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Distinct Upstream Role of Type I IFN Signaling in Hematopoietic Stem Cell-Derived and Epithelial Resident Cells for Concerted Recruitment of Ly-6Chi Monocytes and NK Cells via CCL2-CCL3 Cascade.

Uyangaa E, Kim JH, Patil AM, Choi JY, Kim SB, Eo SK - PLoS Pathog. (2015)

Bottom Line: However, the crucial regulators and cell populations that are affected by IFN-I to establish the early environment of innate cells in HSV-infected mucosal tissues are largely unknown.Also, IFN-I signaling in HSC-derived leukocytes appeared to suppress Ly-6Ghi neutrophil recruitment to ameliorate immunopathology.Therefore, this cascade response of resident-to-hematopoietic-to-resident cells that drives cytokine-to-chemokine-to-cytokine production to recruit orchestrated innate cells is critical for attenuation of HSV replication in inflamed tissues.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Iksan, Republic of Korea.

ABSTRACT
Type I interferon (IFN-I)-dependent orchestrated mobilization of innate cells in inflamed tissues is believed to play a critical role in controlling replication and CNS-invasion of herpes simplex virus (HSV). However, the crucial regulators and cell populations that are affected by IFN-I to establish the early environment of innate cells in HSV-infected mucosal tissues are largely unknown. Here, we found that IFN-I signaling promoted the differentiation of CCL2-producing Ly-6Chi monocytes and IFN-γ/granzyme B-producing NK cells, whereas deficiency of IFN-I signaling induced Ly-6Clo monocytes producing CXCL1 and CXCL2. More interestingly, recruitment of Ly-6Chi monocytes preceded that of NK cells with the levels peaked at 24 h post-infection in IFN-I-dependent manner, which was kinetically associated with the CCL2-CCL3 cascade response. Early Ly-6Chi monocyte recruitment was governed by CCL2 produced from hematopoietic stem cell (HSC)-derived leukocytes, whereas NK cell recruitment predominantly depended on CC chemokines produced by resident epithelial cells. Also, IFN-I signaling in HSC-derived leukocytes appeared to suppress Ly-6Ghi neutrophil recruitment to ameliorate immunopathology. Finally, tissue resident CD11bhiF4/80hi macrophages and CD11chiEpCAM+ dendritic cells appeared to produce initial CCL2 for migration-based self-amplification of early infiltrated Ly-6Chi monocytes upon stimulation by IFN-I produced from infected epithelial cells. Ultimately, these results decipher a detailed IFN-I-dependent pathway that establishes orchestrated mobilization of Ly-6Chi monocytes and NK cells through CCL2-CCL3 cascade response of HSC-derived leukocytes and epithelium-resident cells. Therefore, this cascade response of resident-to-hematopoietic-to-resident cells that drives cytokine-to-chemokine-to-cytokine production to recruit orchestrated innate cells is critical for attenuation of HSV replication in inflamed tissues.

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Critical role of IFN-I signaling on resident CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs in the production of initial CCL2 protein for early migration of CD11b+Ly-6Chi monocytes.(A) Early expression of CCL2 and CCL3 by sorted CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs. Subsets of macrophages (CD11bhiF4/80hi, CD11bhiF4/80int/lo, CD11bloF4/80hi, and CD11bloF4/80lo) and DCs (CD11chiEpCAM+, CD11chiEpCAM−, CD11cloEpCAM+, and CD11cloEpCAM−) were sorted 12 h after mucosal HSV-1 infection, and CCL2 and CCL3 expression was analyzed with real-time qRT-PCR. (B,C) Regulation of CCL2 and CCL3 production from CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs by IFN-I signaling. CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs producing CCL2 (B) and CCL3 (C) were determined by flow cytometric analysis at 12 h pi. Blue line, HSV-1-infected BL/6; Red line, HSV-1-infected IFNAR KO; Gray line, mock-infected. (D) Confocal microscopic analysis of early CCL2 production by resident CD11b+ macrophages and CD11c+ DCs. CD11b+ macrophages and CD11c+ DCs producing CCL2 protein were visualized by confocal microscopy at 12 h pi. CD11b+ macrophages and CD11c+ DCs producing CCL2 protein are denoted by white arrows. (E,F) Essential role of IFN-I signaling in the production of CCL2 protein from CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs. Vaginal CD11bhiF4/80hi macrophages (E) and CD11chiEpCAM+ DCs (F) isolated from WT and IFNAR KO mice were stimulated with recombinant IFN-α (2,000 and 4,000 IU/ml) for 6 h. The expression and production of CCL2 were determined by real-time qRT-PCR and CBA using stimulated cells and collected culture media, respectively. Data represent the average ± SD of values derived from three individual experiments (n = 4–5). *, p<0.05; **, p<0.01; ***, p<0.001 compared with the level of the indicated group.
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ppat.1005256.g007: Critical role of IFN-I signaling on resident CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs in the production of initial CCL2 protein for early migration of CD11b+Ly-6Chi monocytes.(A) Early expression of CCL2 and CCL3 by sorted CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs. Subsets of macrophages (CD11bhiF4/80hi, CD11bhiF4/80int/lo, CD11bloF4/80hi, and CD11bloF4/80lo) and DCs (CD11chiEpCAM+, CD11chiEpCAM−, CD11cloEpCAM+, and CD11cloEpCAM−) were sorted 12 h after mucosal HSV-1 infection, and CCL2 and CCL3 expression was analyzed with real-time qRT-PCR. (B,C) Regulation of CCL2 and CCL3 production from CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs by IFN-I signaling. CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs producing CCL2 (B) and CCL3 (C) were determined by flow cytometric analysis at 12 h pi. Blue line, HSV-1-infected BL/6; Red line, HSV-1-infected IFNAR KO; Gray line, mock-infected. (D) Confocal microscopic analysis of early CCL2 production by resident CD11b+ macrophages and CD11c+ DCs. CD11b+ macrophages and CD11c+ DCs producing CCL2 protein were visualized by confocal microscopy at 12 h pi. CD11b+ macrophages and CD11c+ DCs producing CCL2 protein are denoted by white arrows. (E,F) Essential role of IFN-I signaling in the production of CCL2 protein from CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs. Vaginal CD11bhiF4/80hi macrophages (E) and CD11chiEpCAM+ DCs (F) isolated from WT and IFNAR KO mice were stimulated with recombinant IFN-α (2,000 and 4,000 IU/ml) for 6 h. The expression and production of CCL2 were determined by real-time qRT-PCR and CBA using stimulated cells and collected culture media, respectively. Data represent the average ± SD of values derived from three individual experiments (n = 4–5). *, p<0.05; **, p<0.01; ***, p<0.001 compared with the level of the indicated group.

Mentions: Since early infiltrated HSC-derived leukocytes are considered the main cellular regulators in the recruitment of CD11b+Ly-6Chi monocytes via CCL2, unsolved questions still remain: What induces the initial infiltration of these leukocytes producing CCL2? What are the initial regulators of leukocyte recruitment, including CD11b+Ly-6Chi monocytes? Because infiltrated CD11b+Ly-6Chi monocytes are also the main producers of CCL2 protein, we assumed that the seeded amount of CCL2 produced by certain resident cells in vaginal tract might induce initial recruitment of CD11b+Ly-6Chi monocytes, which subsequently provide amplified recruitment of more CD11b+Ly-6Chi monocytes via CCL2. To this end, we examined CCL2 and CCL3 expression in tissue resident CD11bhi and CD11chi cell subpopulations at 12 h pi, before the recruitment of CD11b+Ly-6Chi monocytes peaked. Interestingly, initial expression of CCL2 was dominantly achieved in CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs sorted from vaginal tract of WT BL/6 mice, compared to those of IFNAR KO mice (Fig 7A). Also, other CD11bhi myeloid cells (CD11bhiF4/80lo) and CD11chi DCs (CD11chiEpCAM−), but not CD11blo and CD11clo cells, showed significantly higher levels of CCL2 expression in WT BL/6 compared to IFNAR KO mice. In addition, CCL3 expression was also apparent in CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs derived from WT BL/6 mice, even though levels of CCL3 expression were much lower than those of CCL2. To confirm these findings, we enumerated CCL2-producing CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs in vaginal tract of both WT BL/6 and IFNAR KO mice at 12 h pi. As expected, increased expression of CCL2 protein in vaginal CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs of WT BL/6 mice was observed, and a higher number of CCL2-producing CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs was detected in vaginal tract of WT BL/6 mice, compared with IFNAR KO mice (Fig 7B). Similarly, vaginal CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs derived from WT BL/6 mice showed higher expression of CCL3 compared to those of IFNAR KO mice (Fig 7C). The production of CCL2 protein by vaginal CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs was further visualized using confocal microscopy (Fig 7D). In consistent, CCL2-producing CD11b+ and CD11c+ cells were detected at a higher frequency in vaginal tract of WT BL/6 mice than IFNAR KO mice. Also, F4/80+ cells producing CCL2 protein were detected at a higher frequency in the vaginal tract of WT BL/6 than IFNAR KO mice (S4 Fig). Since IFN-I signaling plays an important role in inducing CCL2 protein by stimulation with IFN-I proteins produced from epithelial cells through IFI-16 and STING recognition upon HSV-1 infection [34,35], we examined the expression of CCL2 from vaginal CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs sorted from WT BL/6 and IFNAR KO mice upon stimulation with IFN-α protein. As expected, CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs derived from vaginal tract of WT BL/6 mice showed higher expression of CCL2 protein than those of IFNAR KO mice (Fig 7E and 7F), even though CCL2 expression in CD11chiEpCAM+ DCs was detected at lower levels than CD11bhiF4/80hi macrophages. Similarly, other CC chemokines (CCL3, CCL4, CCL5), but not CXC chemokines, were expressed with slightly higher levels in CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs derived from vaginal tract of WT BL/6 mice, compared to those of IFNAR KO (S5 Fig). Collectively, these results suggest that tissue resident CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs are candidates for supplying the initial CCL2 protein upon stimulation by IFN-I produced from infected epithelial cells, thereby inducing early infiltration of CD11b+Ly-6Chi monocytes into mucosal tissues.


Distinct Upstream Role of Type I IFN Signaling in Hematopoietic Stem Cell-Derived and Epithelial Resident Cells for Concerted Recruitment of Ly-6Chi Monocytes and NK Cells via CCL2-CCL3 Cascade.

Uyangaa E, Kim JH, Patil AM, Choi JY, Kim SB, Eo SK - PLoS Pathog. (2015)

Critical role of IFN-I signaling on resident CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs in the production of initial CCL2 protein for early migration of CD11b+Ly-6Chi monocytes.(A) Early expression of CCL2 and CCL3 by sorted CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs. Subsets of macrophages (CD11bhiF4/80hi, CD11bhiF4/80int/lo, CD11bloF4/80hi, and CD11bloF4/80lo) and DCs (CD11chiEpCAM+, CD11chiEpCAM−, CD11cloEpCAM+, and CD11cloEpCAM−) were sorted 12 h after mucosal HSV-1 infection, and CCL2 and CCL3 expression was analyzed with real-time qRT-PCR. (B,C) Regulation of CCL2 and CCL3 production from CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs by IFN-I signaling. CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs producing CCL2 (B) and CCL3 (C) were determined by flow cytometric analysis at 12 h pi. Blue line, HSV-1-infected BL/6; Red line, HSV-1-infected IFNAR KO; Gray line, mock-infected. (D) Confocal microscopic analysis of early CCL2 production by resident CD11b+ macrophages and CD11c+ DCs. CD11b+ macrophages and CD11c+ DCs producing CCL2 protein were visualized by confocal microscopy at 12 h pi. CD11b+ macrophages and CD11c+ DCs producing CCL2 protein are denoted by white arrows. (E,F) Essential role of IFN-I signaling in the production of CCL2 protein from CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs. Vaginal CD11bhiF4/80hi macrophages (E) and CD11chiEpCAM+ DCs (F) isolated from WT and IFNAR KO mice were stimulated with recombinant IFN-α (2,000 and 4,000 IU/ml) for 6 h. The expression and production of CCL2 were determined by real-time qRT-PCR and CBA using stimulated cells and collected culture media, respectively. Data represent the average ± SD of values derived from three individual experiments (n = 4–5). *, p<0.05; **, p<0.01; ***, p<0.001 compared with the level of the indicated group.
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ppat.1005256.g007: Critical role of IFN-I signaling on resident CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs in the production of initial CCL2 protein for early migration of CD11b+Ly-6Chi monocytes.(A) Early expression of CCL2 and CCL3 by sorted CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs. Subsets of macrophages (CD11bhiF4/80hi, CD11bhiF4/80int/lo, CD11bloF4/80hi, and CD11bloF4/80lo) and DCs (CD11chiEpCAM+, CD11chiEpCAM−, CD11cloEpCAM+, and CD11cloEpCAM−) were sorted 12 h after mucosal HSV-1 infection, and CCL2 and CCL3 expression was analyzed with real-time qRT-PCR. (B,C) Regulation of CCL2 and CCL3 production from CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs by IFN-I signaling. CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs producing CCL2 (B) and CCL3 (C) were determined by flow cytometric analysis at 12 h pi. Blue line, HSV-1-infected BL/6; Red line, HSV-1-infected IFNAR KO; Gray line, mock-infected. (D) Confocal microscopic analysis of early CCL2 production by resident CD11b+ macrophages and CD11c+ DCs. CD11b+ macrophages and CD11c+ DCs producing CCL2 protein were visualized by confocal microscopy at 12 h pi. CD11b+ macrophages and CD11c+ DCs producing CCL2 protein are denoted by white arrows. (E,F) Essential role of IFN-I signaling in the production of CCL2 protein from CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs. Vaginal CD11bhiF4/80hi macrophages (E) and CD11chiEpCAM+ DCs (F) isolated from WT and IFNAR KO mice were stimulated with recombinant IFN-α (2,000 and 4,000 IU/ml) for 6 h. The expression and production of CCL2 were determined by real-time qRT-PCR and CBA using stimulated cells and collected culture media, respectively. Data represent the average ± SD of values derived from three individual experiments (n = 4–5). *, p<0.05; **, p<0.01; ***, p<0.001 compared with the level of the indicated group.
Mentions: Since early infiltrated HSC-derived leukocytes are considered the main cellular regulators in the recruitment of CD11b+Ly-6Chi monocytes via CCL2, unsolved questions still remain: What induces the initial infiltration of these leukocytes producing CCL2? What are the initial regulators of leukocyte recruitment, including CD11b+Ly-6Chi monocytes? Because infiltrated CD11b+Ly-6Chi monocytes are also the main producers of CCL2 protein, we assumed that the seeded amount of CCL2 produced by certain resident cells in vaginal tract might induce initial recruitment of CD11b+Ly-6Chi monocytes, which subsequently provide amplified recruitment of more CD11b+Ly-6Chi monocytes via CCL2. To this end, we examined CCL2 and CCL3 expression in tissue resident CD11bhi and CD11chi cell subpopulations at 12 h pi, before the recruitment of CD11b+Ly-6Chi monocytes peaked. Interestingly, initial expression of CCL2 was dominantly achieved in CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs sorted from vaginal tract of WT BL/6 mice, compared to those of IFNAR KO mice (Fig 7A). Also, other CD11bhi myeloid cells (CD11bhiF4/80lo) and CD11chi DCs (CD11chiEpCAM−), but not CD11blo and CD11clo cells, showed significantly higher levels of CCL2 expression in WT BL/6 compared to IFNAR KO mice. In addition, CCL3 expression was also apparent in CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs derived from WT BL/6 mice, even though levels of CCL3 expression were much lower than those of CCL2. To confirm these findings, we enumerated CCL2-producing CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs in vaginal tract of both WT BL/6 and IFNAR KO mice at 12 h pi. As expected, increased expression of CCL2 protein in vaginal CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs of WT BL/6 mice was observed, and a higher number of CCL2-producing CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs was detected in vaginal tract of WT BL/6 mice, compared with IFNAR KO mice (Fig 7B). Similarly, vaginal CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs derived from WT BL/6 mice showed higher expression of CCL3 compared to those of IFNAR KO mice (Fig 7C). The production of CCL2 protein by vaginal CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs was further visualized using confocal microscopy (Fig 7D). In consistent, CCL2-producing CD11b+ and CD11c+ cells were detected at a higher frequency in vaginal tract of WT BL/6 mice than IFNAR KO mice. Also, F4/80+ cells producing CCL2 protein were detected at a higher frequency in the vaginal tract of WT BL/6 than IFNAR KO mice (S4 Fig). Since IFN-I signaling plays an important role in inducing CCL2 protein by stimulation with IFN-I proteins produced from epithelial cells through IFI-16 and STING recognition upon HSV-1 infection [34,35], we examined the expression of CCL2 from vaginal CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs sorted from WT BL/6 and IFNAR KO mice upon stimulation with IFN-α protein. As expected, CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs derived from vaginal tract of WT BL/6 mice showed higher expression of CCL2 protein than those of IFNAR KO mice (Fig 7E and 7F), even though CCL2 expression in CD11chiEpCAM+ DCs was detected at lower levels than CD11bhiF4/80hi macrophages. Similarly, other CC chemokines (CCL3, CCL4, CCL5), but not CXC chemokines, were expressed with slightly higher levels in CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs derived from vaginal tract of WT BL/6 mice, compared to those of IFNAR KO (S5 Fig). Collectively, these results suggest that tissue resident CD11bhiF4/80hi macrophages and CD11chiEpCAM+ DCs are candidates for supplying the initial CCL2 protein upon stimulation by IFN-I produced from infected epithelial cells, thereby inducing early infiltration of CD11b+Ly-6Chi monocytes into mucosal tissues.

Bottom Line: However, the crucial regulators and cell populations that are affected by IFN-I to establish the early environment of innate cells in HSV-infected mucosal tissues are largely unknown.Also, IFN-I signaling in HSC-derived leukocytes appeared to suppress Ly-6Ghi neutrophil recruitment to ameliorate immunopathology.Therefore, this cascade response of resident-to-hematopoietic-to-resident cells that drives cytokine-to-chemokine-to-cytokine production to recruit orchestrated innate cells is critical for attenuation of HSV replication in inflamed tissues.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Iksan, Republic of Korea.

ABSTRACT
Type I interferon (IFN-I)-dependent orchestrated mobilization of innate cells in inflamed tissues is believed to play a critical role in controlling replication and CNS-invasion of herpes simplex virus (HSV). However, the crucial regulators and cell populations that are affected by IFN-I to establish the early environment of innate cells in HSV-infected mucosal tissues are largely unknown. Here, we found that IFN-I signaling promoted the differentiation of CCL2-producing Ly-6Chi monocytes and IFN-γ/granzyme B-producing NK cells, whereas deficiency of IFN-I signaling induced Ly-6Clo monocytes producing CXCL1 and CXCL2. More interestingly, recruitment of Ly-6Chi monocytes preceded that of NK cells with the levels peaked at 24 h post-infection in IFN-I-dependent manner, which was kinetically associated with the CCL2-CCL3 cascade response. Early Ly-6Chi monocyte recruitment was governed by CCL2 produced from hematopoietic stem cell (HSC)-derived leukocytes, whereas NK cell recruitment predominantly depended on CC chemokines produced by resident epithelial cells. Also, IFN-I signaling in HSC-derived leukocytes appeared to suppress Ly-6Ghi neutrophil recruitment to ameliorate immunopathology. Finally, tissue resident CD11bhiF4/80hi macrophages and CD11chiEpCAM+ dendritic cells appeared to produce initial CCL2 for migration-based self-amplification of early infiltrated Ly-6Chi monocytes upon stimulation by IFN-I produced from infected epithelial cells. Ultimately, these results decipher a detailed IFN-I-dependent pathway that establishes orchestrated mobilization of Ly-6Chi monocytes and NK cells through CCL2-CCL3 cascade response of HSC-derived leukocytes and epithelium-resident cells. Therefore, this cascade response of resident-to-hematopoietic-to-resident cells that drives cytokine-to-chemokine-to-cytokine production to recruit orchestrated innate cells is critical for attenuation of HSV replication in inflamed tissues.

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Related in: MedlinePlus