Received 23 Feb 16 Accepted 16 Sep 16 Published 3 Nov 16 DOI: 1.138/ncomms13277 OPEN Splenic differentiation and emergence of CCR5 þ þ CXCL1 þ monocyte-derived dendritic cells in the brain during cerebral malaria Isabella C. Hirako 1,2,, Marco A. Ataide 1,3,, Lucas Faustino 2, Patricia A. Assis 1, Elizabeth W. Sorensen 2, Hisashi Ueta 2, Natalia M. Araújo 1,3, Gustavo B. Menezes 3, Andrew D. Luster 2, & Ricardo T. Gazzinelli 1,3,4, Dendritic cells have an important role in immune surveillance. After being exposed to microbial components, they migrate to secondary lymphoid organs and activate T lymphocytes. Here we show that during mouse malaria, splenic inflammatory monocytes differentiate into monocyte-derived dendritic cells (), which are CD11b þ F4/ þ þ MHCII high DC-SIGN high Ly6c þ and express high levels of CCR5, and CXCL1 (CCR5 þ /1 þ ). We propose that malaria-induced splenic take a reverse migratory route. After differentiation in the spleen, CCR5 þ /1 þ traffic to the brain in a CCR2-independent, CCR5-dependent manner, where they amplify the influx of CD8 þ T lymphocytes, leading to a lethal neuropathological syndrome. 1 Centro de Pesquisas René Rachou, Fundac ão Oswaldo Cruz, Av. Augusto de Lima 1715, Barro Preto, Belo Horizonte MG 319-2, Brazil. 2 Division of Rheumatology, Allergy and Immunology, Massachusetts General Hospital, Harvard Medical School, 149 Thirteenth Street, Charlestown, Massachusetts 2129, USA. 3 Departamento de Bioquímica e Imunologia and Centro de Biologia Gastrointestinal, Departamento de Morfologia, Universidade Federal of Minas Gerais, Av. Antonio Carlos 6627, Belo Horizonte MG 3127-91, Brazil. 4 Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, Massachusetts 1655, USA. These authors contributed equally to this work. These authors jointly supervised this work. Correspondence and requests for materials should be addressed to A.D.L. (email: aluster@mgh.harvard.edu) or to R.T.G. (email: ricardo.gazzinelli@umassmed.edu). NATURE COMMUNICATIONS 7:13277 DOI: 1.138/ncomms13277 www.nature.com/naturecommunications 1
Monocytes, macrophages and dendritic cells (DCs) are heterogeneous cell populations that have critical roles in tissue repair, sensing the presence of invasive microorganisms and initiating protective immune responses. These cell subsets have overlapping functions. DCs are more specialized in antigen presentation and shaping T-cell-mediated immunity, whereas macrophages primarily act as a source of proinflammatory cytokines and phagocytic cells that effectively destroy pathogens. Monocytes are less specialized cells that contribute to the overall production of inflammatory cytokines, anti-microbial effector functions and are the main progenitors for DCs and macrophages 1 3. DCs, monocytes and macrophages are thought to have an important role in host resistance to both mouse and human malaria 4,5. During malaria, DCs are activated through Toll-like receptors (TLRs), primarily TLR9 (refs 6 9), and serve as an important source of interleukin (IL)-12. IL-12 activates natural killer cells to produce interferon-g (IFNg) and promotes differentiation of T-helper type 1 (Th1) lymphocytes that orchestrate acquired protective immunity against Plasmodium infection 1 16. Importantly, uptake of infected erythrocytes seems to inhibit maturation and function of human DCs 17, and a low number of circulating DCs is associated with impairment of antigen-specific T-cell responses in symptomatic patients infected with either Plasmodium falciparum or Plasmodium vivax, suggesting their role in resistance to human disease 18. In addition, inflammatory monocytes in mouse (CCR2 þ Ly6c þ and F4/ þ ) and humans (CD14 þ CD16 high ), as well as in DCs, are important sources of inflammatory cytokines and reactive nitrogen and oxygen intermediates that effectively destroy Plasmodium parasites within phagocytosed infected red blood cells (irbcs) 11,19 21. DCs also contribute to the pathogenesis of mouse malaria. Blockade of T cell and DC interaction prevents a deleterious response that is associated with a wasting syndrome and hypothermia in Plasmodium chabaudi-infected mice 22. DCs are also required for the development of experimental cerebral malaria (ECM) 23. In addition, a subset of Ly6c þ monocytic cells is critical for recruiting CD8 þ T cells to the central nervous system (CNS) and development of ECM 24. Furthermore, a high proportion of inflammatory monocytes and DCs express inflammasomes and on a secondary microbial stimuli release excessive levels of IL-1b, both in mouse and human malaria 25. Although most DC progenitors that enter peripheral tissues at homeostasis are not monocytes, studies have demonstrated that in response to infection or inflammation, monocytes can be induced to differentiate and provide important DC functions 26 36. Monocyte-derived DCs () share many morphological and functional characteristics with conventional DCs (cdcs), including antigen capture and presentation to CD4 þ T lymphocytes, as well cross-presentation to CD8 þ T lymphocytes. Monocytes are more abundant than DCs in the blood and bone marrow (BM), and the in vivo mobilization of this monocyte reservoir to generate potent antigen-presenting DCs is of central importance during microbial infection 31 34,36. Studies have defined markers that enable the distinction of cdcs and inflammatory monocytes from ; however, the role of in mouse malaria, as well as in neuroinflammation observed during ECM, has not been explored. Here we report that emerge as a main splenic DC population during early stages of Plasmodium berghei ANKA (PbA) infection in mice. These are unique in that they express high levels of the chemokine receptor CCR5, as well as the IFN-inducible CXCR3 chemokine ligands (MIG) and CXCL1 (IP1) (CCR5 þ /1 þ ). CCR5 þ /1 þ are the main DC subset in the CNS of mice with cerebral malaria. Importantly, emergence of in the CNS and development of ECM is dependent on MO-DC CCR5 expression and independent of CCR2 expression. Our results reveal a previously unappreciated role of in PbA-induced neuroinflammation and the mechanism by which CCR5 mediates the development of ECM. Results Malaria infection induces. Recent studies have demonstrated that in vivo microbial challenge signal inflammatory monocytes to differentiate into 35,36. Here we evaluated whether emerge during mouse malaria by searching for þ MHC II high CD11b þ F4/ þ DC-SIGN high cells in the spleen, a main site of phagocytic cell interaction with Plasmodium irbcs. For this purpose, we gated double-positive CD11b and F4/ spleen cells for MHC II high DC-SIGN þ þ (ref. 35). The results presented in Fig. 1a indicate that the frequency of in total CD11b þ F4/ þ splenic cells was increased from 18% in uninfected to 74% in PbA-infected mice. In addition, the level of expression, as indicated by the mean fluorescence intensity (MFI), of DC-SIGN and major histocompatibility complex (MHC) II in from infected mice increased threefold. A fraction of these cells also expressed different levels of Ly6c. In contrast, the frequency of CD11b þ F4/ þ DC-SIGN int MHC II Ly6c high cells (inflammatory monocytes) decreased from 19% to 4.4%, suggesting that inflammatory monocytes were converted into. After infection, most monocytes (Gate 3, CD11b þ F4/ þ DC-SIGN MHC II ) became Ly6c high, but as a whole the difference in number of cells was not statistically significant when comparing uninfected with infected wild-type (WT) mice. We also performed the initial gating on þ MHC II high cells and then on the DC-SIGN þ LY6c þ population, and confirmed that over 89% of these cells in PbA-infected mice were CD11b þ F4/ þ (Supplementary Fig. 1A). In addition, our analysis indicated that the frequency of þ MHC II high CD11b F4/ DC SIGN Ly6c cells, which correspond to cdcs, decreased from 48% in uninfected control mice to % of total þ MHC II high in infected mice. To further characterize the, we sorted (95 99% purity) by flow cytometry (Supplementary Fig. 2) and analysed by Giemsa staining. and optical and scanning electronic microscopy (SEM) (Fig. 1b). The results obtained from SEM show that morphology of is heterogeneous. Although a significant proportion of from PbA-infected mice were typical DCs with a smooth surface and dendrites, other cells displayed characteristics of monocytes or transitional morphological phenotypes. Interestingly, we found in the Giemsa staining that a high frequency of purified from infected mice contained haemozoin, which are haem polymers produced by the parasite. To further evaluate the phagocytic capacity of, we incubated spleen cells from uninfected controls and infected mice with erythrocytes infected with a PbA clone that expresses green fluorescent protein (GFP). By flow cytometry, we demonstrated that were highly efficient phagocytic cells, when compared with inflammatory monocytes and monocytes (Fig. 1c). In addition, we observed that unlike inflammatory monocytes and monocytes, express very high levels of CD and CD86 (Fig. 1d and Supplementary Fig. 1B). Furthermore,, but not monocytes, cross-presented soluble antigens (Fig. 1e), suggesting that they play an important role on activating CD8 þ T lymphocytes either in the spleen or in the CNS. Malaria-induced CCR5 þ /1 þ. To further characterize, lysates of highly purified fluorescence- 2 NATURE COMMUNICATIONS 7:13277 DOI: 1.138/ncomms13277 www.nature.com/naturecommunications
activated cell-sorted (Supplementary Fig. 2) were obtained and gene transcripts analysed for 547 immune-related genes plus 14 housekeeping genes using a commercially available set of nanostring probes. We observed an increase in the expression of B18% of the analysed genes in isolated from the spleens of infected mice compared with uninfected mice. Of note, the induction in the expression of (24-fold increase) and CXCL1 (19-fold increase) stood out. In addition, the expression of CCR5 was enhanced sixfold (Fig. 2a). As and CXCL1 have been shown to play an important role on a b c Total number of cells % of the max % of the max Non-infected 1.91 CD11b + F4/ + cells 1 4 1 4 1 3 1 3 5 1 6 4 1 6 3 1 6 2 1 6 1 1 6 CD11b + F4/ + 2.63 1 3 1 4 1 3 1 4 CD11b Inflammatory 1 4 monocytes 8, 4, (gate 2) 1 3 6, 4, Inflammatory monocytes 3, 1, 1 3 1 3 1 4 1 3 1 3 1 4 1 3 1 3 1 4 PbA-GFP (MFI) PbA-GFP (MFI) F4/ Giemsa staining 1 3 1 3 1 4 Comp FITC A :: parasite GFP 3, 2, 1, DC-SIGN (MFI) 2, 1 µm 1 µm 1 3 1 3 1 4 Comp FITC A :: parasite GFP MHC-II (MFI) 2, Scanning electronic microscopy 1 2 3 Non-infected + CD11b + F4/ + DC-SIGN + MHC II high ns Inflammatory monocytes Monocytes 1 3 1 3 1 4 4 Comp FITC A :: parasite GFP + RBC + irbc 6 th + RBC 6 th + irbc Monocytes DC-SIGN Monocytes (gate 3) 1 4 1 3 1 3 19.2 Non-infected 25.5 18.2 1 3 1 4 4.54 74.2 1 3 Total number of cells MFI Absolute number (gate 1) 1 3 1 4 MHC II 3 1 6 2 1 6 1 1 6 1.5 (gate 1) 8, + cells 1 3 Ly6c 1 4 1 3 1 3 1 4 Gate 1 Gate 2 Gate 3 Gate 1 Gate 2 Gate 3 Gate 1 Gate 2 Gate 3 Gate 1 Gate 2 Gate 3 5 1 5 8 1 5 4 1 5 6 1 5 3 1 5 4 1 5 2 1 5 2 1 1 1 5 5 Inflammatory Monocytes monocytes (gate 3) (gate 2) CD + cells 1,5 CD86 + cells 1, 1 1 6 1.5 1 6 1.5 1 5 8 1 5 6 1 5 1. 1 6 1. 1 5 4 1 5 5. 1 5 5. 1 4 2 1 5.. (infected) Inflammatory monocytes (control) Inflammatory monocytes (infected) (control) e % of the max % of the max d 6, 4, 2, Monocytes Cell trace Inflammatory monocytes (control) Inflammatory monocytes (infected) (control) (infected) APC/T cell ratio 1:3 1:1 1:3 9.47 16.7 15.9 33.1 43.2 4, 3, 2, 1, 58.1 Inflammatory monocytes (control) Inflammatory monocytes (infected) (control) % OT-I proliferation IFN-γ (pg ml 1 ) 5 (infected) 15 5 Monocytes 1:3 1:1 Monocytes 1:3 NATURE COMMUNICATIONS 7:13277 DOI: 1.138/ncomms13277 www.nature.com/naturecommunications 3
ECM 37 39, we further investigated the expression of these chemokines in spleens of PbA-infected mice. The results presented in Supplementary Fig. 3A show that expression of and CXCL1 messenger RNA achieved the highest level from 5 to 7 days post infection, coinciding with the peak of MO- DCs in the spleen. We also used the reporting expression of CXCR3 ligands (REX3) transgenic mice that express the red fluorescent protein (RFP) and blue fluorescent protein (BFP) as reporter genes under the control of the and CXCL1 promoters, respectively. We found that a large proportion of the double-positive RFP þ BFP þ cells from infected mice were (Fig. 2b,c). Likewise, over % of these cells were CCR2 þ CCR5 þ, whereas the inflammatory monocytes and from uninfected mice were CCR2 þ, but CCR5 (Fig. 2d). We also asked whether might be directly recruited from the BM. WT mice were infected with PbA and emergence of inflammatory monocytes or in the BM and spleens evaluated side-by-side. Our results presented in Fig. 3a,b indicate that there is an increase of F4/ þ, CD11b þ and MHCII þ in the BM. However, in contrast to spleen, these cells were both CD11C and CCR5. Hence, our results suggest that inflammatory monocytes acquire the MO-DC phenotype elsewhere and cannot be recruited directly from the BM by CCR5 ligands. Importantly, we performed the adoptive cell transfer experiments. Monocytes (F4/ þ CD11b þ DC SIGN MHCII ) were enriched from spleens of uninfected CD45.2 C57BL/6 mice and two million cells transferred to either uninfected or PbA-infected CD45.1 congenic mice. Two days later, we gated F4/ þ CD11b þ cells and looked for distribution of DC-SIGN MHCII, DC-SIGN þ MHCII and DC-SIGN þ MHCII þ, which were either CD45.2 þ or CD45.1 þ. We found that the frequencies of splenic CD45.2 þ MO, inflammatory monocytes and MO-DCS, as well as splenic CD45.1 þ MO, inflammatory monocytes and, were similar, respectively (Fig. 3c). In uninfected mice, we found that migration of F4/ þ CD11b þ CD45.2 þ MO to the spleen was highly reduced and we detected very low frequency of either CD45.1 þ or CD45.2 þ. These data are consistent with the hypothesis that splenic monocytes are differentiating into. CCR5 þ /1 þ in the CNS of PbA-infected mice. We next evaluated the presence of in the CNS of mice undergoing ECM. To analyse the presence of in the CNS of PbA-infected mice, we first gated the CD45 high brain mononuclear cells to discriminate haematopoietic cells from microglia (CD45 interm ). As the cellular infiltrate in ECM is highly enriched for CD8 þ T cells, we then gated on CD45 high CD8 cells. emerge at day 5 and peaked at day 7 post infection with PbA, which coincides with the expression of and CXCL1, and other inflammatory mediators in the CNS (Fig. 4a and Supplementary Figs 3B and 4A). Importantly, most of CD45 þ þ MHC II high cells in the CNS were also CD11b þ F4/ þ, which is consistent with the MO-DC phenotype. We further analysed brain mononuclear cells from PbA-infected mice for, CXCL1 and CCR5 expression. We found no cells expressing and CXCL1 in uninfected REX3 mice (Fig. 4b). In infected REX3 mice, BFP þ RFP þ cells were CD45 high. CD45 high CD8 þ cells were BFP RFP, whereas B% of CD45 high CD8 cells were BFP þ RFP þ (Fig. 4c). Furthermore, % of CD45 high CD8 BFP þ RFP þ cells were CD11b þ Ly6c þ þ MHC II high, consistent with the hypothesis that are also a main haematopoietic source of and CXCL1 in the CNS of infected mice (Fig. 4c). CD45 interm and CD45 cells, most probably microglia and neuron or glial cells, respectively, were both BFP and RFP negative. Finally, we evaluated the expression of CCR2 and CCR5 in derived from CNS of infected mice. The results shown in Fig. 4d demonstrate that B% of brain from infected, but not from control mice, were double positive for CCR2 and CCR5. IFNc-induced expression of and CXCL1 by. A recent study has demonstrated that IFNg is an important mediator of MO-DC differentiation 36. In addition, IFNg has been shown to induce the expression of and CXCL1 in macrophages. Hence, we used IFNg / and REX3IFNgR / mice infected with PbA. We first evaluated the profile of gene expression in highly purified splenic (Supplementary Fig. 2) isolated from infected C57BL/6 and IFNg / mice. From 98 PbA-induced genes, 58 (%) were dependent on endogenous IFNg. Expression of and CXCL1 mrna induced by PbA infection was totally absent in from IFNg / mice, whereas enhanced expression of other genes, such as CCR5, CCL7 and CCL8, was independent of endogenous IFNg (Fig. 5a). Consistently, from REX3, but not from REX3IFNgR / mice, expressed high levels of RFP and BFP (Fig. 5b,c). We also tested whether IFNg is necessary for differentiation of. We noticed an accumulation of inflammatory monocytes and decreased frequency of in REX3/IFNgR / and IFNg / relative to REX3 and C57BL/6 mice infected with PbA (Fig. 5b,c and Supplementary Fig. 5). Together, these results indicate that differentiation of precursor inflammatory Figure 1 Differentiation of splenic in PbA-infected mice. Spleens were harvested 5 days after PbA infection. (a) Splenocytes were first gated for F4/ þ CD11b þ cells and then for DC-SIGN þ MHC II high cells. F4/ þ CD11b þ DC-SIGN high MHC II high cells were checked for þ and Ly6c þ expression. Bar graphs correspond to total number of cells (four mice per group). The data shown are representative of eight independent experiments. Results are expressed as median±s.d. An asterisk indicates that difference is statistically significant when comparing infected and non-infected mice (Po.1) after Mann Whitney U-test. (b) Splenocytes were labelled with fluorescent antibodies and þ CD11b þ F4/ þ DC-SIGN high MHC II high purified by flow cytometry and analysed by Giemsa staining and optical microscopy, as well as SEM. Panel 1 and panels 2, 3 and 4 of SEM illustrate MO-like and DC-like cells, respectively. White arrows indicate haemozoin crystals inside purified (scale bar, 1 mm). (c) Flow cytometry histograms show the MFI results from spleen cells of uninfected (shaded lines) versus infected (open lines) incubated, at 1:1 ratio, with uninfected RBCs (blue) or RBCs infected with GFP-expressing PbA (red). The results indicate the phagocytic activity of, inflammatory monocytes and monocytes, as indicated. Bar graphs correspond to the MFI average of three to four mice per group. The data shown are representative of two independent experiments. (d) Expression of co-stimulatory molecules CD and CD86 on cell surface of monocytes, inflammatory monocytes and. (e) Cell Trace-labelled naive OT-I cells were cultured with monocytes or (495% purity) from PbA-infected mice at indicated APC/T cell ratio in the presence of OVA protein ( mg ml 1 ), and 3 days later the percentage of Cell Trace low CD8 þ T cells was evaluated. Representative histograms on the left and top right graph show average and s.e. of T-cell proliferation. Average and s.e. of IFNg production in the medium of co-cultures at 1:3 ratio measured by enzyme-linked immunosorbent assay are shown in the right bottom graph. Data are representative of two independent experiments with three mice per group, which yielded similar results. Results expressed as mean±s.d. Differences considered statistically significant comparing infected and non-infected mice after two-way analysis of variance, Po.5, Po.1. 4 NATURE COMMUNICATIONS 7:13277 DOI: 1.138/ncomms13277 www.nature.com/naturecommunications
a + CD11b + F4/ + DC-SIGN + MHC II high.5 1. G1 from Wt infected/ Wt non infected G1 from Wt infected/ Wt non infected G1 from Wt infected/ Wt non infected 8. CCL11 CCL12 CCL19 CCL2 CCL CCL22 CCL24 CCL25 CCL26 CCL3 CCL4 CCL5 CCL6 CCL7 CCL8 CCL9 CCL1 CCR2 CCR3 CCR4 CCR5 CCR6 CCR7 CCR8 CCR9 CHUK CX3CL1 CX3CR1 CXCL1 CXCL1 CXCL11 CXCL12 CXCL13 CXCL15 CXCL3 CXCR1 CXCR2 CXCR3 CXCR4 CXCR5 CXCR6 IKBKB IKBKG JAK2 JAK3 MAPK1 NFKB1 NFKB1A PPBP PRKCD PTK2 RELA SRC STAT1 STAT2 STAT3 STAT5B XCL1 XCR1 b c F4/ / CXCL1 / F4/ CXCL1 Follicle Follicle F4/ F4/ CXCL1 µm F4/ / CXCL1 / CXCL1 Follicle Follicle µm CXCL1 Non-infected.47 5.6 1 4 1 3 1 4 1 4 1 3 1 3 1 3 1 4 1 3 1 4 CXCL1 1 88 1 4 9 1 3 5.59 77.6 1 2 3 1 3 1 4 1 3 1 4 1 3 1 4 CD11b MHC II DC-SIGN d Non-infected F4/ 2.83 21.2.1 5.45 1 4 1 4 1 4 1 3 1 3 1 3 1 4 1 3 1 3 1 4 MHC II 2.55 1 2 1 3 71.6 2.81 1 4 1 3 1 2 1 3 1 4 CCR2 1 3 1 3 1 4 72.9 8.72 74.6 1 4 1 3 1 3 3.94 12.7 % of cells CD11b + F4/ + MHC II + + CCR2 + CCR5 + Noninfected 6 days postinfection 1 3 1 4 1 3 1 4 1 3 1 3 1 4 CD11b CCR5 Figure 2 Expression of and CXCL1 by from PbA-infected mice. Splenocytes were harvested at 6 days post PbA infection. þ CD11b þ F4/ þ DC-SIGN high MHC II high were purified by flow cytometry (Supplementary Fig. 2) and analysed for (a) RNA expression of immune-related genes using an ncounter Analysis System. The results indicate fold increase of gene expression, when comparing isolated from three infected mice compared with three non-infected mice. (b) Confocal microscopy of the spleen from an infected REX3 mouse. Top panels shows F4/ þ (APC converted in green), -RFP and CXCL1-BFP staining. Bottom panels show þ (APC converted in green), -RFP and CXCL1-BFP staining. Arrows and arrowheads show double- and triple-positive cells, respectively (scale bar, mm). (c) PbA-infected REX3 mice were killed at day 6 post infection and splenic MO-DC cells analysed for and CXCL1 expression. Spleen cells were first gated on double-positive /CXCL1, and then on F4/ þ CD11b þ and DC-SIGN þ MHC II þ cells. All þ CXCL1 þ F4/ þ CD11b þ DC-SIGN þ MHC II þ cells were þ. (d) Spleen cells harvested 6 days post infection were first gated on F4/ þ CD11b þ and then on þ MHC II þ cells. The majority of from uninfected and PbA-infected mice were CCR2 þ CCR5 and CCR2 þ CCR5 þ, respectively. Representative primary flow cytometric dot plots are shown. The data are representative of three experiments with three mice per group that yielded similar results. Results are expressed as median±s.d. Difference considered statistically significant when comparing infected and non-infected mice (P.1) after Mann Whitney U-test. NATURE COMMUNICATIONS 7:13277 DOI: 1.138/ncomms13277 www.nature.com/naturecommunications 5
a Spleen-gated on CD11b + F4/ + b CD11b + F4/ + cells MHC-II P.b. ANKA 3.43 37.8 1 4 1 3 1 3 1 4 1 4 Bone marrow-gated on CD11b + F4/ + P.b. ANKA 2.25 4.88 1 4 1 3 1 3 1 4 1 4 Absolut number of cells % of CD11b + F4/ + 5 3 1 3 2 1 Bone marrow Spleen BM-uninfected 15 Spleen-uninfected BM-P.b. ANKA Spleen-P.b. ANKA P.b. ANKA MFI 5 Bone marrow CCR5 1 4 CCR5 Spleen c Spleen CD11b + F4/ + monocytes enrichment 2 days Spleen CD45.2 uninfected donors DC-SIGN 1 4 1 3 1 3.36 94.2.25 CD45.1 recipient PbA 3 d.p.i Gated on CD45.2 + donor cells 1 4 MHC-II Gated on CD45.1 + recipient cells P.b. ANKA P.b. ANKA P.b. ANKA F4/ DC-SIGN 1 4 1 3 1 3 CD11b 1 4 1 3 1 3 36.6 1.64 2.31 1 4 1 3 1 3 1 4 1 4 1 4 1 4 14.8 2.99 13. 11.2 87.2 1 4 1 3 16.7 1 3 61.8 16.8 1 4 1 4 1 4 1 4 4.54 7.1 25.1 5.7 Freq. of CD11b + F4/ + cells Donor n.s. Recipient Donor Inflammatory monocytes Recipient Monocytes Donor Recipient MHC-II Figure 3 Differentiation of splenic in mice infected with PbA. Spleens and BM were harvested from mice, either uninfected or 5 days post PbA infection. Splenocytes and BM cells were then labelled and analysed by fluorescence-activated cell sorting. We first gated F4/ þ CD11b þ cells and then þ MHC II high cells, which are the. (a) Representative primary flow cytometric dot plots are shown. Upper bar graph on the right corresponds to the frequency of þ MHC II high cells and botton bar graph to the total number of þ MHC II high cells within the total F4/ þ CD11b þ population. (b) Flow cytometry histograms show the MFI results of CCR5 expression in total F4/ þ CD11b þ cells of uninfected (shaded lines) versus infected (open lines). Bar graph corresponds to MFI average of CCR5 expression. The data shown in a and b are representative of two independent experiments (three mice per group). (c) Enriched splenic F4/ þ CD11b þ DC-SIGN MHC-II cells from uninfected CD45.2 þ donor mice were adoptively transferred i.v. into CD45.1 þ uninfected or PbA-infected mice. Dot plot of enriched monocytes show that o.5% of the cells were þ MHC II high (). After 48 h, spleens of recipient mice were obtained and frequency of CD45.2 þ donor-derived MO-DC, inflammatory monocytes and monocytes were compared with corresponding populations of CD45.1 þ recipient mice. Bar graphs show frequency of MO-DC, inflammatory monocytes and monocytes in F4/ þ CD11b þ populations from CD45.2 þ or CD45.1 þ cells of two pooled experiments that yielded similar results. Results are expressed as mean±s.d. Differences were considered statistically significant when Po.5, as indicated by two-way analysis of variance, Po.1. 6 NATURE COMMUNICATIONS 7:13277 DOI: 1.138/ncomms13277 www.nature.com/naturecommunications
a c day 7 Number of events 15 Absolute number 5 3 1 4 2 1 4 1 1 4 85.1 BMNC Brain CD45 high CD11b + Ly6c + MHCII + + DC SIGN + 5 7 Days post-infection 14.4 1 3 1 4 CD45 (MFI) Number of events CD45 high cells CD8 59.2 1 3 1 3 1 4 CD8 b Non-infected Number of events 15 5 94.8 BMNC 4.57 CD45 high CD8 cells Number of events CD45 high cells CD8 12.8 1 3 1 4 1 3 1 3 1 4 CD45 (MFI) CD8 CXCL1 Number of events 3 1 1,5 1, 5 CD45 low/neg cells CD8 94.3 1 3 1 3 1 4 CD8 1 4 1 3 1 4 1 3 CD45 high CD8 cells CD45 low/neg CD8 cells.98 67.5 94.4 85.6 1 4 1 3 1 3 1 4 Ly6c 1 4 1 3 MHC II 1 4 1 3 1 2 1 3 1 4 CXCL1 1 3 1 4 CXCL1 1 3 1 4 1 3 1 4 CD11b d Non-infected Number of events Number of events 1 9 3 93.8 88 BMNC 6.15 1 3 1 3 1 4 12 1 3 1 3 1 4 CD45 (MFI) Number of events CD8 1.2K 1 4 1 3 9 3 1 4 1 3 CD45 low/neg cells CD8 96.1 1 3 1 3 1 4 CD45 high cells 34.8 CD8 Ly6c 1 4 1 3 1 4 1 3 CD45 low/neg CD8 cells 1.2 CD45 high CD3 CD8 cells 36 84.5 1.72 1 48.9 2 13.8 1 3 1 4 1 3 1 4 1 3 1 4 1 4 1 3 1 3 1 4 58.4 CXCL1 CCR2 CD45 high CD3 CD8 Ly6c + CD11b + cells 43.2 53.9 1 2 2.91 1 3 1 4 1 3 1 4 13 1 4 CD3 CD11b CCR5 1 4 1 3 1 4 1 3 CD45 high CD3 CD8 Ly6c + CD11b + + CCR2 + CCR5 + cells CD45 high CD3 CD8 Ly6c + CD11b + CCR2 + CCR5 + cells Non-infected 1 3 1 4 Figure 4 High levels of in the brain from PbA-infected mice. (a) (CD45 high CD8 CD3 CD11b þ Ly6c þ MHC II þ DC-SIGN high þ ) were quantified by flow cytometric analysis within brain mononuclear cells (BMNCs) isolated at, 5 and 7 days post infection with PbA. (b) BMNCs from uninfected REX3 mice were analysed by flow cytometric analysis. Cells that were first selected on CD45 high and then CD8 cells were negative for (RFP) and CXCL1 (RFP). (c) Cells first selected on CD45 high and then CD8 from BMNCs from PbA-infected REX3 mice were B7% double positive for the and CXCL1 reporters. Eighty percent of CD45 hi CD8 þ CXCL1 þ cells were CD11b þ Ly6c þ þ MHC II þ. CD45 low/negative CD8 BMNCs were all negative for -RFP and CXCL1-BFP. Representative primary flow cytometric dot plots are shown after CD45 high gating for leukocytes. (d) BMNCs were harvested from WT mice at and 7 days post infection and expression of CCR2 and CCR5 analysed in, first selected on CD45 high, gated on CD8 CD3 and then on Ly6c þ CD11b þ. The histogram in the far right represents the MFI of expression on from uninfected and PbA-infected mice. Data represent the average of three or four mice per group and results are representative of three independent experiments. Differences were considered statistically significant when Po.5 or Po.1 as indicated by two-way analysis of variance (values are means±s.d.). (a) or Mann Whitney U-test (values are medians±s.d.) (d). NATURE COMMUNICATIONS 7:13277 DOI: 1.138/ncomms13277 www.nature.com/naturecommunications 7
a + CD11b + F4/ + DC-SIGN + MHC II high.5 1. 8. G1 from C56BL/6 infected G1 from IFNγ / infected Cc111 Cc112 Cc119 Cc12 Cc1 Cc122 Cc124 Cc125 Cc126 Cc13 Cc14 Cc15 Cc16 Cc17 Cc18 Cc19 Ccr1 Ccr2 Ccr3 Ccr4 Ccr5 Ccr6 Ccr7 Ccr8 Ccr9 Chuk Cx3c11 Cx3cr1 Cxc11 Cxc11 Cxc111 Cxc112 Cxc113 Cxc115 Cxc13 Cxc19 Cxcr1 Cxcr2 Cxcr3 Cxcr4 Cxcr5 Cxcr6 Ikbkb Ikbkg Jak2 Jak3 Mapk1 Nfkb1 Nfkbia Ppbp Prkcd Ptk2 Rela Src Stat1 Stat2 Stat3 Stat5b Xc11 Xcr1 c % of cells 5 3 1 b Non-infected Non-infected F4/ F4/ 1 4 1 3 1 4 1 3 1 4 1 3 1 4 1 3 2.34 1 3 1 4 1 3 1 4 Monocytes (Gate 3) 4.76 1.3 69.2 4.82 32.2 24.7 1 4 7.98 G2 - CD11b+ F4+ DC-SIGN int MHC 6 days postinfection Noninfected 1 3 1 4 CD11b Inflammatory monocytes (Gate 2) DC-SIGN DC-SIGN Rex3 transgenic mice 31.1 9.48 17.9.6 13.2 12.2.95 37.6 (Gate 1) 3.4 Rex3 transgenic IFNγR / mice 76.2 1 3 1 4 1 3 1 4 MHC II CXCL1..28 1 3 1 4 1 3 1 4 1 3 1 4 CD11b MHC II CXCL1 % of cells CD11b+ F4/+ 12 Rex3 9 6 Rex3IFNγR 3 % of cells 5 3 1 Noninfected 1 4 1 3 1 2 1 4 1 3 1 2 1 3 1 2 1 3 1 4 1 3 1 4 1 4 1 3 1 2 G3 - CD11b+F4+ DC-SIGN MHC II 6 days postinfection Noninfected % of cells % of cells G1 - CD11b+ F4/+ DC-SIGN+ MHC+ 6 days postinfection Noninfected CD11b+F4/+DC SIGN+ MHC II++CXCL1+ 8 4 1 4 1 3 1 4 1 3 1 4 1 3 1 4 1 3 6 days postinfection Noninfected 1 3 1 4 1 3 1 4 6 days postinfection Figure 5 IFNc-dependent expression of and CXCL1 by from PbA-infected mice. C57BL/6, IFNg /, REX3 or REX3/IFNgR / mice were infected with PbA and their spleens harvested at 6 days later. (a) Gene expression was analysed in total RNA extracted from highly purified (over 98% purity) from infected C57BL/6 and IFNg / mice by ncounter Analysis System. Data represent the average of gene expression of three mice per group. (b) Representative primary flow cytometric dot plots are shown for splenic from REX3 and REX3/IFNgR / mice gated on F4/ þ CD11b þ (left panels), then on DC-SIGN high MHC II high cells (middle panels), and and CXCL1 double-positive cells (right panels), respectively. The percentage of MO-DC (Gate 1), inflammatory monocytes (Gate 2) and monocytes (Gate 3), and þ CXCL1 þ are indicated. (c) Frequency of splenic CD11b þ F4/ þ cell subsets in infected REX3 and REX3/IFNgR / mice are shown. The average and s.d. are representative of two independent experiments with three or four mice per group. Differences were considered statistically significant when Po.1 and Po.1 as indicated by two-way analysis of variance analysis. 8 NATURE COMMUNICATIONS 7:13277 DOI: 1.138/ncomms13277 www.nature.com/naturecommunications
monocytes into is impaired in mice lacking either functional IFNg or IFNgR genes. TLR-induced expression of and CXCL1 by. Cheong et al. 35 demonstrated that lipopolysaccharide induces differentiation of inflammatory monocytes into. It is also well established that Plasmodium infection activates the innate immune system primarily by activation of TLR9 (refs 6 9,41) and other nucleic acid sensors 42,43. The activation of TLR9 during PbA infection induces the production of IL-12 by c CD4 CD8 CD44 1 4 1 3 Copies per β2 microglobulin 14.6 Copies per β2 microglobulin.4.3.2.1..15.1.5..15.1.5. IFNγ RT-PCR (spleen) p=.179 (treated) 5 days 5 days (treated) 7 days 7 days (treated) Naive/effector T cell populations pg ml 1 1 2 1 2 1 2 77.5 23.9 72.6 1 2 1 3 1 4 1 2 1 3 1 4 1 2 1 3 1 4 1 4 1 3 1 2 1.51 79.9 a 1 2 1 3 1 4 d e 1 4 1 3 1 4 1 3 1 2 CD4 CD8 53.9 1 2 1 3 1 4 CD62L 1 4 1 3 63.6 1 4 1 3 Vehicle 24.7 2.16 3.94 1 4 1 3 1 3 1 4 CXCL1 1 3 1 4 (treated) 5 days 1 4 1 3 16.5 2.5 1 2 76.5 1 2 1 3 1 4 5 days (treated) 1 4 1 3 1 4 1 3 b E6446 Vehicle IFNγ CBA (serum) Non infected Day 3 Day 4 Day 5 Day 6 Day 7 8.99 3.5 1 4 1 3 1 4 14.4 1 3 1 4 IFNγ-GFP f 1 4 1 3 1 4 1 3 % CD4 + CD62L low CD44 hi % CD8 + CD62L low CD44 hi 1 3 1 4 1 3 1 3 1 4 1 3 1 4 CXCL1 4. E6446 Vehicle Vehicle pg ml 1 E6446 E6446 1 3 1 4 1 3 1 4 15 5 % CD4 + IFNγ + % CD8 + IFNγ + Non infected % CD4 + CD62L hi CD44 low % CD8 + CD62L hi CD44 low 15 1 5 15 1 5 Vehicle CD11b + F4/ + + MHC II high DCs (vehicle) (E6446) % + CXCL1 + 5 3 1 Vehicle E6446 Vehicle E6446 Vehicle E6446 Vehicle Treated E6446 E6446 (vehicle) Treated (E6446) NATURE COMMUNICATIONS 7:13277 DOI: 1.138/ncomms13277 www.nature.com/naturecommunications 9
DCs, culminating in the production of IFNg, primarily T lymphocytes 6,22,41. Hence, we decided to evaluate whether PbA infection was inducing differentiation of via TLR activation. With this in mind, we used E6446, an antagonist that prevents activation of TLR7 and TLR9 by single-stranded RNA and DNA 41,44. The results presented in Supplementary Fig. 6 show that therapy with E6446 prevented lethality when given in the very early days of infection (from day 1 to 3 post infection). The data presented in Fig. 6a,b show that treatment with E6446 inhibits the expression of IFNg mrna by spleen cells and results in decreased circulating levels of IFNg in sera of mice at 6 days post infection with PbA. In addition, at 5 days post infection, the proportion of activated (CD44 high /CD62L low ) CD4 þ T and CD8 þ T lymphocytes was augmented to 55.5% and 39.9%, when compared with 14.7% and 12.2% in uninfected controls or 27.8% and 1.2% in infected mice treated with E6446, respectively (Fig. 6c). The percentage of resting T cells (CD44 low /CD62L high ) was inversely proportional to activated cells and decreased to 32.9% of CD4 þ T and 37.3% of CD8 þ T lymphocytes in infected mice, as compared with 77.7% and 78.1% in uninfected controls and 59.6% and 64% in infected mice treated with E6446, respectively. Likewise, E6446 inhibited the expression of IFNg by both CD4 þ T and CD8 þ T cells from infected mice. The results presented in Fig. 6d were obtained by using GREAT mice that express a YFP gene under the IFNg promoter. Expression of yellow fluorescent protein (YFP) by CD4 þ T and CD8 þ T cells isolated from controls and infected mice increased from 2.16 to 8.99% and from 3.94% to 14.%, respectively. In GREAT mice infected with PbA and treated with E6446, only 3.5% and 4.% of CD4 þ T and CD8 þ T cells expressed YFP, respectively. As a consequence of inhibition of IFNg production by T lymphocytes, treatment with E6446 also inhibited the expression of and CXCL1 mrna by spleen cells (Fig. 6e), as well as RFP () and BFP (CXCL1) by from infected WT and REX3 mice, respectively (Fig. 6f). Immunocytochemistry analysis revealed the inhibitory effect of E6446 treatment on and CXCL1 expression by spleen cells from PbA-infected REX3 mice (Supplementary Fig. 6B). IFNc-dependent recruitment of to the CNS. The results presented in Supplementary Fig. 3B show that expression of IFNg and chemokine genes (that is, CCL2, CCL3, CCL4, CCL5, CCL8, and CXCL1) is induced and peaks in the brain at 6 7 days postinfection with PbA. Our experiments also demonstrated that expression of these genes is not seen or largely decreased in the brains of PbA-infected IFNg / mice or PbA-infected C57BL/6 mice treated with E6446 (Fig. 7a,b). As express both CCR2 (receptor for CCL2 MCP1) and CCR5 (receptor for CCL3 MIP1a, CCL4 MIP1b and CCL5 RANTES), we evaluated the emergence of in the brain of PbA-infected IFNg / mice and infected C57BL/6 mice treated with E6446. We observed that migration of into the brain of infected IFNg / mice or E6446-treated WT mice was also impaired (Fig. 7c). Consistent with the importance of in recruitment of CD8 þ T cells and development of ECM 37 39, the total number of CD8 þ T lymphocytes was reduced, but not absent, in the CNS of either IFNg / mice or PbA-infected C57BL/6 mice treated with E6446. Based on these results and those published elsewhere 4,45 48, we propose that IFNg is a key cytokine mediating ECM by also promoting the recruitment of into the brain and consequently amplifying recruitment of CD8 þ T cells to the CNS. CCR5-dependent recruitment of to the CNS. We hypothesized that CCL2, CCL3, CCL4 and CCL5 are largely responsible for recruiting to the CNS of PbA-infected mice. The data presented in Fig. 8a provided evidence that isolated (75 85% purity) migrate towards both CCL2 and CCL5 in vitro, although CCL5 appeared to be a more efficacious chemoattractant for. We also noticed an additive effect of the two chemokines. As CCL2 and CCR2 have been shown to be important for recruitment of inflammatory monocytes during infection with Toxoplasma gondii and P. chabaudi 19,49, we evaluated the role of CCR2 in the development of ECM. As expected, we observed a decreased frequency of in the / mice spleen of both uninfected and PbA-infected CCR2 (Supplementary Fig. 7A,B). However, the number of in the CNS of PbA-infected CCR2 / mice was only marginally reduced (Fig. 8c). In contrast, we observed no difference in the frequency or number of splenic in infected CCR5 / mice (Supplementary Fig. 8A,B). Furthermore, we observed no impairment on activation and IFNg production by CD4 þ T and CD8 þ T cells in spleens from CCR5 / mice infected with PbA (Supplementary Fig. 9A,B). Nevertheless, emergence of in the CNS of PbA-infected CCR5 / mice was highly compromised (Fig. 8c). Consistent with numbers of in the CNS, CCR5 / mice, but not CCR2 / mice, were more resistant to the development of ECM than PbA-infected WT mice (Fig. 8d). Importantly, over % of in the CNS of infected C57BL/6 mice expressed CCR5. Less than 1% of CD4 þ T and CD8 þ T lymphocytes expressed CCR5, whereas over 99% expressed CXCR3 (Fig. 9a,b). Hence, we hypothesize that CCR5 is a key receptor for MO-DC recruitment to the CNS, whereas CXCR3 and its ligands, and CXCL1, are responsible for attracting T lymphocytes to the CNS. Consistently, there is a decrease in the absolute number of both and CD8 þ T cells in the CNS of PbA-infected CCR5 / mice (Fig. 9c). We also performed in vivo imaging experiments to evaluate the role of CCR5 in the recruitment of to the CNS. Figure 6 E6446 inhibits expression of IFNc by T cells and and CXCL1 by. C57BL/6, GREAT and REX3 mice were infected with PbA and treated with either E6446 (1 mg kg 1 per day) or vehicle from 1 to 3 days post infection. (a) Spleens were harvested from WT mice at, 5 and 7 post infection, total RNA extracted and expression levels of IFNg mrna analysed by quantitative PCR. The results were normalized to b2-microbulin. (b) Sera from infected C57BL/6 mice were collected at different times post infection and used to measure the levels of IFNg by Cytometric Bead Analysis. The inhibitory effect of E6446 on the levels of circulating IFNg was evaluated at 6 days post infection (right panel). (c) Spleens from untreated and E6446-treated C57BL6 mice were harvested 5 days post infection. Cells were gated on CD44 and CD62L from CD4 þ or CD8 þ. Bar graphs correspond to the percentage of naive (CD62L high CD44 low ) versus activated effector (CD62L low CD44 high ) CD4 þ T (top panels) and CD8 þ T (bottom panels) cells. (d) Splenocytes from uninfected and infected GREAT mice treated with E6446 or left untreated were stimulated with PMA (5 ng ml 1 ) and ionomycin (5 ng ml 1 ) for 4 h in culture containing brefeldin A and analysed by flow cytometry gated on CD4 þ or CD8 þ cells. YFP-positive cells were considered as IFNg producers CD4 þ T (top panels) and CD8 þ T (bottom panels) cells. (e) Quantitative reverse transcriptase PCR (left panels) and (f) flow cytometry analysis revealed that treatment with E6446 resulted in inhibition of and CXCL1 mrna expression in the spleen and RFP and BFP proteins by splenic from PbA-infected C57BL/6 and REX3 mice, respectively. Representative primary flow cytometry dot plots are shown in c f. Results are representative of two to three independent experiments with three to five mice per group. Differences considered statistically significant when Po.5, Po.1, Po.1 or Po.1, as indicated by two-way analysis of variance analysis (values are means±s.d.). 1 NATURE COMMUNICATIONS 7:13277 DOI: 1.138/ncomms13277 www.nature.com/naturecommunications
(75 85% purity) from spleens of PbA-infected WT or CCR5 / mice were transferred into WT or CCR5 / mice at 5 days post infection. Migration of carboxyfluorescein succinimidyl ester (CSFE)-labelled was evaluated from 15 to min after cell transfer. We found high frequency of WT, primarily in the microvasculature. from a c Copies per β2 microglobulin WT WT IFNγ / treated (E6446).4.3.2.1..6.4.2...15.1.5. WT control CCL2 CCL4 IFNγ / control BMNCs WT 7th day IFNγ / 7 th day CD8.4.3.2.1.5 CCL3 CCL2.4.3.2.1..25..15.1.5...15.1.5. CCL5 WT control CD45 high cells CXCL1 IFNγ / control WT 7th day Ly6c IFNγ / 7 th day Copies per β2 microglobulin CD45 high CD3 CD8 cells 25 53.6 96.9 3.6 15 1 4 1 4 1 3 2.75 1 3 1 2 1 3 5 1 2 Day 1 3 1 3 1 4 1 3 1 4 1 3 1 3 1 4 3 82.5 17.1 1 3 1 3 1 4 96.8 88.4 3.24 1 3 1 3 1 4 3 11.6 1 3 1 3 1 4 CD45 1 4 1 3 1 4 1 3 1 4 1 3 58.1 6.43 8.6 11.7 1 3 1 4 53.8 1 3 1 4 53.5 1 3 1 4 CD3 1 4 1 3 1 4 1 3 1 2 1 2 1 4 1 3 1 2 85.6 1 3 1 4 7.55 Day 7 1 3 1 4 27.8 b Day 7 Day 7 1 3 1 4 CD11b...15.1.5..25..15.1.5. CCL4 (treated) WT (day 7) IFNγ / (day 7) 7 days 7 days (treated) WT (E6446) (day 7) 1 3 1 3 1 4 DC-SIGN 1 3 1 3 1 4 WT WT IFNγ /..15.1.5..12.8.4..25..15.1.5..4.3.2.1. (E6446) CCL3 CCL5 CXCL1 IFNγ (treated) 7 days 7 days (treated) Absolute number of cells 2 1 4 4 1 4 6 1 4 CD45 high CD3 CD8 CD11b + + DC-SIGN high Ly6c + CD45 high CD3 + CD8 + Figure 7 IFNc-dependent chemokine expression and emergence of CD45 high haematopoietic cells. CCL2, CCL3, CCL4, CCL5,, CXCL1 and IFNg mrna expression was analysed in brain tissue of uninfected or PbA-infected (a) IFNg / and C57BL/6 mice, as well as (b) C57BL/6 mice treated from day 1 to 3 post infection with vehicle or E6446 (1 mg kg 1 per day), as indicated. The mrna levels were analysed by quantitative PCR and results presented after normalization with b2-microglobulin mrna. (c) Flow cytometric analysis of brain first selected on CD45high, gated on CD3 CD8 and then CD11b þ Ly6c þ ( þ DC-SIGN þ ) from uninfected and PBA-infected WT, IFNg / and WT mice treated with E6446 7 days post infection. The numbers of both CD45 high CD8 þ CD3 þ and CD45 high CD8 CD3 þ CD11b þ DC-SIGN high Ly6c þ cells are significantly decreased in the brain of infected IFNg / and E6446-treated mice. Differences considered statistically significant when Po.5, Po.1, Po.1 and Po.1 as indicated by two-way analysis of variance analysis (values are means±s.d.). NATURE COMMUNICATIONS 7:13277 DOI: 1.138/ncomms13277 www.nature.com/naturecommunications 11
CCR5 / mice were less efficient in migrating to the CNS of infected mice. Furthermore, WT, but not CCR5 / MO DCs, migrated to the CNS of CCR5 /, indicating that expression of CCR5 is critical for MO-DC migration to the CNS (Fig. 9d and Supplementary Movies 1 6). No migration of to the CNS of uninfected mice was observed. Finally, the results presented in a Migrating DC (number of cells) c C57BL/6 day 7 C57BL/6 day 7 CCR5 / 1, 8, 6, 4, 2, ng ml -1 (infected) 15 1 5 3 CCL2 CCL5 CCL2 + CCL5.1 ng ml -1 (infected) 96.6 1 3 1 3 1 4 75.5 1 3 1 3 1 4 3 87. 1 3 1 3 1 4 1 ng ml -1 (infected) 3.39 24.5 13. CD45 (MFI) C57BL/6 C57BL/6 CCR5 / 1 ng ml -1 (infected) CD8 1 4 1 3 1 4 1 3 1 4 1 3 7.14 12, 1 3 1 3 1 4 1 3 1 3 1 4 Comp-alexa fluor 7-A:: Comp-APC-A:: DC SIGN DC-SIGN 8, 4, 1 2 1 3 1 4 1 ng ml -1 (control) Ly6c 1 4 1 3 1 2 1 4 1 3 1 2 1 ng ml -1 (infected) 1 3 1 4 1 3 1 4 29.4 6.61 1 4 1 3 1 2 1 2 1 3 1 4 1 3 1 4 CD3 CD3 CD8 CD11b+ Ly6c+ cells Absolute number 8 1 3 6 1 3 4 1 3 2 1 3 78.9 1 3 1 4 45.2 CD11b WT uninfected WT (7th day) CCR5 / (7th day) b d Percent survival (%) Brain mononuclear cells (BMNC) Abosolute number 1 3 1 3 1 4 DC-SIGN 1 3 1 3 1 4 CD3+CD8 CD11b+Ly6c+ cells 6 1 3 4 1 3 2 1 3 ns WT uninfected WT (7 th day) CCR2 / (7 th day) CCR5 / (n =11) CCR2 / (n =12) WT (n =16) WT uninfected CCR2 / infected (7 th day) WT infected (7 th day) 1 2 3 4 5 6 7 8 9 1 11 12 13 14 Days post infection Figure 8 Enhanced resistance of CCR5 / mice to development of ECM. (a) In vitro migration of enriched towards different concentrations of CCL2, CCL5 or the combination of CCL2 and CCL5. (b) Flow cytometric analysis of in the CNS of WT and CCR2 / mice infected with PbA. A lower, but not significant difference in the frequency of CD45 high þ CD11b þ DC-SIGN high MHCII high Ly6c þ is observed in the brain of PbA-infected CCR2 / mice. (c) Representative primary flow cytometric dot plots gated on CD8 CD3 and then Ly6c þ CD11b þ cells are shown after gating the CD45 high cells. The numbers of CD45 high CD8 CD3 þ CD11b þ DC-SIGN high Ly6c þ are significantly decreased in the brain of PbA-infected CCR5 / mice. (d) Survival curve of PbA-infected C57BL/6, CCR2 / and CCR5 / mice. The results are pool of three independent experiments with three to six mice per group. Differences were considered statistically significant when Po.1 as indicated by one-way analysis of variance (values are means±s.d.). The survival curves were analysed by log-rank test and CCR5 / mice were shown more resistant to PbA infection, when compared with CCR2 / or WT mice (Po.1). 12 NATURE COMMUNICATIONS 7:13277 DOI: 1.138/ncomms13277 www.nature.com/naturecommunications
Fig. 9e show that transfer of enriched WT MO-DC population restored ECM in PbA-infected CCR5 / mice. Discussion We provide compelling evidence that acute infection with PbA stimulates differentiation of splenic inflammatory monocytes into in an IFNg-dependent manner. Malaria-induced possess a strong phagocytic activity, capacity to cross-present antigens to CD8 þ T cells and express high levels of, CXCL1 and the chemokine receptor CCR5. Hence, splenic CCR5 þ /1 þ seem to play an important role in recruiting and activating T lymphocytes. We hypothesize that after differentiation, splenic CCR5 þ /1 þ migrate to the brain in a CCR5-dependent manner and amplify the recruitment and activation of CXCR3 þ CD8 þ T cells, promoting neuroinflammation and ECM (Fig. 1). a c d 1, 9 3 Absolute number 6 1 3 4 1 3 2 1 3 Wild type DCs Y 78.5 Z Y Z X X 21.5 1 3 1 4 CD45 1 3 1 4 MHCII Ly6c CD8 CD4 WT uninfected 1 4 1 3 1 4 1 3 1 4 1 3 46.2 6.6 WT (7 th day) 1 3 1 4.5 CD45 high CCR5 + CD11b + + DC SIGN high Ly6c + CD45 high CCR5 CXCR3 + CD3 + CD8 + CCR5 / (7 th day) X X CD45 high cells CCR5 / DCs Y Z Y Z CXCR3 CXCR3 CXCR3 78.2 1.75 19.8.24 78 8.26 1.89.92 1 3 1 4 1 3 1 4 CD3 CCR5 84.6 12.8 1 2 1 3 1415 21.3 65.8 1 2 1 3 1 4 13 14 15 CD11b CCR5 1 4 1 3 1 4 1 3 1 4 1 3 254µm 1 3 1 4 11 b % CD45 high Day 6 Day 7 35 3 25 15 1 5 4 3 2 1 CFSE + cells per FOV CD3 + CD4 + T cells CD3 + CD8 + T cells Undefined Day 3 Day 4 CXCR3 CCR5 CXCR3 CCR5+ 2, 1,5 1, 5 Day 5 Day 6 CD3+CD4+ T lymphocyte ns WT inf > WT NI WT inf > WT inf CCR5 KO inf > WT inf WT inf > CCR5 KO inf CCR5 KO inf > CCR5 KO inf Day 7 CD3+CD8+ T lymphocyte CXCR3+CCR5+ CXCR3+CCR5 e CCR5 / > CCR5 / (n=8) WT > CCR5 / (n=8) Clinical score Ruffled fur 2 Hunching Unbalancing Limb paralysis Convuctions, coma and death 4 6 8 1 Clinical score 1 8 6 4 2 Percent survival (%) CCR5 / > CCR5 / (n=8) CCR5 / (n=1) WT > CCR5 / (n=8) WT (n=1) 4 5 6 7 8 9 1 11 12 13 14 Days post infection 1 2 3 4 5 6 7 8 9 1 11 12 13 14 Days post infection NATURE COMMUNICATIONS 7:13277 DOI: 1.138/ncomms13277 www.nature.com/naturecommunications 13
IgG2a/IgG2c, which mediate host resistance to Plasmodium infection in mice11,5. In addition, both inflammatory monocytes and DCs can internalize and destroy infected RBCs in the spleens and mediate resistance to P. chabaudi infection in mice19,21. However, depending on experimental setting, DCs may become refractory or impaired for presenting antigen to CD4 þ T and CD8 þ T cells51,52. For instance, virulent P. yoelli strains impair DC functions to prevent the induction of host protective immune responses15. Inflammatory monocytes and DCs also play key roles in the deleterious inflammatory response and pathogenesis DCs and inflammatory monocytes play important roles in host resistance to malaria. Indeed, there is a very dynamic distribution of inflammatory monocytes and DC subpopulations in the spleen of acutely infected mice, which changes with the course of infection, parasite virulence and infective Plasmodium spp.5. For instance, DCs exposed to the erythrocytic stage of P. chabaudi produce high levels of IL-12 and promote the production of IFNg by both T lymphocytes and natural killer cells14. This cytokine activates macrophages to produce toxic metabolites, such as reactive nitrogen intermediates and promotes Ig switch to the CCR5+/1 MO-DC CXCR3 CD4+T CCR5 CCL3 CCL4 CCL5 IFNγ ICAM + CD4 T CXCL1 Endothelial dysfunction IFNγ/TNFα erythrocyte CXCR3 TLR7 TLR9 CD4+T Dendritic cells IFNγ TNFα IFNγ TNFα CD4+T CD4+T CCR5 Inflammatory monocytes CD4+T CCR5+/1 MO-DC Figure 1 CCR5 þ /1 þ mediate cerebral malaria in PbA-infected mice. Infection with PbA leads to activation of DCs and induces IFNg-dependent differentiation of inflammatory monocytes into splenic that express high levels of CCR5, and CXCL1 (CCR5 þ /1 þ ). We hypothesize that after differentiation, CCR5 þ /1 þ migrate to the CNS in response to CCR5 ligands (that is, CCL3, CCL4 and CCL5) amplifying recruitment of CD8 þ T lymphocytes, initiated by endothelial cells expressing CXCL1 and promote development of ECM. Figure 9 CCR5 þ migrate to the CNS and mediate ECM in PbA-infected mice. (a) Brain mononuclear cells (BMNC) were recovered from PbA-infected mice gated on CD45high, then on CD3 þ CD8 þ or CD3 þ CD4 þ and analysed for CXCR3 and CCR5 expression. Alternatively, CD45high cells were gated on CD3 ÿ CD8 ÿ CD11b þ Ly6c þ MHC II þ þ and then analysed for CXCR3 and CCR5 expression. (b) Top graph shows the frequency of CD45high cells (that is,, CD4 þ T and CD8 þ T cells) in total BMNCs isolated from C57BL/6 mice at different time post infection. Bottom panel shows the frequency of, CD4 þ T cells and CD8 þ T cells expressing CXCR3 and CCR5. (c) Numbers of both CD45highCD8 ÿ CD3 ÿ CCR5 þ þ CD11b þ DC-SIGNhighLy6c þ and CD45highCCR5 ÿ CXCR3 þ CD8 þ CD3 þ are significantly decreased in the brain of PbA-infected CCR5 ÿ / ÿ mice. These results are representative of four experiments (values are means±s.d.). (d) MO-DC-enriched populations were obtained from WTand CCR5 ÿ / ÿ mice 5 days post infection and labelled with CSFE transferred i.v. (1 2 million cells per mouse) in the orbital vein into PbA-infected WT or CCR5 ÿ / ÿ mice. As control, cells were also transferred to uninfected mice. The presence of CSFE labeled cells were detected in the microvasculature and brain parenchyma by confocal microscopy 15 min after cell transfer (Supplementary Movies 1 6). Each point represents total number of labelled cells in four field of views per mouse (scale bar, 254 mm). The results are pool of three independent experiments with two or three mice per group. Differences were considered statistically significant when Po.1, as indicated by one-way analysis of variance analysis. (e) Three million splenic derived from either WTor CCR5 ÿ / ÿ mice at 5 days post infection were transferred to CCR5 ÿ / ÿ mice at four days post infection. Scores of disease severity and survival curves of PbA-infected CCR5 ÿ / ÿ mice that received WT or CCR5 ÿ / ÿ are shown in left and right panels, respectively. PbA-infected WT or CCR5 ÿ / ÿ mice that received no were used as controls. The results are pool of two independent experiments with four mice per group. The survival curves were analysed by log-rank test. PbA-infected CCR5 ÿ / ÿ mice that received WT were more susceptible to infection, when compared with PbA-infected CCR5 ÿ / ÿ mice that received CCR5 ÿ / ÿ or PbA-infected CCR5 ÿ / ÿ mice that received no (Po.1, Po.1). 14 NATURE COMMUNICATIONS 7:13277 DOI: 1.138/ncomms13277 www.nature.com/naturecommunications