Name: Murine Model of Epicutaneously-Induced Immunomodulation (Video) | JoVE Journal
Uploaded: 2025-06-24T13:00:00.000+00:00
Duration: 9 min 7 s
Description: Learn epicutaneous immunization for skin-induced immunomodulation using a murine model of allergic contact dermatitis. This needle-free method generates T suppressor cells to regulate immune responses effectively.

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This study was supported by grants from Polish Committee of Scientific Research Grants 3 PO5B 091 25, 2PO5A 204 29 and from National Science Center N N401 3554 33 to M.S. The graphs were drawn using GraphPad Prism software 10.2.3, and the figures 1-3 were created in BioRender. Zemelka-Wiacek, M. (2025): (1) https://BioRender.com/3ux2px ; (2)&#160; https://BioRender.com/s0y8zs7 ; (3) https://BioRender.com/0qziglw. The authors thank Dorota Wo&#378;niak for taking care of the mouse colonies.

Authors have nothing to disclose.

The investigations were conducted on many different animal models and using many mouse strains like BALB/c, C57BL/6, HLADR4-Tg, DBA 1, and B10.PL and SJL demonstrate that EC immunization with an assigned antigen can serve as a versatile and potentially limitless means of creating tolerance and controlling unwanted immune responses. The current protocol outlines two cell transfer models: (1) &#39;adoptive transfer IN,&#39; which is useful for assessing immunomodulation during both the afferent and efferent phases of CHS, ...

This article presents a method for inducing tolerance (referred to as &#39;suppression&#39; throughout this text) in a mouse model of contact hypersensitivity (CHS) and for reversing this tolerance (referred to as &#39;contrasuppression&#39; throughout this text) via epicutaneous (EC) application of an antigen alone or in combination with ligands for pathogen recognition receptors (PRRs), respectively1. In described protocol (a) haptenized mouse immunoglobulins (TNP-Ig) are used as antigen&#160;to suppress contact hypersensitivity or (b) antigen&#160;with pathogen-associated molecular patterns (PAMPs), ligands for Toll-like receptors (TLR) and nucleotide-binding oligomerization domain-containing protein (NOD)-like receptors (NLRs), to reverse skin-induced suppression2,3,4. The findings indicate that EC-induced immunomodulation is mediated by antigen-non-specific T suppressor (Ts) cells and antigen-specific T contrasuppressor (Tcs) cells. These cells can be examined in a mouse model of adoptive cell transfer to determine the mechanism of the tested reactions. The current protocol describes two models of cell transfer, &#34;adoptive transfer IN&#34; and &#34;adoptive transfer OUT&#34;. Adoptive transfer IN is useful for testing the immunomodulation in the afferent and efferent phases of CHS. On the other hand, &#34;adoptive transfer OUT&#34; reflects the effect of immunomodulation only in the efferent phase of CHS.
The article describes a method of skin-induced suppression via the epicutaneous application of an antigen in a murine model of human allergic contact dermatitis (ACD) - contact hypersensitivity (CHS) mediated by T helper type 1 lymphocytes (Th1)5. However, this approach was also tested for its efficacy in regulating (suppressing, tolerance) experimental models of T cytotoxic lymphocyte type 1 (Tc1)-mediated CHS6,7, autoimmune diseases such as collagen-induced arthritis (CIA)8,9 , experimental autoimmune encephalomyelitis (EAE)10,11, and ulcerative colitis12. Moreover, the protocol describes the method of skin-induced contrasuppression (reversed suppression) via EC application of an antigen together with PRR ligands in a murine model of CHS13,14,15,16. The presented results indicate that EC-induced antigen-nonspecific suppression can be reversed by purifying TLR2, TLR3, TLR4, TLR9, and NOD2 ligands. The method of skin-induced contrasuppression was also employed in an animal model of atopic dermatitis (AD), showing that EC treatment with an antigen in the presence of synthetic single-stranded DNA molecules containing unmethylated oligodeoxynucleotides (CpG) - ligand for TLR9 can suppress Th2-mediated responses in an antigen-specific manner (shifts the immune response toward a Th1/Th17 phenotype)17. The described method was also tested in pneumococcal pneumonia, demonstrating that EC immunization with pneumococcal polysaccharide conjugated to bovine serum albumin (PC-BSA), together with CpG, could potentially enhance immunity to Streptococcus pneumoniae18.
A pivotal aspect of EC-induced immunomodulation is the selection of the model and the nature of the antigen. The article includes a table detailing the antigens used, the antigen exposure duration, and the tested models&#39; T-cell specificity (see Table 1). The described method for immunomodulation is targeted towards diseases involving antigen-specific T lymphocytes. The effectiveness of this method was assessed by utilizing a compatible EC-applied antigen in the process. The compatibility of the antigen is crucial to ensure that it effectively interacts with the T lymphocytes involved in the disease process. The application of a protein or peptide patch on intact skin facilitates antigen penetration through the epidermis19,20. This increased antigen penetration is attributed to heightened perspiration beneath the dressing. Alterations in epidermal permeability occur within 4-10 h of patch application, while EC-induced immunomodulation may remain effective for up to two weeks5,14.
Although EC treatment is widely used, the existence of many variations of this method could raise questions. Current understanding suggests that the extent of epidermal damage prior to antigen administration may influence the nature of the resulting immune response21. Superficial epidermal damage triggers the release of anti-inflammatory cytokines by Langerhans cells, favoring the initiation of the Th2 response22. In contrast, more extensive epidermal damage promotes the release of pro-inflammatory cytokines and facilitates antigen delivery to dermal dendritic cells23.The presented protocol highlights (a) the recognition of challenges when applying this method in small and active animals, particularly mice; (b) considering factors such as skin condition after razor shaving, proper contact of substances with the skin, exposure time, and hygienic conditions of the skin during EC treatment which can determine the treatment&#39;s effectiveness. It is crucial to consider the method of skin depilation, whether physical or chemical, before antigen application, as different skin preparations can affect the skin&#39;s condition in various ways. It is acknowledged that all these procedures can activate skin immune cells, potentially leading to skin infections or influencing disease progression. The potential side effects on skin conditions after depilation should be a primary consideration when selecting the depilation method. The researchers described a method of T-cell-mediated immune suppression induced via the EC application of an antigen on razor-shaved dorsal skin, demonstrating its effect on various mouse models of immune responses. The antigen&#39;s EC application, along with PAMPs, induces antigen-specific T contrasuppressor cells. Moreover, the presented method of EC application, particularly with repeated exposure to antigens or PAMPs, does not cause concerning side effects.
The described protocol underlines the importance of various factors in achieving reliable and repeatable results with this method, especially in small animals like mice. The non-invasive nature of this approach typically results in a lower risk of adverse reactions (such as pain, infection, or scarring) compared to intradermal or injectable immunization techniques. Epicutaneous immunization is user-friendly and easily administered in non-clinical settings, improving accessibility and compliance compared to more invasive methods.
Table 1: EC-induced modulation of immune response in different mouse disease models. Abbreviations:&#160;ACD = allergic contact dermatitis; AD = atopic dermatitis; Ag = antigen; CIA = collagen-induced arthritis; CHS = contact hypersensitivity; CFA = complete Freund&#39;s adjuvant; COLL II = type II collagen; DNFB = 2,4-dinitrofluorobenzene; DNP-BSA = dinitrophenyl conjugated to bovine serum albumin; EAE = experimental autoimmune encephalomyelitis; EC = epicutaneous; LPS = lipopolysaccharide; OVA = ovalbumin; PAMP = pathogen-associated molecular pattern; PC-BSA = pneumococcal polysaccharide conjugated to bovine serum albumin; Th1 = T helper type 1 lymphocyte; Tc1 = T cytotoxic type 1 lymphocyte; TNCB = 2,4,6-trinitrochlorobenzene; TNP-Ig = 2,4,6-trinitrophenyl-conjugated mouse immunoglobulins; Ts = T suppressor cells; Tcs = T contrasuppressor cells. <a href="https://www.jove.com/files/ftp_upload/67578/Table 1.xlsx" target="_blank">Please click here to download this Table.</a>

<table><tbody><tr><td>2,4,6-trinitrochlorobenzene (TNCB)</td><td>Tokyo Chemical Industry CO., LTD, Japan</td><td>C0307</td><td></td></tr><tr><td>2,4,6-trinitrophenyl-conjugated mouse immunoglobulin (TNP-Ig)</td><td>Chair of Biomedical Sciences, Faculty of Health Sciences, Jagiellonian University Medical College, Krakow, Poland</td><td></td><td></td></tr><tr><td>acetone (ACS reagent, &amp;ge;99.5%)</td><td>Merck KGaA, Darmstadt, Germany</td><td>179124</td><td></td></tr><tr><td>aluminum foil</td><td>Merck KGaA, Darmstadt, Germany</td><td>Z185140&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>analytical balance&amp;nbsp;</td><td>Sartorius Weighing Technology GmbH, Goettingen, Germany</td><td>PRACTUM224-1s, 29105177</td><td></td></tr><tr><td>bovine serum albumine (BSA)</td><td>Merck KGaA, Darmstadt, Germany</td><td>A9418</td><td></td></tr><tr><td>CBA/J (H-2k)&amp;nbsp;</td><td>Breeding unit of the Chair of Biomedical Sciences, Faculty of Health Sciences, Jagiellonian University Medical College, Krakow, Poland</td><td></td><td></td></tr><tr><td>cell strainer, pore size 70 &amp;mu;m</td><td>BIOLOGIX, China</td><td>15-1070</td><td></td></tr><tr><td>disposable pipettes capacity: 5 mL, 10 mL, 25 mL</td><td>Merck KGaA, Darmstadt, Germany</td><td>Z740301, Z740302, Z740303</td><td></td></tr><tr><td>Dulbecco&amp;prime;s phosphate buffered saline (DPBS)</td><td>ThermoFisher Scientific,&amp;nbsp; Waltham, Massachusetts, USA</td><td>14190144</td><td></td></tr><tr><td>eppendorf Safe-Lock Tubes 1.5ml</td><td>Eppenforf, Germany</td><td>30120086</td><td></td></tr><tr><td>eppendorf Safe-Lock Tubes, 2.0 ml</td><td>Eppenforf, Germany</td><td>30120094</td><td></td></tr><tr><td>ethanol 100% (absolute alcohol)</td><td>Merck KGaA, Darmstadt, Germany</td><td>1.07017</td><td></td></tr><tr><td>ethanol 96%</td><td>Merck KGaA, Darmstadt, Germany</td><td>1.59010</td><td></td></tr><tr><td>fetal bovine serum (FBS)</td><td>ThermoFisher Scientific,&amp;nbsp; Waltham, Massachusetts, USA</td><td>A3160802</td><td></td></tr><tr><td>glass microscope slides (sandblasted single frosted)</td><td>ThermoScientific, Portsmouth, NH, USA</td><td>421-004T</td><td></td></tr><tr><td>Graph Pad Prism</td><td>GraphPad Software Inc.</td><td>v. 10.2.3</td><td></td></tr><tr><td>grey soap (Bialy jelen soap bar)</td><td>Pollena Ostrzesz&amp;oacute;w, Producent Chemii Gospodarczej Sp. Z.o.o. , Sp. K., Poland</td><td>163057</td><td></td></tr><tr><td>hemocytometer</td><td>VWR, Avantor, U.S.A</td><td>612-5719</td><td></td></tr><tr><td>incubator Heracell 150i</td><td>Thermo Electron&amp;nbsp; LED Gmbh, Germany</td><td>41071068</td><td></td></tr><tr><td>isoflurane (Aerrane)</td><td>Baxter, Deerfield, IL, USA</td><td>8AGG9623</td><td></td></tr><tr><td>ketamine 100 mg/ mL, solution for injection</td><td>Biowet Pulawy Sp. z o.o., Pulawy, Poland</td><td>cat.# not avaliable</td><td></td></tr><tr><td>laboratory Centrifuge (Heraeus Fresco 21)</td><td>Thermo Scientific, Germany</td><td>75002425</td><td></td></tr><tr><td>laboratory Centrifuge (Heraeus Megafuge 1.0R)</td><td>&amp;nbsp;Thermo Scientific, Germany</td><td>B00013899</td><td></td></tr><tr><td>lipopolysaccharide (LPS)</td><td>Merck KGaA, Darmstadt, Germany</td><td>L-8274</td><td></td></tr><tr><td>mask (FFP2)</td><td>VWR, Radnor, Pennsylvania, United States</td><td>111-0917</td><td></td></tr><tr><td>micrometer</td><td>Mitutoyo, Tokyo, Japan</td><td>193-111</td><td></td></tr><tr><td>microscope with objectives</td><td>Leica Microsystems CMS GmbH, Germany</td><td>DM1000, 294011-082007</td><td></td></tr><tr><td>propylene glycol&amp;nbsp;</td><td>Chempur, Piekary &lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/67578/67578eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt; Poland</td><td>114489005</td><td></td></tr><tr><td>razor blade</td><td>VWR, Radnor, Pennsylvania, United States</td><td>PERS94-0462</td><td></td></tr><tr><td>transparent document sleeves 30 &amp;micro;m</td><td>SCHEMAT</td><td>5906961126542</td><td></td></tr><tr><td>trypan blue</td><td>Merck KGaA, Darmstadt, Germany</td><td>T8154</td><td></td></tr><tr><td>tubes 15 mL sterile</td><td>Merck KGaA, Darmstadt, Germany</td><td>CLS430055 (Corninig)</td><td></td></tr><tr><td>tubes 50 mL, sterile</td><td>Merck KGaA, Darmstadt, Germany</td><td>CLS430290 (Corning)</td><td></td></tr><tr><td>vials, screw top, clear glass (vial only) 22 mL</td><td>Merck KGaA, Darmstadt, Germany</td><td>27173</td><td></td></tr><tr><td>Viscoplast Polovis, Silk adhesive, 5x0.0125m</td><td>3M Poland, Nadarzyn, Poland</td><td>7100231620</td><td></td></tr><tr><td>water bath&amp;nbsp;</td><td>AJL Electronic, Poland</td><td>LW102</td><td></td></tr><tr><td>xylazine (xylapan 20 mg/ mL) solution for injection</td><td>Vetoquinol Biowet Sp. z o.o., Gorzow Wielkopolski, Poland</td><td>cat.# not avaliable</td><td></td></tr></tbody></table>

murine model of epicutaneously-induced immunomodulation

Murine Model of Epicutaneously-Induced Immunomodulation

The skin, as the largest organ in the human body interfacing with the external environment, plays crucial physiological roles, including (1) protecting the organism from xenobiotics and microorganisms, (2) facilitating thermoregulation, (3) maintaining water and electrolyte balance, (4) synthesizing vitamin D, and (5) participating in immunity. This paper highlights the skin&#39;s pivotal involvement in immunological mechanisms, showcasing its capacity to immunomodulate by suppressing or potentiating the immune response. Previous findings reveal that, in mice, epicutaneous (EC) immunization with an antigen, either alone or in conjunction with pathogen-associated molecular patterns (PAMPs), can suppress or potentiate immune responses, respectively. Specifically, it was observed that EC immunization with an antigen can suppress immune responses in various murine models of autoimmune diseases or allergic contact dermatitis. The results indicate that EC immunization with an antigen combined with PAMPs could serve as an effective strategy to boost immunity against pathogens or alleviate T helper type 2 lymphocyte (Th2)-dependent allergies. This protocol outlines a method for inducing skin suppression via the epicutaneous application of an antigen in a murine model of human allergic contact dermatitis (ACD)-contact hypersensitivity (CHS) mediated by Th1 cells. However, it can also be applied to various murine disorder models. The article presents EC-induced immunomodulation as an attractive murine model for needle-free immunotherapy across various disorders.

This study examines whether antigens applied via skin alone or combined with PAMP can induce Ts or Tcs cells, respectively, which modulate the CHS reaction. The immunization of CBA/J mice via EC with 100 µg of TNP-Ig or TNP-Ig plus 100 µg of LPS induces Ts or Tcs cells, respectively. The mice were then sensitized with 5% TNCB and challenged with the same hapten 4 days later. The reaction was evaluated by measuring ear swelling before and after the challenge. Each experiment has a negative control, which...

The study demonstrates the role of the skin in modulating immune responses in mice through epicutaneous (EC) immunization. EC immunization with a protein antigen can suppress immune responses in contact hypersensitivity or reverse skin-induced suppression when combined with pathogen-associated molecular patterns. EC immunization shows promise as an effective, needle-free approach to immunotherapy.

The skin, as the largest organ in the human body interfacing with the external environment, plays crucial physiological roles, including (1) protecting ...

This protocol outlines a method for skin-induced immunomodulation in a murine model of human allergic contact dermatitis mediated by T helper one lymphocytes. Recent studies showed that epicutaneous immunization with protein antigen alone suppresses T cell-mediated immune responses while coadministration with pumps reverses skin-induced suppression. This highlights the potential of skin-based, needle-free strategies for generating regulatory cells to modulate immune responses.It is well known that the degree of epidermal damage prior to antigen administration may determine the type of the induced immune response. Our study has established that epicutaneously induced immunomodulation is mediated by antigen-nonspecific T suppressor cells and antigen-specific T contrasuppressor cells. Despite widespread use of the epicutaneous immunization, the lack of standardized protocols and variations in application methods create inconsistencies in outcomes.Our protocol addresses the challenges by offering a reproducible and controlled murine model. To begin, shave the back skin of the anesthetized mouse with a razor on day zero. On day one, prepare a one-milligram-per-milliliter solution of antigen 2, 4, 6-trinitrophenyl conjugated mouse immunoglobulin in sterile DPBS.Protect the antigen from light by covering the vial with aluminum foil. Mix the solutions just before use in a two-milliliter vial. Apply the antigen solution to the shaved skin by placing a one-square-centimeter gauze patch soaked in 100 microliters of antigen.Cover this gauze with a two-square-centimeter thin plastic patch and secure it with fabric adhesive tape. Once the mouse recovers from anesthesia, return it to its cage. On day five, after anesthetizing the mouse, use safety scissors with rounded corners to carefully cut the patch.Gently remove the adhesive tape. After the mouse regains consciousness and is returned to its cage, allow it to move freely and clean its skin naturally for at least four hours. On day eight, to test EC-induced T suppressor cells in Th1-mediated contact hypersensitivity reaction, apply 150 microliters of the freshly prepared TNCB hapten solution to the shaved abdominal skin of the anesthetized mouse to induce T effector cells.Four days after inducing T effector cells, evaluate the contact hypersensitivity response by measuring the ear baseline and applying TNCB hapten to both sides of the ears. Measure the ear thickness by using a micrometer 24 hours later. For the isolation of seven-day T suppressor or four-day T effector cells from the donor mice, disinfect the entire abdominal area of the anesthetized mouse with 70%ethanol.Make a one-centimeter incision to expose the peritoneal cavity. Using sterile forceps, isolate the auxiliary and inguinal lymph nodes and spleen into tubes filled with sterile DPBS supplemented with 1%fetal bovine serum, or FBS. Pull the lymph nodes from all donors in one vial and spleens in another and keep the vials on ice.Mash lymph nodes and spleens between the frosted ends of two microscope slides to create a cell suspension. Strain the cell suspension through a cell strainer with a 70-micrometer pore size. Rinse the cells with 30 milliliters of DPBS supplemented with 1%FBS and centrifuge at 300g for 10 minutes at four degrees Celsius.Discard the supernatant and resuspend the pellet in one to five milliliters of DPBS. To count the cell viability, mix 10 microliters of cell suspension with 90 to 990 microliters of trypan blue, depending on the cell count, and count the cells using a hemocytometer. Next, prepare a 1:1 mixture of auxiliary lymph node and spleen cells to obtain 1 to 5 times 10 to the power of 7 T suppressor cells per recipient in one milliliter of DPBS with 1%FBS.For T effector cells, prepare 7 times 10 to the power of 7 cells per recipient in one milliliter of the same buffer. Rinse the isolated cells with 30 milliliters of DPBS, then centrifuge them at 300g for 10 minutes at four degrees Celsius and resuspend the pellet in 200 microliters of DPBS per recipient. For the adoptive transfer in, administer an intravenous injection of the prepared mixture containing 1 to 5 times 10 to the power of 7 T suppressor cells in 200 microliters to the anesthetized recipient before inducing Th1-mediated contact hypersensitivity reaction.On the same day, sensitize anesthetized mice by applying 150 microliters of HAp10 on the previously shaved spot. Four days later, evaluate the contact hypersensitivity response by measuring the ear baseline and applying HAp10 to both sides of the ears. Measure the ear thickness 24 hours later.For the adoptive transfer out, after inducing and isolating T suppressor and T effector cells from donors, divide the T effector cells into two separate vials, labeled as TS test group"and CHS control. Add T suppressor cells to T effector cells for the TS test group and mix gently. Then add DPBS supplemented with 1%FBS only to the T effector cells in the CHS control vial and mix it.Place the vials in a 37-degree Celsius water bath for 30 minutes, mixing the contents gently every five minutes. Rinse each vial with 30 milliliters of DPBS, then centrifuge at 300g for 10 minutes at four degrees Celsius. Decant the supernatant and resuspend the pellet in 200 microliters of DPBS per recipient.Now, pass each cell suspension through a 70-micrometer pore cell strainer. Administer an intravenous injection of the prepared cells into recipient mice. Epicutaneous exposure to antigen with or without pathogen-associated molecular pattern influences the induction and modulation of contact hypersensitivity reaction.Epicutaneous immunization with TNP-Ig antigen significantly suppressed contact hypersensitivity as shown by reduced ear swelling compared to the contact hypersensitivity control group. Coapplication with lipopolysaccharide reversed the suppression, restoring swelling levels similar to the control. Intravenous transfer of lymph node and spleen cells from mice tolerized with TNP-Ig significantly reduced TNCB-induced ear swelling in recipient mice compared to the contact hypersensitivity control, confirming the suppressive role of T suppressor cells.Transfer of T effector cells incubated with T suppressor cells significantly suppressed the contact hypersensitivity response in recipients, while transfer of T effector cells alone triggered the contact hypersensitivity response.

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Learn epicutaneous immunization for skin-induced immunomodulation using a murine model of allergic contact dermatitis. This needle-free method generates T suppressor cells to regulate immune responses effectively.

Murine Model of Epicutaneously-Induced Immunomodulation

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