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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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IFN-I signaling is involved in connected recruitment and activation of NK cells to CD11b+Ly-6Chi monocytes.(A) NK cell infiltration of infected BL/6 or IFNAR KO mice. (B) Total accumulated NK cell number. Cells were prepared from vaginal tract (VT) and iliac LN (ILN) by collagenase digestion at 24, 48, and 72 h pi and used for analysis of NK cells. Values in dot-plots denote the average percentages of NK1.1+DX5+ NK cells derived from four independent samples after gating on CD3-negative cells at 48 h pi. (C) Frequency of IFN-γ or granzyme B-producing cells among NK cells. The production of IFN-γ and granzyme B by CD3−NK1.1+DX5+ NK cells was determined by intracellular staining after stimulation of vaginal NK cells with PMA plus ionomycin at 48 h pi. Values in histograms represent the average percentages of IFN-γ or granzyme B-producing cells among CD3−NK1.1+DX5+ NK cells. (D) Absolute number of IFN-γ or granzyme B-producing NK cells. Total number of IFN-γ or granzyme B-producing CD3−NK1.1+DX5+ NK cells in vaginal tract was enumerated by flow cytometric analysis using intracellular and surface staining at 48 h pi. (E) Secreted IFN-γ levels in vaginal tract. Secreted IFN-γ levels were determined by ELISA at 48 h pi using vaginal lavages. Data in bar chart represent the average ± SD of values derived from three individual experiments (n = 4–5). **, p<0.01; ***, p<0.001 compared with the levels of IFNAR KO mice.
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ppat.1005256.g003: IFN-I signaling is involved in connected recruitment and activation of NK cells to CD11b+Ly-6Chi monocytes.(A) NK cell infiltration of infected BL/6 or IFNAR KO mice. (B) Total accumulated NK cell number. Cells were prepared from vaginal tract (VT) and iliac LN (ILN) by collagenase digestion at 24, 48, and 72 h pi and used for analysis of NK cells. Values in dot-plots denote the average percentages of NK1.1+DX5+ NK cells derived from four independent samples after gating on CD3-negative cells at 48 h pi. (C) Frequency of IFN-γ or granzyme B-producing cells among NK cells. The production of IFN-γ and granzyme B by CD3−NK1.1+DX5+ NK cells was determined by intracellular staining after stimulation of vaginal NK cells with PMA plus ionomycin at 48 h pi. Values in histograms represent the average percentages of IFN-γ or granzyme B-producing cells among CD3−NK1.1+DX5+ NK cells. (D) Absolute number of IFN-γ or granzyme B-producing NK cells. Total number of IFN-γ or granzyme B-producing CD3−NK1.1+DX5+ NK cells in vaginal tract was enumerated by flow cytometric analysis using intracellular and surface staining at 48 h pi. (E) Secreted IFN-γ levels in vaginal tract. Secreted IFN-γ levels were determined by ELISA at 48 h pi using vaginal lavages. Data in bar chart represent the average ± SD of values derived from three individual experiments (n = 4–5). **, p<0.01; ***, p<0.001 compared with the levels of IFNAR KO mice.

Mentions: Together with CD11b+Ly-6Chi monocytes, NK cells are a critical innate cell population for providing protection against mucosal HSV-1 infection via production of IFN-γ [17–19]. Since CD11b+Ly-6Chi monocytes and CD11c+ DCs were shown to be consecutively recruited to the vaginal tract, we were also interested in the recruitment kinetics and activation of NK cells in IFNAR-ablated mice. Before virus infection, the frequency and number of CD3−NK1.1+DX5+ NK cells were comparable in vaginal tract and iliac LN of both BL/6 and IFNAR KO mice, except liver tissue (S3 Fig), which indicates that IFN-I signaling may not contribute to basal recruitment of NK cells in primary target tissues of HSV-1 mucosal infection. However, it was revealed that IFN-I signaling plays a crucial role in recruiting CD3−NK1.1+DX5+ NK cells into vaginal tract after HSV-1 infection. Notably, WT BL/6 mice displayed delayed recruitment of CD3−NK1.1+DX5+ NK cells in the vaginal tract with the levels peaking at 48 h pi, compared to CD11b+Ly-6Chi monocytes, and an increased frequency of NK cells, compared to IFNAR KO mice (Fig 3A). Also, iliac NK cell frequency was moderately increased in WT BL/6 mice compared to IFNAR KO mice. Supporting these findings, total accumulated number of CD3-NK1.1+DX5+ NK cells in vaginal tract and iliac LN was greater in WT BL/6 mice, with levels peaking at 48 h pi, than in IFNAR KO mice (Fig 3B). In addition, when the activation levels of NK cells were examined by analysis of NK cells producing IFN-γ and granzyme B, the ablation of IFN-I signal resulted in reduced production of IFN-γ and granzyme B from CD3−NK1.1+DX5+ NK cells (Fig 3C), and a decrease in the total number of NK cells producing IFN-γ or granzyme B (Fig 3D). Consistent with these findings, lower levels of IFN-γ protein in vaginal lavages of IFNAR KO mice were detected at 48 h pi compared to WT BL/6 mice (Fig 3E). Collectively, these results indicate that NK cell recruitment in vaginal tract follows that of CD11b+Ly-6Chi monocytes like CD11c+ DCs, and that IFN-I signal also has a critical contribution to the early concerted recruitment and activation of CD11b+Ly-6Chi monocytes and NK cells in mucosal tissues following HSV-1 infection.


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)

IFN-I signaling is involved in connected recruitment and activation of NK cells to CD11b+Ly-6Chi monocytes.(A) NK cell infiltration of infected BL/6 or IFNAR KO mice. (B) Total accumulated NK cell number. Cells were prepared from vaginal tract (VT) and iliac LN (ILN) by collagenase digestion at 24, 48, and 72 h pi and used for analysis of NK cells. Values in dot-plots denote the average percentages of NK1.1+DX5+ NK cells derived from four independent samples after gating on CD3-negative cells at 48 h pi. (C) Frequency of IFN-γ or granzyme B-producing cells among NK cells. The production of IFN-γ and granzyme B by CD3−NK1.1+DX5+ NK cells was determined by intracellular staining after stimulation of vaginal NK cells with PMA plus ionomycin at 48 h pi. Values in histograms represent the average percentages of IFN-γ or granzyme B-producing cells among CD3−NK1.1+DX5+ NK cells. (D) Absolute number of IFN-γ or granzyme B-producing NK cells. Total number of IFN-γ or granzyme B-producing CD3−NK1.1+DX5+ NK cells in vaginal tract was enumerated by flow cytometric analysis using intracellular and surface staining at 48 h pi. (E) Secreted IFN-γ levels in vaginal tract. Secreted IFN-γ levels were determined by ELISA at 48 h pi using vaginal lavages. Data in bar chart represent the average ± SD of values derived from three individual experiments (n = 4–5). **, p<0.01; ***, p<0.001 compared with the levels of IFNAR KO mice.
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ppat.1005256.g003: IFN-I signaling is involved in connected recruitment and activation of NK cells to CD11b+Ly-6Chi monocytes.(A) NK cell infiltration of infected BL/6 or IFNAR KO mice. (B) Total accumulated NK cell number. Cells were prepared from vaginal tract (VT) and iliac LN (ILN) by collagenase digestion at 24, 48, and 72 h pi and used for analysis of NK cells. Values in dot-plots denote the average percentages of NK1.1+DX5+ NK cells derived from four independent samples after gating on CD3-negative cells at 48 h pi. (C) Frequency of IFN-γ or granzyme B-producing cells among NK cells. The production of IFN-γ and granzyme B by CD3−NK1.1+DX5+ NK cells was determined by intracellular staining after stimulation of vaginal NK cells with PMA plus ionomycin at 48 h pi. Values in histograms represent the average percentages of IFN-γ or granzyme B-producing cells among CD3−NK1.1+DX5+ NK cells. (D) Absolute number of IFN-γ or granzyme B-producing NK cells. Total number of IFN-γ or granzyme B-producing CD3−NK1.1+DX5+ NK cells in vaginal tract was enumerated by flow cytometric analysis using intracellular and surface staining at 48 h pi. (E) Secreted IFN-γ levels in vaginal tract. Secreted IFN-γ levels were determined by ELISA at 48 h pi using vaginal lavages. Data in bar chart represent the average ± SD of values derived from three individual experiments (n = 4–5). **, p<0.01; ***, p<0.001 compared with the levels of IFNAR KO mice.
Mentions: Together with CD11b+Ly-6Chi monocytes, NK cells are a critical innate cell population for providing protection against mucosal HSV-1 infection via production of IFN-γ [17–19]. Since CD11b+Ly-6Chi monocytes and CD11c+ DCs were shown to be consecutively recruited to the vaginal tract, we were also interested in the recruitment kinetics and activation of NK cells in IFNAR-ablated mice. Before virus infection, the frequency and number of CD3−NK1.1+DX5+ NK cells were comparable in vaginal tract and iliac LN of both BL/6 and IFNAR KO mice, except liver tissue (S3 Fig), which indicates that IFN-I signaling may not contribute to basal recruitment of NK cells in primary target tissues of HSV-1 mucosal infection. However, it was revealed that IFN-I signaling plays a crucial role in recruiting CD3−NK1.1+DX5+ NK cells into vaginal tract after HSV-1 infection. Notably, WT BL/6 mice displayed delayed recruitment of CD3−NK1.1+DX5+ NK cells in the vaginal tract with the levels peaking at 48 h pi, compared to CD11b+Ly-6Chi monocytes, and an increased frequency of NK cells, compared to IFNAR KO mice (Fig 3A). Also, iliac NK cell frequency was moderately increased in WT BL/6 mice compared to IFNAR KO mice. Supporting these findings, total accumulated number of CD3-NK1.1+DX5+ NK cells in vaginal tract and iliac LN was greater in WT BL/6 mice, with levels peaking at 48 h pi, than in IFNAR KO mice (Fig 3B). In addition, when the activation levels of NK cells were examined by analysis of NK cells producing IFN-γ and granzyme B, the ablation of IFN-I signal resulted in reduced production of IFN-γ and granzyme B from CD3−NK1.1+DX5+ NK cells (Fig 3C), and a decrease in the total number of NK cells producing IFN-γ or granzyme B (Fig 3D). Consistent with these findings, lower levels of IFN-γ protein in vaginal lavages of IFNAR KO mice were detected at 48 h pi compared to WT BL/6 mice (Fig 3E). Collectively, these results indicate that NK cell recruitment in vaginal tract follows that of CD11b+Ly-6Chi monocytes like CD11c+ DCs, and that IFN-I signal also has a critical contribution to the early concerted recruitment and activation of CD11b+Ly-6Chi monocytes and NK cells in mucosal tissues following HSV-1 infection.

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.

Show MeSH
Related in: MedlinePlus