and IL-17A dependent. Finally, the results suggest that epicutaneous immunization with OVA and CpG decreases the synthesis of OVA-specific IgE and skin eosinophil peroxidase activity in mice with ongoing skin allergy. CONCLUSION: Epicutaneous application of protein antigen in the presence of adjuvant could be an attractive needle-free and self-administered immunotherapy for allergic diseases.

Expression of activation-induced cytidine deaminase enhances the clearance of pneumococcal pneumonia: evidence of a subpopulation of protective anti-pneumococcal B1a cells.

We describe a protective early acquired immune response to pneumococcal pneumonia that is mediated by a subset of B1a cells. Mice deficient in B1 cells (xid), or activation-induced cytidine deaminase (AID(-/-) ), or invariant natural killer T (iNKT) cells (Jα18(-/-) ), or interleukin-13 (IL-13(-/-) ) had impaired early clearance of pneumococci in the lung, compared with wild-type mice. In contrast, AID(-/-) mice adoptively transferred with AID(+/+) B1a cells, significantly cleared bacteria from the lungs as early as 3 days post infection. We show that this early bacterial clearance corresponds to an allergic contact sensitivity-like cutaneous response, probably due to a subpopulation of initiating B1a cells. In the pneumonia model, these B1a cells were found to secrete higher affinity antigen-specific IgM. In addition, as in contact sensitivity, iNKT cells were required for the anti-pneumococcal B1a cell initiating response, probably through early production of IL-13, given that IL-13(-/-) mice also failed to clear infection. Our study is the first to demonstrate the importance of AID in generating an appropriate B1a cell response to pathogenic bacteria. Given the antibody affinity and pneumonia resistance data, natural IgM produced by conventional B1a cells are not responsible for pneumonia clearance compared with the AID-dependent subset.

[Skin as a place of immunomodulation].

The skin is the largest organ of the human body which plays a key role not only in physiological processes such as thermoregulation and the maintenance of the correct water and electrolyte balance, but also forms an effective barrier against microorganisms, protecting the organism against harmful external factors. Moreover, the skin is an important organ involved in immune mechanisms. The skin is a place of drug application to treat various local and systemic diseases. Those drugs are proteins and peptides sensitive to digestion in gastrointestinal tract, or chemical molecules metabolized in the liver. Epicutaneous immunization (EC) is a new therapeutic method used to treat immunological disorders. The phenomenom of immunomodulation has already been exploited to treat allergic diseases and is called allergen specific immunotherapy. The authors describe the current state of knowledge of the immune regulatory mechanisms that allow to use the EC method to treat immunological diseases mediated by Th1, Tc1 or Th2 lymphocytes. The new aspect of this immunotherapy is immunopotentiation that involves components of the innate immune system, working via the TLRs. The first clinical trials give promising outcomes of the use of EC method in the treatment of multiple sclerosis and respiratory allergic diseases.

Epicutaneous Immunization with Collagen Induces TCRαß Suppressor T Cells That Inhibit Collagen-Induced Arthritis.

BACKGROUND: We have shown previously, in an animal model of multiple sclerosis and in TNBS-induced colitis, that epicutaneous (EC) immunization with protein antigen induces T suppressor cells that strongly inhibit the inflammatory response in contact hypersensitivity reactions. METHODS: EC immunization was performed by applying to the shaved skin of the mouse dorsum a gauze patch soaked with a solution containing various amounts of type II collagen (COLL II) in a volume of 100 µl of PBS on days 0 and 4. On day 7 the patches were removed and mice were intradermally (i.d.) immunized with COLL II to induce collagen-induced arthritis (CIA). RESULTS: Our study shows that EC immunization with 100 or 30 µg of COLL II reduces disease severity, whereas lower doses (10 or 3 µg) do not affect CIA. Decreased disease severity observed after EC immunization with COLL II correlates with reduced myeloperoxidase activity in joint tissue and with reduced production of anti-citrullinated protein and anti-COLL II IgG2a antibodies. Transfer experiments show that EC immunization with COLL II induces suppressor cells that belong to the population of TCRαß lymphocytes and that EC-induced suppression declines with time. Both in vitro and in vivo experiments show that IL-17A plays an important role in EC-induced suppression of CIA. EC application of COLL II at the first signs of CIA also results in disease suppression. CONCLUSIONS: The suppression of inflammatory responses by T suppressor cells induced through EC immunization of a protein antigen may become an attractive noninvasive therapeutic method for a variety of clinical situations.

[Immunological mechanisms involved in obesity and their role in metabolic syndrome]. / Mechanizmy immunologiczne towarzyszace otylosci i ich rola w zaburzeniach metabolizmu.

In the past 50 years, the occurrence of human obesity has risen dramatically across the globe. The WHO reported that at least 1.9 billion (1.9 × 10(9)) adults are overweight and 600 million are obese, and the numbers are expected to rise dramatically in the future without intervention. The recent increase in human obesity is caused by increased energy intake and reduced energy expenditure that results in a massive increase in adipose tissue, which is generally harmful to our health. Indeed, the increase in human obesity is strongly associated with an increase in many diseases such as type 2 diabetes (T2D), biliary disease, cardiovascular disease, hepatic steatosis, airway disease, neurodegeneration and certain cancers. The metabolic and immune systems are closely linked and functionally dependent. As a result, excessive nutrient consumption associated with obesity can be recognized as a harmful, stress-inducing biological event by innate pattern recognition receptors (PRRs). This activates inflammatory and stress responses in various metabolic tissues, leading to the chronic low-grade inflammation called metabolic inflammation or "metainflammation". Adipose tissue is mainly composed of adipocytes, although other cell types contribute to its growth and function, including pre-adipocytes, macrophages, lymphocytes, fibroblasts and vascular cells. Obesity can result in profound changes in the cell composition of fat tissue and can lead to the modulation of individual cell phenotypes. Many factors are involved in development of metainflammation, including hypoxia of adipocytes, oxidative stress, endoplasmic reticulum stress, activation of inflammasomes, adipocyte death, activation of TLR and abnormal gut flora.

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.

Lack of TCRalphabeta+ CD8+ and TCRgammadelta+ lymphocytes ameliorates LPS induced orchitis in mice--preliminary histological observations.

The inflammation of the reproductive system can affect reproduction causing partial or complete infertility. It is well known that lipopolysaccharide (LPS) triggers an inflammatory response in the whole organism, including immunologically privileged organs, e.g. the testicles. Adult male TCRalpha-/-, TCRdelta-/-, CD1d-/- and beta2m-/- on B10.PL (H-2(u)) and B10.PL control mice were intraperitonealy (i.p.) injected with lipopolysaccharide (LPS). The animals were killed 24h and 10 days post LPS treatment and their gonads were prepared for microscopic examination. Histological changes in the testes after LPS injection were found only in control B10PL and CD1d-/- mice. The experiments revealed disturbances in Leydig's glands structure, blood vessel dilatation in the interstitial tissue as well as degeneration of seminal tubule epithelium, disruption ofspermatogenesis and subsequent decrease of sperm cell number in the tubule lumen. These changes were noticed mainly 10 days after LPS treatment. Lack of either TCRalphabeta+ CD8+ or TCRgammadelta+ lymphocytes diminishes the response of testicular macrophages to LPS whereas the absence of CD1d-dependent NKT cells does not affect macrophage reactivity.

Natural killer cell-mediated contact sensitivity develops rapidly and depends on interferon-α, interferon-γ and interleukin-12.

Natural killer (NK) cell-mediated contact sensitivity was recently described in mice. Here, we confirm NK cell-mediated contact sensitivity (CS) in SCID and RAG1(-/-) mice but not in SCIDbeige mice, which have non-functional NK cells that lack NK cell granules. NK cell-mediated CS was transferred by liver mononuclear cells and the DX5(+) fraction of liver cells, confirming that NK cells mediate CS in the absence of T and B cells. Participation of NKT cells and B-1 cells was ruled out using Jα18(-/-) and JH(-/-) mice, respectively. Remarkably, NK cell-mediated CS was observed just 1 hr after immunization and was detectable as early as 30 min after challenge. Further, we examined cytokine requirements for NK cell-mediated CS, and found that liver mononuclear cells from interleukin-12(-/-) , interferon-γ(-/-) and interferon-α receptor(-/-) donors fail to transfer NK cell-mediated CS to naive hosts. Our studies clearly show that dinitrofluorobenzene sensitized NK cells mediate very rapid, antigen-specific cell-mediated immunity, with features of both innate and acquired immune responses.

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.

Down-regulation of macrophage immune activity by natural CD8+ regulatory T cells.

To evaluate the influence of natural regulatory CD8+ T cells on macrophages we investigated in vitro production of cytokines, reactive oxygen intermediates (ROIs) and expression of CD80 surface costimulatory molecules by macrophages (MF) of wild type (WT) B10PL and syngeneic knock-out (KO) strains, TCRalpha-/-, beta2m-/- and CD1d-/- mice. MF of TCR alpha-/- (CD4- and CD8-) and beta2m-/- (CD8-) animals produced higher levels ofTNF-a, IL-6, IL-12 and ROIs and showed increased expression of CD80 costimulatory molecules in comparison to MF of WT or CD1d-/- (NKT-) mice. When MF of these strains were conjugated with TNP hapten and injected i.v. into WT mice to test either induction of contact sensitivity (CS) or tolerance, only TNP-MF of TCRalpha-/- and beta2m-/- animals induced a significant CS reaction, while cells of WT and CD1d-/- strains were tolerogenic. MF of the tested strains can be classified functionally as resembling either proinflammatory (TCRalpha-/- and beta2m-/-mice) or immunosuppressive (WT and CD1d-/-) phenotypes. We suggest the presence of an in vivo regulatory loop in which innate CD8+ Treg cells control the transition between MF phenotypes and thus adjust the magnitude of the inflammatory response to strictly local requirements.

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.

Epicutaneous immunization with DNP-BSA induces CD4+ CD25+ Treg cells that inhibit Tc1-mediated CS.

As we have shown previously that protein antigen applied epicutaneously (EC) in mice inhibits TNP-specific Th1-mediated contact sensitivity (CS), we postulated that the maneuver of EC immunization might also suppress Tc1-dependent CS response. Here we showed that EC immunization of normal mice with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) applied on the skin in the form of a patch induces a state of subsequent unresponsiveness due to regulatory T cells (Treg) that inhibited sensitization and elicitation of effector T-cell responses. Suppression is transferable in vivo by TCRαß(+) CD4(+) CD25(+) lymphocytes harvested from lymph nodes (LNs) of skin-patched animals. Flow cytometry revealed that EC immunization with DNP-BSA increased TCRαß(+) CD4(+) CD25(+) FoxP3(+) lymphocytes in subcutaneous LNs, suggesting that observed suppression was mediated by Treg cells. Further, in vitro experiments showed that EC immunization with DNP-BSA prior to 1-fluoro-2,4-dinitrobenzen sensitization suppressed LN cell proliferation and inhibited production of TNF-α, IL-12 and IFN-γ. Using a transwell system or anti-CTLA-4 mAb, we found that EC induced suppression required direct Treg-effector cell contact and is CTLA-4-dependent.

Contact Hypersensitivity as a Murine Model of Allergic Contact Dermatitis.

Contact hypersensitivity (CHS) is an experimental model of allergic contact dermatitis (ACD) that can be studied in mice. This study aims to present an objective laboratory method that may help to study the CHS reaction in mice, which can be measured and quantified by various tests. To induce CHS, on day "0", mice were sensitized on a previously shaved spot by abdominal skin painting with the hapten 2,4,6-trinitrochlorobenzene (TNCB) in an acetone-ethanol mixture, whereas negative control mice were sham sensitized with vehicle alone-acetone-ethanol mixture. On day "4", the baseline ear thickness was measured with a micrometer prior to the elicitation of CHS (challenge) by painting both ears with diluted TNCB both in the test and control groups. After 24 h, the ear swelling was measured with a micrometer. CHS is an example of a T cell-mediated immune response that causes swelling in inflamed tissue, peaking 24 h after the skin challenge with the same hapten. An increase in ear edema correlated with augmented ear weight, myeloperoxidase (MPO) activity, pro-inflammatory cytokine concentration in the ear extracts, increased thickening of the edematous dermis in the histological examination, and ear vascular permeability. There was also an increase in the concentration of TNP-specific IgG1 antibodies in the sera of the test group when compared with the control mice. Additionally, CHS can be successfully transferred with the CHS-effector cells obtained from donors previously sensitized with TNCB. The CHS-effector cells were administered intravenously into naïve recipient mice, which were subsequently challenged with the same diluted hapten. Ear swelling was measured with a micrometer 24 h later.

Post-operative complications in patients with a significant post-operative decrease in γδT cells.

BACKGROUND: It has previously been shown that appropriate distribution of immune cells between different tissues and organs of the body is required for proper function of the immune system. Our previous work demonstrated that surgical trauma in mice induces γδT lymphocyte migration from peripheral blood to the peritoneal lymphoid organs. These described γδT cells have immunoregulatory activity as they suppress the cell-mediated immune response in vitro. We found a similar phenomenon in patients after different surgical operations. In the current study, we analyse post-operative complications in patients with a significant post-operative decrease in γδT cells. METHODS: We investigated the percentage of γδT cells in peripheral blood of patients undergoing standard surgical procedures (gastric resection, colorectal resection, cholecystectomy and strumectomy) before and 3 days after the operation. The percentage of γδT cells was evaluated by the fluorescence-activated cell sorting cytofluorimeter. Patients were grouped based on the decrease of γδT cells. We compared the number of septic complications in patients with a large and small decrease in γδT cells. RESULTS: After major surgery in the peritoneal cavity (gastric and colorectal surgery), in the group that had a large decrease in γδT cells we found significantly more septic complications than in the group of patients with small γδT decrease. That effect was not visible after less traumatic surgery. CONCLUSION: Surgery results in a decreased percentage of γδT lymphocytes in the peripheral human blood which correlates with the number of septic complications. This observation may help to predict post-operative recovery after gastroabdominal surgery.