Revathi Shanmugasundaram

Regulatory T Cell Properties of Chicken CD4+CD25+ Cells

Chicken CD4+CD25+ cells were characterized for mammalian regulatory T cells’ suppressive and cytokine production properties. Anti-chicken CD25 mAb was produced in mice and conjugated with a fluorescent tag. The specificity of the Ab against chicken CD25 was confirmed by evaluating Con A-induced CD25 upregulation in thymocytes and by quantifying the CD25 mRNA content of positive and negative cells identified by anti-chicken CD25 Ab. The percentage of CD4+CD25+ cells, expressed as a percentage of CD4+ cells, in thymus and blood was ∼3–7%, in spleen was 10%, and in cecal tonsil, lung, and bone marrow was ∼15%. Bursa had no detectable CD4+CD25+ cells. CD25+ cells were mostly CD4+ in the thymus, whereas in every other organ studied, CD25+ cells were distributed between CD4+ and CD4− cells. Chicken thymic CD4+CD25+ cells did not proliferate in vitro in the absence of recombinant chicken IL-2 (rCIL-2). In the presence of rCIL-2, PMA plus ionomycin or Con A stimulated CD4+CD25+ cell proliferation, whereas anti-CD3 plus CD28 did not stimulate CD4+CD25+ cell proliferation. Naive CD4+CD25+ cells had 29-fold more IL-10 mRNA and 15-fold more TGF-β mRNA than the naive CD4+CD25− cells. Naive CD4+CD25+ had no detectable IL-2 mRNA. Both naive and PMA plus ionomycin-stimulated thymic CD4+CD25+ cells suppressed naive T cell proliferation. The suppressive properties were partially contact dependent. Supplementing CD4+CD25+ cell coculture with rCIL-2 reversed the suppressive properties of CD4+CD25+ cells. Chicken CD4+CD25+ cells have suppressive properties similar to that of mammalian regulatory T cells.

Effects of dietary lutein and PUFA on PPAR and RXR isomer expression in chickens during an inflammatory response.

This study investigated the effects of dietary lutein and polyunsaturated fatty acids (PUFA) on the total lutein and lipid levels, the peroxisome proliferators activated receptors (PPAR) alpha and gamma, retinoic acid X receptor (RXR) alpha and gamma and IL-1 mRNA levels in chicken (Gallus gallus) liver and spleen. In experiment I, chickens were fed either 0, 25 or 50mg lutein in a diet with 3% PUFA fat. In experiment II, chickens were fed either 3 or 6% PUFA fat with 25mg lutein. At 23d of age, chickens were injected with LPS. LPS injection decreased the lutein content and increased the fat content in the liver and spleen in both experiments. Increasing dietary PUFA fat to 6% ameliorated the LPS-induced lutein depletion in experiment II. LPS injection increased IL-1 and decreased splenic PPARalpha, PPARgamma, RXRalpha mRNA in experiment I and II. The LPS-induced PPARalpha and RXRalpha downregulation were partially reversed by increasing the dietary lutein content to 50mg/kg feed in experiment I and by increasing the dietary PUFA fat content to 6% in experiment II. Increasing dietary lutein content to 50mg/kg feed increased PPARgamma mRNA amount only in the LPS untreated groups in experiment I. Increasing dietary PUFA fat or LPS injection in the 6% PUFA fat group upregulated PPARgamma mRNA in experiment II. Increasing dietary PUFA fat to 6% blunted the increase in IL-1 mRNA due to LPS. It is concluded that dietary lutein and PUFA fat were anti-inflammatory due to modification of immune tissue lutein content, PPAR, RXR isomers and IL-1beta mRNA levels in liver and spleen.

In vitro human TGF-β treatment converts CD4+CD25- T cells into induced T regulatory like cells.

In mammals, induced T regulatory cells (iTregs) are produced by treating CD4(+)CD25(-) cells in vitro with TGF-beta in the presence of anti-CD3 and IL-2. Chicken CD4(+)CD25(-) cells were treated in vitro with TGF-beta, anti-CD3, anti-CD28 and IL-2 and analyzed for suppressive properties, IL-10 and IL-2 mRNA amounts. TGF-beta treatment decreased (P<0.01) CD4(+)CD25(-) cell proliferation by 45% at 5d of cell culture with no increase in dead cell population. CD4(+)CD25(-) cells treated with 5ng TGF-beta for 5d had 130-fold (P<0.01) more IL-10 and 104-fold (P<0.01) less IL-2 mRNA content than the 0ng TGF-beta treated groups. CD4(+)CD25(-) cells treated with 5ng TGF-beta for 5d suppressed (P<0.01) naïve cell proliferation compared to the 0ng TGF-beta treated groups. The results suggest the presence of iTreg-like cells in avian species.

Lutein supplementation alters inflammatory cytokine production and antioxidant status in F-line turkeys

Effect of dietary lutein supplementation on turkey production parameters, cytokine production, and oxidative status during an acute phase response following lipopolysaccharide (LPS) injection was studied. One-day-old chicks were fed a basal diet supplemented with 3 levels (0, 25, or 50 mg/kg of feed) of lutein. At 50 d of dietary lutein supplementation, turkeys were injected or not injected with LPS. Increasing dietary lutein increased the liver and plasma lutein content in both LPS injected and uninjected groups. In the groups fed 50 mg of lutein, LPS treatment decreased the lutein content of both the liver and the plasma at 48 h post-LPS injection. In the groups fed 0 mg of lutein, LPS treatment decreased the BW gain and feed consumption at 24 and 48 h post-LPS injection. The feed intake and BW gain of the group fed 50 mg of lutein in the LPS injected groups were comparable to those of the group with no LPS injection at both 24 and 48 h post-LPS injection. Treatment with LPS increased IL-1β mRNA content (P = 0.01) in the group fed 0 mg of lutein. In the LPS injected groups, increasing dietary lutein to 50 mg decreased the IL-1β mRNA amount compared with the group fed 0 mg of lutein. In the LPS injected groups, increasing dietary lutein to 50 mg increased IL-10 mRNA content compared with the group fed 0 mg of lutein. Injection of LPS increased the thiobarbituric reactive substances content of the liver in the group fed 0 mg of lutein. Increasing dietary lutein to 50 mg decreased the thiobarbituric reactive substances content of the liver in the LPS injected groups. Dietary lutein supplementation decreased oxidative damage and inflammatory responses post-LPS injection by decreasing IL-1β production and increasing IL-10 production in turkeys.

Effect of yeast cell product (CitriStim) supplementation on broiler performance and intestinal immune cell parameters during an experimental coccidial infection

This experiment studied the effects of whole yeast cell product supplementation on broiler production parameters, fecal coccidial oocyst counts, and local and systemic immune parameters following an experimental coccidial infection. Birds were fed 0, 0.1, or 0.2% whole yeast cell product (CitriStim). At 21 d of age, birds were challenged with live coccidial oocysts. Supplementation with whole yeast cell product increased BW gain between 0 and 12 d (P = 0.01) postcoccidial challenge. Birds supplemented with 0.2% Citristim had better (P = 0.01) feed efficiency between 0 and 12 d postcoccidial infection. Supplementation with whole yeast cell product decreased (P = 0.01) the fecal coccidial oocyst count at 7 d postcoccidial challenge. Citristim supplementation at 0.2% increased (P < 0.01) macrophage nitric oxide production by 93 and 193% at 5 and 12 d postcoccidial challenge. Supplementation with whole yeast cell product at 0.2% increased cecal tonsil interleukin-1 mRNA amounts approximately 4.5- and 3.7-fold at 5 and 12 d postcoccidial challenge, respectively, over the group with no whole yeast cell product supplementation. Citristim supplementation downregulated cecal tonsil interleukin-10 mRNA amounts compared with the unsupplemented groups at both 5 (P = 0.01) and 12 d (P < 0.01) postcoccidial challenge. Supplementation with whole yeast cell product did not alter (P > 0.05) serum anticoccidial IgG contents or cecal tonsil CD4(+) and CD8(+) cell percentages at 5 and 12 d postcoccidial infection. It could be concluded that supplementing whole yeast cell product (CitriStim) to broiler diets can improve production parameters, decrease fecal oocyst count, and increase inflammatory cytokine production postcoccidial infection.

Effect of killed whole yeast cell prebiotic supplementation on broiler performance and intestinal immune cell parameters

Two experiments were conducted to study the effect of CitriStim, a commercial killed whole yeast cell prebiotic, on broiler performance, regulatory T cells, CD4(+) and CD8(+) percentages, and IL-10 and IL-1 mRNA contents of the spleen and cecal tonsils. No immune challenges were imposed in either of the 2 experiments. One-day-old broiler chicks were fed a corn- and soybean meal-based diet supplemented with 0, 0.1, or 0.2% CitriStim (ADM, Decatur, IL) for 35 d. At 21 (P = 0.03) and 35 d (P = 0.02) of age, CitriStim supplementation at 0.2% increased regulatory T cell percentage in the cecal tonsil compared with that of the 0% CitriStim-supplemented group. At 21 (P = 0.08) and 35 d (P = 0.01) of age, CitriStim supplementation at 0.2% increased IL-10 mRNA content of the cecal tonsil compared with that of the 0% CitriStim-supplemented group. At 21 (P = 0.13) and 35 d (P < 0.01) of age, CitriStim supplementation at 0.2% decreased IL-1 mRNA content compared with that of the 0% CitriStim supplemented group. CitriStim supplementation did not (P > 0.05) alter the IL-10 and IL-1 mRNA contents in the spleen. CitriStim supplementation did not (P > 0.05) alter the CD4(+) and CD8(+) cell percentages in the spleen and cecal tonsil at 21 and 35 d of the experiment. CitriStim supplementation increased regulatory T cell percentage and IL-10 mRNA content and decreased IL-1 mRNA content in the cecal tonsil to produce a net antiinflammatory milieu. The immunomodulatory effect of CitriStim supplementation was a local effect rather than a systemic effect.

Effect of yeast cell product supplementation on broiler cecal microflora species and immune responses during an experimental coccidial infection

This experiment was conducted to study the effects of whole yeast (Pichia guilliermondii; CitriStim, ADM, Quincy, IL) cell product supplementation on cecal microflora population and intestinal immune parameters in broilers. In the first experiment, birds were fed 0, 0.1, or 0.2% yeast cell wall product for 42 d. Feeding yeast cell wall products decreased (P = 0.03) the proportion of Escherichia coli in the ceca by 31% compared with the control group. The group fed 0.2% yeast cell wall product had a 20% decrease (P = 0.23) in Salmonella population compared with the control group. In the second experiment, birds were fed yeast cell wall product for 21 d and challenged or not challenged with coccidial oocysts, thus resulting in a 2 (0 and 0.2% whole yeast product) × 2 (coccidial challenge and no coccidial challenge) factorial model. Supplementing whole yeast cell wall product prevented a coccidial infection-induced decrease in the Lactobacillus population (P = 0.09) at 12 d postchallenge. Supplementing yeast cell wall product prevented a coccidial infection-induced increase in the Salmonella population (P = 0.08) and E. coli (P = 0.12) at 12 d postchallenge. At 5 d (P < 0.01) and 12 d (P < 0.01) postcoccidial infection, yeast cell wall product supplementation or coccidial infection increased the regulatory T cell (Treg) percentage in the cecal tonsils, whereas yeast cell wall product supplementation in the coccidial-infected group decreased the increase in Treg percentage. At 5 d postcoccidial infection, coccidial infection increased (P = 0.01) the relative amounts of cecal interferon (IFN)γ mRNA. In addition, the yeast cell wall product supplementation in the coccidial-infected groups further increased (P = 0.15) the IFNγ mRNA. It could be concluded that yeast cell wall product supplementation decreased coccidial-infection-induced increase in E. coli and Salmonella colonization and improved IFNγ mRNA amounts after coccidial infection.

Vitamin D-1α-hydroxylase and vitamin D-24-hydroxylase mRNA studies in chickens.

A series of experiments were conducted to study the basal amounts of vitamin D-1α-hydroxylase and vitamin D-24-hydroxylase mRNA amounts in different organs and the effect of immune stimulation on 1α- and 24-hydroxylase mRNA amounts in chickens. At day of hatch, kidneys had an approximately 66-fold higher amount of 1α-hydroxylase and 550-fold higher amount of 24-hydroxylase mRNA, thigh and breast muscles had an approximately 20-fold higher amount of 1α-hydroxylase mRNA, and the thymus had an approximately 41-fold higher amount of 24-hydroxylase mRNA than the liver. An in vivo LPS injection did not alter the amount of 1α-hydroxylase mRNA in the breast muscle (P=0.60) or in the kidneys (P=0.39). An in vivo LPS injection decreased (P=0.01) the amount of 24-hydroxylase mRNA in the breast muscle at 3 d post-LPS injection. An in vivo LPS injection increased (P=0.01) the amount of 24-hydroxylase mRNA in the kidneys at 2, 3, and 6 d post-LPS injection. An in vitro stimulation altered amounts of 1α- (P=0.01) and 24-hydroxylase (P=0.04) mRNA in CD4+ cells. In conclusion, the distribution of 1α- and 24-hydroxylase mRNA amounts was similar to mammals, and an immune stimulation altered the amounts of 1α- and 24-hydroxylase mRNA in chickens.

25-Hydroxycholecalciferol supplementation improves growth performance and decreases inflammation during an experimental lipopolysaccharide injection

Three experiments were conducted to study the effects of 25-hydroxycholecalciferol supplementation on BW gain, IL-1β, and 1α-hydroxylase mRNA expression in different organs of broiler chickens following a lipopolysaccharide (LPS) injection. In experiment I, birds were fed a basal diet supplemented with either cholecalciferol (3,000 IU/kg) or 25-hydroxycholecalciferol (69 µg/kg). At 21 and 35 d of age, birds were injected with LPS. Post-LPS injection, birds supplemented with 25-hydroxycholecalciferol gained approximately 2.5% (P = 0.03) and 3.8% (P < 0.01), respectively, more BW than the birds supplemented with cholecalciferol over the 24-h period. In experiment II, birds were fed basal diets supplemented with 25-hydroxycholecalciferol at 6.25, 25, and 50 µg/kg of feed or cholecalciferol at 250 IU/kg of feed. At 35 d of age, birds were injected with LPS. Birds fed 25-hydroxycholecalciferol at 25 and 50 µg/kg and injected with LPS had approximately 7-fold and 3-fold less (P = 0.010) IL-1β mRNA in the liver compared with those birds fed 6.25 µg/kg of 25-hydroxycholecalciferol and the cholecalciferol (250 IU/kg) group. In experiment III, birds were fed a basal diet supplemented with either cholecalciferol (3,000 IU/kg) or 25-hydroxycholecalciferol (69 µg/kg). At 28 d of age, birds were fed 25-hydroxycholecalciferol and injected with LPS had 1.1-fold less (P < 0.01) IL-1β mRNA in the liver than the cholecalciferol-fed group. After an LPS injection, birds supplemented with 25-hydroxycholecalciferol had increased 1α-hydroxylase mRNA amounts in the liver (P = 0.07). In conclusion, 25-hydroxycholecalciferol supplementation at higher doses improved growth performance and decreased inflammatory gene IL-1β mRNA amounts in the liver post-LPS injection.

Effects of in vivo injection of anti-chicken CD25 monoclonal antibody on regulatory T cell depletion and CD4+CD25− T cell properties in chickens

Regulatory T cells (Tregs) are defined as CD4(+)CD25(+) cells in chickens. This study examined the effects of an anti-chicken CD25 monoclonal antibody injection (0.5 mg/bird) on in vivo depletion of Tregs and the properties of CD4(+)CD25(-) cells in Treg-depleted birds. The CD4(+)CD25(+) cell percentage in the blood was lower at 8 d post injection than at 0 d. Anti-CD25-mediated CD4(+)CD25(+) cell depletion in blood was maximum at 12 d post injection. The anti-CD25 antibody injection depleted CD4(+)CD25(+) cells in the spleen and cecal tonsils, but not in the thymus, at 12 d post antibody injection. CD4(+)CD25(-) cells from the spleen and cecal tonsils of birds injected with the anti-chicken CD25 antibody had higher proliferation and higher IL-2 and IFNγ mRNA amounts than the controls at 12 d post injection. At 20 d post injection, CD4(+)CD25(+) cell percentages in the blood, spleen and thymus were comparable to that of the 0 d post injection. It could be concluded that anti-chicken CD25 injection temporarily depleted Treg population and increased and IL-2 and IFNγ mRNA amounts in CD4(+)CD25(-) cells at 12d post injection.