Nicotine Protects Kidney from Renal Ischemia/Reperfusion Injury through the Cholinergic Anti-Inflammatory Pathway

Kidney ischemia/reperfusion injury (I/R) is characterized by renal dysfunction and tubular damages resulting from an early activation of innate immunity. Recently, nicotine administration has been shown to be a powerful inhibitor of a variety of innate immune responses, including LPS-induced toxaemia. This cholinergic anti-inflammatory pathway acts via the α7 nicotinic acetylcholine receptor (α7nAChR). Herein, we tested the potential protective effect of nicotine administration in a mouse model of renal I/R injury induced by bilateral clamping of kidney arteries. Renal function, tubular damages and inflammatory response were compared between control animals and mice receiving nicotine at the time of ischemia. Nicotine pretreatment protected mice from renal dysfunction in a dose-dependent manner and through the α7nAChR, as attested by the absence of protection in α7nAChR-deficient mice. Additionally, nicotine significantly reduced tubular damages, prevented neutrophil infiltration and decreased productions of the CXC-chemokine KC, TNF-α and the proinflammatory high-mobility group box 1 protein. Reduced tubular damage in nicotine pre-treated mice was associated with a decrease in tubular cell apoptosis and proliferative response as attested by the reduction of caspase-3 and Ki67 positive cells, respectively. All together, these data highlight that nicotine exerts a protective anti-inflammatory effect during kidney I/R through the cholinergic α7nAChR pathway. In addition, this could provide an opportunity to overcome the effect of surgical cholinergic denervation during kidney transplantation

Donor T-cell development in host thymus after heterotopic limb transplantation in mice.

We developed a mouse model of heterotopic limb transplantation in which we took advantage of Thy1.1 and Thy1.2 congenic strains to track and characterize donor T cells, to determine the role of recipient's thymus in mixed T-cell chimerism induction as well as transplant immunocompetence. The vascularized Thy1.1 limb graft composed of femur, muscle, and skin (VBT) survived long-term in more than 87.5% of Thy1.2 recipients. Percentages of donor-type Thy1.1 T cells increased from day 30 to 90 in thymus and spleen of recipients. Most peripheral donor T cells displayed a naïve phenotype and a few others were regulatory T cells. Thymectomy prevented peripheral T-cell chimerism. Congenic VBT in immunodeficient RAG mice restored their ability to reject skin allografts. These observations suggest that donor T cells differentiated in host thymus might contribute to maintenance of mixed chimerism after transplantation of tissue composite grafts that include vascularized bone.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Critical role of regulatory T cells in Th17-mediated minor antigen-disparate rejection

Th17-mediated immune responses have been recently identified as novel pathogenic mechanisms in a variety of conditions; however, their importance in allograft rejection processes is still debated. In this paper, we searched for MHC or minor Ag disparate models of skin graft rejection in which Th17 immune responses might be involved. We found that T cell-derived IL-17 is critical for spontaneous rejection of minor but not major Ag-mismatched skin grafts. IL-17 neutralization was associated with a lack of neutrophil infiltration and neutrophil depletion delayed rejection, suggesting neutrophils as an effector mechanism downstream of Th17 cells. Regulatory T cells (Tregs) appeared to be involved in Th17 reactivity. We found that in vivo Treg depletion prevented IL-17 production by recipient T cells. An adoptive cotransfer of Tregs with naive monospecific antidonor T cells in lymphopenic hosts biased the immune response toward Th17. Finally, we observed that IL-6 was central for balancing Tregs and Th17 cells as demonstrated by the prevention of Th17 differentiation, the enhanced Treg/Th17 ratio, and a net impact of rejection blockade in the absence of IL-6. In conclusion, the ability of Tregs to promote the Th17/neutrophil-mediated pathway of rejection that we have described should be considered as a potential drawback of Treg-based cell therapy. Copyright © 2010 by The American Association of Immunologists, Inc.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Nicotine-mediated protection against renal I/R is a7nAChoR-dependent.

<p>Serum creatinine levels one day after renal I/R were measured in the following groups from left to the right: saline-treated wild-type α7nAChoR sufficient animals (+/+, black bars, n = 10), nicotine-treated wild type α7nAChoR sufficient animals (+/+, white bars, n = 5), saline-treated α7nAChoR−/− animals (−/−, black bars, n = 8) and nicotine-treated α7nAChoR−/− animals. (−/−, white bars, n = 10). Nicotine was given at the dose of 0.25 mg/kg body weight. Data are expressed as mean ± SEM, (*) p≤0.05.</p

Nicotine pretreatment prevents neutrophil infiltration.

<p>LY-6G immunostaining was performed to assess neutrophil infiltration in the corticomedullary junction 24 hours after I/R (panel A, magnification ×40). Sham operated animals displayed no neutrophil (data not shown). Nicotine administration completely prevented neutrophil infiltration (panel B, magnification ×40). Quantification is represented in panel C. Black bars represent saline-treated animals and white bars nicotine-treated groups after either sham operation (n = 6 in each group) or I/R (n = 8 in each group). Data are expressed as mean ± SEM, (*) p≤0.05.</p

Nicotine reduces tubular epithelial cell apoptosis and subsequent proliferation.

<p>Immunostaining of cleaved caspase-3 (A, B) and Ki67 (D, E) were performed one day and 3 days after I/R, respectively. Panel A represents TEC apoptosis in saline-treated animals (magnification ×40) and panel B represents TEC apoptosis in nicotine pre-treated animals (magnification ×40). Graph C compares the quantification of apoptotic TECs in saline-treated (black bars) or nicotine-treated animals (white bars), either after sham operation (n = 6 in each group) or renal I/R (n = 7 in saline-treated group and n = 10 in nicotine pretreated group). Panels D and E compare Ki-67+ cells between saline-treated animals and nicotine-treated animals (magnification ×40). Graph F compares the quantification of proliferation 3 days after reperfusion (n = 5 in each group). Data are expressed as mean ± SEM, (*) p≤0.05.</p

Nicotine controls tubular damage after renal I/R.

<p>Tubular damage score (A) is shown in sham-operated animals (n = 7 in each group), 1 and 3 days after I/R in either saline-treated (black bars, n = 17 and n = 14 at day 1 and 3, respectively) or nicotine-treated animals (white bars, n = 15 and n = 13 at day 1 and 3, respectively). Histology of the corticomedullary junction 1 day after reperfusion from a sham-operated mouse (B, referred as sham), a saline-treated mouse that underwent I/R (C, referred as IRI-CTRL) and a nicotine-treated mouse (D, referred as IRI-nicotine), PAS staining, original magnification ×40. The PAS positive brush border (BB) is shown in panel B. Tubular casts (TC) and tubular dilatations (TD) are visible in panel C. Similar results were obtained in two separate experiments. Data are expressed as mean ± SEM, (*) p≤0.05.</p

Nicotine protects renal function after I/R in a dose-dependent manner.

<p>A dose range from 0.1 to 1 mg/kg body weight of nicotine has been administrated intraperitoneally 30 minutes before the induction of renal ischemia (A). Only 0.5 mg/kg (n = 12) and 1 mg/kg (n = 15) of nicotine protects renal function 1 day after renal I/R. Doses lower than 0.5 mg/kg such as 0.25 mg/kg (n = 6) and 0.1 mg/kg (n = 7) were not protective. Data are expressed as mean ± SEM, (*) p≤0.05. On panel B, serum creatinine levels were measured in sham-operated animals that received either saline solution (black bars, n = 8) or nicotine at the dose of either 0.5 mg/kg body weight (white bars, n = 6) or 1 mg/kg body weight (n = 6) and compared with saline-treated (black bars, n = 19 at day 1 and n = 15 at day 3) or nicotine treated animals at doses of 0.5 mg/kg (n = 12 at day 1 and n = 15 at day 3) or 1 mg/kg (n = 16 at day 1 and n = 12 at day 3) that underwent renal I/R. Blood samples were harvested 1 and 3 days after reperfusion. Results are representative of three independent experiments. Data are expressed as mean ± SEM, (*) p≤0.05.</p

The cholinergic pathway dampens immune responses in the draining lymph node of skin-grafted and imiquimod-treated female recipients.

<p>(<b>A</b> to <b>D</b>): From <b>A</b> to <b>D</b>, mRNA coding for IL-2, IL-17, IL-13 and IFN-γ were quantified by qPCR in draining lymph nodes 30 days after transplantation. Black bars represent wild type donor-recipient combination and white bars the α7−/− donor-recipient combination. Results are expressed as fold increase (FI = 2^−ΔΔCT) of quantities measured in lymph nodes from either ungrafted α7+/+ or α7−/− mice, n = 4 to 8 mice per group. (*): p<0.05 compared with wild type α7+/+ donor-recipient combination. (<b>E</b> to <b>G</b>): Intracellular IL-17 production in CD4 gated T cells after 4 hours of PMA-ionomycin stimulation is shown in control α7+/+ ungrafted mice (<b>E</b>), wild type donor-recipient combination (<b>F</b>) and α7−/− donor-recipient combinations (<b>G</b>). (<b>H</b> to <b>J</b>): Intracellular IFN-γ production in CD8 gated T cells after 4 hours of PMA-ionomycin stimulation is shown in control α7+/+ ungrafted mice (<b>H</b>), in wild type donor-recipient combination (<b>I</b>) and α7−/− donor-recipient combinations (<b>J</b>). (<b>K,L</b>): Bars summarize the percentages of IL-17+ CD4 T cells (<b>K</b>) and IFN-γ+ CD8 T cells (<b>L</b>) in either the wild type (black bars) or the α7−/− (white bars) donor-recipient combination. Controls have been harvested from α7+/+ ungrafted mice (grey bars). n = 4 to 8 mice/group. No difference regarding IL-17 (Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079984#pone-0079984-g001" target="_blank">figure 1</a>) or IFN-γ production has been observed between α7+/+ and α7−/− ungrafted controls. (**): p<0.01 α7−/− compared to α7+/+ donor-recipient combinations.</p