Monika Majewska-Szczepanik

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.

Partial depletion of natural gut flora by antibiotic aggravates collagen induced arthritis (CIA) in mice

BACKGROUND Rheumatoid arthritis (RA) is a chronic inflammatory disease that affects about 1% of the adult population and occurs twice as frequently among women than men. At present it is accepted that pathogenesis of RA is based on inflammatory response mediated by CD4(+) Th1 and Th17 lymphocytes. The most commonly applied model imitating RA is the collagen induced arthritis (CIA). A growing evidence shows that there is a correlation between microbial dysbiosis and human pathology which includes autoimmunity, allergic diseases, obesity, inflammatory bowel disease (IBD), metabolic syndrome. METHODS Collagen induced arthritis was used to study influence of natural gut flora on course of rheumatoid arthritis. RESULTS Current work employing CIA model showed that partial depletion of natural gut flora with orally administered antibiotic Baytril (enrofloxacin) aggravates disease severity when compared to control mice. Observed partial depletion of both aerobic and anaerobic bacteria did not affect animal body weight. Additionally, in vitro study showed increased production of IFN-? and IL-17A and decreased release of IL-4 by axillary lymph node cells (ALNC) isolated from mice treated with antibiotic and induced CIA when compared to positive control. Furthermore, treatment with antibiotic prior to CIA induction results in augmented production of IFN-?, IL-17A and IL-6 by mesenteric lymph node cells (MLNC). CONCLUSION Presented data suggest that alteration of gut microbiota via use of enrofloxacin may play a role in modulating arthritis symptom severity in this mouse model.

Participation of iNKT cells in the early and late components of Tc1-mediated DNFB contact sensitivity: cooperative role of γδ-T cells.

Prior studies of classical 24 h responses in TNP‐Cl (picryl chloride) allergic contact sensitivity (CS), showed mediation by Th1 cells in CBA mice, and established that 24 h elicitation of responses requires an early 2 h CS‐initiating component dependent on iNKT cells, IL‐4 and B‐1 B cells. Here, we studied the other form of cytotoxic T cell (Tc1) CS in DNFB sensitized BALB/c mice and determined that similar CS‐initiation also is required. We systematically tested each step of the initiation pathway in this model. Thus, DNFB Tc1 CS was significantly impaired in iNKT cell deficient CD1d−/− and Jα18−/− mice, IL4Rα−/− and STAT‐6−/− mice, and also in pan B‐cell deficient JH−/− mice. Further, the Tc1 DNFB CS‐initiating component, like Th1 response to TNP‐Cl, was elicited by only 1‐day after immunization, due to B‐1 cells. In summary, we show that CS‐Initiation also is required in Tc1 CS. Further, we have newly determined regulatory support of both the early and late components of DNFB induced Tc1 CS by iNKT cells and γδ‐T cells. In summary, both iNKT cells and assisting γδ‐T cells are involved in initiating and effector phases of DNFB induced CS.

Stimulatory Lipids Accumulate in the Mouse Liver within 30 min of Contact Sensitization to Facilitate the Activation of Naïve iNKT Cells in a CD1d-Dependent Fashion

Natural killer T cells with invariant αβ‐T cell receptors (TCRs) (iNKT cells) constitute a lipid‐responsive arm of the innate immune system that has been implicated in the regulation or promotion of various immune, infectious and neoplastic processes. Contact sensitivity (CS), also known as contact hypersensitivity or allergic contact dermatitis, is one such immune process that begins with topical sensitization to an allergen and culminates in a localized cutaneous inflammatory response after challenge with the same allergen. CS depends on events initiated early in sensitization by hepatic iNKT cells. We have shown previously that these iNKT cells release IL‐4 early after skin sensitization to activate B‐1 B cells to produce IgM antibodies that aid in local recruitment of the effector T cells. Here, we utilize adoptive transfer techniques in several strains of knockout mice to demonstrate that hepatic lipids isolated 30 min after sensitization are significantly more stimulatory to naïve hepatic iNKT cells than hepatic lipids isolated after sham sensitization. These stimulatory hepatic lipids specifically affect iNKT cells and not B‐1 B cells. The downstream CS response is abrogated with anti‐CD1d‐blocking antibodies, suggesting a critical role of CD1d in the activation of hepatic iNKT cells with these lipids. Hepatocytes may not be essential, as donor hepatic iNKT cells can reconstitute CS without migrating to the recipient mouse liver. Rather, CD1d‐expressing liver mononuclear cells are sufficient for activation of iNKT cells. In conclusion, stimulatory lipids accumulate in the liver soon after sensitization and facilitate iNKT cell activation in a CD1d‐dependent yet potentially hepatocyte‐independent manner.

Inhibitory effect of antidepressant drugs on contact hypersensitivity reaction

BACKGROUND Contact hypersensitivity (CS) reaction in the skin is T-cell mediated immune reaction which plays a major role in the pathogenesis and chronicity of various inflammatory skin disorders and, like other delayed-type hypersensitivity (DTH) reactions, affords immunity against tumor cells and microbes. CS response is a self-limiting reaction, and interleukin (IL)-10 is considered to be a natural suppressant of cutaneous inflammatory response. Recently, it has been demonstrated that major depression is related to activation of the inflammatory response and elevation of some parameters of cell-mediated immunity. It has been suggested that such activation of the immune system may play a role in etiology of depression. If this immunoactivation is involved in etiology of depression, one would expect that antidepressant agents may have negative immunoregulatory effects. To the best of our knowledge, the effect of antidepressants on contact hypersensitivity has not been studied. METHODS The aim of the present study was to establish the effect of prolonged desipramine or fluoxetine treatment on CS reaction to picryl chloride. RESULTS Antidepressants significantly suppressed CS reaction, fluoxetine by 53% whereas desipramine by 47% compared to positive control. Moreover, desipramine and fluoxetine decreased relative weight of auxillary lymph nodes. Desipramine decreased also relative weight of inguinal lymph nodes and spleens whereas desipramine and fluoxetine increased production of IL-10 in comparison to positive control. CONCLUSION The observed effect of antidepressant drugs on CS reaction is consistent with the hypothesis that T-cell mediated immunity is targeted by antidepressants.

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 TCRαβ+ CD4+ CD25+ lymphocytes harvested from lymph nodes (LNs) of skin‐patched animals. Flow cytometry revealed that EC immunization with DNP‐BSA increased TCRαβ+ 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.

IRAK-M Deficiency Promotes the Development of Type 1 Diabetes in NOD Mice

Type 1 diabetes mellitus (T1DM) is an organ-specific autoimmune disease characterized by progressive destruction of insulin-secreting pancreatic β-cells. Both T-cell–mediated adaptive responses as well as innate immune processes are involved in pathogenesis. Interleukin-1 receptor–associated kinase M (IRAK-M) can effectively inhibit the MyD88 downstream signals in Toll-like receptor pathways, while lack of IRAK-M is known to be associated with autoimmunity. Our study showed that IRAK-M–deficient (IRAK-M−/−) nonobese diabetic (NOD) mice displayed early onset and rapid progression of T1DM with impaired glucose tolerance, more severe insulitis, and increased serum anti-insulin autoantibodies. Mechanistic studies showed that the enhanced activation and antigen-presenting function of IRAK-M−/− antigen-presenting cells from IRAK-M−/− mice were responsible for the rapid progression of disease. Moreover, IRAK-M−/− dendritic cells induced enhanced activation of diabetogenic T cells in vitro and the rapid onset of T1DM in vivo in immunodeficient NOD mice when cotransferred with diabetogenic T cells. This study illustrates how the modulation of innate immune pathways through IRAK-M influences the development of autoimmune diabetes.

Inhibition of 2,4-dinitrofluorobenzene-induced contact hypersensitivity reaction by antidepressant drugs

BACKGROUND Contact hypersensitivity (CHS) induced by a topical application of hapten - 2,4-dinitrofluorobenzene (DNFB), is a T cytotoxic (Tc)1-cell-mediated antigen-specific type of skin inflammation. Recently, it has been shown that antidepressant drugs inhibit the T helper (Th)1-mediated CHS reaction induced by picryl chloride. The aim of present study was to establish the effect of two-week desipramine or fluoxetine administration on the CHS reaction induced by DNFB. METHODS Balb/c (H-2(d)) male mice were divided into six groups: 1) vehicle-treated negative control group; 2) desipramine-treated negative control group; 3) fluoxetine-treated negative control group; 4) vehicle-treated DNFB group (positive control group); 5) desipramine-treated DNFB group; 6) fluoxetine-treated DNFB group. T lymphocytes proliferation was determined by incorporation of [(3)H]-thymidine to DNA of concanavalin A stimulated cells. ELISA test was used for estimation of cytokines production. RESULTS The antidepressants significantly suppressed the CHS reaction mediated by Tc1 cells: desipramine by 55% and fluoxetine by 54% compared to the positive control. Moreover, the antidepressants decreased the proliferative activity of splenocytes and the ability of splenocytes to produce interleukin (IL)-6 and interferon (IFN)-γ and increased IL-10 production by the lymph node (LN) cells of DNFB-treated mice. CONCLUSION The results of the present study show that the Tc1-dependent reactivity to DNFB is significantly suppressed by antidepressant drugs, which suggests their inhibitory effect on Tc1 mediated immunity.

Epicutaneous immunization with hapten-conjugated protein antigen alleviates contact sensitivity mediated by three different types of effector cells.

BACKGROUND Allergic contact dermatitis (ACD) is a common clinical condition in industrialized countries and often causes occupational diseases. Animal model of contact sensitivity (CS) is commonly used to study ACD in mice and can be induced by skin application of haptens. It has been previously shown that CS is mediated by CD4(+) or CD(8+) T effector cells. More recently it was found that also liver NK cells can play a role of CS effector cells in mice. METHODS The aim of the present study was to test whether skin-induced suppression could inhibit CS response in vivo. RESULTS Here we show that EC immunization of normal mice with hapten conjugated protein antigen prior to hapten sensitization suppresses Th1, Tc1 and NK mediated CS responses. CONCLUSIONS These data strongly suggest that maneuver of EC immunization may have important implications for designing therapeutic schemes aimed at modulating unwanted immune responses in contact hypersensitivity.

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 &agr;&bgr;+CD4+CD25+FoxP3+ Treg, CD19+B220+CD5+ IL‐10+, IL‐10+ Tr1, and IL‐10+ TCR &ggr;&dgr;+ 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. Graphical abstract Figure. No Caption available.