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CX₃CL1 (fractalkine) and its receptor CX₃CR1 regulate atopic dermatitis by controlling effector T cell retention in inflamed skin.

Staumont-Sallé D, Fleury S, Lazzari A, Molendi-Coste O, Hornez N, Lavogiez C, Kanda A, Wartelle J, Fries A, Pennino D, Mionnet C, Prawitt J, Bouchaert E, Delaporte E, Glaichenhaus N, Staels B, Julia V, Dombrowicz D - J. Exp. Med. (2014)

Bottom Line: CX3CR1 deficiency affected neither antigen presentation nor T cell proliferation in vivo upon skin sensitization, but CX3CR1 expression by both Th2 and Th1 cells was required to induce AD.Surprisingly, unlike in allergic asthma, where CX3CL1 and CX3CR1 regulate the pathology by controlling effector CD4(+) T cell survival within inflamed tissues, adoptive transfer experiments established CX3CR1 as a key regulator of CD4(+) T cell retention in inflamed skin, indicating a new function for this chemokine receptor.Therefore, although CX3CR1 and CX3CL1 act through distinct mechanisms in different pathologies, our results further indicate their interest as promising therapeutic targets in allergic diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale U1011, Institut Pasteur de Lille and Université Lille 2, 59019 Lille, FranceInstitut National de la Santé et de la Recherche Médicale U1011, Institut Pasteur de Lille and Université Lille 2, 59019 Lille, FranceInstitut National de la Santé et de la Recherche Médicale U1011, Institut Pasteur de Lille and Université Lille 2, 59019 Lille, France European Genomic Institute of Diabetes, 59045 Lille, France Department of Dermatology, Claude-Huriez Hospital, 59037 Lille, France.

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CX3CR1 is required for T cell retention in chronically inflamed skin. (A) Equal numbers of LACK-specific CX3CR1gfp/gfp and CX3CR1+/gfp Th2 or Th1 cells were coinjected into WT recipient mice at day 0. Recipients were sensitized with LACK at days 1 and 4 and analyzed at days 3 and 7. Donor cells were analyzed by flow cytometry 3 d later in inguinal draining LNs (dLN) after staining with antibodies against CD45, CD4, Thy1.1, Thy1.2, annexin-V, and 7-AAD. Data show mean frequencies of annexin-V+ cells among donor cells ± SEM. (B) CX3CR1+/gfp and CX3CR1gfp/gfp Th2 cells were infected with a Bcl2 or an empty retroviral vector. Equal numbers of transduced cells were coinjected into recipients that were further sensitized with one round of LACK sensitization. Data show the mean of donor cell frequency in skin (bottom) and inguinal draining LN (top) of individual mice in one representative experiment out of two (n = 5 mice per group). *, P < 0.05. (C) AD was induced in WT mice as described in the legend of Fig. 1, and equal numbers of LACK-specific CX3CR1gfp/gfp and CX3CR1+/gfp Th1 (top) or Th2 (bottom) cells were coinjected at day 41. Donor cells were analyzed in skin, draining LN, and spleen by flow cytometry. Data show representative flow cytometry profiles. Numbers indicate frequencies ± SEM of donor Th1 (top) or Th2 (bottom) cells among the CD4+ T cell population. One representative experiment out of two is shown (n = 12 mice per group). (right) 18 h before sacrifice, recipient mice were injected with BrdU and donor cells were analyzed by flow cytometry after staining with anti-BrdU, anti-Thy1.1, anti-Thy1.2, anti-CD4, or anti-CD45 antibodies. Data show representative cytometry profiles, and numbers indicate the mean (n = 12 mice). One representative experiment out of two is shown. (D) Equal numbers of LACK-specific CX3CR1gfp/gfp and CX3CR1+/gfp effector cells were coinjected into WT recipient mice at day 0. Recipients were sensitized with LACK at days 1 and 4. At day 4, some recipient mice topically received LACK together with CX3-AT. 3 d later, the frequencies of donor cells were analyzed in skin and draining LNs by flow cytometry. Data show mean of donor cell frequency of individual mice in one representative experiment out of two (n = 5 mice per group). *, P < 0.05. (E–G) AD was induced as described in the legend of Fig. 1. During the last round of sensitization, some mice also received CX3-AT (50 µg/mouse) through the patch together with LACK as indicated in the figure. (E) May-Grünwald Giemsa staining of skin sections. (F) Epidermal thickness. (G) Mast cell and eosinophil numbers. Data for E–G show mean of cell numbers in one representative experiment out of two (n = 6 mice per group). *, P < 0.05. (H–J) CX3CR1gfp/gfp mice were injected with polyclonal naive WT or CX3CR1gfp/gfp (KO) CD4+ T cells 24 h before AD induction. During the last round of sensitization, mice also received CX3-AT (50 µg/mouse) through the patch together with LACK as indicated in the figure. (H) Epidermal thickness. (I and J) Mast cell and eosinophil numbers. Data for H–J show mean of cell numbers in one representative experiment (n = 5 mice per group). *, P < 0.05. Error bars indicate SEM.
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fig5: CX3CR1 is required for T cell retention in chronically inflamed skin. (A) Equal numbers of LACK-specific CX3CR1gfp/gfp and CX3CR1+/gfp Th2 or Th1 cells were coinjected into WT recipient mice at day 0. Recipients were sensitized with LACK at days 1 and 4 and analyzed at days 3 and 7. Donor cells were analyzed by flow cytometry 3 d later in inguinal draining LNs (dLN) after staining with antibodies against CD45, CD4, Thy1.1, Thy1.2, annexin-V, and 7-AAD. Data show mean frequencies of annexin-V+ cells among donor cells ± SEM. (B) CX3CR1+/gfp and CX3CR1gfp/gfp Th2 cells were infected with a Bcl2 or an empty retroviral vector. Equal numbers of transduced cells were coinjected into recipients that were further sensitized with one round of LACK sensitization. Data show the mean of donor cell frequency in skin (bottom) and inguinal draining LN (top) of individual mice in one representative experiment out of two (n = 5 mice per group). *, P < 0.05. (C) AD was induced in WT mice as described in the legend of Fig. 1, and equal numbers of LACK-specific CX3CR1gfp/gfp and CX3CR1+/gfp Th1 (top) or Th2 (bottom) cells were coinjected at day 41. Donor cells were analyzed in skin, draining LN, and spleen by flow cytometry. Data show representative flow cytometry profiles. Numbers indicate frequencies ± SEM of donor Th1 (top) or Th2 (bottom) cells among the CD4+ T cell population. One representative experiment out of two is shown (n = 12 mice per group). (right) 18 h before sacrifice, recipient mice were injected with BrdU and donor cells were analyzed by flow cytometry after staining with anti-BrdU, anti-Thy1.1, anti-Thy1.2, anti-CD4, or anti-CD45 antibodies. Data show representative cytometry profiles, and numbers indicate the mean (n = 12 mice). One representative experiment out of two is shown. (D) Equal numbers of LACK-specific CX3CR1gfp/gfp and CX3CR1+/gfp effector cells were coinjected into WT recipient mice at day 0. Recipients were sensitized with LACK at days 1 and 4. At day 4, some recipient mice topically received LACK together with CX3-AT. 3 d later, the frequencies of donor cells were analyzed in skin and draining LNs by flow cytometry. Data show mean of donor cell frequency of individual mice in one representative experiment out of two (n = 5 mice per group). *, P < 0.05. (E–G) AD was induced as described in the legend of Fig. 1. During the last round of sensitization, some mice also received CX3-AT (50 µg/mouse) through the patch together with LACK as indicated in the figure. (E) May-Grünwald Giemsa staining of skin sections. (F) Epidermal thickness. (G) Mast cell and eosinophil numbers. Data for E–G show mean of cell numbers in one representative experiment out of two (n = 6 mice per group). *, P < 0.05. (H–J) CX3CR1gfp/gfp mice were injected with polyclonal naive WT or CX3CR1gfp/gfp (KO) CD4+ T cells 24 h before AD induction. During the last round of sensitization, mice also received CX3-AT (50 µg/mouse) through the patch together with LACK as indicated in the figure. (H) Epidermal thickness. (I and J) Mast cell and eosinophil numbers. Data for H–J show mean of cell numbers in one representative experiment (n = 5 mice per group). *, P < 0.05. Error bars indicate SEM.

Mentions: The higher frequency of CX3CR1-proficient CD4+ effector cells in skin could be explained by several hypotheses: the preferential late recruitment of CX3CR1+ effector T cells into the skin, their prolonged survival, or their selective advantage for residence in the inflamed skin. As CX3CR1 is involved in effector T cell survival in allergic lung inflammation (Mionnet et al., 2010), and as the role of CX3CR1–CX3CL1 in microglial cell (Meucci et al., 1998) and monocyte survival (Landsman et al., 2009) has also been reported, we next assessed whether this was also the case in inflamed skin. WT mice were coinjected with CX3CR1-proficient and -deficient, antigen-specific Th1 or Th2 donor cells and sensitized by epicutaneous antigen administration, and the frequencies of apoptotic and/or necrotic donor cells were measured. Similar frequencies of annexin-V+ and/or 7-aminoactinomycin D+ (7-AAD+) CX3CR1+/gfp and CX3CR1gfp/gfp donor cells were found in both skin and draining LNs (Fig. 5 A and not depicted), suggesting that CX3CR1 is not involved in T cell survival. However, as Th2 donor cells were difficult to monitor in skin because of their low frequencies, additional experiments were performed to confirm these data. CX3CR1+/gfp and CX3CR1gfp/gfp antigen-specific Th2 cells were transduced with a retroviral construct leading to the expression of antiapoptotic BCL-2 (an empty vector was used as control) and transferred into WT recipients that were sensitized by LACK. Although overexpression of BCL-2 led to a decrease and, respectively, an increase in the recovery of transduced cells in the skin and draining LNs, it did not affect the ratio between CX3CR1-proficient and -deficient Th2 cells within these tissues, ruling out a role of CX3CR1 in T cell survival (Fig. 5 B).


CX₃CL1 (fractalkine) and its receptor CX₃CR1 regulate atopic dermatitis by controlling effector T cell retention in inflamed skin.

Staumont-Sallé D, Fleury S, Lazzari A, Molendi-Coste O, Hornez N, Lavogiez C, Kanda A, Wartelle J, Fries A, Pennino D, Mionnet C, Prawitt J, Bouchaert E, Delaporte E, Glaichenhaus N, Staels B, Julia V, Dombrowicz D - J. Exp. Med. (2014)

CX3CR1 is required for T cell retention in chronically inflamed skin. (A) Equal numbers of LACK-specific CX3CR1gfp/gfp and CX3CR1+/gfp Th2 or Th1 cells were coinjected into WT recipient mice at day 0. Recipients were sensitized with LACK at days 1 and 4 and analyzed at days 3 and 7. Donor cells were analyzed by flow cytometry 3 d later in inguinal draining LNs (dLN) after staining with antibodies against CD45, CD4, Thy1.1, Thy1.2, annexin-V, and 7-AAD. Data show mean frequencies of annexin-V+ cells among donor cells ± SEM. (B) CX3CR1+/gfp and CX3CR1gfp/gfp Th2 cells were infected with a Bcl2 or an empty retroviral vector. Equal numbers of transduced cells were coinjected into recipients that were further sensitized with one round of LACK sensitization. Data show the mean of donor cell frequency in skin (bottom) and inguinal draining LN (top) of individual mice in one representative experiment out of two (n = 5 mice per group). *, P < 0.05. (C) AD was induced in WT mice as described in the legend of Fig. 1, and equal numbers of LACK-specific CX3CR1gfp/gfp and CX3CR1+/gfp Th1 (top) or Th2 (bottom) cells were coinjected at day 41. Donor cells were analyzed in skin, draining LN, and spleen by flow cytometry. Data show representative flow cytometry profiles. Numbers indicate frequencies ± SEM of donor Th1 (top) or Th2 (bottom) cells among the CD4+ T cell population. One representative experiment out of two is shown (n = 12 mice per group). (right) 18 h before sacrifice, recipient mice were injected with BrdU and donor cells were analyzed by flow cytometry after staining with anti-BrdU, anti-Thy1.1, anti-Thy1.2, anti-CD4, or anti-CD45 antibodies. Data show representative cytometry profiles, and numbers indicate the mean (n = 12 mice). One representative experiment out of two is shown. (D) Equal numbers of LACK-specific CX3CR1gfp/gfp and CX3CR1+/gfp effector cells were coinjected into WT recipient mice at day 0. Recipients were sensitized with LACK at days 1 and 4. At day 4, some recipient mice topically received LACK together with CX3-AT. 3 d later, the frequencies of donor cells were analyzed in skin and draining LNs by flow cytometry. Data show mean of donor cell frequency of individual mice in one representative experiment out of two (n = 5 mice per group). *, P < 0.05. (E–G) AD was induced as described in the legend of Fig. 1. During the last round of sensitization, some mice also received CX3-AT (50 µg/mouse) through the patch together with LACK as indicated in the figure. (E) May-Grünwald Giemsa staining of skin sections. (F) Epidermal thickness. (G) Mast cell and eosinophil numbers. Data for E–G show mean of cell numbers in one representative experiment out of two (n = 6 mice per group). *, P < 0.05. (H–J) CX3CR1gfp/gfp mice were injected with polyclonal naive WT or CX3CR1gfp/gfp (KO) CD4+ T cells 24 h before AD induction. During the last round of sensitization, mice also received CX3-AT (50 µg/mouse) through the patch together with LACK as indicated in the figure. (H) Epidermal thickness. (I and J) Mast cell and eosinophil numbers. Data for H–J show mean of cell numbers in one representative experiment (n = 5 mice per group). *, P < 0.05. Error bars indicate SEM.
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fig5: CX3CR1 is required for T cell retention in chronically inflamed skin. (A) Equal numbers of LACK-specific CX3CR1gfp/gfp and CX3CR1+/gfp Th2 or Th1 cells were coinjected into WT recipient mice at day 0. Recipients were sensitized with LACK at days 1 and 4 and analyzed at days 3 and 7. Donor cells were analyzed by flow cytometry 3 d later in inguinal draining LNs (dLN) after staining with antibodies against CD45, CD4, Thy1.1, Thy1.2, annexin-V, and 7-AAD. Data show mean frequencies of annexin-V+ cells among donor cells ± SEM. (B) CX3CR1+/gfp and CX3CR1gfp/gfp Th2 cells were infected with a Bcl2 or an empty retroviral vector. Equal numbers of transduced cells were coinjected into recipients that were further sensitized with one round of LACK sensitization. Data show the mean of donor cell frequency in skin (bottom) and inguinal draining LN (top) of individual mice in one representative experiment out of two (n = 5 mice per group). *, P < 0.05. (C) AD was induced in WT mice as described in the legend of Fig. 1, and equal numbers of LACK-specific CX3CR1gfp/gfp and CX3CR1+/gfp Th1 (top) or Th2 (bottom) cells were coinjected at day 41. Donor cells were analyzed in skin, draining LN, and spleen by flow cytometry. Data show representative flow cytometry profiles. Numbers indicate frequencies ± SEM of donor Th1 (top) or Th2 (bottom) cells among the CD4+ T cell population. One representative experiment out of two is shown (n = 12 mice per group). (right) 18 h before sacrifice, recipient mice were injected with BrdU and donor cells were analyzed by flow cytometry after staining with anti-BrdU, anti-Thy1.1, anti-Thy1.2, anti-CD4, or anti-CD45 antibodies. Data show representative cytometry profiles, and numbers indicate the mean (n = 12 mice). One representative experiment out of two is shown. (D) Equal numbers of LACK-specific CX3CR1gfp/gfp and CX3CR1+/gfp effector cells were coinjected into WT recipient mice at day 0. Recipients were sensitized with LACK at days 1 and 4. At day 4, some recipient mice topically received LACK together with CX3-AT. 3 d later, the frequencies of donor cells were analyzed in skin and draining LNs by flow cytometry. Data show mean of donor cell frequency of individual mice in one representative experiment out of two (n = 5 mice per group). *, P < 0.05. (E–G) AD was induced as described in the legend of Fig. 1. During the last round of sensitization, some mice also received CX3-AT (50 µg/mouse) through the patch together with LACK as indicated in the figure. (E) May-Grünwald Giemsa staining of skin sections. (F) Epidermal thickness. (G) Mast cell and eosinophil numbers. Data for E–G show mean of cell numbers in one representative experiment out of two (n = 6 mice per group). *, P < 0.05. (H–J) CX3CR1gfp/gfp mice were injected with polyclonal naive WT or CX3CR1gfp/gfp (KO) CD4+ T cells 24 h before AD induction. During the last round of sensitization, mice also received CX3-AT (50 µg/mouse) through the patch together with LACK as indicated in the figure. (H) Epidermal thickness. (I and J) Mast cell and eosinophil numbers. Data for H–J show mean of cell numbers in one representative experiment (n = 5 mice per group). *, P < 0.05. Error bars indicate SEM.
Mentions: The higher frequency of CX3CR1-proficient CD4+ effector cells in skin could be explained by several hypotheses: the preferential late recruitment of CX3CR1+ effector T cells into the skin, their prolonged survival, or their selective advantage for residence in the inflamed skin. As CX3CR1 is involved in effector T cell survival in allergic lung inflammation (Mionnet et al., 2010), and as the role of CX3CR1–CX3CL1 in microglial cell (Meucci et al., 1998) and monocyte survival (Landsman et al., 2009) has also been reported, we next assessed whether this was also the case in inflamed skin. WT mice were coinjected with CX3CR1-proficient and -deficient, antigen-specific Th1 or Th2 donor cells and sensitized by epicutaneous antigen administration, and the frequencies of apoptotic and/or necrotic donor cells were measured. Similar frequencies of annexin-V+ and/or 7-aminoactinomycin D+ (7-AAD+) CX3CR1+/gfp and CX3CR1gfp/gfp donor cells were found in both skin and draining LNs (Fig. 5 A and not depicted), suggesting that CX3CR1 is not involved in T cell survival. However, as Th2 donor cells were difficult to monitor in skin because of their low frequencies, additional experiments were performed to confirm these data. CX3CR1+/gfp and CX3CR1gfp/gfp antigen-specific Th2 cells were transduced with a retroviral construct leading to the expression of antiapoptotic BCL-2 (an empty vector was used as control) and transferred into WT recipients that were sensitized by LACK. Although overexpression of BCL-2 led to a decrease and, respectively, an increase in the recovery of transduced cells in the skin and draining LNs, it did not affect the ratio between CX3CR1-proficient and -deficient Th2 cells within these tissues, ruling out a role of CX3CR1 in T cell survival (Fig. 5 B).

Bottom Line: CX3CR1 deficiency affected neither antigen presentation nor T cell proliferation in vivo upon skin sensitization, but CX3CR1 expression by both Th2 and Th1 cells was required to induce AD.Surprisingly, unlike in allergic asthma, where CX3CL1 and CX3CR1 regulate the pathology by controlling effector CD4(+) T cell survival within inflamed tissues, adoptive transfer experiments established CX3CR1 as a key regulator of CD4(+) T cell retention in inflamed skin, indicating a new function for this chemokine receptor.Therefore, although CX3CR1 and CX3CL1 act through distinct mechanisms in different pathologies, our results further indicate their interest as promising therapeutic targets in allergic diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale U1011, Institut Pasteur de Lille and Université Lille 2, 59019 Lille, FranceInstitut National de la Santé et de la Recherche Médicale U1011, Institut Pasteur de Lille and Université Lille 2, 59019 Lille, FranceInstitut National de la Santé et de la Recherche Médicale U1011, Institut Pasteur de Lille and Université Lille 2, 59019 Lille, France European Genomic Institute of Diabetes, 59045 Lille, France Department of Dermatology, Claude-Huriez Hospital, 59037 Lille, France.

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