Anti-Inflammatory Activity of a Cyclic Tetrapeptide in Mouse and Human Experimental Models

A cyclic tetrapeptide Pro-Pro-Pheβ3ho-Phe (4B8M) was tested for immunosuppressive activity and potential therapeutic utility in several in vitro and in vivo mouse and human models. The tetrapeptide was less toxic for mouse splenocytes in comparison to cyclosporine A (CsA) and a parent cyclolinopeptide (CLA). The tetrapeptide demonstrated potent anti-inflammatory properties in antigen-specific skin inflammatory reactions to oxazolone and toluene diisocyanate as well to nonspecific irritants such as salicylic acid. It also inhibited inflammatory processes in an air pouch induced by carrageenan. In addition, 4B8M proved effective in amelioration of animal models corresponding to human diseases, such as nonspecific colon inflammation induced by dextran sulfate and allergic pleurisy induced by ovalbumin (OVA) in sensitized mice. The tetrapeptide lowered expression of EP1 and EP3 but not EP2 and EP4 prostaglandin E2 (PGE2) receptors on lipopolysaccharide-stimulated Jurkat T cells and ICAM-1 expression on human peripheral blood mononuclear cells (PBMC). Its anti-inflammatory property in the carrageenan reaction was blocked by EP3 and EP4 antagonists. In addition, 4B8M induced an intracellular level of PGE2 in a human KERTr keratinocyte cell line. In conclusion, 4B8M is a low toxic and effective inhibitor of inflammatory disorders with potential therapeutic use, affecting the metabolism of prostanoid family molecules.

o C, in 5% CO2 (NuAire, Plymouth, MN, USA). Trypsin-EDTA solution (Institute of Immunology and Experimental Therapy, Wrocław) was used to collect the cells.
The peptide 4B8M was synthesized and delivered by one of us (J.Z.), Łódź University of Technology. The synthesis of the peptide was described in US patent [15]. The peptide was initially dissolved in DMSO and subsequently in the culture medium at concentration of 1 mg/ml. The peptide solution was stored frozen at -20 o C until use. For topical administration the peptide was dissolved by means of 1h stirring in a 50:50 weight mixture of vaseline and paraffin (obtained from a drug store). A microscopic evaluation showed no presence of crystals in the mixture containing up to 1% of the peptide.

Statistics
The calculations and statistical analysis of the data were performed using the licensed Microsoft ® Excel XP and STATISTICA ® 7 programs for Windows. The statistical significance of differences between mean values were tested by means of variance analysis (ANOVA) for the one-factor classification with post hoc analysis (multi comparisons) by RIR Tukey's test. The evaluation of normality of the variables distribution was performed by W Shapiro-Wilk test, and the evaluation of the homogenous variance assumption by Brown-Forsyth's test. In addition, the occurrence of dependencies between group means and variances were checked. Fulfillment of the above assumptions (normal or symmetric distribution of variables, homogeneity of variance, lack of dependencies between means and variances) is a condition for application of the variance analysis for testing the mean equality hypothesis. The lack of fulfillment of these assumption was a basis for application of nonsymmetrical Kruskal-Wallis' test. The significance was set at P<0.05. NS -not significant.

Isolation and proliferation of mouse splenocytes
The spleens were pressed against a plastic screen into 0.83% NH4Cl solution to lyze erythrocytes (5 min incubation at room temperature). The cells were then washed twice with Hanks' medium, passed through a glass wool column to remove debris, and re-suspended in the culture medium, referred to below as the culture medium, consisting of RPMI-1640, supplemented with 10% FCS, L-glutamine, sodium pyruvate, 2-mecraptoethanol and antibiotics. The cells were then distributed into 96-well flat-bottom tissue culture plates (Nunc) at a density of 2×10 5 /100 µl/well. 2.5 µg/ml concanavalin A (Con A) or 2.5 µg/ml pokeweed mitogen (PWM) were added to induce cell proliferation. The compounds were added to the cultures at doses of 1-100 µg/ml. After a 3-day incubation, the cell proliferation was determined using a colorimetric MTT assay [Hansen et al. 1989]. The results are presented as the mean optical density (OD) at 550 nm with the reference wavelenght of 630 nm ± standard error (SE) from quadruplicate determinations.
Isolation and proliferation of human PBMC Venous blood from a single donor was withdrawn into heparinized syringes and diluted twice with PBS. PBMC were isolated by centrifugation on Ficoll-uropoline gradient (density 1.077 g/ml) and centrifuged at 800×g for 20 min at 4 o C. The interphase cells, consisting of lymphocytes (20%) and monocytes (80%) were then washed three times with Hanks' medium and re-suspended in the culture medium at density of 2×10 6 cells/ml. The isolated PBMC were distributed into 96-well flat-bottom plates in 100 µl aliquots (2×10 5 cells/well). PHA was added at a concentration of 5 µg/ml. The compounds were tested at doses 1, 10 and 100 µg/ml. DMSO at appropriate dilutions served as control. After a four-day incubation in a cell culture incubator, the proliferative response of the cells was determined by the colorimetric MTT method [Hansen et al. 1989]. The data are presented as a mean OD value from quadruplicate wells ± SE.
Colorimetric MTT assay for cell growth and kill The assay was performed according to Hansen et al. 1989. Briefly, 25 µl of MTT (5 mg/ml) stock solution was added per well at the end of cell incubation and the plates were incubated for 3h in a cell culture incubator. Then, 100 µl of the extraction buffer (20% SDS with 50% DMF, pH 4.7) was added. After additional overnight incubation, the optical density (OD) was measured at 550 nm with the reference wavelength of 630 nm (spectrophotometer Dynatech 5000).

Determination of cytokine activities
Human whole blood was diluted 5× with RPMI-1640 medium and distributed to 24-well culture plates in 1 ml aliquots. LPS from E. coli, was added to the culture at concentration of 1 µg/ml. The studies compounds were added at concentration of 1, 10 and 100 µg/ml. After overnight incubation the supernatants were harvested and frozen at -20 o C until cytokine determination. TNF-α and interleukin-6 were measured using bioassays [Espevik and Nissen-Meyer 1986;van Snick et al. 1986].
IL-6 bioassay IL-6-dependent murine B cell hybridoma (7TD1 line) was incubated (2000 cells/well) in 96well plates with serial dilutions of supernatants in quadruplicate. Three days later the number of proliferating cells was estimated by the MTT colorimetric method. The detection limit of the assay was about 1.5 pg/ml. One unit of IL-6 activity was defined as an inverse of the supernatant dilution which caused half-maximal (50%) proliferation of the indicator cell line 7TD1.

TNF-α bioassay
The indicator cell line WEHI 164.13 were distributed into 96-flat-bottom plates (Nunc) at a density 2×10 4 /well. Serially diluted supernatants (samples) were added (100 μl/well) to the cultures of WEHI 164.13 cells in duplicate. The cells were incubated overnight in the culture medium (RPMI-1640 plus supplements plus FCS) in the presence of actinomycin D (1 µg/ml). The amount of viable cells was measured by MTT colorimetric method. The activity of TNF-α was expressed in pg/ml (10 pg/ml corresponds to one unit of TNF-α). The sensitivity limit for the assay is 2.5 pg/ml. One unit of TNF-α activity is defined as an inverse of supernatant dilution where 50% cell death takes place.
Humoral immune response to SRBC Secondary humoral immune response in vitro: Mice were sensitized i.p. with 0.2 ml of 5% SRBC suspension. After four days spleens from these mice were isolated, splenocyte single cell suspension prepared and suspended in the culture medium at a density of 5×10 6 /ml. The cells were distributed to 24-well culture plates in 1 ml aliquots and 0.05 ml of 0.005% SRBC was added as antigen. The compounds were added to the cultures in the beginning of the fourday incubation period at concentration range 10 and 100 µg/ml. The number of antibodyforming cells (AFC) in the cultures were determined using a method of local hemolysis in agar gel according to Mishel and Dutton 1967. The number of viable cells in the suspension was determined in a Bürker hemocytometer in Trypan blue dye. Next, a suspension of SRBC (the antigen) was prepared for the test. To a tube placed in a water bath (45 o C) the following ingredients were added: 0.05 ml of 10× Hanks' concentrate. 0.05 ml of heat inactivated FCS, 0.4 ml of hot (90 o C) 0.6% agarose and (after cooling the solution to the bath temperature) 0.05 ml of 30% SRBC in Hanks' medium. Then, to a tube containing 0.1 ml of complement 0.1 ml of splenocyte suspension was added, warmed for 30 sec in the water bath and mixed with 0.55 ml of the SRBC suspension. The mixture was stirred and poured onto microscopic glasses. After solidification of the mixture the glasses were placed in humidified boxes at 37 o C for 3h. After the incubation the plaques (places of lysis) were counted by use of an inverted microscope (10× magnification). The results were shown as a number of AFC per 10 6 viable cells used for the test.
Primary humoral immune response to SRBC in vivo: Mice were immunized with 0.2 ml 5% SRBC suspension i.p. and 2h after immunization mice were given i.p. 4B8M peptide (10 or 100 µg/mouse) and other mice received CsA (10 or 100 µg/mouse). Control mice received appropriately diluted DMSO. After four days mice were sacrificed, spleen were isolated and pressed against plastic screens by use of syringe pistons to Hanks' medium to prepare a single cell suspension (4-5×10 4 cells/0.1 ml). The spleen cell suspensions were kept in an ice bath before performance of the test. Next the number of AFC in the spleens was determined using a method of local hemolysis in agar gel as described above.

Delayed type hypersensivity test
The test was performed according to Lagrange et al. 1974. Mice were sensitized subcutaneously with 5 µg ovalbumin (OVA) emulsified in Freund's complete adjuvant in the tail base. After 4 days the mice were challenged with 50 µg OVA in Freund's incomplete adjuvant in the hind footpads. Following next twenty four hours the footpad thickness was measured using a caliper. Control (background response mice) were not sensitized but received the challenging dose of OVA. 4B8M was administered to mice in two 100 µg i.p. doses, 2h before and 24h after the sensitizing dose of antigen. The results were presented as a mean value of antigen-specific increase of footpad thickness and expressed in DTH units (one DTH unit = 10 -2 cm) ± SE. One DTH unit corresponds to 0.1 mm. Antigen-specific increase means that background (BG) values measured in mice given only the eliciting dose of antigen were subtracted.

Preliminary studies -Results
Effect of the 4B8M peptide on mitogen-induced proliferation of mouse and human lymphocytes The effect of 4B8M peptide on mitogen-induced proliferation of mouse splenocytes is shown in Fig. S1AB. CsA and native CLA served as reference drugs. The inhibitory effects of the compounds were compared with appropriate dilutions of the solvent -DMSO. The results indicate that the peptide inhibited Con A-induced cell proliferation in a dose-dependent manner (Fig. S1A). Its activity was lower as compared to that of native CLA although quite significant at high doses (75% at 75 µg/ml and about 80% at 100 µg/ml). In addition, the peptide strongly inhibited PWM-induced proliferation of splenocytes at 100 µg/ml (Fig.   S1B).     Effect of the peptide on LPS-induced production of TNF-α and IL-6 by human whole blood cell cultures Effects of 4B8M peptide on LPS-inducible production of TNF-α and IL-6 are presented in Fig. S3AB. The peptide inhibited production of TNF-α production by more than 50% at all concentrations. The inhibitory action of CsA was even stronger, particularly at 10 and 100 µg/ml (Fig. S3A). 4B8M showed a dose-dependent increase in IL-6 production. Similar, but a stronger increase was observed with CsA at 1 and 10 µg/ml doses but not at 100 µg/ml (Fig.   S3B).  Compound/Concentration IL-6 (pg/ml)

Effect of the 4B8M peptide on the secondary humoral immune response to SRBC in vitro
The suppressive effects of 4B8M peptide at 10 and 100 µg/ml doses, are shown in Fig. S4A.
The results indicate that the suppressive action of the peptide at 100 µg/ml concentration was comparable to those of CsA and CLA. 10 µg/ml concentration of the peptide exerted a weaker suppression as compared to the reference compounds.   Mice were immunized with SRBC as described in the Materials and Methods and after 2h were given 10 or 100 µg of the peptide. CsA served as a reference drug. The number of antibody-forming cells to SRBC was measured after four days. The results (Fig. S4B) showed that the peptide was strongly inhibitory at both doses. CsA was less effective.  The results showed that 4B8M peptide 100 µg/dose, given in two doses, 2h before and 24h after the immunization of mice, strongly inhibited the delayed type hypersensitivity reaction to OVA. That suppressive action was stronger than those of CLA and CsA (Fig. S5).

Contact sensitivity to oxazolone -Materials and Methods
Sensitization and elicitation of the contact sensitivity The test was performed according to Noonan and Halliday, 1978 with some modifications.
Mice were shaved on the abdomen (2×2 cm area). On a next day 100 µl of 0.5% oxazolone dissolved in acetone was applied on the skin of abdomen (the sensitizing dose of antigen).
After 5 days 50 µl of 1% oxazolone was applied on both sides of the auricles (the eliciting dose of antigen).

Treatment of mice with the preparations
The peptide was applied in a form of 0.1% ointment onto both sides of the auricles (0.1 ml volume), twice during 2h. The reference preparations (Protopic ® and Elidel ® ) were used in a similar way. The auricles of control mice were treated with the creme base. The preparations were applied after 24h following the eliciting dose of antigen, so at the time of the highest manifestation of the cellular inflammatory response. 4B8M peptide was therefore applied as a therapeutic for the contact sensitivity reaction. In another experiment 1% creme Hydrocortisonum ® was topically used as a reference drug. 24h after application of the therapeutic agents (48h following elicitation of the reaction) the parameters of the inflammatory reaction were determined.

Measurement of the ear edema
The ear edema (ear thickness) was measured by means of a spring caliper with the accuracy of 0.05 mm (Mitutoyo, Japan) after 48h from application to mice of the eliciting dose of antigen (24h after application of therapeutic preparations). The results were presented as antigen-specific increase of the ear thickness, i.e. BG values were subtracted (background values were measured in mice given only the eliciting dose of antigen).

Cell isolation from lymph nodes
After measurement of the ear edema the mice were sacrificed by cervical dislocation and salivary and mandibular lymph nodes were isolated, placed in PBS and pressed against plastic screen to obtain a single-cell suspension. The cells were washed in PBS in 15 ml tubes by centrifugation at 800×g.

Determination of lymph node cell number and viability
The cells were re-suspended in PBS containing 0.   Effect of the peptide of number and percentage of viable and dead lymphocytes in the draining lymph nodes The number of dead cells in the draining lymph nodes after application of Protopic ® increased significantly, whereas 4B8M and Elidel ® decreased the number of dead cells as compared to non-treated control group (Fig. S6B).  Dead cells

Viable cells
The analysis of the percentage of viable and dead dells in the draining lymph nodes showed that the peptide did not increase the content of dead cells in relation to the untreated control ( Fig. S6C). However, a 2× increase was observed with application of Elidel ® . Protopic ® , in turn, increased the % of dead cells up to 95%.  Therapeutic effect of 4B8M in comparison with 1% Hydrocortisonum ® (HC) The aim of the next experiment was to topically apply the peptide (0.1% ointment) as a therapeutic agent preventing the contact sensitivity reaction in comparison with Hydrocortisonum ® 1% creme as a reference drug. Fig. S7 shows that both compounds inhibited the ear swelling to a similar degree. 4B8M peptide and HC were also very effective in reducing draining lymph node cell number (Fig. S8). On the other hand the treatment with hydrocortisone led to a significant increase of the neutrophil (bands and segments) content in the peripheral blood, and the blood cell composition in 4B8M group was comparable to BG mice ( Fig. S9).  The normalizing effects of the peptide on the inflammatory skin histological changes induced by oxazolone, are presented in Fig. S10 and Fig. S11. The picture presents extensive inflammatory cell infiltrations, composed mainly of neutrophils and to a lesser degree of macrophages and lymphocytes. In some places, in the zone adjacent to perichondrium, a loosening of the connective, fibrous tissue was observed, probably associated with perivascular swelling, 400× magnification.  In the histochemical studies numerous mastocytes were found, at different functional stages, grouped around blood vessels. The intensity of mastocytes concentration was proportional to the intensity of cell infiltration, 400× magnification. The results of quantitative analysis of the inflammatory cell types in high-power field are presented in Fig. S11. The histological examinations revealed that the cellular composition at 24h after treatment with 4B8M peptide resembled that of naive mice. In contrast, Protopic ® did not change the content of neutrophils and the number of macrophages and lymphocytes exceeded those in the control mice. The treatment with Elidel ® led to about 50% reduction in neutrophil numbers, however, more mastocytes could be found in that area.

Skin inflammation to toluene diisocyanate -Materials and Methods
The test was performed according to Yamamoto et al. 2006, with minor modifications. The mice were shaved on the abdomen (2×2 cm) and after 24h 100 µl of 3% TDI in acetone for 3 subsequent days were applied (the sensitizing dose of antigen). After 14 days the antigenspecific skin reaction was elicited by five applications of 20 µl of 0.3% TDI (the eliciting dose of antigen) on both sides of the auricles in 3-day intervals: days 14, 17, 20, 23 and 27. 5h and 24h following the last elicitation of the reaction (27 th and 28 th days of the test, respectively) the parameters of the inflammatory reaction were determined.
Application of the peptide and the reference compounds 4B8M peptide was applied in a form of 0.1% ointment, topically, on both sides of the auricles (total volume of 100 µl), 1h after each elicitation of the reaction by TDI (days: 14,17,20,23 and 27 of the test). The reference drugs (Protopic ® and Elidel ® ) were applied in the same manner.

Determination of leukocyte number
After 5h (27th day of the test) and after 24h (28th day of the test) following the last elicitation of the reaction, the mice were subjected to halothane anesthesia and bled from the retroorbital plexus, followed by sacrifice by cervical dislocation. The number of leukocytes was determined after 20× dilution of blood in Türk's solution and enumeration of cells in a Bürker camera. The results were presented as the number of cells per 1 µl of blood as mean values ±SE.

Determination of cell blood composition
After 24h (27th day of the test) mice were bled and blood smears were made on microscopic glasses, the preparations were dried and stained with Giemsa and May-Grünwald reagents.
The blood picture was evaluated in a light microscope at 1000× magnification (in immersion oil). Up to 100 cells was counted per preparation. The results were shown as a percentage (mean values of respective cell types: mature neutrophils, band forms of neutrophils, eosinophils, lymphocytes and monocytes).

Determination of cell numbers in the draining lymph nodes
After sacrifice of mice (24h following the last elicitation of the reaction -28th day of the test) the superficial parotid, mandibular and accessory mandibular lymph nodes were isolated, homogenized by pressing against a stainless screen into PBS, washed 2× with PBS and resuspended in PBS containing 0.2% Trypan blue. The total and nonviable cell numbers were counted using a light microscope and Bürker's hemocyctometer.

Blood vessel permeability test
The test was performed according to Katayama et al, 1978 with some modifications. 5h following the last elicitation of the reaction (27 th day of the test) the mice were given 1 mg of Evens blue in 0.2 ml of 0.9% NaCl, intravenously. After 30 min the mice were sacrificed, the auricles were cut off, weighed and immersed in 50 µl of 1 M KOH for 18h at 37 o C. The dye was extracted from the ears using 450 µl of 0.2 M phosphoric acid and acetone (5:13 ratio).
The samples were centrifuged at 3,000 rpm for 15 min. OD of the supernatants were measured at 550/630 nm. The amount of Evens blue (µg/ml) was determined based on a standard curve. The results were presented as the amount of Evans blue per 100 mg of wet tissue. Mice treated only with the eliciting dose of antigen served as a BG control.
Histological analysis and statistics as described above.

Therapeutic effect of 4B8M peptide on skin inflammation to toluene diisocyanate
Effect of the peptide on peripheral blood leukocyte number Effects of the 4B8M peptide and the reference compounds on the number of circulating leukocytes was shown in Fig. S12. Blood for analysis derived from mice 5h after last (5 th ) application of the eliciting dose of antigen. A complete return to the BG number of leukocytes in mice treated with the 4B8M peptide was indicated, and a lesser efficacy for Elidel ® and Protopic ® (not significant changes). The changes in blood picture of mice (24h measurement from the last application of the eliciting dose of antigen) were shown in Fig. S13. In the blood picture of inflammed mice not treated with the compounds (Control group) an increased level of eosinophils (to 7.2%) was noted as compared to 2.2% in BG group. The application of 4B8M reduced the % of eosinophils to 1.3%. Protopic ® and Elidel ® also decreased the eosinophil content although to lesser degree than 4B8M (to 2.2 and 2.0%, respectively).

Effect of the peptide on permeability of capillary blood vessels
The effects of the compounds on the permeability of skin capillary vessels of the auricles (5h measurement from the last application of the eliciting dose of antigen) were shown in Fig.   S14. The results indicate that all compounds were highly effective in suppression of the blood vessel permeability, measured as amount of Evans blue dye penetrating the tissue.  The effects of the peptide and reference compounds on the histological changes of auricles (5h measurement from the last application of the eliciting dose of antigen) are presented in photographs (Fig. S15) and figures S16 and S17. toluidine blue staining. The epidermis is thin with distinctly visible basal and spinous layers and weakly separated granular, lucid and corneal layers. In the perivascular zone single lymphocytes and macrophages can be found. Single neutrophils were restricted mainly to the interior of blood vessels. The mastocytes occurred in a condensed form within the subepithelial connective tissue and around blood vessels.    toluidine blue staining. The auricles of mice treated with Elidel ® presented a strong inflammation. An increased proliferation of the epidermal basal layer, significantly thicker spinous layer may indicate akanthosis but thicker corneal layer proves about hyperkeratosis of epidermis. In the lower parts of epidermis focal spongiosis (enlarged spaces between keratinocytes) was registered and infiltration of single lymphocytes was revealed. In the dermis the inflammatory changes were more strongly pronounced. Vast infiltrations, consisting mainly of mastocytes and neutrophils, and to a lesser degree of macrophages and lymphocytes, were observed. Toluidine blue staining showed a significant increase of the mastocyte numbers as compared to other groups. Mastocytes formed clusters around blood vessels, in the zone of connective, subepithelial tissue and around glands. The cells were packed with granules and the majority of cells were in a degranulation phase. Also, a loosening of the architecture of the collagen fibers was noted which could explain the swelling of the connective tissue. In Fig. S16 the cell composition of the inflamed auricle tissue (5h measurement from the last application of the eliciting dose of antigen) is presented. In control mice, not treated with the compounds, a significant increase of the neutrophil number (infiltration) and mastocytes, as compared to the normal picture, was observed. A diminution of the neutrophil infiltration, as well as mastocytes, is best seen upon application of Protopic ® . 4B8M peptide showed somewhat weaker action. Elidel ® , in turn, significantly increased infiltration of mastocytes, macrophages and lymphocytes. Fig. S17 illustrates changes regarding the thickness of the epidermis (5h measurement). 4B8M peptide and Protopic ® exhibited comparable action, whereas Elidel ® caused about 2-fold increase of the epidermis.

Non specific skin inflammation induced by salicylic acid -Materials and Methods
We followed a method described elsewhere [Patrick et al. 1987]. 20% salicylic acid (SA) solution in 96% ethanol was prepared and was applied for 3 days (5× per day every 30 min) on both sides of the auricles (20 µl portions). Mice from the BG group were given only 96% ethanol. After last application of SA on a given day (following 30 min of last SA dose), the auricles were smeared with the vehiculum (the BG and control groups), 0.1% 4B8M in ointment, Protopic ® or Elidel ® . 24h after the last SA application (day 3rd of the test) the parameters of the inflammatory reaction were determined.

Determination of skin vessel permeability
The test was performed according to Katayama et al, 1978 with minor modifications. Mice were given 1 mg of Evans blue in 0.2 ml of 0.9% NaCl, intravenously, on day 3 of the test.
After 30 min the mice were sacrificed by cervical dislocation, the auricles cut off and immersed in 50 µl of 1 M KOH for 18h at 37 o C. The dye was extracted from the tissue using a mixture of 450 µl of 0.2 M phosphoric acid and acetone (5:13 ratio). The samples were centrifuged at 3,000 rpm for 15 min. OD of the supernatants was measured at 550/630 nm.
The amount of Evans blue (µg/ml) was determined from a standard curve. The results were presented as amount of the dye per 100 mg of wet tissue.

Therapeutic effect of 4B8M peptide on skin inflammation induced by salicylic acid
Effect of the preparations on ear edema

Effect of the preparations on blood leukocyte numbers
Effect of the preparations on blood leukocyte numbers was shown in Fig. S19. The leukocyte numbers were strongly elevated in the control group. 4B8M most effectively lowered the leukocyte number (by 57%), even below the values observed in the BG group. The reference compounds Protopic ® and Elidel ® were less effective (about 35% inhibition).  In control inflammed mice an increase of band forms (immature neutrophils) and eosinophils was found. 4B8M peptide almost totally reduced the percentage of band forms and eosinophils (statistical significance). Similar effects were observed in Protopic ® -treated mice.
Treatment of mice with Elidel ® led to considerable distortion of the blood picture (an increase of bands and eosinophils).

Effect of the preparations on lymph node cell numbers
Effects of the preparations of cell numbers in draining lymph nodes were presented in Fig.   S21. In mice with induced inflammation (control group) more than 2-fold increase of the cell numbers was observed. A significant decrease of cell numbers were noted in mice treated with the 4B8M tetrapeptide and a somewhat weaker in mice treated with Protopic ® , Elidel ® was without effect.  Effect of the preparations on skin blood vessel permeability Effect of the preparations on permeability of skin vessels of the auricles was presented in Fig.   S22. The measurement of that parameter revealed a 4-fold increase in the control group as compared to the BG naive group. The best inhibitory effects showed 4B8M peptide and a slightly weaker Protopic ® , Elidel ® was less effective. The normalizing effects of the peptide on the inflammatory skin histological changes induced by SA, are presented in Fig. S23.    toluidine blue staining. In mice treated with Elidel ® the histological picture of auricles presents an inflammatory state in the dermis, however less expressed than in the control group because of a fewer number of neutrophils in the connective tissue of the dermis. No increase of mastocyte numbers was noted, which were in majority in a condensed state. The inflammatory process was strongly expressed in the epidermis. Exocytosis of neutrophils and lymphocytes into the epidermis was observed. A presence of neutrophilic pustules in various development stages were registered. In some areas a disruption of the epidermal integrity was found. Similarly as in the control group, the areas of epidermal necrosis occurred. Generally, 4B8M peptide had a similar potency as Protopic ® in the SA-induced inflammation model, with a more beneficial effects of Protopic ® in histological analysis. However, 4B8M had a better influence on blood picture and was devoid of toxicity in contrast to Protopic ® (tacrolimus). Elidel ® (pimecrolimus) exhibited undesirable effects.

Carrageenan-induced inflammation in air pouch -Materials and Methods
A method described by Bottomley et al. 1988 was used. The model of carrageenan-induced inflammation in the air pouch is useful for evaluation of potential anti-inflammatory properties of compounds. An advantage of the test is a moderate inflammation process and occurrence of granuloma. The air pouch was formed in CBA mice by a s.c. injection into the dorsal region (in a halothane anesthesia) of 5 ml of air (needle 23G×11/4.5 ml syringe). On a next day the air pouch was given an additional 1 ml of air and an inflammatory process was elicited by an injection of 1% of carrageenan in PBS. The tetrapeptide (100 µg/0.2 ml) was given i.p. into mice. Indomethacin was diluted in 0.9% NaCl and administered at a dose of 100 µg/mouse and dexamethasone was diluted in 0.9% NaCl and administered at a dose of 100 µg/mouse i.p. Aspirin was dissolved in 0.9% NaCl and given at a dose of 6 mg/mouse, per os using a stomach tube. The compounds were administered at 24h following formation of the air pouch (day 1 of the test). After 90 min carrageenan was injected into the air pouch.
The mice form the BG group were given 0.9% NaCl into the air pouches. Control mice were given carrageenan into the air pouches and appropriately diluted DMSO i.p.
On a next day after carrageenan injection (2 nd day of the test) mice were subjected to halothane anesthesia and blood was taken from the retroorbital plexus. From the blood smears were made, dried and stained with May-Grünwald and Giemsa reagents. The blood was diluted with Türk's solution for determination of circulating leukocytes. Exudates cells were harvested by injection of 1 ml of 0.9% NaCl into the air pouches and a subsequent aspiration by means of a syringe. In the exudates the cell numbers were counted and after exudates centrifugation the cell smears were performed form the cell pellets for cell types differentiation. The smears were dried and stained with May-Grünwald and Giemsa reagents.

Therapeutic effect of 4B8M peptide on carrageenan-induced inflammation in air pouch
Effect of the preparations on the number of cells in the air pouches Fig. S24 presents effects of administration of the peptide and the reference drugs (aspirin, indomethacin and dexamethasone), on the number of cells in the air pouches. In the control, carrageenan-treated mice, a very significant increase of the cell number in the exudates was observed (from 0.48×10 6 cells in the BG group to 1.7×10 7 in the control). The applied preparations strongly and significantly decreased cell numbers (48BM to 4.02×10 6 , dexamethasone to 2.25×10 6 , indomethacin to 1.84×10 6 and aspirin to 2.23×10 6 ).  Effect of the preparations on degranulation of mastocytes in the air pouches Carrageenan reaction is initiated by degranulation of skin mastocytes [Alves et al. 2017]. Fig.   S25 illustrates effects of the studied compounds on degranulation of mastocytes in the air pouches. In mice not treated with carrageenan (BG group) all mastocytes were intact. In turn, in the control group with non-treated inflammation, the contribution of degranulated mastocytes was 67%. Among the applied compounds only dexomethasone did not significantly affect the percentage of degranulated mastocytes. The highest increase of nondegranulated mastocytes occurred after application of the peptide (75.5%). Indomethacin and aspirin increased the content of intact mastocytes to 64.8% and 54.7%, respectively.  Effect of the preparations on the circulating blood cell composition   In this layer mainly: histiocytes, fibroblasts, fibrocytes and rare mastocytes, located among collagen bundles, were observed ( Fig. S28C and Fig. S28D). Locally, in the granuloma, areas of collagen necrobiosis were seen, disintegration of collagen bundles were registered and changes in collagen staining (an increased basophilicity) (Fig. S28D). Also, the surfaces of necrobiosis were surrounded by numerous histiocytes, arranged in a form of cascades. In the interstitium of the connective tissue mucin deposits were also observed being a proof of mucoid edema. The deposits were formed due to accumulation of acid glycosaminoglycans in the connective tissue and in H&E staining they appeared as granular or strip-like material ( Fig. S28C). Toluidine blue staining, revealing acidic glycosaminoglycans, confirmed the presence of mucins in the dermis (Fig. S28D). In the control group a very strong accumulation of mucins in the dermis was observed (Fig. S28D). The mucin deposits were seen in the dermis connective tissue (around blood vessels and skin appendages), in the connective tissue of subdermis, in the connective tissue of granuloma and in the connective, intermuscular tissue; 100× magnification. toluidine blue staining. In this group of mice the thickness of the granuloma was the smallest.
Within the granuloma a development of a fibrous zone was observed ( Fig. S28E and Fig.   S28F). A diminution of mucin content in the dermis was noted, particularly around hair follicles. Toluidine blue staining revealed weaker metachromasia in this area (Fig. S28F). The content of mucin in the connective, intermuscular tissue and in the fatty stroma, was comparable with that in the aspirin treated-mice; 100×magnification.

Thickness of the granuloma -a comparative study
Beside of the histological analysis of the skin also histometric measurements of the granuloma thickness were performed (Fig. S29). The granuloma thickness was best reduced in the group of mice treated with the peptide. Aspirin treatment was least effective and dexomethasone did not affect the thickness of the granuloma tissue.  The results from the air pouch model in mice with carrageenan-induced inflammation showed that the tetrapeptide was more efficient, as compared to the reference drugs (aspirin and dexomethasone), in diminishing symptoms of inflammation. The tetrapeptide normalized more effectively cell compositions in the air pouch exudates and circulating blood. On the other hand, it was less effective in comparison with the reference drugs in lowering numbers of exudates cells and circulating leukocytes. In the histological picture the tetrapeptide exhibited a strong anti-inflammatory and repair actions, comparable with that of aspirin. In addition, the tetrapeptide displayed the strongest action with regard to diminution of the granuloma thickness, as compared with the reference compounds.

Methods
The test was performed according to Melgar et al. 2005, with some modifications. C57Bl/6 mice were given 4% solution of dextran sulphate in tap drinking water acidified to pH 3.5 (with hydrochloric acid to limit growth of Pseudomonas species), for 6 days. Then, the mice were given only the acidified water. The tetrapeptide 4B8M was administered to mice intragastrically, by means of a stomach tube, at a dose of 250 μg in 0.2 ml, daily on days 1-5.
Pentasa ® (5-ASA) was given in the same way at a dose of 1.5 mg (0.15 ml). The control group received appropriately diluted DMSO solution (as in 4B8M preparation). On day 12 of the experiment the mice were subjected to halothane anesthesia and bled via retroorbital plexus, followed by cervical dislocation. The thymuses and colon were isolated.
Determination of the level of IL-1β in the colonic tissue Colon was placed in disposable Petri dishes. Approximately 0.1 g of colonic mucosa was added to 1 ml of sterile, cold PBS with 10% FCS and protease inhibitor (cOmplete Protease inhibitor Coctail, Roche Diagnostics, Poland) and the mixture was pulverised homogeneously by passing through a nylon mesh. The samples were centrifuged and the supernatants analyzed by ELISA kit (eBioscience) for IL-1β concentration.

Therapeutic effect of 4B8M peptide on non-specific colon inflammation induced by dextran sulfate
The colonic tissue homogenate level of IL-1β was significantly lower in control group (Fig.   S30). 4B8M peptide, and to a lesser degree 5-ASA, reversed that fall in the cytokine level.

Allergic pleurisy induced by ovalbumin -Materials and Methods
The previously described method was used (Sampaio et al. 2000). The mice were sensitized i.p. with 50 μg of ovalbumin (OVA) in Maalox (aluminii hydroxydum 3.5 g, magnesi hydroxydum 4.0 g in 100 ml; Rhone-Poulenc Rorer) as adjuvant. After 14 days the mice were injected with 12.5 μg OVA in 50 μl of 0.9% NaCl into pleural cavity by means of a syringe with 2 mm needle length. Mice from BG group were given only second (eliciting) dose of OVA. 2h before administration of the eliciting dose of antigen the mice were given per os (by gastic gavage) 250 μg of 4B8M in olive oil. The reference compounds -dexamethasone (125 μg/mouse) and indometacin (250 μg/mouse) were also given per os. On the next day mice were subjected to halothane anesthesia and bled via retroorbital plexus, followed by cervical dislocation. The draining lymph nodes and exudates from inflammed pleural cavity were also isolated.

Therapeutic effect of 4B8M peptide on OVA-induced allergic pleurisy
Effect of the preparations on the number of circulating leukocytes An increase of the number of circulating leukocytes in the group of BG mice and a further increase in the control group (2× increase as compared with naive mice) was indicated (Fig.   S32). The administration of 4B8M lowered the leukocyte number to a level registered in naive mice and to the level of BG mice by dexamethasone. Indometacin drastically lowered the leukocyte number (below the values noted in naive mice).

Effect of the preparations on degranulation of mastocytes in the pleural exudates
In the BG group the percentage of non-degranulated mastocytes in pleural exudates was high (55.2% of all isolated cells) and low in the control (inflammed) group (20.6%) (Fig. S33). The investigated compounds in a various degree increased the content of non-degranulated mastocytes: 4B8M to 70.3%, dexamethasone to 31.7% and indometacin to 63.2%.

Effect of the preparations on the number of cells in draining lymph nodes
The cell number in draining lymph nodes (Fig. S34) was significantly elevated in inflammed control mice (1.4×10 7 vs 5.5×10 6 in naive mice and 8.8×10 6 in the BG group). The applied compounds evenly lowered the cell numbers below BG levels and near the naive mice values.  The protective effects of the 4B8M peptide on the histological changes in inflammed lungs from mice with pleurisy are presented in Fig. S35.  S35CD. Lungs from inflammed control mice group; H&E staining. In the lungs of animals from control group severe pulmonary edema was observed, as indicated by the presence of eosinophilic, edematous fluid in the alveolar septa and inside the alveoli. The dominant histological finding was interstitial pulmonary edema. The alveolar septa were strongly thickened by the edematous fluid accumulating within them, and the pulmonary capillaries were dilated. In addition, there was also perivascular edema. Alveolar pulmonary edema has been reported mainly in the subpleural zone and near large pulmonary vessels.
Within the edematous lesions, numerous siderophages (hemosiderin-laden macrophages) and haemosiderin deposits were observed, both in the alveolar septa and inside the alveoli; hemosyderophages indicated by arrows; magnification 100× (B) and 400× (C). In the lungs of dexamethasone-treated animals, the edematous lesions were weaker as compared to the control group with full blown inflammation. Foci of weak interstitial edema ( Fig. S35E) and moderate, alveolar edema in the subpleural area (Fig. S35F) were observed.
In addition, there were areas of lung congestion with local haemorrhages into the interstitium and alveoli (Fig. S35E). Numerous siderophages and haemosiderin deposits were observed in the edematous lesions as well as in the area of hyperemia and haemorrhages; hemosyderophages indicated by arrows; magnification 100× (C) and 400× (D).