Marginal zone CD169+ macrophages coordinate apoptotic cell-driven cellular recruitment and tolerance - PubMed (original) (raw)

Marginal zone CD169+ macrophages coordinate apoptotic cell-driven cellular recruitment and tolerance

Buvana Ravishankar et al. Proc Natl Acad Sci U S A. 2014.

Abstract

Tolerance to apoptotic cells is essential to prevent inflammatory pathology. Though innate responses are critical for immune suppression, our understanding of early innate immunity driven by apoptosis is lacking. Herein we report apoptotic cells induce expression of the chemokine CCL22 in splenic metallophillic macrophages, which is critical for tolerance. Systemic challenge with apoptotic cells induced rapid production of CCL22 in CD169(+) (metallophillic) macrophages, resulting in accumulation and activation of FoxP3(+) Tregs and CD11c(+) dendritic cells, an effect that could be inhibited by antagonizing CCL22-driven chemotaxis. This mechanism was essential for suppression after apoptotic cell challenge, because neutralizing CCL22 or its receptor, reducing Treg numbers, or blocking effector mechanisms abrogated splenic TGF-β and IL-10 induction; this promoted a shift to proinflammatory cytokines associated with a failure to suppress T cells. Similarly, CCR4 inhibition blocked long-term, apoptotic cell-induced tolerance to allografts. Finally, CCR4 inhibition resulted in a systemic breakdown of tolerance to self after apoptotic cell injection with rapid increases in anti-dsDNA IgG and immune complex deposition. Thus, the data demonstrate CCL22-dependent chemotaxis is a key early innate response required for apoptotic cell-induced suppression, implicating a previously unknown mechanism of macrophage-dependent coordination of early events leading to stable tolerance.

Keywords: autoimmunity; migration; regulation; spleen; transplantation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Apoptotic cells induce CCL22 expression in splenic CD169+ metallophillic macrophages. (A) B6 mice were injected with 107 apoptotic thymocytes i.v. and at indicated time points, whole-spleen lysates were tested for CCL22. (B) At 4 h after apoptotic cell injection as in A, phagocytes were sorted based on indicated markers by FACS, and CCL22 message was measured by sqPCR. (C) B6.CD169DTR mice were depleted of MMΦs as described (13) and injected with apoptotic thymocytes as described in A. At 24 h later, CCL22 protein was measured in whole-spleen lysates by ELISA. Immunofluorescence shows representative spleen sections from B6.CD169DTR and littermate control groups 48 h after last injection with diphtheria toxin stained for B-cell markers (αB220, red) and αCD169 (green). (D) Splenic CD169+ cells were analyzed by FACS for uptake of Pkh26-labeled apoptotic cells 30 min after injection with 107 cells i.v. (E) CD169+ and CD11c+ cells were sorted from B6 mice and cultured in complete RPMI with apoptotic thymocytes at a 10:1 apoptotic cell/phagocyte ratio for 4 h. CCL22 message was then measured by sqPCR as described. (F) At 4 h after apoptotic cell injection as in A, CD169+ MΦs were sorted by FACS and chemokine message for the species indicated was measured by sqPCR. (G) FACS-purified splenic CD169+ macrophages were incubated with apoptotic cells at a 10:1 ratio. At 24 h, supernatant from the cultures were analyzed for chemotactic activity against MACS-purified CD4+CD25+ Tregs. In some wells, neutralizing αCCL22 antibody or CCR4 antagonist was added to confirm the dependence of migration on CCL22-dependent mechanisms as described in Materials and Methods. Bars represent mean value for five mice ± SD (A, C, and D), pooled samples from three mice (B and F), or triplicate wells (E and G). *P val ≤ 0.05; **P val < 0.01 as determined by Student t test. Experiments were repeated at least three times with similar results. ND, not detected.

Fig. 2.

Fig. 2.

Apoptotic cell exposure drives rapid Treg and DC migration into the splenic follicle. B6 mice were pretreated with CCR4 antagonist as described in

SI Materials and Methods

6 h before injection of 107 apoptotic thymocytes i.v. Four hours after apoptotic cell administration, the spleen was collected for analysis. (A) Splenocytes analyzed by flow cytometry to quantify CD4+FoxP3+ Treg accumulation after exposure to apoptotic cells. (B) Representative immunofluorescence staining of splenic sections to determine localization of CD11c+ DCs (red) and FoxP3+ Tregs (green) 4 h after apoptotic cell challenge. Rp, red pulp; wp, white pulp. (C) Image analysis of the number of FoxP3+ cells per follicle in splenic sections from mice treated as described in B. (D) Representative FACS analysis histograms showing surface expression of the markers indicated in CD25+FoxP3+ Tregs 4 h after apoptotic cell challenge. (E and F) Image analysis of splenic sections from mice treated as in B for semiquantitative analysis of follicular CD11c+ DC accumulation and Treg/DC interactions after apoptotic cell challenge. Distance between Tregs and DCs considered contacts was 0.02 μm or less. Distance was quantified by Applied Precision Software (Softworx) on images captured as described in Materials and Methods. (G) Histogram analysis of CCR4 expression in the DC populations indicated (i.e., CD11c+CD8α+CD103+/−). (H) Chemotaxis of DCs in response to apoptotic cell/MMΦ-conditioned media was done as described in Fig. 1_F_ using FACS-purified DCs with the phenotype indicated. Bars represent mean value for triplicate samples (H) or five or more mice per group (A and G) ± SD. Images in B are representative for five or more mice and are 200× magnification. *P val < 0.05 and **P val < 0.01 as determined by Student t test. Experiments were repeated three times with similar results.

Fig. 3.

Fig. 3.

CCR4 inhibition promotes an apoptotic cell-driven proinflammatory cytokine response in the spleen. (A) B6 mice were injected i.p. with CCR4 antagonist as described in

SI Materials and Methods

. At 12 h later, the mice were challenged with 107 apoptotic syngeneic thymocytes i.v., and 18 h after apoptotic cell administration, spleens were collected and cytokine protein concentrations were determined on whole-spleen lysate by ELISA. (B) B6 mice were treated with CCR4 antagonist and injected with apoptotic cells as in A. At 4 h after apoptotic cell challenge, CD169+ MMΦs and CD11c+CD8α+ DCs were sorted by FACS, and cytokine message for the indicated species was determined by sqPCR. (C) CD25+ Treg numbers were depleted by administration of 250 μg of αCD25 monoclonal antibody (clone PC61) as described in Materials and Methods. At 24 h later, mice were challenged with 107 apoptotic cells i.v., and macrophages and DCs were analyzed for cytokine message induction as in B. For A, bars represent mean value for five mice ± SD, and for B and C, bars represent the value for pooled samples from three mice. **P val < 0.01 as determined by Student t test. Experiments were repeated at least three times with similar results.

Fig. 4.

Fig. 4.

Apoptotic cell-mediated allograft tolerance requires an intact CD169+ MMΦ population and CCR4 function. (A) Female B6.CD169DTR mice were injected 3× with saline ± 100 ng diphtheria toxin (DT) to deplete the MMΦ population. At 48 h after the last DT injection, 107 male B6 thymocytes were adoptively transferred i.v. followed by male skin engraftment 7 d later (as described in Materials and Methods). (B) Female B6 mice were injected with CCR4 antagonist and challenged with male apoptotic cells receiving male skin allografts as in A. (C) Female mice were injected with apoptotic cells ± CCR4 antagonist pretreatment, and 7 d later 107 splenocytes were adoptively transferred i.v. to secondary recipient female B6 mice. At 1 d after the transfer, mice received male B6 skin. For A_–_C, graphs represent the cumulative survival of the skin allografts over a 50-d time period posttransplantation (n = 7–10 mice per group). Significance determined as described. (D) B6 mice were injected once per week (3× total) i.v. with apoptotic cells and i.p. with CCR4 antagonist as described in Materials and Methods. The serum was collected at the indicated time points, and concentrations of total IgG and IgG3 reactive against dsDNA as a marker of systemic autoimmunity were determined by ELISA. (E) Kidneys from mice in D were collected after the terminal bleed, and frozen sections were stained with α-mouse IgG to measure immune complex deposition. For D, each data point represents the mean value for five mice per group ± SD. *P val <0.05 and **P val < 0.01 as determined by Student t test. Images in C are representative images shown at 200× magnification. Experiments were repeated three times with similar results.

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